WO2026128564A1

WO2026128564A1 - Methods and compositions for editing a gene associated with galactosemia

Info

Publication number: WO2026128564A1
Application number: PCT/US2025/058927
Authority: WO
Inventors: Ayan Banerjee; Tom FERNANDEZ
Original assignee: Beam Therapeutics Inc
Current assignee: Beam Therapeutics Inc
Priority date: 2024-12-11
Filing date: 2025-12-10
Publication date: 2026-06-18
Anticipated expiration: 2027-06-11

Abstract

Compositions and methods for treating galactosemia by introducing one or more alterations into a galactose-1-phosphate uridylyltransferase (GALT) polynucleotide in a cell. In particular embodiments, the disclosure provides a base editor system (e.g., a fusion protein or complex comprising a programable DNA binding protein, a nucleobase editor, and gRNA) for modifying a GALT polynucleotide, where the modification is associated with an increase in the level or activity of the GALT polypeptide encoded by the polynucleotide.

Description

ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

METHODS AND COMPOSITIONS FOR EDITING A GENE ASSOCIATED WITH GALACTOSEMIA

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U. S. Provisional Applications No. 63/730,807 filed December 11, 2024, and 63/853,947, filed July 30, 2025, the entire contents of each of which are hereby incorporated by reference in their entirety.

SEQUENCE LISTING

This application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The Sequence Listing XML file, created on December 10, 2025, is named 180802-047402PCT_SL.xml and is 1,335,782 bytes in size.

BACKGROUND

Galactosemia is characterized by an inability to metabolize galactose that results in build up of galactose and galactose metabolites (e.g., galactitol or galactose- 1-P) in a subject, which may be caused by deficient activity of galactose- 1 -phosphate uridylyltransferase (GALT).

Galactose is a component of lactose, which is the sugar found in milk. Untreated newborns having galactosemia experience vomiting, lethargyjaundice, cataracts, sepsis, multisystem organ failure, and/or death. In adults, galactosemia results in neurocognitive impairment, cataracts, liver failure, poor muscle tone, premature menopause in women, and motor disorders. Over 2,250 patients in north America suffer from galactosemia, and over 50 new patients are diagnosed with galactosemia each year. To date, the only treatment available for galactosemia is to avoid foods that contain galactose or lactose for life. Many galactosemia patients find it challenging to adhere to such a diet. Galactose is also endogenously produced via de novo synthesis; thus, dietary restriction alone is not sufficient to prevent the progression of long-term complications.

Therefore, there is a present need for improved compositions and methods for treatment of galactosemia.

SUMMARY

As described below, the disclosure features compositions and methods for treating galactosemia by introducing one or more alterations into a galactose- 1 -phosphate ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

uridylyltransferase (GALT) polynucleotide in a cell. In particular embodiments, the disclosure provides a base editor system (e.g., a fusion protein or complex comprising a programable DNA binding protein, a nucleobase editor, and gRNA) for modifying a GALT polynucleotide, where the modification is associated with an increase in activity of the GALT polypeptide encoded by the polynucleotide.

In one aspect, the disclosure provides a method of editing a nucleobase of a galactose- 1-phosphate uridylyltransferase (GALT) polynucleotide. The method involves contacting the GALT polynucleotide with a guide RNA, or a polynucleotide encoding the guide RNA, and a base editor containing a deaminase domain capable deaminating cytidine in DNA and a nucleic acid programmable DNA binding protein (napDNAbp) domain, or one or more polynucleotides encoding the base editor. The guide RNA targets the base editor to effect an alteration to a nucleobase in codon 188 of the GALT polynucleotide encoding an R amino acid. The alteration to the nucleobase changes codon 188 to a codon encoding a Q amino acid. Also, a) the deaminase domain contains an amino acid sequence with at least 85% sequence identity to a deaminase selected from one or more of RrA3f, T1.52, and 6b; and/or b) the guide RNA contains a spacer, where the spacer contains at least 19 contiguous nucleotides of a sequence selected from one or more of: CCCUUACCCGGCAGUGGGGG (SEQ ID NO: 699), UUACCCGGCAGUGGGGGUG (SEQ ID NO: 793), CCUUACCCGGCAGUGGGGGU (SEQ ID NO: 701), CUUACCCGGCAGUGGGGGUG (SEQ ID NO: 703), UUACCCGGCAGUGGGGGUGG (SEQ ID NO: 705), UACCCGGCAGUGGGGGUGGG (SEQ ID NO: 707), CUGACACCCUUACCCGGCAG (SEQ ID NO: 709), UGACACCCUUACCCGGCAGU (SEQ ID NO: 711), GACACCCUUACCCGGCAGUG (SEQ ID NO: 713), ACACCCUUACCCGGCAGUGG (SEQ ID NO: 715), CCCUUACCCGGCAGUGGGGGU (SEQ ID NO: 717), and ACCCGGCAGUGGGGGUGGGG (SEQ ID NO: 719).

In another aspect, the disclosure provides a method for producing a cell, the method involves editing a GALT polynucleotide in the cell according to the method of any aspect or embodiment of the disclosure.

In another aspect, the disclosure provides a method for treating galactosemia in a subject in need thereof. The subject contains a galactose- 1 -phosphate uridylyltransferase (GALT) polynucleotide encoding a GALT polypeptide having a Q188R mutation. The method involves administering to the subject a base editor system containing a guide RNA, or a polynucleotide encoding the guide RNA, and a base editor containing a deaminase domain capable of deaminating cytidine in DNA and a nucleic acid programmable DNA binding protein (napDNAbp) domain, or one or more polynucleotides encoding the base editor. The guide RNA ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

targets the base editor to effect an alteration to a nucleobase in codon 188 of the GALT polynucleotide, where the alteration to the nucleobase changes codon 188 to a codon encoding a Q amino acid. Also a) the deaminase domain contains an amino acid sequence with at least 85% sequence identity to a deaminase selected from one or more of RrA3f, T1.52, and 6b; and/or b) the guide RNA contains a spacer, where the spacer contains at least 19 contiguous nucleotides of a sequence selected from one or more of: CCCUUACCCGGCAGUGGGGG (SEQ ID NO: 699), UUACCCGGCAGUGGGGGUG (SEQ ID NO: 793), CCUUACCCGGCAGUGGGGGU (SEQ ID NO: 701), CUUACCCGGCAGUGGGGGUG (SEQ ID NO: 703), UUACCCGGCAGUGGGGGUGG (SEQ ID NO: 705), UACCCGGCAGUGGGGGUGGG (SEQ ID NO: 707), CUGACACCCUUACCCGGCAG (SEQ ID NO: 709), UGACACCCUUACCCGGCAGU (SEQ ID NO: 711), GACACCCUUACCCGGCAGUG (SEQ ID NO: 713), ACACCCUUACCCGGCAGUGG (SEQ ID NO: 715), CCCUUACCCGGCAGUGGGGGU (SEQ ID NO: 717), and ACCCGGCAGUGGGGGUGGGG (SEQ ID NO: 719).

In another aspect, the disclosure provides a base editor system. The base editor system contains a guide RNA, or a polynucleotide encoding the guide RNA, and a base editor containing a deaminase domain capable of deaminating cytidine in DNA and a nucleic acid programmable DNA binding protein (napDNAbp) domain, or one or more polynucleotides encoding the base editor. The guide RNA targets the base editor to effect an alteration to a nucleobase in codon 188 of a GALT polynucleotide encoding an R amino acid, where the alteration to the nucleobase changes codon 188 to a codon encoding a Q amino acid. Also a) the deaminase domain contains an amino acid sequence with at least 85% sequence identity to a deaminase selected from one or more of RrA3f, T1.52, and 6b; and/or b) the guide RNA contains a spacer, where the spacer contains at least 19 contiguous nucleotides of a sequence selected from one or more of: CCCUUACCCGGCAGUGGGGG (SEQ ID NO: 699), UUACCCGGCAGUGGGGGUG (SEQ ID NO: 793), CCUUACCCGGCAGUGGGGGU (SEQ ID NO: 701), CUUACCCGGCAGUGGGGGUG (SEQ ID NO: 703), UUACCCGGCAGUGGGGGUGG (SEQ ID NO: 705), UACCCGGCAGUGGGGGUGGG (SEQ ID NO: 707), CUGACACCCUUACCCGGCAG (SEQ ID NO: 709), UGACACCCUUACCCGGCAGU (SEQ ID NO: 711), GACACCCUUACCCGGCAGUG (SEQ ID NO: 713), ACACCCUUACCCGGCAGUGG (SEQ ID NO: 715), CCCUUACCCGGCAGUGGGGGU (SEQ ID NO: 717), and ACCCGGCAGUGGGGGUGGGG (SEQ ID NO: 719).

In another aspect, the disclosure provides a polynucleotide or set of polynucleotides encoding the base editor system of any aspect or embodiment of the disclosure, or a component thereof. ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

In another aspect, the disclosure provides a vector or set of vectors containing the base editor system, or the polynucleotide or set of polynucleotides of any aspect or embodiment of the disclosure.

In another aspect, the disclosure provides a lipid nanoparticle containing the base editor system, or the polynucleotide or set of polynucleotides of any aspect or embodiment of the disclosure.

In another aspect, the disclosure provides a cell containing the base editor system of any aspect or embodiment of the disclosure.

In another aspect, the disclosure provides a pharmaceutical composition containing an effective amount of the base editor system, the polynucleotide or set of polynucleotides, the vector or set of vectors, the lipid nanoparticle, the composition, the guide polynucleotide, or the cell of any aspect or embodiment of the disclosure, and a pharmaceutically acceptable excipient.

In another aspect, the disclosure provides a kit containing the base editor system of any aspect or embodiment of the disclosure, or a component thereof, the polynucleotide or set of polynucleotides of any aspect or embodiment of the disclosure, the vector or set of vectors of any aspect or embodiment of the disclosure, the lipid nanoparticle of any aspect or embodiment of the disclosure, the composition of any aspect or embodiment of the disclosure, the guide polynucleotide of any aspect or embodiment of the disclosure, or the pharmaceutical composition of any aspect or embodiment of the disclosure, disposed within a container.

In another aspect, the disclosure provides a guide polynucleotide containing a spacer or gRNA nucleotide sequence listed in Table 1.

In another aspect, the disclosure provides a method of editing a nucleobase of a galactose- 1 -phosphate uridylyltransferase (GALT) polynucleotide. The method involves contacting the GALT polynucleotide with a guide RNA and mRNA encoding a base editor. The base editor contains a deaminase domain containing a deaminase selected from one or more of RrA3f, T1.52, and 6b, and a nucleic acid programmable DNA binding protein (napDNAbp) domain. The napDNAbp is an SpRY, an SpRYc, an saCas9, an spCas9, or an spCas9 variant having specificity for a protospacer-adjacent motif (PAM) selected from one or more of: NG, NRRH, and NRTH, where “N” indicates A, C, G, or T, “R” indicates A or G, “Y” indicates C or T, and “H” indicates A, C, or T. The guide RNA targets (i.e., “directs”) the base editor to effect an alteration to a nucleobase in codon 188 of the GALT polynucleotide encoding an R amino acid. The alteration to the nucleobase changes codon 188 to a codon encoding a Q amino acid. The guide RNA contains a spacer where the spacer contains or contains only a sequence selected from one or more of: CCCUUACCCGGCAGUGGGGG (SEQ ID NO: 699), ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

UUACCCGGCAGUGGGGGUG (SEQ ID NO: 793), CCUUACCCGGCAGUGGGGGU (SEQ ID NO: 701), CUUACCCGGCAGUGGGGGUG (SEQ ID NO: 703), UUACCCGGCAGUGGGGGUGG (SEQ ID NO: 705), UACCCGGCAGUGGGGGUGGG (SEQ ID NO: 707), CUGACACCCUUACCCGGCAG (SEQ ID NO: 709), UGACACCCUUACCCGGCAGU (SEQ ID NO: 711), GACACCCUUACCCGGCAGUG (SEQ ID NO: 713), ACACCCUUACCCGGCAGUGG (SEQ ID NO: 715), CCCUUACCCGGCAGUGGGGGU (SEQ ID NO: 717), and ACCCGGCAGUGGGGGUGGGG (SEQ ID NO: 719).

In another aspect, the disclosure provides a base editor system containing a guide RNA and mRNA encoding a base editor. The base editor contains a deaminase domain containing a deaminase selected from one or more of RrA3f, T1.52, and 6b, and a nucleic acid programmable DNA binding protein (napDNAbp) domain. The napDNAbp is an SpRY, an SpRYc, an saCas9, an spCas9, or an spCas9 variant having specificity for a protospacer-adjacent motif (PAM) selected from one or more of: NG, NRRH, and NRTH, where “N” indicates A, C, G, or T, “R” indicates A or G, “Y” indicates C or T, and “H” indicates A, C, or T. The guide RNA targets the base editor to effect an alteration to a nucleobase in codon 188 of the GALT polynucleotide encoding an R amino acid, where the alteration to the nucleobase changes codon 188 to a codon encoding a Q amino acid. The guide RNA contains a spacer, where the spacer contains or contains only a sequence selected from one or more of: CCCUUACCCGGCAGUGGGGG (SEQ ID NO: 699), UUACCCGGCAGUGGGGGUG (SEQ ID NO: 793), CCUUACCCGGCAGUGGGGGU (SEQ ID NO: 701), CUUACCCGGCAGUGGGGGUG (SEQ ID NO: 703), UUACCCGGCAGUGGGGGUGG (SEQ ID NO: 705), UACCCGGCAGUGGGGGUGGG (SEQ ID NO: 707), CUGACACCCUUACCCGGCAG (SEQ ID NO: 709), UGACACCCUUACCCGGCAGU (SEQ ID NO: 711), GACACCCUUACCCGGCAGUG (SEQ ID NO: 713), ACACCCUUACCCGGCAGUGG (SEQ ID NO: 715), CCCUUACCCGGCAGUGGGGGU (SEQ ID NO: 717), and ACCCGGCAGUGGGGGUGGGG (SEQ ID NO: 719).

In another aspect, the disclosure provides a lipid nanoparticle containing the base editor system of any aspect or embodiment of the disclosure.

In another aspect, the disclosure provides a composition. The composition contains a guide RNA and mRNA encoding a base editor. The base editor contains a deaminase domain containing a deaminase selected from one or more of RrA3f, T1.52, and 6b, and a nucleic acid programmable DNA binding protein (napDNAbp) domain. The napDNAbp is an SpRY, an SpRYc, an saCas9, an spCas9, or an spCas9 variant having specificity for a protospacer-adjacent motif (PAM) selected from one or more of: NG, NRRH, and NRTH, where “N” indicates A, C, G, or T, “R” indicates A or G, “Y” indicates C or T, and “H” indicates A, C, or T. The guide ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

RNA targets the base editor to effect an alteration to a nucleobase in codon 188 of the GALT polynucleotide encoding an R amino acid, where the alteration to the nucleobase changes codon 188 to a codon encoding a Q amino acid. The guide RNA contains a spacer, where the spacer contains or contains only a sequence selected from one or more of:

CCCUUACCCGGCAGUGGGGG (SEQ ID NO: 699), UUACCCGGCAGUGGGGGUG (SEQ ID NO: 793), CCUUACCCGGCAGUGGGGGU (SEQ ID NO: 701), CUUACCCGGCAGUGGGGGUG (SEQ ID NO: 703), UUACCCGGCAGUGGGGGUGG (SEQ ID NO: 705), UACCCGGCAGUGGGGGUGGG (SEQ ID NO: 707), CUGACACCCUUACCCGGCAG (SEQ ID NO: 709), UGACACCCUUACCCGGCAGU (SEQ ID NO: 711), GACACCCUUACCCGGCAGUG (SEQ ID NO: 713), ACACCCUUACCCGGCAGUGG (SEQ ID NO: 715), CCCUUACCCGGCAGUGGGGGU (SEQ ID NO: 717), and ACCCGGCAGUGGGGGUGGGG (SEQ ID NO: 719).

In another aspect, the disclosure provides a lipid nanoparticle (LNP) composition containing a guide RNA and mRNA encoding a base editor. The base editor contains a deaminase domain contains a deaminase selected from one or more of RrA3f, T1.52, and 6b, and a nucleic acid programmable DNA binding protein (napDNAbp) domain. The napDNAbp is an SpRY, an SpRYc, an saCas9, an spCas9, or an spCas9 variant having specificity for a protospacer-adjacent motif (PAM) selected from one or more of: NG, NRRH, and NRTH, where “N” indicates A, C, G, or T, “R” indicates A or G, “Y” indicates C or T, and “H” indicates A, C, or T. The guide RNA targets the base editor to effect an alteration to a nucleobase in codon 188 of the GALT polynucleotide encoding an R amino acid, where the alteration to the nucleobase changes codon 188 to a codon encoding a Q amino acid. The guide RNA contains a spacer, where the spacer contains or contains only a sequence selected from one or more of: CCCUUACCCGGCAGUGGGGG (SEQ ID NO: 699), UUACCCGGCAGUGGGGGUG (SEQ ID NO: 793), CCUUACCCGGCAGUGGGGGU (SEQ ID NO: 701), CUUACCCGGCAGUGGGGGUG (SEQ ID NO: 703), UUACCCGGCAGUGGGGGUGG (SEQ ID NO: 705), UACCCGGCAGUGGGGGUGGG (SEQ ID NO: 707), CUGACACCCUUACCCGGCAG (SEQ ID NO: 709), UGACACCCUUACCCGGCAGU (SEQ ID NO: 711), GACACCCUUACCCGGCAGUG (SEQ ID NO: 713), ACACCCUUACCCGGCAGUGG (SEQ ID NO: 715), CCCUUACCCGGCAGUGGGGGU (SEQ ID NO: 717), and ACCCGGCAGUGGGGGUGGGG (SEQ ID NO: 719).

In another aspect, the disclosure provides a method of treatment. The method of treatment involves administering to a subject in need thereof, where the subject contains a galactose- 1 -phosphate uridylyltransferase (GALT) polynucleotide encoding a GALT polypeptide having a Q188R mutation, a lipid nanoparticle (LNP). The LNP contains a guide RNA and mRNA encoding a base editor. The base editor contains a deaminase domain containing a ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

deaminase selected from one or more of RrA3f, T1.52, and 6b, and a nucleic acid programmable DNA binding protein (napDNAbp) domain. The napDNAbp is an SpRY, an SpRYc, an saCas9, an spCas9, or an spCas9 variant having specificity for a protospacer-adjacent motif (PAM) selected from one or more of: NG, NRRH, and NRTH, where “N” indicates A, C, G, or T, “R” indicates A or G, “Y” indicates C or T, and “H” indicates A, C, or T. The guide RNA targets the base editor to effect an alteration to a nucleobase in codon 188 of the GALT polynucleotide encoding an R amino acid, where the alteration to the nucleobase changes codon 188 to a codon encoding a Q amino acid. The guide RNA contains a spacer, where the spacer contains or contains only a sequence selected from one or more of: CCCUUACCCGGCAGUGGGGG (SEQ ID NO: 699), UUACCCGGCAGUGGGGGUG (SEQ ID NO: 793), CCUUACCCGGCAGUGGGGGU (SEQ ID NO: 701), CUUACCCGGCAGUGGGGGUG (SEQ ID NO: 703), UUACCCGGCAGUGGGGGUGG (SEQ ID NO: 705), UACCCGGCAGUGGGGGUGGG (SEQ ID NO: 707), CUGACACCCUUACCCGGCAG (SEQ ID NO: 709), UGACACCCUUACCCGGCAGU (SEQ ID NO: 711), GACACCCUUACCCGGCAGUG (SEQ ID NO: 713), ACACCCUUACCCGGCAGUGG (SEQ ID NO: 715), CCCUUACCCGGCAGUGGGGGU (SEQ ID NO: 717), and ACCCGGCAGUGGGGGUGGGG (SEQ ID NO: 719).

In another aspect, the disclosure provides a lipid nanoparticle (LNP) containing a guide RNA, or a polynucleotide encoding the guide RNA, an mRNA molecule, or a polynucleotide encoding the mRNA molecule, and an ionizable lipid, or a pharmaceutically acceptable salt thereof. The guide RNA contains a spacer containing a nucleotide sequence selected from UUACCCGGCAGUGGGGGUGG (SEQ ID NO: 705); UUACCCGGCAGUGGGGGUG (SEQ ID NO: 793), and those listed in Table 1. The mRNA molecule encodes a base editor polypeptide containing a nucleic acid programmable DNA binding protein (napDNAbp) domain and a deaminase domain. The deaminase domain contains an amino acid sequence with at least 90% sequence identity to a deaminase selected from one or more of 6b, RrA3f, and T1.52. The ionizable lipid has any one of the following formulas:

i) a compound of Formula (I):

where:

each L¹ and L^1' is independently -C(O)- or -OC(O)-; ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

each L² and L^2' is independently an optionally substituted bivalent saturated or unsaturated, straight or branched C1-12 hydrocarbon chain;

L³ is a covalent bond, -O-, -C(O)O-, -OC(O)-, or -OC(O)O-;

O-R^a

i__LCyA__c A r^LRa~(

R¹ is optionally substituted C1-20 aliphatic, ‘ ^y, or O-R^a;

L^CyA is a covalent bond or an optionally substituted bivalent saturated or unsaturated, straight or branched C1-12 hydrocarbon chain;

Cy^A is an optionally substituted ring selected from 3- to 7-membered saturated or partially unsaturated carbocyclyl, 1-adamantyl, 2-adamantyl, sterolyl, and phenyl;

L^Ra is an optionally substituted bivalent saturated or unsaturated, straight or branched C1-12 hydrocarbon chain;

each R^a and R¹ is independently optionally substituted C1-20 aliphatic;

Y¹ is -C(O)- or -C(O)O-;

Y² is an optionally substituted bivalent saturated or unsaturated, straight or branched C1-6 hydrocarbon chain;

Y³ is optionally substituted C1-20 aliphatic;

X¹ is a covalent bond, -O-, or -NR-;

X² is an optionally substituted bivalent saturated or unsaturated, straight or branched C1-12 hydrocarbon chain, where 1-3 methylene units are optionally and independently replaced with -O- or -NR-;

X³ is hydrogen or an optionally substituted ring selected from 3- to 7- membered saturated or partially unsaturated carbocyclyl, phenyl, 3- to 7-membered heterocyclyl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or 5- to 6-membered heteroaryl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and

each R is independently hydrogen or optionally substituted C1-6 aliphatic;

ii) a compound of formula A’:

A³

O R

1 /Ll

y c) R

L²

R<.bk,/^R"

A’

or its N-oxide, or a pharmaceutically acceptable salt thereof, where

L¹ is absent, C1-6 alkylenyl, or C2-6 heteroalkylenyl; ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

each L² is independently optionally substituted C2-15 alkylenyl, or optionally substituted C3-15 heteroalkyl enyl;

L is Ci-io alkylenyl, or C2-10 heteroalkyl enyl;

X² is -OC(O)-, -C(O)O-, or -OC(O)O-;

X is absent, -OC(O)-, -C(O)O-, or -OC(O)O-;

O

|_3a y^ I o^R⁶

o^x ^R^a %

Ax³ _a

R” is hydrogen,

O R, O R⁶, or an optionally substituted group selected from C6-20 aliphatic, 3- to 12-membered cycloaliphatic, 7- to 12-membered bridged bicyclic containing 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 1- adamantyl, 2-adamantyl, sterolyl, and phenyl;

each of R and R^a is independently hydrogen, or an optionally substituted group selected from Ce- 20 aliphatic, 3- to 12-membered cycloaliphatic, 7- to 12-membered bridged bicyclic containing 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 1- adamantyl, 2-adamantyl, sterolyl, and phenyl

each of L³ and L^3a is independently absent, optionally substituted C1-10 alkylenyl, or optionally substituted C2-10 heteroalkylenyl;

R¹ is hydrogen, optionally substituted phenyl, optionally substituted 3- to 7-membered cycloaliphatic, optionally substituted 3- to 7-membered heterocyclyl containing 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, optionally substituted 5- to 6-membered monocyclic heteroaryl containing 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, optionally substituted 8- to 10-membered bicyclic heteroaryl containing 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, -OR², -C(O)OR², -C(O)SR², -OC(O)R², -OC(O)OR², -CN,

-N(R²)₂, -C(O)N(R²)₂, -S(O)₂N(R²)₂, -NR²C(O)R², -OC(O)N(R²)₂, -N(R²)C(O)OR², -NR²S(O)₂R², -NR²C(O)N(R²)2, -NR²C(S)N(R²)2, -NR²C(NR²)N(R²)2, -NR²C(CHR²)N(R²)2, -N(OR²)C(O)R², -N(OR²)S(O)₂R², -N(OR²)C(O)OR², -N(OR²)C(O)N(R²)2, -N(OR²)C(S)N(R²)2, -N(OR²)C(NR²)N(R²)2, -N(OR²)C(CHR²)N(R²)2, -C(NR²)N(R²)2, -C(NR²)R², -C(O)N(R²)OR², -C(R²)N(R²)₂C(O)OR², -CR²(R³)2, -OP(O)(OR²)2, or -P(O)(OR²)₂; or ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

R² R²

/ \

R

¹ is ° O, or a ring selected from 3- to 7-membered cycloaliphatic and 3- to 7- membered heterocyclyl containing 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, where the cycloaliphatic or heterocyclyl ring is optionally substituted with 1-4 R² or R³ groups;

each R² is independently hydrogen, oxo, -CN, -NO2, -OR⁴, -S(O)2R⁴, -S(O)2N(R⁴)2, -(CH2)n-R⁴, or an optionally substituted group selected from C1-6 aliphatic, phenyl, 3- to 7-membered cycloaliphatic, 5- to 6-membered monocyclic heteroaryl containing 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and 3- to 7-membered heterocyclyl containing 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; or two occurrences of R², taken together with the atom(s) to which they are attached, form optionally substituted 4- to 7-membered heterocyclyl containing 0-1 additional heteroatom selected from nitrogen, oxygen, and sulfur;

each R³ is independently -(CH2)n-R⁴; or

two occurrences of R³, taken together with the atom(s) to which they are attached, form optionally substituted 5- to 6-membered heterocyclyl containing 0-1 additional heteroatom selected from nitrogen, oxygen, and sulfur;

each R⁴ is independently hydrogen, -OR⁵, -N(R⁵)2, -OC(O)R⁵, -OC(O)OR⁵, -CN, -C(O)N(R⁵)2, -NR⁵C(O)R⁵, -OC(O)N(R⁵)2, -N(R⁵)C(O)OR⁵, -NR⁵S(O)₂R⁵, -NR⁵C(O)N(R⁵)2,

R⁵ R⁵

/ \

R⁵ *

-Z —

-

NR⁵C(S)N(R⁵)2, -NR⁵C(NR⁵)N(R⁵)2, or O O ■

each R⁵ is independently hydrogen, or optionally substituted C1-6 aliphatic; or

two occurrences of R⁵, taken together with the atom(s) to which they are attached, form optionally substituted 4- to 7-membered heterocyclyl containing 0-1 additional heteroatom selected from nitrogen, oxygen, and sulfur;

each R⁶ is independently C4-12 aliphatic; and

each n is independently 0 to 4; or

x) a compound of Formula I: ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

or a pharmaceutically acceptable salt thereof, where:

L¹ is a covalent bond, -C(O)-, or -OC(O)-;

L² is a covalent bond, an optionally substituted bivalent saturated or unsaturated, straight or HH-^cy^A-H

branched C1-C12 hydrocarbon chain, or ^{m m};

Cy^A is an optionally substituted ring selected from phenylene and 3- to 7-membered saturated or partially unsaturated carbocyclene;

each m is independently 0, 1, or 2;

L³ is a covalent bond, -C(O)-, -C(O)O-, -OC(O)-, -O-, or -OC(O)O-;

R, is H4 ^p-C'^{, B}, an optionally substituted saturated or unsaturated, straight or branched

C1-C20 hydrocarbon chain where 1-3 methylene units are optionally and independently

, O-A¹

H^L ₂

replaced with -O- or -NR-, or O^_A;

Cy^B is an optionally substituted ring selected from 3- to 12-membered saturated or partially

unsaturated carbocyclyl, 1-adamantyl, 2-adamantyl,

, sterolyl, and phenyl; p is 0, 1, 2, or 3;

each L⁴ is independently a bivalent saturated or unsaturated, straight or branched Ci-Ce hydrocarbon chain;

each A¹ and A² is independently an optionally substituted C1-C20 aliphatic or -L⁵-R⁵;

or A¹ and A², together with their intervening atoms, may form an optionally substituted ring:

^#K2) X.

where

x is selected from 1 or 2; and

# represents the point of attachment to L⁴;

each L⁵ is independently a bivalent saturated or unsaturated, straight or branched C1-C20 hydrocarbon chain, where 1-3 methylene units are optionally and independently replaced with -O- or -NR-; ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

each R⁵ is independently an optionally substituted group selected from a 5- to 10-membered aryl ring or a 3- to 8-membered carbocyclic ring;

X¹ is a covalent bond, -O-, or -NR-;

X² is a covalent bond or an optionally substituted, bivalent saturated or unsaturated, straight or branched, C1-C12 hydrocarbon chain, where 1-3 methylene units are optionally and independently replaced with -O-, -NR-, or -Cy^c-;

Cy^c is an optionally substituted ring selected from 3- to 7- membered saturated or partially unsaturated carbocyclene, phenylene, 3- to 7-membered saturated or partially unsaturated heterocyclene having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and 5- to 6-membered heteroarylene having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur;

X³ is hydrogen or an optionally substituted ring selected from 3- to 7-membered saturated or partially unsaturated carbocyclyl, phenyl, 3- to 7-membered saturated or partially unsaturated heterocyclyl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or 5- to 6-membered heteroaryl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and

each R is independently hydrogen or an optionally substituted Ci-Ce aliphatic group;

FH-cy^B

provided that when L is a covalent bond, then R must be ^p

In another aspect, the disclosure provides a lipid nanoparticle (LNP) containing a guide RNA, or a polynucleotide encoding the guide RNA, an mRNA molecule, or a polynucleotide encoding the mRNA molecule, and an ionizable lipid, or a pharmaceutically acceptable salt thereof. The guide RNA is capable of directing a base editor polypeptide to alter a nucleotide in a galactose- 1 -phosphate uridylyltransferase (GALT) polynucleotide. The mRNA molecule encodes a base editor polypeptide containing a nucleic acid programmable DNA binding protein (napDNAbp) domain and a deaminase domain, where the deaminase domain containing an amino acid sequence with at least 90% sequence identity to a deaminase selected from one or more of 6b, RrA3f, and T1.52. The an ionizable lipid has Formula (I):

where:

ELECTRONIC DEPOSIT DATE: December 10, 2025

L³ is a covalent bond, -O-, -C(O)O-, -OC(O)-, or -OC(O)O-;

O-R^a

i__LCyA__c A r^LRa~(

R¹ is optionally substituted C1-20 aliphatic, ‘ ^y, or O-R^a;

each R^a and R¹ is independently optionally substituted C1-20 aliphatic;

Y¹ is -C(O)- or -C(O)O-;

Y³ is optionally substituted C1-20 aliphatic;

X¹ is a covalent bond, -O-, or -NR-;

each R is independently hydrogen or optionally substituted C1-6 aliphatic.

In another aspect, the disclosure provides a lipid nanoparticle (LNP) containing a guide RNA, a base editor polypeptide, or a polynucleotide encoding the base editor polypeptide, and an ionizable lipid, or a pharmaceutically acceptable salt thereof. The guide RNA has a nucleotide sequence selected from: mUsmUsmAsCCCGGCAGUGGGGGUGGGUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmCmC mGmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmC mAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 746); and mUsmUsmAsCCCGGCAGUGGGGGUGGUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmCmCm GmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCm ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025 AmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 791), where “mN” indicates a 2'-OMe modification of the nucleotide “N”, and “Ns” indicates that the nucleotide “N” is linked to the following nucleotide by a phosphorothioate (PS). The base editor polypeptide contains a nucleic acid programmable DNA binding protein (napDNAbp) domain and a deaminase domain containing a deaminase domain containing a deaminase selected from one or more of 6b, RrA3f, and T1.52. The ionizable lipid has the following structure:

H

. CL. N

In another aspect, the disclosure provides a method for modifying a target nucleobase in a galactose- 1 -phosphate uridylyltransferase (GALT) polynucleotide in a cell. The method involves contacting the cell with the lipid nanoparticle, the base editor system, or the polynucleotide or set of polynucleotides of any aspect of the disclosure, or embodiments thereof, thereby modifying the target nucleobase in the GALT polynucleotide.

In another aspect, the disclosure provides a method of treating a disease in a subject in need thereof. The disease is associated with a pathogenic mutation in a galactose- 1 -phosphate uridylyltransferase (GALT) polynucleotide in the subject. The method involves administering to the subject the lipid nanoparticle, the base editor system, or the polynucleotide or set of polynucleotides of any aspect of the disclosure, or embodiments thereof, thereby altering a target nucleobase in the GALT polynucleotide in the subject.

In any aspect or embodiment of the disclosure, the guide RNA contains a spacer, where the spacer contains at least 19 contiguous nucleotides of a sequence selected from one or more of: CCCUUACCCGGCAGUGGGGG (SEQ ID NO: 699), UUACCCGGCAGUGGGGGUG (SEQ ID NO: 793), CCUUACCCGGCAGUGGGGGU (SEQ ID NO: 701), CUUACCCGGCAGUGGGGGUG (SEQ ID NO: 703), UUACCCGGCAGUGGGGGUGG (SEQ ID NO: 705), UACCCGGCAGUGGGGGUGGG (SEQ ID NO: 707), CUGACACCCUUACCCGGCAG (SEQ ID NO: 709), UGACACCCUUACCCGGCAGU (SEQ ID NO: 711), GACACCCUUACCCGGCAGUG (SEQ ID NO: 713), ACACCCUUACCCGGCAGUGG (SEQ ID NO: 715), CCCUUACCCGGCAGUGGGGGU (SEQ ID NO: 717), and ACCCGGCAGUGGGGGUGGGG (SEQ ID NO: 719). In any aspect or embodiment of the disclosure, the guide RNA contains a spacer, where the spacer contains only a sequence selected from one or more of: CCCUUACCCGGCAGUGGGGG (SEQ ID NO: 699), UUACCCGGCAGUGGGGGUG (SEQ ID NO: 793), CCUUACCCGGCAGUGGGGGU (SEQ ID NO: ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

701), CUUACCCGGCAGUGGGGGUG (SEQ ID NO: 703), UUACCCGGCAGUGGGGGUGG (SEQ ID NO: 705), UACCCGGCAGUGGGGGUGGG (SEQ ID NO: 707), CUGACACCCUUACCCGGCAG (SEQ ID NO: 709), UGACACCCUUACCCGGCAGU (SEQ ID NO: 711), GACACCCUUACCCGGCAGUG (SEQ ID NO: 713), ACACCCUUACCCGGCAGUGG (SEQ ID NO: 715), CCCUUACCCGGCAGUGGGGGU (SEQ ID NO: 717), and ACCCGGCAGUGGGGGUGGGG (SEQ ID NO: 719). In any aspect or embodiment of the disclosure, the guide RNA contains a spacer, where the spacer contains only a sequence selected from one or more of:

UACCCGGCAGUGGGGGUGGG(SEQ ID NO: 707), UUACCCGGCAGUGGGGGUG (SEQ ID NO: 793), ACACCCUUACCCGGCAGUGG (SEQ ID NO: 715), CUUACCCGGCAGUGGGGGUG (SEQ ID NO: 703), and UUACCCGGCAGUGGGGGUGG (SEQ ID NO: 705). In any aspect or embodiment of the disclosure, the guide RNA contains a sequence selected from one or more of:

End-mod spCas9 guide polynucleotide mNsmNsmNsNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAG UCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsmUsmUsmU (SEQ ID NO: 765); End-mod saCas9 guide polynucleotide mNsmNsmNsNNNNNNNNNNNNNNNNNNGUUUUAGUACUCUGUAAUGAAAAUUACAGAAUCUACU AAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUsmUsmUsmU (SEQ ID NO: 766); and

LONGEST: mNsmNsmNsNNNNNNNNNNNNNNNNNGUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmCmC mGmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmC mAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 769), where “N” represents any nucleotide, “mN” indicates a 2'-OMe modification of the nucleotide “N”, and “Ns” indicates that the nucleotide “N” is linked to the following nucleotide by a phosphorothioate (PS), and where the number of N nucleotides is between 19 and 25. In any aspect or embodiment of the disclosure, the guide RNA contains a sequence selected from one or more of

mC smC smC sUUACCCGGCAGUGGGGGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAG UCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCmUsmUsmUsU (SEQ ID NO: 728); mC smC smUsUACCCGGCAGUGGGGGUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAG UCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCmUsmUsmUsU (SEQ ID NO: 729); mCsmUsmUsACCCGGCAGUGGGGGUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAG UCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCmUsmUsmUsU (SEQ ID NO: 730); ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025 mUsmUsmAsCCCGGCAGUGGGGGUGGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAG UCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCmUsmUsmUsU (SEQ ID NO: 731); mUsmAsmC s CCGGCAGUGGGGGUGGGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAG UCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCmUsmUsmUsU (SEQ ID NO: 732); mCsmUsmGsACACCCUUACCCGGCAGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAG UCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCmUsmUsmUsU (SEQ ID NO: 733); mUsmGsmAsCACCCUUACCCGGCAGUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAG UCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCmUsmUsmUsU (SEQ ID NO: 734); mGsmAsmCsACCCUUACCCGGCAGUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAG UCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCmUsmUsmUsU (SEQ ID NO: 735); mAsmC smAs CCCUUACCCGGCAGUGGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAG UCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCmUsmUsmUsU (SEQ ID NO: 736); mC smC smC sUUACCCGGCAGUGGGGGUGUUUUAGUACUCUGUAAUGAAAAUUACAGAAUCUACU AAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUsmUsmUsmU (SEQ ID NO: 737);

mAsmC smC s CGGCAGUGGGGGUGGGGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAG UCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCmUsmUsmUsU (SEQ ID NO: 738); mA s mC s mA s C C CUUAC C CGGC AGUGGGUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmCmC mGmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmC mAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 739); mCsmCsmCsUUACCCGGCAGUGGGGGGUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmCmC mGmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmC mAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 740); mCsmCsmUsUACCCGGCAGUGGGGGUGUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmCmC mGmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmC mAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 741);

mC s mU s mG s AC AC C CUUAC C CGGC AGGUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmCmC mGmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmC mAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 742); mCsmUsmUsACCCGGCAGUGGGGGUGGUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmCmC mGmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmC mAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 743); ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

mG s mA s mC s C C CUUAC C CGGC AGUGGUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmCmC mGmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmC mAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 744); mUsmGsmAsCACCCUUACCCGGCAGUGUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmCmC mGmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmC mAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 745); mUsmUsmAsCCCGGCAGUGGGGGUGGGUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmCmC mGmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmC mAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 746); and mUsmUsmAsCCCGGCAGUGGGGGUGGUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmCmCm GmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCm AmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 791);

where “mN” indicates a 2'-OMe modification of the nucleotide “N”, and “Ns” indicates that the nucleotide “N” is linked to the following nucleotide by a phosphorothioate (PS). In any aspect or embodiment of the disclosure, the guide RNA contains a sequence selected from one or more of: mUsmAsmC s CCGGCAGUGGGGGUGGGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAG UCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCmUsmUsmUsU (SEQ ID NO: 732); mA s mC s mA s C C CUUAC C CGGC AGUGGGUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmCmC mGmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmC mAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 739); mCsmUsmUsACCCGGCAGUGGGGGUGGUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmCmC mGmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmC mAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 743); mUsmUsmAsCCCGGCAGUGGGGGUGGUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmCmCm GmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCm AmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 791); and mUsmUsmAsCCCGGCAGUGGGGGUGGGUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmCmC mGmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmC mAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 746); where “mN” indicates a 2'-OMe modification of the nucleotide “N”, and “Ns” indicates that the nucleotide “N” is linked to the following nucleotide by a phosphorothioate (PS).

In any aspect or embodiment of the disclosure, the deaminase domain is inserted within the napDNAbp domain. In any aspect or embodiment of the disclosure, the deaminase is inserted between amino acid positions 906 and 907 of the napDNAbp referenced to SEQ ID NO: 197. ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

In any aspect or embodiment of the disclosure, the napDNAbp has specificity for a protospacer-adjacent motif (PAM) selected from one or more of: NGG, NNGRRT, NRTH, NRN, NG, or RY, where “N” indicates A, C, G, or T, “R” indicates A or G, “Y” indicates C or T, and “H” indicates A, C, or T. In any aspect or embodiment of the disclosure, the napDNAbp is a Cas9. In any aspect or embodiment of the disclosure, the Cas9 is spCas9, saCas9, or a variant thereof. In any aspect or embodiment of the disclosure, the napDNAbp is SpRY or SpRYc. In any aspect or embodiment of the disclosure, the napDNAbp is a nickase. In any aspect or embodiment of the disclosure, the nickase contains a D10A amino acid alteration referenced to SEQ ID NO: 197. In any aspect or embodiment of the disclosure, the Cas9 contains an amino acid sequence selected from one or more of:

spCas9 DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLK RTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYH EKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYN QLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFD LAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASM IKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDG TEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIP YYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSL LYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAH LFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFK EDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQT TQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRL SDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKF DNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSK LVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKS EQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSM PQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGK SKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLI IKLPKYSLFELENGRKRMLASA GELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEI IEQISEFSKRVI LADANLDKVLSAYNKHRDKPIREQAENI IHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDA TLIHQSITGLYETRIDLSQLGGD (SEQ ID NO: 783);

saCas9 ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

KRNY I LGLD I G I TS VGYG I ID YETRDVIDAGVRLFKEANVENNEGRRS KRGARRLKRRRRHR I Q RVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTG NELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQL DQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYN ALNDLNNLVITRDENEKLEYYEKFQI IENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKP EFTNLKVYHDIKDITARKEI IENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISN LKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPV VKRS F I QS I KV I NA 11 KKYGL PND HIE LARE KNS KDAQKM I NEMQKRNRQTNER I E E 11 RTTG KENAKYL I EKI KLHDMQEGKCL YS LEAI PLEDLLNNPFNYE VDH 11 PRS VS FDNS FNNKVLVKQ EENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDF INRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDA LI IANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDY KYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHD PQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDY PNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAE FIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRI IKTIASKTQ S IKKYSTDILGNLYEVKSKKHPQI IKKG (SEQ ID NO: 784);

Nureki DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLK RTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYH EKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYN QLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFD LAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASM IKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDG TEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIP YYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSL LYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAH LFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFK EDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQT TQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRL SDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKF DNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSK LVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKS EQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSM ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025 PQVNIVKKTEVQTGGFSKESIRPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGK SKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLI IKLPKYSLFELENGRKRMLASA RFLQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEI IEQISEFSKRVI LADANLDKVLSAYNKHRDKPIREQAENI IHLFTLTNLGAPRAFKYFDTTIDRKVYRSTKEVLDA TLIHQSITGLYETRIDLSQLGGD (SEQ ID NO: 785);

SpRYc EKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTNRKSIKKNLMGALLFDSGETAEATRLK RTARRRYTRRKNRIRYLQEIFANEMAKLDDSFFQRLEESFLVEEDKKNERHPIFGNLADEVAYH RNYPTIYHLRKKLADSPEKADLRLIYLALAHI IKFRGHFLIEGKLNAENSDVAKLFYQLIQTYN QLFEESPLDEIEVDAKGILSARLSKSKRLEKLIAVFPNEKKNGLFGNI IALALGLTPNFKSNFD LTEDAKLQLSKDTYDDDLDELLGQIGDQYADLFSAAKNLSDAILLSDILRSNSEVTKAPLSASM VKRYDEHHQDLALLKTLVRQQFPEKYAEIFKDDTKNGYAGYVGADKKLRKRSGKLATEEEFYKF IKPILEKMDGAEELLAKLNRDDLLRKQRTFDNGSIPHQIHLKELHAILRRQEEFYPFLKENREK IEKILTFRIPYYVGPLARGNSRFAWLTRKSEEAITPWNFEEVVDKGASAQSFIERMTNFDEQLP NKKVLPKHSLLYEYFTVYNELTKVKYVTERMRKPEFLSGEQKKAIVDLLFKTNRKVTVKQLKED YFKKIECFDSVEI IGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVLTLTLFEDREM IEERLKTYAHLFDDKVMKQLKRRHYTGWGRLSRKMINGIRDKQSGKTILDFLKSDGFSNRNFMQ LIHDDSLTFKEEIEKAQVSGQGDSLHEQIADLAGSPAIKKGILQTVKIVDELVKVMGHKPENIV IEMARENQTTTKGLQQSRERKKRIEEGIKELESQILKENPVENTQLQNEKLYLYYLQNGRDMYV DQELDINRLSDYDVDHIVPQSFIKDDSIDNKVLTRSVENRGKSDNVPSEEVVKKMKNYWRQLLN AKL I TQRKFDNLTKAERGGLS EADKAGF I KRQLVETRQ I TKHVAR I LDSRMNTKRDKNDKP I RE VKVITLKSKLVSDFRKDFQLYKVRDINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVY DVRKMIAKSEQEIGKATAKRFFYSNIMNFFKTEVKLANGEIRKRPLIETNGETGEVVWNKEKDF ATVRKVLAMPQVNIVKKTEVQTGGFSKESIRPKRNSDKLIARKKDWDPKKYGGFLWPTVAYSVL VVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLI IKLPKYSLFELEN GRKRMLASAKQLQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEI IEQ ISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENI IHLFTLTRLGAPRAFKYFDTTIDPKQY RSTKEVLDATLIHQSITGLYETRIDLSQLGGD (SEQ ID NO: 786);

SpRY DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAERTRLK RTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYH EKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYN QLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFD LAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASM IKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDG ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025 TEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIP YYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSL LYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAH LFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFK EDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQT TQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRL SDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKF DNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSK LVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKS EQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSM PQVNIVKKTEVQTGGFSKESIRPKRNSDKLIARKKDWDPKKYGGFLWPTVAYSVLVVAKVEKGK SKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLI IKLPKYSLFELENGRKRMLASA KQLQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEI IEQISEFSKRVI LADANLDKVLSAYNKHRDKPIREQAENI IHLFTLTRLGAPRAFKYFDTTIDPKQYRSTKEVLDA TLIHQSITGLYETRIDLSQLGGD (SEQ ID NO: 787);

spCas9 NRRH DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLK RTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYH EKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYN QLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFD LAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASM VKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDG TEELLVKLNREDLLRKQRTFDNGI IPHQIHLGELHAILRRQGDFYPFLKDNREKIEKILTFRIP YYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSL LYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAH LFDDKVMKQLKRLRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFK EDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGGHKPENIVIEMARENQT TQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRL SDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKF DNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSK LVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKS EQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSM PQVNIVKKTEVQTGGFSKESILPKGNSDKLIARKKDWDPKKYGGFNSPTAAYSVLVVAKVEKGK ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025 SKKLKSVKELLGITIMERSSFEKNPIGFLEAKGYKEVKKDLI IKLPKYSLFELENGRKRMLASA GVLHKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEI IEQISEFSKRVI LADANLDKVLSAYNKHRDKPIREQAENI IHLFTLTNLGVPAAFKYFDTTIDKKRYTSTKEVLDA TLIHQSITGLYETRIDLSQLGGD (SEQ ID NO: 788); and

spCas9 NRTH DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLK RTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYH EKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYN QLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFD LAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASM VKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDG TEELLVKLNREDLLRKQRTFDNGI IPHQIHLGELHAILRRQGDFYPFLKDNREKIEKILTFRIP YYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSL LYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAH LFDDKVMKQLKRLRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFK EDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGGHKPENIVIEMARENQT TQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRL SDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKF DNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSK LVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKS EQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSM PQVNIVKKTEVQTGGFSKESILPKGNSDKLIARKKDWDPKKYGGFNSPTVAYSVLVVAKVEKGK SKKLKSVKELLGITIMERSSFEKNPIGFLEAKGYKEVKKDLI IKLPKYSLFELENGRKRMLASA SVLHKGNELALPSKYVNFLYLASHYEKLKGSSEDNKQKQLFVEQHKHYLDEI IEQISEFSKRVI LADANLDKVLSAYNKHRDKPIREQAENI IHLFTLTNLGASAAFKYFDTTIGRKLYTSTKEVLDA TLIHQSITGLYETRIDLSQLGGD (SEQ ID NO: 789).

In any aspect or embodiment of the disclosure, the base editor further contains two uracil glycosylase inhibitor (UGI) domains. In any aspect or embodiment of the disclosure, the base editor further contains a nuclear localization signal (NLS).

In any aspect or embodiment of the disclosure, the nucleobase in codon 188 corresponds to the lowercase “c” in the following nucleotide sequence:

CTGACACCCTTACCcGGCAGTGGGGGTGGGGGTTAG (SEQ ID NO: 692).

In any aspect or embodiment of the disclosure, the deaminase domain contains an amino acid sequence with at least 90% sequence identity to a sequence selected from one or more of: ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

T1.52 SEVEYSHEYWMRHALTLAKRARDERHVPVGAVLVLNNRVIGEGWNRAKGLHDPTAHAEIMALRQ GGLVMQNYRLWGATL YTTFE PCVMCAGAM IHSR I GRVVFGVCNAKTHACMS LMDVLGHPGMPHR VEITEGILADECEALLCRFFRMPRRVFNAQKKAQSSTD (SEQ ID NO: 773);

6b SSEVEFSHEYWMRHALTLAKRARDEGEAPVGAVLVLNNRVIGEGWVRRIGLHDPTAHAEIMALR QGGLVMQNPRL IDATLYVTFE PCVMCAGAM INSR I GRVVFGVRNS KRGAAGS LMNVLNYPGMNH RVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN (SEQ ID NO: 790); and

RrA3f

KPQIRDHRPNPMEAMYPHIFYFHFENLEKAYGRNETWLCFTVEI IKQYLPVPWKKGVFRNQVDP ETHCHAEKCFLSWFCNNTLSPKKNYQVTWYTSWSPCPECAGEVAEFLAEHSNVKLTIYTARLYY FWDTDYQEGLRSLSEEGASVEIMDYEDFQYCWENFVYDDGEPFKRWKGLKYNFQSLTRRLREIL

Q (Positions 2 to 193 of SEQ ID NO: 67).

In any aspect or embodiment of the disclosure, the deaminase domain contains an amino acid sequence selected from one or more of SEQ ID NOs: 773, 790, and 67.

In any aspect or embodiment of the disclosure, the base editor contains, in order from N-terminus to C-terminus, the deaminase domain, a peptide linker, and the napDNAbp domain, where the deaminase domain is separated from the napDNAbp domain in the base editor by less than about 15 amino acids. In any aspect or embodiment of the disclosure, the peptide linker contains or contains only an amino acid sequence selected from one or more of:

EGSSSKEEEEPG (SEQ ID NO: 647), NSISSSNGQK (SEQ ID NO: 648), GEEGEGSGGGEK (SEQ ID NO: 649), EGEGGKESGSSE (SEQ ID NO: 650), GGGGSSKSPGSE (SEQ ID NO: 651), PIGSDQDD (SEQ ID NO: 652), TEKGQVPHGS (SEQ ID NO: 653), ESGEGGGGSEKK (SEQ ID NO: 654), EEGKPKEGEGSG (SEQ ID NO: 655), ASREPKDSS (SEQ ID NO: 656), KQGSEHDE (SEQ ID NO: 657), SESKSEKGSSEK (SEQ ID NO: 658), QYDSGERSDQ (SEQ ID NO: 659), PGANEEIPGQ (SEQ ID NO: 660), NSPTDEK (SEQ ID NO: 661), EGANEEIPGQ (SEQ ID NO: 662), EGEKEKKKSGES (SEQ ID NO: 663), PGRHEEVPGQ (SEQ ID NO: 664), SKHQTEQDDS (SEQ ID NO: 665), ESEDDSSGRK (SEQ ID NO: 666), KESEKKESESKS (SEQ ID NO: 667), KGEGKSSIKD (SEQ ID NO: 668), DRSQKQDQQD (SEQ ID NO: 669), GPSSTSSS (SEQ ID NO: 670), GSSGEKEEGEPS (SEQ ID NO: 671), GEPKSKKSGSGS (SEQ ID NO: 672), SSGEGGKSESGP (SEQ ID NO: 673), SPQPTSSD (SEQ ID NO: 674), EGGSEEEEESGS (SEQ ID NO: 675), KGPKPKKEESEK (SEQ ID NO: 676), SKSQQFVTYE (SEQ ID NO: 677), TGNSKYQTGK (SEQ ID NO: 678), PQPIPHTNPT (SEQ ID NO: 679), ANAHSDISTG (SEQ ID NO: 680), KSQQTEDQSK (SEQ ID NO: 681), QSQDQKQKEH (SEQ ID NO: 682), NQQRPSSD ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

(SEQ ID NO: 683), TTKDTSPKPQ (SEQ ID NO: 684), EGKDNQQTGE (SEQ ID NO: 685), and EPQPDSSE (SEQ ID NO: 686).

In any aspect or embodiment of the disclosure, the base editor contains an amino acid sequence having at least 85% or 90% identity to a sequence selected from one or more of: MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRL KRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAY HEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTY NQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNF DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMD GTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRI PYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHS LLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTF KEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQ TTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINR LSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK FDNLTKAERGGSSGSETPGTSESATPESSGSEVEYSHEYWMRHALTLAKRARDERHVPVGAVLV LNNRVIGEGWNRAKGLHDPTAHAEIMALRQGGLVMQNYRLWGATLYTTFEPCVMCAGAMIHSRI GRVVFGVCNAKTHACMSLMDVLGHPGMPHRVEITEGILADECEALLCRFFRMPRRVFNAQKKAQ SSTDGSSGSGSSGSGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVI TLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVR KVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAK VEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLI IKLPKYSLFELENGRKR MLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEI IEQISEF SKRVILADANLDKVLSAYNKHRDKPIREQAENI IHLFTLTNLGAPAAFKYFDTTIDRKRYTSTK EVLDATLIHQSITGLYETRIDLSQLGGDSGGSGGSGGSTNLSDIIEKETGKQLVIQESILMLPE EVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSGGS GGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDA PEYKPWALVIQDSNGENKIKMLEGADKRTADGSEFESPKKKRKV (SEQ ID NO: 774);

MKRTADGSEFESPKKKRKVSSEVEFSHEYWMRHALTLAKRARDEGEAPVGAVLVLNNRVIGEGW VRRIGLHDPTAHAEIMALRQGGLVMQNPRLIDATLYVTFEPCVMCAGAMINSRIGRVVFGVRNS KRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSINSGGSSG ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025 GSSGSETPGTSESATPESSGGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTD RHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEE SFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGH FLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPG EKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKN LSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGY AGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAIL RRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGAS AQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDL LFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDI LEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTI LDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKV VDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQ NEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP S EE VVKKMKNYWRQLLNAKL I TQRKFDNLTKAERGGLS ELDKAGF I KRQLVETRQ I TKHVAQ I L DSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIK KYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLI ETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWD PKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVK KDLI IKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQK QLFVEQHKHYLDEI IEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENI IHLFTLTNLG APAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSGGSGGSTNLSD I IEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWAL VIQDSNGENKIKMLSGGSGGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDI LVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSKRTADGSEFESPKKKRK

V (SEQ ID NO: 775);

MKRTADGSEFESPKKKRKVMKPQIRDHRPNPMEAMYPHIFYFHFENLEKAYGRNETWLCFTVEI IKQYLPVPWKKGVFRNQVDPETHCHAEKCFLSWFCNNTLSPKKNYQVTWYTSWSPCPECAGEVA EFLAEHSNVKLTIYTARLYYFWDTDYQEGLRSLSEEGASVEIMDYEDFQYCWENFVYDDGEPFK RWKGLKYNFQSLTRRLREILQSGGSSGGSSGSETPGTSESATPESSGGSSGGSDKKYSIGLAIG TNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRK NRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRK KLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSK DTYDDDLDNLLAQ I GDQ YADLFLAAKNLSDAI LLSD I LRVNTE I TKAPLS ASM I KRYDEHHQDL ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025 TLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRE DLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNS RFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNEL TKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFN ASLGTYHDLLKI IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLK RRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQ GDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRER MKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQ SFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGL SELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQF YKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKY FFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV QTGGFSKESIRPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELL GITIMERSSFEKNPIDFLEAKGYKEVKKDLI IKLPKYSLFELENGRKRMLASARFLQKGNELAL PS KYVNFL YLASHYEKLKGS PEDNEQKQLFVEQHKHYLDE 11 EQ I S E FS KRVI LADANLDKVLS AYNKHRDKPIREQAENIIHLFTLTNLGAPRAFKYFDTTIDRKVYRSTKEVLDATLIHQSITGLY ETRIDLSQLGGDSGGSGGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILV HTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSGGSGGSTNLSDI IEKETGK QLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGE NKIKMLSGGSKRTADGSEFESPKKKRKV (SEQ ID NO: 776);

MKPQIRDHRPNPMEAMYPHIFYFHFENLEKAYGRNETWLCFTVEI IKQYLPVPWKKGVFRNQVD PETHCHAEKCFLSWFCNNTLSPKKNYQVTWYTSWSPCPECAGEVAEFLAEHSNVKLTI YEARLY YFWDTDYQEGLRSLSEEGASVEIMDYEDFQYCWENFVYDDGEPFKRWKGLKYNFQSLTRRLREI LQSGGSSGGSSGSETPGTSESATPESSGGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKK FKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDD SFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALA HMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYA DLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIF FDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIH LGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFE EVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGE QKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI IKDKDFL DNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIR DKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKK ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025 GILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEH PVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKN RGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQI TKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNA VVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANG EIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKL IARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLE AKGYKEVKKDLI IKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKG S PEDNEQKQLFVEQHKHYLDE 11 EQ I S E FS KRVI LADANLDKVLS AYNKHRDKP I REQAENI IH LFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSGGS GGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDA PEYKPWALVIQDSNGENKIKMLSGGSGGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVI GNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSKRTADGSEF ESPKKKRKV (SEQ ID NO: 777);

MKPQIRDHRPNPMEAMYPHIFYFHFENLEKAYGRNETWLCFTVEI IKQYLPVPWKKGVFRNQVD PETHCHAEKCFLSWFCNNTLSPKKNYQVTWYTSWSPCPECAGEVAEFLAEHSNVKLTI YEARLY YFWDTDYQEGLRSLSEEGASVEIMDYEDFQYCWENFVYDDGEPFKRWKGLKYNFQSLTRRLREI LQSGGSSGGSSGSETPGTSESATPESSGGSSGGSGKRNYILGLAIGITSVGYGI IDYETRDVID AGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVK GLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERL KKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFG WKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQI TEN VFKQKKKPTLKQ TAKE I LVNEED I KGYRVTSTGKPE FTNLKVYHD I KD I TARKE 11 ENAELLDQ IAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQ IAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIEL AREKNSKDAQKMINEMQKRNRQTNERIEEI IRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIP LEDLLNNPFNYEVDHI IPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYETFKK HILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLD VKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFE EKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGN TLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETG NYLTKYS KKDNGPVI KKI KYYGNKLNAHLD I TDD YPNSRNKVVKLS LKP YRFDVYLDNGVYKFV TVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLN RIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKGG SPKKKRKVSSDYKDHDGDYKDHDIDYKDDDDKSGGSGGSGGSTNLSDIIEKETGKQLVIQESIL ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025 MLPEEVEEVIGNKPESDILVHEAYDESEDENVMLLESDAPEYKPWALVIQDSNGENKIKMLSGG SGGSGGSENLSDIIEKEEGKQLVIQESILMLPEEVEEVIGNKPESDILVHEAYDESEDENVMLL ESDAPEYKPWALVIQDSNGENKIKMLSGGSKREADGSEFESPKKKRKV (SEQ ID NO: 778); MKPQIRDHRPNPMEAMYPHIFYFHFENLEKAYGRNEEWLCFEVEI IKQYLPVPWKKGVFRNQVD PEEHCHAEKCFLSWFCNNELSPKKNYQVEWYESWSPCPECAGEVAEFLAEHSNVKLEI YEARLY YFWDEDYQEGLRSLSEEGASVEIMDYEDFQYCWENFVYDDGEPFKRWKGLKYNFQSLERRLREI LQSGGSSGGSSGSEEPGESESAEPESSGGSSGGSEKKYSIGLDIGENSVGWAVIEDDYKVPSKK FKVLGNENRKSIKKNLMGALLFDSGEEAEAERLKREARRRYERRKNRIRYLQEIFANEMAKLDD SFFQRLEESFLVEEDKKNERHPIFGNLADEVAYHRNYPEIYHLRKKLADSPEKADLRLIYLALA

H 11 KFRGHFL I EGKLNAENSDVAKLF YQL I QEYNQLFEES PLDE I E VDAKG I LS ARLS KS KRLE KLIAVFPNEKKNGLFGNI IALALGLEPNFKSNFDLEEDAKLQLSKDEYDDDLDELLGQIGDQYA DLFSAAKNLSDAILLSDILRSNSEVEKAPLSASMVKRYDEHHQDLALLKELVRQQFPEKYAEIF KDDEKNGYAGYVGADKKLRKRSGKLAEEEEFYKFIKPILEKMDGAEELLAKLNRDDLLRKQREF DNGSIPHQIHLKELHAILRRQEEFYPFLKENREKIEKILEFRIPYYVGPLARGNSRFAWLERKS EEAIEPWNFEEVVDKGASAQSFIERMENFDEQLPNKKVLPKHSLLYEYFEVYNELEKVKYVEER MRKPEFLSGEQKKAIVDLLFKENRKVEVKQLKEDYFKKIECFDSVEI IGVEDRFNASLGEYHDL LKI IKDKDFLDNEENEDILEDIVLELELFEDREMIEERLKEYAHLFDDKVMKQLKRRHYEGWGR LSRKMINGIRDKQSGKEILDFLKSDGFSNRNFMQLIHDDSLEFKEEIEKAQVSGQGDSLHEQIA DLAGSPAIKKGILQEVKIVDELVKVMGHKPENIVIEMARENQEEEKGLQQSRERKKRIEEGIKE LESQILKENPVENEQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFIKDDSIDN KVLERSVENRGKSDNVPSEEVVKKMKNYWRQLLNAKLIEQRKFDNLEKAERGGLSEADKAGFIK RQLVEERQIEKHVARILDSRMNEKRDKNDKPIREVKVIELKSKLVSDFRKDFQLYKVRDINNYH HAHDAYLNAVVGEALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKAEAKRFFYSNIMNFF KEEVKLANGEIRKRPLIEENGEEGEVVWNKEKDFAEVRKVLAMPQVNIVKKEEVQEGGFSKESI RPKRNSDKLIARKKDWDPKKYGGFLWPEVAYSVLVVAKVEKGKSKKLKSVKELLGIEIMERSSF EKNPIDFLEAKGYKEVKKDLI IKLPKYSLFELENGRKRMLASAKQLQKGNELALPSKYVNFLYL ASHYEKLKGS PEDNEQKQLFVEQHKHYLDE 11 EQ I S E FS KRVI LADANLDKVLS AYNKHRDKP I REQAENIIHLFTLTRLGAPRAFKYFDTTIDPKQYRSTKEVLDATLIHQSITGLYETRIDLSQLG GDSGGSGGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDE NVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSGGSGGSTNLSDIIEKETGKQLVIQESILM LPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGS KRTADGSEFESPKKKRKV (SEQ ID NO: 779);

MKPQIRDHRPNPMEAMYPHIFYFHFENLEKAYGRNETWLCFTVEI IKQYLPVPWKKGVFRNQVD PETHCHAEKCFLSWFCNNTLSPKKNYQVTWYTSWSPCPECAGEVAEFLAEHSNVKLTI YEARLY YFWDEDYQEGLRSLSEEGASVEIMDYEDFQYCWENFVYDDGEPFKRWKGLKYNFQSLERRLREI ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025 LQSGGSSGGSSGSETPGTSESATPESSGGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKK FKVLGNTDRHSIKKNLIGALLFDSGETAERTRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDD SFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALA HMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYA DLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIF FDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIH LGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFE EVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGE QKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI IKDKDFL DNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIR DKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKK GILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEH PVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKN RGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQI TKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNA VVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANG EIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESIRPKRNSDKL IARKKDWDPKKYGGFLWPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLE AKGYKEVKKDLI IKLPKYSLFELENGRKRMLASAKQLQKGNELALPSKYVNFLYLASHYEKLKG S PEDNEQKQLFVEQHKHYLDE 11 EQ I S E FS KRVI LADANLDKVLS AYNKHRDKP I REQAENI IH LFTLTRLGAPRAFKYFDTTIDPKQYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSGGS GGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDA PEYKPWALVIQDSNGENKIKMLSGGSGGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVI GNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSKRTADGSEF ESPKKKRKV (SEQ ID NO: 780);

MKPQIRDHRPNPMEAMYPHIFYFHFENLEKAYGRNETWLCFTVEI IKQYLPVPWKKGVFRNQVD PETHCHAEKCFLSWFCNNTLSPKKNYQVTWYTSWSPCPECAGEVAEFLAEHSNVKLTI YEARLY YFWDTDYQEGLRSLSEEGASVEIMDYEDFQYCWENFVYDDGEPFKRWKGLKYNFQSLTRRLREI LQSGGSSGGSSGSETPGTSESATPESSGGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKK FKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDD SFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALA HMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYA DLFLAAKNLSDAILLSDILRVNTEITKAPLSASMVKRYDEHHQDLTLLKALVRQQLPEKYKEIF ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025 FDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGI IPHQIH LGELHAILRRQGDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFE EVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGE QKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI IKDKDFL DNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRLRYTGWGRLSRKLINGIR DKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKK GILQTVKVVDELVKVMGGHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEH PVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKN RGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQI TKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNA VVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANG EIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKGNSDKL IARKKDWDPKKYGGFNSPTAAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIGFLE AKGYKEVKKDLI IKLPKYSLFELENGRKRMLASAGVLHKGNELALPSKYVNFLYLASHYEKLKG S PEDNEQKQLFVEQHKHYLDE 11 EQ I S E FS KRVI LADANLDKVLS AYNKHRDKP I REQAENI IH LFTLTNLGVPAAFKYFDTTIDKKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSGGS GGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDA PEYKPWALVIQDSNGENKIKMLSGGSGGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVI GNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSKRTADGSEF ESPKKKRKV (SEQ ID NO: 781);

MKRTADGSEFESPKKKRKVMKPQIRDHRPNPMEAMYPHIFYFHFENLEKAYGRNETWLCFTVEI IKQYLPVPWKKGVFRNQVDPETHCHAEKCFLSWFCNNTLSPKKNYQVTWYTSWSPCPECAGEVA EFLAEHSNVKLTIYTARLYYFWDTDYQEGLRSLSEEGASVEIMDYEDFQYCWENFVYDDGEPFK RWKGLKYNFQSLTRRLREILQSGGSSGGSSGSETPGTSESATPESSGGSSGGSDKKYSIGLAIG TNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRK NRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRK KLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSK DTYDDDLDNLLAQ I GDQ YADLFLAAKNLSDAI LLSD I LRVNTE I TKAPLS ASMVKRYDEHHQDL TLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRE DLLRKQRTFDNGI IPHQIHLGELHAILRRQGDFYPFLKDNREKIEKILTFRIPYYVGPLARGNS RFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNEL TKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFN ASLGTYHDLLKI IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLK RLRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQ ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025 GDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGGHKPENIVIEMARENQTTQKGQKNSRER MKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQ SFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGL SELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQF YKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKY FFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV QTGGFSKESILPKGNSDKLIARKKDWDPKKYGGFNSPTVAYSVLVVAKVEKGKSKKLKSVKELL GITIMERSSFEKNPIGFLEAKGYKEVKKDLI IKLPKYSLFELENGRKRMLASASVLHKGNELAL PSKYVNFLYLASHYEKLKGSSEDNKQKQLFVEQHKHYLDEI IEQISEFSKRVILADANLDKVLS AYNKHRDKPIREQAENIIHLFTLTNLGASAAFKYFDTTIGRKLYTSTKEVLDATLIHQSITGLY ETRIDLSQLGGDSGGSGGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILV HTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSGGSGGSTNLSDI IEKETGK QLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGE NKIKMLSGGSKRTADGSEFESPKKKRKV (SEQ ID NO: 782); and MKRTADGSEFESPKKKRKVssevef sheywmrhaltlakrardegeapvgavlvlnnrvigegw vrriglhdptahaeimalrqgglvmqnprlidatlyvtf epcvmcagaminsrigrvvf gvrns krgaagslmnvlnypgmnhrveitegiladecaallcdfyrmprqvf naqkkaqssinEGGSEE EEESGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETA EATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIV DEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQ LVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPN FKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKA PLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPI LEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKI LTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKV LPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKK IECFDSVEISGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHD DSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEM ARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQE LDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKL I TQRKFDNLTKAERGGLS ELDKAGF I KRQLVETRQ I TKHVAQ I LDSRMNTKYDENDKL I RE VKV ITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVR KMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATV RKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVA ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025 KVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLI IKLPKYSLFELENGRK RMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEI IEQISE FSKRVILADANLDKVLSAYNKHRDKPIREQAENI IHLFTLTNLGAPAAFKYFDTTIDRKRYTST KEVLDATLIHQSITGLYETRIDLSQLGGDSGGSGGSGGSTNLSDIIEKETGKQLVIQESILMLP EEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSGG SGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSD APEYKPWALVIQDSNGENKIKMLSGGSKRTADGSEFESPKKKRKV (SEQ ID NO: 792).

In any aspect or embodiment of the disclosure, the GALT polynucleotide is in a cell. In any aspect or embodiment of the disclosure, the cell is a mammalian cell. In any aspect or embodiment of the disclosure, the subject is a mammal. In any aspect or embodiment of the disclosure, the mammal is a human.

In any aspect or embodiment of the disclosure, the method results in lowering of the concentration of galactose- 1-P in the blood of the subject relative to an untreated subject. In any aspect or embodiment of the disclosure, the concentration of galactose-l-P in the blood of the subject is lowered to a concentration of less than 5 mg/dL.

In any aspect or embodiment of the disclosure, the method results in an increase in GALT polypeptide activity in a hepatocyte of the subject.

In any aspect or embodiment of the disclosure, the method further involves administering to a subject a lipid nanoparticle containing the base editor system.

In any aspect or embodiment of the disclosure, a target nucleotide is deaminated with an efficiency of about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%.

In any aspect or embodiment of the disclosure, the deaminase domain is modified to omit an N-terminal methionine.

In any aspect of the disclosure, or embodiments thereof, the lipid nanoparticle contains an ionizable lipid having the following structure, or a pharmaceutically acceptable salt thereof:

In any aspect of the disclosure, or embodiments thereof, the LNP contains a molar ratio of ionizable nitrogen atoms in an ionizable lipid to the total negative charge in the payload (N: P ratio) of between about 5:1 and 7:1. In any aspect of the disclosure, or embodiments thereof, the LNP contains an N: P ratio of about 6:1. ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

In any aspect of the disclosure, or embodiments thereof, the guide RNA contains a scaffold with the following nucleotide sequence:

GUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmCmCmGmGmCAAGUUAAAAUAAGGCUAGUC CGUU AU C AmAmCmU mU mGmAmAmAm AmAmGmU mGmGmCm AmCmCmGm AmGmU mCmGmGmU mGm CmUsmUsmUsmU (SEQ ID NO: 794), where “mN” indicates a 2'-OMe modification of the nucleotide “N”, and “Ns” indicates that the nucleotide “N” is linked to the following nucleotide by a phosphorothioate (PS). In any aspect of the disclosure, or embodiments thereof, the guide RNA contains a spacer containing the nucleotide sequence UUACCCGGCAGUGGGGGUGG (SEQ ID NO: 705) or UUACCCGGCAGUGGGGGUG (SEQ ID NO: 793). In any aspect of the disclosure, or embodiments thereof, the guide RNA contains a nucleotide sequence selected from those listed in Table 2. In any aspect of the disclosure, or embodiments thereof, the guide RNA contains one of the following nucleotide sequences: mUsmUsmAsCCCGGCAGUGGGGGUGGGUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmCmC mGmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmC mAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 746); mUsmUsmAsCCCGGCAGUGGGGGUGGUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmCmCm GmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCm AmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 791);

where “mN” indicates a 2'-0Me modification of the nucleotide “N”, and “Ns” indicates that the nucleotide “N” is linked to the following nucleotide by a phosphorothioate (PS). In any aspect of the disclosure, or embodiments thereof, the guide RNA contains a spacer selected from those listed in Table 1.

In any aspect of the disclosure, or embodiments thereof, the napDNAbp is an SpCas9, an SpRY, an SpRYc, an saCas9, or an spCas9 variant having specificity for a protospacer-adjacent motif (PAM) selected from one or more of: NG, NRRH, and NRTH, where “N” indicates A, C, G, or T, “R” indicates A or G, “Y” indicates C or T, and “FT indicates A, C, or T. In any aspect of the disclosure, or embodiments thereof, the napDNAbp is a nickase.

In any aspect of the disclosure, or embodiments thereof, the base editor polypeptide contains an amino acid sequence with at least about 90% identity to a sequence listed in Table 2.

In any aspect of the disclosure, or embodiments thereof, the cell is in a subject.

In any aspect of the disclosure, or embodiments thereof, the disease is galactosemia.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this disclosure belongs. The ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

following references provide one of skill with a general definition of many of the terms used in this disclosure: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

By “6b polypeptide” is meant a deaminase capable of deaminating cytidine in DNA and comprising an amino acid sequence with at least 85% identity to the following amino acid sequence:

MSSEVEFSHEYWMRHALTLAKRARDEGEAPVGAVLVLNNRVIGEGWVRRIGLHDPTAHAEIMAL RQGGLVMQNPRL IDATL YVTFE PCVMCAGAM INSR I GRVVFGVRNS KRGAAGS LMNVLNYPGMN HRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN (SEQ ID NO: 771), or a functional fragment thereof. In various embodiments, the 6b polypeptide may be inserted within a Cas9 polypeptide between amino acid positions 906 and 907 referenced to SEQ ID NO: 197 (where a base editor with a deaminase inserted at this position may be referred to as an “IBE34” base editor). In some embodiments, the 6b polypeptide lacks an N-terminal methionine.

By “6b polynucleotide” is meant a polynucleotide encoding an 6b polypeptide.

By “galactose- 1 -phosphate uridylyltransferase (GALT) polypeptide” is meant a protein with at least about 85% amino acid sequence identity to GenBank Accession No. AAC83409.1, which is provided below, or a fragment thereof. A functional GALT polypeptide is capable of metabolizing the conversion of galactose-l-P to glucose-l-P (see, e.g., FIG. 1). In some embodiments, GALT contains a pathogenic Q188R mutation that prevents functional protein activity required to break down galactose in a subject, which may be associated with a toxic accumulation of galactose metabolites (e.g., galacitol or Galactose-l-P) in a cell or subject. Amino acid position 188 is underlined in the below sequence. In various embodiment, the methods of the disclosure involve altering a GALT polypeptide to replace an R at amino acid position 188 with a Q.

> AAC83409.1 galactose- 1 -phosphate uridyl transferase [Homo sapiens] MSRSGTDPQQRQQASEADAAAATFRANDHQHIRYNPLQDEWVLVSAHRMKRPWQGQVEPQLLKT VPRHDPLNPLCPGAIRANGEVNPQYDSTFLFDNDFPALQPDAPSPGPSDHPLFQAKSARGVCKV MCFHPWSDVTLPLMSVPEIRAVVDAWASVTEELGAQYPWVQIFENKGAMMGCSNPHPHCQVWAS SFLPDIAQREERSQQAYKSQHGEPLLMEYSRQELLRKERLVLTSEHWLVLVPFWATWPYQTLLL PVGHVRRLPELTPAERDDLASIMKKLLTKYDNLFETSFPYSMGWHGAPTGSEAGANWNHWQLHA ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025 HYYPPLLRSATVRKFMVGYEMLAQAQRDLTPEQAAERLRALPEVHYHLGQKDRETATIA (SEQ ID NO: 687)

By “galactose- 1 -phosphate uridylyltransferase (GALT) polynucleotide” is meant a nucleic acid molecule encoding a GALT polypeptide, as well as the introns, exons, 3' untranslated regions, 5' untranslated regions, and regulatory sequences associated with its expression, or fragments thereof. In embodiments, a GALT polynucleotide is the genomic sequence, cDNA, mRNA, or gene associated with and/or required for GALT expression. An exemplary GALT polynucleotide sequence is provided at ENSEMBL Gene Accession No. ENSG00000213930, which is provided below. A further exemplary GALT nucleotide sequence from Homo Sapiens is provided below (GenBank Accession No.: M60091.1:29-1168). In various embodiments, the methods of the disclosure involve deaminating a “C” complementary to a “G” at the nucleotide position corresponding to the position of the “A” underlined in the below sequences (i.e., deaminating the C complementary to coding position 563 (c. G563)).

>chromosome: GRCh38:9:34637533:34651635:l (ENSG000002139300, where GALT exons are shown in bold CTCTGCCCGCTCACCAGCGTACTGCCGCGCCAACTGCGCTATCTCTTCGCGCTGGAAGACGAAG CTCTGCGTACCCAGCCAGAGCCAGACGACCTGGGTCAGCACCGCTGCGACAGCCAGGAGCAGCG CGGCCCACGCCCACCGCCGGCCCACGGCCCACTGCATCCCGGCGGGCGGCCTGGCACGGCGCAG CTCAGGAGGGAGCCGGGGCCTGAGGCTTTGCGCTCACGGCCTCGGAGCCCGCCGGCTGCCCACG AAGCGCTCCCCCGCCACCGCCGTGGTACGGCTCGCCCTGACCAATCGGAGCGGCCCGGGCACCT TGGCACCGCCCTTCGGTTGAACCATTTCCTCGGGGGGAGCACGGAGGAGTGGGGCGGTGCCGCG CAAGGGAGGCAAGGGGGCGGGGCCAAGTCGCCCAGCGGCAGATGTCAGGGTCTTCAGGCGAGGT CTGACCTAGCTAGCGCCACTCTACATACGGATAGGTGTGGGGATAGTGAGACCCAGAATGGACG GGCTCGCCCGAGCTCACTGGCTGGTGAAGCAGACGAGCCCGGAAGTGAAGCCTTCTGGGACTCA ACGATTGTTAAGGCCCGCCTTTACCAGGACTGAAGCGGAGAGGAGGCAGTTCAAGCTGGGAGCC CCAGGGTGGGCATTCCAGAGGCGGTGACACCTGAGCAGTGTCTTTAAGAGCTCGCAGAAGGGGC ATGCCAGCCCACAGGGACCAGCCCCTAGAGCAAAGGCCTCCGAGGAGAGACAGCTTGGGGTCTT CAGGGGCCTACAATTCCTTCTCTGTTCTGCGGCACAAAATGTGAGAGGAGGCCTTGTAAACCAG CTGAGGGTTATCAGCAAGGGAGGAAGGGGATTAGATTTTGCCACCGAGTGGCACAGGACTGTCG AGTGGAGGGGAGCAAGATGAGTCAGGGAGACCATGACTGTGGGCCAGGTAAGAGGGAAGGTCTG TGTCAGTGGAGATGGAAGAAAGAAATGAGCTTAACAACTCTGGAAATGGAGTTGGCAGGGCTTG ATCTCTTCTTCCTGGAATACTCTTTCCCCCTTTATCCTATCCCCACTTTCCCCTTATTTGGCTA ACATCCTTTGCATGTCTAGTGGGCAGTTGAGTGTCCAGGTCTCGAAGCTCAGCTCCAGTCTGGA GATAGAGCTGGGAGTTGTCATTCATTCAGATACTTTGTTGGCACTTGTATGTGCCAAGTTCTGT ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025 GGTGGCCATTAGGGATATGGCTAGAAACAATGGTCCTACTCCCCAGCTTAGAGATCTCACTGGA GAGTCTACAGAAAACCTCTAAGAATGGATGAAATCTCACAGGGAGAGAGAATAGAGTAAGATAA GGCTAAAGACAGAGCCCTGGAGAGCACCAACAGCAAAGGGTAGGCAGAGGAAGAAGAAAGCAAA AAGGAAAAGAAACAAAAGAGAAAAGAGGTAGCATTGTGCAGTGATTAAGAAATAAACTCTGGAG CTGGACTGCCTGAGTTCAAGGCCTGGCTCCTGCTACCTACTAGTTGTCTGATCTTGGGCAGTTT ACTTAAAATCTCTGTAACTTAGTTTTCTTACCTGTAAAATGGAATGGAGATAAGTACCTACCTC ATAGTACATATCTTCATTGTGATGAGATTAGATGGGTTAATATTTATAAAGCAGTTAGTGCCTC ACATGGTGAGAATTTTATGTGATGGTTATATATATAAAACATAAAGGAACTGGAAAAAAGTAGC CTCATACATTTTTGACCTTAAAGAAAGAAACTGAGGCCGGGCGAGGTGGCTCATAACTGTAATC CCAACACTTTGGGAGGCTGAGGCAGACGGAACACTTGAGTCCAGGTATTCGAGACCAGCCTTGG CAACATGGTGAAACCCCATCTCTGCAAAAAGTACAAAAATTAGTCAGGAGTGGGGGCGCACACC TGTAGTCCCAGCTACTCAGGAGGCCGAAGTGGGAGGATCGCTTGAGCCTGGGAAGTGGAGGTTG CAGTTGAGCCAACAGAGGGAGACTCTGCCTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGGC CAGGCACAGTGGCACACACCTGTAGTCCCAGCACTTTGGGAGGCCAAGGTGGGCAGGCAGATTT CCTGAGCTCAGAAGTTCGAGACCACCCTGGGCAACATGGTGAAACCTCGTCTCTACTAAAATAC AAAAAATTAGCCGGGCGTGGTGGCAGGTGCCTGTAGTCCCAGCTACTCGGGAGGCTGAAGCAGA AGAATCGCTTGAACCCAGAAGGTGGATGTTGCAATGAGCCAAGATCGTGCCACTGCACTCCAGC CTGGACAACAGAGGGAGACCCTGTCTCTAAAAAAAAAAGAAAAGAAAAAGGGGCCGGGCACTGT GGCTCACACCTGCAATCCCAGCACTTTGGGAGGCCGAGGCGGGTGGATCACCTGAGGTTGGGAA TTTGAGACCAGCCTAACCAACATGGAGAAACCCTGTCTCTACTAAAAATACAAAATTAGCTGGG CGTGGTGGCGCATGCTTGTAATCCCAGCTACTCAGGAGGCTGAGGCAGGAGAATCACTTGAACC CAGGAGGCAGAGGTTGCGGTGAGCCGAGATCACGCCATTGCACTGCAGCCTCAGTGTGAAACAG CAAAACTCCGTCTCAAAAAAAACAAAACAGGCTGGGCGGGGTGGCTCACGCCTGTAATCCCAGC GCTTTGGGAGGCCGAGGCAGGTGGATCACGTGGTCAGGAGTTCGAGACCAGCCTGACCAACATG GTGAAACCCCATCTCTACTAAAAAATACAAAAATTAGCCGGGCGCGGTGGCGCGTGCCACCCAC TTGGCCCAGCTACTTGGGGGGCTGAGGCAGGAGAATCGTTTGAACCCGGGAGGTGGAGGTTGCA GTGAGTGGAGACTGCACCATTGCACTCCAGCCTGGGCGACAGAGTGAGACTCTGTCTCAAAAAA GAAAAACACACAAACAAAAACAACAGTGAGGAAAAAAAAAAGCTGAGGCAAAATTAATATAGAG AGTTTATTTGGGCCAAGGTAGAGAACGACTGCTCGGGACACTTTTCCAAGTTGCCTTGGGGAAT GTTGATACAAGCATTTTTTATTTTTATATGTGGTTTTTATTTTTATTTTTTATTTTCTTGGAGA TGGATTCTTACTCTGTTGCCTAGGCTGGAATGCAGTAGTGCAATCTTGGCTCACTGCAACTCCC GCCTCCCAGGTTCAAGCTATCCTCCTCCCTCAGCCTCCCGAGTAGCTGGGACCACAGGTGTGCA CCACCATGCCTGGCTAATTTTTGTAGTTTTAGTGGAGACAGGTTTTTGCCATGTTGGCCAGGCT GGTCTCTAACTCCTGACCTCAGGTGATCCACACTCCTCGGCCTCCCAAAGTGTGGGATTACATG TGTGAGCCACTGCACCTGGCCACTTTATTTTAACTTAATTTAATTTTATTTTTTTTGAGATGGT ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025 GTCTCCACTGTCGCCCAGGCTGGAGTGCAGTGGCGCAATCTCGGCTCACTGCAACCTCTGCCTC CCAGTTCAAGAAATTCTCCTGCCTCAGCCTCCCGAGTAGCTGGGACTACAGGCACCTGCCACCA CATCCAGCTAATTTTTTTGTATTTTTAGTAGAGATGGGGTTTCACCATGTTGGCCAGGCTGGTC TTGAACTCTGACCTCGTGATTTGCCCGCCTCGGCCTCCCAAAGTGCTGGGATTATAGGCGTGAG CCACCGCGCCCGGCCTATTTTTATTTTTTGAGACCGGATCTTGCTCTGTTACCTAGGCTGGAGT TCAGTGGCACGATCACAGCTCATTGCAGCCTTGACCTCTTGGGCTCAAGGGATCATCCTGCCTT GGCCTCCCAAAGTGCTGGAATTACAGGTATGAACCATCATGCCCAGCCACAAGCAAGTTTTTAA AGGCAAAAGAGGACAAGGAGTGGGCTGACACAAAATTGTTTAACAGGAATTCTCATTGGCTTAC AGAAACAACATTGATTAGTGATTGGCTATACCTTGTTGAACTATAGGGTATGAGTTATGGTGTC TAGACTATGGCATTTTATGGCTATGTGGTATCAATTTAGAGCCCACATAGCAAGTGGCTTCAAG GGGTAATTATTTGGCTCCATGGGGGTGGGGGAAGGTGAGATATGACTGCTGTTACATGCTGTCA _{CTTTTTTTTTTTTTTTTTTTTGAGACTGAGTCATACTCTGTCGCCCAGGCTGGAGTGCAGTGGC} GCAATCTCGGCTCACTGCAACCTCCGCCTCCCGGGTTGAAGCGATTCTCCTGCCTCCGCCACCC AAGTAGCTGGAATTACAGGCACCTGCCACTATGCTCAGCTTTTTGTATTTTTAGTAGTGACAGG GTTTCACCATAATGGCCAGGCTGGTCTCAAACTCCTGACCTCAAGTGATCCCGCCCGCCTTGGT TTCCCAGTGTGCTGGGATTACAAGTGTGAGCCACTGCACCCGGCCTTTTTTTTTCTTTTTAAAA TTATTATTATTATTATTATTATTATTTTGAGACGGAGTCTCTTGCACTGTCGCCGGGGCTGGAG TGCAGTGGCATGATCCTGGCTCACTGCAACCTCTGCCTCCCAGGTTCAAGCAATTCTCCTGCCT CAGCCTCCTGAGTAGCTGGGATTACAGGCGCCCGCCACCATGCCCAACTAATTTTTTGTATTTT TAGTAGAGACAGGGTTTCACCATGTTGGCCAGGCTGGTCTCGAACTCCTGACCTCGTGATTCGC CCACCTTGGCCTCCCAAATTGCTGGGATTACAGGCGTGAGCCACCGCGCCAGGCCCATGCTGTC ACATTTTAATGCCTCTCTGGGCCTGATAATTAAAAGAGGCTGGCATTTCTCAGATAAAAAGTTT CTTTTCTTTCTCAAGAGTGATGGAATGGGCCAGGCACGGTGGCTCACACCTGTAATCCCAGCAC TTTGGGAGGCCGAGGTAGGGGAATCACCTGAGGTTGCCAGTTCAAGATCCAGCCTGACCAACAT GGAGAAACCCCATCTCTTCTAAAAATACAAAATTAGCCAGGCGTGGTGGCGCATACCTGTAATC CCAGCTACTCGGGAGGCTGAGGCAGGAGAATCGCTTGAACCTGGGAGGCGGAGGTTATGGTGAG CTGAGAGCACGCCATTGCACTCCAGTTTGGGCAATGAGAGCGAAACTCCATCTAAAAAAATTAA AAAAAGAGTGATGGAATGAAGGGTTAACACAGTGAAATATAATGGTGAGATTCAATGAGATGAT GGGAAATGATCATTAGACTTCTCAGTAGGGGCATGTGGCAGAGACAATGCTATACATTCACAAA TCCATTTCCTTTTGCTCTTGGACAGCTAGATTACATTCCACAGTCTACTCTGGATCATGTGATT GCATTCTGGCCAATGAAATATAGATAGAAATGATGTATGACATTTCTAGGGCTGGCTCCTAAAG TCTCCTCTGAAATTCTTTTTTTTTTTTTTCTCTTTCTCTTTTTAAGAGACAGGATTGTGCTATG TCACTCCCAGACTGGTATCAAACTCTTGGGCTCAAGTGATCTCCCACCCTGGCTTCCCAAGAGC TGGGGGACTACAGGCACATAGTACTATGCCCAGCTTGAAGTTCTTTACATTTTCTTTCTTCCCA TGTCTATTACTGTAGTTATAGAGGATCTCTAGTGGAGTATTCTGAGACCCTAGGACATGGCATA ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025 GCTACTAGATGGATGGCACCTGGATCCCTAAATACTGCTTGGAACAGAGCTCCCTGTTACCCCC AGAATTGACTGTAGTGCTAAGCCATTGAGTTTTGGGAGTGTTTGTTACAGCAATTAAGATTACT TACCCTGACAAATATAGGGCAAAAGATGTGTTACAATATACCATGCAGCTAAGAGTAGAGACCA GAAAGATGGCCTCTGTTTTTAAGAAACTTGGCAGGCCGGGCACGGTGGCTCACGCCTGTAATCC CAATACTTTGGGAGGCAGAGGCAGGCGGATCACCTGAGGTCAGGAATTTGGGACCAGCCTGGCC AACGTGGTGAAACCCTGTCTCTACTACAAATACAAAAATTAGCTGGGTGTGGTGGTGCACGCCT GTAATCCCAGCTACAGGGGAGGCTGAGGCAGGAGAATCACTTGAATCAGGAGGCGGATGTTGCA GTGAGCCTAGATTGCACCACTGTACTCCAGCCTGGGCGACAGAGCGAGACTCCATCTAAAAAAA AAAAAAAAAAAAAAAAAAAAGAAACTTGGCAGAGAAATTAAGGATAAAGAAAGGATTATCTAGA GGAGAATACAGGATTGAGGGAACATGTTTTTGTATTGGTTCTGGCTTCTGTGGCATTCCCGACT CGACAATTCACTAAGAACCCTGGGCAGCAGGGATTAGCCCCCAAATTTGGTCTCTGCTCCCCAT TCCAGGAACAGCTTTCTGGGATCATCTGGGCCTACACCTCCCCTATCTGGTCTTTCAGAAAGCT TTTCCAGGCCTTTCCAGGTGTGGTGACTTATGTCTGTAATCCCAGCACTTTGGGAAGGCCGAGA TGGACAGATTACCTGAGGTCAGGCGTTCAAGACCAGCCTGGCCAACATGGTGAAACTCTGTCTC TAAAAAATACAAAAATTAGCTAGTCATGATGGTGGGTGCCTGTAATTCCAGCTACTCGGGAGGC TGAGGCAGGAGAATCACTTGAACCTGGGAGGCAGAGGTTGCGGTGAGCCGAGATCAAGCCACTG CACTCCAGCCTGGGCAACAGAGTGAAACTCCGTCTTAAAAAAAAAAAATTATATATATATATAT ATATATATATATATATATATATATATATATATATATATATATATAAATGGATAACCAGCAGCCC TCAGGGGTGCTCTGTCTATGGAGTGGCCATTTTTTTATTCCTTCACTTTCCTGATAAACTTGCT TTCACTTCACACTATGGACTCACCTTGAATTCTTTCTTGTGTGAGATCCAAGAATCCTCTCTTG GGGTCTGGATTGGGATCTCTTTACTGTAACAACACCTCTGGGATCTGGCAGGGAGGGAGCTGGT TAGGCCCATCTTAGTCATTCTCCTCTTCCTCCAAAAAATGACCTTGGGGAGTCATAGAGGAGAA TTATGGAGATGTTCCTGAAGAGATGTTCCTGATGGGGGTCAGGATTTAATTAGCATGATTCAGG GGGCGGCACTGAAGCACTGGTGTTCTTGGGAAAATGGAGCTGTCTACAAGCTCTAGTCACTCAA ACAAATATAAATGAGAGAATGGAAAAGCAGATCTCCTGTTCCTGTCTCACCTCTTTGGGTAATA TGAACAGCCAGTGGGACAGGTAAAACCCCTGTTTCTCAGTCCTCAGCTCCCTGCCCCCTACCTC TGAAGGTAGCCTCTTCCTATTCCAGTCTGAGGACCCTAGGGCAGAGATCAAGTAAATGACCCAT GACCTAGTTTTTGCAGCTCAATTGCAAAACACCTGGCTTGGCTTGGTCTTGATTGGGGGAAGGG ATGTCCCTGCTATGGCTCAACTTCTAACTCACTGGGTCTCTGTCCTGCATTCCACTGAAGCCTC AGAGGCATCTCACAGCACTGGGAAAAGCCTTGGAAACTTGCATTCAAATCTCAGCTTGGCCACT AATTCTCTGGGTGACCTTGAGTCCTCTGGGCTCCAATGTGAAATGGGGAAGAGGTTTAGACAAT ATTTAGATGGCTCTTTAAAAGTACTGGGGGCCAGCTGTTTGGGAGGCTGAGGCAGGAGGATCAC TTGAAGTCAGGAGTTCAAGACCAGCCTGGCCAACATGTGAAACCCCGTCTCTACTAAAAATACA TAAATTAGCCCAGTGTGGTGGCACACAGCTCTAATTCCAGCTACTTGGAGGCTGAGGCAGGAGA ATCGCTTGAACCTGGGAGGTGGAGGTTGCAGTGAGCCAAGATCACGCCACTGCACTCCAGCCTG ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025 GGTGGCAGAGTGAGACTCTCTCTCAAACAAACAAACAAACAAAAGTACCAGGGGAGGAATTAAT TTGAATTTTATCCTAGTGTTAGCCAATTGGTCCCATCCAAGGAAAATTTAGAAAAGGGAAGGGG ATGTGTAAAGGAAACACTAGGCCCCACCTAGATGGTGGCTGGAGCTTCTGATAGTCCTGTACTC TCCACATTTTTAGACTTTCTTGTACTTTTTTTTTTTTTTTTGTGACGGAGTCTGCTCTGTCGCC AGGCTAGAGTGCATGGGCACAATCTCGGCTCACTGCAACCTCTGCCTCCTGGGTTCAAGTGATT CTCCTGCCTCAGCCTCCCGAGTAGGTGGGACTACAGGTGCACGCCACCAAGACCAGCTAATTTT _{TTTTTTTTTTTTTTTTACTAGAGACGGGGTTTCACCATGTTGGCCAGGATGGTCTCAATTTCTT} GACCTTGTGATCCGCCTGCCTTGGTCTCCCAAAGTGTTGGGATTACAGGCGTGAGCCACCGAGC CCGGCCATTTTTAGATTTTTTTTTATAGCCCTTTTACCCCTCCCACTTCTTGTGCTGTCTTGGT GTGTGTATGAAAAAGAGAGAGAGAGAGAGAGGAAGGAGAGCAAGAGTGAGAGAAAAAGAGAATA TGGTTACTTAGCCCTTTTTCCCAAATCTGTTTTTGCTTTTGGGGATAGGATATGGTTATGGTGA AGTACTCCACACAGACCCCACCAAGTTTGCCTCTCCTGCGCTACAGCCACTGAGCAGGTGACTG AGGCGCTGTTGATCCAGGGTCATATCCTTACTCGCCCTAGAATGTAAGCTCAGGCAGGGCAGAG GCCATGCCTGACATGTGCATCCCAATGCTTTACACAGCCTAGGTGCCTAGCACATGCTAGATGC TTGGTAAATATTTGCTGAATGAAAGATCAAATGAATGATTGCAGCAAGCAAGTCCTGTAGGCAT CCTGGAGCCCAAGGATTCTGCAGTAGGCAGCTTTCACAGAGGTTCTTCCAGTGTAGTGGCTCTA GCTCTGGGTGAAGTAGGATCATCAATGTCGGCCCCCAGGGTTCACAGCTGTTCTGAGCCCCGCC CCCAGGTGGCAGGGCAGCCCAGTCAGTCAGTCACGTGCTGGCGGCTGGCCAATCATCGGGGGCG GCGCGGGGAGGGGTGGTGTGGACGGAGAAAGTGAAAGGTGAGGCACGGCCCTGCAGATTTTCCA GCGGATCCCCCGGTGGCCTCATGTCGCGCAGTGGAACCGATCCTCAGCAACGCCAGCAGGCGTC AGAGGCGGACGCCGCAGCAGCAACCTTCCGGGCAAACGGTAACTGCACCGCGGCAGGGACTCGC TGGGGCGCGGAGCCGAGCCCTCCCCTTCCTTAGGAAGCTTTCGTCCCCTCCGAAGGTTGGAACG CTCATCCCGAGCCAGACCGACAAGGCGTACAGTCTGCAGGCCTGTACGAGCAGCAGGCCAATTG GCGCTGGGAAAGTCCAATCCTGGGCCTCTAGCTCCTGAGCGGGACAGGGCCGAGAGGGCGCTCC CGAGCTTGGGCCTGCTGGTGGGTGAGACCCAGGAGAGAGGGAGCTAGAGAGCTCTGAGGACTGA TCTTGACTGTCTGCCCCCAGACCATCAGCATATCCGCTACAACCCGCTGCAGGATGAGTGGGTG CTGGTGTCAGCTCACCGCATGAAGCGGCCCTGGCAGGGTCAAGTGGAGCCCCAGCTTCTGAAGA CAGTGCCCCGCCATGACCCTCTCAACCCTCTGTGTCCTGGGGCCATCCGAGCCAACGGAGAGGT AAGCCTGTAGAGCCCTGCATCTGCAGGCTGGGCCACGGGGAGTAGTTCCCTCTTAGAACTGTCC TCCACCCACAGGGATAGTGAACCTCCTTCTGGGTCATATCCCACCAAGCTTTTTGGTCCCCTAG GGTGGGCCTTCCCTACTCCCTTGTAGCCTGTCCAGTCTTTGAAGCCCACCAGGTAACTGGTGGT ATGGGGCAGTGAGTGCTTCTAGCCTATCCTTGTCGGTAGGTGAATCCCCAGTACGATAGCACCT TCCTGTTTGACAACGACTTCCCAGCTCTGCAGCCTGATGCCCCCAGTCCAGGTAACCTGGCTCC AACTGCTGCTGGGGAGGAGGGTGGCTAGACCTCTTGAGGGACTTCTGCTGCAGAGAGTGATACT CCTTTACCTCAGGACCCAGTGATCATCCCCTTTTCCAAGCAAAGTCTGCTCGAGGAGTCTGGTA ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025 ACTATGGATTTCCCCTCTTACAACTTTCAAACCAGAGTTGGAGACTCAGCATTGGGGTTCGGCC CTGCCCGTAGCACAGCCAAGCCCTACCTCTCGGTTATCTTTTCTCCCGTCACCACCCAGTAAGG TCATGTGCTTCCACCCCTGGTCGGATGTAACGCTGCCACTCATGTCGGTCCCTGAGATCCGGGC TGTTGTTGATGCATGGGCCTCAGTCACAGAGGAGCTGGGTGCCCAGTACCCTTGGGTGCAGGTT TGTGAGGTCGCCCCTTCCCCTGGATGGGCAGGGAGGGGGTGATGAAGCTTTGGTTCTGGGGAGT AACATTTCTGTTTCCACAGGGTGTGGTCAGGAGGGAGTTGACTTGGTGTCTTTTGGCTAACAGA GCTCCGTATCCCTATCTGATAGATCTTTGAAAACAAAGGTGCCATGATGGGCTGTTCTAACCCC CACCCCCACTGCCAGGTAAGGGTGTCAGGGGCTCCAGTGGGTTTCTTGGCTGAGTCTGAGCCAG CACTGTGGACATGGGAACAGGATTAATGGATGGGACAGAGGAAATATGCCAATGATGTGGAGGC TTGGAGGTAAAGGACCTGCCTGTTCTTCTCTGCTTTTGCCCCTTGACAGGTATGGGCCAGCAGT TTCCTGCCAGATATTGCCCAGCGTGAGGAGCGATCTCAGCAGGCCTATAAGAGTCAGCATGGAG AGCCCCTGCTAATGGAGTACAGCCGCCAGGAGCTACTCAGGAAGGTGGGAGAGAGCCAAGCCCT GTGTCCCCAAGGAGTCCCTAACTTTCTTATCCCATGAGAGAGGTGTGTAAAGGAGAAAGCTAGA GGTGAACTAGTAGAGAGAGACTTGCTAGGAGGCCTTAGCAATAATCCAGTAATCTAAAGGAAAG ATGATGGTGACTTAGACTCGGGTGGTTAGTGGTAGAGGTGGTGAGAAGACATCAGATCCTGGGC ACATTCTTTTCTTCTGCTTCCCTTGCCTATTTGCTGACCACACTCCGGCTCCTATGTCACCTTG ATGACTTCCTATCCATTCTGTCTTCCTAGGAACGTCTGGTCCTAACCAGTGAGCACTGGTTAGT ACTGGTCCCCTTCTGGGCAACATGGCCCTACCAGACACTGCTGCTGCCCCGTCGGCATGTGCGG CGGCTACCTGAGCTGACCCCTGCTGAGCGTGATGGTCAGTCTCCCAAGTAGGATCCTGGGGCTA GGCACTGGATGGAGGTTGCTCCCAGTAGGGTCAGCATCTGGACCCCAGGCTGAGAGTCAGGCTC TGATTCCAGATCTAGCCTCCATCATGAAGAAGCTCTTGACCAAGTATGACAACCTCTTTGAGAC GTCCTTTCCCTACTCCATGGGCTGGCATGGTGAGGCTTTTCAAGTACCTATATTTAGCCCCAAC ACCATTTCTGGGCTCCTGGGGCTCAGCCTAGTGAACTGCAACCTCAAAGGAGCAAGCCTTGAAA CAGTTGCTGGGGGAAGTGGCCAGAGTAGAGATGCTGGGACTGAGGGTGGAGCAGCAAACTTGGT GAAACTACATCTCCAATGTGCTTTCTAATCTCCTGCCAGCTCTTCTCAAGCAGGGGATCCTGGG AGATGTAGTTTTCAGATACCTGGTTGGGTTTGGGAGTAGGTGCTAACCTGGATAACTGTAAAAG GGCTCTCTCTCCCCACTGTCTCTCTTCTTTCTGTCAGGGGCTCCCACAGGATCAGAGGCTGGGG CCAACTGGAACCATTGGCAGCTGCACGCTCATTACTACCCTCCGCTCCTGCGCTCTGCCACTGT CCGGAAATTCATGGTTGGCTACGAAATGCTTGCTCAGGCTCAGAGGGACCTCACCCCTGAGCAG GTCAGGACTCAGAACAGTCTGGCGTCTCCAGACTCTCACATGCAGTATGTGCAGGCACCTGATA CTTCTGTTGCCCTTGTGCTCCAGTCATTGCACAAGGCAGAAAACAGCTCTGGCAGGAAGGGACT GCCAAAGTTAGGAGCCCTAGGGCCTGGAAGGAGAGTATGGTCCTCAGATCCCCCTTCTCTCCTG CTTCCTCCAGGGAACCCAACAGTCATGACCCTGATAGTTTCCCATAACAACCTGGGCATTCCTT GGGACTCAGGAGCTGCTAAACTCTTTCATCCCCTGGTGGCTTCAGCAGTCCTTATCACCAGCCT CACAATCCCACAGGCCCACCCCCAGTGGGCCTGTGGCATTCATATTTCATATTCATATTTCAAA ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025 CCACAATATCCAGCAAAATGTCTCCTGAGCACCCAGAACTCCATACCATCGGCCGGGTGTGGTG GCTCATGCCTTAATCCCAGCACTTTGGGAGGTCAAGATGGGAGGATTGCTTGAGCCCAGAAGTT CGAGACTAGCCTGGGAAACATAGGAAGCCCTCGTCTCTACAAAAAAAATTTAAAAAGTTAGCCA GGTATGGTGGCATATACTTGTGGTCCCAGATACTTGGGAGGCTGAGATAGGATCACTTGGGCCC AGGAGTTTGAGGCTGCAGTGAGCCATCATCATGGCATCATTGCATTCCAGCCTGGGCAACAGAG CAAGACCTCGTCTCAAAAAAAAAAAAAAAATGAAGTCCATGCCACCATTCTTGGCAGCCCAGCC CTTATCCTCCTTAATTGCTCCCTGTCCCTTTTCCAGGCTGCAGAGAGACTAAGGGCACTTCCTG AGGTTCATTACCACCTGGGGCAGAAGGACAGGGAGACAGCAACCATCGCCTGACCACGCCGACC ACAGGGCCTTGAATCCTTTTTTGTTTTCAACAGTCTTGCTGAATTAAGCAGAAAGGGCCTTGAA TCCTGGCCTGGAATTTGGGCAGATATAGCATTAATAAAACTGTGCATCTCAAACTTTTATCACA TACTCTAATATCAGAGGAGTGTGAACCTTCAGAGATCTAGGGTTAAAAGCTAAAGGCATAGCTC CAGCTACAACTTTTCTTTGTCTACAGATGTGTAAAATCTTGATGCAAAAATCTACCCCAAATTT CTGAACAAAATTAATATTCAGTATTATATCTAGCCTATAGATTCTCTTACTCTTGGTGTAATGA CAGCATTCCCAGTTTAGGAAACTGTTTTAAAATCTTGTGTCTTGGTTGTGGCTGAGGCTTTGGG AAGGCCAGAGCCCCCTTTGCCACCACCCCTAGGGTGTCAGCTCCAAGCCTGGGCCTGAGGGTTT GATGGGGAGCGGGTGAGCTGGATCCTGATCCCAGTGGTCAGTGGCCAGATCCTGGGCTGCTGGC CAGAACCCTGGCTTTGTGAGTGGCTGCAGCTTGGCTCTGCTCCAGCCCACAGGGGCTAAAGGGG CGGGTAGGGAGTGTGTAGGGAGCGTGTACGTGGCTTTGTGTCCTTGGCTCCTCCAGACTCTCAC ATGTGCGGGCACCATATACTTCTGCTGCCCTTGTGCCCCAATCATTGCACAAACAGAAACAGCT CTGACAGAGAAGGGACTGCTAGAGTTAGGAACCCTAGGGCCTGGAAGGAAGGTATGGCTCTCAG AGTGACCTGGGGTCTATTCAGTCCCCTCTGGACCTGTTTCCCTGGCAACGCTGTGGGAACAACA GTCTATGCAACACAGGAGACCTGATGCCACTGGTGTCCTCCAAATTTTAAGGCGAAGGCAGGCA TTGTGAATGATGCAGGGATGGCCAGAATGGTTTTTGCCTTCGTTCTTTCCCCAGGGGAACCAAG CCTGTGGGTGGGGAAATCACACTAAGGCTGGACAGTGACTGAATCTGGCCCAGGAGAAACTTGG CTGCCTGCCTTCCAGACCAGGGTTTTTCTTCCCTGAGATCCTGGAGGGGAAGTACCTGGAACTT GGGAGTGTTGCCCTGGTCTGGAAGATCTGATAGACATAGTATGCACACATTTCCCTCGAGCTTC CCATGACTGGGGAGTAGATGAGA (SEQ ID NO: 688)

> M60091.1:29-l 168 Homo sapiens galactose- 1 -phosphate uridyl transferase (GALT) mRNA, complete cds ATGTCGCGCAGTGGAACCGATCCTCAGCAACGCCAGCAGGCGTCAGAGGCGGACGCCGCAGCAG CAACCTTCCGGGCAAACGACCATCAGCATATCCGCTACAACCCGCTGCAGGATGAGTGGGTGCT GGTGTCAGCTCACCGCATGAAGCGGCCCTGGCAGGGTCAAGTGGAGCCCCAGCTTCTGAAGACA GTGCCCCGCCATGACCCTCTCAACCCTCTGTGTCCTGGGGCCATCCGAGCCAACGGAGAGGTGA ATCCCCAGTACGATAGCACCTTCCTGTTTGACAACGACTTCCCAGCTCTGCAGCCTGATGCCCC ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025 CAGTCCAGGACCCAGTGATCATCCCCTTTTCCAAGCAAAGTCTGCTCGAGGAGTCTGTAAGGTC ATGTGCTTCCACCCCTGGTCGGATGTAACGCTGCCACTCATGTCGGTCCCTGAGATCCGGGCTG TTGTTGATGCATGGGCCTCAGTCACAGAGGAGCTGGGTGCCCAGTACCCTTGGGTGCAGATCTT TGAAAACAAAGGTGCCATGATGGGCTGTTCTAACCCCCACCCCCACTGCCAGGTATGGGCCAGC AGTTTCCTGCCAGATATTGCCCAGCGTGAGGAGCGATCTCAGCAGGCCTATAAGAGTCAGCATG GAGAGCCCCTGCTAATGGAGTACAGCCGCCAGGAGCTACTCAGGAAGGAACGTCTGGTCCTAAC CAGTGAGCACTGGTTAGTACTGGTCCCCTTCTGGGCAACATGGCCCTACCAGACACTGCTGCTG CCCGTCGGCCATGTGCGGCGGCTACCTGAGCTGACCCCTGCTGAGCGTGATGATCTAGCCTCCA TCATGAAGAAGCTCTTGACCAAGTATGACAACCTCTTTGAGACGTCCTTTCCCTACTCCATGGG CTGGCATGGGGCTCCCACAGGATCAGAGGCTGGGGCCAACTGGAACCATTGGCAGCTGCACGCT CATTACTACCCTCCGCTCCTGCGCTCTGCCACTGTCCGGAAATTCATGGTTGGCTACGAAATGC TTGCTCAGGCTCAGAGGGACCTCACCCCTGAGCAGGCTGCAGAGAGACTAAGGGCACTTCCTGA GGTTCATTACCACCTGGGGCAGAAGGACAGGGAGACAGCAACCATCGCCTGA (SEQ ID NO: 689)

By “adenine” or “ 9Z7-Purin-6-amine” is meant a purine nucleobase with the molecular

formula C5H5N5, having the structure, and corresponding to CAS No. 73-24-5.

By “adenosine” or “ 4-Amino-l-[(2A,3A,45,5A)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]pyrimidin-2(177)-one” is meant an adenine molecule attached to a NH₂

ribose sugar via a glycosidic bond, having the structure

OH OH corresponding to CAS No. 65-46-3. Its molecular formula is C10H13N5O4.

By “adenosine deaminase” or “adenine deaminase” is meant a polypeptide or fragment thereof capable of catalyzing the hydrolytic deamination of adenine or adenosine. In some embodiments, the deaminase or deaminase domain is an adenosine deaminase catalyzing the hydrolytic deamination of adenosine to inosine or deoxy adenosine to deoxyinosine. In some ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

embodiments, the adenosine deaminase catalyzes the hydrolytic deamination of adenine or adenosine in deoxyribonucleic acid (DNA). The adenosine deaminases (e.g., engineered adenosine deaminases, evolved adenosine deaminases) provided herein may be from any organism (e.g., eukaryotic, prokaryotic), including but not limited to algae, bacteria, fungi, plants, invertebrates (e.g., insects), and vertebrates (e.g., amphibians, mammals). In some embodiments, the adenosine deaminase is an adenosine deaminase variant with one or more alterations and is capable of deaminating both adenine and cytosine in a target polynucleotide (e.g., DNA, RNA) and may be referred to as a “dual deaminase”. Non-limiting examples of dual deaminases include those described in PCT/US22/22050. In some embodiments, the target polynucleotide is single or double stranded. In some embodiments, the adenosine deaminase variant is capable of deaminating both adenine and cytosine in DNA. In some embodiments, the adenosine deaminase variant is capable of deaminating both adenine and cytosine in singlestranded DNA. In some embodiments, the adenosine deaminase variant is capable of deaminating both adenine and cytosine in RNA. In embodiments, the adenosine deaminase variant is selected from those described in PCT/US2020/018192, PCT/US2020/049975, PCT/US2017/045381, PCT/US2021/016827, PCT/US2022/073781, PCT/US24/34189, or PCT/US2020/028568, the full contents of which are each incorporated herein by reference in their entireties for all purposes. Further non-limiting examples of adenosine deaminases include those disclosed or referenced in Rufflow, etal., “Design of highly functional genome editors by modeling of the universe of CRISPR-Cas Sequences,” bioRxiv, posted April 22, 2024, doi: 10.1101/2024.04.22.590591, the disclosure of which is incorporated herein by reference in its entirety for all purposes, which were designed using artificial intelligence. Further exemplary adenosine deaminase amino acid sequences include: TadA-8e (SEQ ID NO: 470), Tadl (SEQ ID NO: 471), Tad2 (SEQ ID NO: 472), Tad3 (SEQ ID NO: 473), Tad4 (SEQ ID NO: 474), Tad6 (SEQ ID NO: 475), Tad6-SR (SEQ ID NO: 476), TadA9 (SEQ ID NO: 477), TadA20 (SEQ ID NO: 478), Staphylococcus aureus Tad A (SEQ ID NO: 479), Bacillus sublilis Tad A (SEQ ID NO: 480), Salmonella typhimurium Tad A (SEQ ID NO: 481), Shewanella putrefaciens (SEQ ID NO: 482), Haemophilus influenzae F3031 Tad A (SEQ ID NO: 483), Caulobacter crescentus TadA (SEQ ID NO: 484), Geobacter sulfiureducens TadA (SEQ ID NO: 485), Streptococcus pyogenes TadA (SEQ ID NO: 486), Aquifex aeolicus TadA (SEQ ID NO: 487), and E. coll TadA deaminase (ecTadA) (SEQ ID NO: 488).

By “adenosine deaminase activity” is meant catalyzing the deamination of adenine or adenosine to guanine in a polynucleotide. ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

By “Adenosine Base Editor (ABE)” is meant a base editor comprising an adenosine deaminase.

By “Adenosine Base Editor (ABE) polynucleotide” is meant a polynucleotide encoding an ABE.

“Administering” is referred to herein as providing one or more compositions described herein to a patient or a subject. By way of example and without limitation, composition administration (e.g., injection) can be performed by intravenous (i.v.) injection, sub-cutaneous (s.c.) injection, intradermal (i.d.) injection, intraperitoneal (i.p.) injection, or intramuscular (i.m.) injection. One or more such routes can be employed. Parenteral administration can be, for example, by bolus injection or by gradual perfusion over time. In some embodiments, parenteral administration includes infusing or injecting intravascularly, intravenously, intramuscularly, intraarterially, intrathecally, intratumorally, intradermally, intraperitoneally, transtracheally, subcutaneously, subcuticularly, intraarticularly, subcapsularly, subarachnoidly and intrasternally. Alternatively, or concurrently, administration can be by the oral route.

By “agent” is meant any small molecule chemical compound, antibody, lipid nanoparticle, nucleic acid molecule, polypeptide, polypeptide complex, or functional fragments thereof. In an embodiment, the agent is a base editor system described herein or a component thereof. In some embodiments, the agent is a lipid nanoparticle of the disclosure. In some embodiments, the lipid nanoparticle contains a base editor system of the disclosure. The base editor system contains a base editor, or a polynucleotide (e.g., an mRNA molecule) encoding the base editor, and a guide RNA, or a polynucleotide encoding the guide RNA.

By “alteration” is meant a change in the level, structure, or activity of an analyte, gene or polypeptide as detected by standard art known methods such as those described herein. As used herein, an alteration includes a change (e.g., increase or reduction) in expression levels. In embodiments, the increase or reduction in expression levels is by 10%, 25%, 40%, 50% or greater. In some embodiments, an alteration includes an insertion, deletion, or substitution of a nucleobase or amino acid (by, e.g., genetic engineering).

By “ameliorate” is meant reduce, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease. In embodiments, the disease is galactosemia, an autosomal recessive inborn error of metabolism associated with a deficiency of galactose- 1-phosphate uridylyltransferase (GALT). Editing of a GALT gene in a subject having galactosemia restores GALT activity, thereby ameliorating symptoms associated with the disease.

The term “aliphatic” or “aliphatic group”, as used herein, means a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely ATTORNEY DOCKET NO. 180802-47402/PCT

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saturated or that contains one or more units of unsaturation, or a monocyclic hydrocarbon or bicyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic (also referred to herein as “carbocycle,” “carbocyclic”, “cycloaliphatic” or “cycloalkyl”), that has a single point of attachment to the rest of the molecule. Unless otherwise specified, aliphatic groups contain 1-6 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-5 carbon atoms. In some embodiments, aliphatic groups contain 1-4 carbon atoms. In some embodiments, aliphatic groups contain 1-3 carbon atoms, and in some embodiments, aliphatic groups contain 1-2 carbon atoms. In some embodiments, “carbocyclic” (or “cycloaliphatic” or “carbocycle” or “cycloalkyl”) refers to an optionally substituted monocyclic Cs-Cs hydrocarbon, or an optionally substituted C6-C12 bicyclic hydrocarbon, that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single point of attachment to the rest of the molecule. Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

As used herein, the term “alkenyl” refers to an alkyl group, as defined herein, having one or more double bonds. In some embodiments, the term “alkenyl”, used alone or as part of a larger moiety, refers to an optionally substituted straight or branched hydrocarbon chain having at least one double bond and having (unless otherwise specified) 2-20, 2-18, 2-16, 2-14, 2-12, 2-10, 2-8, 2-6, 2-4, or 2-3 carbon atoms (e.g., C2-20, C2-18, C2-16, C2-14, C2-12, C2-10, C2-8, C2-6, C2-4, or C2-3). Exemplary alkenyl groups include ethenyl, propenyl, butenyl, pentenyl, hexenyl, and heptenyl.

The term “alkenylene” refers to a bivalent alkenyl group. A substituted alkenylene chain is a polymethylene group containing at least one double bond in which one or more hydrogen atoms are replaced with a substituent. Suitable substituents include those described below for a substituted aliphatic group.

As used herein, the term “alkyl” is given its ordinary meaning in the art and may include saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. In some embodiments, alkyl has 1-100 carbon atoms. In certain embodiments, a straight chain or branched chain alkyl has about 1-20 carbon atoms in its backbone (e.g., C1-C20 for straight chain, C2-C20 for branched chain), and alternatively, about 1-10. In some embodiments, a cycloalkyl ring has from about 3-10 carbon atoms in their ring structure where such rings are monocyclic or bicyclic, and alternatively about 5, 6 or 7 carbons in the ring ATTORNEY DOCKET NO. 180802-47402/PCT

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structure. In some embodiments, an alkyl group may be a lower alkyl group, wherein a lower alkyl group comprises 1-4 carbon atoms (e.g., C1-C4 for straight chain lower alkyls).

The term “alkylenyl” or “alkylene” refers to a bivalent alkyl group (i.e., a bivalent saturated hydrocarbon chain) that is a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted. Any of the above mentioned monovalent alkyl groups may be an alkylenyl by abstraction of a second hydrogen atom from the alkyl. In some embodiments, an “alkylenyl” is a polymethylene group, i.e., -(CH2)n-, wherein n is a positive integer, preferably from 1 to 10, from 1 to 9, from 1 to 8, from 1 to 7, from 1 to 6, from 1 to 5, from 1 to 4, from 1 to 3, from 1 to 2, from 2 to 5, or from 4 to 8. A substituted alkylenyl is a polymethylene group in which one or more methylene hydrogen atoms are replaced with a substituent. Suitable substituents include those described below for a substituted aliphatic group.

As used herein, the term “alkynyl” refers to an alkyl group, as defined herein, having one or more triple bonds. In some embodiments, the term “alkynyl”, used alone or as part of a larger moiety, refers to an optionally substituted straight or branched chain hydrocarbon group having at least one triple bond and having (unless otherwise specified) 2-20, 2-18, 2-16, 2-14, 2-12, 2-10, 2-8, 2-6, 2-4, or 2-3 carbon atoms (e.g., C2-20, C2-18, C2-16, C2-14, C2-12, C2-10, C2-8, C2-6, C2-4, or C2-3). Exemplary alkynyl groups include ethynyl, propynyl, butynyl, pentynyl, hexynyl, and heptynyl.

By “analog” is meant a molecule that is not identical but has analogous functional or structural features. For example, a polypeptide analog retains the biological activity of a corresponding naturally-occurring polypeptide, while having certain biochemical modifications that enhance the analog’s function relative to a naturally occurring polypeptide. Such biochemical modifications could increase the analog’s protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding. An analog may include an unnatural amino acid.

The term “aryl” refers to monocyclic and bicyclic ring systems having a total of six to fourteen ring members (e.g., C6-14), wherein at least one ring in the system is aromatic and wherein each ring in the system contains three to seven ring members. The term “aryl” may be used interchangeably with the term “aryl ring”. In some embodiments, “aryl” refers to an aromatic ring system which includes, but is not limited to, phenyl, naphthyl, anthracyl and the like, which may bear one or more substituents. Unless otherwise specified, “aryl” groups are hydrocarbons.

By “base editor (BE),” or “nucleobase editor polypeptide (NBE)” is meant an agent that binds a polynucleotide and has nucleobase modifying activity. In various embodiments, the base ATTORNEY DOCKET NO. 180802-47402/PCT

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editor comprises a nucleobase modifying polypeptide (e.g., a deaminase) and a polynucleotide programmable nucleotide binding domain (e.g., Cas9 or Cpfl). Representative nucleic acid and protein sequences of base editors include those sequences having about or at least about 85% sequence identity to any base editor sequence provided in the sequence listing, such as those corresponding to SEQ ID NOs: 2-11.

By “BE4 cytidine deaminase (BE4) polypeptide,” is meant a base editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) domain, a cytidine deaminase domain, and two uracil glycosylase inhibitor domains (UGIs). In embodiments, the napDNAbp is a Cas9n (DIO A) polypeptide. Non-limiting examples of cytidine deaminase domains include rAPOBEC, ppAPOBEC, RrA3F, AmAPOBECl, and SsAPOBEC3B.

By “BE4 cytidine deaminase (BE4) polynucleotide,” is meant a polynucleotide encoding a BE4 polypeptide.

By “base editing activity” is meant acting to chemically alter a base within a polynucleotide. In one embodiment, a first base is converted to a second base. In one embodiment, the base editing activity is cytidine deaminase activity, e.g., converting target OG to T»A. In another embodiment, the base editing activity is adenosine or adenine deaminase activity, e.g., converting A»T to G»C.

By “base editing efficiency” is meant the total percent of one or more target bases in a sample that have been modified using a base editor. In some cases, the base editing efficiency is calculated as the total percent of target polynucleotides in a sample containing a modified target base. In some instances, the base editing efficiency is calculated as the total percent of target polynucleotides in a sample containing a modification to one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10) of 2, 3, 4, 5, 6, 7, 8, 9, or 10 target bases. Methods for measuring base editing efficiency for a base editor are known in the art (see, e.g., Gaudelli, etal. Nature 551:464-471 (2017), the disclosure of which is incorporated herein in its entirety for all purposes). In some cases a base editing efficiency is a median base editing efficiency calculated across 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more target sites.

By “base editing window” for a base editor is meant bases within a target polynucleotide sequence that can be modified using the base editor. In some embodiments, the position of the nucleobases in the target polynucleotide sequence are numbered relative to a protospacer adjacent motif (PAM) for which a nucleic acid programmable DNA binding protein (napDNAbp) domain of the base editor has specificity, where base 1 corresponds to the base immediately adjacent to the PAM. In some embodiments, the position of the nucleobases in the target polynucleotide sequence are numbered relative to the 5' or 3' end of a spacer of a guide ATTORNEY DOCKET NO. 180802-47402/PCT

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polynucleotide used to guide a nucleic acid programmable DNA binding protein (napDNAbp) domain of the base editor to a target site, where base 1 corresponds to the 5' or 3' terminal base of the spacer.

The term “base editor system” refers to an intermolecular complex for editing a nucleobase of a target nucleotide sequence. In various embodiments, the base editor (BE) system comprises (1) a polynucleotide programmable nucleotide binding domain, a deaminase domain (e.g., cytidine deaminase or adenosine deaminase) for deaminating nucleobases in the target nucleotide sequence; and (2) one or more guide polynucleotides (e.g., guide RNA) in conjunction with the polynucleotide programmable nucleotide binding domain. In various embodiments, the base editor (BE) system comprises a nucleobase editor domain selected from an adenosine deaminase or a cytidine deaminase, and a domain having nucleic acid sequence specific binding activity. In some embodiments, the base editor system comprises (1) a base editor (BE) comprising a polynucleotide programmable DNA binding domain and a deaminase domain for deaminating one or more nucleobases in a target nucleotide sequence; and (2) one or more guide RNAs in conjunction with the polynucleotide programmable DNA binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable DNA binding domain. In some embodiments, the base editor is a cytidine base editor (CBE). In some embodiments, the base editor is an adenine or adenosine base editor (ABE). In some embodiments, the base editor is an adenine or adenosine base editor (ABE) or a cytidine or cytosine base editor (CBE). In some embodiments, the base editor system (e.g., a base editor system comprising a cytidine deaminase) comprises a uracil glycosylase inhibitor or other agent or peptide (e.g., a uracil stabilizing protein such as provided in WO2022015969, the disclosure of which is incorporated herein by reference in its entirety for all purposes) that inhibits the inosine base excision repair system.

As used herein, the term “bivalent” refers to a chemical moiety with two points of attachment. For example, a “bivalent Ci-s (or Ci-e) saturated or unsaturated, straight or branched, hydrocarbon chain”, refers to bivalent alkylene, alkenylene, and alkynylene chains that are straight or branched as defined herein.

As used herein, the term “bridged bicyclic” refers to any bicyclic ring system, i.e. carbocyclic or heterocyclic, saturated or partially unsaturated, having at least one bridge. As defined by IUPAC, a “bridge” is an unbranched chain of atoms or an atom or a valence bond connecting two bridgeheads, where a “bridgehead” is any skeletal atom of the ring system which is bonded to three or more skeletal atoms (excluding hydrogen). In some embodiments, a bridged bicyclic group has 7-12 ring members and 0-4 heteroatoms independently selected from ATTORNEY DOCKET NO. 180802-47402/PCT

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nitrogen, oxygen, or sulfur. Such bridged bicyclic groups are well known in the art and include those groups set forth below where each group is attached to the rest of the molecule at any substitutable carbon or nitrogen atom. Unless otherwise specified, a bridged bicyclic group is optionally substituted with one or more substituents as set forth for aliphatic groups.

Additionally or alternatively, any substitutable nitrogen of a bridged bicyclic group is optionally substituted. Exemplary bridged bicyclics include but are not limited to:

The terms “carbocyclyl,” “carbocycle,” and “carbocyclic ring” as used herein, refer to saturated or partially unsaturated cyclic aliphatic monocyclic, bicyclic, or polycyclic ring systems, as described herein, having from 3 to 14 members, wherein the aliphatic ring system is optionally substituted as described herein. Carbocyclic groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, norbornyl, adamantyl, and cyclooctadienyl. In some embodiments, “carbocyclyl” (or “cycloaliphatic”) refers to an optionally substituted monocyclic Cs-Cs hydrocarbon, or an optionally substituted C6-C12 bicyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single point of attachment to the rest of the molecule. The term “cycloalkyl” refers to an optionally substituted saturated ring system of about 3 to about 10 ring carbon atoms. In some embodiments, cycloalkyl groups have 3-6 carbons. Exemplary monocyclic cycloalkyl rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. The term “cycloalkenyl” refers to an optionally substituted non-aromatic monocyclic or multicyclic ring ATTORNEY DOCKET NO. 180802-47402/PCT

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system containing at least one carbon-carbon double bond and having about 3 to about 10 carbon atoms. Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl, and cycloheptenyl.

The term “Cas9” or “Cas9 domain” refers to an RNA guided nuclease comprising a Cas9 protein, or a fragment thereof (e.g., a protein comprising an active, inactive, or partially active DNA cleavage domain of Cas9, and/or the gRNA binding domain of Cas9). A Cas9 nuclease is also referred to sometimes as a casnl nuclease or a CRISPR (clustered regularly interspaced short palindromic repeat) associated nuclease.

The term “conservative amino acid substitution” or “conservative mutation” refers to the replacement of one amino acid by another amino acid with a common property. A functional way to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz, G. E. and Schirmer, R. EL, Principles of Protein Structure, Springer-Verlag, New York (1979)). According to such analyses, groups of amino acids can be defined where amino acids within a group exchange preferentially with each other, and therefore resemble each other most in their impact on the overall protein structure (Schulz, G. E. and Schirmer, R. EL, supra). Nonlimiting examples of conservative mutations include amino acid substitutions of amino acids, for example, lysine for arginine and vice versa such that a positive charge can be maintained; glutamic acid for aspartic acid and vice versa such that a negative charge can be maintained; serine for threonine such that a free -OH can be maintained; and glutamine for asparagine such that a free -NH2 can be maintained.

Amino acids generally can be grouped into classes according to the following common side- chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

In some embodiments, conservative substitutions can involve the exchange of a member of one of these classes for another member of the same class. In some embodiments, nonconservative amino acid substitutions can involve exchanging a member of one of these classes for another class. ATTORNEY DOCKET NO. 180802-47402/PCT

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The term “coding sequence” or “protein coding sequence” as used interchangeably herein refers to a segment of a polynucleotide that codes for a protein. Coding sequences can also be referred to as open reading frames. The region or sequence is bounded nearer the 5' end by a start codon and nearer the 3' end with a stop codon. Stop codons useful with the base editors described herein include the following: TAG, TAA, and TGA.

By “complex” is meant a combination of two or more molecules whose interaction relies on inter-molecular forces. Non-limiting examples of inter-molecular forces include covalent and non-covalent interactions. Non-limiting examples of non-covalent interactions include hydrogen bonding, ionic bonding, halogen bonding, hydrophobic bonding, van der Waals interactions (e.g., dipole-dipole interactions, dipole-induced dipole interactions, and London dispersion forces), and 7t-effects. In an embodiment, a complex comprises polypeptides, polynucleotides, or a combination of one or more polypeptides and one or more polynucleotides. In one embodiment, a complex comprises one or more polypeptides that associate to form a base editor (e.g., base editor comprising a nucleic acid programmable DNA binding protein, such as Cas9, and a deaminase) and a polynucleotide (e.g., a guide RNA). In an embodiment, the complex is held together by hydrogen bonds. It should be appreciated that one or more components of a base editor (e.g., a deaminase, or a nucleic acid programmable DNA binding protein) may associate covalently or non-covalently. As one example, a base editor may include a deaminase covalently linked to a nucleic acid programmable DNA binding protein (e.g., by a peptide bond).

Alternatively, a base editor may include a deaminase and a nucleic acid programmable DNA binding protein that associate noncovalently (e.g., where one or more components of the base editor are supplied in trans and associate directly or via another molecule such as a protein or nucleic acid). In an embodiment, one or more components of the complex are held together by hydrogen bonds.

By “cytosine” or “4-Aminopyrimidin-2(U7)-one” is meant a purine nucleobase with the o

N^NH

molecular formula C4H5N3O, having the structure

2, _anc[ corresponding to CAS No. 71-30-7. ATTORNEY DOCKET NO. 180802-47402/PCT

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By “cytidine” is meant a cytosine molecule attached to a ribose sugar via a glycosidic

bond, having the structure, and corresponding to CAS No. 65-46-3. Its molecular formula is C9H13N3O5.

By “Cytidine Base Editor (CBE)” is meant a base editor comprising a cytidine deaminase. Non-limiting examples of cytidine deaminase base editor amino acid sequences include amino acid sequences for BE4max (SEQ ID NO: 553), YE1-BE4 (SEQ ID NO: 554), YE2-BE4 (SEQ ID NO: 555), YEE-BE4 (SEQ ID NO: 556), EE-BE4 (SEQ ID NO: 557), R33A-BE4 (SEQ ID NO: 558), R33A+K34A-BE4 (SEQ ID NO: 559), APOBEC3A (A3A)-BE4 (SEQ ID NO: 560), APOBEC3B (A3B)-BE4 (SEQ ID NO: 561), APOBEC3G (A3G)-BE4 (SEQ ID NO: 562), AID-BE4 (SEQ ID NO: 563), CDA-BE4 (SEQ ID NO: 564), FERNY-BE4 (SEQ ID NO: 565), evolved APOBEC3A (eA3A)-BE4 (SEQ ID NO: 566), AALN-BE4 (SEQ ID NO: 567), BE4max modified with SpCas9-NG (SEQ ID NO: 568), YEl-SpCas9-NG (YE1-NG) (SEQ ID NO: 569), YE2-SpCas9-NG (SEQ ID NO: 570), YEE-SpCas9-NG (SEQ ID NO: 571), EE-SpCas9-NG (SEQ ID NO: 572), R33A+K34A-SpCas9-NG (SEQ ID NO: 573), YE1-CP1028 (YE1-BE4-CP1028, or YE1-CP) (SEQ ID NO: 574), YE2-CP1028 (YE2-BE4-CP1028) (SEQ ID NO: 575), YEE-CP1028 (YEE-BE4-CP1028) (SEQ ID NO: 576), EE-CP1028 (EE-BE4-CP1028) (SEQ ID NO: 577), R33A+K34A-CP1028 (R33A+K34A-BE4-CP1028) (SEQ ID NO: 578), BE4max (with nickase) (SEQ ID NO: 597), BE4 (SEQ ID NO: 598), BE4 with His tag (SEQ ID NO: 599), BE4max (SEQ ID NO: 600), AncBE4max 689 (SEQ ID NO: 601), and AncBE4max 687 (SEQ ID NO: 602). Exemplary CBE sequences are also provided at SEQ ID NOs: 774 to 782, and 792.

By “Cytidine Base Editor (CBE) polynucleotide” is meant a polynucleotide encoding a CBE. Non-limiting examples of polynucleotide sequences encoding cytidine deaminase base editors include those encoding BE4max (SEQ ID NO: 616), AncBE4max689 (SEQ ID NO: 617), and AncBE4max687 (SEQ ID NO: 618).

By “cytidine deaminase” or “cytosine deaminase” is meant a polypeptide or fragment thereof capable of deaminating cytidine or cytosine. In embodiments, the cytidine or cytosine is present in a polynucleotide. In one embodiment, the cytidine deaminase converts cytosine to ATTORNEY DOCKET NO. 180802-47402/PCT

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uracil or 5-methylcytosine to thymine. In some embodiments, a cytidine deaminase is 6b, RrA3f, or T1.52. The terms “cytidine deaminase” and “cytosine deaminase” are used interchangeably throughout the application. Petromyzon marinus cytosine deaminase 1 (PmCDAl) (SEQ ID NO: 12-13), Activation-induced cytidine deaminase (AICDA) (SEQ ID NOs: 14-20), and APOBEC (SEQ ID NOs: 21-61) are exemplary cytidine deaminases. Further exemplary cytidine deaminase (CD A) sequences are provided in the Sequence Listing as SEQ ID NOs: 62-66 and SEQ ID NOs: 67-189. Non-limiting examples of cytidine deaminases include those described in PCT/US20/16288, PCT/US2018/021878, 180802-021804/PCT, PCT/US2018/048969, PCT/US2016/058344, PCT/US2020/062428, and PCT/US2019/033848, the disclosures of which are incorporated herein by reference in their entireties for all purposes. Non-limiting examples of cytidine deaminase amino acid sequences include amino acid sequences for Rat APOBEC 1 (SEQ ID NO: 579), Human APOBEC1 (SEQ ID NO: 580), Human APOBEC3 (SEQ ID NO: 581), Human APOBEC3B (SEQ ID NO: 582), Human APOBEC3G (SEQ ID NO: 583), evoAPOBEC3A(eA3A) (SEQ ID NO: 584), evoCDA (SEQ ID NO: 585), evoAPOBECl (SEQ ID NO: 586), YE1 (SEQ ID NO: 587), YE2 (SEQ ID NO: 588), YEE (SEQ ID NO: 589), EE (SEQ ID NO: 590), R33A (SEQ ID NO: 591), R33A+K34A (SEQ ID NO: 592), AALN (SEQ ID NO: 593), FERNY (SEQ ID NO: 594), evoFERNY (SEQ ID NO: 595), APOBEC (SEQ ID NO: 619), Anc686 APOBEC (SEQ ID NO: 620), Human APOBEC-3GD316R D317R (SEQ ID NO: 621), Human APOBEC-3G chain A (SEQ ID NO: 622), Human APOBEC3-G chain A D120R D121R (SEQ ID NO: 623), Mouse APOBEC3 (SEQ ID NO: 624), Rat APOBEC3 (SEQ ID NO: 625), Rhesus macaque APOBEC-3G (SEQ ID NO: 626), Chimpanzee APOBEC-3G (SEQ ID NO: 627), Green Monkey APOBEC-3G (SEQ ID NO: 628), Human APOBEC-3G (SEQ ID NO: 629), Human APOBEC-3F (SEQ ID NO: 630), Human APOBEC-3B (SEQ ID NO: 631), Rat APOBEC-3B (SEQ ID NO: 632), Bovine APOBEC-3B (SEQ ID NO: 633), Chimpanzee APOBEC-3B (SEQ ID NO: 634), Gorilla APOBEC-3C (SEQ ID NO: 635), Human APOBEC-3A (SEQ ID NO: 636), Rhesus macaque APOBEC-3A (SEQ ID NO: 637), Bovine APOBEC-3A (SEQ ID NO: 638), Human APOBEC-3H (SEQ ID NO: 639), Human APOBEC-3D (SEQ ID NO: 640), Rat ABOPEC1 (SEQ ID NO: 641), Anc689 APOBEC (SEQ ID NO: 642), Anc687 APOBEC (SEQ ID NO: 643), Anc686 APOBEC (SEQ ID NO: 644), Anc655 APOBEC (SEQ ID NO: 645), and Anc733 APOBEC (SEQ ID NO: 646).

By “cytidine deaminase polynucleotide” is meant a polynucleotide encoding a cytidine deaminase. Non-limiting examples of polynucleotide sequences encoding cytidine deaminase domains include those encoding Rat APOBEC 1 (SEQ ID NO: 604), Anc689 APOBEC (SEQ ID NO: 605), Anc687 APOBEC (SEQ ID NO: 606), Anc686 APOBEC (SEQ ID NO: 607), Anc655 ATTORNEY DOCKET NO. 180802-47402/PCT

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APOBEC (SEQ ID NO: 608), Anc733 APOBEC (SEQ ID NO: 609), Rat APOBEC1 (SEQ ID NO: 610), Anc689 APOBEC (SEQ ID NO: 611), Anc687 APOBEC (SEQ ID NO: 612), Anc686 APOBEC (SEQ ID NO: 613), Anc655 APOBEC (SEQ ID NO: 614), and Anc733 APOBEC (SEQ ID NO: 615).

By “cytosine deaminase activity” is meant catalyzing the deamination of cytosine or cytidine. In one embodiment, a polypeptide having cytosine deaminase activity converts an amino group to a carbonyl group. In an embodiment, a cytosine deaminase converts cytosine to uracil (/.<., C to U) or 5-methylcytosine to thymine (/.<., 5mC to T). In some embodiments, a cytosine deaminase as provided herein has increased cytosine deaminase activity (e.g., at least 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or more) relative to a reference cytosine deaminase.

The term “deaminase” or “deaminase domain,” as used herein, refers to a protein or fragment thereof that catalyzes a deamination reaction.

The term “detect” refers to identifying the presence, absence or amount of the analyte to be detected. In one embodiment, a sequence alteration in a polynucleotide or polypeptide is detected. In another embodiment, the presence of indels is detected. In one embodiment, galactose- 1 -phosphate uridylyltransferase (GALT) activity is detected. In another embodiment, levels of galactose and/or galactose metabolites (e.g., galactitol or galactose- 1-P) in a biological sample (e.g., blood, serum, plasma, tissue) are detected.

By “disease” is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Exemplary diseases include galactosemia.

By “effective amount” is meant the amount of an agent (e.g., a base editor, cell) as described herein, that is required to ameliorate the symptoms of a disease relative to an untreated patient or an individual without disease, i.e., a healthy individual, or is the amount of the agent sufficient to elicit a desired biological response. The effective amount of active compound(s) used to practice embodiments of the present disclosure for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an “effective” amount. In one embodiment, an effective amount is the amount of a base editor of the disclosure sufficient to introduce an alteration in a gene of interest in a cell (e.g., a cell in vitro or in vivo). In one embodiment, an effective amount is the amount of a base editor required to achieve a therapeutic effect. Such therapeutic effect need not be sufficient to alter a pathogenic gene in all cells of a subject, tissue or organ, but only to alter the pathogenic gene in about 1%, 5%, 10%, 25%, 50%, ATTORNEY DOCKET NO. 180802-47402/PCT

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75% or more of the cells present in a subject, tissue or organ. In one embodiment, an effective amount is sufficient to ameliorate one or more symptoms of a disease. For example, an effective amount of a base editor described herein is delivered to a tissue of a patient where it increases the level of expression of functional galactose- 1 -phosphate uridylyltransferase (GALT), thereby reducing the accumulation of galactose or galactose metabolites (e.g., galactitol or galactose- 1-P) in the serum of the patient. In some cases, an effective amount of a base editor described herein is delivered to a tissue of a patient where it increases the level of expression galactose- 1-phosphate uridylyltransferase (GALT) activity, thereby reducing the accumulation of galactose and/or galactose metabolites (e.g., galactitol or galactose- 1-P) in the serum of the patient.

The term “encapsulated” is used herein to refer to substances that are completely surrounded by another material. In an embodiment, an mRNA molecule encoding a base editor and/or a guide polynucleotide is encapsulated by a lipid nanoparticle of the disclosure.

By “fragment” is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids. In some embodiments, the fragment is a functional fragment.

By “glycome” is meant the complete set of carbohydrates contained in an organism, cell, tissue, or organ).

By “guide polynucleotide” is meant a polynucleotide or polynucleotide complex which is specific for a target sequence and can form a complex with a polynucleotide programmable nucleotide binding domain protein (e.g., Cas9 or Cpfl). In an embodiment, the guide polynucleotide is a guide RNA (gRNA). gRNAs can exist as a complex of two or more RNAs, or as a single RNA molecule.

The term “haloaliphatic” refers to an aliphatic group substituted by one or more halogen atoms (e.g., one, two, three, four, five, six, or seven halo, such as fluoro, iodo, bromo, or chloro). In some embodiments, haloaliphatic groups contain 1-7 halogen atoms. In some embodiments, haloaliphatic groups contain 1-5 halogen atoms. In some embodiments, haloaliphatic groups contain 1-3 halogen atoms. ATTORNEY DOCKET NO. 180802-47402/PCT

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The term “haloalkyl” refers to an alkyl group substituted by one or more halogen atoms (e.g., one, two, three, four, five, six, or seven halo, such as fluoro, iodo, bromo, or chloro). In some embodiments, haloalkyl groups contain 1-7 halogen atoms. In some embodiments, haloalkyl groups contain 1-5 halogen atoms. In some embodiments, haloalkyl groups contain 1-3 halogen atoms.

The term “heteroalkylenyl” or “heteroalkylene”, as used herein, denotes an optionally substituted straight-chain (/.<?., unbranched), or branched bivalent alkyl group (i.e., bivalent saturated hydrocarbon chain) having, in addition to carbon atoms, from one to five heteroatoms. The term “heteroatom” is described below. In some embodiments, heteroalkylenyl groups contain 2-10 carbon atoms wherein 1-3 carbon atoms are optionally and independently replaced with heteroatoms selected from oxygen, nitrogen, and sulfur. In some embodiments, heteroalkylenyl groups contain 2-8 carbon atoms wherein 1-3 carbon atoms are optionally and independently replaced with heteroatoms selected from oxygen, nitrogen, and sulfur. In some embodiments, heteroalkylenyl groups contain 4-8 carbon atoms, wherein 1-3 carbon atoms are optionally and independently replaced with heteroatoms selected from oxygen, nitrogen, and sulfur. In some embodiments, heteroalkylenyl groups contain 2-5 carbon atoms, wherein 1-2 carbon atoms are optionally and independently replaced with heteroatoms selected from oxygen, nitrogen, and sulfur. In yet other embodiments, heteroalkylenyl groups contain 1-3 carbon atoms, wherein 1 carbon atom is optionally and independently replaced with a heteroatom selected from oxygen, nitrogen, and sulfur. Suitable heteroalkylenyl groups include, but are not limited to -CH2O-, -(CH₂)₂O-, -CH₂OCH₂-, -O(CH₂)₂-, -(CH₂)₃O-, -(CH₂)₂OCH₂-, -CH₂O(CH₂)₂-, -O(CH₂)₃-, -(CH₂)₄O-, -(CH₂)₃OCH₂-, -CH₂O(CH₂)₃-, -(CH₂)₂O(CH₂)₂-, -O(CH₂)4-. Unless otherwise specified, Cx heteroalkylenyl refers to heteroalkylenyl having x number of carbon atoms prior to replacement with heteroatoms.

The terms “heteroaryl” and “heteroar-”, used alone or as part of a larger moiety, e.g., “heteroaralkyl”, or “heteroaralkoxy”, refer to monocyclic or bicyclic ring groups having 5 to 10 ring atoms (e.g., 5- to 6-membered monocyclic heteroaryl or 9- to 10-membered bicyclic heteroaryl); having 6, 10, or 147t electrons shared in a cyclic array; and having, in addition to carbon atoms, from one to five heteroatoms. Exemplary heteroaryl groups include, without limitation, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridonyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, pteridinyl, imidazo[l,2-a]pyrimidinyl, imidazo[l,2-a]pyridinyl, thienopyrimidinyl, triazol opyridinyl, and benzoisoxazolyl. The terms “heteroaryl” and “heteroar-”, as used herein, also include groups in ATTORNEY DOCKET NO. 180802-47402/PCT

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which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring (i.e., a bicyclic heteroaryl ring having 1 to 3 heteroatoms). Nonlimiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzothiazolyl, benzothiadiazolyl, benzoxazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, H quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, pyrido[2,3-b]-l,4-oxazin-3(4H)-one, and benzoisoxazolyl. The term “heteroaryl” may be used interchangeably with the terms “heteroaryl ring”, “heteroaryl group”, or “heteroaromatic”, any of which terms include rings that are optionally substituted.

The term “heteroatom” means one or more of oxygen, sulfur, nitrogen, phosphorus, or silicon (including, any oxidized form of nitrogen, sulfur, phosphorus, or silicon; the quatemized form of any basic nitrogen or; a substitutable nitrogen of a heterocyclic ring, for example N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or NR⁺ (as in N-substituted pyrrolidinyl)).

The terms “heterocycle”, “heterocyclyl”, “heterocyclic radical”, and “heterocyclic ring” are used interchangeably herein, and refer to a stable 3- to 8-membered monocyclic, a 7- to 12-membered bicyclic, or a 10- to 16-membered polycyclic heterocyclic moiety that is either saturated or partially unsaturated, and having, in addition to carbon atoms, one or more, such as one to four, heteroatoms, as defined above. When used in reference to a ring atom of a heterocycle, the term “nitrogen” includes a substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur or nitrogen, the nitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or NR⁺ (as in N-substituted pyrrolidinyl). A heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure and any of the ring atoms can be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include, without limitation, azetidinyl, oxetanyl, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, piperidinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, tetrahydropyranyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, thiamorpholinyl, and

. A heterocyclyl group may be mono-, bi-, tri-, or polycyclic, preferably mono-, bi-, or tricyclic, more preferably mono- or bicyclic. The term “heterocyclylalkyl” refers to an alkyl group substituted by a heterocyclyl, wherein the alkyl and heterocyclyl portions independently are optionally substituted. A bicyclic heterocyclic ring also includes groups in which the heterocyclic ring is fused to one or more aryl, heteroaryl, or cycloaliphatic rings. Exemplary ATTORNEY DOCKET NO. 180802-47402/PCT

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bicyclic heterocyclic groups include indolinyl, isoindolinyl, benzodi oxolyl, 1,3-dihydroisobenzofuranyl, 2,3-dihydrobenzofuranyl, and tetrahydroquinolinyl. A bicyclic heterocyclic ring can also be a spirocyclic ring system (e.g., 7- to 11-membered spirocyclic fused heterocyclic ring having, in addition to carbon atoms, one or more heteroatoms as defined above (e.g., one, two, three or four heteroatoms)). A bicyclic heterocyclic ring can also be a bridged ring system (e.g., 7- to 11-membered bridged heterocyclic ring having one, two, or three bridging atoms.

The term “hairpin” as used herein describes a duplex of nucleic acids that is created when a nucleic acid strand folds and forms base pairs with another section of the same strand. A hairpin may form a structure that comprises a loop or a U-shape. In some embodiments, a hairpin may be comprised of an RNA loop. Hairpins can be formed with two complementary sequences in a single nucleic acid molecule bind together, with a folding or wrinkling of the molecule. In some embodiments, hairpins comprise stem or stem loop structures. In the context of modified gRNAs described herein, a “hairpin region” refers to hairpin 1 and hairpin 2 from the 5' end to the 3’ end of the gRNA. A conserved portion of a gRNA locates between hairpin 1 and hairpin 2 of the gRNA.

“Hybridization” means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.

By “increase” is meant a positive alteration of at least 10%, 25%, 50%, 75%, or 100%, or about 1.5 fold, about 2 fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, or about 100-fold. In embodiments, expression of a base editor described herein increases expression of functional galactose- 1 -phosphate uridylyltransferase (GALT) in a tissue of a subject. In embodiments, expression of a base editor described herein increases galactose- 1 -phosphate uridylyltransferase (GALT) activity in a tissue of a subject (e.g., in the liver).

The terms “inhibitor of base repair”, “base repair inhibitor”, “IBR” or their grammatical equivalents refer to a protein that is capable in inhibiting the activity of a nucleic acid repair enzyme, for example a base excision repair enzyme.

The terms “isolated,” “purified,” or “biologically pure” refer to material that is free to varying degrees from components which normally accompany it as found in its native state. “Isolate” denotes a degree of separation from original source or surroundings. “Purify” denotes a ATTORNEY DOCKET NO. 180802-47402/PCT

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degree of separation that is higher than isolation. A “purified” or “biologically pure” protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this disclosure is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term “purified” can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.

By “isolated polynucleotide” is meant a nucleic acid molecule that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the disclosure is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.

By an “isolated polypeptide” is meant a polypeptide of the disclosure that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. In embodiments, the preparation is at least 75%, at least 90%, or at least 99%, by weight, a polypeptide of the disclosure. An isolated polypeptide of the disclosure may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

The term “linker”, as used herein, refers to a molecule that links two moieties. In one embodiment, the term “linker” refers to a covalent linker (e.g., covalent bond) or a non-covalent linker. ATTORNEY DOCKET NO. 180802-47402/PCT

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As used herein, a “lipid nanoparticle (LNP) composition” is a nanoparticle composition comprising one or more ionizable lipids. Exemplary LNP compositions include, but are not limited to, phospholipids, conjugate-linker lipids (e.g., PEG-lipids), sterols, and ionizable lipids.

The term “loop” as used herein describes a region of nucleotides that do not base pair (i.e., are not complementary) that may cap a stem. For example, a “tetraloop” describes a loop of 4 nucleotides. In some embodiments, the upper stem of a modified gRNA may comprise a tetraloop. In some embodiments, GAAA is the GNR. A tetraloop that is used to lock the structure of the hairpin. In some embodiments, the tetraloop is ANYA, CUYG, GNRA, UNAC, or UNCG.

By “marker” is meant any protein or polynucleotide having an alteration in expression, level, structure, or activity that is associated with a disease or disorder. In an embodiment, the level of galactose or galactose metabolites (e.g., galactitol or galactose- 1-P) in a sample (e.g., blood, serum, plasma, tissue) is a marker of galactosemia. In some embodiments, an aberrant glycome relative to a healthy subject or cell is a marker of galactosemia.

The term “mutation,” as used herein, refers to a substitution of a residue within a sequence, e.g., a nucleic acid or amino acid sequence, with another residue, or a deletion or insertion of one or more residues within a sequence. Mutations are typically described herein by identifying the original residue followed by the position of the residue within the sequence and by the identity of the newly substituted residue. Various methods for making the amino acid substitutions (mutations) provided herein are well known in the art, and are provided by, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual (4^th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (2012)).

As used herein, “N: P ratio” is the molar ratio of ionizable nitrogen atoms in a lipid(s) to phosphate groups in a nucleic acid molecular entity(ies). In some embodiments, the lipids are lipids of a lipid nanoparticle and the nucleic acid molecular entity(ies) is a payload of the lipid nanoparticle. In various embodiments the N: P ratio is about or at least about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In various embodiments the N: P ratio less than about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In some embodiments, the nitrogen atoms are ionizable within a physiological pH range. In some embodiments, the molecular entities in a nanoparticle composition include a lipid component and an RNA. Ionizable nitrogen atoms can include, for example, nitrogen atoms that can be protonated at about pH 1, about pH 2, about pH 3, about pH 4, about pH 4.5, about pH 5, about pH 5.5, about pH 6, about pH 6.5, about pH 7, about pH 7.5, or about pH 8 or higher. The physiological pH range can include, for example, the pH range of different cellular compartments (such as organs, tissues, and cells) and bodily fluids (such as blood, CSF, gastric juice, milk, bile, saliva, tears, and urine). In certain specific embodiments, ATTORNEY DOCKET NO. 180802-47402/PCT

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the physiological pH range refers to the pH range of blood in a mammal, for example, from about 7.35 to about 7.45. Similarly, for phosphate charge neutralizers that have one or more ionizable nitrogen atoms, the N/P ratio can refer to a molar ratio of ionizable nitrogen atoms in the phosphate charge neutralizer to the phosphate groups in a nucleic acid. In some embodiments, ionizable nitrogen atoms refer to those nitrogen atoms that are ionizable within a pH range between 5 and 14.

For a payload that does not contain a phosphate group, the N: P ratio can refer to a molar ratio of ionizable nitrogen atoms in a lipid to the total negative charge in the payload. For example, the N: P ratio of an LNP composition can refer to a molar ratio of the total ionizable nitrogen atoms in the LNP composition to the total negative charge in the payload that is present in the composition.

As used herein, the term “nanoparticle” refers to a particle having a diameter of less than 1000 nanometers (nm). In some embodiments, a nanoparticle has a diameter of less than 300 nm, as defined by the National Science Foundation. In some embodiments, a nanoparticle has a diameter of less than 100 nm as defined by the National Institutes of Health. In some embodiments, nanoparticles are micelles in that they comprise an enclosed compartment, separated from the bulk solution by a micellar membrane, typically comprised of amphiphilic entities which surround and enclose a space or compartment (e.g., to define a lumen). In some embodiments, a micellar membrane is comprised of at least one polymer, such as for example a biocompatible and/or biodegradable polymer. In some embodiments, lipid nanoparticles described herein can have an average hydrodynamic diameter from about 30 to about 170 nm. In some embodiments, lipid nanoparticles described herein can have an average hydrodynamic diameter that is about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 155 nm, 160 nm, 165 nm, 170 nm, 170 nm, 175 nm, 180 nm, 185 nm, 190 nm, 195 nm, 200 nm, 205 nm, 210 nm, 215 nm, 220 nm, or any range having endpoints defined by any two of the aforementioned values. For example, in some embodiments, lipid nanoparticles described herein have an average hydrodynamic diameter from between 50 nm to 100 nm. For example, in some embodiments, compositions, preparations, nanoparticles, and/or nanomaterials described herein have an average hydrodynamic diameter from between 50 nm to 90 nm. In some embodiments, lipid nanoparticles described herein have an average hydrodynamic diameter from about 60 to about 80 nm. For example, in some embodiments, lipid nanoparticles described herein have an average hydrodynamic diameter from between 50 nm to 70 nm. ATTORNEY DOCKET NO. 180802-47402/PCT

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The terms “nucleic acid” and “nucleic acid molecule,” as used herein, refer to a compound comprising a nucleobase and an acidic moiety, e.g., a nucleoside, a nucleotide, or a polymer of nucleotides. Typically, polymeric nucleic acids, e.g., nucleic acid molecules comprising three or more nucleotides are linear molecules, in which adjacent nucleotides are linked to each other via a phosphodiester linkage. In some embodiments, “nucleic acid” refers to individual nucleic acid residues (e.g., nucleotides and/or nucleosides). In some embodiments, “nucleic acid” refers to an oligonucleotide chain comprising three or more individual nucleotide residues. As used herein, the terms “oligonucleotide” and “polynucleotide” can be used interchangeably to refer to a polymer of nucleotides (e.g, a string of at least three nucleotides). In some embodiments, “nucleic acid” encompasses RNA as well as single and/or doublestranded DNA. Nucleic acids may be naturally occurring, for example, in the context of a genome, a transcript, an mRNA, tRNA, rRNA, siRNA, snRNA, a plasmid, cosmid, chromosome, chromatid, or other naturally occurring nucleic acid molecule. On the other hand, a nucleic acid molecule may be a non-naturally occurring molecule, e.g, a recombinant DNA or RNA, an artificial chromosome, an engineered genome, or fragment thereof, or a synthetic DNA, RNA, DNA/RNA hybrid, or including non-naturally occurring nucleotides or nucleosides.

Furthermore, the terms “nucleic acid,” “DNA,” “RNA,” and/or similar terms include nucleic acid analogs, e.g., analogs having other than a phosphodiester backbone. Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic acids comprise nucleoside analogs such as analogs having chemically modified bases or sugars, and backbone modifications. A nucleic acid sequence is presented in the 5' to 3' direction unless otherwise indicated. In some embodiments, a nucleic acid is or comprises natural nucleosides (e.g. adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2 -thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and/or modified phosphate groups (e.g., phosphorothioates and 5'-A-phosphoramidite linkages). In some cases, a guide RNA of the present disclosure includes a modification selected from 2'-O-methyl modified nucleotides (e.g., 2'-O-methyl 3'-phosphorothioate (MS) nucleotide, ATTORNEY DOCKET NO. 180802-47402/PCT

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2'-O-methyl 3 '-thioPACE (MSP) nucleotide), 2’-F modified nucleotides, locked nucleic acid, MOE (methoxy ethyl), DNA nucleotides functionalized for conjugation, and a combination thereof.

The term “nuclear localization sequence,” “nuclear localization signal,” or “NLS” refers to an amino acid sequence that promotes import of a protein into the cell nucleus. Nuclear localization sequences are known in the art and described, for example, in Plank et al., International PCT application, PCTZEP2000/011690, filed November 23, 2000, published as W 0/2001/038547 on May 31, 2001, the contents of which are incorporated herein by reference for their disclosure of exemplary nuclear localization sequences. In other embodiments, the NLS is an optimized NLS described, for example, by Koblan etal., Nature Biotech. 2018 doi:10.1038/nbt.4172. In some embodiments, an NLS comprises the amino acid sequence KRTADGSEFESPKKKRKV (SEQ ID NO: 190), KRPAATKKAGQAKKKK (SEQ ID NO: 191), KKTELQTTNAENKTKKL (SEQ ID NO: 192), KRGINDRNFWRGENGRKTR (SEQ ID NO: 193), RKSGKIAAIVVKRPRK (SEQ ID NO: 194), PKKKRKV (SEQ ID NO: 195), MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 196), PKKKRKVEGADKRTADGSEFESPKKKRKV (SEQ ID NO: 328), or RKSGKIAAIVVKRPRKPKKKRKV (SEQ ID NO: 329).

The term “nucleobase,” “nitrogenous base,” or “base,” used interchangeably herein, refers to a nitrogen-containing biological compound that forms a nucleoside, which in turn is a component of a nucleotide. The ability of nucleobases to form base pairs and to stack one upon another leads directly to long-chain helical structures such as ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). Five nucleobases - adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U) - are called primary or canonical. Adenine and guanine are derived from purine, and cytosine, uracil, and thymine are derived from pyrimidine. DNA and RNA can also contain other (non-primary) bases that are modified. Non-limiting exemplary modified nucleobases can include hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine (m5C), and 5-hydromethylcytosine. Hypoxanthine and xanthine can be created through mutagen presence, both of them through deamination (replacement of the amine group with a carbonyl group). Hypoxanthine can be modified from adenine. Xanthine can be modified from guanine. Uracil can result from deamination of cytosine. A “nucleoside” consists of a nucleobase and a five carbon sugar (either ribose or deoxyribose). Examples of a nucleoside include adenosine, guanosine, uridine, cytidine, 5-methyluridine (m5U), deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine, and deoxycytidine. Examples of a nucleoside with a modified nucleobase includes inosine (I), xanthosine (X), 7-methylguanosine (m7G), ATTORNEY DOCKET NO. 180802-47402/PCT

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dihydrouridine (D), 5-methylcytidine (m5C), and pseudouridine (T). A “nucleotide” consists of a nucleobase, a five carbon sugar (either ribose or deoxyribose), and at least one phosphate group. Non-limiting examples of modified nucleobases and/or chemical modifications that a modified nucleobase may include are the following: pseudo-uridine, 5-Methyl-cytosine, 2'-O-methyl-3'-phosphonoacetate, 2'-O-methyl thioPACE (MSP), 2'-O-methyl-PACE (MP), 2'-fluoro RNA (2'-F-RNA), constrained ethyl (S-cEt), 2'-O-methyl (‘M’), 2'-O-methyl-3'-phosphorothioate (‘MS’), 2'-O-methyl-3'-thiophosphonoacetate (‘MSP’), 5-methoxyuridine, phosphorothioate, and N1 -Methylpseudouridine.

The term “nucleic acid programmable DNA binding protein” or “napDNAbp” may be used interchangeably with “polynucleotide programmable nucleotide binding domain” to refer to a protein that associates with a nucleic acid (e.g., DNA or RNA), such as a guide nucleic acid or guide polynucleotide (e.g., gRNA), that guides the napDNAbp to a specific nucleic acid sequence. In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable DNA binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable RNA binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain is a Cas9 protein. In some embodiments, the polynucleotide programmable nucleotide binding domain is an SpRY, an SpRYc, an saCas9, an spCas9, or an spCas9 variant having specificity for a protospacer-adjacent motif (PAM) selected from one or more of: NG, NRRH, and NRTH, where “N” indicates A, C, G, or T, “R” indicates A or G, “Y” indicates C or T, and “H” indicates A, C, or T. A Cas9 protein can associate with a guide RNA that guides the Cas9 protein to a specific DNA sequence that is complementary to the guide RNA. In some embodiments, the napDNAbp is a Cas9 domain, for example a nuclease active Cas9, a Cas9 nickase (nCas9), or a nuclease inactive Cas9 (dCas9). Non-limiting examples of nucleic acid programmable DNA binding proteins include, Cas9 (e.g., dCas9 and nCas9), Casl2a/Cpfl, Casl2b/C2cl, Casl2c/C2c3, Casl2d/CasY, Casl2e/CasX, Casl2g, Casl2h, Casl2i, and Casl2j/Cas< I> (Casl2j/Casphi). Non-limiting examples of Cas enzymes include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (also known as Csnl or Csxl2), CaslO, CaslOd, Casl2a/Cpfl, Casl2b/C2cl, Casl2c/C2c3, Casl2d/CasY, Casl2e/CasX, Casl2g, Casl2h, Casl2i, Casl2j/Cas< I>, Cpfl, Csyl, Csy2, Csy3, Csy4, Csel, Cse2, Cse3, Cse4, Cse5e, Cscl, Csc2, Csa5, Csnl, Csn2, Csml, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, CsxlS, Csxll, Csfl, Csf2, CsO, Csf4, Csdl, Csd2, Cstl, Cst2, Cshl, Csh2, Csal, Csa2, Csa3, Csa4, Csa5, Type II Cas effector proteins, Type V Cas ATTORNEY DOCKET NO. 180802-47402/PCT

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effector proteins, Type VI Cas effector proteins, CARF, DinG, homologues thereof, or modified or engineered versions thereof. Other nucleic acid programmable DNA binding proteins are also within the scope of this disclosure, although they may not be specifically listed in this disclosure. See, e.g., Makarova et al. “Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?” CRISPRJ. 2018 Oct; 1:325-336. doi: 10.1089/crispr.2018.0033; Yan eta/., “Functionally diverse type V CRISPR-Cas systems” Science. 2019 Jan 4;363(6422):88-91. doi: 10.1126/science.aav7271, the entire contents of each are hereby incorporated by reference.

Exemplary nucleic acid programmable DNA binding proteins and nucleic acid sequences encoding nucleic acid programmable DNA binding proteins are provided in the Sequence Listing as SEQ ID NOs: 197-231, 232-245, 254-257, 260, and 378. In some embodiments, the napDNAbp is the (CRISPR-associated system) Cas9 endonuclease, for example, Cas9 (Csnl) from Streptococcus pyogenes (e.g., SEQ ID NO: 197), Cas9 from Neisseria meningitidis (NmeCas9; SEQ ID NO: 208), Nme2Cas9 (SEQ ID NO: 209), Streptococcus constellatus (ScoCas9), or derivatives thereof (e.g., a sequence with at least about 85% sequence identity to a Cas9, such as Nme2Cas9 or spCas9). Further non-limiting examples of nucleic acid programmable DNA binding proteins include those disclosed or referenced in Rufflow, et al., “Design of highly functional genome editors by modeling of the universe of CRISPR-Cas Sequences,” bioRxiv, posted April 22, 2024, doi: 10.1101/2024.04.22.590591, the disclosure of which is incorporated herein by reference in its entirety for all purposes, which were designed using artificial intelligence. In some embodiments, the napDNAbp is OpenCRISPR-1, or a variant thereof (e.g., a variant comprising a D10A amino acid alteration and/or lacking an N-terminal methionine). Further non-limiting examples of nucleic acid programmable DNA binding proteins include those disclosed in International Patent Application No.

PCT/US2019/047996.

“Patient in need thereof’ or “subject in need thereof’ is referred to herein as a patient diagnosed with, at risk or having, predetermined to have, or suspected of having a disease or disorder.

The terms “pathogenic mutation”, “pathogenic variant”, “disease causing mutation”, “disease causing variant”, “deleterious mutation”, or “predisposing mutation” refers to a genetic alteration or mutation that is associated with a disease or disorder or that increases an individual’s susceptibility or predisposition to a certain disease or disorder. In some embodiments, the pathogenic mutation comprises at least one wild-type amino acid substituted by at least one pathogenic amino acid in a protein encoded by a gene. In some embodiments, the pathogenic mutation is in a terminating region (e.g., stop codon). In some embodiments, the ATTORNEY DOCKET NO. 180802-47402/PCT

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pathogenic mutation is in a non-coding region (e.g., intron, promoter, etc.). In some embodiments, the pathogenic mutation is a Q188R amino acid alteration in a GALT polypeptide.

The term “pharmaceutically acceptable salt”, as used herein, refers to salts of such compounds that are appropriate for use in pharmaceutical contexts, /.<., salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977).

As used herein, a “phospholipid” can refer to a lipid that includes a phosphate moiety and one or more carbon chains, such as unsaturated fatty acid chains. A phospholipid may include one or more multiple (e.g., double or triple) bonds. In some embodiments, a phospholipid may facilitate fusion to a membrane. For example, a cationic phospholipid may interact with one or more negatively charged phospholipids of a membrane (e.g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane may allow one or more elements of an LNP to pass through the membrane, i.e., delivery of the one or more elements to a cell.

The terms “protein”, “peptide”, “polypeptide”, and their grammatical equivalents are used interchangeably herein, and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. A protein, peptide, or polypeptide can be naturally occurring, recombinant, or synthetic, or any combination thereof.

The term “fusion protein” as used herein refers to a hybrid polypeptide which comprises protein domains from at least two different proteins.

The term “recombinant” as used herein in the context of proteins or nucleic acids refers to proteins or nucleic acids that do not occur in nature but are the product of human engineering. For example, in some embodiments, a recombinant protein or nucleic acid molecule comprises an amino acid or nucleotide sequence that comprises at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations as compared to any naturally occurring sequence.

By “reduces” is meant a negative alteration of at least 10%, 25%, 50%, 75%, or 100%. In embodiments, a base editor system of the invention reduces and/or normalizes levels of galactose or galactose metabolites (e.g., galactitol or galactose- 1-P) in a biological sample of the subject.

By “reference” is meant a standard or control condition. In one embodiment, the reference is the level of galactose or galactose metabolites (e.g., galactitol or galactose- 1-P) ATTORNEY DOCKET NO. 180802-47402/PCT

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present in the blood of a subject not having galactosemia. In another embodiment, the reference is the level of galactose or galactose metabolites (e.g., galactitol or galactose- 1-P) present in the blood of a subject having galactosemia prior to treatment. In another embodiment, the reference is a number that describes an ideal range for galactose or galactose metabolites (e.g., galactitol or galactose- 1-P) levels in blood.. In other embodiments and without limitation, a reference is an untreated cell that is not subjected to a test condition, or is subjected to placebo or normal saline, medium, buffer, and/or a control vector that does not harbor a polynucleotide of interest. In embodiments, a reference is a base editor system containing a base editor polypeptide and/or guide polynucleotide that differs from that of a base editor system of interest.

A “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 16 amino acids, at least about 20 amino acids, at least about 25 amino acids, about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, at least about 60 nucleotides, at least about 75 nucleotides, about 100 nucleotides or about 300 nucleotides or any integer thereabout or therebetween. In some embodiments, a reference sequence is a wild-type sequence of a protein of interest. In other embodiments, a reference sequence is a polynucleotide sequence encoding a wild-type protein.

By “RrA3f polypeptide” is meant a deaminase capable of deaminating cytidine in DNA and comprising an amino acid sequence with at least 85% identity to the following amino acid sequence:

MKPQIRDHRPNPMEAMYPHIFYFHFENLEKAYGRNETWLCFTVEI IKQYLPVPWKKGVFRNQVD PETHCHAEKCFLSWFCNNTLSPKKNYQVTWYTSWSPCPECAGEVAEFLAEHSNVKLTI YEARLY YFWDEDYQEGLRSLSEEGASVEIMDYEDFQYCWENFVYDDGEPFKRWKGLKYNFQSLERRLREI LQ (SEQ ID NO: 67), or a functional fragment thereof. In various embodiments, the RrA3f polypeptide may be inserted within a Cas9 polypeptide between amino acid positions 906 and 907 referenced to SEQ ID NO: 197 (where a base editor with a deaminase inserted at this position may be referred to as an “IBE34” base editor). In some embodiments, the RrA3f polypeptide lacks an N-terminal methionine.

By “RrA3f polynucleotide” is meant a polynucleotide encoding an RrA3f polypeptide. By “ Streptococcus pyogenes Cas9 (spCas9) polypeptide” is meant a nucleic acid programmable DNA binding domain (napDNAbp) comprising an amino acid sequence with at ATTORNEY DOCKET NO. 180802-47402/PCT

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least 85% identity to the following amino acid sequence:

MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRL KRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAY HEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTY NQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNF DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMD GTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRI PYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHS LLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTF KEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQ TTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINR LSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK FDNLTKAERGGLS ELDKAGF I KRQLVETRQ I TKHVAQ I LDSRMNTKYDENDKL I RE VKVI TLKS KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAK SEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG KSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLI IKLPKYSLFELENGRKRMLAS AGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEI IEQISEFSKRV ILADANLDKVLSAYNKHRDKPIREQAENI IHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD (SEQ ID NO: 197), or a functional fragment thereof. In some embodiments, the spCas9 polypeptide lacks an N-terminal methionine. In some embodiments, spCas9 polypeptide comprises alterations, as described herein. In some embodiments, spCas9 contains a D10A amino acid alteration. In some embodiments, the SpCas9 polypeptide further comprises the amino acid alterations LI 111R, DI 135V, G1218R, E1219F, A1322R, R1335V, and T1337R referenced to SEQ ID NO: 197 (i.e., is a “Nureki” SpCas9 variant) and has protospacer-adjacent motif (PAM) specificity for a PAM containing the nucleotide sequence NG, where “N” is A, C, T, or G. In some embodiments the SpCas9 polypeptide has PAM specificity for a PAM containing the nucleotide sequence NGG, where “N” is A, C, T, or G. In some embodiments, the SpCas9 polypeptide further comprises the amino acid alterations I322V, S409I, E427G, R654L, R753G, R1114G, DI 135N, VI 139A, DI 180G, E1219V, Q1221H, A1320V, and R1333K referenced to SEQ ID NO: 197 and has protospacer-adjacent motif (PAM) specificity for a PAM containing the nucleotide sequence NRRH, where ATTORNEY DOCKET NO. 180802-47402/PCT

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“N” is A, C, T, or G, “R” is A or G, and “H” is A, C, or T. In some embodiments, the SpCas9 polypeptide further comprises the amino acid alterations I322V, S409I, E427G, R654L, R753G, R1114G, D1135N, D1180G, G1218S, E1219V, Q1221H, P1249S, E1253K, P1321S, D1332G, and R1335L referenced to SEQ ID NO: 197 and has protospacer-adjacent motif (PAM) specificity for a PAM containing the nucleotide sequence NRTH, where “N” is A, C, T, or G, “R” is A or G, and “FT is A, C, or T.

By “ Streptococcus pyogenes Cas9 (spCas9) polynucleotide” is meant a polynucleotide encoding an spCas9 polypeptide.

By “ Staphylococcus aureus Cas9 (saCas9) polypeptide” is meant a nucleic acid programmable DNA binding domain (napDNAbp) comprising an amino acid sequence with at least 85% identity to the following amino acid sequence:

MKRNYILGLAIGITSVGYGI IDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRI QRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDT GNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQ LDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLY NALNDLNNLVITRDENEKLEYYEKFQI IENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGK PEFTNLKVYHDIKDITARKEI IENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQIS NLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSP VVKRS FIQSIKVINAII KKYGL PND HIE LARE KNS KDAQKM I NEMQKRNRQTNER I E E 11 RTT GKENAKYL I EKI KLHDMQEGKCL YS LEAI PLEDLLNNPFNYE VDH 11 PRS VS FDNS FNNKVLVK QEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKD FINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAED ALI IANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKD YKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHH DPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDD YPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQA EFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRI IKTIASKT QSIKKYSTDILGNLYEVKSKKHPQIIKKG (SEQ ID NO: 218), or a functional fragment thereof. In some embodiments, the saCas9 polypeptide lacks an N-terminal methionine. In some embodiments, saCas9 polypeptide comprises alterations, as described herein. In some embodiments, saCas9 contains a D10A amino acid alteration. In some embodiments the SaCas9 polypeptide has PAM specificity for a PAM containing the nucleotide sequence NNGRRT, where “N” is A, C, T, or G, and where “R” is A or G.

By “ Staphylococcus aureus Cas9 (saCas9) polynucleotide” is meant a polynucleotide encoding an saCas9 polypeptide. ATTORNEY DOCKET NO. 180802-47402/PCT

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By “SpRY polypeptide” is meant a nucleic acid programmable DNA binding domain comprising an amino acid sequence with at least 85% identity to the following amino acid sequence:

MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRL KRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAY HEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTY NQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNF DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMD GTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRI PYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHS LLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTF KEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQ TTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINR LSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK FDNLTKAERGGLS ELDKAGF I KRQLVETRQ I TKHVAQ I LDSRMNTKYDENDKL I RE VKVI TLKS KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAK SEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG KSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLI IKLPKYSLFELENGRKRMLAS AGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEI IEQISEFSKRV ILADANLDKVLSAYNKHRDKPIREQAENI IHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD (SEQ ID NO: 197) and comprises the following amino acid alterations referenced to SEQ ID NO: 197: A61R, LI 111R, DI 135L, SI 136W, G1218K, E1219Q, N1317R, A1322R, R1333P, R1335Q, and T1337R, or a functional fragment thereof In some embodiments, the SpRY polypeptide lacks an N-terminal methionine. In some embodiments, SpRY polypeptide comprises alterations, as described herein. SpRY is described in Walton, et al. “Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants,” Science, 368:290-296 (2020), the disclosure of which is hereby incorporated by reference in its entirety for all purposes. In some embodiments, an SpRY polypeptide is a nucleic acid programmable DNA binding protein with specificity for protospacer-adjacent motifs having the sequence NRN and/or NYN, where “N” represents A, C, T, or G, “R” represents a purine (A or G), and “Y” represents a pyrimidine (C or T). In some embodiments, SpRY can recognize ATTORNEY DOCKET NO. 180802-47402/PCT

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almost all PAMs. In some embodiments the SpRY polypeptide has PAM specificity for a PAM containing the nucleotide sequence NRN, where “N” is A, C, T, or G, and where “R” is A or G.

By “SpRY polynucleotide” is meant a polynucleotide encoding an SpRY polypeptide. By “nSpRY polypeptide” is meant an SpRY polypeptide that is a nickase. In various embodiments, an nSpRY polypeptide is an SpRY polypeptide comprising aDlOA amino acid alteration.

By “nSpRY polynucleotide” is meant a polynucleotide encoding an nSpRY polypeptide. By “SpRYc polypeptide” is meant a nucleic acid programmable DNA binding domain comprising an amino acid sequence with at least 85% identity to the following amino acid sequence:

MEKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTNRKSIKKNLMGALLFDSGETAEATRL KRTARRRYTRRKNRIRYLQEIFANEMAKLDDSFFQRLEESFLVEEDKKNERHPIFGNLADEVAY HRNYPTIYHLRKKLADSPEKADLRLIYLALAHI IKFRGHFLIEGKLNAENSDVAKLFYQLIQTY NQLFEESPLDEIEVDAKGILSARLSKSKRLEKLIAVFPNEKKNGLFGNI IALALGLTPNFKSNF DLTEDAKLQLSKDTYDDDLDELLGQIGDQYADLFSAAKNLSDAILLSDILRSNSEVTKAPLSAS MVKRYDEHHQDLALLKTLVRQQFPEKYAEIFKDDTKNGYAGYVGADKKLRKRSGKLATEEEFYK FIKPILEKMDGAEELLAKLNRDDLLRKQRTFDNGSIPHQIHLKELHAILRRQEEFYPFLKENRE KIEKILTFRIPYYVGPLARGNSRFAWLTRKSEEAITPWNFEEVVDKGASAQSFIERMTNFDEQL PNKKVLPKHSLLYEYFTVYNELTKVKYVTERMRKPEFLSGEQKKAIVDLLFKTNRKVTVKQLKE DYFKKIECFDSVEI IGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVLTLTLFEDRE MIEERLKTYAHLFDDKVMKQLKRRHYTGWGRLSRKMINGIRDKQSGKTILDFLKSDGFSNRNFM QLIHDDSLTFKEEIEKAQVSGQGDSLHEQIADLAGSPAIKKGILQTVKIVDELVKVMGHKPENI VIEMARENQTTTKGLQQSRERKKRIEEGIKELESQILKENPVENTQLQNEKLYLYYLQNGRDMY VDQELDINRLSDYDVDHIVPQSFIKDDSIDNKVLTRSVENRGKSDNVPSEEVVKKMKNYWRQLL NAKL I TQRKFDNLTKAERGGLS EADKAGF I KRQLVETRQ I TKHVAR I LDSRMNTKRDKNDKP I R EVKVITLKSKLVSDFRKDFQLYKVRDINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKV YDVRKMIAKSEQEIGKATAKRFFYSNIMNFFKTEVKLANGEIRKRPLIETNGETGEVVWNKEKD FATVRKVLAMPQVNIVKKTEVQTGGFSKESIRPKRNSDKLIARKKDWDPKKYGGFLWPTVAYSV LVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLI IKLPKYSLFELE NGRKRMLASAKQLQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEI IE QISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENI IHLFTLTRLGAPRAFKYFDTTIDPKQ YRSTKEVLDATLIHQSITGLYETRIDLSQLGGD (SEQ ID NO: 772), or a functional fragment thereof. In some embodiments, the SpRYc polypeptide lacks anN-terminal methionine. In some embodiments, SpRYc polypeptide comprises alterations, as described herein. In some embodiments, SpRYc contains a D10A amino acid alteration. In some embodiments the SpRYc ATTORNEY DOCKET NO. 180802-47402/PCT

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polypeptide has PAM specificity for a PAM containing the nucleotide sequence NNN, where “N” is A, C, T, or G.

By “SpRYc polynucleotide” is meant a polynucleotide encoding an SpRYc polypeptide. As used herein, the term “stem loop” describes a secondary structure of nucleotides that form a base-paired “stem” that ends in a loop of unpaired nucleic acids. A stem may be formed when two regions of the same nucleic acid strand are at least partially complementary in sequence when read in opposite directions.

The term “sterolyl,” as used herein, refers to a 17-membered fused polycyclic ring moiety that is either saturated or partially unsaturated and substituted with at least one hydroxyl group, and has a single point of attachment to the rest of the molecule at any substitutable carbon or oxygen atom. In some embodiments, a sterolyl group is a cholesterolyl group, or a variant or derivative thereof. In some embodiments, a cholesterolyl group is modified. In some embodiments, a cholesterolyl group is an oxidized cholesterolyl group (e.g., oxidized on the beta-ring structure or on the hydrocarbon tail structure). In some embodiments, a cholesterolyl group is an esterified cholesterolyl group. In some embodiments, a sterolyl group is a phytosterolyl group. Exemplary sterolyl groups include but are not limited to 25-hydroxycholesterolyl (25-OH), 20a-hydroxycholesterolyl (20a-OH), 27-hydroxycholesterolyl, 6-keto-5a-hydroxycholesterolyl, 7-ketocholesterolyl, 7P-hydroxycholesterolyl, 7a-hydroxycholesterolyl, 7P-25-dihydroxycholesterolyl, beta-sitosterolyl, stigmasterolyl, brassicasterolyl, and campesterolyl.

By “subject” or “patient” is meant a mammal, including, but not limited to, a human or non-human mammal. In embodiments, the mammal is a bovine, equine, canine, ovine, rabbit, rodent, nonhuman primate, or feline. In an embodiment, “patient” refers to a mammalian subject with a higher than average likelihood of developing a disease or a disorder. Exemplary patients can be humans, non-human primates, cats, dogs, pigs, cattle, cats, horses, camels, llamas, goats, sheep, rodents (e.g., mice, rabbits, rats, or guinea pigs) and other mammalians that can benefit from the therapies disclosed herein. Exemplary human patients can be male and/or female.

As described herein, compounds of this disclosure may be described as “substituted” or “optionally substituted”. That is, compounds may contain optionally substituted and/or substituted moieties. In general, the term “substituted,” whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. “Substituted” applies to one or more hydrogens that are either

'NH -FR¹ -j-R¹ explicit or implicit from the structure (e.g.,

refers to at least

; and ATTORNEY DOCKET NO. 180802-47402/PCT

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1 l^^NH

refers to at least

, ^R JO^NH, ^R, or CA ^R ). Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. Combinations of substituents envisioned by this disclosure are preferably those that result in the formation of stable or chemically feasible compounds. The term “stable,” as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein. Groups described as being “substituted” preferably have between 1 and 4 substituents, more preferably 1 or 2 substituents. Groups described as being “optionally substituted” may be unsubstituted or be “substituted” as described above.

Suitable monovalent substituents include halogen; -(CH₂)o ₄R°; -(CH₂)o 4OR⁰; -0(CH₂)o-₄R°, -0-(CH₂)O ₄C(O)OR^O; -(CH₂)O ₄CH(OR^O)₂; -(CH₂)O ₄Ph, which may be substituted with R°; -(CH₂)o ₄0(CH₂)o iPh which may be substituted with R°; -CH=CHPh, which may be substituted with R°; -(CH₂)o-40(CH₂)o-i-pyridyl which may be substituted with R°; -NO₂; -CN; -N₃; -(CH₂)O ₄N(R^O)₂; -(CH₂)O ₄N(R^O)C(O)R°; -N(R°)C(S)R°; -(CH₂)O-₄N(R^O)C(O)NR^O ₂; -N(R^O)C(S)NR°₂; -(CH₂)O ₄N(R^O)C(O)OR°; -N(R°)N(R°)C(O)R°; -N(R°)N(R°)C(O)NR°₂; -N(R°)N(R°)C(O)OR°; -(CH₂)O ₄C(O)R^O; -C(S)R°; -(CH₂)O-₄C(O)OR^O; -(CH₂)O ₄C(O)SR^O; -(CH₂)O ^C(O)OSiR°₃; -(CH₂)o ₄OC(O)R°; -OC(0)(CH₂)o ₄SR⁰-, -SC(S)SR°; -(CH₂)O ₄SC(O)R°; -(CH₂)O ₄C(O)NR^O ₂; -C(S)NR^O ₂; -C(S)SR°; -SC(S)SR°, -(CH₂)O ₄OC(O)NR^O ₂; -C(O)N(OR°)R°; -C(O)C(O)R°; -C(O)CH₂C(O)R^O; -C(NOR°)R°; -(CH₂)O ₄SSR°; -(CH₂)O ₄S(O)₂R^O; -(CH₂)O ₄S(O)₂OR^O; -(CH₂)O ₄OS(O)₂R^O; -S(O)₂NR°₂; -(CH₂)O ₄S(O)R^C; -N(R^O)S(O)₂NR°₂; -N(R^O)S(O)₂R°; -N(OR°)R°; -C(NH)NR^O ₂; -P(O)₂R°; -P(O)R^O ₂; -OP(O)R^O ₂; -OP(O)(OR^O)₂; -SiR°₃; -OSiR°₃; -(Ci ₄ straight or branched alkylene)O-N(R°)₂; or -(Ci-₄ straight or branched alkylene)C(O)O-N(R°)₂, wherein each R° may be substituted as defined below and is independently hydrogen, C1-6 aliphatic, -CH₂Ph, -0(CH₂)o iPh, -CH₂-(5-6 membered heteroaryl ring), or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R°, taken together with their intervening atom(s), form a 3-12-membered saturated, partially unsaturated, ATTORNEY DOCKET NO. 180802-47402/PCT

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or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be substituted as defined below.

Suitable monovalent substituents on R° (or the ring formed by taking two independent occurrences of R° together with their intervening atoms), are independently halogen, -(CH₂)o-₂R*, -(haloR*), -(CH₂)O 2OH, -(CH₂)o-₂OR*, -(CH₂)o-₂CH(OR*)₂; -O(haloR’), -CN, -N3, -(CH₂)O ₂C(O)R*, -(CH₂)O ₂C(O)OH, -(CH₂)O ₂C(O)OR*, -(CH₂)O ₂C(O)NH₂, -(CH₂)O-₂C(O)NHR*, -(CH₂)O ₂C(O)NR*₂, -(CH₂)O ₂SR*, -(CH₂)O ₂SH, -(CH₂)O ₂NH₂, -(CH₂)O ₂NHR*, -(CH₂)O-₂NR*₂, -NO₂, -SiR*3, -OSiR*3, -C(O)SR* -(Ci^i straight or branched alkylene)C(O)OR*, or -SSR* wherein each R* is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently selected from Ci-4 aliphatic, -CH₂Ph, -0(CH₂)o iPh, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents on a saturated carbon atom of R° include =0 and =S.

Suitable divalent substituents include the following: =0, =S, =NNR*₂, =NNHC(O)R*, =NNHC(O)OR*, =NNHS(O)₂R*, =NR*, =N0R*, -O(C(R*₂))₂ 3O-, or -S(C(R*₂))₂ 3S-, wherein each independent occurrence of R* is selected from hydrogen, C1-6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents that are bound to vicinal substitutable carbons of an “optionally substituted” group include: -O(CR*₂)₂ 3O-, wherein each independent occurrence of R* is selected from hydrogen, C1-6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R* include halogen, -R*, -(haloR*), -OH, -OR*, -O(haloR’), -CN, -C(O)OH, -C(O)OR*, -NH₂, -NHR*, -NR*₂, or -NO₂, wherein each R* is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently Ci 4 aliphatic, -CH₂Ph, -0(CH₂)o iPh, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In some embodiments, suitable substituents on a substitutable nitrogen include -R¹', - Rt₂, -C(O)R^t, -C(O)OR^t, -C(O)C(O)R^t, -C(O)CH₂C(O)R^t, -S(O)₂Rt, -S(O)₂NRt₂, -C(S)NR^t ₂, -C(NH)NRJ₂, or -^R^S^)^; wherein each R¹' is independently hydrogen, C1-6 aliphatic which may be substituted as defined below, unsubstituted -OPh, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently ATTORNEY DOCKET NO. 180802-47402/PCT

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selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R¹', taken together with their intervening atom(s) form an unsubstituted 3-12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R^: are independently halogen, -R*, -(haloR*), -OH, -OR*, -O(haloR’), -CN, -C(O)OH, -C(O)OR*, -NH₂, -NHR*, -NR*₂, or -NO₂, wherein each R* is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently Ci 4 aliphatic, -CH₂Ph, -0(CH₂)o iPh, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

By “substantially identical” is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence. In one embodiment, a reference sequence is a wild-type amino acid or nucleic acid sequence. In another embodiment, a reference sequence is any one of the amino acid or nucleic acid sequences described herein. In one embodiment, such a sequence is at least about 60%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or even 99.99%, identical at the amino acid level or nucleic acid level to the sequence used for comparison.

Nucleic acid molecules useful in the methods of the disclosure include any nucleic acid molecule that encodes a polypeptide of the disclosure or a functional fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a doublestranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the disclosure include any nucleic acid molecule that encodes a polypeptide of the disclosure or a functional fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. By “hybridize” is meant pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507). ATTORNEY DOCKET NO. 180802-47402/PCT

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By “TadA*8.19” is meant a deaminase capable of deaminating adenine in DNA and containing an amino acid sequence with at least 85% identity to SEQ ID NO: 1 having the amino acid alterations V82S, Y123H, Y147R, and Q154R, or a functional fragment thereof. In some embodiments, TadA*8.19 lacks anN-terminal methionine.

By “TadA*8.19 polynucleotide” is meant a polynucleotide encoding a TadA*8.19 polypeptide.

By “TadA*8.20” is meant a deaminase capable of deaminating adenine in DNA and containing an amino acid sequence with at least 85% identity to SEQ ID NO: 1 having the amino acid alterations I76Y, V82S, Y123H, Y147R, and Q154R, or a functional fragment thereof. In some embodiments, TadA*8.20 lacks an N-terminal methionine.

By “TadA*8.20 polynucleotide” is meant a polynucleotide encoding a TadA*8.20 polypeptide.

By “T1.52 polypeptide” is meant a deaminase capable of deaminating cytidine in DNA and comprising an amino acid sequence with at least 85% identity to the following amino acid sequence and further comprising the amino acid alterations F6Y, E27H, I49K, I76W, D77G, V82T, R107C, G112H, A114C, G115M, H122G, Y123H, N127P, A142E, Y147R, Q154R: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR QGGLVMQNYRL IDATL YVTFE PCVMCAGAM IHSR I GRWFGVRNAKTGAAGS LMDVLHYPGMNH RVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 1), or a functional fragment thereof. In various embodiments, the T1.52 polypeptide may be inserted within a Cas9 polypeptide between amino acid positions 906 and 907 referenced to SEQ ID NO: 197 (where a base editor with a deaminase inserted at this position may be referred to as an “IBE34” base editor). In some embodiments, the T1.52 polypeptide lacks an N-terminal methionine. A representative T1.52 polypeptide sequence is provided below:

SEVEYSHEYWMRHALTLAKRARDERHVPVGAVLVLNNRVIGEGWNRAKGLHDPTAHAEIMALRQ GGLVMQNYRLWGATLYTTFE PCVMCAGAM IHSR I GRVVFGVCNAKTHACMS LMDVLGHPGMPHR VEITEGILADECEALLCRFFRMPRRVFNAQKKAQSSTD (SEQ ID NO: 773)

By “T1.52 polynucleotide” is meant a polynucleotide encoding an T1.52 polypeptide. The term “target site” refers to a nucleotide sequence or nucleobase of interest within a nucleic acid molecule that is modified. In embodiments, the modification is deamination of a base. The deaminase can be a cytidine or an adenine deaminase.

As used herein, the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith or obtaining a desired pharmacologic and/or physiologic effect. It will be appreciated that, although not precluded, ATTORNEY DOCKET NO. 180802-47402/PCT

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treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated. In some embodiments, the effect is therapeutic, /.<., without limitation, the effect partially or completely reduces, diminishes, abrogates, abates, alleviates, reduces the intensity of, or cures a disease and/or adverse symptom attributable to the disease. In some embodiments, the effect is preventative, /.<., the effect protects or prevents an occurrence or reoccurrence of a disease or condition. To this end, the presently disclosed methods comprise administering a therapeutically effective amount of a composition as described herein.

By “uracil glycosylase inhibitor” or “UGI” is meant an agent that inhibits the uracil-excision repair system. Base editors comprising a cytidine deaminase convert cytosine to uracil, which is then converted to thymine through DNA replication or repair. In various embodiments, a uracil DNA glycosylase (UGI) prevent base excision repair which changes the U back to a C. In some instances, contacting a cell and/or polynucleotide with a UGI and a base editor prevents base excision repair which changes the U back to a C. An exemplary UGI comprises an amino acid sequence as follows:

>splP14739IUNGI_BPPB2 Uracil-DNA glycosylase inhibitor MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPE YKPWALVIQDSNGENKIKML (SEQ ID NO: 231).

In some embodiments, the agent inhibiting the uracil-excision repair system is a uracil stabilizing protein (USP). See, e.g., WO 2022015969 Al, incorporated herein by reference.

As used herein, the term “vector” refers to a means of introducing a nucleic acid molecule into a cell, resulting in a transformed cell. Vectors include plasmids, transposons, phages, viruses, liposomes, lipid nanoparticles, and episomes.

Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

All terms are intended to be understood as they would be understood by a person skilled in the art. Unless defined otherwise, all technical and scientific terms used herein have the same ATTORNEY DOCKET NO. 180802-47402/PCT

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meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains

In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. In this application, the use of “or” means “and/or” unless stated otherwise. Furthermore, use of the term “including” as well as other forms, such as “include”, “includes,” and “included,” is not limiting.

As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended. This wording indicates that specified elements, features, components, and/or method steps are present, but does not exclude the presence of other elements, features, components, and/or method steps. Any embodiments specified as “comprising” a particular component(s) or element(s) are also contemplated as “consisting of’ or “consisting essentially of’ the particular component(s) or element(s) in some embodiments. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the present disclosure, and vice versa. Furthermore, compositions of the present disclosure can be used to achieve methods of the present disclosure.

The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, z.e., the limitations of the measurement system.

Reference in the specification to “some embodiments,” “an embodiment,” “one embodiment” or “other embodiments” means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the present disclosures.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 provides a schematic diagram providing a description of the reaction catalyzed by galactose- 1 -phosphate uridylyltransferase (GALT) and the metabolic context of the reaction. In FIG. 1 the terms “Type I”, “Type II”, “Type III”, and “Type IV” indicate the galactosemia type caused by a pathogenic mutation in the indicated enzyme.

FIG. 2 provides a schematic diagram showing a region of the galactose- 1 -phosphate uridylyltransferase (GALT) polynucleotide targeted for base editing, where the target cytidine for ATTORNEY DOCKET NO. 180802-47402/PCT

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base editing is underlined. The nucleotide sequence of FIG. 2 is a reverse complement of the coding strand of the GALT polynucleotide. The nucleotide sequence in FIG. 2 is CCTGACACCCTTACCCGGCAGTGGGGGTGGGGGTTAGAACAGCCCAT (SEQ ID NO: 690).

FIG. 3 provides a schematic diagram showing a region of the GALT polynucleotide targeted for base editing, where the target cytidine (C) for base editing (“target”) and its complementary guanine (G) are highlighted and shown in lowercase highlighted text, a C and its complementary G corresponding to a synonymous bystander edit (“synonymous”) are shown in ITALICS UPPERCASE TEXT, a C and its complementary G corresponding to a splice site disruption bystander edit (“splice”) are shown in BOLD UPPERCASE TEXT, a C and its complementary G corresponding to a nonsynonymous bystander edit (“nonsynonymous”) are shown in BOLD ITALICIZED UPPERCASE TEXT, and nucleotides corresponding to an intron are shown in HIGHLIGHTED UPPERCASE TEXT. In FIG. 3, “sense” refers to the coding strand of a GALT polynucleotide, and “antisense” refers to the non-coding strand of the GALT polynucleotide. The two nucleotide sequences shown in FIG. 3, are CTAACCCCCACCCCCACTGCCgGGTAAGGGTGTCAG (SEQ ID NO: 691; lowercase g is complementary to the target C) and CTGACACCCTTACCcGGCAGTGGGGGTGGGGGTTAG (SEQ ID NO: 692; target C is shown in lowercase), which are reverse complements of one another.

FIG. 4 provides a stacked bar graph showing the allelic distribution of the indicated base edits combinations, which are described in part in FIG. 3, for fibroblast cells from two different donors (GM00440 and GM00441) (n=3 for cells from each donor) contacted with a base editor system containing a combination of an mRNA encoding a base editor and a guide RNA, as indicated along the x-axis. The sequences for the guide RNAs are provided in Table 1. The number following the hyphen after each guide RNA number represents a lot number. A description of the base editors encoded by each mRNA is provided in FIG. 4, where RrA3f, T1.52, or 6b identify the deaminase domain of each cytidine base editor (CBE), IBE34 indicates a base editor where the deaminase domain is inserted within the nucleic acid programmable DNA binding protein domain (napDNAbp) of the base editor, and the terms NNGRRT, NRTH, NG-Nureki, SpRY, SpRYc, NRRH, and NGG indicate the identity of the Cas9 variant serving as the napDNAbp of each base editor and/or the protospacer-adjacent motif (PAM) specificity of the Cas9 variant, where “N” indicates any nucleotide, “R” indicates A or G, “Y” indicates C or T, and “H” indicates A, C, or T. Each stacked bar of FIG. 4 represents, from top to bottom, the alleles corresponding to the base edits “other”, “intronic or synonymous bystanders”, “splice_or_nonsynonymous”, “target_and_splice”, “target_and_nonsynonymous,”

“target and synonymous,” and “target alone”, where “intronic” indicates the base editing of an ATTORNEY DOCKET NO. 180802-47402/PCT

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intronic C that is not within a splice site or exon, “nonsynonymous bystanders” or “nonsynonymous” indicates base editing of a C that results in the change of an encoded amino acid, “synonymous bystanders” or “synonymous” indicates base editing of a C that results in a silent mutation, “splice” indicates base editing of a C in a splice site, “target” indicates base editing of the target C, and “target alone” indicates editing of only the target C, and “other” indicates remaining alleles that do not meet the criteria of any of the aforementioned categories. Each stacked bar represents means of from 6 biological replicates, and the bars represent standard deviation. The bars shown within the boxed region correspond to base editor systems containing the guide RNA gRNA9843, where the target site (“protospacer”) for gRNA9843 is surrounded by a dashed box within the sequence provided in FIG. 4. The nucleotide sequence shown in FIG. 4 is CTGACACCCTTACCcGGCAGTGGGGGTGGGGGTTAG (SEQ ID NO: 692). The numbers following the hyphen after each guide RNA identifier in FIG. 4 refer to lot numbers.

FIG. 5 provides a stacked bar graph showing the allelic distribution of the indicated base edits combinations, which are described in part in FIG. 3, for cells from two different donors (GM00440 and GM00441) (n=3 for cells from each donor) contacted with a base editor system containing a combination of an mRNA encoding a base editor and a guide RNA, as indicated along the x-axis. The sequences for the guide RNAs are provided in Table 1. The number following the hyphen after each guide RNA number represents a lot number. A description of the base editors encoded by each mRNA is provided in FIG. 5, where RrA3f, T1.52, or 6b identify the deaminase domain of each cytidine base editor (CBE), IBE34 indicates a base editor where the deaminase domain is inserted within the nucleic acid programmable DNA binding protein domain (napDNAbp) of the base editor, and the terms NNGRRT, NRTH, NG-Nureki, SpRY, SpRYc, NRRH, and NGG indicate the identity of the Cas9 variant serving as the napDNAbp of each base editor and/or the protospacer-adjacent motif (PAM) specificity of the Cas9 variant, where “N” indicates any nucleotide, “R” indicates A or G, “Y” indicates C or T, and “H” indicates A, C, or T. Each stacked bar of FIG. 5 represents, from top to bottom, the alleles corresponding to the base edits “other”, “intronic or synonymous bystanders”, “splice_or_nonsynonymous”, “target_and_splice”, “target_and_nonsynonymous,”

“target and synonymous,” and “target alone”, where “intronic” indicates the base editing of an intronic C that is not within a splice site or exon, “nonsynonymous bystanders” or “nonsynonymous” indicates base editing of a C that results in the change of an encoded amino acid, “synonymous bystanders” or “synonymous” indicates base editing of a C that results in a silent mutation, “splice” indicates base editing of a C in a splice site, “target” indicates base ATTORNEY DOCKET NO. 180802-47402/PCT

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editing of the target C, and “target alone” indicates editing of only the target C, and “other” indicates remaining alleles that do not meet the criteria of any of the aforementioned categories. Each stacked bar represents means of from 6 biological replicates, and the bars represent standard deviation. The bars shown within the boxed region correspond to base editor systems containing the guide RNA gRNA9843, where the target site (“protospacer”) for gRNA9846 is surrounded by a dashed box within the sequence provided in FIG. 5. The nucleotide sequence shown in FIG. 5 is CTGACACCCTTACCcGGCAGTGGGGGTGGGGGTTAG (SEQ ID NO: 692). The numbers following the hyphen after each guide RNA identifier in FIG. 5 refer to lot numbers. FIG. 5 presents the same data as shown in FIG. 4.

FIG. 6 provides a bar graph presenting an alternative presentation of the data from FIGs.

4 and 5 and showing maximum OT percent base editing for each base editor system used to transfect the fibroblasts from both donors (n=6). The x-axis of FIG. 6 identifies the base editor of the base editor system corresponding to each bar, where the base editors are described in FIGs. 4 and 5, and the guides corresponding to each bar are indicated by the letter above each bar. The sequences for the guide RNAs are provided in Table 1. The number following the hyphen after each guide RNA number represents a lot number.

FIG. 7 provides a bar graph presenting the same data as shown in FIG. 6, but where the bars are clustered by base editor.

FIG. 8 provides a stacked bar graph presenting an alternative presentation of data from FIGs. 4 and 5 where only data corresponding to fibroblasts from donor GM00440 (n=3) is shown. A description of the base edits represented in each stacked bar and of the base editor systems indicated along the x-axis corresponding to each stacked bar is provided in FIGs. 4 and 5.

FIG. 9 provides a stacked bar graph presenting an alternative presentation of data from FIGs. 4 and 5 where only data corresponding to fibroblasts from donor GM00441 (n=3) is shown. A description of the base edits represented in each stacked bar and of the base editor systems indicated along the x-axis corresponding to each stacked bar is provided in FIGs. 4 and 5.

FIG. 10 provides a stacked bar graph providing an alternative presentation of the same data as presented in FIG. 8 where the edits stacked within each bar are grouped differently. In FIG. 10, each stacked bar represents from top-to-bottom the alleles corresponding to the base edits “other”, “silent”, “bystander”, and “target”, where “silent” indicates a base edit that does not alter a splice site and that does not result in a missense mutation, “bystander” represents a base edit that results in a missense mutation, and “target” represents base editing of the target C. ATTORNEY DOCKET NO. 180802-47402/PCT

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FIG. 11 provides a stacked bar graph providing an alternative presentation of the same data as presented in FIG. 9 where the edits stacked within each bar are grouped differently. In FIG. 11, each stacked bar represents from top-to-bottom the alleles corresponding to the base edits “other”, “silent”, “bystander”, and “target”, where “silent” indicates a base edit that does not alter a splice site and that does not result in a missense mutation, “bystander” represents a base edit that results in a missense mutation, and “target” represents base editing of the target C.

FIG. 12 provides a schematic diagram showing the hairpin structures of exemplary end-modified sgRNA, standard heavily modified sgRNA modification and a LONGEST gRNA design including 5 base pair in the upper stem of the gRNA. The chemical modified sgRNAs (end mod. and heavily mod.) include only 4 base pairs in the upper stem. The sequences disclosed in FIG. 12 correspond, in order of occurrence, to SEQ ID NOs: 694 to 695. The first 20 nucleotides from the 5’ end of the sgRNA sequences of FIG. 12 represent a spacer sequence, which in various embodiments may be replaced with any spacer of interest, where the length of the spacer may be greater than or less than the spacer listed in the sequences of FIG. 12. The nucleotides following the spacer sequence correspond to a scaffold sequence.

FIG. 13 provides a schematic diagram showing the hairpin structures of gRNA 4 (standard end-modifications), gRNA 5 (GOLD modification) and gRNA 6(LONGEST-GOLD modifications). The sequences disclosed in FIG. 13 correspond, in order of occurrence, to SEQ ID NOs: 696 to 697. The N’s at the 5’ end of the gRNA sequences of FIG. 13 represent a spacer sequence, which may be replaced with any spacer of interest, where the length of the spacer may be greater than or less than the spacer listed in the sequences of FIG. 13. The nucleotides following the spacer sequence correspond to a scaffold sequence.

FIG. 14 provides a plot showing maximum C-to-T (C> T) base-editing efficiency for bystander mutation installation in a wild-type GALT gene in Huh-7 cells from a wild-type donor (i.e., a donor containing a GALT gene encoding a GALT polypeptide having a Q at position 188 (Q188)) administered the indicated doses (i.e., lipid nanoparticle (LNP) concentrations) of lipid nanoparticles containing a base editor system (i.e., LNPL A or LNPL B).

FIG. 15 provides a plot showing maximum C-to-T (C> T) base-editing efficiency for altering codon 188 in a mutant GALT gene encoding an R such that the codon encodes a Q (i.e., correction of a Q188R mutation) in fibroblasts from human donor GM00441 administered the indicated doses (i.e., lipid nanoparticle (LNP) concentrations) of lipid nanoparticles containing a base editor system (i.e., LNPL A or LNPL B). Donor GM00441 contained a GALT gene encoding a GALT polypeptide having an R at position 188 (Q188R). ATTORNEY DOCKET NO. 180802-47402/PCT

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DETAILED DESCRIPTION

Provided herein are base editors and base editor systems for use in editing, modifying, or altering a target polynucleotide. In particular embodiments, a base editor or endonuclease of the present disclosure modifies a galactose- 1 -phosphate uridylyltransferase (GALT) polynucleotide. In particular embodiments, a base editor of the invention deaminates a pathogenic nucleobase in a GALT polynucleotide to increase or restore activity of the GALT polypeptide encoded by the same.

The invention is based, at least in part, upon the discovery that base editor systems (e.g., base editor systems containing a cytidine base editor selected from CBE RrA3f NNGRRT, CBE RrA3f NGG, CBE RrA3f NRTH, CBE RrA3f NG - Nureki, CBE RrA3f SpRY, CBE RrA3F SpRYc, CBE RrA3F NRRH, CBE-T1.52 IBE34 NGG, and CBE6b NGG and a guide RNA containing a sequence selected from those listed in Table 1) may be used to correct a Q188R pathogenic mutation in a GALT polynucleotide.

In embodiments, the methods of the present disclosure include altering a GALT polynucleotide to restore or increase activity of a GALT polypeptide encoded thereby. For example, the base editors or base editor systems provided herein can be used for editing a nucleobase in an exon of the GALT polynucleotide. In some embodiments, the target sequence is or contains codon 188 of the GALT polynucleotide. In some embodiments, the deamination of an A or C nucleobase in the exon results in a restoration or increase of activity of a GALT polypeptide. In some embodiments, the subject has or has the potential to develop galactosemia.

In some instances, the methods of the present disclosure include modifying a GALT polynucleotide to increase levels or activity of the GALT polynucleotide and/or polypeptide. The alterations can be effected by a base editor system, such as those described herein.

In some embodiments, the present disclosure provides base editor systems that efficiently generate an intended mutation, such as an alteration of a nucleotide, in a nucleic acid molecule (e.g., a nucleic acid within a genome of a subject) without generating a significant number of unintended mutations, such as unintended point mutations. In some embodiments, an intended mutation is a mutation that is generated by a specific base editor (e.g., an adenosine base editor or a cytidine base editor) bound to a guide polynucleotide (e.g., gRNA) specifically designed to generate the intended mutation. In some embodiments, the intended mutation is an adenine (A) to guanine (G) point mutation within the coding region of a gene. In some embodiments, the intended mutation is a cytosine (C) to thymine (T) point mutation within the coding region of a gene. In some embodiments, the intended mutation is a mutation of a nucleobase corresponding to codon 188 of a GALT polynucleotide, where in some embodiments codon 188 encodes an ATTORNEY DOCKET NO. 180802-47402/PCT

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arginine (R) amino acid prior to base editing. In some instances, the intended mutation results in a R188Q amino acid alteration in the GALT polypeptide encoded by the GALT polynucleotide. In some embodiments, the intended mutation is a cytidine (C) to thymine (T) point mutation in codon 188 of the GALT polynucleotide. In some embodiments, the intended mutation is a missense mutation. In some cases, the intendent mutation results in the correction of a Q188R pathogenic mutation in a GALT polypeptide.

In some embodiments, any of the base editors provided herein are capable of generating a ratio of intended mutations to unintended mutations (e.g., intended point mutations: unintended point mutations) that is greater than 1: 1. In some embodiments, any of the base editors provided herein are capable of generating a ratio of intended mutations to unintended mutations (e.g., intended point mutations: unintended point mutations) that is at least 1.5: 1, at least 2: 1, at least 2.5: 1, at least 3: 1, at least 3.5: 1, at least 4: 1, at least 4.5: 1, at least 5: 1, at least 5.5: 1, at least 6: 1, at least 6.5: 1, at least 7: 1, at least 7.5: 1, at least 8: 1, at least 10: 1, at least 12: 1, at least 15: 1, at least 20: 1, at least 25: 1, at least 30: 1, at least 40: 1, at least 50: 1, at least 100: 1, at least 150: 1, at least 200: 1, at least 250: 1, at least 500: 1, or at least 1000: 1, or more.

In some embodiments, editing of a plurality of nucleobase pairs in one or more genes using the methods provided herein results in formation of at least one intended mutation. In some embodiments, the formation of the at least one intended mutation is in the exon of a disease-associated gene and results in a restoration or increase in activity of a polypeptide encoded by the disease-associated gene. It should be appreciated that multiplex editing can be accomplished using any method or combination of methods provided herein.

The present disclosure provides methods for the treatment of a subject diagnosed with a galactosemia. For example, in some embodiments, a method is provided that comprises administering to a subject having or having a propensity to develop galactosemia, an effective amount of a nucleobase editor (e.g., an adenosine deaminase base editor or a cytidine deaminase base editor) to effect an alteration in a GALT polynucleotide sequence, thereby reducing levels of galactose and/or galactose metabolites (e.g., galactitol or galactose- 1-P) in a biological sample of the subject.

GALACTOSEMIA

Galactosemia is an autosomal recessive disorder that results from a deficiency of any 1 of the 4 enzymes catalyzing the conversion of galactose to glucose: galactose- 1 -phosphate uridylyltransf erase (GALT; FIG. 1), galactokinase, uridine diphosphate galactose-4-epimerase, and galactose mutarotase. In some cases, galactosemia is caused by a Q188R mutation in GALT. ATTORNEY DOCKET NO. 180802-47402/PCT

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GALT deficiency is the most common cause (-75% of all galactosemia patients) of galactosemia and is often referred to as classic galactosemia or Type I classic galactosemia (see, e.g., Elsas 2^nd and Lai, “The molecular biology of galactosemia,” Genet Med, 1:40-48 (1998) and Wang, et al., “Molecular and biochemical basis of galactosemia,” Mol Genet Metab, 63:263-269 (1998)). About 2250 patients suffer from classic galactosemia in the United States as of 2024, and over 50 new patients are diagnosed each year. The complete or near-complete deficiency of GALT enzyme is life-threatening if left untreated. Complications in the neonatal period include failure to thrive, liver failure, sepsis, and death.

Galactosemia is treated by a galactose-restricted diet, which allows for rapid recovery from the acute symptoms and a generally good prognosis. Despite adequate treatment from an early age, individuals with galactosemia remain at increased risk for developmental delays, speech problems, and motor function abnormalities. Female patients with galactosemia are at increased risk for premature ovarian failure. Based upon reports by newborn screening programs, the frequency of classic galactosemia in the United States is approximately 1 in 30,000, although literature reports range from 1 in 10,000 to 1 in 60,000 live births.

Galactose- 1 -phosphate (Gal IP) accumulates in the erythrocytes of patients with galactosemia. The quantitative measurement of Gal IP is useful for monitoring compliance with dietary therapy. Gal IP is thought to be the causative factor for development of liver disease in these patients. Because of this, patients should maintain low levels and be monitored on a regular basis.

The method by which potentially affected individuals are detected may include the measurement of total galactose (galactose and Gal IP) and/or determining the activity of the GALT enzyme. The diagnosis of galactosemia is established by follow-up quantitative measurement of GALT enzyme activity. If enzyme level is less than 24.5 nmol/h/mg of hemoglobin, sequencing of the GALT gene is performed.

In various embodiments, galactosemia may be diagnosed using a genetic screen and/or using a galactosemia screen. In a galactosemia screen, blood levels of GALT are measured in a subject, where, in various embodiments, a level less than about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nmol/h/mg Hb selects a patient as having galactosemia, where one of skill in the art understands 1 nmol/h/mg indicates the amount of GALT that catalyzes the reaction of 1 nmol of galactose per hour per mg of hemoglobin.

Accordingly, the present disclosure provides compositions and methods for use in the treatment of galactosemia in a subject. In embodiments, the methods of the disclosure are associated with an increase in GALT activity in a tissue (e.g., a liver) of a subject. In various ATTORNEY DOCKET NO. 180802-47402/PCT

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embodiments, the GALT activity in a biological sample (e.g., a blood sample) from the subject is increased to a level that is greater than about 19 nmol/h/mg Hb, 20 nmol/h/mg Hb, 21, nmol/h/mg Hb, 22 nmol/h/mg Hb, 23 nmol/h/mg Hb, 24 nmol/h/mg Hb, 25 nmol/h/mg Hb, 26 nmol/h/mg Hb, 27 nmol/h/mg Hb, 29 nmol/h/mg Hb, 29 nmol/h/mg Hb, or 30 nmol/h/mg Hb. Methods for diagnosing galactosemia are known in the art and described; for example, Mayo Clinic Laboratories “Galactosemia Reflex, Blood,” Pediatric Catalog, pediatric.testcatalog.org/show/GCT, Accessed 11 November 2024.

In some embodiments, the methods of the disclosure result in decreased levels of circulating glactose-l-P (Gallp) in the blood of a subject. In some cases, the levels of Gallp in the blood of the subject is decreased to a level that is less than 10 mg/dL, 5 mg/dL, 4.9 mg/dL, 4.8 mg/dL, 4.7 mg/dL, 4.6 mg/dL, 4.5 mg/dL, 4.25 mg/dL, 4 mg/dL, 3 mg/dL, 2 mg/dL, 1 mg/dL, 0.9 mg/dL, 0.8 mg/dL, 0.7 mg/dL, 0.6 mg/dL, or 0.5 mg/dL.

EDITING OF TARGET GENES

To produce the gene edits described herein, cells (e.g., hepatocytes) are contacted with one or more guide RNAs and a nucleobase editor polypeptide (e.g., by transfecting the cells with an mRNA encoding a nucleobase editor polypeptide) comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a cytidine deaminase or adenosine deaminase or comprising one or more deaminases with cytidine deaminase and/or adenosine deaminase activity (e.g., a “dual deaminase” which has cytidine and adenosine deaminase activity). In some embodiments, cells to be edited are contacted with at least one nucleic acid, wherein the at least one nucleic acid encodes one or more guide RNAs and a nucleobase editor polypeptide comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a cytidine deaminase. In some embodiments, the gRNA comprises nucleotide analogs. In some instances, the gRNA is added directly to a cell. These nucleotide analogs can inhibit degradation of the gRNA from cellular processes.

In various instances, it is advantageous for a spacer sequence to include a 5' and/or a 3' “G” nucleotide. In some cases, for example, any spacer sequence or guide polynucleotide provided herein comprises or further comprises a 5' “G”, where, in some embodiments, the 5' “G” is or is not complementary to a target sequence. In some embodiments, the 5' “G” is added to a spacer sequence that does not already contain a 5' “G ” For example, it can be advantageous for a guide RNA to include a 5' terminal “G” when the guide RNA is expressed under the control of a U6 promoter or the like because the U6 promoter prefers a “G” at the transcription start site (see Cong, L. et al. “Multiplex genome engineering using CRISPR/Cas systems. Science ATTORNEY DOCKET NO. 180802-47402/PCT

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339:819-823 (2013) doi: 10.1126/science.l231143). In some cases, a 5' terminal “G” is added to a guide polynucleotide that is to be expressed under the control of a promoter but is optionally not added to the guide polynucleotide if or when the guide polynucleotide is not expressed under the control of a promoter.

Exemplary guide polynucleotide sequences and target site nucleotide sequences are provided in the following Table 1, and exemplary base editor sequences are provided in the following Table 2. Variants of the spacer sequences listed in Table 1 comprising 1, 2, 3, 4, or 5 nucleobase alterations are contemplated. For example, variation of a target polynucleotide sequence within a population (e.g., single nucleotide polymorphisms) may require said alterations to a spacer sequence to allow the spacer to better bind a variant of a target sequence in a subject.

Table 1 Exemplary guide polynucleotide sequences.

Guide Target Target Site (protospacer SEQ ID Spacer Sequence SEQ ID ID Site ID adjacent motifs (PAMs) are in NO NO bold)

gRNA9 TSBTxl CCCTTACCcGGCAGTGGGGGTG 698 CCCUUACCCGGCAG 699 828 1431 UGGGGG

gRNA9 TSBTxl CCTTACCcGGCAGTGGGGGTGG 700 CCUUACCCGGCAGU 701 829 1432 GGGGGU

gRNA9 TSBTxl CTTACCcGGCAGTGGGGGTGGG 702 CUUACCCGGCAGUG 703 830 1433 GGGGUG

gRNA9 TSBTxl TTACCcGGCAGTGGGGGTGGGG 704 UUACCCGG CAGUGG 705 831 1434 GGGUGG

gRNA9 TSBTxl TACCcGGCAGTGGGGGTGGGGG 706 UACCCGGCAGUGGG 707 832 1435 GGUGGG

gRNA9 TSBTxl CTGACACCCTTACCcGGCAGTG 708 CUGACACCCUUACC 709 833 1436 G CGGCAG

gRNA9 TSBTxl TGACACCCTTACCcGGCAGTGG 710 UGACACCCUUACCC 711 834 1437 G GGCAGU

gRNA9 TSBTxl GACACCCTTACCcGGCAGTGGG 712 GACACCCUUACCCG 713 835 1438 G GCAGUG

gRNA9 TSBTxl ACACCCTTACCcGGCAGTGGGG 714 ACACCCUUACCCGG 715 836 1439 G CAGUGG

gRNA9 TSBTxl CCCTTACCcGGCAGTGGGGGTG 716 CCCUUACCCGGCAG 717 837 1444 GGGGT UGGGGGU

gRNA9 TSBTxl ACCcGGCAGTGGGGGTGGGGGT 718 ACCCGGCAGUGGGG 719 838 1448 T GUGGGG

gRNA9 TSBTxl ACACCCTTACCcGGCAGTGGGG 720 ACACCCUUACCCGG 715 839 1439 G CAGUGG

gRNA9 TSBTxl CCCTTACCcGGCAGTGGGGGTG 721 CCCUUACCCGGCAG 699 840 1431 UGGGGG

gRNA9 TSBTxl CCTTACCcGGCAGTGGGGGTGG 722 CCUUACCCGGCAGU 701 841 1432 GGGGGU

gRNA9 TSBTxl CTGACACCCTTACCcGGCAGTG 723 CUGACACCCUUACC 709

842 1436 G CGGCAG ATTORNEY DOCKET NO. 180802-47402/PCT

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gRNA9 TSBTxl CTTACCcGGCAGTGGGGGTGGG 724 CUUACCCGGCAGUG 703 843 1433 GGGGUG

gRNA9 TSBTxl GACACCCTTACCcGGCAGTGGG 725 GACACCCUUACCCG 713 844 1438 G GCAGUG

gRNA9 TSBTxl TGACACCCTTACCcGGCAGTGG 726 UGACACCCUUACCC 711 845 1437 G GGCAGU

gRNA9 TSBTxl TTACCcGGCAGTGGGGGTGGGG 727 UUACCCGGCAGUGG 705 846 1434 GGGUGG

gRNAl TTACCCGGCAGTGGGGGTG 795 UUACCCGGCAGUGG 793

0320 GGGUG

Table 1 (CONTINUED).¹

Guide ID gRNA nucleotide sequence SEQ ID NO

gRNA9828 mCsmCsmCsUUACCCGGCAGUGGGGGGUUUUAGAGCUAGAAAUAGCAAGUUA 728

AAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCmU smUsmUsU

gRNA9829 mCsmCsmUsUACCCGGCAGUGGGGGUGUUUUAGAGCUAGAAAUAGCAAGUUA 729 AAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCmU smUsmUsU

gRNA9830 mCsmUsmUsACCCGGCAGUGGGGGUGGUUUUAGAGCUAGAAAUAGCAAGUUA 730 AAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCmU smUsmUsU

gRNA9831 mUsmUsmAsCCCGGCAGUGGGGGUGGGUUUUAGAGCUAGAAAUAGCAAGUUA 731

AAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCmU smUsmUsU

gRNA9832 mUsmAsmCsCCGGCAGUGGGGGUGGGGUUUUAGAGCUAGAAAUAGCAAGUUA 732 AAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCmU smUsmUsU

gRNA9833 mCsmUsmGsACACC CUU AC C CGG C AGGUUUU AG AG CU AG AAAU AG C AAGUU A 733 AAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCmU smUsmUsU

gRNA9834 mUsmGsmAsCACCCUUACCCGGCAGUGUUUUAGAGCUAGAAAUAGCAAGUUA 734 AAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCmU smUsmUsU

gRNA9835 mGsmAsmCsACCCUUACCCGGCAGUGGUUUUAGAGCUAGAAAUAGCAAGUUA 735 AAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCmU smUsmUsU

gRNA9836 mAs mC smAs CC CUUACC CGGC AGUGGGUUUUAG AG CU AG AAAU AG CAAGUUA 736 AAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCmU smUsmUsU

gRNA9837 mCsmCsmCsUUACCCGGCAGUGGGGGUGUUUUAGUACUCUGUAAUGAAAAUU 737 ACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUU GGCGAGAUsmUsmUsmU

gRNA9838 mAsmCsmCsCGGCAGUGGGGGUGGGGGUUUUAGAGCUAGAAAUAGCAAGUUA 738

AAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCmU

smUsmUsU

¹ “mN” indicates a 2'-OMe modification of the nucleotide “N”, and “Ns” indicates that the nucleotide “N” is linked to the following (i.e., 3') nucleotide by a phosphorothioate (PS). ATTORNEY DOCKET NO. 180802-47402/PCT

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Guide ID gRNA nucleotide sequence SEQ ID NO

gRNA9839 mAsmCsmAsCCCUUACCCGGCAGUGGGUUUUAGAmGmCmCmGmGmCmGmGmA 739 mAmAmCmGmCmCmGmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmU mUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU smUsmUsmU

gRNA9840 mCsmCsmCsUUACCCGGCAGUGGGGGGUUUUAGAmGmCmCmGmGmCmGmGmA 740 mAmAmCmGmCmCmGmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmU mUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU smUsmUsmU

gRNA9841 mCsmCsmUsUACCCGGCAGUGGGGGUGUUUUAGAmGmCmCmGmGmCmGmGmA 741 mAmAmCmGmCmCmGmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmU mUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU smUsmUsmU

gRNA9842 mC s mU smG s AC AC CCUUAC CCGG CAGGUUUUAG AmGmCmCmGmGmCmGmGmA 742 mAmAmCmGmCmCmGmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmU mUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU smUsmUsmU

gRNA9843 mCsmUsmUsACCCGGCAGUGGGGGUGGUUUUAGAmGmCmCmGmGmCmGmGmA 743 mAmAmCmGmCmCmGmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmU mUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU smUsmUsmU

gRNA9844 mGsmAsmCsACCCUUACCCGGCAGUGGUUUUAGAmGmCmCmGmGmCmGmGmA 744 mAmAmCmGmCmCmGmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmU mUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU smUsmUsmU

gRNA9845 mUsmGsmAsCACCCUUACCCGGCAGUGUUUUAGAmGmCmCmGmGmCmGmGmA 745 mAmAmCmGmCmCmGmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmU mUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU smUsmUsmU

gRNA9846 mUsmUsmAsCCCGGCAGUGGGGGUGGGUUUUAGAmGmCmCmGmGmCmGmGmA 746 mAmAmCmGmCmCmGmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmU mUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU smUsmUsmU

gRNA 10320 mUsmUsmAsCCCGGCAGUGGGGGUGGUUUUAGAmGmCmCmGmGmCmGmGmAm 791 AmAmCmGmCmCmGmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUm

UmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUs

mUsmUsmU

Table 2: Exemplary base editor amino acid sequences.²

Base Editor Encoding Sequence SEQ Name mRNA ID ID NO CBE-T1.52 MRNA74 MDKKYS I GLAI GTNS VGWAVI TDEYKVPSKKFKVLGNTDRHS I KKN 774

IBE34 NGG 89 LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLOEIFSNEMAK

² Amino acid sequences corresponding to Cas9 domains are shown as DOUBLE UNDERLINED ALL CAPS TEXT, amino acid sequences corresponding to deaminase domains are shown as lowercase plain text, amino acid sequences corresponding to linkers are shown as ITALIC ALL CAPS TEXT, amino acid sequences corresponding to nuclear localization signals (NLS) are shown as UNDERLINED ALL CAPS TEXT, and amino acid sequences corresponding to uracil ATTORNEY DOCKET NO. 180802-47402/PCT

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Base Editor Encoding Sequence SEQ Name mRNA ID ID NO VDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHL RKKLVDS TDKADLRL I YLALAHM I KFRGH FL I EGDLNPDNS DVDKL

F 10LV0TYN0L FEENP I NASGVDAKAI LS ARLS KS RRLENL I AOL P GEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLOLSKDTYDDDLD NLLAO I GDO YADL FLAAKNLS DAI LLS D I LRVNTE I TKAPLS ASM I KRYDEHHODLTLLKALVROOL PEKYKE I F FDOS KNGYAG Y I DGGAS OEEFYKFIKPI LEKMDGTEELLVKLNREDLLRKORTFDNGS I PHO I HLGELHAILRROEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNS RFAWMTRKSEETITPWNFEEWDKGASAOSFIERMTNFDKNLPNEK VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEOKKAIVDLL FKTNRKVTVKOLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLL KI IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFD DKVMKOLKRRR YTGWGRLS RKL I NG I RDKOSGKTI LD FLKS DG FAN RNFMOL I HDDS LTFKED I OKAOVSGOGDS LHEH I ANLAGS PAI KKG ILOTVKWDELVKVMGRHKPENIVIEMARENOTTOKGOKNSRERMK R I EEG I KELGS 01 LKEH PVENTOLONEKL YL YYLONGRDMYVDOEL DINRLSDYDVDHIVPOSFLKDDSIDNKVLTRSDKNRGKSDNVPSEE WKKMKNYWRQLLNAKL I TQRKFDNLTKAERGGSS GSETPGTSESA TPESSGseveysheywmrhaltlakrarderhvpvgavlvlnnrvi gegwnrakglhdptahaeimalrqgglvmqnyrlwgatlyttf epc vmcagamihsrigrvvf gvcnakthacmslmdvlghpgmphrveit egiladeceallcrf f rmprrvfnaqkkaqsstdGSSGSGSSGSGL SELDKAGFIKROLVETROITKHVAOILDSRMNTKYDENDKLIREVK VITLKSKLVSDFRKDFOFYKVREINNYHHAHDAYLNAWGTALIKK Y PKLE SE FVYGDYKVYDVRKM I AKS EOE I GKATAKYF FYSN I MNF F KTE I TLANGE I RKRPLI ETNGETGE I VWDKGRDFATVRKVLSMPOV N I VKKTE VOTGG F S KE S I L PKRNS DKL I ARKKD WD PKKYGG FD S PT VAYSVLWAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEA KGYKEVKKDLI IKLPKYSLFELENGRKRMLASAGELOKGNELALPS KYVNFLYLASHYEKLKGSPEDNEOKOLFVEOHKHYLDEI IEOISEF

SKRVILADANLDKVLSAYNKHRDKPIREOAENI IHLFTLTNLGAPA

glycosylase inhibitor (UGI) domains are shown as BOLD ALL CAPS TEXT. The term “CBE” in each base editor name indicates a base editor containing a deaminase domain capable of deaminating cytidine in DNA (i.e., a “cytidine base editor”, where a base editor containing a “cytidine adenosine base editor” is considered a “cytidine base editor”). The terms “T1.52”, “6b”, “RrA3f ’ in each base editor name identifies the deaminase of the base editor. The terms “NGG”, “NG”, “NNGRRT”, “NNN”, “NRN”, “NRRH”, and “NRTH” in the base editor names indicate the protospacer-adjacent motif specificity of the Cas9 domain of the base editor, where “N” indicates A, C, G, or T, “R” indicates A or G, “Y” indicates C or T, and “H” indicates A, C, or T. The terms SpRYc, SpRY, and Nureki in the base editor names indicate the Cas9 domain of the base editor. The term “IBE34” in a base editor name indicates an internal base editor where the deaminase domain has been inserted within the Cas9 domain between amino acid positions 906 and 907 referenced to SEQ ID NO: 197. In some embodiments, the N-terminal methionine of the RrA3f domain is absent. ATTORNEY DOCKET NO. 180802-47402/PCT

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Base Editor Encoding Sequence SEQ Name mRNA ID ID NO AFKYFDTTI DRKRYTSTKEVLDATL I HOS I TGLYETR I DLSOLGGD SGGSGGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKP ESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKM LSGGSGGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNK PESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIK MLEGADKRTADGSEFESPKKKRKV CBE6b MRNA71 MKRTADGSEFESPKKKRKVs s eve f sheywmrhal 11 akrardege 775 NGG 67 or apvgavlvlnnrvigegwvrriglhdptahaeimalrqgglvmqnp MRNA92 rlidatlyvtf epcvmcagaminsrigrvvf gvrnskrgaagslmn 78 vlnypgmnhrveitegiladecaallcdf yrmprqvf naqkkaqss lnSGGSSGGSSGSETPGTSESATPESSGGSSGGSDKK. YSIGLAI. GT NSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETA EATRLKRTARRRYTRRKNR I CYLOE I FSNEMAKVDDS FFHRLEES F LVEEDKKHERHPI FGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADL RLI YLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIOLVOTYNOLFE ENPINASGVDAKAILSARLSKSRRLENLIAOLPGEKKNGLFGNLIA LSLGLTPNFKSNFDLAEDAKLOLSKDTYDDDLDNLLAOIGDOYADL FLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHODLTLL KALVROOLPEKYKEI FFDOSKNGYAGYIDGGASOEEFYKFIKPILE KMDGTEELLVKLNREDLLRKORTFDNGS I PHOI HLGELHAI LRROE DFYPFLKDNREKIEKILTFRI PYYVGPLARGNSRFAWMTRKSEETI TPWNFEEWDKGASAOSFIERMTNFDKNLPNEKVLPKHSLLYEYFT VYNELTKVKYVTEGMRKPAFLSGEOKKAIVDLLFKTNRKVTVKOLK EDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI IKDKDFLDNEE NED I LED I VLTLTLFEDREMI EERLKTYAHLFDDKVMKOLKRRRYT GWGRLSRKLINGIRDKOSGKTILDFLKSDGFANRNFMOLIHDDSLT FKE D 10 KAO VS GOGDSLHEHI ANLAGS PA I KKG I LOTVKWDE LVK VMGRHKPENIVIEMARENOTTOKGOKNSRERMKRIEEGIKELGSOI LKEHPVENTOLONEKLYLYYLONGRDMYVDOELDINRLSDYDVDHI VPOSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEWKKMKNYWROLL NAKLITORKFDNLTKAERGGLSELDKAGFIKROLVETROITKHVAO ILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFOFYKVREIN NYHHAHDAYLNAWGTAL I KKYPKLES E FVYGD YKVYDVRKM I AKS EOEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGE IVWDKGRDFATVRKVLSMPOVNIVKKTEVOTGGFSKESILPKRNSD KLIARKKDWDPKKYGGFDSPTVAYSVLWAKVEKGKSKKLKSVKEL LG I TI MERS S FEKNP I D FLEAKG YKEVKKDL 11 KL PKYS LFELENG RKRMLASAGELOKGNELALPSKYVNFLYLASHYEKLKGSPEDNEOK OLFVEOHKHYLDE 11 EO I SEFSKRVI LADANLDKVLS AYNKHRDKP I REOAEN 11 HL FTLTNLGAPAAFKY FDTT I DRKRYTS TKEVLDATL

I HOS I TGLYETRI DLSOLGGDSGGSGGSGGSTNLS DIIEKETGKQL VIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDA PEYKPWALVIQDSNGENKIKMLSGGSGGSGGSTNLSDI IEKETGKQ LVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSD APEYKPWALVIQDSNGENKIKMLSGGSKRTADGSEFESPKKKRKV

RrA3fNG MRNA62 MKRTADGSEFESPKKKRKVmkpqirdhrpnpmeamyphi f y f hf en 776 (Nureki) 63 lekaygrnetwlcf tveiikqylpvpwkkgvf rnqvdpethchaek cf Iswf cnntlspkknyqvtwytswspcpecagevaef laehsnvk Itiytarlyyfwdtdyqeglrslseegasveimdyedfqycwenfv y ddgep f krwkgl kynf qsltrrlrei 1 qSGGSSGGSSGSETPGTS ESATPESSGGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKF KVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNR I CYLOE I FSNEMAKVDDS FFHRLEES FLVEEDKKHERHP I FGNI VD

E VAYHEKYPTI YHLRKKLVDS TDKADLRL I YLALAHM I KFRGH FL I ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

Base Editor Encoding Sequence SEQ Name mRNA ID ID NO EGDLNPDNSDVDKLFIOLVOTYNOLFEENPINASGVDAKAILSARL SKSRRLENLIAOLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDA KLOLS KDTYDDDLDNLLAO I GDO YADL FLAAKNLS DAI LLS D I LRV NTE I TKAPLSASM I KRYDEHHODLTLLKALVROOLPEKYKE I FFDO SKNGYAGYIDGGASOEEFYKFIKPILEKMDGTEELLVKLNREDLLR KORTFDNGSIPHOIHLGELHAILRROEDFYPFLKDNREKIEKILTF RIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEWDKGASAOSFI ERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPA FLSGEOKKAIVDLLFKTNRKVTVKOLKEDYFKKIECFDSVEISGVE DRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVLTLTLFEDRE M I EERLKTYAHLFDDKVMKOLKRRR YTGWGRLS RKL I NG I RDKOSG KTILDFLKSDGFANRNFMOLIHDDSLTFKEDIOKAOVSGOGDSLHE H I ANLAGS PAI KKGI LOTVKVVDELVKVMGRHKPENI VI EMARENO TTOKGOKNS RERMKR I EEG I KELGS 01 LKEH PVENTOLONEKL YL Y YLONGRDMYVDOELDINRLSDYDVDHIVPOSFLKDDSIDNKVLTRS DKNRGKSDNVPSEEWKKMKNYWROLLNAKLITORKFDNLTKAERG GLSELDKAGFIKROLVETROITKHVAOILDSRMNTKYDENDKLIRE VKVITLKSKLVSDFRKDFOFYKVREINNYHHAHDAYLNAWGTALI KKY PKLE SE FVYGDYKVYDVRKM I AKS EOE I GKATAKYF FYSN I MN FFKTE ITLANGEI RKRPLI ETNGETGE I VWDKGRDFATVRKVLSMP

0 VN I VKKTE VOTGG F S KE S I R PKRNS DKL I ARKKD WD PKKYGG FVS PTVAYSVLWAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFL EAKGYKEVKKDLI IKLPKYSLFELENGRKRMLASARFLOKGNELAL PSKYVNFLYLASHYEKLKGSPEDNEOKOLFVEOHKHYLDEI IEOIS E FS KRVI LADANLDKVLS AYNKHRDKP I REOAENI I HLFTLTNLGA PRAFKYFDTTI DRKVYRSTKEVLDATL I HOS I TGLYETR I DLSOLG GDSGGSGGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGN KPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKI KMLSGGSGGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIG NKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENK I KMLSGGSKRTADGS E FES PKKKRKV

RrA3fNGG MRNA61 mkpqi rdhrpnpmeamyphi f y f hf enl ekaygrne t wl c f t ve i i 777

60 kqy Ipvpwkkgvf rnqvdpe t hchaekc f 1 s wf cnnt 1 spkknyqv twytswspcpecagevaef laehsnvkltiytarlyyfwdtdyqeg Irslseegasveimdyedf qycwenfvyddgepf krwkglkynf qs Itrrlreil CjSGGSSGGSSGSETPGTSESATPESSGGSSGGSDKKY SIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL LFDSGETAEATRLKRTARRRYTRRKNR I CYLOE I FSNEMAKVDDS F FHRLEESFLVEEDKKHERHPI FGNIVDEVAYHEKYPTIYHLRKKLV DSTDKADLRLI YLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIOLV OTYNOLFEENPINASGVDAKAILSARLSKSRRLENLIAOLPGEKKN GLFGNLIALSLGLTPNFKSNFDLAEDAKLOLSKDTYDDDLDNLLAO IGDOYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDE HHODLTLLKALVROOLPEKYKEI FFDOSKNGYAGYIDGGASOEEFY KFI KP I LEKMDGTEELLVKLNREDLLRKORTFDNGS I PHOI HLGEL HAILRROEDFYPFLKDNREKIEKILTFRI PYYVGPLARGNSRFAWM TRKSEET I TPWNFEE WDKGAS AOS F I ERMTNFDKNL PNEKVL PKH SLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEOKKAIVDLLFKTNR KVTVKOLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI IKD KDFLDNEENED I LED I VLTLTLFEDREMI EERLKTYAHLFDDKVMK OLKRRRYTGWGRLSRKLINGIRDKOSGKTILDFLKSDGFANRNFMO LIHDDSLTFKEDIOKAOVSGOGDSLHEHIANLAGSPAIKKGILOTV KWDELVKVMGRHKPENIVIEMARENOTTOKGOKNSRERMKRIEEG

IKELGSOILKEHPVENTOLONEKLYLYYLONGRDMYVDOELDINRL ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

Base Editor Encoding Sequence SEQ Name mRNA ID ID NO SDYDVDHIVPOSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEWKKM KNYWROLLNAKLITORKFDNLTKAERGGLSELDKAGFIKROLVETR OITKHVAOILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFO FYKVREINNYHHAHDAYLNAWGTALIKKYPKLESEFVYGDYKVYD VRKMIAKSEOEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLI ETNGETGEIVWDKGRDFATVRKVLSMPOVNIVKKTEVOTGGFSKES ILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLWAKVEKGKSK KLKS VKELLG I TI MERS S FEKNP I D FLEAKG YKEVKKDL 11 KL PKY SLFELENGRKRMLASAGELOKGNELALPSKYVNFLYLASHYEKLKG S PEDNEOKOLFVEOHKHYLDE 11 EG I SEFSKRVI LADANLDKVLS A YNKHRDKP I REQAEN 11 HL FTLTNLGAPAAFKY FDTT I DRKRYTS T KEVLDATLIHOSITGLYETRIDLSOLGGDSGGSGGSGGSTNLSDI I EKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDEN VML LTSDAPE YKPWALV I QDSNGENK I KMLSGGSGGSGGSTNL SD I IEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDE NVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSKRTADGSEFES PKKKRKV

RrA3f MRNA39 mkpqi rdhrpnpmeamyphi fyfhf enl ekaygrne t wl c f t ve i i 778 NNGRRT 88 kqy Ipvpwkkgvf rnqvdpe t hchaekc f 1 s wf cnnt 1 spkknyqv twytswspcpecagevaef laehsnvkltiytarlyyfwdtdyqeg Irslseegasveimdyedf qycwenfvyddgepf krwkglkynf qs Itrrlreil qSGGSSGGSSGSETPGTSESATPESSGGSSGGSGKRN Y I LGLAI G I TS VG YG 11 DYETRDVI DAGVRL FKEANVENNEGRRS K RGARRLKRRRRHRIORVKKLLFDYNLLTDHSELSGINPYEARVKGL SOKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEOISRNS KALEEKYVAELOLERLKKDGEVRGSINRFKTSDYVKEAKOLLKVOK AYHOLDOS F I DTY I DLLETRRTYYEGPGEGS PFGWKD I KEWYEMLM GHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEK FO 11 ENVFKOKKKPTLKO I AKE I LVNEED I KGYRVTS TGKPE FTNL KVYHDIKDITARKEI IENAELLDOIAKILTI YOSSEDIOEELTNLN SELTOEEIEOISNLKGYTGTHNLSLKAINLILDELWHTNDNOIAIF NRLKLVPKKVDLSOOKEIPTTLVDDFILSPWKRSFIOSIKVINAI I KKYGLPND 111 ELAREKNSKDAQKMINEMQKRNRQTNERI EE HR TTGKENAKYLIEKIKLHDMOEGKCLYSLEAI PLEDLLNNPFNYEVD HIIPRSVSFDNSFNNKVLVKOEENSKKGNRTPFOYLSSSDSKISYE T FKKH I LNL AKGKGR I S KTKKE YLLEE RD INRFSVOKDFI NRNLVD TRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKER NKGYKHHAEDALI IANADFI FKEWKKLDKAKKVMENQMFEEKQAES MPEIETEOEYKEI FITPHQIKHIKDFKDYKYSHRVDKKPNRELIND TLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHH DPOTYOKLKLIMEOYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIK KIKYYGNKLNAHLDITDDYPNSRNKWKLSLKPYRFDVYLDNGVYK FVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNOAEFIASFYNND LIKINGELYRVI G VNNDLLNR I E VNM I D I TYRE YLENMNDKR P PR I I KT I AS KTQ S I KKYS TD I LGNL YE VKS KKH PQ 11 KKGGS PKKKRKV S SDYKDHDGDYKDHD I DYKDDDDKSGGSGGSGGSTNLSD I 1 EKETG KQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLT SDAPEYKPWALVI QDSNGENK IKMLSGGSGGSGGSTNLSD I I EKET GKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLL TSDAPE YKPWALV I QDSNGENK I KMLSGGSKRTADGSEFESPKKKR KV

RrA3fNNN MRNA65 mkpqi rdhrpnpmeamyphi fyfhf enl ekaygrne twl c f t ve i i 779 (SpRYc) 09 kqy Ipvpwkkgvf rnqvdpe t hchaekc f 1 s wf cnnt 1 spkknyqv

twytswspcpecagevaef laehsnvkltiytarlyyfwdtdyqeg ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

Base Editor Encoding Sequence SEQ Name mRNA ID ID NO

Irslseegasveimdyedf qycwenfvyddgepf krwkglkynf qs Itrrlreil gSGGSSGGSSGSETPGTSESATPESSGGSSGGSEKKY SIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTNRKSIKKNLMGAL L FDSGETAE ATRLKRTARRRYTRRKNR I R YLOE I FANEMAKLDDS F FORLEESFLVEEDKKNERHPI FGNLADEVAYHRNYPTIYHLRKKLA DSPEKADLRLI YLALAHI IKFRGHFLIEGKLNAENSDVAKLFYOLI OTYNOLFEESPLDEIEVDAKGILSARLSKSKRLEKLIAVFPNEKKN GLFGNI IALALGLTPNFKSNFDLTEDAKLOLSKDTYDDDLDELLGO IGDOYADLFSAAKNLSDAILLSDILRSNSEVTKAPLSASMVKRYDE HHODLALLKTLVROOFPEKYAEI FKDDTKNGYAGYVGADKKLRKRS GKLATEEEFYKFIKPILEKMDGAEELLAKLNRDDLLRKORTFDNGS I PHOI HLKELHAI LRROEEFYPFLKENREKI EKILTFRI PYYVGPL ARGNSRFAWLTRKSEEAITPWNFEEWDKGASAOSFIERMTNFDEO LPNKKVLPKHSLLYEYFTVYNELTKVKYVTERMRKPEFLSGEOKKA I VDLL FKTNRKVTVKOLKE DYFKKIECFDSVEI I G VE DR FNAS LGT YHDLLKI I KDKDFLDNEENED I LED I VLTLTLFEDREMI EERLKTY AHLFDDKVMKOLKRRHYTGWGRLSRKMINGIRDKOSGKTILDFLKS DGFSNRNFMOLIHDDSLTFKEEIEKAOVSGOGDSLHEOIADLAGSP AIKKGILOTVKIVDELVKVMGHKPENIVIEMARENOTTTKGLOOSR ERKKRIEEGIKELESOILKENPVENTOLONEKLYLYYLONGRDMYV DOELDINRLSDYDVDHIVPOSFIKDDSIDNKVLTRSVENRGKSDNV PSEEWKKMKNYWROLLNAKLITORKFDNLTKAERGGLSEADKAGF I KROLVETRO I TKHVAR I LDS RMNTKRDKNDKP I REVKVI TLKSKL VSD FRKD FOLYKVRD INNYHHAHDAYLNAWGTAL I KKY PKLE SE F VYGDYKVYDVRKM I AKS EOE I GKATAKRF FYSN I MNF FKTE VKLAN GET RKRPL I ETNGETGE WWNKEKD FATVRKVLAM POVN I VKKTE V OTGGFSKESIRPKRNSDKLIARKKDWDPKKYGGFLWPTVAYSVLW AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKK DLI IKLPKYSLFELENGRKRMLASAKOLOKGNELALPSKYVNFLYL ASHYEKLKGSPEDNEOKOLFVEOHKHYLDEI IEOISEFSKRVILAD ANLDKVLSAYNKHRDKPIREOAENI IHLFTLTRLGAPRAFKYFDTT I DPKO YRSTKE VLDATL I HQS I TGL YETR I DLS QEGGDSGGSGGSG GSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHT AYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSGGS GGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVH TAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSKR TADGSEFESPKKKRKV

RrA3fNRN MRNA62 mkpqi rdhrpnpmeamyphi f y f hf enl ekaygrne t wl c f t ve i i 780 (SpRY) 76 kqy Ipvpwkkgvf rnqvdpe t hchaekc f 1 s wf cnnt 1 spkknyqv twytswspcpecagevaef laehsnvkltiytarlyyfwdtdyqeg Irslseegasveimdyedf qycwenfvyddgepf krwkglkynf qs Itrrlreil aSGGSSGGSSGSETPGTSESATPESSGGSSGGSDKK. Y SIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL LFDSGETAERTRLKRTARRRYTRRKNR I CYLOE I FSNEMAKVDDS F FHRLEESFLVEEDKKHERHPI FGNIVDEVAYHEKYPTIYHLRKKLV DSTDKADLRLI YLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIOLV OTYNOLFEENPINASGVDAKAILSARLSKSRRLENLIAOLPGEKKN GLFGNLIALSLGLTPNFKSNFDLAEDAKLOLSKDTYDDDLDNLLAO IGDOYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDE HHODLTLLKALVROOLPEKYKEI FFDOSKNGYAGYIDGGASOEEFY KFI KP I LEKMDGTEELLVKLNREDLLRKORTFDNGS I PHOI HLGEL HAI LRROEDFYPFLKDNREKI EKILTFRI PYYVGPLARGNSRFAWM TRKSEET I TPWNFEE WDKGAS AOS F I ERMTNFDKNL PNEKVL PKH

SLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEOKKAIVDLLFKTNR ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

Base Editor Encoding Sequence SEQ Name mRNA ID ID NO KVTVKOLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI IKD KDFLDNEENED I LED I VLTLTLFEDREMI EERLKTYAHLFDDKVMK OLKRRRYTGWGRLSRKLINGIRDKOSGKTILDFLKSDGFANRNFMO LIHDDSLTFKEDIOKAOVSGOGDSLHEHIANLAGSPAIKKGILOTV KWDELVKVMGRHKPENIVIEMARENOTTOKGOKNSRERMKRIEEG IKELGSOILKEHPVENTOLONEKLYLYYLONGRDMYVDOELDINRL SDYDVDHIVPOSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEWKKM KNYWROLLNAKLITORKFDNLTKAERGGLSELDKAGFIKROLVETR OITKHVAOILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFO FYKVREINNYHHAHDAYLNAWGTALIKKYPKLESEFVYGDYKVYD VRKMIAKSEOEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLI ETNGETGEIVWDKGRDFATVRKVLSMPOVNIVKKTEVOTGGFSKES IRPKRNSDKLIARKKDWDPKKYGGFLWPTVAYSVLWAKVEKGKSK KLKS VKELLG I TI MERE S FEKNP I D FLEAKG YKEVKKDL 11 KL PKY SLFELENGRKRMLASAKOLOKGNELALPSKYVNFLYLASHYEKLKG S PEDNEOKOLFVEOHKHYLDE 11 EO I SEFSKRVI LADANLDKVLS A YNKHRDKP I REOAEN 11 HL FTLTRLGAPRAFKY FDTT I D PKOYRS T KEVLDATLIHOSITGLYETRIDLSOLGGDSGGSGGSGGSTNLSDI I EKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDEN VML LTSDAPE YKPWALV I QDSNGENK I KMLSGGSGGSGGSTNL SD I IEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDE NVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSKRTADGSEFES PKKKRKV

RrA3f MRNA62 mkpqi rdhrpnpmeamyphi f y f hf enl ekaygrne t wl c f t ve i i 781 NRRH 78 kqy Ipvpwkkgvf rnqvdpe t hchaekc f 1 s wf cnnt 1 spkknyqv twytswspcpecagevaef laehsnvkltiytarlyyfwdtdyqeg Irslseegasveimdyedf qycwenfvyddgepf krwkglkynf qs Itrrlreil CjSGGSSGGSSGSETPGTSESATPESSGGSSGGSDKKY SIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL LFDSGETAEATRLKRTARRRYTRRKNR I CYLOE I FSNEMAKVDDS F FHRLEESFLVEEDKKHERHPI FGNIVDEVAYHEKYPTIYHLRKKLV DSTDKADLRLI YLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIOLV OTYNOLFEENPINASGVDAKAILSARLSKSRRLENLIAOLPGEKKN GLFGNLIALSLGLTPNFKSNFDLAEDAKLOLSKDTYDDDLDNLLAO IGDOYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMVKRYDE HHODLTLLKALVROOLPEKYKEI FFDOSKNGYAGYIDGGASOEEFY KFIKPILEKMDGTEELLVKLNREDLLRKORTFDNGI I PHOIHLGEL HAILRROGDFYPFLKDNREKIEKILTFRI PYYVGPLARGNSRFAWM TRKSEET I TPWNFEE WDKGAS AOS F I ERMTNFDKNL PNEKVL PKH SLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEOKKAIVDLLFKTNR KVTVKOLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI IKD KDFLDNEENED I LED I VLTLTLFEDREMI EERLKTYAHLFDDKVMK OLKRLRYTGWGRLSRKLINGIRDKOSGKTILDFLKSDGFANRNFMO LIHDDSLTFKEDIOKAOVSGOGDSLHEHIANLAGSPAIKKGILOTV KWDELVKVMGGHKPENIVIEMARENOTTOKGOKNSRERMKRIEEG IKELGSOILKEHPVENTOLONEKLYLYYLONGRDMYVDOELDINRL SDYDVDHIVPOSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEWKKM KNYWROLLNAKLITORKFDNLTKAERGGLSELDKAGFIKROLVETR OITKHVAOILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFO FYKVREINNYHHAHDAYLNAWGTALIKKYPKLESEFVYGDYKVYD VRKMIAKSEOEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLI ETNGETGEIVWDKGRDFATVRKVLSMPOVNIVKKTEVOTGGFSKES I LPKGNSDKLI ARKKDWDPKKYGGFNS PTAAYSVLWAKVEKGKSK

KLKS VKELLG I TI MERE S FEKNP I G FLEAKG YKEVKKDL 11 KL PKY ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

Base Editor Encoding Sequence SEQ Name mRNA ID ID NO SLFELENGRKRMLASAGVLHKGNELALPSKYVNFLYLASHYEKLKG S PEDNEOKOLFVEOHKHYLDE 11 EO I SEFSKRVI LADANLDKVLS A YNKHRDKP I REOAEN 11 HL FTLTNLGVPAAFKY FDTT I DKKRYTS T KEVLDATLIHOSITGLYETRIDLSOLGGDSGGSGGSGGSTNLSDI I EKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDEN VML LTSDAPE YKPWALV I QDSNGENK I KMLSGGSGGSGGSTNL SD I IEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDE NVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSKRTADGSEFES PKKKRKV

RrA3f MRNA62 MKRTADGSEFESPKKKRKVmkpqirdhrpnpmeamyphi f y f hf en 782 NRTH 60 lekaygrnetwlcf tveiikqylpvpwkkgvf rnqvdpethchaek cf Iswf cnntlspkknyqvtwytswspcpecagevaef laehsnvk Itiytarlyyfwdtdyqeglrslseegasveimdyedfqycwenfv y ddgep f krwkgl kynf qsltrrlrei 1 qSGGSSGGSSGSETPGTS ESATPESSGGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKF KVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNR I CYLOE I FSNEMAKVDDS FFHRLEES FLVEEDKKHERHP I FGNI VD E VAYHEKYPTI YHLRKKLVDS TDKADLRL I YLALAHM I KFRGH FL I EGDLNPDNSDVDKLFIOLVOTYNOLFEENPINASGVDAKAILSARL SKSRRLENLIAOLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDA KLOLS KDTYDDDLDNLLAO I GDO YADL FLAAKNLS DAI LLS D I LRV NTEITKAPLSASMVKRYDEHHODLTLLKALVROOLPEKYKEIFFDO SKNGYAGYIDGGASOEEFYKFIKPILEKMDGTEELLVKLNREDLLR KORTFDNGI IPHOIHLGELHAILRROGDFYPFLKDNREKIEKILTF RIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEWDKGASAOSFI ERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPA FLSGEOKKAIVDLLFKTNRKVTVKOLKEDYFKKIECFDSVEISGVE DRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVLTLTLFEDRE M I EERLKTYAHLFDDKVMKOLKRLR YTGWGRLS RKL I NG I RDKOSG KTILDFLKSDGFANRNFMOLIHDDSLTFKEDIOKAOVSGOGDSLHE H I ANLAGS PAI KKGI LOTVKVVDELVKVMGGHKPENI VI EMARENO TTOKGOKNS RERMKR I EEG I KELGS 01 LKEH PVENTOLONEKL YL Y YLONGRDMYVDOELDINRLSDYDVDHIVPOSFLKDDSIDNKVLTRS DKNRGKSDNVPSEEWKKMKNYWROLLNAKLITORKFDNLTKAERG GLSELDKAGFIKROLVETROITKHVAOILDSRMNTKYDENDKLIRE VKVITLKSKLVSDFRKDFOFYKVREINNYHHAHDAYLNAWGTALI KKY PKLE SE FVYGDYKVYDVRKM I AKS EOE I GKATAKYF FYSN I MN FFKTE ITLANGEI RKRPLI ETNGETGE I VWDKGRDFATVRKVLSMP

0 VN I VKKTE VOTGG F S KE S I L PKGNS DKL I ARKKD WD PKKYGG FNS PTVAYSVLWAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIGFL EAKGYKEVKKDLI IKLPKYSLFELENGRKRMLASASVLHKGNELAL PSKYVNFLYLASHYEKLKGSSEDNKOKOLFVEOHKHYLDEI IEOIS E FS KRVI LADANLDKVLS AYNKHRDKP I REOAENI I HLFTLTNLGA S AAFKYFDTTI GRKLYTSTKEVLDATL I HOS I TGLYETR I DLSOLG

CrDSGGSGGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGN KPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKI KMLSGGSGGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIG NKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENK I KMLSGGSKRTADGS E FES PKKKRKV CBE6b_NGG MRNA92 MKRTADGSEFESPKKKRKVs s eve f sheywmrhal 11 akrardege 792 (version 2) 79 apvgavlvlnnrvigegwvrriglhdptahaeimalrqgglvmqnp rlidatlyvtf epcvmcagaminsrigrvvf gvrnskrgaagslmn vlnypgmnhrveitegiladecaallcdf yrmprqvf naqkkaqss

i nEGGSEEEEESGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFK ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

Base Editor Encoding Sequence SEQ Name mRNA ID ID NO VLGNTDRHS I KKNL I GALL FDSGETAE ATRLKRTARRRYTRRKNR I CYLOEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDE VAYHEKYPTIYHLRKKLVDSTDKADLRLI YLALAHMIKFRGHFLIE GDLNPDNSDVDKLFIOLVOTYNOLFEENPINASGVDAKAILSARLS KSRRLENLIAOLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAK LOLSKDTYDDDLDNLLAOIGDOYADLFLAAKNLSDAILLSDILRVN TEITKAPLSASMIKRYDEHHODLTLLKALVROOLPEKYKEI FFDOS KNG YAGY I DGGAS GEE F YKF I KP I LEKMDGTEELLVKLNREDLLRK ORTFDNGS I PHQI HLGELHAI LRRQEDFYPFLKDNREKI EKILTFR I PYYVGPLARGNSRFAWMTRKSEETITPWNFEEWDKGASAOSFIE RMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAF LSGEOKKAIVDLLFKTNRKVTVKOLKEDYFKKIECFDSVEISGVED RFNASLGTYHDLLKI I KDKDFLDNEENED I LED I VLTLTLFEDREM IEERLKTYAHLFDDKVMKOLKRRRYTGWGRLSRKLINGIRDKOSGK TILDFLKSDGFANRNFMOLIHDDSLTFKEDIOKAOVSGOGDSLHEH I ANLAGS PAI KKG I LOTVKWDELVKVMGRHKPENI VI EMARENOT TOKGOKNSRERMKRIEEGIKELGSOILKEHPVENTOLONEKLYLYY LONGRDMYVDOELDINRLSDYDVDHIVPOSFLKDDSIDNKVLTRSD KNRGKSDNVPSEEWKKMKNYWROLLNAKLITORKFDNLTKAERGG LSELDKAGFIKROLVETROITKHVAOILDSRMNTKYDENDKLIREV KVITLKSKLVSDFRKDFOFYKVREINNYHHAHDAYLNAWGTALIK KYPKLESEFVYGDYKVYDVRKMIAKSEOEIGKATAKYFFYSNIMNF FKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPO VNI VKKTEVOTGG FS KE S I LPKRNS DKL I ARKKDWDPKKYGGFDS P TVAYS VL WAKVEKGKS KKLKS VKELLG I TI MERE S FEKNP I D FLE AKGYKEVKKDLI IKLPKYSLFELENGRKRMLASAGELOKGNELALP SKYVNFLYLASHYEKLKGS PEDNEOKOLFVEOHKHYLDE 11 EO I SE FSKRVILADANLDKVLSAYNKHRDKPIREOAENI IHLFTLTNLGAP AAFKY FDTT I DRKRYTS TKEVLDATL I HOS I TGLYETR I DLSOLGG

gSGGSGGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNK PESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIK MLSGGSGGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGN KPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKI

KML SGGSKRTADGSE FE S PKKKRKV

sg23/sgRNA_088 spacer sequence: CAGGAUCCGCACAGACUCCA (SEQ ID NO: 747) (target gene: ALAS1). The target sequence corresponding to sg23 is as follows, where the PAM sequence is in bold: CAGGATCCGCACAGACTCCAGGG (SEQ ID NO: 748).

NUCLEOBASE EDITORS

Useful in the methods and compositions described herein are nucleobase editors that edit, modify or alter a target nucleotide sequence of a polynucleotide. Nucleobase editors described herein typically include a polynucleotide programmable nucleotide binding domain and a nucleobase editing domain (e.g., adenosine deaminase, cytidine deaminase, or a dual deaminase). A polynucleotide programmable nucleotide binding domain, when in conjunction with a bound ATTORNEY DOCKET NO. 180802-47402/PCT

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guide polynucleotide (e.g., gRNA), can specifically bind to a target polynucleotide sequence and thereby localize the base editor to the target nucleic acid sequence desired to be edited.

Polynucleotide Programmable Nucleotide Binding Domain

Polynucleotide programmable nucleotide binding domains bind polynucleotides (e.g, RNA, DNA). A polynucleotide programmable nucleotide binding domain of a base editor can itself comprise one or more domains (e.g., one or more nuclease domains). In some embodiments, the nuclease domain of a polynucleotide programmable nucleotide binding domain comprises an endonuclease or an exonuclease.

Disclosed herein are base editors comprising a polynucleotide programmable nucleotide binding domain comprising all or a portion (e.g., a functional portion) of a CRISPR protein (i.e., a base editor comprising as a domain all or a portion (e.g., a functional portion) of a CRISPR protein (e.g., a Cas protein), also referred to as a “CRISPR protein-derived domain” of the base editor). A CRISPR protein-derived domain incorporated into a base editor can be modified compared to a wild-type or natural version of the CRISPR protein. A CRISPR protein-derived domain can comprise one or more mutations, insertions, deletions, rearrangements and/or recombinations relative to a wild-type or natural version of the CRISPR protein.

Cas proteins that can be used herein include class 1 and class 2. Non-limiting examples of Cas proteins include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas9 (also known as Csnl or Csxl2), CaslO, Csyl, Csy2, Csy3, Csy4, Csel, Cse2, Cse3, Cse4, Cse5e, Cscl, Csc2, Csa5, Csnl, Csn2, Csml, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, CsxlS, Csfl, Csf2, CsO, Csf4, Csdl, Csd2, Cstl, Cst2, Cshl, Csh2, Csal, Csa2, Csa3, Csa4, Csa5, Casl2a/Cpfl, Casl2b/C2cl (e.g, SEQ ID NO: 232), Casl2c/C2c3, Casl2d/CasY, Casl2e/CasX, Casl2g, Casl2h, Casl2i, and Casl2j/Cas< I>, CARF, DinG, Turbo Cas9 (i.e., an SpCas9 with the amino acid alterations Q844R, V842L, F846Y, L847M, and I852F), homologues thereof, or modified versions thereof. A CRISPR enzyme can direct cleavage of one or both strands at a target sequence, such as within a target sequence and/or within a complement of a target sequence. For example, a CRISPR enzyme can direct cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence.

A vector that encodes a CRISPR enzyme that is mutated to with respect to a corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence can be used. A ATTORNEY DOCKET NO. 180802-47402/PCT

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Cas protein (e.g., Cas9, Cast 2) or a Cas domain (e.g., Cas9, Cast 2) can refer to a polypeptide or domain with at least or at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and/or sequence homology to a wild-type exemplary Cas polypeptide or Cas domain. Cas (e.g., Cas9, Cas 12) can refer to the wild-type or a modified form of the Cas protein that can comprise an amino acid change such as a deletion, insertion, substitution, variant, mutation, fusion, chimera, or any combination thereof.

In some embodiments, a CRISPR protein-derived domain of a base editor can include all or a portion (e.g., a functional portion) of Cas9 from Corynebacterium ulcerans (NCBI Refs:

NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasma taiwanense (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1); Psychroflexus torquis (NCBI Ref: NC 018721.1); Streptococcus thermophilus (NCBI Ref: YP 820832.1); Listeria innocua (NCBI Ref: NP 472073.1); Campylobacter jejuni (NCBI Ref:

YP_002344900.1); Neisseria meningitidis (NCBI Ref: YP_002342100.1), Streptococcus pyogenes, or Staphylococcus aureus.

Some aspects of the disclosure provide high fidelity Cas9 domains. High fidelity Cas9 domains are known in the art and described, for example, in KI einstiver, B. P., et al. “High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects.” Nature 529, 490-495 (2016); and Slaymaker, I. M., et al. “Rationally engineered Cas9 nucleases with improved specificity.” Science 351, 84-88 (2015); the entire contents of each of which are incorporated herein by reference. An Exemplary high fidelity Cas9 domain is provided in the Sequence Listing as SEQ ID NO: 233.

In some embodiments, any of the Cas9 fusion proteins or complexes provided herein comprise one or more of a D10A, N497X, a R661X, a Q695X, and/or a Q926X mutation, or a corresponding mutation in any of the amino acid sequences provided herein, wherein X is any amino acid..

Typically, Cas9 proteins, such as Cas9 from S. pyogenes (spCas9), require a “protospacer adjacent motif (PAM)” or PAM-like motif, which is a 2-6 base pair DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system. The presence of an NGG PAM sequence is required to bind a particular nucleic acid region, where the “N” in “NGG” is adenosine (A), thymidine (T), or cytosine (C), and the G is guanosine. In some embodiments, any of the fusion proteins or complexes provided herein may contain a Cas9 domain that is capable of binding a nucleotide sequence that does not ATTORNEY DOCKET NO. 180802-47402/PCT

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contain a canonical (e.g., NGG) PAM sequence. Cas9 domains that bind to non-canonical PAM sequences have been described in the art and would be apparent to the skilled artisan. For example, Cas9 domains that bind non-canonical PAM sequences have been described in Kleinstiver, B. P., et al., “Engineered CRISPR-Cas9 nucleases with altered PAM specificities” Nature 523, 481-485 (2015); and Kleinstiver, B. P., etal., “Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition” Nature Biotechnology 33, 1293-1298 (2015); the entire contents of each are hereby incorporated by reference.

In some embodiments, the napDNAbp is a circular permutant (e.g., SEQ ID NO: 238). In some embodiments, the polynucleotide programmable nucleotide binding domain comprises a nickase domain. Herein the term “nickase” refers to a polynucleotide programmable nucleotide binding domain comprising a nuclease domain that is capable of cleaving only one strand of the two strands in a duplexed nucleic acid molecule (e.g., DNA). For example, where a polynucleotide programmable nucleotide binding domain comprises a nickase domain derived from Cas9, the Cas9-derived nickase domain can include a D10A mutation and a histidine at position 840. In another example, a Cas9-derived nickase domain comprises an H840A mutation, while the amino acid residue at position 10 remains a D.

In some embodiments, a Cas9 nuclease has an inactive (e.g., an inactivated) DNA cleavage domain, that is, the Cas9 is a nickase, referred to as an “nCas9” protein (for “nickase” Cas9; SEQ ID NO: 201). The Cas9 nickase may be a Cas9 protein that is capable of cleaving only one strand of a duplexed nucleic acid molecule (e.g., a duplexed DNA molecule). In some embodiments the Cas9 nickase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the Cas9 nickases provided herein. Additional suitable Cas9 nickases will be apparent to those of skill in the art based on this disclosure and knowledge in the field and are within the scope of this disclosure.

Also provided herein are base editors comprising a polynucleotide programmable nucleotide binding domain which is catalytically dead (i.e., incapable of cleaving a target polynucleotide sequence). For example, in the case of a base editor comprising a Cas9 domain, the Cas9 can comprise both a D10A mutation and an H840A mutation. In further embodiments, a catalytically dead polynucleotide programmable nucleotide binding domain comprises a point mutation (e.g., D10A or H840A) as well as a deletion of all or a portion (e.g., a functional portion) of a nuclease domain. dCas9 domains are known in the art and described, for example, in Qi et al., “Repurposing CRISPR as an RNA-guided platform for sequence-specific control of ATTORNEY DOCKET NO. 180802-47402/PCT

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gene expression.” Cell. 2013; 152(5): 1173-83, the entire contents of which are incorporated herein by reference.

The term “protospacer adjacent motif (PAM)” or PAM-like motif refers to a 2-6 base pair DNA sequence immediately following the DNA sequence targeted by a nucleic acid programmable DNA binding protein. In some embodiments, the PAM can be a 5' PAM (z.e., located upstream of the 5' end of the protospacer). In other embodiments, the PAM can be a 3' PAM (z.e., located downstream of the 5' end of the protospacer). The PAM sequence can be any PAM sequence known in the art. Suitable PAM sequences include, but are not limited to, NGG, NGA, NGC, NGN, NGT, NGTT, NGCG, NGAG, NGAN, NGNG, NGCN, NGCG, NGTN, NNGRRT, NNNRRT, NNGRR(N), TTTV, TYCV, TYCV, TATV, NNNNGATT, NNAGAAW, or NAAAAC. Y is a pyrimidine; N is any nucleotide base; W is A or T.

A base editor provided herein can comprise a CRISPR protein-derived domain that is capable of binding a nucleotide sequence that contains a canonical or non-canonical protospacer adjacent motif (PAM) sequence.

In some embodiments, the PAM is an “NRN” PAM where the “N” in “NRN” is adenine (A), thymine (T), guanine (G), or cytosine (C), and the R is adenine (A) or guanine (G); or the PAM is an “NYN” PAM, wherein the “N” in NYN is adenine (A), thymine (T), guanine (G), or cytosine (C), and the Y is cytidine (C) or thymine (T), for example, as described in R. T. Walton etal., 2020, Science, 10.1126/science.aba8853 (2020), the entire contents of which are incorporated herein by reference.

Several PAM variants are described in Table 3 below.

Table 3. Cas9 proteins and corresponding PAM sequences. N is A, C, T, or G; and V is A, C, or G.

Variant PAM

spCas9 NGG

spCas9-VRQR NGA

spCas9-VRER NGCG

xCas9 (sp) NGN

saCas9 NNGRRT

saCas9-KKH NNNRRT

spCas9-MQKSER NGCG

spCas9-MQKSER NGCN

spCas9-LRKIQK NGTN

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Variant PAM

spCas9-LRVSQK NGTN

spCas9-LRVSQL NGTN

spCas9-MQKFRAER NGC

Cpfl 5 ' (TTTV)

SpyMac 5 ' -NAA-3 '

In some embodiments, the PAM is NGC. In some embodiments, the NGC PAM is recognized by a Cas9 variant. In some embodiments, the NGC PAM Cas9 variant includes one or more amino acid substitutions selected from DI 135M, SI 136Q, G1218K, E1219F, A1322R, D1332A, R1335E, and T1337R (collectively termed “MQKFRAER”) of spCas9 (SEQ ID No: 197), or a corresponding mutation in another Cas9. In some embodiments, the Cas9 variant contains one or more amino acid substitutions selected from DI 135V, G1218R, R1335Q, and T1337R (collectively termed VRQR) of spCas9 (SEQ ID No: 197), or a corresponding mutation in another Cas9. In some embodiments, the Cas9 variant contains one or more amino acid substitutions selected from DI 135V, G1218R, R1335E, and T1337R (collectively termed VRER) of spCas9 (SEQ ID No: 197), or a corresponding mutation in another Cas9. In some embodiments, the Cas9 variant contains one or more amino acid substitutions selected from E782K, N968K, and R1015H (collectively termed KHH) of saCas9 (SEQ ID NO: 218). In some embodiments, the Cas9 variant includes one or more amino acid substitutions selected from D1135M, S1136Q, G1218K, E1219S, R1335E, and T1337R (collectively termed “MQKSER”) of spCas9 (SEQ ID No: 197), or a corresponding mutation in another Cas9.

In some cases, a Cas9 variant has specificity for the PAM 5 " -NGC-3 ". In some embodiments, a Cas9 variant includes one or more amino acid substitutions selected from D1135M, S1136Y, G1218K, E1219F, A1322R, D1332A, R1335E, and T1337K of spCas9 (SEQ ID No: 197), or a corresponding mutation in another Cas9. In some embodiments, the Cas9 variant includes one or more amino acid substitutions selected from DI 135L, SI 136Y, G1218K, E1219F, A1322R, D1332A, R1335E, and T1337R of spCas9 (SEQ ID No: 197), or a corresponding mutation in another Cas9. In some embodiments, a Cas9 variant includes one or more amino acid substitutions selected from D1135M, S1136Y, G1218K, E1219F, A1322R, D1332A, R1335E, and T1337K of spCas9 (SEQ ID No: 197), or a corresponding mutation in another Cas9. In some embodiments, a Cas9 variant includes one or more amino acid substitutions selected from DI 135L, S1136Y, G1218K, E1219F, A1283D, A1322R, D1332A, R1335E, and T1337K of spCas9 (SEQ ID No: 197), or a corresponding mutation in another ATTORNEY DOCKET NO. 180802-47402/PCT

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Cas9. In some embodiments, a Cas9 variant includes one or more amino acid substitutions selected from A61R, LI 111R, DI 135L, SI 136W, G1218K, E1219Q, N1317R, A1322R, R1333P, R1335Q, and T1337R of spCas9 (SEQ ID No: 197) (SpRY), or a corresponding mutation in another Cas9. In some embodiments, a Cas9 variant includes one or more amino acid substitutions selected from D1135L, S1136Q, G1218K, E1219F, E1250K, A1283D, A1322R, D1332A, R1335E, and T1337K of spCas9 (SEQ ID No: 197), or a corresponding mutation in another Cas9. In some embodiments, a Cas9 variant includes one or more amino acid substitutions selected from D1135M, S1136Y, G1218K, E1219F, E1250K, A1283D, A1322R, D1332A, R1335E, and T1337R of spCas9 (SEQ ID No: 197), or a corresponding mutation in another Cas9. In some embodiments, a Cas9 variant includes one or more amino acid substitutions selected from R765A, Q768A, D1135L, S1136Y, G1218K, A1283D, E1219F, A1322R, D1332A, R1335E, and T1337K of spCas9 (SEQ ID No: 197), or a corresponding mutation in another Cas9. In some embodiments, any of the Cas9 proteins provided herein, including an SpCas9 comprises any one, two, three, four, five, six, seven, eight, nine, or ten of the following amino acid substitutions in a corresponding residue: R765A, Q768A, W1126R, R1359W, E1250K, A1239T, A1239V, A1283D, R1335D, D1135L, D1135M, D1135R, D1135W, S1136H, S1136Q, S1136Y, G1218D, G1218K, G1218R, G1218E, G1218L, E1219F, E1219K, E1219N, A1322A, A1322R, A1322K, D1332A, R1335V, T1337K, T1337T, D1332A, DI 135V and T1337R.

In some embodiments, a CRISPR protein-derived domain of a base editor comprises all or a portion (e.g., a functional portion) of a Cas9 protein with a canonical PAM sequence (NGG). In other embodiments, a Cas9-derived domain of a base editor can employ a non-canonical PAM sequence. Such sequences have been described in the art and would be apparent to the skilled artisan. For example, Cas9 domains that bind non-canonical PAM sequences have been described in Kleinstiver, B. P., etal., “Engineered CRISPR-Cas9 nucleases with altered PAM specificities” Nature 523, 481-485 (2015); and Kleinstiver, B. P., etal., “Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition” Nature Biotechnology 33, 1293-1298 (2015); R. T. Walton et al. “Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants” Science 10.1126/science.aba8853 (2020); Hu et al. “Evolved Cas9 variants with broad PAM compatibility and high DNA specificity,” Nature, 2018 Apr. 5, 556(7699), 57-63; Miller etal., “Continuous evolution of SpCas9 variants compatible with non-G PAMs” Nat. Biotechnol., 2020 Apr;38(4):471-481; the entire contents of each are hereby incorporated by reference. ATTORNEY DOCKET NO. 180802-47402/PCT

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Fusion Proteins or Complexes Comprising a NapDNAbp and a Cytidine Deaminase and/or Adenosine Deaminase

Some aspects of the disclosure provide fusion proteins or complexes comprising a Cas9 domain or other nucleic acid programmable DNA binding protein (e.g., Cast 2) and one or more cytidine deaminase, adenosine deaminase, or cytidine adenosine deaminase domains. It should be appreciated that the Cas9 domain may be any of the Cas9 domains or Cas9 proteins (e.g., dCas9 or nCas9) provided herein. In some embodiments, any of the Cas9 domains or Cas9 proteins (e.g., dCas9 or nCas9) provided herein may be fused with any of the cytidine deaminases and/or adenosine deaminases provided herein. The domains of the base editors disclosed herein can be arranged in any order.

In some embodiments, the fusion proteins or complexes comprising a cytidine deaminase or adenosine deaminase and a napDNAbp (e.g., Cas9 or Casl2 domain) do not include a linker sequence. In some embodiments, a linker is present between the cytidine or adenosine deaminase and the napDNAbp. In some embodiments, cytidine or adenosine deaminase and the napDNAbp are fused via any of the linkers provided herein. For example, in some embodiments the cytidine or adenosine deaminase and the napDNAbp are fused via any of the linkers provided herein.

It should be appreciated that the fusion proteins or complexes of the present disclosure may comprise one or more additional features. For example, in some embodiments, the fusion protein or complex may comprise inhibitors, cytoplasmic localization sequences, export sequences, such as nuclear export sequences, or other localization sequences, as well as sequence tags that are useful for solubilization, purification, or detection of the fusion proteins or complexes. Suitable protein tags provided herein include, but are not limited to, biotin carboxylase carrier protein (BCCP) tags, myc-tags, calmodulin-tags, FLAG-tags, hemagglutinin (HA)-tags, polyhistidine tags, also referred to as histidine tags or His-tags, maltose binding protein (MBP)-tags, nus-tags, glutathione-S-transferase (GST)-tags, green fluorescent protein (GFP)-tags, thioredoxin-tags, S-tags, Softags (e.g., Softag 1, Softag 3), strep-tags, biotin ligase tags, FlAsH tags, V5 tags, and SBP-tags. Additional suitable sequences will be apparent to those of skill in the art. In some embodiments, the fusion protein or complex comprises one or more His tags.

Exemplary, yet nonlimiting, fusion proteins are described in International PCT Application Nos. PCT/US2017/045381, PCT/US2019/044935, and PCT/US2020/016288, each of which is incorporated herein by reference for its entirety. ATTORNEY DOCKET NO. 180802-47402/PCT

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Fusion Proteins or Complexes with Internal Insertions

Provided herein are fusion proteins or complexes comprising a heterologous polypeptide fused to a nucleic acid programmable nucleic acid binding protein, for example, a napDNAbp. The heterologous polypeptide can be fused to the napDNAbp at a C-terminal end of the napDNAbp, an N-terminal end of the napDNAbp, or inserted at an internal location of the napDNAbp. In some embodiments, the heterologous polypeptide is a deaminase (e.g., cytidine or adenosine deaminase) or a functional fragment thereof. For example, a fusion protein can comprise a deaminase flanked by an N- terminal fragment and a C-terminal fragment of a Cas9 or Casl2 (e.g., Casl2b/C2cl), polypeptide.

The deaminase can be a circular permutant deaminase. In some embodiments, the deaminase is a circular permutant TadA, circularly permutated at amino acid residue 116, 136, or 65 as numbered in a TadA reference sequence.

The fusion protein or complexes can comprise more than one deaminase. The fusion protein or complex can comprise, for example, 1, 2, 3, 4, 5 or more deaminases. The deaminases in a fusion protein or complex can be adenosine deaminases, cytidine deaminases, or a combination thereof.

In some embodiments, the napDNAbp in the fusion protein or complex contains a Cas9 polypeptide or a fragment thereof. The Cas9 polypeptide can be a variant Cas9 polypeptide. The Cas9 polypeptide can be a circularly permuted Cas9 protein.

The heterologous polypeptide (e.g., deaminase) can be inserted in the napDNAbp (e.g., Cas9 or Casl2 (e.g., Casl2b/C2cl)) at a suitable location, for example, such that the napDNAbp retains its ability to bind the target polynucleotide and a guide nucleic acid. A deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase (dual deaminase)) can be inserted into a napDNAbp without compromising function of the deaminase (e.g., base editing activity) or the napDNAbp (e.g., ability to bind to target nucleic acid and guide nucleic acid).

In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted in regions of the Cas9 polypeptide comprising higher than average B-factors (e.g., higher B factors compared to the total protein or the protein domain comprising the disordered region). Cas9 polypeptide positions comprising a higher than average B-factor can include, for example, residues 768, 792, 1052, 1015, 1022, 1026, 1029, 1067, 1040, 1054, 1068, 1246, 1247, and 1248 as numbered in SEQ ID NO: 197. Cas9 polypeptide regions comprising a higher than average B-factor can include, for example, residues 792-872, 792-906, and 2-791 as numbered in SEQ ID NO: 197. ATTORNEY DOCKET NO. 180802-47402/PCT

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In some embodiments, a heterologous polypeptide (e.g., deaminase) is inserted in a flexible loop of a Cas9 polypeptide. The flexible loop portions can be selected from the group consisting of 530-537, 569-570, 686-691, 943-947, 1002-1025, 1052-1077, 1232-1247, or 1298-1300 as numbered in SEQ ID NO: 197, or a corresponding amino acid residue in another Cas9 polypeptide. The flexible loop portions can be selected from the group consisting of: 1-529, 538-568, 580-685, 692-942, 948-1001, 1026-1051, 1078-1231, or 1248-1297 as numbered in SEQ ID NO: 197, or a corresponding amino acid residue in another Cas9 polypeptide.

A heterologous polypeptide (e.g., adenine deaminase) can be inserted into a Cas9 polypeptide region corresponding to amino acid residues: 1017-1069, 1242-1247, 1052-1056, 1060-1077, 1002 - 1003, 943-947, 530-537, 568-579, 686-691, 1242-1247, 1298 - 1300, 1066-1077, 1052-1056, or 1060-1077 as numbered in SEQ ID NO: 197, or a corresponding amino acid residue in another Cas9 polypeptide.

A heterologous polypeptide (e.g., adenine deaminase) can be inserted in place of a deleted region of a Cas9 polypeptide. The deleted region can correspond to an N-terminal or C-terminal portion of the Cas9 polypeptide. Exemplary internal fusions base editors are provided in Table 4 below:

Table 4: Insertion loci in Cas9 proteins

BE ID Modification Other ID IBE001 Cas9 Tad A ins 1015 ISLAY01 IBE002 Cas9 Tad A ins 1022 ISLAY02 IBE003 Cas9 Tad A ins 1029 ISLAY03 IBE004 Cas9 Tad A ins 1040 ISLAY04 IBE005 Cas9 TadA ins 1068 ISLAY05 IBE006 Cas9 TadA ins 1247 ISLAY06 IBE007 Cas9 TadA ins 1054 ISLAY07 IBE008 Cas9 TadA ins 1026 ISLAY08 IBE009 Cas9 TadA ins 768 ISLAY09 IBE020 delta HNH TadA 792 ISLAY20 IBE021 N-term fusion single TadA helix truncated 165-end ISLAY21 IBE029 TadA-Circular Permutantl 16 insl067 ISLAY29 IBE031 TadA- Circular Permutant 136 insl248 ISLAY31 IBE032 TadA- Circular Permutant 136ins 1052 ISLAY32 IBE035 delta 792-872 TadA ins ISLAY35 IBE036 delta 792-906 TadA ins ISLAY36 IBE043 TadA-Circular Permutant 65 insl246 ISLAY43

IBE044 TadA ins C-term truncate2791 ISLAY44

A heterologous polypeptide (e.g., deaminase) can be inserted within a structural or functional domain of a Cas9 polypeptide. A heterologous polypeptide (e.g., deaminase) can be ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

inserted between two structural or functional domains of a Cas9 polypeptide. A heterologous polypeptide (e.g., deaminase) can be inserted in place of a structural or functional domain of a Cas9 polypeptide, for example, after deleting the domain from the Cas9 polypeptide. The structural or functional domains of a Cas9 polypeptide can include, for example, RuvC I, RuvC II, RuvC III, Reel, Rec2, PI, or HNH.

A fusion protein can comprise a linker between the deaminase and the napDNAbp polypeptide. The linker can be a peptide or a non-peptide linker. For example, the linker can be an XTEN, (GGGS)n (SEQ ID NO: 246), SGGSSGGS (SEQ ID NO: 330), (GGGGS)n (SEQ ID NO: 247), (G)n, (EAAAK)n (SEQ ID NO: 248), (GGS)n, SGSETPGTSESATPES (SEQ ID NO: 249). In some embodiments, the fusion protein comprises a linker between the N-terminal Cas9 fragment and the deaminase. In some embodiments, the fusion protein comprises a linker between the C-terminal Cas9 fragment and the deaminase. In some embodiments, the N-terminal and C-terminal fragments of napDNAbp are connected to the deaminase with a linker. In some embodiments, the N-terminal and C-terminal fragments are joined to the deaminase domain without a linker. In some embodiments, the fusion protein comprises a linker between the N-terminal Cas9 fragment and the deaminase but does not comprise a linker between the C-terminal Cas9 fragment and the deaminase. In some embodiments, the fusion protein comprises a linker between the C-terminal Cas9 fragment and the deaminase but does not comprise a linker between the N-terminal Cas9 fragment and the deaminase.

In some embodiments, the napDNAbp in the fusion protein or complex is a Casl2 polypeptide, e.g., Casl2b/C2cl, or a functional fragment thereof capable of associating with a nucleic acid (e.g., a gRNA) that guides the Casl2 to a specific nucleic acid sequence. The Casl2 polypeptide can be a variant Casl2 polypeptide. In other embodiments, the N- or C-terminal fragments of the Casl2 polypeptide comprise a nucleic acid programmable DNA binding domain or a RuvC domain. In other embodiments, the fusion protein contains a linker between the Cast 2 polypeptide and the catalytic domain. In other embodiments, the amino acid sequence of the linker is GGSGGS (SEQ ID NO: 250) or GSSGSETPGTSESATPESSG (SEQ ID NO: 251). In other embodiments, the linker is a rigid linker. In other embodiments of the above aspects, the linker is encoded by GGAGGCTCTGGAGGAAGC (SEQ ID NO: 252) or GGCTCTTCTGGATCTGAAACACCTGGCACAAGCGAGAGCGCCACCCCTGAGAGCTCTGGC (SEQ ID NO: 253).

In other embodiments, the fusion protein or complex contains a nuclear localization signal (e.g., a bipartite nuclear localization signal). In other embodiments, the amino acid sequence of the nuclear localization signal is MAPKKKRKVGIHGVPAA (SEQ ID NO: 261). In ATTORNEY DOCKET NO. 180802-47402/PCT

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other embodiments of the above aspects, the nuclear localization signal is encoded by the following sequence:

ATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCC (SEQ ID NO: 262). In other embodiments, the Casl2b polypeptide contains a mutation that silences the catalytic activity of a RuvC domain. In other embodiments, the Cast 2b polypeptide contains D574A, D829A and/or D952A mutations.

In some embodiments, the fusion protein or complex comprises a napDNAbp domain (e.g., Casl2-derived domain) with an internally fused nucleobase editing domain (e.g., all or a portion (e.g., a functional portion) of a deaminase domain, e.g, an adenosine deaminase domain). In some embodiments, the napDNAbp is a Casl2b. In some embodiments, the base editor comprises a BhCasl2b domain with an internally fused TadA*8 domain inserted at the loci provided in Table 5 below.

Table 5: Insertion loci in Casllb proteins

BhCasl2b Insertion site Inserted between aa

position 1 153 PS

position 2 255 KE

position 3 306 DE

position 4 980 DG

position 5 1019 KL

position 6 534 FP

position 7 604 KG

position 8 344 HF

BvCasl2b Insertion site Inserted between aa

position 1 147 PD

position 2 248 GG

position 3 299 PE

position 4 991 GE

position 5 1031 KM

AaCasl2b Insertion site Inserted between aa

position 1 157 PG

position 2 258 VG

position 3 310 DP

position 4 1008 GE

position 5 1044 GK

In some embodiments, the base editing system described herein is an ABE with TadA inserted into a Cas9. Polypeptide sequences of relevant ABEs with TadA inserted into a Cas9 are provided in the attached Sequence Listing as SEQ ID NOs: 263-308. ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

Exemplary, yet nonlimiting, fusion proteins are described in International PCT Application Nos. PCT/US2020/016285 and U. S. Provisional Application Nos. 62/852,228 and 62/852,224, the contents of which are incorporated by reference herein in their entireties.

Cto T Editing

In some embodiments, a base editor disclosed herein comprises a fusion protein or complex comprising cytidine deaminase capable of deaminating a target cytidine (C) base of a polynucleotide to produce uridine (U), which has the base pairing properties of thymine. In some embodiments, for example where the polynucleotide is double-stranded (e.g., DNA), the uridine base can then be substituted with a thymidine base (e.g., by cellular repair machinery) to give rise to a C: G to a T: A transition. In other embodiments, deamination of a C to U in a nucleic acid by a base editor cannot be accompanied by substitution of the U to a T.

The deamination of a target C in a polynucleotide to give rise to a U is a non-limiting example of a type of base editing that can be executed by a base editor described herein. In another example, a base editor comprising a cytidine deaminase domain can mediate conversion of a cytosine (C) base to a guanine (G) base. For example, a U of a polynucleotide produced by deamination of a cytidine by a cytidine deaminase domain of a base editor can be excised from the polynucleotide by a base excision repair mechanism e.g., by a uracil DNA glycosylase (UDG) domain), producing an abasic site. The nucleobase opposite the abasic site can then be substituted (e.g., by base repair machinery) with another base, such as a C, by for example a translesion polymerase. Although it is typical for a nucleobase opposite an abasic site to be replaced with a C, other substitutions (e.g., A, G or T) can also occur.

Accordingly, in some embodiments a base editor described herein comprises a deamination domain (e.g., cytidine deaminase domain) capable of deaminating a target C to a U in a polynucleotide. Further, as described below, the base editor can comprise additional domains which facilitate conversion of the U resulting from deamination to, in some embodiments, a T or a G. For example, a base editor comprising a cytidine deaminase domain can further comprise a uracil glycosylase inhibitor (UGI) domain to mediate substitution of a U by a T, completing a C-to-T base editing event. In another example, the base editor may comprise a uracil stabilizing protein as described herein. In another example, a base editor can incorporate a translesion polymerase to improve the efficiency of C-to-G base editing, since a translesion polymerase can facilitate incorporation of a C opposite an abasic site (i.e., resulting in incorporation of a G at the abasic site, completing the C-to-G base editing event). ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

A base editor comprising a cytidine deaminase as a domain can deaminate a target C in any polynucleotide, including DNA, RNA and DNA-RNA hybrids.

In some embodiments, a cytidine deaminase of a base editor comprises all or a portion (e.g., a functional portion) of an apolipoprotein B mRNA editing complex (APOBEC) family deaminase. APOBEC is a family of evolutionarily conserved cytidine deaminases. Members of this family are C-to-U editing enzymes. The N-terminal domain of APOBEC like proteins is the catalytic domain, while the C-terminal domain is a pseudocatalytic domain. More specifically, the catalytic domain is a zinc dependent cytidine deaminase domain and is important for cytidine deamination. APOBEC family members include APOBEC 1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D (“APOBEC3E” now refers to this), APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4, and Activation-induced (cytidine) deaminase.

Other exemplary deaminases that can be fused to Cas9 according to aspects of this disclosure are provided below. In embodiments, the deaminases are activation-induced deaminases (AID). It should be understood that, in some embodiments, the active domain of the respective sequence can be used, e.g., the domain without a localizing signal (nuclear localization sequence, without nuclear export signal, cytoplasmic localizing signal).

Some aspects of the present disclosure are based on the recognition that modulating the deaminase domain catalytic activity of any of the fusion proteins or complexes described herein, for example by making point mutations in the deaminase domain, affect the processivity of the fusion proteins (e.g., base editors) or complexes. For example, mutations that reduce, but do not eliminate, the catalytic activity of a deaminase domain within a base editing fusion protein or complexes can make it less likely that the deaminase domain will catalyze the deamination of a residue adjacent to a target residue, thereby narrowing the deamination window. The ability to narrow the deamination window can prevent unwanted deamination of residues adjacent to specific target residues, which can reduce or prevent off-target effects.

In some embodiments, an APOBEC deaminase incorporated into a base editor can comprise one or more mutations selected from the group consisting of R33A, K34A, E63A, H102P, D104N, H121R, H122R, H122L, D124N; R126A, R126E, R118A, W90A, W90Y, and R132E of rAPOBECl; D316R, D317R, R320A, R320E, R313A, W285A, W285Y, and R326E of hAPOBEC3G; and any alternative mutation at the corresponding position, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, an APOBEC deaminase incorporated into a base editor can comprise one or more combinations of mutations selected from K34A, H122L, and D124N (AALN); H102P and D104N (evoFERNY derived from FERNY); W90Y and R126E (YE1); W90Y and R132E (YE2); R126E and R132E (EE); ATTORNEY DOCKET NO. 180802-47402/PCT

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W90Y, R126E, and R132E (YEE), or rAPOBECl; and any alternative mutation at the corresponding positions, or one or more corresponding mutations in another APOBEC deaminase.

A number of modified cytidine deaminases are commercially available, including, but not limited to, SaBE3, SaKKH-BE3, VQR-BE3, EQR-BE3, VRER-BE3, YE1-BE3, EE-BE3, YE2-BE3, and YEE-BE3, which are available from Addgene (plasmids 85169, 85170, 85171, 85172, 85173, 85174, 85175, 85176, 85177). In some embodiments, a deaminase incorporated into a base editor comprises all or a portion (e.g., a functional portion) of an APOBEC1 deaminase.

In some embodiments, the fusion proteins or complexes of the disclosure comprise one or more cytidine deaminase domains. In some embodiments, the cytidine deaminases provided herein are capable of deaminating cytosine or 5-methylcytosine to uracil or thymine. In some embodiments, the cytidine deaminases provided herein are capable of deaminating cytosine in DNA. The cytidine deaminase may be derived from any suitable organism. In some embodiments, the cytidine deaminase is a naturally-occurring cytidine deaminase that includes one or more mutations corresponding to any of the mutations provided herein. One of skill in the art will be able to identify the corresponding residue in any homologous protein, e.g., by sequence alignment and determination of homologous residues. Accordingly, one of skill in the art would be able to generate mutations in any naturally-occurring cytidine deaminase that corresponds to any of the mutations described herein. In some embodiments, the cytidine deaminase is from a prokaryote. In some embodiments, the cytidine deaminase is from a bacterium. In some embodiments, the cytidine deaminase is from a mammal (e.g., human).

In some embodiments, the cytidine deaminase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the cytidine deaminase amino acid sequences set forth herein. It should be appreciated that cytidine deaminases provided herein may include one or more mutations (e.g., any of the mutations provided herein). Some embodiments provide a polynucleotide molecule encoding the cytidine deaminase nucleobase editor polypeptide of any previous aspect or as delineated herein. In some embodiments, the polynucleotide is codon optimized.

In embodiments, a fusion protein of the disclosure comprises two or more nucleic acid editing domains.

Details of C to T nucleobase editing proteins are described in International PCT Application No. PCT/US2016/058344 (WO2017/070632) and Komor, A. C., eta!., “Programmable editing of a target base in genomic DNA without double-stranded DNA ATTORNEY DOCKET NO. 180802-47402/PCT

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cleavage” Nature 533, 420-424 (2016), the entire contents of which are hereby incorporated by reference. Further non-limiting examples of C to T nucleobase editing proteins are described in PCT Applications No. PCT/US2020/062428 and PCT/US2019/033848, the entire contents of which are hereby incorporated by reference.

Cytidine Adenosine Base Editors (CABEs)

In some embodiments, a base editor described herein comprises an adenosine deaminase variant that has increased cytidine deaminase activity. Such base editors may be referred to as “cytidine adenosine base editors (CABEs)” or “cytosine base editors derived from TadA* (CBE-Ts),” and their corresponding deaminase domains may be referred to as “TadA* acting on DNA cytosine (TADC)” domains or TadA-derived cytidine deaminases (TadA-CD). Base editors containing adenosine deaminase variants having both cytidine deaminase and adenosine deaminase activity (i.e., TadA-Dual deaminases) may be referred to as TadA-based dual editors (TadDE). In some instances, an adenosine deaminase variant has both adenine and cytosine deaminase activity (i.e., is a dual deaminase). In some embodiments, the adenosine deaminase variants deaminate adenine and cytosine in DNA. In some embodiments, the adenosine deaminase variants deaminate adenine and cytosine in single-stranded DNA. In some embodiments, the adenosine deaminase variants deaminate adenine and cytosine in RNA. In some embodiments, the adenosine deaminase variant predominantly deaminates cytosine in DNA and/or RNA (e.g., greater than 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of all deaminations catalyzed by the adenosine deaminase variant, or the number of cytosine deaminations catalyzed by the variant is about or at least about 2-fold, 3 -fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 25-fold, 50-fold, 75-fold, 100-fold, 500-fold, or 1,000-fold greater than the number adenine deaminations catalyzed by the variant). In some embodiments, the adenosine deaminase variant has approximately equal cytosine and adenosine deaminase activity (e.g., the two activities are within about 10% or 20% of each other). In some embodiments, the adenosine deaminase variant has predominantly cytosine deaminase activity, and little, if any, adenosine deaminase activity. In some embodiments, the adenosine deaminase variant has cytosine deaminase activity, and no significant or no detectable adenosine deaminase activity. In some embodiments, the target polynucleotide is present in a cell in vitro or in vivo. In some embodiments, the cell is a bacteria, yeast, fungi, insect, plant, or mammalian cell.

Examples of adenosine deaminase variants having increased cytidine deaminase activity include those described in International Patent Application Publications No. WO 2024/040083 and WO ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

2022/204574, the disclosures of which are hereby incorporated by reference in their entireties for all purposes.

In some embodiments, the CABE comprises a bacterial TadA deaminase variant (e.g., ecTadA). In some embodiments, the CABE comprises a truncated TadA deaminase variant. In some embodiments, the CABE comprises a fragment of a TadA deaminase variant. In some embodiments, the CABE comprises a TadA*8.20 variant.

In some embodiments, an adenosine deaminase variant of the disclosure is a TadA adenosine deaminase comprising one or more alterations that increase cytosine deaminase activity (e.g, at least about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold or more increase) while maintaining adenosine deaminase activity (e.g., at least about 30%, 40%, 50% or more of the activity of a reference adenosine deaminase (e.g., TadA*8.20 or TadA*8.19)). In some instances, the adenosine deaminase variant comprises one or more alterations that increase cytosine deaminase activity (e.g., at least about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold or more increase) relative to the activity of a reference adenosine deaminase and comprise undetectable adenosine deaminase activity or adenosine deaminase activity that is less than 30%, 20%, 10%, or 5% of that of a reference adenosine deaminase. In some embodiments, the reference adenosine deaminase is Tad A* 8.20 or TadA* 8.19.

In some embodiments, the adenosine deaminase variant is an adenosine deaminase comprising two or more alterations at an amino acid position selected from the group consisting of 2, 4, 6, 8, 13, 17, 23, 27, 29, 30, 47, 48, 49, 67, 76, 77, 82, 84, 96, 100, 107, 112, 114, 115, 118, 119, 122, 127, 142, 143, 147, 149, 158, 159, 162 165, 166, and 167, of an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or greater identity to SEQ ID NO: 1, or a corresponding alteration in another deaminase.

In some embodiments, the adenosine deaminase variant is an adenosine deaminase comprising one or more alterations selected from the group consisting of S2H, V4K, V4S, V4T, V4Y, F6G, F6H, F6Y, H8Q, R13G, T17A, T17W, R23Q, E27C, E27G, E27H, E27K, E27Q, E27S, E27G, P29A, P29G, P29K, V30F, V30I, R47G, R47S, A48G, I49K, I49M, I49N, I49Q, I49T, G67W, I76H, I76R, I76W, Y76H, Y76R, Y76W, F84A, F84M, H96N, G100A, G100K, T111H, G112H, Al 14C, G115M, Ml 18L, H122G, H122R, H122T, N127I, N127K, N127P, A142E, R147H, A158V, Q159S, A162C, A162N, A162Q, and S165P of an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or greater identity to SEQ ID NO: 1, or a corresponding alteration in another deaminase. ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

In some embodiments, the adenosine deaminase variant is an adenosine deaminase comprising an amino acid alteration or combination of amino acid alterations selected from those listed in any of Tables 6A-6F.

The residue identity of exemplary adenosine deaminase variants that are capable of deaminating adenine and/or cytidine in a target polynucleotide (e.g., DNA) is provided in Tables 6A-6F below. Further examples of adenosine deaminase variants include the following variants of 1.17 (see Table 6 A): 1.17+E27H; 1.17+E27K; 1.17+E27S; 1.17+E27S+I49K; 1.17+E27G; 1.17+I49N; 1.17+E27G+I49N; and 1.17+E27Q. In some embodiments, any of the amino acid alterations provided herein are substituted with a conservative amino acid. Additional mutations known in the art can be further added to any of the adenosine deaminase variants provided herein.

In some embodiments, the base editor systems comprising a CABE provided herein have at least about a 30%, 40%, 50%, 60%, 70% or more C to T editing activity in a target polynucleotide (e.g., DNA). In some embodiments, a base editor system comprising a CABE as provided herein has an increased C to T base editing activity (e.g., increased at least about 30-fold, 40-fold, 50-fold, 60-fold, 70-fold or more) relative to a reference base editor system comprising a reference adenosine deaminase (e.g., TadA*8.20 or TadA*8.19).

Table 6A. Adenosine Deaminase Variants. Mutations are indicated with reference to TadA*8.20. “S” indicates “Surface,” and “NAS” indicates “Near Active Site.”

location in N/A s s S hi NAS NAS NAS NAS s structure hl hl

Amino Acid

No. (* START 2 8 13 17 27 47 48 49 67 76 77 Met is AA#1)

TadA*8.20 S H R T E R A I G Y D TadA*8.19 I

1.1 H I

1.2 H K I

1.3 S K I

1.4 S K I

1.5 K

1.6 K

1.7 H I

1.8 S K W

1.9 T W

1.10 C I

1.11 G Q

1.12 A H M I

1.13 Q I

1.14 H K I ATTORNEY DOCKET NO. 180802-47402/PCT

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location in N/A s S S hi NAS NAS NAS NAS S structure hl hl

Amino Acid

No. (* START 2 8 13 17 27 47 48 49 67 76 77 Met is AA#1)

TadA*8.20 S H R T E R A I G Y D TadA*8.19 I 1.15 S

1.16 Q Q I 1.17 A G

1.18 G

1.19 G N

1.20 G G Table 6A (continued). Adenosine Deaminase Variants. Mutations are indicated with reference to TadA*8.20. “I” indicates “Internal,” “S” indicates “Surface,” and “NAS” indicates “Near Active Site.”

location in I NAS NAS s S s S s structure

Amino Acid

No. (* START 82 84 96 107 112 115 118 119 127 142 162 165 Met is AA#1)

TadA*8.20 S F H R G G M D N A A S TadA*8.19

1.1 M

1.2

1.3

1.4 N 1.5

1.6 N

1.7

1.8

1.9 N

1.10 N

1.11 K

1.12 L

1.13 M

1.14 H

1.15 C

1.16

1.17 T E

1.18

1.19

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Table 6B. Adenosine deaminase variants. Mutations are indicated with reference to TadA*8.20.

Position 27 29 30 49 82 84 107 112 115 142 No.

TadA*8.20 E p V I s F R G G A Alterations G/S/H G/A/K I/L/F K T L/A C H M E Evaluated

Sl.l S K T

S1.2 S K T C

S1.3 s K T H

S1.4 s K T M

S1.5 s K T E S1.6 s K T c H

S1.7 s K T c M

S1.8 s K T c E S1.9 s K T H E Sl.lO s K T M E Sl.ll s K T c H M E S1.12 s I K T

SI.13 s I K T c

SI.14 s I K T H

SI.15 s I K T M

SI.16 s I K T E SI.17 s I K T c H

SI.18 s I K T c M

SI.19 s I K T c E S1.20 s I K T H E S1.21 s I K T M E S1.22 s I K T c H M E S1.23 s L K T

S1.24 s L K T c

S1.25 s L K T H

S1.26 s L K T M

S1.27 s L K T E S1.28 s L K T c H

S1.29 s L K T c M

S1.30 s L K T c E S1.31 s L K T H E S1.32 s L K T M E S1.33 s L K T c H M E S1.34 s F K T A

S1.35 s F K T A c

S1.36 s F K T A H

SI.37 s F K T A M

S1.38 s F K T A E

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Position 27 29 30 49 82 84 107 112 115 142 No.

S1.40 S F K T A C M S1.41 S F K T A c E S1.42 s F K T A H E S1.43 s F K T A M E S1.44 s F K T A c H M E S1.45 s K T L

S1.46 s K T L c

S1.47 s K T L H

S1.48 s K T L M S1.49 s K T L E S1.50 s K T L c H

S1.51 s K T L c M S1.52 s K T L c E S1.53 s K T L H E S1.54 s K T L M E S1.55 s K T L c H M E S1.56 s I K T L

S1.57 s I K T L c

S1.58 s I K T L H

S1.59 s I K T L M S1.60 s I K T L E S1.61 s I K T L c H

S1.62 s I K T L c M S1.63 s I K T L c E S1.64 s I K T L H E S1.65 s I K T L M E S1.66 s I K T L c H M E S1.67 s G K T

S1.68 s G K T c

S1.69 s G K T H

SI. 70 s G K T M S1.71 s G K T E S1.72 s G K T c H

S1.73 s G K T c M SI.74 s G K T c E S1.75 s G K T H E S1.76 s G K T M E S1.77 s G K T c H M E S1.78 G K T

S1.79 G K T c

S1.80 G K T H

S1.81 G K T M

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Position 27 29 30 49 82 84 107 112 115 142 No.

S1.83 G K T C H

S1.84 G K T c M S1.85 G K T c E S1.86 G K T H E S1.87 G K T M E S1.88 G K T c H M E S1.89 K K T

S1.90 K K T c

S1.91 k K T H

S1.92 K K T M S1.93 k K T E S1.94 K K T c H

S1.95 k K T c M S1.96 K K T c E S1.97 K K T H E S1.98 k K T M E S1.99 K K T c H M E SI. 100 k I K T

SI. 101 K I K T c

SI. 102 K I K T H

S1.103 k i K T M SI. 104 K I K T E SI. 105 k I K T c H

SI. 106 K I K T c M SI. 107 k I K T c E SI. 108 K I K T H E SI. 109 K I K T M E si.iio k i K T c H M E SI. I l l K K T L

SI. 112 k K T L c

S1.113 K K T L H

SI. 114 K K T L M si.l 15 k K T L E SI. 116 K K T L c H

SI. 117 k K T L c M SI. 118 K K T L c E SI. 119 k K T L H E si.iio K K T L M E SI. 121 K K T L c H M E si. 122 k I K T L

SI. 123 K I K T L c

SI.124 k I K T L H

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Position 27 29 30 49 82 84 107 112 115 142 No.

SI. 126 k I K T L E SI. 127 K I K T L C H

SI. 128 K I K T L c M si.129 k i K T L c E SI. 130 K i K T L H E SI. 131 k i K T L M E SI. 132 K i K T L c H M E SI. 133 G K T

SI. 134 G K T c

SI. 135 G K T H

SI. 136 G K T M SI. 137 G K T E SI. 138 G K T c H

S1.139 G K T c M SI. 140 G K T c E SI. 141 G K T H E SI. 142 G K T M E SI. 143 G K T c H M E SI. 144 H K T

SI. 145 H K T c

S1.146 H K T H

SI. 147 H K T M

S H K T

SiI.114489 E H K T c H

SI. 150 H K T c M S1.151 H K T c E SI. 152 H K T H E S1.153 H K T M E SI. 154 H K T c H M E SI. 155 S T

SI. 156 S T c

SI. 157 S T H

SI. 158 S T M SI. 159 S T E SI. 160 S T c H

SI. 161 S T c M SI. 162 S T c E S1.163 S T H E SI. 164 S T M E SI. 165 S T c H M E SI. 166 A T

SI. 167 A T c

SI.168 A T H ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

Position 27 29 30 49 82 84 107 112 115 142

No.

SI.169 A T M

SI.170 A T E

SI.171 A T C H

81.172 A T c M

SI.173 A T c E

SI.174 A T H E

SI.175 A T M E

SI.176 A T c H M E

SI.177 S I T

SI.178 S I T c

SI.179 s I T H

SI.180 s I T M

SI.181 s I T E

S1.182 s I T c H

SI.183 s I T c M

SI.184 s I T c E

SI.185 s I T H E

SI.186 s I T M E

SI.187 s I T c H M E

SI.188 A I T L

S1.189 A i T L c

SI.190 A i T L H

SI.191 A i T L M

SI.192 A i T L E

SI.193 A i T L c H

SI.194 A i T L c M

SI.195 A i T L c E

S1.196 A i T L H E

SI.197 A i T L M E

SI.198 A i T L c H M E

SI.199 s A L K T L c H M E

Table 6C. Adenosine deaminase variants. Mutations are indicated with reference to variant 1.2 (Table 6 A).

Residue identity (START Met is amino

acid #1)

Variant Name Alternative Variant 4 6 17 23 76 77 1 1 1

Names 0 1 1

0 1 4 Reference 1.2 (see Table 6 A) V F T R I D G T A TadAC2.1 pDKL-135; 2.1 K C

TadAC2.2 pDKL-136; 2.2 K G ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

Residue identity (START Met is amino acid #1)

Variant Name Alternative Variant 4 6 17 23 76 77 1 1 1

Names 0 1 1

0 1 4 Reference 1.2 (see Table 6 A) V F T R I D G T A TadAC2.3 pDKL-137; 2.3 Y A TadAC2.4 pDKL-138; 2.4 T R

TadAC2.5 pDKL-139; 2.5 Y W

TadAC2.6 pDKL-140; 2.6 Y

TadAC2.7 pDKL-141; 2.7 Y C TadAC2.8 pDKL-142; 2.8 Y

TadAC2.9 pDKL-143; 2.9 K W

TadAC2.10 pDKL-144; 2.10 G R K TadAC2.11 pDKL-145; 2.11 H

TadAC2.12 pDKL-146; 2.12 C TadAC2.13 pDKL-147; 2.13 Y H

TadAC2.14 pDKL-148; 2.14

TadAC2.15 pDKL-149; 2.15 Q R

TadAC2.16 pDKL-150; 2.16 H

TadAC2.17 pDKL-151; 2.17 Y H TadAC2.18 pDKL-152; 2.18 W

TadAC2.19 pDKL-153; 2.19 H TadAC2.20 pDKL-154; 2.20

TadAC2.21 pDKL-155; 2.21 Y R

TadAC2.22 pDKL-156; 2.22 W H

TadAC2.23 pDKL-157; 2.23 S Y

TadAC2.24 pDKL-158; 2.24

Table 6C. (CONTINUED)

Residue identity (START Met is amino acid #1)

Variant Name Alternative Variant 1 1 1 1 1 1 1 1 16

Names 1 2 2 4 4 5 5 6 6

9 2 7 3 7 8 9 2 Reference 1.2 (see Table 6 A) D H N A R A Q A T TadAC2.1 pDKL-135; 2.1

TadAC2.2 pDKL-136; 2.2

TadAC2.3 pDKL-137; 2.3 R

TadAC2.4 pDKL-138; 2.4 G

TadAC2.5 pDKL-139; 2.5

TadAC2.6 pDKL-140; 2.6 N

TadAC2.7 pDKL-141; 2.7

TadAC2.8 pDKL-142; 2.8 ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

Residue identity (START Met is

amino acid #1)

Variant Name Alternative Variant 1 1 1 1 1 1 1 1 16

Names 1 2 2 4 4 5 5 6 6

9 2 7 3 7 8 9 2

Reference 1.2 (see Table 6 A) D H N A R A Q A T TadAC2.9 pDKL-143; 2.9 T

TadAC2.10 pDKL-144; 2.10

TadAC2.11 pDKL-145; 2.11 N

TadAC2.12 pDKL-146; 2.12

TadAC2.13 pDKL-147; 2.13 R I TadAC2.14 pDKL-148; 2.14 P

TadAC2.15 pDKL-149; 2.15

TadAC2.16 pDKL-150; 2.16 R V

TadAC2.17 pDKL-151; 2.17

TadAC2.18 pDKL-152; 2.18

TadAC2.19 pDKL-153; 2.19 G C

TadAC2.20 pDKL-154; 2.20 E

TadAC2.21 pDKL-155; 2.21

TadAC2.22 pDKL-156; 2.22 G V

TadAC2.23 pDKL-157; 2.23 E s

TadAC2.24 pDKL-158; 2.24 I

Q

Table 6D. Adenosine deaminase variants. Mutations are indicated with reference to TadA*8.20.

AA Positions 6 27 49 76 77 82 107 112 114 115 119 122 127 142 143

TadA*8.20 F E I Y D S R G A G D H N A A

SI.154 F H K Y D T C H M E Alterations

from Table

6C Y W G C N G P E S2.1 Y H K W T C H M E S2.2 Y H K G T C H M E S2.3 Y H K T C H C M E S2.4 Y H K T C H M N E S2.5 Y H K T C H M G E S2.6 Y H K T C H M P E S2.7 Y H K T C H M E E S2.8 Y H K T C H M A E S2.9 Y H K W G T C H M E

S2.10 Y H K W T C H C M E ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

AA Positions 6 27 49 76 77 82 107 112 114 115 119 122 127 142 143

TadA*8.20 F E I Y D S R G A G D H N A A S2.11 Y H K W T C H M N E S2.12 Y H K W T C H M G E S2.13 Y H K W T C H M P E S2.14 Y H K W T C H M E E S2.15 Y H K W T C H M A E S2.16 Y H K G T C H C M E S2.17 Y H K G T C H M N E S2.18 Y H K G T C H M G E S2.19 Y H K G T C H M P E S2.20 Y H K G T C H M E E S2.21 Y H K G T C H M A E S2.22 Y H K T C H C M N E S2.23 Y H K T C H C M G E S2.24 Y H K T C H C M P E S2.25 Y H K T C H M N G E S2.26 Y H K T C H M N P E S2.27 Y H K T C H M G P E S2.28 Y H K W G T C H C M E S2.29 Y H K W G T c H M N E S2.30 Y H K W G T c H M G E S2.31 Y H K W G T c H M P E S2.32 Y H K W G T c H M E E S2.33 Y H K W G T c H M A E S2.34 Y H K W T c H C M N E S2.35 Y H K W T c H C M G E S2.36 Y H K W T c H C M P E S2.37 Y H K W T c H C M E E S2.38 Y H K W T c H C M A E S2.39 Y H K W T c H M N G E S2.40 Y H K W T c H M N P E S2.41 Y H K W T c H M G P E S2.42 Y H K W T c H C M N G E S2.43 Y H K W T c H C M N P E S2.44 Y H K W T c H c M G P E S2.45 Y H K W G T c H c M N E S2.46 Y H K W G T c H c M G E

S2.47 Y H K W G T c H c M P E ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

AA Positions 6 27 49 76 77 82 107 112 114 115 119 122 127 142 143

TadA*8.20 F E I Y D S R G A G D H N A A S2.48 Y H K W G T C H C M E E S2.49 Y H K W G T C H C M A E S2.50 Y H K W G T C H C M N G E S2.51 Y H K W G T C H C M N P E S2.52 Y H K W G T C H C M G P E S2.53 Y H K W T C H C M N G P E E S2.54 Y H K W T C H C M N G P A E S2.55 Y H K W G T C H C M N G P E E

S2.56 Y H K W G T C H C M N G P A E

Table 6E. Hybrid constructs. Mutations are indicated with reference to TadA*7.10.

TadA amino acid subsitutions

76 82 109 111 119 122 123 147 149 154 166 167 TadA*7.10 I V A T D H Y Y F Q T D TadA*8e s R N N D Y I N TadA*8.20 Y S H R R

Tad A* 8.17 S R

pNMG-B878 Y S H D R

pNMG-B879 Y S H R Y R

PNMG-B880 Y S H R R I pNMG-B881 Y S H R R N PNMG-B882 Y s H D Y R I N pNMG-B883 Y s R N H R R

PNMG-B884 Y s s R N N H R R

pNMG-B885 Y s s H R R

PNMG-B886 Y s R H R R

PNMG-B887 Y s N H R R

pNMG-B888 Y s N H R R

PNMG-B889 Y s s R H R R

PNMG-B890 Y s N N H R R

PNMG-B891 Y s s R N N H D Y R I N

Table 6F. Base editor variants. Mutations are indicated with reference to TadA*8.19/8.20.

AA positions: 17 27 48 49 76 82 84 118 142 147 149 166 167

T E A I Y/I S F M A Y F T D ABE8.19m/8.20m

1.1 + 8e(B879) H I M Y

1.2 + 8e(B879) H K I Y

1.12 + 8e(B879) A H M I L Y

1.17 + 8e(B879) A G T E Y

1.18 + 8e(B879) G Y ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

AA positions: 17 27 48 49 76 82 84 118 142 147 149 166 167

T E A I Y/I S F M A Y F T D ABE8.19m/8.20m

1.19 + 8e(B879) G N Y

1.1 + 8e(B882) H I M D Y I N 1.2 + 8e(B882) H K I D Y I N 1.12 + 8e(B882) A H M I L D Y I N 1.17 + 8e(B882) A G T E D Y I N 1.18 + 8e(B882) G D Y I N

G N D Y I N

1.19 + 8e(B882)

A TadA-derived cytidine deaminase (e.g., TadA-CD), according to certain embodiments, comprises an amino acid sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, and at least 99.5% identical to the amino acid sequence of SEQ ID NO: 489, wherein residue 27 of SEQ ID NO: 489 is any amino acid expect for E (glutamic acid). TadA-CDs with other sequence homologies are also possible. For example, in certain embodiments, the TadA-derived cytidine deaminase (e.g., TadA-CD) comprises an amino acid sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, and at least 99.5% identical to the amino acid sequence of SEQ ID NO: 489, wherein residue 28 of SEQ ID NO: 489 is any amino acid expect for V (valine). In another exemplary embodiment, the TadA-derived cytidine deaminase is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, and at least 99.5% identical to the amino acid sequence of SEQ ID NO: 489, wherein residue 96 of SEQ ID NO: 489 is any amino acid expect for H (histidine). In another exemplary embodiment, the TadA-derived cytidine deaminase is at least 80% identical, at least 85% identical, at. least. 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, and at least 99.5% identical to the amino acid sequence of SEQ ID NO: 489, wherein residue 26 of SEQ ID NO: 489 is any amino acid expect for R (arginine). In various embodiments, the TadA-derived cytidine deaminase comprises an alteration at one or more of positions 26, 27, 28, 48, 73, or 96 compared to SEQ ID NO: 489.

As will be appreciated by those of skill in the art, TadA-derived cytidine deaminases (e.g., TadA-CD) may comprise a plurality of mutations relative to the parent adenosine deaminase (e.g., TadA-8e). In some embodiments, the deaminase of the instant, application (e g., TadA-CD) comprises mutations at residues E27, V28, and H96. In some embodiments, the disclosed deaminase further comprises at least one mutation at. a residue selected from R26, ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

M61, Y73, 176, M151, Q154, and Al 58, in the amino acid sequence of SEQ ID NO: 489, or corresponding mutations in a homologous adenosine deaminase.

In some embodiments, the deaminase comprises at least one mutation selected from E27A, E27K, V28G, V28A, and H96N, and further comprises at least one mutation at a residue selected from R26G, M61I, Y73H, Y73S, Y73C, I76F, M151I, Q154R, Q154H, and A158S, in the amino acid sequence of SEQ ID NO: 489, or a corresponding mutation in a homologous adenosine deaminase. Other mutations are also possible. For example, in certain embodiments, the TadA-CD enzyme comprises mutations selected from E27A, V28G, and H96N, and further comprises at least one mutation selected from R26G, M61I, Y73H, Y73S, Y73C, I76F, M151I, Q 154R, Q154H, and A158S, in the amino acid sequence of SEQ ID NO: 489, or corresponding mutations in a homologous adenosine deaminase.

Other exemplary embodiments may include (1) deaminases comprising mutations E27K, V28G, and H96N, and further comprising at least one mutation selected from R26G, M61I, Y73H, Y73S, Y73C, I76F, M151I, Q154R, Q154H, and A158S, in the amino acid sequence of SEQ ID NO: 489 or corresponding mutations in a homologous adenosine deaminase, (2) deaminases comprising mutations E27A, V28A, and H96N, and further comprising at least one mutation selected from R26G, M61I, Y73H, Y73S, Y73C, I76F, M151I, Q154R, Q154H, and A158S, in the amino acid sequence of SEQ ID NO: 489, or corresponding mutations in a homologous adenosine deaminase; (3) deaminases comprising mutations E27K, V28A, and H96N, and further comprising at least one mutation selected from R26G, M61I, Y73H, Y73S, Y73C, I76F, Ml 511, Q 154R, Q 154H, and Al 58S, in the amino acid sequence of SEQ ID NO: 489, or corresponding mutations in a homologous adenosine deaminase.

In some embodiments, the TadA-derived cytidine deaminases (TadA-CD) comprise at least two mutations at residues selected from R26, M61, Y73, 176, M151, Q154, and A158 (relative to a reference adenosine deaminase). In other embodiments, the TadA-CD comprises at least two mutations at residues selected from R26G, M61I, Y73H, I76F, M151I, Q154H, Q154R, and A158S.

In some embodiments, the addition of a V106W mutation improves the selectivity by suppressing A deamination to a greater extent than C deamination.

In some embodiments, a TadA-based dual editor comprises an adenosine deaminase variant comprising one, two, three, four, or five mutations selected from R26G, V28A, A48R, Y73S, and H96N (e g... SEQ ID NO: 495).

As such, in some embodiments, provided herein are deaminases that comprise mutations at residues R26, V28, A48, and Y73 in the amino acid sequence of SEQ ID NO: 489, or ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

corresponding mutations in a homologous adenosine deaminase. Further provided herein are deaminases that comprise mutations at residues R26, E27, V28, A48, and Y73 (e.g., further comprise a mutation at E27) in the amino acid sequence of SEQ ID NO: 489. In particular embodiments, these deaminases comprise the mutations R26G, V28A, A48R, Y73S, and H96N. In some embodiments, these deaminases comprise the mutations R26G, V28G, A48R, and Y73C.

TadA-CD variants may comprise at least one mutation selected from R26G, E27A, V28G, I76F, H96N, and Ml 5 II (e.g, TadA-CDa, SEQ ID NO: 490), R26G, E27A, V28G, I76F, H96N, and A158S (e.g, TadA-CDb, SEQ ID NO: 491); R26G, E27A, V28G, I76F, H96N, QI 54R, and A158S (e.g, TadA-CDc, SEQ ID NO: 492); E27A, V28G, Y73H, H96N, QI54H, and A158S (e.g., TadA-CDd, SEQ ID NO: 493); R26G, V28A, A48R, Y73S, and H96N (e.g., TadA-CDe, SEQ ID NO: 494); V28A, A48R, and Y73S (e.g, TadA-CDf, SEQ ID NO: 495), and R26G, V28G, A48R, and Y73C (e.g, TadA-CDg, SEQ ID NO: 496).

In some preferred embodiments, the deaminase comprises the mutations R26G, E27A, V28G, I76F, H96N, and A158S (e.g., TadA-CDa, SEQ ID NO: 490), R26G, E27A, V28G, I76F, H96N, Q154R, and A158S (e.g., TadA-CDb, SEQ ID NO: 491), R26G, E27A, V28G, I76F, H96N, and M15II (e.g., TadA-CDc, SEQ ID NO: 492), E27K, V28A, M611, and H96N (e.g., TadA-CDd, SEQ ID NO: 493), E27A, V28G, Y73FI, H96N, Q154H, and A158S (e.g., TadA- CDe, SEQ ID NO: 494), R26G, V28A, A48R, Y73S, and H96N (e.g., TadA-CDf, SEQ ID NO: 495), and R26G, V28G, A48R, and Y73C (e.g., TadA-CDg, SEQ ID NO: 496).

In some embodiments, the TadA-CD variants described above and herein may also comprises a VI06W mutation.

In some embodiments, the TadA-CD variants comprise at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% to any of the amino acid sequences of SEQ ID NOs: 489-496.

In some embodiments, the evolved TadA-Dual deaminase comprises the mutations R26G, V28A, N46I, A48R, Y73P, and H96N (TadA-CD- 1, SEQ ID NO: 497) relative to the amino acid sequence of SEQ ID NO: 489. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations R26G, V28A, N46T, A48R, Y73P, and H96N (TadA-CD-2, SEQ ID NO: 498) relative to the amino acid sequence of SEQ ID NO: 489 In some embodiments, the evolved TadA- Dual deaminase comprises the mutations R26G, V28A, N46T, A48R, Y73S, and H96N (TadA-CD- 3, SEQ ID NO: 499) relative to the amino acid sequence of SEQ ID NO: 489. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations R26G, V28A, N46V, A48R, Y73S, and H96N (TadA-CD-4, SEQ ID NO: 500) ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

relative to the amino acid sequence of SEQ ID NO: 489. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations R26G, V28A, N46V, A48R, Y73P, and H96N (TadA-CD-5, SEQ ID NO: 501) relative to the amino acid sequence of SEQ ID NO: 489 In some embodiments, the evolved TadA-Dual deaminase comprises the mutations R26G, V28A, N46L, A48R, Y73P, and H96N (Tad A -CD-6, SEQ ID NO: 502) relative to the amino acid sequence of SEQ ID NO: 489. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations V2.8A, N46L, A48P, and Y73P (TadA-CD-7, SEQ ID NO: 503) relative to the amino acid sequence of SEQ ID NO: 489. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations V28A, N46C, A48P, and Y73P (TadA-CD-8, SEQ ID NO: 504) relative to the amino acid sequence of SEQ ID NO: 489. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations R26G, V28A, N46V, A48R, Y73P, and H96N (TadA-CD-9, SEQ ID NO: 505) relative to the amino acid sequence of SEQ ID NO: 489. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations R26G, V28A, N46V, A48R, Q71H, Y73P, and H96N (TadA-CD- 10, SEQ ID NO: 506) relative to the amino acid sequence of SEQ ID NO: 489. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations R26G, V2.8A, N46L, A48R, Y73P, and H96N (TadA-CD- 11, SEQ ID NO: 507) relative to the amino acid sequence of SEQ ID NO: 489. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations R26G, V28A, N46C, A48R, Y73P, and H96N (TadA-CD-12, SEQ ID NO: 508) relative to the amino acid sequence of SEQ ID NO: 489. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations R26G, V28A, N46C, A48R, Y73P, H96N, and Al 62V (TadA-CD- 13, SEQ ID NO: 509) relative to the amino acid sequence of SEQ ID NO: 489.

In some embodiments, the evolved TadA-Dual deaminase comprises the mutations R26G, V28A, N46I, A48R, Y73S, and H96N (TadA-CD-14, SEQ ID NO: 510) relative to the amino acid sequence of SEQ ID NO: 489. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations R26G, V28A, A48R, Q71S, Y73S, and H96N (TadA-CD- 15, SEQ ID NO: 511) relative to the amino acid sequence of SEQ ID NO: 489. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations R26G, V28A, N46L, A48R, and Y73P (TadA-CD- 16, SEQ ID NO: 512) relative to the amino acid sequence of SEQ ID NO: 489. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations R26G, V28A, N46L, A48R, Y73P, and H96N (TadA-CD-17, SEQ ID NO: 513) relative to the amino acid sequence of SEQ ID NO: 489. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations R26G, V28A, Y73P, and H96N (TadA-CD-18, SEQ ID NO: 514) relative to the amino acid sequence of SEQ ID NO: 489. In some embodiments, the evolved ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

TadA-Dual deaminase comprises the mutations R26G, V28A, N46V, A48R, Y73S, and H96N (TadA-CD-19, SEQ ID NO: 515) relative to the amino acid sequence of SEQ ID NO: 489. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations R26G, V28A, N46V, A48R, Y73P, and H96N (TadA-CD-20, SEQ ID NO: 516) relative to the amino acid sequence of SEQ ID NO: 489. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations R26G and N46L (TadA-CD-21, SEQ ID NO: 517) relative to the amino acid sequence of SEQ ID NO: 489. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations R26G, V28A, N46I, A48R, Y73P, and H96N (TadA-CD-22, SEQ ID NO: 518) relative to the amino acid sequence of SEQ ID NO: 489. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations R26G, V28A, N46V, A48R, Y73P, and H96N (TadA-CD-23, SEQ ID NO: 519) relative to the amino acid sequence of SEQ ID NO: 489. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations R26G, V28A, A48P, Y73H, T79P, and H96N (TadA-CD-24, SEQ ID NO: 520) relative to the amino acid sequence of SEQ ID NO: 489. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations R26G, N46I, and H96N (TadA-CD-25, SEQ ID NO: 521) relative to the amino acid sequence of SEQ ID NO: 489.

In some embodiments, the evolved TadA-Dual deaminase comprises the mutations R26G, V28A, N46V, A48R, Y73P, and H96N (TadA-CD-26, SEQ ID NO: 522) relative to the amino acid sequence of SEQ ID NO: 489. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations R26G, V28A, N46L, A48R, Y73S, and H96N (TadA-CD-27, SEQ ID NO: 523) relative to the amino acid sequence of SEQ ID NO: 489. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations R26G, V28A, N46C, A48R, H96N, and A162V (TadA-CD-28, SEQ ID NO: 524) relative to the amino acid sequence of SEQ ID NO: 489. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations R26G, V28A, N46V, A48R, Q71H, Y73P, and H96N (TadA-CD- 29, SEQ ID NO: 525) relative to the amino acid sequence of SEQ ID NO: 489. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations R26G, V28A, N46C, A48R, Y73P, and H96N (TadA-CD-30, SEQ ID NO: 526) relative to the amino acid sequence of SEQ ID NO: 489 In some embodiments, the evolved TadA-Dual deaminase comprises the mutations R26G, V28A, N46C, A48R, Y73P, H96N, and Al 62V (TadA-CD-31, SEQ ID NO: 527) relative to the amino acid sequence of SEQ ID NO: 489.

In some embodiments, the evolved TadA-Dual deaminase comprises the mutations R26G, V28A, N46V, A48R, Y73P, and H96N (TadA-CD-32, SEQ ID NO: 528) relative to the amino acid sequence of SEQ ID NO: 489. In some embodiments, the evolved TadA-Dual ATTORNEY DOCKET NO. 180802-47402/PCT

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deaminase comprises the mutations R26G, V28A, N46V, A48R, Y73S, and H96N (TadA-CD-33, SEQ ID NO: 529) relative to the amino acid sequence of SEQ ID NO: 489. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations R26G, V28A, N46V, A48P, Y73S, and H96N (TadA-CD-34, SEQ ID NO: 530) relative to the amino acid sequence of SEQ ID NO: 489. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations R26G, V28A, N46C, A48R, Y73P, and H96N (TadA-CD-35, SEQ ID NO: 531) relative to the amino acid sequence of SEQ ID NO: 489. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations R26G, V28A, L34M, N46L, A48R, Y73P, and H96N (TadA-CD-36, SEQ ID NO: 532) relative to the amino acid sequence of SEQ ID NO: 489. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations R26G, V28A, N46L, A48R, Y73P, and H96N (TadA-CD-37, SEQ ID NO: 533) relative to the amino acid sequence of SEQ ID NO: 489 In some embodiments, the evolved TadA-Dual deaminase comprises the mutations R26G, V28A, N46L, A48P, R64K, Y73P, and H96N (TadA-CD- 38, SEQ ID NO: 534) relative to the amino acid sequence of SEQ ID NO: 489.

In some embodiments, the evolved TadA-Dual deaminase comprises the mutations N46I, S73P, and H154Q (TadA-CD-1, SEQ ID NO: 497) relative to the amino acid sequence of SEQ ID NO: 495. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations N46T (TadA-CD-2, SEQ ID NO: 498) relative to the amino acid sequence of SEQ ID NO: 495. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations N46T and H154Q (TadA-CD-3, SEQ ID NO: 499) relative to the amino acid sequence of SEQ ID NO: 495. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations N46V and H154Q (TadA-CD-4, SEQ ID NO: 500) relative to the amino acid sequence of SEQ ID NO: 495. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations N46V, S73P, G105S, and H154Q (TadA-CD-5, SEQ ID NO: 501) relative to the amino acid sequence of SEQ ID NO: 495. In some embodiments, the evolved TadA- Dual deaminase comprises the mutations N46L, S73P, and H154Q (TadA-CD-6, SEQ ID NO: 502) relative to the amino acid sequence of SEQ ID NO: 495. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations G26RN46L, R48P, S73P, N96H, and H154Q (TadA-CD-7, SEQ ID NO: 503) relative to the amino acid sequence of SEQ ID NO: 495. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations N46C, N96H, and H154Q (TadA-CD-8, SEQ ID NO: 504) relative to the amino acid sequence of SEQ ID NO: 495. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations N46V, S73P, and H154Q (TadA-CD-9, SEQ ID NO: 505) relative to the amino acid sequence of SEQ ID NO: 495. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations N46V, ATTORNEY DOCKET NO. 180802-47402/PCT

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Q71H, S73P, and H154Q (TadA-CD-IO, SEQ ID NO: 506) relative to the amino acid sequence of SEQ ID NO: 495. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations N46L and H154Q (TadA-CD-11, SEQ ID NO: 507) relative to the amino acid sequence of SEQ ID NO: 495. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations N46C, S73P, and H154Q (TadA- CD-12, SEQ ID NO: 508) relative to the amino acid sequence of SEQ ID NO: 495. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations N46C, S73P, H154Q, and A 162V (TadA-CD-13, SEQ ID NO: 509) relative to the amino acid sequence of SEQ ID NO: 495.

In some embodiments, the evolved TadA-Dual deaminase comprises the mutations N46I and H154Q (TadA-CD-14, SEQ ID NO: 510) relative to the amino acid sequence of SEQ ID NO: 495. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations Q71S and H154Q (TadA-CD-15, SEQ ID NO: 511) relative to the amino acid sequence of SEQ ID NO: 489. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations N46L, S73P, N79T, and N96H (TadA-CD-16, SEQ ID NO: 512) relative to the amino acid sequence of SEQ ID NO: 495. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations N46L, S73P, N79T (TadA-CD-17, SEQ ID NO: 513) relative to the amino acid sequence of SEQ ID NO: 495. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations R48A, S73P, and N79T (TadA-CD-18, SEQ ID NO: 514) relative to the amino acid sequence of SEQ ID NO: 495. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations N46V and N79T (TadA-CD-19, SEQ ID NO: 515) relative to the amino acid sequence of SEQ ID NO: 495. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations N46V, S73P, and N79T (TadA-CD-20, SEQ ID NO: 516) relative to the amino acid sequence of SEQ ID NO: 495 In some embodiments, the evolved TadA-Dual deaminase comprises the mutations A28V, N46L, R48A, S73Y, N79T, and N96H (TadA-CD-21, SEQ ID NO: 517) relative to the amino acid sequence of SEQ ID NO: 495. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations N46I, S73P, and N79T (TadA-CD-22, SEQ ID NO: 518) relative to the amino acid sequence of SEQ ID NO: 495. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations N46V, S73P, N79T, and G106S (TadA-CD-23, SEQ ID NO: 519) relative to the amino acid sequence of SEQ ID NO: 495. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations R48P, S73H, and N79P (TadA-CD-24, SEQ ID NO: 520) relative to the amino acid sequence of SEQ ID NO: 495. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations A28V, N46I, R48A, S73Y, and N79T (TadA-CD-25, SEQ ID NO: 521) relative to the amino acid sequence of SEQ ID NO: 495. ATTORNEY DOCKET NO. 180802-47402/PCT

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In some embodiments, the evolved TadA-Dual deaminase comprises the mutations N46V and S73P (TadA-CD-26, SEQ ID NO: 522) relative to the amino acid sequence of SEQ ID NO: 495 In some embodiments, the evolved TadA-Dual deaminase comprises the mutation N46L (TadA-CD-27, SEQ ID NO: 523) relative to the amino acid sequence of SEQ ID NO: 495. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations N46C, S73Y, and A162V (TadA-CD-28, SEQ ID NO: 524) relative to the amino acid sequence of SEQ ID NO: 495. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations N46V, Q71H, and S73P (TadA-CD-29, SEQ ID NO: 525) relative to the amino acid sequence of SEQ ID NO: 495. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations N46C and S73P (TadA-CD-30, SEQ ID NO: 526) relative to the amino acid sequence of SEQ ID NO: 495. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations N46C, S73P, and A 162V (TadA-CD-31, SEQ ID NO: 527) relative to the amino acid sequence of SEQ ID NO: 495. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations N46V and S73P (TadA-CD-32, SEQ ID NO: 528) relative to the amino acid sequence of SEQ ID NO: 495. In some embodiments, the evolved TadA-Dual deaminase comprises the mutation N46V (TadA-CD-33, SEQ ID NO: 529) relative to the amino acid sequence of SEQ ID NO: 495. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations N46V and R48P(TadA-CD-34, SEQ ID NO: 530) relative to the amino acid sequence of SEQ ID NO: 495. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations N46CV and S73P (TadA-CD-35, SEQ ID NO: 531) relative to the amino acid sequence of SEQ ID NO: 495. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations L34M, N46L and S73P (TadA-CD- 36, SEQ ID NO: 532) relative to the amino acid sequence of SEQ ID NO: 495. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations N46L and S73P (TadA-CD-37, SEQ ID NO: 533) relative to the amino acid sequence of SEQ ID NO: 495. In some embodiments, the evolved TadA-Dual deaminase comprises the mutations N46L, R48P, R64K and S73P (TadA-CD-38, SEQ ID NO: 534) relative to the amino acid sequence of SEQ ID NO: 495.

In some embodiments, the TadA-CDs evolved from TadA-dual comprise at least 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% identical to any of the amino acid sequences of SEQ ID NOs: 39, 41-54, and 359-38.3

Exemplary TadA-derived cytosine base editor amino acid sequences include: TadA-CDa base editor (SpCas9n napDNAbp domain) (TadCBEa) (SEQ ID NO: 535), TadA-CDb base editor (SpCas9n napDNAbp domain) (TadCBEb) (SEQ ID NO: 536), TadA-CDc base editor (SpCas9n napDNAbp domain) (TadCBEc) (SEQ ID NO: 537), TadA-CDd base editor (SpCas9n ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

napDNAbp domain) (TadCBEd) (SEQ ID NO: 538), TadA-CDe base editor (SpCas9n napDNAbp domain) (TadCBEe) (SEQ ID NO: 539), TadA-CDa(V106W) base editor (SpCas9n napDNAbp domain) (TadCBEa(V106W)) (SEQ ID NO: 540), TadA-CDd(V106W) base editor (SpCas9n napDNAbp domain) (TadCBEd(V106W)) (SEQ ID NO: 541), TadA-CDf base editor (SpCas9n napDNAbp domain) (TadCBEf) (SEQ ID NO: 542), TadA-CDg base editor (SpCas9n napDNAbp domain) (TadCBEg) (SEQ ID NO: 543), TadA-CDa:eNme2Cas9 base editor (SEQ ID NO: 544), TadA-CDa: SaCas9 base editor (SEQ ID NO: 545), TadA-CDa: SpCas9-NGbase editor (SEQ ID NO: 546), TadA-CDa:enCjCas9 base editor (SEQ ID NO: 547).

Exemplary polynucleotides encoding TadA-derived cytosine base editors of the disclosure include: TadCBEa-eNme2-C-BE4max vector (SEQ ID NO: 548), TadCBEa-enCjCas9-BE4max vector (SEQ ID NO: 549), TadCBEa-SpCas9-BE4max vector (SEQ ID NO: 550), TadCBEa-SaCas9-BE4max vector (SEQ ID NO: 551), TadCBEa-SpCas9-NG-BE4max vector (SEQ ID NO: 552).

Guide Polynucleotides

A polynucleotide programmable nucleotide binding domain, when in conjunction with a bound guide polynucleotide (e.g., gRNA), can specifically bind to a target polynucleotide sequence (/.<., via complementary base pairing between bases of the bound guide nucleic acid and bases of the target polynucleotide sequence) and thereby localize the base editor to the target nucleic acid sequence desired to be edited. In some embodiments, the target polynucleotide sequence comprises single-stranded DNA or double-stranded DNA. In some embodiments, the target polynucleotide sequence comprises RNA. In some embodiments, the target polynucleotide sequence comprises a DNA-RNA hybrid.

In an embodiment, a guide polynucleotide described herein can be RNA or DNA. In one embodiment, the guide polynucleotide is a gRNA.

In some embodiments, the guide polynucleotide is at least one single guide RNA (“sgRNA” or “gRNA”). In some embodiments, a guide polynucleotide comprises two or more individual polynucleotides, which can interact with one another via for example complementary base pairing (e.g., a dual guide polynucleotide, dual gRNA). For example, a guide polynucleotide can comprise a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA) or can comprise one or more trans-activating CRISPR RNA (tracrRNA).

A guide polynucleotide may include natural or non-natural (or unnatural) nucleotides (e.g., peptide nucleic acid or nucleotide analogs). In some cases, the targeting region of a guide ATTORNEY DOCKET NO. 180802-47402/PCT

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nucleic acid sequence (e.g., a spacer) can be at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

In some embodiments, the methods described herein can utilize an engineered Cas protein. A guide RNA (gRNA) is a short synthetic RNA composed of a scaffold sequence necessary for Cas-binding and a user-defined ~20 nucleotide spacer that defines the genomic target to be modified. Exemplary gRNA scaffold sequences are provided in the sequence listing as SEQ ID NOs: 317-327 and 425. Thus, a skilled artisan can change the genomic target of the Cas protein specificity is partially determined by how specific the gRNA targeting sequence is for the genomic target compared to the rest of the genome. In embodiments, the spacer is about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, or more nucleotides in length. The spacer of a gRNA can be or can be about 19, 20, or 21 nucleotides in length.

A gRNA or a guide polynucleotide can target any exon or intron of a gene target. In some embodiments, a composition comprises multiple gRNAs that all target the same exon or multiple gRNAs that target different exons. An exon and/or an intron of a gene can be targeted. A gRNA or a guide polynucleotide can target a nucleic acid sequence of about 20 nucleotides or less than about 20 nucleotides (e.g., at least about 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 nucleotides), or anywhere between about 1-100 nucleotides (e.g., 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, 100). A target nucleic acid sequence can be or can be about 20 bases immediately 5' of the first nucleotide of the PAM. A gRNA can target a nucleic acid sequence. A target nucleic acid can be at least or at least about 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, or 1-100 nucleotides.

The guide polynucleotides can comprise standard ribonucleotides, modified ribonucleotides (e.g., pseudouridine), ribonucleotide isomers, and/or ribonucleotide analogs.

In some embodiments, a base editor system may comprise multiple guide polynucleotides, e.g., gRNAs. For example, the gRNAs may target to one or more target loci (e.g., at least 1 gRNA, at least 2 gRNA, at least 5 gRNA, at least 10 gRNA, at least 20 gRNA, at least 30 g RNA, at least 50 gRNA) comprised in a base editor system. The multiple gRNA sequences can be tandemly arranged and may be separated by a direct repeat.

Modified Polynucleotides

To enhance expression, stability, and/or genomic/base editing efficiency, and/or reduce possible toxicity, the base editor-coding sequence (e.g., mRNA) and/or the guide polynucleotide (e.g., gRNA) can be modified to include one or more modified nucleotides and/or chemical modifications, e.g. using pseudo-uridine, 5-Methyl-cytosine, 2'-O-methyl-3'-phosphonoacetate, ATTORNEY DOCKET NO. 180802-47402/PCT

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2'-(9-methyl thioPACE (MSP), 2'-(9-methyl-PACE (MP), 2'-fluoro RNA (2'-F-RNA), =constrained ethyl (S-cEt), 2'-O-methyl (‘M’), 2'-O-methyl-3'-phosphorothioate (‘MS’), 2'-O-methyl-3'-thiophosphonoacetate (‘MSP’), 5-methoxyuridine, phosphorothioate, andNl-Methylpseudouridine. Chemically protected gRNAs can enhance stability and editing efficiency in vivo and ex vivo. Methods for using chemically modified mRNAs and guide RNAs are known in the art and described, for example, by Jiang et al., Chemical modifications of adenine base editor mRNA and guide RNA expand its application scope. Nat Commun 11, 1979 (2020). doi.org/10.1038/s41467-020-15892-8, Callum et al., Al -Methylpseudouridine substitution enhances the performance of synthetic mRNA switches in cells, Nucleic Acids Research, Volume 48, Issue 6, 06 April 2020, Page e35, and Andries et al., Journal of Controlled Release, Volume 217, 10 November 2015, Pages 337-344, each of which is incorporated herein by reference in its entirety. Examples of modified guide RNAs suitable for use in various embodiments or aspects of the disclosure include those described in International Patent Application No. PCT/US2024/026601 or in Riesenberg, et al. Nature Communications 13:489 (2022) doi: 10.1038/s41467-022-28137-7, the disclosures of which are hereby incorporated by reference in its entirety for all purposes.

Methods for synthesizing modified guide RNAs are familiar to one of skill in the art and include as non-limiting examples, those methods described in International Patent Application No. PCT/US2024/026601, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.

In some embodiments, the guide polynucleotide comprises one or more modified nucleotides at the 5' end and/or the 3' end of the guide. In some embodiments, the guide polynucleotide comprises two, three, four or more modified nucleosides at the 5' end and/or the 3' end of the guide. In some embodiments, the guide polynucleotide comprises two, three, four or more modified nucleosides at the 5' end and/or the 3' end of the guide.

In some embodiments, a guide RNA (gRNA) comprises one or more modified nucleosides comprising a modified sugar moiety. Such a modified gRNA containing one or more sugar-modified nucleosides may have desirable properties, such as enhanced nuclease stability or increased binding affinity with a target nucleic acid relative to guide RNA lacking such sugar-modified nucleosides. In certain embodiments, modified sugar moieties are linearly modified sugar moieties. In certain embodiments, modified sugar moieties are bicyclic or tricyclic sugar moieties. In certain embodiments, modified sugar moieties are sugar surrogates. Such sugar surrogates may comprise one or more substitutions corresponding to those of substituted sugar moieties. ATTORNEY DOCKET NO. 180802-47402/PCT

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In some embodiments, the guide comprises at least about 50%-75% modified nucleotides. In some embodiments, the guide comprises at least about 85% or more modified nucleotides. In some embodiments, at least about 1-5 nucleotides at the 5' end of the gRNA are modified and at least about 1-5 nucleotides at the 3' end of the gRNA are modified. In some embodiments, at least about 3-5 contiguous nucleotides at each of the 5' and 3' termini of the gRNA are modified. In some embodiments, at least about 20% of the nucleotides present in a direct repeat or anti-direct repeat are modified. In some embodiments, at least about 50% of the nucleotides present in a direct repeat or anti-direct repeat are modified. In some embodiments, at least about 50-75% of the nucleotides present in a direct repeat or anti-direct repeat are modified. In some embodiments, at least about 100 of the nucleotides present in a direct repeat or antidirect repeat are modified. In some embodiments, at least about 20% or more of the nucleotides present in a hairpin present in the gRNA scaffold are modified. In some embodiments, at least about 50% or more of the nucleotides present in a hairpin present in the gRNA scaffold are modified. In some embodiments, the guide comprises a variable length spacer. In some embodiments, the guide comprises a 20-40 nucleotide spacer. In some embodiments, the guide comprises a spacer comprising at least about 20-25 nucleotides or at least about 30-35 nucleotides. In some embodiments, the spacer comprises modified nucleotides. In some embodiments, the guide comprises two or more of the following:

• at least about 1-5 nucleotides at the 5' end of the gRNA are modified and at least about 1- 5 nucleotides at the 3' end of the gRNA are modified;

• at least about 20% of the nucleotides present in a direct repeat or anti-direct repeat are modified;

• at least about 50-75% of the nucleotides present in a direct repeat or anti-direct repeat are modified;

• at least about 20% or more of the nucleotides present in a hairpin present in the gRNA scaffold are modified;

• a variable length spacer; and

• a spacer comprising modified nucleotides.

In embodiments, the gRNA contains numerous modified nucleotides and/or chemical modifications. Such modifications can increase base editing ~2 fold in vivo or in vitro. In embodiments, the gRNA comprises 2'-O-methyl or phosphorothioate modifications. In an embodiment, the gRNA comprises 2'-O-methyl and phosphorothioate modifications. In an embodiment, the modifications increase base editing by at least about 2 fold. ATTORNEY DOCKET NO. 180802-47402/PCT

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A guide polynucleotide can comprise one or more modifications to provide a nucleic acid with a new or enhanced feature. A guide polynucleotide can comprise a nucleic acid affinity tag. A guide polynucleotide can comprise synthetic nucleotide, synthetic nucleotide analog, nucleotide derivatives, and/or modified nucleotides.

A gRNA or a guide polynucleotide can also be modified by 5' adenylate, 5' guanosinetriphosphate cap, 5' N7-Methylguanosine-triphosphate cap, 5' triphosphate cap, 3' phosphate, 3' thiophosphate, 5' phosphate, 5' thiophosphate, Cis-Syn thymidine dimer, trimers, C12 spacer, C3 spacer, C6 spacer, dSpacer, PC spacer, rSpacer, Spacer 18, Spacer 9, 3 '-3' modifications, -O-methyl thioPACE (MSP), 2'-O-methyl-PACE (MP), and constrained ethyl (S-cEt), 5 '-5' modifications, abasic, acridine, azobenzene, biotin, biotin BB, biotin TEG, cholesteryl TEG, desthiobiotin TEG, DNP TEG, DNP-X, DOTA, dT-Biotin, dual biotin, PC biotin, psoralen C2, psoralen C6, TINA, 3' DABCYL, black hole quencher 1, black hole quencher 2, DABCYL SE, dT -DABCYL, IRDye QC-1, QSY-21, QSY-35, QSY-7, QSY-9, carboxyl linker, thiol linkers, 2'-deoxyribonucleoside analog purine, 2'-deoxyribonucleoside analog pyrimidine, ribonucleoside analog, 2'-O-methyl ribonucleoside analog, sugar modified analogs, wobble/universal bases, fluorescent dye label, 2'-fluoro RNA, 2'-O-methyl RNA, methylphosphonate, phosphodiester DNA, phosphodiester RNA, phosphothioate DNA, phosphorothioate RNA, UNA, pseudouridine-5 '-triphosphate, 5 '-methylcytidine-5 '-triphosphate, or any combination thereof.

In some cases, a phosphorothioate enhanced RNA gRNA can inhibit RNase A, RNase Tl, calf serum nucleases, or any combinations thereof. These properties can allow the use of PS-RNA gRNAs to be used in applications where exposure to nucleases is of high probability in vivo or in vitro. For example, phosphorothioate (PS) bonds can be introduced between the last 3-5 nucleotides at the 5'- or 3 '-end of a gRNA which can inhibit exonuclease degradation. In some cases, phosphorothioate bonds can be added throughout an entire gRNA to reduce attack by endonucleases.

In certain embodiments, modified sugar moieties are linearly modified sugar moieties comprising a furanosyl ring with one or more acyclic substituent, including but not limited to substituents at the 2' and/or 5' positions. Examples of 2 '-substituent groups suitable for linearly modified sugar moieties include but are not limited to: 2'-F, 2'-OCH3 (“Ome” or “O-methyl”), and 2'-O(CH2)2OCH3 (“MOE”). In certain embodiments, 2 '-substituent groups are selected from among: halo, allyl, amino, azido, SH, CN, OCN, CF3, OCF3, O — C1-C10 alkoxy, O — Ci-C 10 substituted alkoxy, O — C1-C10 alkyl, O — Ci-C 10 substituted alkyl, S-alkyl, N(R_m)-alkyl, O-alkenyl, S-alkenyl, N(R_m)-alkenyl, O-alkynyl, S-alkynyl, N(R_m)-alkynyl, O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH2)2SCH3, O(CH2)2ON(R_m)(Rn) or ATTORNEY DOCKET NO. 180802-47402/PCT

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OCH2C(=O) — N(Rm)(Rn), where each R_mand Rnis, independently, H, an amino protecting group, or substituted or unsubstituted C1-C10 alkyl. Certain embodiments of these 2'-substituent groups can be further substituted with one or more substituent groups independently selected from among: hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy, thioalkyl, halogen, alkyl, aryl, alkenyl and alkynyl. Examples of 5 '-substituent groups suitable for linearly modified sugar moi eties include but are not limited to: 5 '-methyl (R or S), 5 '-vinyl, and 5 '-methoxy. In certain embodiments, linearly modified sugars comprise more than one nonbridging sugar substituent, for example, 2'-F-5 '-methyl sugar moi eties (see, e.g., PCT International Application WO 2008/101157, the disclosure of which is hereby incorporated by reference in its entirety for all purposes, for additional 2', 5 '-bis substituted sugar moi eties and nucleosides).

In certain embodiments, a 2'-substituted nucleoside or 2'-linearly modified nucleoside comprises a sugar moiety comprising a linear 2 '-substituent group selected from: F, NH2, N3, OCF3, 0CH3, O(CH₂)₃NH2, CH₂CH=CH₂, OCH₂CH=CH₂, OCH2CH2OCH3, O(CH₂)₂SCH3, O(CH₂)2ON(Rm)(Rn), O(CH2)2O(CH₂)2N(CH₃)2, and N-substituted acetamide (OCH₂C(=O)— N(R_m)(Rn)), where each R_mand Rnis, independently, H, an amino protecting group, or substituted or unsubstituted C1-C10 alkyl.

In certain embodiments, a 2'-substituted nucleoside or 2'-linearly modified nucleoside comprises a sugar moiety comprising a linear 2'-substituent group selected from: F, OCF3, 0CH3, OCH2CH2OCH3, O(CH₂)₂SCH3, 0(CH₂)2ON(CH₃)2, O(CH2)2O(CH₂)2N(CH₃)2, and OCH₂C(=O)— N(H)CH₃ (“NMA”).

In certain embodiments, a 2'-substituted nucleoside or 2'-linearly modified nucleoside comprises a sugar moiety comprising a linear 2'-substituent group selected from: F, OCH3, and OCH2CH2OCH3.

Certain modified sugar moieties comprise a bridging sugar substituent that forms a second ring resulting in a bicyclic sugar moiety. In certain such embodiments, the bicyclic sugar moiety comprises a bridge between the 4' and the 2' furanose ring atoms. Examples of such 4' to 2' bridging sugar substituents include but are not limited to: 4'-CH2-2', 4'-(CH2)2-2', 4'-(CH2)3-2', 4'-CH₂— O-2' (“LNA”), 4'-CH₂— S-2', 4'-(CH₂)₂— O-2' (“ENA”), 4'-CH(CH₃)— 0-2' (referred to as “constrained ethyl” or “cEf ’ when in the S configuration), 4'-CH2 — O — CH2-2', 4'-CH2 — NI-2', 4'-CH(CH2OCH3) — 0-2' (“constrained MOE” or “cMOE”) and analogs thereof (see, e.g., U. S. Pat. No. 7,399,845, the disclosure of which is hereby incorporated by reference in its entirety for all purposes), 4'-C(CH3)(CH3) — 0-2' and analogs thereof (see, e.g., W02009/006478, the disclosure of which is hereby incorporated by reference in its entirety for ATTORNEY DOCKET NO. 180802-47402/PCT

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all purposes), 4'-CH2 — N(OCH3)-2' and analogs thereof (see, e.g., W02008/150729), 4'-CH2 — O— N(CH₃)-2' (see, e.g., US2004/0171570), 4'-CH₂— C(H)(CH₃)-2' (see, e.g., Chattopadhyaya, et al., J. Org. Chem., 2009, 74, 118-134, the disclosure of which is hereby incorporated by reference in its entirety for all purposes), 4'-CH2 — C(=CH2)-2' and analogs thereof (see, published PCT International Application WO 2008/154401, the disclosure of which is hereby incorporated by reference in its entirety for all purposes), 4'-C(RaRbI(R) — O-2', 4'-C(RaRiO — N(R)-2', 4'-H₂— O— N(R)-2', I 4'-CH₂— N(R)— O-2', wherein each R, Ra, and Rbis, independently, H, a protecting group, or C1-C12 alkyl (see, e.g. U. S. Pat. No. 7,427,672, the disclosure of which is hereby incorporated by reference in its entirety for all purposes).

In certain embodiments, such 4' to 2' bridges independently comprise from 1 to 4 linked groups independently selected from: — [C(Ra)(Rb)]n —, — [C(Ra)(Rb)]n — O —, — C(Ra)=C(Rb) —, — C(Ra)=N—, — C(=NRa)—, — C(=O)—, — C(=S)—, — O—, — Si(Ra)₂—, — S(=O)x—, and — N(Ra) —; wherein X is 0, 1 or 2 and n is 1, 2, 3 or 4.

In some embodiments, bicyclic sugar moieties and nucleosides incorporating such bicyclic sugar moieties are further defined by isomeric configuration. For example, an LNA nucleoside (described above) may be in the a-L configuration or in the P-D configuration. In other embodiments, modified sugar moieties are sugar surrogates. In certain such embodiments, the oxygen atom of the sugar moiety is replaced, e.g., with a sulfur, carbon or nitrogen atom. In certain such embodiments, such modified sugar moieties also comprise bridging and/or nonbridging substituents as described above. For example, certain sugar surrogates comprise a d'sulfur atom and a substitution at the 2'-position (see, e.g., US2005/0130923, the disclosure of which is hereby incorporated by reference in its entirety for all purposes) and/or the 5' position.

In some embodiments, the modification in the sugar group can be a modification at the 2' position of the ribose group. In some instances, the modification at the 2' position of the ribose group is selected from the group consisting of 2'-O-methyl, 2'-fluoro, 2'-deoxy, and 2'-O-(2-methoxy ethyl).

In one embodiment, the one or more modifications in the modified gRNA comprises 2’0Me modification, such as 2'-O-methyl (M) nucleotides, 2'-O-methyl 3'-phosphorothioate (MS) nucleotides, 2'-O-methyl 3 '-thioPACE (MSP) nucleotides, or combinations thereof.

In some embodiments, the modified gRNA comprise one or more nucleoside comprising a modified nucleobase. In certain embodiments, the modified gRNA comprise one or more nucleoside that does not comprise a nucleobase, referred to as an abasic nucleoside.

In some embodiments, modified nucleobases are selected from: 5-substituted pyrimidines, 6-azapyrimidines, alkyl or alkynyl substituted pyrimidines, alkyl substituted ATTORNEY DOCKET NO. 180802-47402/PCT

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purines, and N-2, N-6 and 0-6 substituted purines. In certain embodiments, modified nucleobases are selected from: 2-aminopropyladenine, 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-N-methylguanine, 6-N-methyladenine, 2-propyladenine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl ( — C=C — CH3) uracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-ribosyluracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, 8-aza and other 8-substituted purines, 5-halo, particularly 5-bromo, 5-trifluoromethyl, 5-halouracil, and 5-halocytosine, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine, 7-deazaguanine, 7-deazaadenine, 3 -deazaguanine, 3 -deazaadenine, 6-N-benzoyladenine, 2-N-isobutyrylguanine, 4-N-benzoylcytosine, 4-N-benzoyluracil, 5-methyl 4-N-benzoylcytosine, 5-methyl 4-N-benzoyluracil, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases. Further modified nucleobases include tricyclic pyrimidines, such as 1,3-diazaphenoxazine-2-one, l,3-diazaphenothiazine-2-one and 9-(2-aminoethoxy)-l,3-diazaphenoxazine-2-one (G-clamp). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example, 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.

In some embodiments, nucleosides of modified gRNAs described herein, may be linked together using any internucleoside linkage. The two main classes of internucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleoside linkages include but are not limited to phosphates, which contain a phosphodiester bond (“P=O”) (also referred to as unmodified or naturally occurring linkages), phosphotriesters, methylphosphonates, phosphoramidates, and phosphorothioates (“P=S”), and phosphorodithioates (“HS — P=S”). Representative non-phosphorus containing internucleoside linking groups include but are not limited to methylenemethylimino ( — CH2 — N(CH3) — O — CH2 — ), thiodiester ( — O — C(=O) — S — ), thionocarbamate ( — O — C(=0)(NH) — S — ); siloxane ( — O — SiFh — O — ); and N, N'-dimethylhydrazine ( — CH2 — NfCFh) — NfCFh) — ). Modified internucleoside linkages, compared to naturally occurring phosphate linkages, can be used to alter, typically increase, nuclease resistance of the oligonucleotide. In certain embodiments, intemucleoside linkages having a chiral atom can be prepared as a racemic mixture, or as separate enantiomers. Representative chiral internucleoside linkages include but are not limited to alkylphosphonates and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing internucleoside linkages are well known to those skilled in the art. ATTORNEY DOCKET NO. 180802-47402/PCT

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Neutral internucleoside linkages include, without limitation, phosphotriesters, methylphosphonates, MMI (3'-CH2 — N(CH₃) — 0-5'), amide-3 (3'-CH2 — C(=O) — N(H)-5'), amide-4 (3'-CH2 — N(H) — C(=O)-5'), formacetal (3'-0 — CH2 — 0-5'), methoxypropyl, and thioformacetal (3'-S — CH2 — 0-5'). Further neutral internucleoside linkages include nonionic linkages comprising siloxane (dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonate ester and amides (See for example: Carbohydrate Modifications in Antisense Research,' Y. S. Sanghvi and P. D. Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65, the disclosure of which is hereby incorporated by reference in its entirety for all purposes). Further neutral internucleoside linkages include nonionic linkages comprising mixed N, O, S and CH2 component parts.

In some embodiments, the one or more chemical modification in the phosphate group can be a phosphorothioate modification.

In some embodiments, a modified gRNA comprises one modified nucleotide at the 5 '-end (e.g., the terminal nucleotide at the 5'-end) or near the 5'-end (e.g., within 1, 2, 3, 4, or 5 nucleotides of the terminal nucleotide at the 5 '-end) of the modified gRNA nucleotide sequence and/or one modified nucleotide at the 3'-end (e.g., the terminal nucleotide at the 3'-end) or near the 3'-end (e.g., within 1, 2, 3, 4, or 5 nucleotides of the 3'-end) of the modified gRNA nucleotide sequence.

In some embodiments, a modified gRNA comprises two consecutive or non-consecutive modified nucleotides starting at the 5'-end (e.g., the terminal nucleotide at the 5'-end) or near the 5 '-end (e.g., within 1, 2, 3, 4, or 5 nucleotides of the terminal nucleotide at the 5 '-end) of the modified gRNA nucleotide sequence and/or two consecutive or non-consecutive modified nucleotides starting at the 3 '-end (e.g., the terminal nucleotide at the 3 '-end) or near the 3 '-end (e.g., within 1, 2, 3, 4, or 5 nucleotides of the 3'-end) of the modified gRNA nucleotide sequence.

Long/ extended upper stem

In one aspect, modified gRNAs provided herein are modified single guide RNAs (sgRNAs) comprising a long (or extended) upper stem including more than 4 base pairs formed by complementary nucleotides, wherein one or more nucleotides of the upper stem are chemically modified. Exemplary modifications include 2’0Me modification, such as 2'-O-methyl (M) nucleotides, 2'-O-methyl 3 '-phosphorothioate (MS) nucleotides, 2'-O-methyl 3'-thioPACE (MSP) nucleotides, or combinations thereof. Non-limiting examples of modified gRNAs and descriptions of regions of a guide RNA referenced by the terms “end mod”, “upper stem”, “hairpin 1”, and “hairpin 2” are provided in FIGs. 12 and 13. ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

In some embodiments, a modified sgRNA described herein comprises a long upper stem region comprising 5-15 base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides). In one embodiment, the long upper stem region comprises 5 base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides). In one embodiment, the long upper stem region comprises 6 base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides). In one embodiment, the long upper stem region comprises 7 base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides). In one embodiment, the long upper stem region comprises 8 base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides). In one embodiment, the long upper stem region comprises 9 base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides). In one embodiment, the long upper stem region comprises 10 base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides). In one embodiment, the long upper stem region comprises 11 base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides). In one embodiment, the long upper stem region comprises 12 base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides). In one embodiment, the long upper stem region comprises 13 base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides). In one embodiment, the long upper stem region comprises 14 base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides). In one embodiment, the long upper stem region comprises 15 base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides).

In some embodiments, the nucleotides of the upper stem of the modified sgRNA described herein are all modified nucleotides. In other embodiments, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the nucleotides of the upper stem of the modified sgRNA are chemically modified nucleotides.

In some embodiments, the sgRNA described herein comprises an upper stem modification, wherein the upper stem modification comprises a modification to any one or more of nucleotides in the upper stem region.

In some embodiments, the sgRNA comprises an upper stem modification, wherein the upper stem modification comprises a modification of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in the upper stem region.

In some embodiments, the gRNA comprises an upper stem modification, wherein the upper stem modification comprises a modification of about 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1- ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

12, 1-14, 1-16, 2-4, 2-6, 2-8, 2-10, 2-12, 2-16, 2-18, 2-20, 4-8, 4-10, 6-12, 6-18, 6-20, 8-10, 8-18, 8-20, or 10-20 nucleotides in the upper stem region.

In some embodiments, the modified nucleotides within the upper stem region of the sgRNA comprise the same chemical modifications. In other embodiments, the modified nucleotides within the upper stem region of the sgRNA comprise different chemical modifications.

In some embodiments, the sgRNA comprises an upper stem modification, wherein the upper stem modification comprises a 2’-OMe modified nucleotide. In some embodiments, the upper stem modification comprises a 2’-0-M0E modified nucleotide. In some embodiments, the upper stem modification comprises a 2’-F modified nucleotide. In some embodiments, the upper stem modification comprises a 2’-0me modified nucleotide, a 2’-0-M0E modified nucleotide, a 2’-F modified nucleotide, and/or combinations thereof.

Stable hairpins

In some embodiments, a modified gRNA is further modified to comprise a locked hairpin. In some cases, a modified gRNA described herein further comprises one or more modified nucleotides within the hairpin 1 and hairpin 2 regions. As noted above, FIGs. 12 and 13 provide examples of modified gRNAs and descriptions of regions of gRNAs referenced by the terms “hairpin 1” and “hairpin 2”. In some embodiments, all the nucleotides within the hairpins 1 and 2 regions are modified nucleotides. In other embodiments, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% and/or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more of the nucleotides within the hairpins 1 and 2 regions of the modified sgRNA are chemically modified nucleotides. In some embodiments, all the nucleotides within the hairpins 1 are 2 are chemically modified.

In some embodiments, a modified gRNA further comprises a modified stable hairpin 1 region, wherein the modified stable hairpin 1 region comprises an extended stem region comprising more than 4 base pairs and wherein the loop of hairpin 1 comprises locked nucleic acids. In some embodiments, the extended stem region of the modified stable hairpin comprises 8 base pairs. In some embodiments, the extended stem region of the modified stable hairpin comprises 8 base pairs formed by chemically modified nucleotides. As a non-limiting example, all the nucleotides of the hairpin comprise a 2’-O-methyl modification.

The highly stable hairpin of a sgRNA refers to a “locked” hairpin. The locked hairpin comprises a locked backbone, e.g., by incorporating one or more locked nucleic acid. As used ATTORNEY DOCKET NO. 180802-47402/PCT

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herein, the term “locked nucleic acid” refers to a bicyclic RNA analogue in which the ribose is locked in a C3'-endo conformation by introduction of a 2'-O,4'-C methylene bridge.

Exemplary LNA monomers and their method of synthesis also are disclosed in U. S. Pat. Nos. 6,043,060, 6,268,490, PCT Publications WO 01/07455, WO 01/00641, WO 98/39352, WO 00/56746, WO 00/56748 and WO 00/66604 as well as in the following papers: Morita et al., Bioorg. Med. Chem. Lett. 12(l):73-76, 2002; Hakansson et al., Bioorg. Med. Chem. Lett.

11(7):935-938, 2001; Koshkin et al., J. Org. Chem. 66(25):8504-8512, 2001; Kvaerno et al., J. Org. Chem. 66(16):5498-5503, 2001; Halkansson et al., J. Org. Chem. 65(17):5161-5166, 2000; Kvaerno et al., J. Org. Chem. 65(17):5167-5176, 2000; Pfundheller et al., Nucleosides Nucleotides 18(9):2017-2030, 1999; and Kumar et al, Bioorg. Med. Chem. Lett. 8(16):2219-2222, 1998.

In some examples, the hairpin region comprises locked nucleic acids or LNAs containing the 2'-O, 4'-C-methylene ribonucleoside (structure A) wherein the ribose sugar moiety is in a “locked” conformation. The hairpin region comprises at least one 2',4'-C-bridged 2' deoxyribonucleoside (CDNA, structure B). See, e.g., U. S. Pat. No. 6,403,566 and Wang et al. (1999) Bioorganic and Medicinal Chemistry Letters, Vol. 9: 1147-1150, both of which are herein incorporated by reference in their entireties. Locked nucleic acids (LNA™) are a class of high-affinity RNA analogs in which the ribose ring is “locked” in the ideal conformation for Watson-Crick binding. LNA™ oligonucleotides exhibit high thermal stability when hybridized to a complementary DNA or RNA strand. In addition, LNA™ oligonucleotides can be made shorter than traditional DNA or RNA oligonucleotides and still retain a high Tm. LNA™ oligonucleotides can consist of a mixture of LNA™ and DNA or RNA. Incorporation of LNA™ into oligonucleotides has been shown to improve sensitivity and specificity for many hybridization-based technologies including PCR, microarray and in situ hybridization.

Other regions and modifications

In some embodiments, the modified gRNA comprises modified nucleotides at the 5' terminus. In some embodiments, the 5' terminus of the sgRNA comprises a spacer or guide region that functions to direct a Cas protein, e.g., a Cas9 protein, to a target nucleotide sequence. In some embodiments, the 5' terminus does not comprise a guide region. In some embodiments, the 5' terminus comprises a spacer and additional nucleotides that do not function to direct a Cas protein to a target nucleotide region.

The sgRNA has a 3’ end, which is the last nucleotide of the sgRNA. The 3’ terminus region includes the last 1-7 nucleotides from the 3’ end. In some embodiments, the 3’ end is the ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

end of hairpin 2. In some embodiments, the sgRNA comprises nucleotides after the hairpin region(s). In some embodiments, the sgRNA includes a 3’ tail region, in which case the last nucleotide of the 3’ tail is the 3’ terminus. In some embodiments, the 3’ tail comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 or more nucleotides, e.g., that are not associated with the secondary structure of a hairpin. In some embodiments, the 3’ tail region comprises 1, 2, 3, or 4 nucleotides that are not associated with the secondary structure of a hairpin. In some embodiments, the 3 ’ tail region comprises 4 nucleotides that are not associated with the secondary structure of a hairpin. In some embodiments, the 3’ tail region comprises 1, 2, or 3 nucleotides that are not associated with the secondary structure of a hairpin.

In some embodiments, the modified gRNA further comprises a targeting nucleic acid sequence that is complementary to a sequence of a target nucleic acid. The complementary sequence is about 20 nucleotides in length. In some embodiments, at least two, three, four, five, six, seven, eight, nine, ten, or more of the nucleotides in the complementary nucleotide sequence are modified nucleotides. In certain instances, about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 of the nucleotides in the complementary nucleotide sequence (e.g., a first nucleotide sequence of about 20 nucleotides in length) are modified nucleotides. In other instances, all the nucleotides in the complementary nucleotide sequence (e.g., a complementary nucleotide sequence of about 20 nucleotides in length) are modified nucleotides. In some instances, the modified nucleotides are located at the 5'-end (e.g., the terminal nucleotide at the 5 '-end) or near the 5 '-end (e.g., within 1, 2, 3, 4, or 5 nucleotides of the terminal nucleotide at the 5 '-end) of the complementary nucleotide sequence and/or at internal positions within the complementary nucleotide sequence. In other instances, from about 10% to about 30% of the nucleotides in the first nucleotide sequence are modified nucleotides.

Exemplary gRNA sequences

In some embodiments, the present disclosure provides a LONGEST gRNA. Table 7A and 7B list some exemplary gRNAs and gRNA scaffolds with chemical modifications that are suitable for use in various aspects or embodiments of the disclosure.

Table 7A. Exemplary LONGEST guide RNAs

Name Sequence

LONGEST 1 mN smNs mN sNNNNNNNNNNNNNNNNNGUUUUAGAmGmCmCmGmGmCmGmGmAmAmAm CmGmCmCmGmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA mAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU

(SEQ ID NO: 749) ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

LONGEST 2 mN smNs mN sNNNNNNNNNNNNNNNNNGUUUUAGAmGmCmGmCmGmGmAmAmAmCmGm CmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCmAmAmCmUmUmGmAmAmAmAmAmGm UmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 750) LONGEST 3 mN smNs mN sNNNNNNNNNNNNNNNNNGUUUUAGAmGmCmUmAmUmGmCmGmAmAmAm GmCmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA mAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 751)

LONGEST 4 mN smNs mN sNNNNNNNNNNNNNNNNNGUUUUAGAmGmCmUmAmUmGmCmUmGmGmAm AmAmCmAmGmCmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmU mGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUs mU (SEQ ID NO: 752)

“N” represents any nucleotide, “mN” indicates a 2'-OMe modification of the nucleotide “N”, and “Ns” indicates that the nucleotide “N” is linked to the following nucleotide by a phosphorothioate (PS), and where the number of N nucleotides is between 15 and 25.

Table 7B: Exemplary guide RNA scaffold sequences

Scaffold Sequence (5 '-3’) GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAG UCGGUGCUUUU (SEQ ID NO: 753)

GUUUUAGAGCUANxi -_nGAAANyi. nUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 754)

yn (n=2-10)

xn (n=2-10)

GUUUUAGAmGmCmUmAm (N_xi-_n) mGmAmAmAm (Nyi- _n ) mUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCmAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmC mAmCmCmGmAmGmUmCmGmGmUmGmCmUmUmUmU (SEQ ID NO: 755)

yn (n=2-10)

xn (n=2-10) GUUUUAGAN_xnGAAANy_nAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCG GUGC(hairpin2)UUUU (SEQ ID NO: 756)

yn (n= 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)

xn (n= 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)

GUUUUAGAm (N_xn) mGmAmAmAm (Nyn) AAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAm AmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUmUmUmU (SEQ ID NO: 757) yn (n=5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)

xn (n=5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) GUUUUAGAGCGCGGAAACGCGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCG AGUCGGUGCUUUU (SEQ ID NO: 758) GUUUUAGAmGmCmGmCmGmGmAmAmAmCmGmCmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCm UmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUmUmUmU (SEQ ID NO: GUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmCmCmGmGmCAAGUUAAAAUAAGGCUAGUCCGUUA UCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 759) GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCmAmAmCmUmU mGmGmAmCmUmUmUmGmGmUmCmCmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUmUm UmU (SEQ ID NO: 760) GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCmAmAmCmUmU mGmGmAmCmUmUmUmGmGmUmCmCmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUmUm

UmU (SEQ ID NO: 760) ATTORNEY DOCKET NO. 180802-47402/PCT

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Scaffold Sequence (5 '-3’) GUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmCmCmGmGmCAAGUUAAAAUAAGGCUAGUCCGUUA UCmAmAmCmUmUmGmGmAmCmUmUmUmGmGmUmCmCmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGm GmUmGmCmUmUmUmU (SEQ ID NO: 761) _ GUUUUAGANxnGAAANynAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGGACUUCGGUCCAAGUGGCA CCGAGUCGGUGCUUUU (SEQ ID NO: 762)

yn (n=5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)

_ xn (n=5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) _ AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU

(SEQ ID NO: 763) _

AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGGACUUCGGUCCAAGUGGCACCGAGUCGG

UGCUUUU (SEQ ID NO: 764) _

“mN” indicates a 2'-OMe modification of the nucleotide “N”, and “Ns” indicates that the nucleotide “N” is linked to the following nucleotide by a phosphorothioate (PS), and where the number of N nucleotides is between 15 and 25.

In any aspect of the disclosure, or embodiments thereof, the one or more guide polynucleotides contain a modified nucleotide. In any aspect of the disclosure, or embodiments thereof, the one or more guide polynucleotides contain a sequence selected from one or more of End-mod spCas9 guide polynucleotide mNsmNsmNsNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAG UCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsmUsmUsmU (SEQ ID NO: 765); End-mod saCas9 guide polynucleotide mNsmNsmNsNNNNNNNNNNNNNNNNNNGUUUUAGUACUCUGUAAUGAAAAUUACAGAAUCUACU AAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUsmUsmUsmU (SEQ ID NO: 766);

HM01: mNsmNsmNsNNNNNNNNNNNNNNNNNGUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCTYAGUUA AAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmG mUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 767);

HM07: mNsmNsmNsmNmNmNmNmNmNmNNNNNNNNNNNmGUUUUAGmAmGmCmUmAmGmAmAmAmUmAm GmCmAmAGUUmAAmAAmUAmAmGmGmCmUmAGUmCmCGUUAmUmCTYAmCmUmUmGmAmAmAmAm AmGmUmGGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 767); NLS (bpsv40): mNsmNsmNsNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAG UCCGUUAUCAACUUGAAAAAGUGGCAGCGAGUCGGUGCmUsmUsmUsmU-NHC6-CrossL-ac-CKRTADGSEFESPKKKRKV (SEQ ID NOs: 767 and 768); ATTORNEY DOCKET NO. 180802-47402/PCT

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LONGEST: mNsmNsmNsNNNNNNNNNNNNNNNNNGUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmCmC mGmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmC mAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 769);

NLS + LONGEST: mNsmNsmNsNNNNNNNNNNNNNNNNNGUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmCmC mGmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmC mAmCmCmGmAmGmUmCmGmGmUmGmCmU s mU s mU s mU-NHC 5 -Cros sL-CKRTADGSEFESPKKKRKV (SEQ ID NOs: 769and 768); and

LONGEST+: mNsmNsmNsNNNNNNNNNNNNNNNNNGUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmCmC mGmGmCAAGUUAAAAUAAGGCUAGUCCGUUAmUmCAAmCmUmUGGACUUCGGUCCmAmAmGUGG mCmAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 770);

where “N” represents any nucleotide, “mN” indicates a 2'-OMe modification of the nucleotide “N”, and “Ns” indicates that the nucleotide “N” is linked to the following nucleotide by a phosphorothioate (PS), and where the number of N nucleotides is between 15 and 25.

Fusion Proteins or Complexes Comprising a Nuclear Localization Sequence (NLS)

In some embodiments, the fusion proteins or complexes provided herein further comprise one or more (e.g., 2, 3, 4, 5) nuclear targeting sequences, for example a nuclear localization sequence (NLS). In one embodiment, a bipartite NLS is used. In some embodiments, a NLS comprises an amino acid sequence that facilitates the importation of a protein, that comprises an NLS, into the cell nucleus (e.g., by nuclear transport). In some embodiments, the NLS is fused to the N-terminus or the C-terminus of the fusion protein. In some embodiments, the NLS is fused to the C-terminus or N-terminus of an nCas9 domain or a dCas9 domain. In some embodiments, the NLS is fused to the N-terminus or C-terminus of the Casl2 domain. In some embodiments, the NLS is fused to the N-terminus or C-terminus of the cytidine or adenosine deaminase. In some embodiments, the NLS is fused to the fusion protein via one or more linkers. In some embodiments, the NLS is fused to the fusion protein without a linker. In some embodiments, the NLS comprises an amino acid sequence of any one of the NLS sequences provided or referenced herein. Additional nuclear localization sequences are known in the art and would be apparent to the skilled artisan. For example, NLS sequences are described in Plank et al., PCTZEP2000/011690, the contents of which are incorporated herein by reference for their disclosure of exemplary nuclear localization sequences. ATTORNEY DOCKET NO. 180802-47402/PCT

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In some embodiments, the NLS is present in a linker or the NLS is flanked by linkers, for example described herein. A bipartite NLS comprises two basic amino acid clusters, which are separated by a relatively short spacer sequence (hence bipartite - 2 parts, while monopartite NLSs are not). The NLS of nucleoplasmin, KR [PAATKKAGQA] KKKK (SEQ ID NO: 191), is the prototype of the ubiquitous bipartite signal: two clusters of basic amino acids, separated by a spacer of about 10 amino acids. The sequence of an exemplary bipartite NLS follows:

PKKKRKVEGADKRTADGSEFESPKKKRKV (SEQ ID NO: 328).

In some embodiments, any of the fusion proteins or complexes provided herein comprise an NLS comprising the amino acid sequence EGADKRTADGSEFESPKKKRKV (amino acids 8 to 29 of SEQ ID NO 328). In some embodiments, any of the adenosine base editors provided herein comprise an NLS comprising the amino acid sequence EGADKRTADGSEFESPKKKRKV (amino acids 8 to 29 of SEQ ID NO: 328). In some embodiments, the NLS is at a C-terminal portion of the adenosine base editor. In some embodiments, the NLS is at the C-terminus of the adenosine base editor.

Additional Domains

A base editor described herein can include any domain which helps to facilitate the nucleobase editing, modification or altering of a nucleobase of a polynucleotide. In some embodiments, a base editor comprises a polynucleotide programmable nucleotide binding domain (e.g., Cas9), a nucleobase editing domain (e.g., deaminase domain), and one or more additional domains. In some embodiments, the additional domain can facilitate enzymatic or catalytic functions of the base editor, binding functions of the base editor, or be inhibitors of cellular machinery (e.g., enzymes) that could interfere with the desired base editing result. In some embodiments, a base editor comprises a nuclease, a nickase, a recombinase, a deaminase, a methyltransferase, a methylase, an acetylase, an acetyltransferase, a transcriptional activator, or a transcriptional repressor domain.

In some embodiments, a base editor comprises an uracil glycosylase inhibitor (UGI) domain. In some cases, a base editor is expressed in a cell in trans with a UGI polypeptide. In some embodiments, cellular DNA repair response to the presence of U: G heteroduplex DNA can be responsible for a reduction in nucleobase editing efficiency in cells. In such embodiments, uracil DNA glycosylase (UDG) can catalyze removal of U from DNA in cells, which can initiate base excision repair (BER), mostly resulting in reversion of the U: G pair to a C: G pair. In such embodiments, BER can be inhibited in base editors comprising one or more domains that bind the single strand, block the edited base, inhibit UGI, inhibit BER, protect the edited base, and /or ATTORNEY DOCKET NO. 180802-47402/PCT

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promote repairing of the non-edited strand. Thus, this disclosure contemplates a base editor fusion protein or complex comprising a UGI domain and/or a uracil stabilizing protein (USP) domain.

BASE EDITOR SYSTEM

Provided herein are systems, compositions, and methods for editing a nucleobase using a base editor system. In some embodiments, the base editor system comprises (1) a base editor (BE) comprising a polynucleotide programmable nucleotide binding domain and a nucleobase editing domain (e.g., a deaminase domain) for editing the nucleobase; and (2) a guide polynucleotide (e.g, guide RNA) in conjunction with the polynucleotide programmable nucleotide binding domain. In some embodiments, the base editor system is a cytidine base editor (CBE) or an adenosine base editor (ABE). In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable DNA or RNA binding domain. In some embodiments, the nucleobase editing domain is a deaminase domain. In some embodiments, a deaminase domain can be a cytidine deaminase or an cytosine deaminase. In some embodiments, a deaminase domain can be an adenine deaminase or an adenosine deaminase. In some embodiments, the adenosine base editor can deaminate adenine in DNA. In some embodiments, the base editor is capable of deaminating a cytidine in DNA.

Use of the base editor system provided herein comprises the steps of: (a) contacting a target nucleotide sequence of a polynucleotide (e.g, double- or single stranded DNA or RNA) of a subject with a base editor system comprising a nucleobase editor (e.g., an adenosine base editor or a cytidine base editor) and a guide polynucleotide (e.g., gRNA), wherein the target nucleotide sequence comprises a targeted nucleobase pair; (b) inducing strand separation of said target region; (c) converting a first nucleobase of said target nucleobase pair in a single strand of the target region to a second nucleobase; and (d) cutting no more than one strand of said target region, where a third nucleobase complementary to the first nucleobase base is replaced by a fourth nucleobase complementary to the second nucleobase. It should be appreciated that in some embodiments, step (b) is omitted. In some embodiments, said targeted nucleobase pair is a plurality of nucleobase pairs in one or more genes. In some embodiments, the base editor system provided herein is capable of multiplex editing of a plurality of nucleobase pairs in one or more genes. In some embodiments, the plurality of nucleobase pairs is located in the same gene. In some embodiments, the plurality of nucleobase pairs is located in one or more genes, wherein at least one gene is located in a different locus. ATTORNEY DOCKET NO. 180802-47402/PCT

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The components of a base editor system (e.g., a deaminase domain, a guide RNA, and/or a polynucleotide programmable nucleotide binding domain) may be associated with each other covalently or non-covalently. For example, in some embodiments, the deaminase domain can be targeted to a target nucleotide sequence by a polynucleotide programmable nucleotide binding domain, optionally where the polynucleotide programmable nucleotide binding domain is complexed with a polynucleotide (e.g., a guide RNA). In some embodiments, a polynucleotide programmable nucleotide binding domain can be fused or linked to a deaminase domain. In some embodiments, a polynucleotide programmable nucleotide binding domain can target a deaminase domain to a target nucleotide sequence by non-covalently interacting with or associating with the deaminase domain. For example, in some embodiments, the nucleobase editing component (e.g., the deaminase component) comprises an additional heterologous portion or domain that is capable of interacting with, associating with, or capable of forming a complex with a corresponding heterologous portion, antigen, or domain that is part of a polynucleotide programmable nucleotide binding domain and/or a guide polynucleotide (e.g., a guide RNA) complexed therewith. In some embodiments, the polynucleotide programmable nucleotide binding domain, and/or a guide polynucleotide (e.g., a guide RNA) complexed therewith, comprises an additional heterologous portion or domain that is capable of interacting with, associating with, or capable of forming a complex with a corresponding heterologous portion, antigen, or domain that is part of a nucleobase editing domain (e.g., the deaminase component). In some embodiments, the additional heterologous portion may be capable of binding to, interacting with, associating with, or forming a complex with a polypeptide. In some embodiments, the additional heterologous portion may be capable of binding to, interacting with, associating with, or forming a complex with a polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding to a guide polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding to a polypeptide linker. In some embodiments, the additional heterologous portion is capable of binding to a polynucleotide linker. An additional heterologous portion may be a protein domain. In some embodiments, an additional heterologous portion comprises a polypeptide, such as a 22 amino acid RNA-binding domain of the lambda bacteriophage antiterminator protein N (N22p), a 2G12 IgG homodimer domain, an AB I, an antibody (e.g. an antibody that binds a component of the base editor system or a heterologous portion thereof) or fragment thereof (e.g. heavy chain domain 2 (CH2) of IgM (MHD2) or IgE (EHD2), an immunoglobulin Fc region, a heavy chain domain 3 (CH3) of IgG or IgA, a heavy chain domain 4 (CH4) of IgM or IgE, an Fab, an Fab2, miniantibodies, and/or ZIP antibodies), a bamase-barstar dimer domain, a Bcl-xL domain, a ATTORNEY DOCKET NO. 180802-47402/PCT

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Calcineurin A (CAN) domain, a Cardiac phospholamban transmembrane pentamer domain, a collagen domain, a Com RNA binding protein domain e.g. SfMu Com coat protein domain, and SfMu Com binding protein domain), a Cyclophilin-Fas fusion protein (CyP-Fas) domain, a Fab domain, an Fe domain, a fibritin foldon domain, an FK506 binding protein (FKBP) domain, an FKBP binding domain (FRB) domain of mTOR, a foldon domain, a fragment X domain, a GAI domain, a GID1 domain, a Glycophorin A transmembrane domain, a GyrB domain, a Halo tag, an HIV Gp41 trimerisation domain, an HPV45 oncoprotein E7 C-terminal dimer domain, a hydrophobic polypeptide, a K Homology (KH) domain, a Ku protein domain (e.g., a Ku heterodimer), a leucine zipper, a LOV domain, a mitochondrial antiviral-signaling protein CARD filament domain, an MS2 coat protein domain (MCP), a non-natural RNA aptamer ligand that binds a corresponding RNA motif/aptamer, a parathyroid hormone dimerization domain, a PP7 coat protein (PCP) domain, a PSD95-Dlgl-zo-l (PDZ) domain, a PYL domain, a SNAP tag, a SpyCatcher moiety, a SpyTag moiety, a streptavidin domain, a streptavidin-binding protein domain, a streptavidin binding protein (SBP) domain, a telomerase Sm7 protein domain e.g. Sm7 homoheptamer or a monomeric Sm-like protein), and/or fragments thereof. In embodiments, an additional heterologous portion comprises a polynucleotide (e.g., an RNA motif), such as an MS2 phage operator stem-loop e.g., an MS2, an MS2 C-5 mutant, or an MS2 F-5 mutant), a non-natural RNA motif, a PP7 operator stem-loop, an SfMu phate Com stemloop, a steril alpha motif, a telomerase Ku binding motif, a telomerase Sm7 binding motif,, and/or fragments thereof. Non-limiting examples of additional heterologous portions include polypeptides with at least about 85% sequence identity to any one or more of SEQ ID NOs: 380, 382, 384, 386-388, or fragments thereof. Non-limiting examples of additional heterologous portions include polynucleotides with at least about 85% sequence identity to any one or more of SEQ ID NOs: 379, 381, 383, 385, or fragments thereof.

In some instances, components of the base editing system are associated with one another through the interaction of leucine zipper domains (e.g., SEQ ID NOs: 387 and 388). In some cases, components of the base editing system are associated with one another through polypeptide domains (e.g., FokI domains) that associate to form protein complexes containing about, at least about, or no more than about 1, 2 (i.e., dimerize), 3, 4, 5, 6, 7, 8, 9, 10 polypeptide domain units, optionally the polypeptide domains may include alterations that reduce or eliminate an activity thereof.

In some instances, components of the base editing system are associated with one another through the interaction of multimeric antibodies or fragments thereof (e.g., IgG, IgD, IgA, IgM, IgE, a heavy chain domain 2 (CH2) of IgM (MHD2) or IgE (EHD2), an immunoglobulin Fc ATTORNEY DOCKET NO. 180802-47402/PCT

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region, a heavy chain domain 3 (CH3) of IgG or IgA, a heavy chain domain 4 (CH4) of IgM or IgE, an Fab, and an Fab2). In some instances, the antibodies are dimeric, trimeric, or tetrameric. In embodiments, the dimeric antibodies bind a polypeptide or polynucleotide component of the base editing system.

In some cases, components of the base editing system are associated with one another through the interaction of a polynucleotide-binding protein domain(s) with a polynucleotide(s). In some instances, components of the base editing system are associated with one another through the interaction of one or more polynucleotide-binding protein domains with polynucleotides that are self-complementary and/or complementary to one another so that complementary binding of the polynucleotides to one another brings into association their respective bound polynucleotide-binding protein domain(s).

In some instances, components of the base editing system are associated with one another through the interaction of a polypeptide domain(s) with a small molecule(s) (e.g., chemical inducers of dimerization (CIDs), also known as “dimerizers”). Non-limiting examples of CIDs include those disclosed in Amara, et al., “A versatile synthetic dimerizer for the regulation of protein-protein interactions,” PNAS, 94:10618-10623 (1997); and VoB, etal. “Chemically induced dimerization: reversible and spatiotemporal control of protein function in cells,” Current Opinion in Chemical Biology, 28:194-201 (2015), the disclosures of each of which are incorporated herein by reference in their entireties for all purposes. In some embodiments, the base editor inhibits base excision repair (BER) of the edited strand. In some embodiments, the base editor protects or binds the non-edited strand. In some embodiments, the base editor comprises UGI activity or USP activity. In some embodiments, the base editor comprises a catalytically inactive inosine-specific nuclease.

The base editors of the present disclosure can comprise any domain, feature or amino acid sequence which facilitates the editing of a target polynucleotide sequence. For example, in some embodiments, the base editor comprises a nuclear localization signal (NLS). In some embodiments, an NLS of the base editor is localized between a deaminase domain and a polynucleotide programmable nucleotide binding domain. In some embodiments, an NLS of the base editor is localized C-terminal to a polynucleotide programmable nucleotide binding domain.

Protein domains included in the fusion protein can be a heterologous functional domain. Non-limiting examples of protein domains which can be included in the fusion protein include a deaminase domain (e.g., cytidine deaminase and/or adenosine deaminase), a uracil glycosylase inhibitor (UGI) domain, epitope tags, and reporter gene sequences. ATTORNEY DOCKET NO. 180802-47402/PCT

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In some embodiments, the adenosine base editor (ABE) can deaminate adenine in DNA. In some embodiments, ABE is generated by replacing APOBEC1 component of BE3 with natural or engineered E. coli TadA, human ADAR2, mouse ADA, or human ADAT2. In some embodiments, ABE comprises an evolved TadA variant. In some embodiments, the base editor is ABE8.1, which comprises or consists essentially of the following sequence or a fragment thereof having adenosine deaminase activity: SEQ ID NO: 331. Other ABE8 sequences are provided in the attached sequence listing (SEQ ID NOs: 332-354).

In some embodiments, the base editor includes an adenosine deaminase variant comprising an amino acid sequence, which contains alterations relative to an ABE 7*10 reference sequence, as described herein. The term “monomer” as used in Table 8 refers to a monomeric form of TadA*7.10 comprising the alterations described. The term “heterodimer” as used in Table 8 refers to the specified wild-type E. coli TadA adenosine deaminase fused to a TadA*7.10 comprising the alterations as described.

Table 8. Adenosine Deaminase Base Editor Variants

Adenosine Adenosine Deaminase Description ABE

Deaminase

ABE-605m MSP605 monomer TadA*7.10 + V82G + Y147T + Q154S

ABE-680m MSP680 monomer TadA*7.10 + I76Y + V82G + Y147T + Q154S ABE-823m MSP823 monomer_TadA*7.10 + L36H + V82G + Y147T + Q154S +

N157K

ABE-824m MSP824 monomer_TadA*7.10 + V82G + Y147D + F149Y + Q154S +

D167N

ABE-825m MSP825 monomer_TadA*7.10 + L36H + V82G + Y147D + F149Y +

Q154S + N157K + D167N

ABE-827m MSP827 monomer_TadA*7.10 + L36H + I76Y + V82G + Y147T + Q154S + N157K

ABE-828m MSP828 monomer_TadA*7.10 + I76Y + V82G + Y147D + F149Y + Q154S + D167N

ABE-829m MSP829 monomer_TadA*7.10 + L36H + I76Y + V82G + Y147D + F149Y + Q154S +N157K + D167N

ABE-605d MSP605 heterodimer (WT)+(TadA*7.10 + V82G + Y147T + Q154S) ABE-680d MSP680 heterodimer_(WT)+(TadA*7.10 + I76Y + V82G + Y147T +

Q154S)

ABE-823d MSP823 heterodimer_(WT)+(TadA*7.10 + L36H + V82G + Y147T +

Q154S + N157K)

ABE-824d MSP824 heterodimer_(WT)+(TadA*7.10 + V82G + Y147D + F149Y + Q154S + D167N)

ABE-825d MSP825 heterodimer_(WT)+(TadA*7.10 + L36H + V82G + Y147D +

F149Y + Q154S + N157K + D167N)

ABE-827d MSP827 heterodimer_(WT)+(TadA*7.10 + L36H + I76Y + V82G + Y147T

+ Q154S + N157K) ATTORNEY DOCKET NO. 180802-47402/PCT

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Adenosine Adenosine Deaminase Description ABE

Deaminase

ABE-828d MSP828 heterodimer_(WT)+(TadA*7.10 + I76Y + V82G + Y147D +

F149Y + Q154S + D167N)

ABE-829d MSP829 heterodimer_(WT)+(TadA*7.10 + L36H + I76Y + V82G + Y147D

+ F149Y + Q154S + N157K + D167N)

In some embodiments, the base editor comprises a domain comprising all or a portion (e.g., a functional portion) of a uracil glycosylase inhibitor (UGI) or a uracil stabilizing protein (USP) domain.

Linkers

In certain embodiments, linkers may be used to link any of the peptides or peptide domains of the disclosure. The linker may be as simple as a covalent bond, or it may be a polymeric linker many atoms in length. In certain embodiments, the linker is a polypeptide or based on amino acids. In other embodiments, the linker is not peptide-like. In certain embodiments, the linker is a covalent bond (e.g., a carbon-carbon bond, disulfide bond, carbonheteroatom bond, etc..

In some embodiments, any of the fusion proteins provided herein, comprise a cytidine or adenosine deaminase and a Cas9 domain that are fused to each other via a linker. Various linker lengths and flexibilities between the cytidine or adenosine deaminase and the Cas9 domain can be employed (e.g., ranging from very flexible linkers of the form (GGGS ) _n (SEQ ID NO: 246), (GGGGS)n (SEQ ID NO: 247), and (G)n to more rigid linkers of the form (EAAAK)_n (SEQ ID NO: 248), (SGGS)n (SEQ ID NO: 355), SGSETPGTSESATPES (SEQ ID NO: 249) (see, e.g., Guilinger JP, et al. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat. Biotechnol. 2014; 32(6): 577-82; the entire contents are incorporated herein by reference) and (XP)n) in order to achieve the optimal length for activity for the cytidine or adenosine deaminase nucleobase editor. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the linker comprises a (GGS)n motif, wherein n is 1, 3, or 7. In some embodiments, cytidine deaminase or adenosine deaminase and the Cas9 domain of any of the fusion proteins provided herein are fused via a linker comprising the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 249), which can also be referred to as the XTEN linker.

In some embodiments, the domains of the base editor are fused via a linker that comprises the amino acid sequence of:

SGGSSGSETPGTSESATPESSGGS (SEQ ID NO: 356), ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025 SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 357), GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEG SAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGGSGGS (SEQ ID NO: 358), EGGSEEEEESGS (SEQ ID NO: 468), or KGPKPKKEESEK (SEQ ID NO: 469).

In some embodiments, domains of the base editor are fused via a linker comprising the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 249), which may also be referred to as the XTEN linker. In some embodiments, a linker comprises the amino acid sequence SGGS (SEQ ID NO: 355). In some embodiments, the linker is 24 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPES (SEQ ID NO: 359). In some embodiments, the linker is 40 amino acids in length. In some embodiments, the linker comprises the amino acid sequence:

SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGS (SEQ ID NO: 360). In some embodiments, the linker is 64 amino acids in length. In some embodiments, the linker comprises the amino acid sequence:

SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGSSGSETPGTSESATPESSGGSSGGS

(SEQ ID NO: 361). In some embodiments, the linker is 92 amino acids in length. In some embodiments, the linker comprises the amino acid sequence:

PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTS TEPSEGSAPGTSESATPESGPGSEPATS (SEQ ID NO: 362).

In some embodiments, a linker comprises a plurality of proline residues and is 5-21, 5-14, 5-9, 5-7 amino acids in length, e.g., PAPAP (SEQ ID NO: 363), PAPAPA (SEQ ID NO: 364), PAPAPAP (SEQ ID NO: 365), PAPAPAPA (SEQ ID NO: 366), P(AP)₄ (SEQ ID NO: 367), P(AP)7 (SEQ ID NO: 368), P(AP)₁₀ (SEQ ID NO: 369) (see, e.g., Tan J, Zhang F, Karcher D, Bock R. Engineering of high-precision base editors for site-specific single nucleotide replacement. Nat Commun. 2019 Jan 25;10(l):439; the entire contents are incorporated herein by reference). Such proline-rich linkers are also termed “rigid” linkers.

In various embodiments, a linker of the disclosure comprises 8, 9, 10, 11, 12, 13, 14, or 15 amino acids. In some cases, the linker comprises a sequence selected from one or more of EGSSSKEEEEPG (SEQ ID NO: 647), NSISSSNGQK (SEQ ID NO: 648), GEEGEGSGGGEK (SEQ ID NO: 649), EGEGGKESGSSE (SEQ ID NO: 650), GGGGSSKSPGSE (SEQ ID NO: 651), PIGSDQDD (SEQ ID NO: 652), TEKGQVPHGS (SEQ ID NO: 653), ESGEGGGGSEKK (SEQ ID NO: 654), EEGKPKEGEGSG (SEQ ID NO: 655), ASREPKDSS (SEQ ID NO: 656), KQGSEHDE (SEQ ID NO: 657), SESKSEKGSSEK (SEQ ID NO: 658), QYDSGERSDQ (SEQ ID NO: 659), PGANEEIPGQ (SEQ ID NO: 660), NSPTDEK (SEQ ID NO: 661), EGANEEIPGQ (SEQ ID NO: ATTORNEY DOCKET NO. 180802-47402/PCT

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662), EGEKEKKKSGES (SEQ ID NO: 663), PGRHEEVPGQ (SEQ ID NO: 664), SKHQTEQDDS (SEQ ID NO: 665), ESEDDSSGRK (SEQ ID NO: 666), KESEKKESESKS (SEQ ID NO: 667), KGEGKSSIKD (SEQ ID NO: 668), DRSQKQDQQD (SEQ ID NO: 669), GPSSTSSS (SEQ ID NO: 670), GSSGEKEEGEPS (SEQ ID NO: 671), GEPKSKKSGSGS (SEQ ID NO: 672), SSGEGGKSESGP (SEQ ID NO: 673), SPQPTSSD (SEQ ID NO: 674), EGGSEEEEESGS (SEQ ID NO: 675), KGPKPKKEESEK (SEQ ID NO: 676), SKSQQFVTYE (SEQ ID NO: 677), TGNSKYQTGK (SEQ ID NO: 678), PQPIPHTNPT (SEQ ID NO: 679), ANAHSDISTG (SEQ ID NO: 680), KSQQTEDQSK (SEQ ID NO: 681), QSQDQKQKEH (SEQ ID NO: 682), NQQRPSSD (SEQ ID NO: 683), TTKDTSPKPQ (SEQ ID NO: 684), EGKDNQQTGE (SEQ ID NO: 685), or EPQPDSSE (SEQ ID NO: 686).

Nucleic Acid Programmable DNA Binding Proteins with Guide RNAs

Provided herein are compositions and methods for base editing in cells. Further provided herein are compositions comprising a guide polynucleotide sequence, e.g., a guide RNA sequence, or a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more guide RNAs as provided herein. In some embodiments, a composition for base editing as provided herein further comprises a polynucleotide that encodes a base editor, e.g., a C-base editor or an A-base editor. For example, a composition for base editing may comprise a mRNA sequence encoding a BE, a BE4, an ABE, and a combination of one or more guide RNAs as provided. A composition for base editing may comprise a base editor polypeptide and a combination of one or more of any guide RNAs provided herein. Such a composition may be used to effect base editing in a cell through different delivery approaches, for example, electroporation, nucleofection, viral transduction or transfection. In some embodiments, the composition for base editing comprises an mRNA sequence that encodes a base editor and a combination of one or more guide RNA sequences provided herein for electroporation.

Some aspects of this disclosure provide systems comprising any of the fusion proteins or complexes provided herein, and a guide RNA bound to a nucleic acid programmable DNA binding protein (napDNAbp) domain (e.g., a Cas9 (e.g., a dCas9, a nuclease active Cas9, or a Cas9 nickase) or Casl2) of the fusion protein or complex. These complexes are also termed ribonucleoproteins (RNPs). In some embodiments, the guide nucleic acid (e.g., guide RNA) is from 15-100 nucleotides long and comprises a sequence of at least 10 contiguous nucleotides that is complementary to a target sequence. In some embodiments, the target sequence is a DNA sequence. In some embodiments, the target sequence is an RNA sequence. In some embodiments, the target sequence is a sequence in the genome of a bacteria, yeast, fungi, insect, ATTORNEY DOCKET NO. 180802-47402/PCT

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plant, or animal. In some embodiments, the target sequence is a sequence in the genome of a human. In some embodiments, the 3' end of the target sequence is immediately adjacent to a canonical PAM sequence (NGG). In some embodiments, the 3' end of the target sequence is immediately adjacent to a non-canonical PAM sequence (e.g., a sequence listed in Table 3 or 5'-NAA-3'). In some embodiments, the guide nucleic acid (e.g., guide RNA) is complementary to a sequence in a gene of interest (e.g., a gene associated with a disease or disorder).

Some aspects of this disclosure provide methods of using the fusion proteins, or complexes provided herein. For example, some aspects of this disclosure provide methods comprising contacting a DNA molecule with any of the fusion proteins or complexes provided herein, and with at least one guide RNA, wherein the guide RNA is about 15-100 nucleotides long and comprises a sequence of at least 10 contiguous nucleotides that is complementary to a target sequence.

The domains of the base editor disclosed herein can be arranged in any order.

A defined target region can be a deamination window. A deamination window can be the defined region in which a base editor acts upon and deaminates a target nucleotide. In some embodiments, the deamination window is within a 2, 3, 4, 5, 6, 7, 8, 9, or 10 base regions. In some embodiments, the deamination window is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 bases upstream of the PAM.

The base editors of the present disclosure can comprise any domain, feature or amino acid sequence which facilitates the editing of a target polynucleotide sequence.

Methods of Using Fusion Proteins or Complexes Comprising a Cytidine or Adenosine Deaminase and a Cas9 Domain

Some aspects of this disclosure provide methods of using the fusion proteins, or complexes provided herein. For example, some aspects of this disclosure provide methods comprising contacting a DNA molecule with any of the fusion proteins or complexes provided herein, and with at least one guide RNA described herein.

In some embodiments, a fusion protein or complex of the disclosure is used for editing a target gene of interest. In particular, a cytidine deaminase or adenosine deaminase nucleobase editor described herein is capable of making multiple mutations within a target sequence. These mutations may affect the function of the target. For example, when a cytidine deaminase or adenosine deaminase nucleobase editor is used to target a regulatory region the function of the regulatory region is altered and the expression of the downstream protein is reduced or eliminated. ATTORNEY DOCKET NO. 180802-47402/PCT

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Base Editor Efficiency

In some embodiments, the purpose of the methods provided herein is to alter a gene and/or gene product via gene editing. The nucleobase editing proteins provided herein can be used for gene editing-based human therapeutics in vitro or in vivo. It will be understood by the skilled artisan that the nucleobase editing proteins provided herein, e.g., the fusion proteins or complexes comprising a polynucleotide programmable nucleotide binding domain (e.g., Cas9) and a nucleobase editing domain (e.g., an adenosine deaminase domain or a cytidine deaminase domain) can be used to edit a nucleotide from A to G or C to T.

Advantageously, base editing systems as provided herein provide genome editing without generating double-strand DNA breaks, without requiring a donor DNA template, and without inducing an excess of stochastic insertions and deletions as CRISPR may do. In some embodiments, the present disclosure provides base editors that efficiently generate an intended mutation, such as a STOP codon, in a nucleic acid (e.g., a nucleic acid within a genome of a subject) without generating a significant number of unintended mutations, such as unintended point mutations.

The base editors of the disclosure advantageously modify a specific nucleotide base encoding a protein without generating a significant proportion of indels (i.e., insertions or deletions). Such indels can lead to frame shift mutations within a coding region of a gene.

In some embodiments, the base editors provided herein are capable of generating a ratio of intended mutations to indels (i.e., intended point mutations:unintended point mutations) that is greater than 1: 1. In some embodiments, the base editors provided herein are capable of generating a ratio of intended mutations to indels that is at least 1.5:1, at least 2:1, at least 2.5:1, at least 3:1, at least 3.5:1, at least 4:1, at least 4.5:1, at least 5:1, at least 5.5:1, at least 6:1, at least 6.5:1, at least 7:1, at least 7.5:1, at least 8:1, at least 10:1, at least 12:1, at least 15:1, at least 20:1, at least 25:1, at least 30:1, at least 40:1, at least 50:1, at least 100:1, at least 200:1, at least 300:1, at least 400:1, at least 500:1, at least 600:1, at least 700:1, at least 800:1, at least 900:1, or at least 1000:1, or more. The number of intended mutations and indels may be determined using any suitable method.

In some embodiments, the base editors provided herein can limit formation of indels in a region of a nucleic acid. In some embodiments, the region is at a nucleotide targeted by a base editor or a region within 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of a nucleotide targeted by a base editor. In some embodiments, any of the base editors provided herein can limit the formation of indels at a region of a nucleic acid to less than 1%, less than 1.5%, less than 2%, less than 2.5%, ATTORNEY DOCKET NO. 180802-47402/PCT

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less than 3%, less than 3.5%, less than 4%, less than 4.5%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, less than 10%, less than 12%, less than 15%, or less than 20%.

Base editing is often referred to as a “modification”, such as, a genetic modification, a gene modification and modification of the nucleic acid sequence and is clearly understandable based on the context that the modification is a base editing modification. A base editing modification is therefore a modification at the nucleotide base level, for example as a result of the deaminase activity discussed throughout the disclosure, which then results in a change in the gene sequence and may affect the gene product.

In some embodiments, the modification, e.g., single base edit results in about or at least about a 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% reduction, or reduction to an undetectable level, of the gene targeted expression.

The disclosure provides adenosine deaminase variants (e.g, ABE8 variants) that have increased efficiency and specificity. In particular, the adenosine deaminase variants described herein are more likely to edit a desired base within a polynucleotide and are less likely to edit bases that are not intended to be altered (e.g, “bystanders”).

In some embodiments, any of the base editing system comprising one of the ABE8 base editor variants described herein has reduced bystander editing or mutations by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% compared to a base editor system comprising an ABE7 base editor, e.g., ABE7.10.

In some embodiments, any of the ABE8 base editor variants described herein has higher base editing efficiency compared to the ABE7 base editors. In some embodiments, any of the ABE8 base editor variants described herein have at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 450%, or 500% higher base editing efficiency compared to an ABE7 base editor, e.g., ABE7.10.

The ABE8 base editor variants described herein may be delivered to a host cell via a plasmid, a vector, a LNP complex, or an mRNA. In some embodiments, any of the ABE8 base editor variants described herein is delivered to a host cell as an mRNA. ATTORNEY DOCKET NO. 180802-47402/PCT

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In some embodiments, the method described herein, for example, the base editing methods has minimum to no off-target effects. In some embodiments, the method described herein, for example, the base editing methods, has minimal to no chromosomal translocations.

In some embodiments, the base editing method described herein results in about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of a cell population that have been successfully edited.

In some embodiments, the percent of viable cells in a cell population following a base editing intervention is greater than at least 60%, 70%, 80%, or 90% of the starting cell population at the time of the base editing event. In some embodiments, the percent of viable cells in a cell population following editing is about 70%. In some embodiments, the percent of viable cells in a cell population following editing is about 75%. In some embodiments, the percent of viable cells in a cell population following editing is about 80%. In some embodiments, the percent of viable cells in a cell population as described above is about 85%. In some embodiments, the percent of viable cells in a cell population as described above is about 90%, or about 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99%, or 100% of the cells in the population at the time of the base editing event.

In embodiments, the cell population is a population of cells contacted with a base editor, complex, or base editor system of the present disclosure.

The number of intended mutations and indels can be determined using any suitable method, for example, as described in International PCT Application Nos. PCT/US2017/045381 (WO2018/027078) and PCT/US2016/058344 (WO2017/070632); Komor, A. C., etal, “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016); Gaudelli, N. M., etal., “Programmable base editing of A»T to G»C in genomic DNA without DNA cleavage” Nature 551, 464-471 (2017); and Komor, A. C., et al, “Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C: G-to-T: A base editors with higher efficiency and product purity” Science Advances 3:eaao4774 (2017); the entire contents of which are hereby incorporated by reference.

In some embodiments, to calculate indel frequencies, sequencing reads are scanned for exact matches to two 10-bp sequences that flank both sides of a window in which indels can occur. If no exact matches are located, the read is excluded from analysis. If the length of this indel window exactly matches the reference sequence the read is classified as not containing an indel. If the indel window is two or more bases longer or shorter than the reference sequence, then the sequencing read is classified as an insertion or deletion, respectively. In some embodiments, the base editors provided herein can limit formation of indels in a region of a ATTORNEY DOCKET NO. 180802-47402/PCT

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nucleic acid. In some embodiments, the region is at a nucleotide targeted by a base editor or a region within 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of a nucleotide targeted by a base editor.

Expression of Fusion Proteins or Complexes in a Host Cell

Fusion proteins or complexes of the disclosure comprising a deaminase may be expressed in virtually any host cell of interest, including but not limited to bacteria, yeast, fungi, insects, plants, and animal cells using routine methods known to the skilled artisan.

A polynucleotide encoding a polypeptide described herein can be obtained by chemically synthesizing the polynucleotide, or by connecting synthesized partly overlapping oligo short chains by utilizing the PCR method and the Gibson Assembly method to construct a polynucleotide (e.g., DNA) encoding the full length thereof. The advantage of constructing a full-length polynucleotide by chemical synthesis or a combination of PCR method or Gibson Assembly method is that the codons to be used can be selected according to the host into which the polynucleotide is to be introduced. In the expression from a heterologous DNA molecule, the protein expression level is expected to increase by converting the DNA sequence thereof to a codon highly frequently used in the host organism. Codon use data for a host cell (e.g., codon use data available at kazusa.or.jp/codon/index.html) can be used to guide codon optimization for a polynucleotide sequence encoding a polypeptide. Codons having low use frequency in the host may be converted to a codon coding the same amino acid and having high use frequency.

An expression vector containing a polynucleotide encoding a base editor system of the disclosure or a component thereof (e.g., a base editor fusion protein) can be produced, for example, by linking the DNA to the downstream of a promoter in a suitable expression vector.

Anypromoter appropriate for a host to be used for gene expression can be used. For example, when the host is an animal cell, an SR.alpha. promoter, SV40 promoter, LTR promoter, cytomegalovirus (CMV) promoter, Rous sarcoma virus (RSV) promoter, Moloney mouse leukemia virus (MoMuLV), LTR, herpes simplex virus thymidine kinase (HSV-TK) promoter, and the like can be used. Of these, CMV promoter, SR.alpha. promoter and the like may be used.

Expression vectors for use in the present disclosure, besides those mentioned above, can comprise an enhancer, a splicing signal, a terminator, a polyA addition signal, a selection marker such as drug resistance gene, an auxotrophic complementary gene and the like, a replication origin, and the like can be used.

An RNA encoding a protein domain described herein can be prepared by, for example, in vitro transcription of a nucleic acid sequence encoding any of the fusion proteins or complexes disclosed herein. ATTORNEY DOCKET NO. 180802-47402/PCT

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Animal cells contemplated in the present disclosure include, but are not limited to, cell lines such as monkey COS-7 cells, monkey Vero cells, Chinese hamster ovary (CHO) cells, dhfr gene-deficient CHO cells, mouse L cells, mouse AtT-20 cells, mouse myeloma cells, rat GH3 cells, human FL cells and the like, pluripotent stem cells such as iPS cells, ES cells derived humans and other mammals, and primary cultured cells prepared from various tissues.

Furthermore, zebrafish embryo, Xenopus oocyte, and the like can also be used.

An expression vector can be introduced by a known method (e.g, the lysozyme method, the competent method, the PEG method, the CaCh coprecipitation method, electroporation, microinjection, particle gun method, lipofection, dg/ /cVe/v/z/zz-rnediated delivery, etc.) according to the kind of the host.

A vector can be introduced into an animal cell according to the methods described in, for example, Cell Engineering additional volume 8, New Cell Engineering Experiment Protocol, 263-267 (1995) (published by Shujunsha), and Virology, 52, 456 (1973).

A cell comprising a vector can be cultured according to a known method according to the kind of the host.

DELIVERY SYSTEMS

Nucleic Acid-Based Delivery of Base Editor Systems

Nucleic acid molecules encoding a base editor system according to the present disclosure can be administered to subjects or delivered into cells in vitro or in vivo by art-known methods or as described herein. For example, a base editor system comprising a deaminase (e.g., cytidine or adenine deaminase) can be delivered by vectors (e.g., viral or non-viral vectors), or by naked DNA, DNA complexes, lipid nanoparticles, or a combination of the aforementioned compositions. A base editor system may be delivered to a cell using any methods available in the art including, but not limited to, physical methods (e.g., electroporation, particle gun, calcium phosphate transfection), viral methods, non-viral methods (e.g., liposomes, cationic methods, lipid nanoparticles, polymeric nanoparticles), or biological non-viral methods (e.g., attenuated bacterial, engineered bacteriophages, mammalian virus-like particles, biological liposomes, erythrocyte ghosts, exosomes).

Nanoparticles, which can be organic or inorganic, are useful for delivering a base editor system or component thereof. Nanoparticles are well known in the art and any suitable nanoparticle can be used to deliver a base editor system or component thereof, or a nucleic acid molecule encoding such components. In one example, organic (e.g, lipid and/or polymer) nanoparticles are suitable for use as delivery vehicles in certain embodiments of this disclosure. ATTORNEY DOCKET NO. 180802-47402/PCT

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Non-limiting examples of lipid nanoparticles suitable for use in the methods of the present disclosure include those described in International Patent Application Publications No.

WO 2025/038864, WO 2025/038855, WO 2024/019936, WO 2023/121975, WO 2023/121964, WO 2023/121971, WO 2023/121970, WO 2022/140239, WO 2022/140252, WO 2022/140238, WO 2022/159421, WO 2022/159472, WO 2022/159475, WO 2022/159463, WO 2021/113365, WO 2024/019936, and WO 2021/141969, the disclosures of each of which is incorporated herein by reference in its entirety for all purposes.

Lipid Nanoparticle (LNP) Compositions

The pharmaceutical compositions for gene modification described herein may be encapsulated in a lipid nanoparticle (LNP). LNP compositions or formulations, as contemplated herein, are typically sized on the order of micrometers or smaller and may include a lipid bilayer. Nanoparticle compositions encompass lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes. For example, a nanoparticle composition or formulation as contemplated herein may be a liposome having a lipid bilayer with a diameter of 500 nm or less. A LNP as described herein may have a mean diameter of from about 20 nm to about 1000 nm, from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 50 nm to 90 nm, from about 55 nm to 85 nm, from about 55 nm to 75 nm, from about 50 nm to about 80 nm, from about 60 nm to about 80 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, or from about 70 nm to about 80 nm. The LNPs described herein can have a mean diameter of about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, or greater. In one embodiment the mean diameter of the LNP is about 70 nm +/- 10 nm, 70 nm +/- 5 nm, 65 nm +/- 10 nm, 65 nm +/- 5 nm, 60 nm +/- 10 nm, or 60 nm +/- 5 nm.

Described herein are LNP compositions comprising an ionizable lipid, a phospholipid, a conjugate-linker lipid (e.g., PEG lipid), a sterol (e.g., cholesterol), or a derivative thereof, a payload, or any combination thereof. Each component is described in more detail below.

In the preparation of LNP compositions comprising the ionizable lipid, phospholipid, conjugate-linker lipid, and sterol, a desired molar ratio of the four excipients may be dissolved in a water miscible organic solvent, such as ethanol. The homogenous lipid solution may then be rapidly in-line mixed with an aqueous buffer containing nucleic acid payload to form the lipid ATTORNEY DOCKET NO. 180802-47402/PCT

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nanoparticle encapsulating the nucleic acid payload(s). After rapid in-line mixing, the LNPs thus formed may undergo further downstream processing including concentration and buffer exchange to achieve the final LNP pharmaceutical composition with near neutral pH for administration into cell line or animal diseases model for evaluation, or to administer to human subjects.

The LNP payload comprises a guide RNA targeting the GALT gene and an mRNA encoding a base editor protein. In some embodiments, the guide RNA to mRNA ratio in the acidic aqueous buffer and in the final formulation is 6:1, 5:1, 4:1, 3:1, 2.5:1, 2:1, 1.5:1, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:5 or 1:6 by weight. In some embodiments, the guide RNA to mRNA ratio in the acidic aqueous buffer and in the final formulation is about 1: 1 by weight. In some embodiment the mRNA encodes an adenosine base editor protein. In some other embodiments the mRNA encodes cytosine or a cytidine base editor protein.

Exemplary LNP compositions are described in Example 2 below.

Ionizable lipids

Among other things, the present disclosure describes compositions, preparations, nanoparticles, and/or nanomaterials that comprise one or more ionizable lipids as described herein. An ionizable lipid as used herein comprises one or more ionizable nitrogen atoms or amine containing groups. In some embodiments, the ionizable nitrogen atom or amine containing group is on the head group of the ionizable lipid.

For example, in some embodiments, compositions, preparations, nanoparticles, and/or nanomaterials having an ionizable lipid that is at about 50 mole percent or less, based on total moles of components of the lipid nanoparticle, may be useful for functional activity of lipid nanoparticles such as desired tropisms, stabilization, and drug delivery efficacy as described herein.

In some embodiments, an ionizable lipid is or comprises a compound described herein In some embodiments, an ionizable lipid is present in a lipid nanoparticle (LNP) preparation from about 30 mole percent to about 70 mole percent, based on total moles of components of the lipid nanoparticle. In some embodiments, an ionizable lipid is present from about 33 mole percent to about 60 mole percent, based on total moles of components of the lipid nanoparticle. In some embodiments, an ionizable lipid is present from about 34 mole percent to about 55 mole percent, based on total moles of components of the lipid nanoparticle. In some embodiments, an ionizable lipid is present from about 33 mole percent to about 51 mole percent, based on total moles of components of the lipid nanoparticle. In some embodiments, an ionizable lipid is ATTORNEY DOCKET NO. 180802-47402/PCT

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present at about 34.7 mole percent, based on total moles of components of the lipid nanoparticle. In some embodiments, an ionizable lipid is present at about 50 mole percent, based on total moles of components of the lipid nanoparticle. In some embodiments, an ionizable lipid is present at about 40 mole percent to about 60 mole percent or about 45 mole percent to about 55 mole percent, based on total moles of components of the lipid nanoparticle

Exemplary, non-limiting ionizable lipids suitable for the compositions described herein include those described herein.

Exemplary Lipids of WO2022140252

In another aspect, an ionizable lipid is according to any formula or structure, or a pharmaceutically acceptable salt or solvate thereof, as described in International Publication No. WO2022140252, which is hereby incorporated by reference in its entirety. In some embodiments, an ionizable lipid has a structure according to any of Formulae A’, A, I”, I’, I, II”, IF, II, III’, III, r-a, I’-a, I-a, r-a-z, F’-a-zz, I”-a-zzz, I”-b, I’-b, I-b, I”-b-z, I”-b-zz, I”-b-zzz, I”-c, I’-c, I-c, I”-c-z, I”-c-zz, I”-c-zzz, I’-d, I-d, I’-d-z, Il-a, Il-a-z, Ill-a, and III-a-z of WO2022140252, or a pharmaceutically acceptable salt or solvate thereof. Exemplary ionizable lipids also include any of the lipids of Table 1 of WO2022140252, including any of the lipids represented by Examples 7-1 to 7-253 and Examples 8-1 to 8-106, or a pharmaceutically acceptable salt or solvate thereof.

In some embodiments, an ionizable lipid is according to Formula A’ of WO2022140252:

J ³

O R

1 /^L- y c) R

L²

R¹ hE. R"

A’

or its N-oxide, or a pharmaceutically acceptable salt thereof, wherein

L¹ is absent, Ci-6 alkylenyl, or C2-6 heteroalkylenyl;

L is Ci-10 alkylenyl, or C2-10 heteroalkyl enyl;

X² is -OC(O)-, -C(O)O-, or -OC(O)O-;

X is absent, -OC(O)-, -C(O)O-, or -OC(O)O-; ATTORNEY DOCKET NO. 180802-47402/PCT

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O

L^3a I

O'" ^R^a %

s ' L'pa

R” is hydrogen,

O R, O R⁶, or an optionally substituted group selected from C6-20 aliphatic, 3- to 12-membered cycloaliphatic, 7- to 12-membered bridged bicyclic comprising 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 1- adamantyl, 2-adamantyl, sterolyl, and phenyl;

each of R and R^a is independently hydrogen, or an optionally substituted group selected from Ce- 20 aliphatic, 3- to 12-membered cycloaliphatic, 7- to 12-membered bridged bicyclic comprising 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 1- adamantyl, 2-adamantyl, sterolyl, and phenyl

each of L³ and L^3a is independently absent, optionally substituted Ci-io alkylenyl, or optionally substituted C2-10 heteroalkylenyl;

R¹ is hydrogen, optionally substituted phenyl, optionally substituted 3- to 7-membered cycloaliphatic, optionally substituted 3- to 7-membered heterocyclyl comprising 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, optionally substituted 5- to 6-membered monocyclic heteroaryl comprising 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, optionally substituted 8- to 10-membered bicyclic heteroaryl comprising 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, -OR², -C(O)OR², -C(O)SR², -OC(O)R², -OC(O)OR², -CN,

-N(R²)₂, -C(O)N(R²)₂, -S(O)₂N(R²)₂, -NR²C(O)R², -OC(O)N(R²)₂, -N(R²)C(O)OR², -NR²S(O)₂R², -NR²C(O)N(R²)2, -NR²C(S)N(R²)2, -NR²C(NR²)N(R²)2, -NR²C(CHR²)N(R²)2, -N(OR²)C(O)R², -N(OR²)S(O)₂R², -N(OR²)C(O)OR², -N(OR²)C(O)N(R²)2, -N(OR²)C(S)N(R²)2, -N(OR²)C(NR²)N(R²)2, -N(OR²)C(CHR²)N(R²)2, -C(NR²)N(R²)2, -C(NR²)R², -C(O)N(R²)OR², -C(R²)N(R²)₂C(O)OR², -CR²(R³)2, -OP(O)(OR²)2, or -P(O)(OR²)₂; or

R² R²

/ \

R

¹ is ⁰ O, or a ring selected from 3- to 7-membered cycloaliphatic and 3- to 7- membered heterocyclyl comprising 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, wherein the cycloaliphatic or heterocyclyl ring is optionally substituted with 1-4 R² or R³ groups; ATTORNEY DOCKET NO. 180802-47402/PCT

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each R² is independently hydrogen, oxo, -CN, -NO2, -OR⁴, -S(O)2R⁴, -S(O)2N(R⁴)2, -(CH2)n-R⁴, or an optionally substituted group selected from C1-6 aliphatic, phenyl, 3- to 7-membered cycloaliphatic, 5- to 6-membered monocyclic heteroaryl comprising 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and 3- to 7-membered heterocyclyl comprising 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; or two occurrences of R², taken together with the atom(s) to which they are attached, form optionally substituted 4- to 7-membered heterocyclyl comprising 0-1 additional heteroatom selected from nitrogen, oxygen, and sulfur;

each R³ is independently -(CH2)n-R⁴; or

two occurrences of R³, taken together with the atom(s) to which they are attached, form optionally substituted 5- to 6-membered heterocyclyl comprising 0-1 additional heteroatom selected from nitrogen, oxygen, and sulfur;

R⁵ R⁵

/ \

_XN N-A.

^r W *

-

NR⁵C(S)N(R⁵)2, -NR⁵C(NR⁵)N(R⁵)2, or O — K O _;

two occurrences of R⁵, taken together with the atom(s) to which they are attached, form optionally substituted 4- to 7-membered heterocyclyl comprising 0-1 additional heteroatom selected from nitrogen, oxygen, and sulfur;

each R⁶ is independently C4-12 aliphatic; and

each n is independently 0 to 4.

In some embodiments, an ionizable lipid is according to Formula Ill-a of WO2022140252:

°Y^LY°'IX^R

_LY °'Y

^R 1 Ji.0^

V^N'iZ Y o Y O.._L,_Z ^LR'

Ill-a

or its N-oxide, or a pharmaceutically acceptable salt thereof, wherein each of R, R¹, L, L¹, L², L³ is as defined therein for any of Formulae A’, A, III’, and III, and described in classes and subclasses above and herein, both singly and in combination. In embodiments of Formula Ill-a, each of R, R¹, L, L¹, L², L³ is as defined herein for Formula A’ above. ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

In some embodiments, an ionizable lipid is according to Formula III-a-z of WO2022140252:

^QY^LY°'R

|_2 R

, Ji.0.,

^RV^N'Y Y Y ^r

⁰ °'R

III-a-z’

or its N-oxide, or a pharmaceutically acceptable salt thereof, wherein each of R, R¹, L, L¹, and L² is as defined therein for any of Formulae A’, A, III’, and III, and described in classes and subclasses above and herein, both singly and in combination. In embodiments of Formula III-a-z, each of R, R¹, L, L¹, and L² is as defined herein for Formula A’ above.

In some embodiments, an ionizable lipid is selected from any of the lipids described in Table 1 of WO2022140252, or its N-oxide, or a pharmaceutically acceptable salt thereof. In embodiments, an ionizable lipid is selected from the group consisting of:

ATTORNEY DOCKET NO. 180802-47402/PCT

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ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

8 X i- o55 8-57

Q'^xX^x/=\/ z — <

J ⁰ J o

1 ⁰

HO. \Z \z _N. x/ \Z o" \Z /x Y.0. \Z /x X. Z /x -. - _ /x -.

< O _H0 ^/xx-' ^N — - — ^°Y^z\^_C;^X^x^/\ < O

0^ /\ Z\ _ X\ 0

8-58 p < < o 8-59

1 < >^o-_x O^^sx/\/^^s^'

10

° / \ / J o

[

0

< n

8-60 8-61

i O _O'^Xx^\X=\x^'

— -'P)^^xxx^xxxx'^^'xx^ H Z - V

< O

< > < o

_HO^ - - -X - ^_oA^A_o-^x^x^ " p A

8-62 8-63 ^z ° /

y 0°⁼ \

11 p₀A^ \ a °x^^_^ 2 o \ (o o=—

J Cx /X /X_/\

x

0

o 7-232

X

o p₀A^°x^/^^

HOx^/Xxz- O

7-233 7-234

o''"'' —

J °

^H °-x^\x ^N

o

7-235 7-236

ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

7-237 7-238 c °

( O

QXXXZ^X^/

X.z^XzX /^

J ⁰

[ ⁰

7-239

0

i

o

1 \

_HO ^{0 0}

-^xx N X/X/XZZXQ zAxzy C'XZXXZX ^H N A^ ^

8-67 / \ ° ° — 8-68

^z ° /

o \° ^X 0 \

p_oA^< /^{o o==} zyzO_zx_z^

J < X zx _ z-x J ^{/ q} / \ <o o= <—X z-X _ X

[ ⁰

_HO z-\ Xz z- N v x/zX\zzX\zzX ₀,. A \z zX\zzXyz°^ -zX - z --\

< X z-x _ x (K z^X _ zZX

8-69 8-70

o

I 0

p_c. A^/yO^zxx-X/ p_C)A^zyO_z^z^z J ^CXZXZ=X/

r ⁰

HO zx — zC x/zX\zzX\z/X ₀„A x^ zXx.z^Xyz°\ Xzz-'XX./Z'=\ xz/ ^HO\x^z^ N A^\z^/^°^^^xZ 'XX/^Xz- 8-71 8-72

7-243 7-244

ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

In some embodiments, an ionizable lipid is Example 7-1, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 7-2, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 7-19, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 7-20, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 7-22, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 7-24, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 7-25, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 8-1, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 8-2, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 8-3, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 8-4, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 8-5, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 8-13, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 8-14, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 8-17, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 8-18, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 8-19, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 8-20, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 8-55, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 8-57, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 8-58, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 8-59, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 8-60, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 8-61, or a pharmaceutically acceptable salt ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

thereof. In some embodiments, an ionizable lipid is Example 8-62, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 8-63, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 7-232, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 7-233, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 7-234, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 7-235, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 7-236, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 7-237, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 7-238, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 7-239, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 8-67, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 8-68, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 8-69, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 8-70, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 8-71, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 8-72, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 7-243, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 7-244, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 7-245, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 7-246, or a pharmaceutically acceptable salt thereof.

Exemplary Lipids of WO2022159472

In another aspect, an ionizable lipid is according to any formula or structure, or a pharmaceutically acceptable salt or solvate thereof, as described in International Publication No. WO2022159472, which is hereby incorporated by reference in its entirety. In some embodiments, an ionizable lipid has a structure according to any of Formulae I, II, III, IIIA, IIIB, IIIC, IV, V, VA, VI, VIA, VII, and VIIA of WO2022159472, or a pharmaceutically acceptable salt or solvate thereof. Exemplary ionizable lipids also include any of the lipids of Table 1 of WO2022159472, including any of the lipids represented by Examples 4-1 to 4-86, or a pharmaceutically acceptable salt or solvate thereof.

In some embodiments, an ionizable lipid is according to Formula I of WO2022159472: ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

or a pharmaceutically acceptable salt thereof, wherein:

L¹ is a covalent bond, -C(O)-, or -OC(O)-;

branched C1-C12 hydrocarbon chain, or ^{m m};

each m is independently 0, 1, or 2;

L³ is a covalent bond, -C(O)-, -C(O)O-, -OC(O)-, -O-, or -OC(O)O-;

R is

^p, an optionally substituted saturated or unsaturated, straight or branched

C1-C20 hydrocarbon chain wherein 1-3 methylene units are optionally and independently, O-A¹

H^L ₂

replaced with -O- or -NR-, or O^_A;

unsaturated carbocyclyl, 1-adamantyl, 2-adamantyl,

, sterolyl, and phenyl; p is 0, 1, 2, or 3;

^#H2) X.

where

x is selected from 1 or 2; and

# represents the point of attachment to L⁴;

each L⁵ is independently a bivalent saturated or unsaturated, straight or branched C1-C20 hydrocarbon chain, wherein 1-3 methylene units are optionally and independently replaced with -O- or -NR.-; ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

X¹ is a covalent bond, -O-, or -NR-;

X² is a covalent bond or an optionally substituted, bivalent saturated or unsaturated, straight or branched, C1-C12 hydrocarbon chain, wherein 1-3 methylene units are optionally and independently replaced with -O-, -NR-, or -Cy^c-;

FH-cy^B

provided that when L is a covalent bond, then R must be ^p

In some embodiments, an ionizable lipid is according to Formula VI of WO2022159472:

'O'

Al

A².

O'

or a pharmaceutically acceptable salt thereof, wherein n is 1, 2, 3 or 4, and L², R¹, A¹, A², X¹, X², and X³ are as defined therein for Formula I and also described in classes and subclasses therein, both singly and in combination. In embodiments, L², R¹, A¹, A², X¹, X², and X³ are as defined herein for Formula I above.

In some embodiments, an ionizable lipid is according to Formula VIA of WO2022159472:

'O'

A¹

A²

O'

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ELECTRONIC DEPOSIT DATE: December 10, 2025

or a pharmaceutically acceptable salt thereof, wherein n is 1, 2, 3 or 4, and L², R¹, A¹, A², X², and X³ are as defined therein for Formula I and also described in classes and subclasses therein, both singly and in combination. In embodiments, L², R¹, A¹, A², X², and X³ are as defined herein for Formula I above.

In some embodiments, an ionizable lipid is selected from any of the lipids described in Table 1 of WO2022159472, or a pharmaceutically acceptable salt thereof. In embodiments, an ionizable lipid is selected from the group consisting of:

Example 4-62

Example 4-63

Example 4-64

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Example 4-68

Example 4-69

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Example 4-73

Example 4-74

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Example 4-76

Example 4-79

ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

Example 4-80

Example 4-81

Example 4-82

Example 4-83

ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

Example 4-84

Example 4-85

Example 4-86

In some embodiments, an ionizable lipid is Example 4-62, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 4-63, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 4- 64, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 4-65, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 4-66, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 4-67, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 4-68, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 4-69, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 4-70, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 4-71, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 4- 72, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 4-73, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 4-74, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 4-75, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 4-76, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 4-77, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 4-78, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 4-79, or a ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 4-80, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 4-81, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 4-82, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 4-83, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 4-84, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 4-85, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 4-86, or a pharmaceutically acceptable salt thereof.

Exemplary Lipids of WO 2024/019936

In another aspect, an ionizable lipid is according to any formula or structure, or a pharmaceutically acceptable salt or solvate thereof, as described in WO 2024/019936, which is hereby incorporated by reference in its entirety. In some embodiments, an ionizable lipid has a structure according to any of Formulae I of WO 2024/019936, or a pharmaceutically acceptable salt or solvate thereof. Exemplary ionizable lipids also include any of the lipids represented by the Examples of WO 2024/019936.

In some embodiments, an ionizable lipid is according to a compound of Formula I of WO 2024/019936:

or a pharmaceutically acceptable salt thereof, wherein:

each L¹ and L^1' is independently -C(O)- or -OC(O)-;

L³ is a covalent bond, -O-, -C(O)O-, -OC(O)-, or -OC(O)O-;

O-R^a

|__LCyA__c A M

R¹ is optionally substituted C1-20 aliphatic, » ^y, or O-R^a;

Cy^A is an optionally substituted ring selected from 3- to 7-membered saturated or partially unsaturated carbocyclyl, 1-adamantyl, 2-adamantyl, sterolyl, and phenyl; ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

each R^a and R¹ is independently optionally substituted C1-20 aliphatic;

Y¹ is -C(O)- or -C(O)O-;

Y³ is optionally substituted C1-20 aliphatic;

X¹ is a covalent bond, -O-, or -NR-;

X² is an optionally substituted bivalent saturated or unsaturated, straight or branched C1-12 hydrocarbon chain, wherein 1-3 methylene units are optionally and independently replaced with -O- or -NR-;

each R is independently hydrogen or optionally substituted C1-6 aliphatic.

In some embodiments, the present disclosure provides a compound of Formula I of WO 2024/019936, wherein:

each L¹ and L¹ is -C(O)-;

each L² and L² is independently a bivalent saturated or unsaturated, straight or branched C1-12 hydrocarbon chain;

L³ is a covalent bond, -C(O)O-, or -OC(O)-;

, O-R^a

|__L ^Ra_^

R¹ is C1-20 alkyl, C2-20 alkenyl, o

r O-R^a;

each L^Ra and R¹ is independently C1-20 alkyl or C2-20 alkenyl;

Y¹ is -C(O)- or -C(O)O-;

Y² is a bivalent saturated or unsaturated, straight or branched C1-6 hydrocarbon chain;

Y³ is Ci -20 alkyl or C2-20 alkenyl;

X¹ is -O- or -NR-;

X² is a bivalent saturated or unsaturated, straight or branched C1-12 hydrocarbon chain, wherein 1-3 methylene units are optionally and independently replaced with -NR-;

X³ is hydrogen or a 3- to 7-membered heterocyclyl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur and optionally substituted with C1-6 alkyl or C2-6 alkenyl; and ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

each R is independently hydrogen or Ci-6 alkyl or C2-6 alkenyl.

In some embodiments, the present disclosure provides a compound of Formula II of WO 2024/019936:

0

or a pharmaceutically acceptable salt thereof, wherein each of L², L², L³, R¹, R¹, Y¹, Y², Y³, X¹, X², and X³ is as described above and in classes and subclasses herein, both singly and in combination.

In some embodiments, the present disclosure provides a compound of Formula III of WO 2024/019936:

O

or a pharmaceutically acceptable salt thereof, wherein each of L¹, L², L², L³, R¹, R¹, Y², Y³, X¹, X², and X³ is as described above and in classes and subclasses herein, both singly and in combination.

In some embodiments, the present disclosure provides a compound of Formula III-A of WO 2024/019936:

O

or a pharmaceutically acceptable salt thereof, wherein each of L¹, L², L², L³, R¹, R¹, Y², Y³, X², and X³ is as described above and in classes and subclasses herein, both singly and in combination.

In some embodiments, the present disclosure provides a compound of Formula III-B of WO 2024/019936: ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

0

or a pharmaceutically acceptable salt thereof, wherein each of L¹, L², L², L³, R¹, R¹, R, Y², Y³, X², and X³ is as described above and in classes and subclasses herein, both singly and in combination.

In some embodiments, the present disclosure provides a compound of Formula III-B-z of WO 2024/019936:

0

In some embodiments, the present disclosure provides a compound of Formula IV of WO 2024/019936:

O

In some embodiments, the present disclosure provides a compound of Formula IV-A of WO 2024/019936:

O

ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

In some embodiments, the present disclosure provides a compound of Formula IV-B of WO 2024/019936:

O

In some embodiments, the present disclosure provides a compound of Formula IV-B-z of WO 2024/019936:

O

In some embodiments, the present disclosure provides a compound of Formula V of WO 2024/019936:

In some embodiments, the present disclosure provides a compound of Formula V-A of WO 2024/019936: ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

In some embodiments, the present disclosure provides a compound of Formula V-B of WO 2024/019936:

In some embodiments, the present disclosure provides a compound of Formula V-B-z of WO 2024/019936:

In some embodiments, the present disclosure provides a compound of Formula VI of WO 2024/019936:

or a pharmaceutically acceptable salt thereof, wherein each of L¹, L², L², L³, R¹, R¹, Y², Y³, X¹, X², and X³ is as described above and in classes and subclasses herein, both singly and in combination. ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

In some embodiments, the present disclosure provides a compound of Formula VI-A of WO 2024/019936:

In some embodiments, the present disclosure provides a compound of Formula VI-B of WO 2024/019936:

In some embodiments, the present disclosure provides a compound of Formula VI-B-z of WO 2024/019936:

In some embodiments, the present disclosure provides a compound of Formula VII of WO 2024/019936:

O

ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

or a pharmaceutically acceptable salt thereof, wherein each of L², L², L³, R¹, R¹, Y², Y³, X¹, X², and X³ is as described above and in classes and subclasses herein, both singly and in combination.

In some embodiments, the present disclosure provides a compound of Formula VILA of WO 2024/019936:

_R<^L-_L2^o

1 ^R,;o 2

' I A ^L

(VILA)

or a pharmaceutically acceptable salt thereof, wherein each of L², L², L³, R¹, R¹, Y², Y³, X², and X³ is as described above and in classes and subclasses herein, both singly and in combination.

In some embodiments, the present disclosure provides a compound of Formula VII-B of WO 2024/019936:

_R<^L<_L2^O

^R,;o 2

1' I A ^L

(VILB)

or a pharmaceutically acceptable salt thereof, wherein each of L², L², L³, R¹, R¹, R, Y², Y³, X², and X³ is as described above and in classes and subclasses herein, both singly and in combination.

In some embodiments, the present disclosure provides a compound of Formula VILB-z of WO 2024/019936:

_R<^L<_L2^< O

1 ^R,;o 2

' I A ^L

(vn-B-z)

In some embodiments, the present disclosure provides a compound of Formula VIII of WO 2024/019936: ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

o

In some embodiments, the present disclosure provides a compound of Formula VIII-A of WO 2024/019936:

O

(VIII-A)

In some embodiments, the present disclosure provides a compound of Formula VIII-B of WO 2024/019936:

O

or a pharmaceutically acceptable salt thereof, wherein each of L², L², L³, R¹, R¹, Y², Y³, R, X², and X³ is as described above and in classes and subclasses herein, both singly and in combination.

In some embodiments, the present disclosure provides a compound of Formula VIII-B-z of WO 2024/019936:

(VIII-B-z) ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

In some embodiments, the present disclosure provides a compound of Formula IX of WO 2024/019936:

or a pharmaceutically acceptable salt thereof, wherein each of L², L², R¹, R¹, Y², Y³, X¹, X², and X³ is as described above and in classes and subclasses herein, both singly and in combination.

In some embodiments, the present disclosure provides a compound of Formula IX-A of WO 2024/019936:

or a pharmaceutically acceptable salt thereof, wherein each of L², L², R¹, R¹, Y², Y³, X², and X³ is as described above and in classes and subclasses herein, both singly and in combination.

In some embodiments, the present disclosure provides a compound of Formula IX-B of WO 2024/019936:

or a pharmaceutically acceptable salt thereof, wherein each of L², L², R¹, R¹, R, Y², Y³, X², and X³ is as described above and in classes and subclasses herein, both singly and in combination. ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

In some embodiments, the present disclosure provides a compound of Formula IX-B-z of WO 2024/019936:

In some embodiments, the present disclosure provides a compound of Formula X of WO 2024/019936:

In some embodiments, the present disclosure provides a compound of Formula X-A of WO 2024/019936:

O

In some embodiments, the present disclosure provides a compound of Formula X-B of WO 2024/019936: ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

R

N., X³

^X²

(X-B)

or a pharmaceutically acceptable salt thereof, wherein each of L², L², R¹, R¹, R, Y², Y³, X², and X³ is as described above and in classes and subclasses herein, both singly and in combination.

In some embodiments, the present disclosure provides a compound of Formula X-B-z WO 2024/019936:

X³

(X-B-z)

In some embodiments of any Formulae of WO 2024/019936 described herein, L¹ is -C(O)- or -OC(O)-. In some embodiments, L¹ is -C(O)-. In some embodiments, L¹ is -OC(O)-.

In some embodiments, the present disclosure provides a compound of Formula I of WO 2024/019936, wherein: each L¹ and L¹ is -C(O)-.

In some embodiments, the present disclosure provides a compound of Formula I of WO 2024/019936, wherein: each L² and L² is independently a bivalent saturated or unsaturated, straight or branched C1-12 hydrocarbon chain.

In some embodiments of any Formulae of WO 2024/019936 described herein, L² is an optionally substituted bivalent saturated or unsaturated, straight or branched C1-12 hydrocarbon chain. In some embodiments, L² is an optionally substituted bivalent saturated or unsaturated, straight or branched C4-8 hydrocarbon chain. In some embodiments, L² is an optionally substituted bivalent saturated or unsaturated, straight or branched C1-6 hydrocarbon chain. In some embodiments, L² is an optionally substituted bivalent saturated, straight or branched C1-12 hydrocarbon chain. In some embodiments, L² is an optionally substituted bivalent saturated, ATTORNEY DOCKET NO. 180802-47402/PCT

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straight or branched C4-8 hydrocarbon chain. In some embodiments, L² is an optionally substituted bivalent saturated, straight or branched C1-6 hydrocarbon chain. In some embodiments, L² is a bivalent saturated, straight or branched C1-12 hydrocarbon chain. In some embodiments, L² is a bivalent saturated, straight or branched C4-8 hydrocarbon chain. In some embodiments, L² is a bivalent saturated, straight or branched C1-6 hydrocarbon chain. In some embodiments, L² is -CH2-. In some embodiments, L² is -(CH2)4- or -(CH2)s-. In some embodiments, L² is -(CH2)e- or -(CFb)?-.

In some embodiments of any Formulae of WO 2024/019936 described herein, L² is an optionally substituted bivalent saturated or unsaturated, straight or branched C1-12 hydrocarbon chain. In some embodiments, L² is an optionally substituted bivalent saturated or unsaturated, straight or branched C1-6 hydrocarbon chain. In some embodiments, L² is an optionally substituted bivalent saturated or unsaturated, straight or branched C1-3 hydrocarbon chain. In some embodiments, L² is an optionally substituted bivalent saturated, straight or branched C1-12 hydrocarbon chain. In some embodiments, L² is an optionally substituted bivalent saturated, straight or branched C1-6 hydrocarbon chain. In some embodiments, L² is an optionally substituted bivalent saturated, straight or branched C1-3 hydrocarbon chain. In some embodiments, L² is a bivalent saturated, straight or branched C1-12 hydrocarbon chain. In some embodiments, L² is a bivalent saturated, straight or branched C1-6 hydrocarbon chain. In some embodiments, L² is a bivalent saturated, straight or branched C1-3 hydrocarbon chain. In some embodiments, L² is -(CH2)2-.

In some embodiments of any Formulae of WO 2024/019936 described herein, L³ is a covalent bond, -O-, -C(O)O-, -OC(O)-, or -OC(O)O-. In some embodiments, L³ is a covalent bond, -C(O)O-, or -OC(O)-. In some embodiments, L³ is a covalent bond. In some embodiments, L³ is -O-, -C(O)O-, -OC(O)-, or -OC(O)O-. In some embodiments, L³ is -C(O)O-or -OC(O)-. In some embodiments, L³ is -O-. In some embodiments, L³ is -C(O)O-. In some embodiments, L³ is -OC(O)-. In some embodiments, L³ is -OC(O)O-.

In some embodiments of any Formulae of WO 2024/019936 described herein, R¹ is O-R^a

|__LCyA__c A r^LRa~(

optionally substituted C1-20 aliphatic, * ^y, or O-R^a. in some embodiments, R¹

is optionally substituted C1-20 aliphatic. In some embodiments, R¹ is

^Cy. In some O-R^a O-R^a I — [_Ra — | — [_^Ra —

embodiments, R¹ is O-R^a. in some embodiments, R¹ is C1-20 aliphatic or O-R^a. In some embodiments, R¹ is optionally substituted C6-12 aliphatic. In some embodiments, R¹ is ATTORNEY DOCKET NO. 180802-47402/PCT

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optionally substituted C6-12 alkyl. In some embodiments, R¹ is optionally substituted C6-12 alkenyl. In some embodiments, R¹ is optionally substituted C12-20 aliphatic. In some embodiments, R¹ is optionally substituted C12-20 alkyl. In some embodiments, R¹ is optionally substituted C12-20 alkenyl. In some embodiments, R¹ is C1-20 aliphatic. In some embodiments, R¹ is C6-12 aliphatic. In some embodiments, R¹ is C6-12 alkyl. In some embodiments, R¹ is C6-12 alkenyl. In some embodiments, R¹ is C12-20 aliphatic. In some embodiments, R¹ is C12-20 alkyl. In some embodiments, R¹ is C12-20 alkenyl. In some embodiments, R¹ is

In some embodiments of any Formulae of WO 2024/019936 described herein, L^CyA is a covalent bond or an optionally substituted bivalent saturated or unsaturated, straight or branched C1-12 hydrocarbon chain.

In some embodiments of any Formulae of WO 2024/019936 described herein, Cy^A is an optionally substituted ring selected from 3- to 7-membered saturated or partially unsaturated carbocyclyl, 1-adamantyl, 2-adamantyl, sterolyl, and phenyl.

In some embodiments of any Formulae of WO 2024/019936 described herein, L^Ra is an optionally substituted bivalent saturated or unsaturated, straight or branched C1-12 hydrocarbon chain. In some embodiments, L^Ra is a bivalent saturated or unsaturated, straight or branched Ci-12 hydrocarbon chain. In some embodiments, L^Ra is an optionally substituted bivalent saturated or unsaturated, straight or branched C1-6 hydrocarbon chain. In some embodiments, L^Ra is a bivalent saturated or unsaturated, straight or branched C1-6 hydrocarbon chain. In some embodiments, L^Ra is an optionally substituted bivalent saturated or unsaturated, straight or branched C1-3 hydrocarbon chain. In some embodiments, L^Ra is a bivalent saturated or unsaturated, straight or branched C1-3 hydrocarbon chain. In some embodiments, L^Ra is -CH2- or -(CH₂)₂-.

In some embodiments of any Formulae of WO 2024/019936 described herein, each R^a is independently optionally substituted C1-20 aliphatic. In some embodiments, each R^a is independently optionally substituted C1-12 aliphatic. In some embodiments, each R^a is independently optionally substituted C1-12 alkyl. In some embodiments, each R^a is independently ATTORNEY DOCKET NO. 180802-47402/PCT

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optionally substituted C1-12 alkenyl. In some embodiments, each R^a is independently optionally substituted C4-10 aliphatic. In some embodiments, R^a is optionally substituted C6-12 aliphatic. In some embodiments, R^a is optionally substituted C6-12 alkyl. In some embodiments, R^a is optionally substituted C6-12 alkenyl. In some embodiments, R^a is optionally substituted C7-9 aliphatic. In some embodiments, R^a is optionally substituted C7-9 alkyl. In some embodiments, R^a is optionally substituted C7-9 alkenyl. In some embodiments, each R^a is independently C1-20 aliphatic. In some embodiments, R^a is C6-12 aliphatic. In some embodiments, R^a is C6-12 alkyl. In some embodiments, R^a is C6-12 alkenyl. In some embodiments, R^a is C7-9 aliphatic. In some embodiments, R^a is C7-9 alkyl. In some embodiments, R^a is C7-9 alkenyl. In some embodiments, R^a is

In some embodiments of any Formulae of WO 2024/019936 described herein, each R¹ is independently optionally substituted C1-20 aliphatic. In some embodiments, R¹ is optionally substituted C6-12 aliphatic. In some embodiments, R¹ is optionally substituted C6-12 alkyl. In ATTORNEY DOCKET NO. 180802-47402/PCT

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some embodiments, R¹ is optionally substituted C6-12 alkenyl. In some embodiments, R¹ is optionally substituted C7-9 aliphatic. In some embodiments, R¹ is optionally substituted C7-9 alkyl. In some embodiments, R¹ is optionally substituted C7-9 alkenyl. In some embodiments, each R¹ is independently C1-20 aliphatic. In some embodiments, R¹ is C6-12 aliphatic. In some embodiments, R¹ is C6-12 alkyl. In some embodiments, R¹ is C6-12 alkenyl. In some embodiments, R¹ is C7-9 aliphatic. In some embodiments, R¹ is C7-9 alkyl. In some embodiments, R¹ is C7-9 alkenyl. In some embodiments, R¹ is

or

In some embodiments of any Formulae of WO 2024/019936 described herein, Y¹ is -C(O)- or -C(O)O-. In some embodiments, Y¹ is -C(O)-. In some embodiments, Y¹ is -C(O)O-.

In some embodiments of any Formulae of WO 2024/019936 described herein, Y² is an optionally substituted bivalent saturated or unsaturated, straight or branched C1-6 hydrocarbon chain. In some embodiments, Y² is an optionally substituted bivalent saturated or unsaturated, straight or branched C1-3 hydrocarbon chain. In some embodiments, Y² is an optionally substituted bivalent saturated, straight or branched C1-6 hydrocarbon chain. In some embodiments, Y² is an optionally substituted bivalent saturated, straight or branched C1-3 hydrocarbon chain. In some embodiments, Y² is a bivalent saturated or unsaturated, straight or branched C1-6 hydrocarbon chain. In some embodiments, Y² is a bivalent saturated, straight or branched C1-6 hydrocarbon chain. In some embodiments, Y² is a bivalent saturated, straight or branched C1-3 hydrocarbon chain. In some embodiments, Y²is -CH2- or -(CH2)2-.

In some embodiments of any Formulae of WO 2024/019936 described herein, Y³ is optionally substituted C1-20 aliphatic. In some embodiments, Y³ is C1-20 aliphatic. In some embodiments, Y³ is optionally substituted C1-12 aliphatic. In some embodiments, Y³ is optionally substituted C1-12 alkyl. In some embodiments, Y³ is optionally substituted C4-8 aliphatic. In some embodiments, Y³ is optionally substituted C4-8 alkyl. In some embodiments, Y³ is optionally substituted C1-6 alkyl. In some embodiments, Y³ is -CH3, -CH2CH3, -(CFb^CFb, -(CH₂)₃CH3, -(CH₂)₄CH3, -(CH₂)₅CH3, -(CH₂)₆CH3, or -(CH₂)7CH₃. In some embodiments, Y³ is -CH3. In some embodiments, Y³ is -CH2CH3. In some embodiments, Y³ is -(CFb^CFb. In some embodiments, Y³ is -(CFb^CFb. In some embodiments, Y³ is -(C b^C b. In some embodiments, Y³ is -(CFb^CFb. In some embodiments, Y³ is -(CFb^CFb. In some embodiments, Y

³ is -(CFb^CFb.

In some embodiments of any Formulae of WO 2024/019936 described herein, X¹ is a covalent bond, -O-, or -NR-. In some embodiments, X¹ is a covalent bond. In some ATTORNEY DOCKET NO. 180802-47402/PCT

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embodiments, X¹ is -O- or -NR.-. In some embodiments, X¹ is -O-. In some embodiments, X¹ is -NR.-.

In some embodiments of any Formulae of WO 2024/019936 described herein, X² is an optionally substituted bivalent saturated or unsaturated, straight or branched C1-12 hydrocarbon chain, wherein 1-3 methylene units are optionally and independently replaced with -O- or -NR.-. In some embodiments, X² is an optionally substituted bivalent saturated or unsaturated, straight or branched C1-6 hydrocarbon chain, wherein 1-2 methylene units are optionally and independently replaced with -O- or -NR.-. In some embodiments, X² is an optionally substituted bivalent saturated or unsaturated, straight or branched C1-12 hydrocarbon chain. In some embodiments, X² is an optionally substituted bivalent saturated or unsaturated, straight or branched C1-6 hydrocarbon chain. In some embodiments, X² is an optionally substituted bivalent saturated or unsaturated, straight or branched C1-3 hydrocarbon chain. In some embodiments, X² is an optionally substituted bivalent saturated, straight or branched C1-12 hydrocarbon chain. In some embodiments, X² is an optionally substituted bivalent saturated, straight or branched C1-6 hydrocarbon chain. In some embodiments, X² is an optionally substituted bivalent saturated, straight or branched C1-3 hydrocarbon chain. In some embodiments, X² is a bivalent saturated, straight or branched C1-12 hydrocarbon chain. In some embodiments, X² is a bivalent saturated, straight or branched C1-6 hydrocarbon chain. In some embodiments, X² is a bivalent saturated, straight or branched C1-3 hydrocarbon chain. In some embodiments, X² is an optionally substituted bivalent saturated or unsaturated, straight or branched C1-12 hydrocarbon chain, wherein 1-3 methylene units are independently replaced with -O- or -NR.-. In some embodiments, X² is an optionally substituted bivalent saturated or unsaturated, straight or branched C4-8 hydrocarbon chain, wherein 1-2 methylene units are independently replaced with -O- or -NR-. In some embodiments, X² is an optionally substituted bivalent saturated or unsaturated, straight or branched C4-8 hydrocarbon chain, wherein 2 methylene units are independently replaced with -NR.-. In some embodiments, X² is an optionally substituted bivalent saturated or unsaturated, straight or branched C4-8 hydrocarbon chain, wherein 1 methylene unit is replaced with -NR-. In some embodiments, X² is a bivalent saturated or unsaturated, straight or branched C1-12 hydrocarbon chain, wherein 1-3 methylene units are optionally and independently replaced with -NR-. In some embodiments, X² is a bivalent saturated or unsaturated, straight or branched C1-12 hydrocarbon chain, wherein 1-2 methylene units are optionally and independently replaced with -NR-. In some embodiments, X² is a bivalent saturated or unsaturated, straight or branched C4-8 hydrocarbon chain, wherein 1 methylene unit is replaced with -NR-. In some embodiments, X² is an optionally substituted ATTORNEY DOCKET NO. 180802-47402/PCT

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bivalent saturated, straight or branched C1-12 hydrocarbon chain, wherein 1-3 methylene units are independently replaced with -O- or -NR.-. In some embodiments, X² is an optionally substituted bivalent saturated, straight or branched C4-8 hydrocarbon chain, wherein 1-2 methylene units are independently replaced with -O- or -NR.-. In some embodiments, X² is an optionally substituted bivalent saturated, straight or branched C4-8 hydrocarbon chain, wherein 2 methylene units are independently replaced with -NR.-. In some embodiments, X² is an optionally substituted bivalent saturated, straight or branched C4-8 hydrocarbon chain, wherein 1 methylene unit is replaced with -NR-. In some embodiments, X² is a bivalent saturated, straight or branched C4-8 hydrocarbon chain, wherein 1 methylene unit is replaced with -NR.-.

In some embodiments of any Formulae of WO 2024/019936 described herein, X³ is hydrogen or an optionally substituted ring selected from 3- to 7- membered saturated or partially unsaturated carbocyclyl, phenyl, 3- to 7-membered heterocyclyl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or 5- to 6-membered heteroaryl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, X³ is hydrogen or a 3- to 7-membered heterocyclyl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur and optionally substituted with C1-6 aliphatic. In some embodiments, X³ is hydrogen. In some embodiments, X³ is an optionally substituted ring selected from 3- to 7- membered saturated or partially unsaturated carbocyclyl, phenyl, 3- to 7-membered heterocyclyl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or 5- to 6-membered heteroaryl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, X³ is optionally substituted 3- to 7-membered heterocyclyl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, X³ is optionally substituted 5- to 6-membered heterocyclyl having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, X³ is 5- to 6-membered heterocyclyl having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, X³ is 5- to 6-membered heterocyclyl, having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur, substituted with -R°, wherein R° is C1-6 aliphatic (e.g., methyl or ethyl). In some embodiments, X³ is optionally substituted 5-membered heterocyclyl having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, X³ is 5-membered heterocyclyl having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, X³ is 5-membered heterocyclyl, having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur, substituted with -R°, wherein R° is C1-6 aliphatic (e.g., methyl or ethyl). In some embodiments, X³ is optionally ATTORNEY DOCKET NO. 180802-47402/PCT

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substituted 5-membered heterocyclyl having 1-2 nitrogen atoms. In some embodiments, X³ is 5-membered heterocyclyl having 1-2 nitrogen atoms. In some embodiments, X³ is 5-membered heterocyclyl, having 1-2 nitrogen atoms, substituted with -R°, wherein R° is Ci-6 aliphatic (e.g., methyl or ethyl). In some embodiments, X³ is optionally substituted 6-membered heterocyclyl having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, X³ is 6-membered heterocyclyl having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, X³ is 6-membered heterocyclyl, having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur, substituted with -R°, wherein R° is Ci-6 aliphatic (e.g., methyl or ethyl). In some embodiments, X³ is optionally substituted pyrrolidinyl, piperidinyl, piperazinyl, or morpholinyl. In some embodiments, X³ is pyrrolidinyl, piperidinyl, piperazinyl, or morpholinyl. In some embodiments, X³ is pyrrolidinyl, piperidinyl, piperazinyl, or morpholinyl substituted with -R°, wherein R° is Ci-6 aliphatic (e.g., methyl or ethyl).

In some embodiments, -X²-X³ is

In some embodiments, at least one of X² and X³ comprises at least one ionizable nitrogen atom.

In some embodiments of any Formulae of WO 2024/019936 described herein, each R is independently hydrogen or optionally substituted Ci-6 aliphatic. In some embodiments, each R is independently hydrogen or Ci-6 aliphatic. In some embodiments, R is hydrogen. In some embodiments, R is optionally substituted Ci-6 aliphatic. In some embodiments, R is Ci-6 aliphatic. In some embodiments, R is optionally substituted C1-3 aliphatic. In some embodiments, R is C1-3 aliphatic. In some embodiments, R is -CH3.

In some embodiments, R° is methyl. In some embodiments, R° is ethyl.

In some embodiments of any Formulae of WO 2024/019936 described herein, the present disclosure provides compounds comprising an ionizable nitrogen atom, wherein the pKa of the conjugate acid thereof is between about 4 and about 12.

In some embodiments, the present disclosure provides a compound selected from Table 1 of WO 2024/019936. In various embodiments, the compound is selected from the group consisting of: ATTORNEY DOCKET NO. 180802-47402/PCT

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ATTORNEY DOCKET NO. 180802-47402/PCT

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ATTORNEY DOCKET NO. 180802-47402/PCT

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ATTORNEY DOCKET NO. 180802-47402/PCT

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ATTORNEY DOCKET NO. 180802-47402/PCT

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ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

₀ o r

A_N A

^O

T H

7-38 L.0

0 0

o rA

oA^O

T H

7-39

o °>

o 0

0 0

1 II M

7-40 n k A/x°Jk ^/xr/A Y A 'A /

_/^_=/^/^,0

0 O

O _H

7-41

o <

o o

o _H

7-42 O VO^A^Y°Y^N^-^N^

^o

o o

I || n

7-43 ^xA Q — ^r^/°^x|f^N^^/^N^ o < /\ ^o 1^

o o

I II I-*

7-44 ^xA _O \^X_QA — -YO_YN^_N^ _xo 1^

ATTORNEY DOCKET NO. 180802-47402/PCT

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)

7-45 /Z- — 0 0 ~ O

O IZ IZ

> L°

o o o

o o

7-46

^^^ o o o===

^^ O o o o= O /= /

o o

o O o O ob——— '

Pf o/ o/== D O O 3—— \

7-4 O/ O/C== O ^O ° o/<=⁼=

9 =

C( ° ^O=⁼

(IZ3)

/ / / / ° °

\

\ \ y < y ) ° ° ° / / / / ° / ° / °

) \ ) ° °

O \ / O \ / o \ / o \ / o \ /

7-50

p 1

p <

7-51

, or a pharmaceutically acceptable salt thereof.

It will be understood that, unless otherwise specified or prohibited by the foregoing definition of any of Formulae I, II, III, III- A, III-B, III-B-z, IV, IV- A, IV-B, IV-B-z, V, V-A, V-B, V-B-z, VI, VI-A, VI-B, VI-B-z, VII, VILA, VILB, VII-B-z, VIII, VIILA, VIILB, VIILB-z, IX, IX-A, IX-B, IX-B-z, X, X-A, X-B, and X-B-z of WO 2024/019936, embodiments of variables L¹, L¹, L², L², L³, L^CyA, Cy^A, L^Ra, R^a, R¹, R¹, R, Y¹, Y², Y³, X¹, X², and X³ as defined above and described in classes and subclasses herein, apply to compounds of any of Formulae I, II, III, III-A, IILB, III-B-z, IV, IV-A, IV-B, IV-B-z, V, V-A, V-B, V-B-z, VI, VI-A, VLB, VI-B-z, VII, VII-A, VILB, VII-B-z, VIII, VIILA, VIILB, VIILB-z, IX, IX-A, IX-B, IX-B-z, X, X-A, X-B, and X-B-z of WO 2024/019936, both singly and in combination.

In some embodiments, provided compounds are provided and/or utilized in a salt form (e.g., a pharmaceutically acceptable salt form). Reference to a compound provided herein is understood to include reference to salts thereof, unless otherwise indicated. ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

It will be appreciated that throughout the present disclosure, unless otherwise indicated, reference to a compound of Formula I of WO 2024/019936 is intended to also include any of Formulae II, III, IILA, IILB, III-B-z, IV, IV-A, IV-B, IV-B-z, V, V-A, V-B, V-B-z, VI, VLA, VI-B, VI-B-z, VII, VILA, VILB, VII-B-z, VIII, VIII-A, VIILB, VIII-B-z, IX, IX-A, IX-B, IX-B-z, X, X-A, X-B, and X-B-z of WO 2024/019936, and compound species of such formulae disclosed herein.

Conjugate-linker lipids

Among other things, the present disclosure describes compositions, preparations, nanoparticles, and/or nanomaterials that comprise one or more conjugate-linker lipids as described herein.

In some embodiments, a conjugate-linker lipid is or comprises a polyethylene glycol (PEG)-lipid or PEG-modified lipid. In some embodiments, PEG or PEG-modified lipids may be alternately referred to as PEGylated lipids or PEG-lipids. Inclusion of a PEGylating lipid can be used to enhance lipid nanoparticle colloidal stability in vitro and circulation time in vivo. In some embodiments, the PEGylation is reversible in that the PEG moiety is gradually released in blood circulation. Exemplary PEG-lipids include but are not limited to PEG conjugated to saturated or unsaturated alkyl chains having a length of C6-C20. PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides (PEG-CER), PEG-modified dialkylamines, PEG-modified diacylglycerols (PEG-DAG), PEG-modified dialkylglycerols, and mixtures thereof. For example, in some embodiments, a PEG lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPE, PEG-DSG or a PEG-DSPE lipid.

In some embodiments, a conjugate-linker lipid comprises a polyethylene glycol lipid. In some embodiments, the conjugate-linker lipid comprises DiMystyrl Glycerol (DMG), 1,2-Dipalmitoyl-rac-glycerol, methoxypolyethylene Glycol (DPG-PEG), or 1,2-Distearoyl-rac-glycero-3 -methylpolyoxyethylene (DSG - PEG). In some embodiments, a conjugate-linker lipid has an average molecular mass from about 500 Da to about 5000 Da. In some embodiments, a conjugate-linker lipid has an average molecular mass of about 2000 Da.

In some embodiments, a LNP preparation comprises from about 0 mole percent to about 5 mole percent conjugate-linker lipid. In some embodiments, a LNP preparation comprises about 1.5 mole percent conjugate-linker lipid. In some embodiments, a LNP preparation comprises about 2.0 mole percent conjugate-linker lipid. In some embodiments, a LNP preparation comprises about 2.5 mole percent conjugate-linker lipid. In some embodiments, a ATTORNEY DOCKET NO. 180802-47402/PCT

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LNP preparation comprises about 3 mole percent conjugate-linker lipid. In some embodiments, a LNP preparation comprises about 3.5 mole percent conjugate-linker lipid.

Phospholipids

Among other things, the present disclosure describes compositions, preparations, nanoparticles, and/or nanomaterials that comprise one or more phospholipids as described herein. In some embodiments, the present disclosure describes compositions, preparations, nanoparticles, and/or nanomaterials that comprise one or more (poly)unsaturated lipids.

In some embodiments, one or more phospholipids may assemble into one or more lipid bilayers. In some embodiments, one or more phospholipids may include a phospholipid moiety. In some embodiments, one or more phospholipids may include one or more fatty acid moieties. In some embodiments, one or more phospholipids may include a phospholipid moiety and one or more fatty acid moieties. In some embodiments, a phospholipid moiety includes but is not limited to phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and sphingomyelin. In some embodiments, a fatty acid moiety includes but is not limited to lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alphalinolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

Exemplary phospholipids include but are not limited to l,2-distearoyl- w-glycero-3-phosphocholine (DSPC), l,2-dioleoyl- w-glycero-3 -phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sw-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sw-glycerophosphocholine (DMPC), l,2-dioleoyl- w-glycero-3 -phosphocholine (DOPC), l,2-dipalmitoyl-sw-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sw-glycerophosphocholine (DUPC), l-palmitoyl-2-oleoyl-sw-glycero-3-phosphocholine (POPC), l,2-di-O-octadecenyl-sw-glycero-3-phosphocholine (18:0 Diether PC), l-oleoyl-2-cholesterylhemisuccinoy l- w-glycero-3-phosphocholine (OChemsPC), 1 -hexadecyl snglycero-3 -phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sw-glycero-3 -phosphocholine, 1,2-diarachidonoyl- w-glycero-3 -phosphocholine, l,2-didocosahexaenoyl-sw-glycero-3 -phosphocholine, l,2-diphytanoyl- w-glycero-3-phosphoethanolamine (ME 16.0 PE), l,2-distearoyl-sw-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sw-glycero-3 -phosphoethanolamine, l,2-dilinolenoyl-sw-glycero-3-phosphoethanolamine, l,2-diarachidonoyl-sw-glycero-3 -phosphoethanolamine, 1,2-didocosahexaenoyl-sw-glycero-3 -phosphoethanolamine, l,2-dioleoyl-sw-glycero-3-phospho-rac-(1 -glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), ATTORNEY DOCKET NO. 180802-47402/PCT

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palmitoyloleoylphosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), l-stearoyl-2-oleoyl-phosphatidy ethanolamine (SOPE), l-stearoyl-2 oleoylphosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), or combinations thereof. In some embodiments, a phospholipid is DSPC. In some embodiments, a phospholipid is DMPC.

In some embodiments, the phospholipid comprises l,2-distearoyl- w-glycero-3-phosphocholine (DSPC), l,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl) (succinyl -DPPE), 1,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), l,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), l,2-dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC), or a combination thereof.

In some embodiments, a LNP preparation comprises from about 5 mole percent to 25 mole percent of phospholipid. In some embodiments, a LNP preparation comprises about 5 mole percent to 15 mole percent of phospholipid. In some embodiments, a LNP preparation comprises about 7 mole percent to 11 mole percent of phospholipid. In some embodiments, a LNP preparation comprises about 7.5 mole percent of phospholipid.

Sterols

Among other things, the present disclosure describes compositions, preparations, nanoparticles, and/or nanomaterials that comprise one or more sterols as described herein.

In some embodiments, a sterol is a cholesterol, or a variant or derivative thereof. In some embodiments, a cholesterol is modified. In some embodiments, a cholesterol is an oxidized cholesterol. In some embodiments, a cholesterol is esterified cholesterol. Unmodified cholesterol can be acted upon by enzymes to form variants that are side-chain or ring oxidized. In some embodiments, a cholesterol can be oxidized on the beta-ring structure or on the hydrocarbon tail structure. In some embodiments, a sterol is a phytosterol. Exemplary sterols that are considered for use in the disclosed lipid nanoparticles include but are not limited to 25-hydroxycholesterol (25-OH), 20a-hydroxycholesterol (20a-OH), 27-hydroxycholesterol, 6-keto-5a-hydroxycholesterol, 7-ketocholesterol, 7P-hydroxycholesterol, 7a-hydroxycholesterol, 7P-25-dihydroxycholesterol, beta-sitosterol, stigmasterol, brassicasterol, campesterol, or combinations thereof. In some embodiments, a side-chain oxidized cholesterol can enhance payload delivery ATTORNEY DOCKET NO. 180802-47402/PCT

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relative to other cholesterol variants. In some embodiments, a cholesterol is an unmodified cholesterol.

In some embodiments, a LNP preparation comprises from about 25 mole percent to about 45 mole percent sterol. In some embodiments, a LNP preparation comprises from about 30 mole percent to about 45 mole percent sterol. In some embodiments, a LNP preparation comprises about 40 mole percent sterol. In some embodiments, a LNP preparation comprises about 35 mole percent sterol. In some embodiments, a LNP preparation comprises about 32.5 mole percent cholesterol.

LNP Formulations

Particular formulation of a nanoparticle composition comprising one or more described lipids is described herein. The present invention provides for compositions, preparations, nanoparticles, and/or nanomaterials that comprise lipid nanoparticles.

In some embodiments, a lipid nanoparticle preparation comprises about 30 mole percent to about 70 mole percent ionizable lipid, about 5 mole percent to about 25 mole percent phospholipid, about 25 mole percent to about 45 mole percent sterol, and about 0 mole percent to about 5 mole percent conjugate-linker lipid.

In some embodiments, a lipid nanoparticle preparation comprises about 47.5 mole percent ionizable lipid, about 10 mole percent phospholipid, about 40 mole percent sterol, and about 2.5 mole percent conjugate-linker lipid.

In some embodiments, a lipid nanoparticle preparation comprises about 51.25 mole percent ionizable lipid, about 10 mole percent phospholipid, about 35.75 mole percent sterol, and about 3 mole percent conjugate-linker lipid.

In some embodiments, the lipid nanoparticle preparation comprises about 55 mole percent ionizable lipid, about 10 mole percent phospholipid, about 32.5 mole percent sterol, and about 2.5 mole percent conjugate-linker lipid.

In some embodiments, the lipid nanoparticle preparation comprises about 55 mole percent ionizable lipid, about 7.5 mole percent phospholipid, about 35 mole percent sterol, and about 2.5 mole percent conjugate-linker lipid.

In some embodiments, a lipid nanoparticle preparation comprises about 45 mole percent to about 60 mole percent ionizable lipid of any provided compound, about 7 mole percent to about 11 mole percent l-2-distearoyl-sn-glycero-3-phosphocholine (DSPC), about 1 mole percent to about 5 mole percent PEG2000-DMG, and about 30 mole percent to about 45 mole percent cholesterol, based on the total moles of these four ingredients. ATTORNEY DOCKET NO. 180802-47402/PCT

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In some embodiments, a lipid nanoparticle preparation is the formulation in Example 2. In some embodiments, a lipid nanoparticle (LNP) preparation comprises about 55 mole percent ionizable lipid IZ4, about 7.5 mole percent l-2-distearoyl-sn-glycero-3-phosphocholine (DSPC), about 2.5 mole percent l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2000-DMG), and about 35 mole percent cholesterol, based on the total moles of these four ingredients, an N: P ratio of 6: 1, and a payload comprising mRNA encoding CBE6b NGG (version 2) of Table 2, and guide RNA gRNA9846 of Table 1. In some embodiments, the molar ratio of mRNA to gRNA in the LNP preparation is about 1:1.

In some embodiments, a lipid nanoparticle (LNP) preparation comprises about 55 mole percent ionizable lipid IZ4, about 7.5 mole percent l-2-distearoyl-sn-glycero-3-phosphocholine (DSPC), about 2.5 mole percent l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2000-DMG), and about 35 mole percent cholesterol, based on the total moles of these four ingredients, an N: P ratio of 6: 1, and a payload comprising mRNA encoding CBE6b_NGG of Table 2, and guide RNA gRNA10320 of Table 1. In some embodiments, the molar ratio of mRNA to gRNA is about 1:1.

The described lipid nanoparticle compositions are capable of delivering a therapeutic agent such as an RNA to a particular cell, tissue, organ, or system or group thereof in a mammal’s body. Physiochemical properties of nanoparticle compositions may be altered in order to increase selectivity for particular bodily targets. For instance, particle sizes may be adjusted based on the fenestration sizes of different organs.

Weight Ratio of Lipid Component to Therapeutic Agent

The amount of a therapeutic agent or drug substance (e.g., the mRNA that encodes for the base editor and the guide RNA) in an LNP composition may depend on the size, composition, desired target and/or application, or other properties of the nanoparticle composition. For example, the amount of an RNA comprised in a nanoparticle composition may depend on the size, sequence, and other characteristics of the RNA. The relative amounts of a therapeutic agent and other elements (e.g., lipids) in a nanoparticle composition may also vary. In some embodiments, the wt/wt ratio of the lipid component to a therapeutic agent in a nanoparticle composition may be from about 5:1 to about 60:1, such as about 5:1. 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, and 60: 1. For example, the wt/wt ratio of the lipid component to a therapeutic agent may be from about 10:1 to about 40: 1. In certain embodiments, the wt/wt ratio is about 20: 1. The ATTORNEY DOCKET NO. 180802-47402/PCT

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amount of a therapeutic agent in a nanoparticle composition can be measured using absorption spectroscopy (e.g., ultraviolet-visible spectroscopy).

N: P Ratio

In some embodiments, an LNP formulation comprises one or more nucleic acids such as RNAs. In some embodiments, the one or more RNAs, lipids, and amounts thereof may be selected to provide a specific N: P ratio. The N: P ratio can be selected from about 1 to about 30. In some embodiments, the N: P ratio is from about 2 to about 15, from about 2 to about 10, from about 2 to about 8, from about 2 to about 6, from about 3 to about 15, from about 3 to about 10, from about 3 to about 8, from about 3 to about 6, from about 4 to about 15, from about 4 to about 10, from about 4 to about 8, or from about 4 to about 6. In some embodiments, the N: P ratio is about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 9, or about 10. In some embodiments, the N: P ratio is from about 5 to about 7. In some embodiments, the N: P ratio is about 6. In each of the above N: P ratios and any other recitation of a single number for an N: P ratio, the moles or parts P is 1 and is not recited for brevity.

Polydispersity

Among other things, the present disclosure describes compositions, preparations, nanoparticles, and/or nanomaterials that have a poly dispersity index (PDI) of about 0.01 to about 0.3. In some embodiments, compositions, preparations, nanoparticles, and/or nanomaterials described herein have a PDI that is about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, or any range having endpoints defined by any two of the aforementioned values. For example, in some embodiments, compositions, preparations, nanoparticles, and/or nanomaterials described herein have a PDI from about 0.05 to about 0.2, about 0.06 to about 0.1, or about 0.07 to about 0.09.

Encapsulation efficiency

Among other things, the present disclosure describes compositions, preparations, nanoparticles, and/or nanomaterials, wherein encapsulation efficiency of provided compositions, preparations, nanoparticles, and/or nanomaterials is from about 80% to about 100%. In some embodiments, encapsulation efficiency of compositions, preparations, nanoparticles, and/or nanomaterials described herein is about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 100%, or any range having endpoints ATTORNEY DOCKET NO. 180802-47402/PCT

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defined by any two of the aforementioned values. For example, in some embodiments, encapsulation efficiency of compositions, preparations, nanoparticles, and/or nanomaterials described herein is from about 90% to about 100%, about 95% to about 100%, about 95% to about 98%, or about 95.5% to about 97.5%. In some embodiments, encapsulation efficiency of compositions, preparations, nanoparticles, and/or nanomaterials described herein is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

pKa

Among other things, the present disclosure describes compositions, preparations, nanoparticles, and/or nanomaterials that have a pKa from about 5 to about 9. In some embodiments, compositions, preparations, nanoparticles, and/or nanomaterials described herein have a pKa that is about 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, or any range having endpoints defined by any two of the aforementioned values. In some embodiments, compositions, preparations, nanoparticles, and/or nanomaterials described herein have a pKa that is about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, or any range having endpoints defined by any two of the aforementioned values.

Payload

The LNPs described herein can be designed to deliver a payload, such as one or more therapeutic agent(s) or drug substances(s) to a target cell or organ of interest. In some embodiments, a LNP described herein encloses one or more components of a base editor system as described herein. For example, a LNP may enclose one or more of a guide RNA, a nucleic acid encoding the guide RNA, a vector encoding the guide RNA, a base editor fusion protein, a nucleic acid encoding the base editor fusion protein, a programmable DNA binding domain, a nucleic acid encoding the programmable DNA binding domain, a deaminase, a nucleic acid encoding the deaminase, or all or any combination thereof. In some embodiments, the nucleic acid is a DNA. In some embodiments, the nucleic acid is a RNA, for example, a mRNA and/or a guide RNA. In some embodiments, the said nucleic acid(s) is/are chemically modified.

In some embodiments, the payload comprises one or more nucleic acid(s) (i.e., one or more nucleic acid molecular entities). In some embodiments, the nucleic acid is a singlestranded nucleic acid. In some embodiments, single-stranded nucleic acid is an RNA. In some embodiments, the nucleic acid is a double-stranded nucleic acid. In some embodiments, the ATTORNEY DOCKET NO. 180802-47402/PCT

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double-stranded nucleic acid is a DNA. In some embodiments, the nucleic acid is a messenger RNA (mRNA).

Methods of manufacturing LNPs

Methods of manufacturing lipid nanoparticles (LNPs) are known in the art., and the following methods may be used to make lipid nanoparticles of the invention. To achieve size reduction and/or to increase the homogeneity of size in the particles, the skilled person may use the method steps set out below, experimenting with different combinations. Additionally, the skilled person could employ sonication, filtration or other sizing techniques which are used in liposomal formulations.

The process for making an LNP composition of the invention typically comprises providing an aqueous solution comprising a biologically active agent in a first reservoir, providing a second reservoir comprising an organic solution, such as an organic alcohol, for example ethanol, of the lipid(s) and then mixing the aqueous solution with the organic lipid solution. The first reservoir is optionally in fluid communication with the second reservoir. The mixing step is optionally followed by an incubation step, a filtration or dialysis step, and a dilution and/or concentration step. The incubation step comprises allowing the solution from the mixing step to stand in a vessel for a period of time (preferably about 0 to about 24 hours) at about room temperature and optionally protected from light. In certain embodiments, the temperature may be about 4 °C during incubation. In one embodiment, a dilution step follows the incubation step. The dilution step may involve dilution with aqueous buffer (e.g. citrate buffer or pure water) e.g., using a pumping apparatus (e.g. a peristaltic pump). The filtration step may be tangential flow filtration, ultrafiltration or dialysis.

There is herein disclosed an apparatus for making a composition of the present invention. The apparatus typically includes a first reservoir for holding an aqueous solution comprising a biologically active agent and a second reservoir for holding an organic lipid solution. The apparatus also typically includes a pump mechanism configured to pump the aqueous and the organic lipid solutions into a mixing region or mixing chamber at substantially equal flow rates. In one embodiment, the mixing region or mixing chamber comprises a T coupling or equivalent thereof, which allows the aqueous and organic fluid streams to combine as input into the T connector and the resulting combined aqueous and organic solutions to exit out of the T connector into a collection reservoir or equivalent thereof. In other embodiments, a microfluidic device can be used for making an LNP composition provided by the invention. ATTORNEY DOCKET NO. 180802-47402/PCT

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PHARMACEUTICAL COMPOSITIONS

In some aspects, the present disclosure provides a pharmaceutical composition comprising any of the cells, polynucleotides, vectors, base editors, base editor systems, guide polynucleotides, fusion proteins, complexes, or the fusion protein-guide polynucleotide complexes described herein.

The pharmaceutical compositions of the present disclosure can be prepared in accordance with known techniques. See, e.g., Remington, The Science And Practice of Pharmacy (21st ed.

2005). In general, the cell, or population thereof is admixed with a suitable carrier prior to administration or storage, and in some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers generally comprise inert substances that aid in administering the pharmaceutical composition to a subject, aid in processing the pharmaceutical compositions into deliverable preparations, or aid in storing the pharmaceutical composition prior to administration. Pharmaceutically acceptable carriers can include agents that can stabilize, optimize or otherwise alter the form, consistency, viscosity, pH, pharmacokinetics, solubility of the formulation. Such agents include buffering agents, wetting agents, emulsifying agents, diluents, encapsulating agents, and skin penetration enhancers. For example, carriers can include, but are not limited to, saline, buffered saline, dextrose, arginine, sucrose, water, glycerol, ethanol, sorbitol, dextran, sodium carboxymethyl cellulose, and combinations thereof.

In some embodiments, the pharmaceutical composition is formulated for delivery to a subject. Suitable routes of administrating the pharmaceutical composition described herein include, without limitation: topical, subcutaneous, transdermal, intradermal, intralesional, intraarticular, intraperitoneal, intravesical, transmucosal, gingival, intradental, intracochlear, transtympanic, intraorgan, epidural, intrathecal, intramuscular, intravenous, intravascular, intraosseus, periocular, intratumoral, intracerebral, and intracerebroventricular administration.

In some embodiments, the pharmaceutical composition described herein is administered locally to a diseased site (e.g., a liver). In some embodiments, the pharmaceutical composition described herein is administered to a subject by injection, by means of a catheter, by means of a suppository, or by means of an implant, the implant being of a porous, non-porous, or gelatinous material, including a membrane, such as a sialastic membrane, or a fiber.

In some embodiments, any of the fusion proteins, gRNAs, and/or complexes described herein are provided as part of a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises any of the fusion proteins or complexes provided herein. In some embodiments pharmaceutical composition comprises a gRNA, a nucleic acid ATTORNEY DOCKET NO. 180802-47402/PCT

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programmable DNA binding protein, a cationic lipid, and a pharmaceutically acceptable excipient. In embodiments, pharmaceutical compositions comprise a lipid nanoparticle and a pharmaceutically acceptable excipient. In embodiments, the lipid nanoparticle contains a gRNA, a base editor, a complex, a base editor system, or a component thereof of the present disclosure, and/or one or more polynucleotides encoding the same. Pharmaceutical compositions can optionally comprise one or more additional therapeutically active substances.

The compositions, as described above, can be administered in effective amounts. The effective amount will depend upon the mode of administration, the particular condition being treated, and the desired outcome. It may also depend upon the stage of the condition, the age and physical condition of the subject, the nature of concurrent therapy, if any, and like factors well-known to the medical practitioner. For therapeutic applications, it is that amount sufficient to achieve a medically desirable result.

In some embodiments, compositions in accordance with the present disclosure can be used for treatment of any of a variety of diseases, disorders, and/or conditions.

METHODS OF TREATMENT

Some aspects of the present disclosure provide methods of treating a subject in need, the method comprising administering to a subject in need an effective therapeutic amount of a pharmaceutical composition as described herein. More specifically, the methods of treatment include administering to a subject in need thereof a lipid nanoparticle containing a guide RNA molecule and an mRNA molecule encoding a base editor of the disclosure. In other embodiments, the methods of the disclosure comprise expressing or introducing into a cell a base editor polypeptide and one or more guide RNAs capable of targeting a nucleic acid molecule encoding at least one polypeptide.

One of ordinary skill in the art would recognize that multiple administrations of the pharmaceutical compositions contemplated in particular embodiments may be required to affect the desired therapy. For example, a composition may be administered to the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times over a span of 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 5, years, 10 years, or more.

Administration of the pharmaceutical compositions contemplated herein may be carried out using conventional techniques including, but not limited to, infusion, transfusion, or parenterally. In some embodiments, parenteral administration includes infusing or injecting intravascularly, intravenously, intramuscularly, intraarterially, intrathecally, intratumorally, ATTORNEY DOCKET NO. 180802-47402/PCT

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intradermally, intraperitoneally, transtracheally, subcutaneously, subcuticularly, intraarticularly, subcapsularly, subarachnoidly and intrastemally.

KITS

The disclosure provides kits for the treatment of galactosemia in a subject. In some embodiments, the kit further includes a base editor system or a polynucleotide encoding a base editor system, wherein the base editor polypeptide system a nucleic acid programmable DNA binding protein (napDNAbp), a deaminase, and a guide RNA. In some embodiments, the napDNAbp is a Cas9 polypeptide. In some embodiments, the polynucleotide encoding the base editor is a mRNA sequence. In some embodiments, the deaminase is a cytidine deaminase or an adenosine deaminase. In some embodiments, the kit comprises an edited cell and instructions regarding the use of such cell.

The kits may further comprise written instructions for using a base editor, base editor system and/or edited cell as described herein. In other embodiments, the instructions include at least one of the following: precautions; warnings; clinical studies; and/or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container. In a further embodiment, a kit comprises instructions in the form of a label or separate insert (package insert) for suitable operational parameters. In yet another embodiment, the kit comprises one or more containers with appropriate positive and negative controls or control samples, to be used as standard(s) for detection, calibration, or normalization. The kit can further comprise a second container comprising a pharmaceutically-acceptable buffer, such as (sterile) phosphate-buffered saline, Ringer’s solution, or dextrose solution. It can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

The practice of embodiments of the present disclosure employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture” (Freshney, 1987); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Current Protocols in Molecular Biology” (Ausubel, 1987); “PCR: The ATTORNEY DOCKET NO. 180802-47402/PCT

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Polymerase Chain Reaction”, (Mullis, 1994); “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the disclosure, and, as such, may be considered in making and practicing embodiments of the disclosure. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the disclosure, and are not intended to limit the scope of what the inventors regard as their invention.

EXAMPLES

Example 1: Base editing of a galactose-l-phosphate uridylyltransf erase (GALT) gene to correct a Q188R mutation

Experiments were undertaken to demonstrate the use of base editor systems to correct a Q188R mutation (i.e., change the R at amino acid position 188 back to a Q) in a GALT polypeptide encoded by a GALT gene in fibroblast cells from two different human donors (GM00440 and GM00441). About 15,000 fibroblast cells from each donor (n=3 for each donor) were added to wells of a 96-well plate (total media volume in each well was 100 pL) and transfected with base editor systems (40 total) containing 100 ng of a guide RNA listed in Table 9 (see Table 1 for full guide RNA sequences) and 200 ng of mRNA encoding a cytidine base editor selected from CBE RrA3f NNGRRT, CBE RrA3f NGG, CBE RrA3f NRTH, CBE RrA3f NG - Nureki, CBE RrA3f SpRY, CBE RrA3F SpRYc, CBE RrA3F NRRH, CBE-T1.52 IBE34 NGG, and CBE6b NGG, where RrA3f, T1.52, or 6b identify the deaminase domain of each cytidine base editor (CBE), IBE34 indicates a base editor where the deaminase domain is inserted within the nucleic acid programmable DNA binding protein domain (napDNAbp) of the base editor positions 906 and 907 referenced to SEQ ID NO: 197, and the terms NNGRRT, NGG, NRTH, NG-Nureki, SpRY, SpRYc, and NRRH indicate the identity of the Cas9 variant serving as the napDNAbp of each base editor and/or the protospacer-adjacent motif (PAM) specificity of the Cas9 variant, where “N” indicates any nucleotide, “R” indicates A or G, “Y” indicates C or T, and “H” indicates A, C, or T. The guide RNAs contained a chemical modification scheme selected from “End-mod” and “LONGEST”. The deaminase domain T1.52 was a cytidine adenosine base editor (CABE) domain. Nucleotide sequences for the site targeted for base editing by each base editor system are provided in Tables 1 and 9 and in FIGs. 2-5, where Table 9 lists particular cytidine nucleotides that were observed in at least some instances ATTORNEY DOCKET NO. 180802-47402/PCT

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as being edited the base editor systems. The cells were transfected with the base editor systems using LIPOFECTAMINE™. Following transfection, the cells were harvested and genomic DNA was then extracted from the fibroblast cells and base editing rates were measured using nextgeneration sequencing (NGS).

Table 9. Target sites corresponding to guide RNA sequences. The nucleotide positions of the referenced “C” nucleotides are indicated relative to the corresponding target site sequence. The target C is shown in lowercase within each target site sequence.

Guide Target Target Site (intronic SEQ Target C C C Name site nucleotides are in ID NO C corresp correspo correspon (number name bold text; exonic onding nding to ding to following nucleotides are in to a a splice non- the plain text) synony site synonymo hyphen mous us indicates bystan bystander a lot der edit number) edit

gRNA983 TSBTx ACACCCTTACCcGGC 714 12 11 10 15 9-11080 11439 AGTGGGGG

gRNA983 TSBTx ACCcGGCAGTGGGGG 718 4 3 2 7 8-11063 11448 TGGGGGTT

gRNA984 TSBTx CCCTTACCcGGCAGT 698 9 8 7 12 0-11081 11431 GGGGGTG

gRNA983 TSBTx CCCTTACCcGGCAGT 716 9 8 7 12 7-11099 11444 GGGGGTGGGGGT

gRNA984 TSBTx CCTTACCcGGCAGTG 700 8 7 6 11 1-11082 11432 GGGGTGG

gRNA984 TSBTx CTGACACCCTTACCc 708 15 14 13 18 2-11083 11436 GGCAGTGG

gRNA984 TSBTx CTTACCcGGCAGTGG 702 7 6 5 10 3-11084 11433 GGGTGGG

gRNA984 TSBTx GACACCCTTACCcGG 712 13 12 11 16 4-11085 11438 CAGTGGGG

gRNA983 TSBTx TACCcGGCAGTGGGG 706 5 4 3 8 2-11062 11435 GTGGGGG

gRNA984 TSBTx TGACACCCTTACCcG 710 14 13 12 17 5-11086 11437 GCAGTGGG

gRNA984 TSBTx TTACCcGGCAGTGGG 704 6 5 4 9

6-11087 11434 GGTGGGG

The allelic distribution of base edits measured in the cells is shown in FIGs. 4, 5, and 8- 11, and maximum OT base editing rates are shown in FIGs. 6 and 7. FIGs.2 and 3 provide a description of the different base edits achieved using the base editor systems. The base editor systems containing gRNA9843 and the cytidine base editor CBE6b, or gRNA9846 and the cytidine base editor CBE6b were associated a high level of editing of the target C (FIGs. 4 and 5). ATTORNEY DOCKET NO. 180802-47402/PCT

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Control experiments were also undertaken demonstrating that base editing rates were similar irrespective of the donor origin of the fibroblast cells and that the fibroblast cells were successfully transfected with the base editor systems. The fibroblast cells from donor GM00440 or GM00441 were transfected with base editor systems containing 300 ng or 200 ng of the guide polynucleotide sgRNA_088 and 100 ng of mRNA encoding the adenosine base editor ABE9.51 (i.e., an adenosine base editor containing a TadA*8.20 adenosine deaminase domain having an S82T (V82T referenced to TadA*7.10) alteration) or a cytidine base editor selected from CBE RrA3f NNGRRT, CBE RrA3f NGG, CBE RrA3f NRTH, CBE RrA3f NG - Nureki, CBE RrA3f SpRY, CBE RrA3F SpRYc, CBE RrA3F NRRH, CBE-T1.52 IBE34 NGG, and CBE6b NGG, where RrA3f, T1.52, or 6b identify the cytidine deaminase domain of each cytidine base editor (CBE), IBE34 indicates a base editor where the cytidine deaminase domain is inserted within the nucleic acid programmable DNA binding protein domain (napDNAbp) of the base editor, and the terms NNGRRT, NGG, NRTH, NG-Nureki, SpRY, SpRYc, and NRRH indicate the identity of the Cas9 variant serving as the napDNAbp of each base editor and/or the protospacer-adjacent motif (PAM) specificity of the Cas9 variant, where “N” indicates any nucleotide, “R” indicates A or G, “Y” indicates C or T, and “H” indicates A, C, or T. The cells were transfected using LIPOFECTAMINE™. Similar base editing rates were observed using either 300 ng or 200 ng of mRNA encoding the base editor systems. Accordingly, base editing rates were minimally influenced by the donor origin of the fibroblasts, if at all, and the fibroblast cells were efficiently transfected with the base editor systems.

Example 2: Use of lipid nanoparticles to deliver to cells a base editor system for altering a galactose-l-phosphate uridylyltransf erase (GALT) gene to correct a Q188R mutation Experiments were undertaken to demonstrate the use of lipid nanoparticles for the delivery of a base editor system to cells to correct a Q188R mutation encoded by a GALT gene.

In the experiments, lipid nanoparticles (LNPL A or LNPL B) containing a base editor system were administered to either A) fibroblast cells from a classical galactosemia patient (GM00441) containing a GALT gene encoding a GALT protein having a Q188R mutation, or to B) wild-type Huh-7 cells. The base editor system of LNPL A contained the guide RNA gRNA9846 and mRNA MRNA9279 encoding a cytidine base editor, and the base editor system of LNPL B contained the guide RNA gRNA10320 and mRNA MRNA9278 encoding a cytidine base editor. The ratio of mRNA to gRNA in LNPL A and LNPL B was 1:1. The N: P ratio of LNPL A and LNPL B was 6:1. The lipid compositions of LNPL A and LPNL B are provided in Table 10 below. Editing at codon 188 of the GALT gene to change a codon encoding R to a ATTORNEY DOCKET NO. 180802-47402/PCT

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codon encoding Q was evaluated in the QM00441 donor cells, and altering of nucleotides near or within codon 188 of the GALT gene (i.e., alteration of bystander nucleotides) was assessed in the Huh-7 cells. Cells were dispensed in tissue culture-treated 96-well plates at a density of 21,000 cells per well, maintained in appropriate cell culture media, and pre-conditioned with 1 pg/mL apolipoprotein E (ApoE) for two hours prior to being contacted with the lipid nanoparticles (LNPs). The LNPs were administered at concentrations ranging from 0.0006 pg/mL to 10 pg/mL, and each concentration was evaluated in triplicate. The cultures were maintained for a period of 72 hours prior to measurement of maximum C> T editing efficiency in the GALT gene using next-generation sequencing (NGS).

Table 10. Lipid composition of LNPL A and LNPL B.

Mol

Component Description

%

3 -((4,4-bi s(((Z)-oct-5 -en- 1 - yl)oxy)butanoyl)oxy)-2-(((7-((2- Ionizable

butyloctanoyl)oxy)heptanoyl)oxy)methyl)propyl 55

lipid

4-(((2-(pyrrolidin- 1 - yl)ethyl)carbamoyl)oxy)decanoate (IZ4)

l,2-distearoyl-sn-glycero-3-phosphocholine

Phospholipid 7.5

(DSPC)

l,2-dimyristoyl-rac-glycero-3- PEG-lipid methoxypolyethylene glycol-2000 (PEG2000- 2.5

DMG)

Sterol Cholesterol 35

Base editing at the intended GALT nucleotide positions was observed in both of the cell types evaluated across all doses evaluated, thereby validating the efficacy of both LNPL A and LNPL B (FIGs. 14 and 15). Cell viability was also maintained post-dosing.

OTHER EMBODIMENTS

From the foregoing description, it will be apparent that variations and modifications may be made to the aspects or embodiments described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof. ATTORNEY DOCKET NO. 180802-47402/PCT

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All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.

Claims

ATTORNEY DOCKET NO. 180802-47402/PCT ELECTRONIC DEPOSIT DATE: December 10, 2025 CLAIMS What is claimed is:

1. A method of editing a nucleobase of a galactose- 1 -phosphate uridylyltransferase (GALT) polynucleotide, the method comprising contacting the GALT polynucleotide with a guide RNA, or a polynucleotide encoding the guide RNA, and a base editor comprising a deaminase domain capable deaminating cytidine in DNA and a nucleic acid programmable DNA binding protein (napDNAbp) domain, or one or more polynucleotides encoding the base editor, wherein said guide RNA targets said base editor to effect an alteration to a nucleobase in codon 188 of the GALT polynucleotide encoding an R amino acid, wherein the alteration to the nucleobase changes codon 188 to a codon encoding a Q amino acid, and wherein:

a) the deaminase domain comprises an amino acid sequence with at least 85% sequence identity to a deaminase selected from the group consisting of 6b, RrA3f, and T1.52; and/or b) the guide RNA comprises a spacer, wherein the spacer comprises at least 19 contiguous nucleotides of a sequence selected from the group consisting of:

UUACCCGGCAGUGGGGGUGG (SEQ ID NO: 705); UUACCCGGCAGUGGGGGUG (SEQ ID NO: 793), CCCUUACCCGGCAGUGGGGG (SEQ ID NO: 699), CCUUACCCGGCAGUGGGGGU (SEQ ID NO: 701), CUUACCCGGCAGUGGGGGUG (SEQ ID NO: 703),, UACCCGGCAGUGGGGGUGGG (SEQ ID NO: 707), CUGACACCCUUACCCGGCAG (SEQ ID NO: 709), UGACACCCUUACCCGGCAGU (SEQ ID NO: 711), GACACCCUUACCCGGCAGUG (SEQ ID NO: 713), ACACCCUUACCCGGCAGUGG (SEQ ID NO: 715), CCCUUACCCGGCAGUGGGGGU (SEQ ID NO: 717), and ACCCGGCAGUGGGGGUGGGG (SEQ ID NO: 719).

2. A method for treating galactosemia in a subject in need thereof, wherein the subject comprises a galactose- 1 -phosphate uridylyltransferase (GALT) polynucleotide encoding a GALT polypeptide having a Q188R mutation, the method comprising: administering to the subject a base editor system comprising a guide RNA, or a polynucleotide encoding the guide RNA, and a base editor comprising a deaminase domain capable of deaminating cytidine in DNA and a nucleic acid programmable DNA binding protein (napDNAbp) domain, or one or more polynucleotides encoding the base editor, wherein said guide RNA targets said base editor to effect an alteration to a nucleobase in codon 188 of the GALT polynucleotide, wherein the alteration to the nucleobase changes codon 188 to a codon encoding a Q amino acid, and wherein: ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

UUACCCGGCAGUGGGGGUGG (SEQ ID NO: 705); UUACCCGGCAGUGGGGGUG (SEQ ID NO: 793), CCCUUACCCGGCAGUGGGGG (SEQ ID NO: 699), CCUUACCCGGCAGUGGGGGU (SEQ ID NO: 701), CUUACCCGGCAGUGGGGGUG (SEQ ID NO: 703), UACCCGGCAGUGGGGGUGGG (SEQ ID NO: 707), CUGACACCCUUACCCGGCAG (SEQ ID NO: 709), UGACACCCUUACCCGGCAGU (SEQ ID NO: 711), GACACCCUUACCCGGCAGUG (SEQ ID NO: 713), ACACCCUUACCCGGCAGUGG (SEQ ID NO: 715), CCCUUACCCGGCAGUGGGGGU (SEQ ID NO: 717), and ACCCGGCAGUGGGGGUGGGG (SEQ ID NO: 719).

3. The method of claim 2 further comprising administering to the subject a lipid nanoparticle comprising the base editor system.

4. A method of editing a nucleobase of a galactose- 1 -phosphate uridylyltransferase (GALT) polynucleotide, the method comprising contacting the GALT polynucleotide with a guide RNA and mRNA encoding a base editor comprising a deaminase domain comprising a deaminase selected from the group consisting of 6b, RrA3f, and T1.52, and a nucleic acid programmable DNA binding protein (napDNAbp) domain, wherein the napDNAbp is an SpCas9, an SpRY, an SpRYc, an saCas9, or an spCas9 variant having specificity for a protospacer-adjacent motif (PAM) selected from the group consisting of: NG, NRRH, and NRTH, wherein “N” indicates A, C, G, or T, “R” indicates A or G, “Y” indicates C or T, and “H” indicates A, C, or T, wherein said guide RNA targets said base editor to effect an alteration to a nucleobase in codon 188 of the GALT polynucleotide encoding an R amino acid, wherein the alteration to the nucleobase changes codon 188 to a codon encoding a Q amino acid, and wherein the guide RNA comprises a spacer, wherein the spacer consists of a sequence selected from the group consisting of:

UUACCCGGCAGUGGGGGUGG (SEQ ID NO: 705); UUACCCGGCAGUGGGGGUG (SEQ ID NO: 793), CCCUUACCCGGCAGUGGGGG (SEQ ID NO: 699), CCUUACCCGGCAGUGGGGGU (SEQ ID NO: 701), CUUACCCGGCAGUGGGGGUG (SEQ ID NO: 703), UACCCGGCAGUGGGGGUGGG (SEQ ID NO: 707), CUGACACCCUUACCCGGCAG (SEQ ID NO: 709), UGACACCCUUACCCGGCAGU (SEQ ID NO: 711), GACACCCUUACCCGGCAGUG (SEQ ID NO: 713), ACACCCUUACCCGGCAGUGG (SEQ ID NO: 715), CCCUUACCCGGCAGUGGGGGU (SEQ ID NO: 717), and ACCCGGCAGUGGGGGUGGGG (SEQ ID NO: 719). ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

5. A method of treatment comprising administering to a subject in need thereof, wherein the subject comprises a galactose- 1 -phosphate uridylyltransferase (GALT) polynucleotide encoding a GALT polypeptide having a Q188R mutation, a lipid nanoparticle (LNP) comprising: a guide RNA and mRNA encoding a base editor comprising a deaminase domain comprising a deaminase selected from the group consisting of 6b, RrA3f, and T1.52, and a nucleic acid programmable DNA binding protein (napDNAbp) domain, wherein the napDNAbp is an SpCas9, an SpRY, an SpRYc, an saCas9, or an spCas9 variant having specificity for a protospacer-adjacent motif (PAM) selected from the group consisting of: NG, NRRH, and NRTH, wherein “N” indicates A, C, G, or T, “R” indicates A or G, “Y” indicates C or T, and “H” indicates A, C, or T, wherein said guide RNA targets said base editor to effect an alteration to a nucleobase in codon 188 of the GALT polynucleotide encoding an R amino acid, wherein the alteration to the nucleobase changes codon 188 to a codon encoding a Q amino acid, and wherein the guide RNA comprises a spacer, wherein the spacer consists of a sequence selected from the group consisting of: UUACCCGGCAGUGGGGGUGG (SEQ ID NO: 705);

UUACCCGGCAGUGGGGGUG (SEQ ID NO: 793, CCCUUACCCGGCAGUGGGGG (SEQ ID NO: 699), CCUUACCCGGCAGUGGGGGU (SEQ ID NO: 701), CUUACCCGGCAGUGGGGGUG (SEQ ID NO: 703), UACCCGGCAGUGGGGGUGGG (SEQ ID NO: 707), CUGACACCCUUACCCGGCAG (SEQ ID NO: 709), UGACACCCUUACCCGGCAGU (SEQ ID NO: 711), GACACCCUUACCCGGCAGUG (SEQ ID NO: 713), ACACCCUUACCCGGCAGUGG (SEQ ID NO: 715), CCCUUACCCGGCAGUGGGGGU (SEQ ID NO: 717), and ACCCGGCAGUGGGGGUGGGG (SEQ ID NO: 719).

6. The method of any one of claims 1-5, wherein the guide RNA comprises a sequence selected from the group consisting of: mUsmUsmAsCCCGGCAGUGGGGGUGGGUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmCmC mGmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmC mAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 746); mUsmUsmAsCCCGGCAGUGGGGGUGGUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmCmCm GmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCm AmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 791);

mC smC smC sUUACCCGGCAGUGGGGGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAG UCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCmUsmUsmUsU (SEQ ID NO: 728); ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

mC smC smUsUACCCGGCAGUGGGGGUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAG UCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCmUsmUsmUsU (SEQ ID NO: 729); mCsmUsmUsACCCGGCAGUGGGGGUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAG UCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCmUsmUsmUsU (SEQ ID NO: 730); mUsmUsmAsCCCGGCAGUGGGGGUGGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAG UCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCmUsmUsmUsU (SEQ ID NO: 731); mUsmAsmC s CCGGCAGUGGGGGUGGGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAG UCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCmUsmUsmUsU (SEQ ID NO: 732); mCsmUsmGsACACCCUUACCCGGCAGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAG UCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCmUsmUsmUsU (SEQ ID NO: 733); mUsmGsmAsCACCCUUACCCGGCAGUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAG UCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCmUsmUsmUsU (SEQ ID NO: 734); mGsmAsmCsACCCUUACCCGGCAGUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAG UCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCmUsmUsmUsU (SEQ ID NO: 735); mAsmC smAs CCCUUACCCGGCAGUGGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAG UCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCmUsmUsmUsU (SEQ ID NO: 736); mC smC smC sUUACCCGGCAGUGGGGGUGUUUUAGUACUCUGUAAUGAAAAUUACAGAAUCUACU AAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUsmUsmUsmU (SEQ ID NO: 737);

mC s mU s mG s AC AC C CUUAC C CGGC AGGUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmCmC mGmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmC mAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 742); ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025 mCsmUsmUsACCCGGCAGUGGGGGUGGUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmCmC mGmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmC mAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 743);

mG s mA s mC s AC C CUUAC C CGGC AGUGGUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmCmC mGmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmC mAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 744); and mUsmGsmAsCACCCUUACCCGGCAGUGUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmCmC mGmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmC mAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 745);

wherein “mN” indicates a 2'-OMe modification of the nucleotide “N”, and “Ns” indicates that the nucleotide “N” is linked to the following nucleotide by a phosphorothioate (PS).

7. A method for producing a cell, the method comprising editing a GALT polynucleotide in a cell according to the method of claim 1 or claim 4.

8. A lipid nanoparticle (LNP) comprising:

A) a guide RNA, or a polynucleotide encoding the guide RNA, wherein the guide RNA comprises a spacer comprising a nucleotide sequence selected from UUACCCGGCAGUGGGGGUGG (SEQ ID NO: 705); UUACCCGGCAGUGGGGGUG (SEQ ID NO: 793), and those listed in Table 1;

B) an mRNA molecule, or a polynucleotide encoding the mRNA molecule, wherein the mRNA molecule encodes a base editor polypeptide comprising a nucleic acid programmable DNA binding protein (napDNAbp) domain and a deaminase domain, wherein the deaminase domain comprises an amino acid sequence with at least 90% sequence identity to a deaminase selected from the group consisting of 6b, RrA3f, and T1.52; and

C) an ionizable lipid according to any one of the following formulas, or a pharmaceutically acceptable salt thereof:

i) a compound of Formula (I):

J ³ Ak

R¹ L² O Y³ O

.. I Y¹ JI JL ^X² ^R^_O ^Y² O X¹ X³ R¹' 1 zL¹ _x

>> L²' °

_(I) wherein: ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

each L¹ and L^1' is independently -C(O)- or -OC(O)-;

L³ is a covalent bond, -O-, -C(O)O-, -OC(O)-, or -OC(O)O-;

O-R^a

l__LCyA__c A M

R¹ is optionally substituted C1-20 aliphatic, 1, or O-R^a;

each R^a and R¹ is independently optionally substituted C1-20 aliphatic;

Y¹ is -C(O)- or -C(O)O-;

Y³ is optionally substituted C1-20 aliphatic;

X¹ is a covalent bond, -O-, or -NR-;

each R is independently hydrogen or optionally substituted C1-6 aliphatic;

ii) a compound of formula A’:

. L³

O R

L^o R

/X²

L²

RV bk ^R"

A’ ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

or its N-oxide, or a pharmaceutically acceptable salt thereof, wherein

L¹ is absent, C1-6 alkylenyl, or C2-6 heteroalkylenyl;

L is Ci-10 alkylenyl, or C2-10 heteroalkyl enyl;

X² is -OC(O)-, -C(O)O-, or -OC(O)O-;

X is absent, -OC(O)-, -C(O)O-, or -OC(O)O-;

O

y^o^A _R6

L^3a [

O'" ^R^a %

vk

R” is hydrogen,

O R _a

, O R⁶, or an optionally substituted group selected from C6-20 aliphatic, 3- to 12-membered cycloaliphatic, 7- to 12-membered bridged bicyclic comprising 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 1- adamantyl, 2-adamantyl, sterolyl, and phenyl;

ELECTRONIC DEPOSIT DATE: December 10, 2025

R² R

/ \²

R

¹ is ° O, or a ring selected from 3- to 7-membered cycloaliphatic and 3- to 7- membered heterocyclyl comprising 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, wherein the cycloaliphatic or heterocyclyl ring is optionally substituted with 1-4 R² or R³ groups;

each R³ is independently -(CH2)n-R⁴; or

R⁵ R⁵

/ \

R⁵ *

-Z —

-

NR⁵C(S)N(R⁵)2, -NR⁵C(NR⁵)N(R⁵)2, or O O ■

each R⁶ is independently C4-12 aliphatic; and

each n is independently 0 to 4; or

iii) a compound of Formula I: ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

or a pharmaceutically acceptable salt thereof, wherein:

L¹ is a covalent bond, -C(O)-, or -OC(O)-;

branched C1-C12 hydrocarbon chain, or ^{m m};

each m is independently 0, 1, or 2;

L³ is a covalent bond, -C(O)-, -C(O)O-, -OC(O)-, -O-, or -OC(O)O-;

H^L ₂

replaced with -O- or -NR-, or O^_A;

unsaturated carbocyclyl, 1-adamantyl, 2-adamantyl,

, sterolyl, and phenyl; p is 0, 1, 2, or 3;

^#K2) X.

where

x is selected from 1 or 2; and

# represents the point of attachment to L⁴;

each L⁵ is independently a bivalent saturated or unsaturated, straight or branched C1-C20 hydrocarbon chain, wherein 1-3 methylene units are optionally and independently replaced with -O- or -NR-; ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

X¹ is a covalent bond, -O-, or -NR-;

FH-cy^B

provided that when L is a covalent bond, then R must be ^p

9. A lipid nanoparticle (LNP) comprising:

A) a guide RNA, or a polynucleotide encoding the guide RNA, wherein the guide RNA is capable of directing a base editor polypeptide to alter a nucleotide in a galactose- 1 -phosphate uridylyltransferase (GALT) polynucleotide;

C) an ionizable lipid according to Formula (I), or a pharmaceutically acceptable salt thereof: ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

wherein:

each L¹ and L^1' is independently -C(O)- or -OC(O)-;

L³ is a covalent bond, -O-, -C(O)O-, -OC(O)-, or -OC(O)O-;

O-R^a

i__LCyA__c A |“^LRa~(

R¹ is optionally substituted C1-20 aliphatic, ‘ ^y, or O-R^a;

each R^a and R¹ is independently optionally substituted C1-20 aliphatic;

Y¹ is -C(O)- or -C(O)O-;

Y³ is optionally substituted C1-20 aliphatic;

X¹ is a covalent bond, -O-, or -NR-;

each R is independently hydrogen or optionally substituted C1-6 aliphatic. ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

10. The LNP of claim 8 or claim 9, wherein the lipid nanoparticle comprises an ionizable lipid having the following structure, or a pharmaceutically acceptable salt thereof:

11. The LNP of claim 8 or claim 9, wherein the LNP comprises a molar ratio of ionizable nitrogen atoms in an ionizable lipid to the total negative charge in the payload (N: P ratio) of between about 5: 1 and 7:1.

12. The LNP of claim 11, wherein the LNP comprises an N: P ratio of about 6: 1.

13. The LNP of claim 8 or claim 9, wherein the guide RNA comprises a scaffold with the following nucleotide sequence:

GUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmCmCmGmGmCAAGUUAAAAUAAGGCUAGUC CGUU AU C AmAmCmU mU mGmAmAmAm AmAmGmU mGmGmCm AmCmCmGm AmGmU mCmGmGmU mGm CmUsmUsmUsmU (SEQ ID NO: 794), wherein “mN” indicates a 2'-0Me modification of the nucleotide “N”, and “Ns” indicates that the nucleotide “N” is linked to the following nucleotide by a phosphorothioate (PS).

14. The LNP of claim 8 or claim 9, wherein the guide RNA comprises a spacer comprising the nucleotide sequence UUACCCGGCAGUGGGGGUGG (SEQ ID NO: 705) or UUACCCGGCAGUGGGGGUG (SEQ ID NO: 793).

15. The LNP of claim 8 or claim 9, wherein the guide RNA comprises a nucleotide sequence selected from those listed in Table 2.

16. The LNP of claim 8 or claim 9, wherein the guide RNA comprises one of the following nucleotide sequences: mUsmUsmAsCCCGGCAGUGGGGGUGGGUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmCmC mGmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmC mAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 746); ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025 mUsmUsmAsCCCGGCAGUGGGGGUGGUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmCmCm GmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCm AmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 791);

17. The LNP of claim 9, wherein the guide RNA comprises a spacer selected from those listed in Table 1.

18. The LNP of claim 8 or claim 9, wherein the napDNAbp is an SpCas9, an SpRY, an SpRYc, an saCas9, or an spCas9 variant having specificity for a protospacer-adjacent motif (PAM) selected from the group consisting of NG, NRRH, and NRTH, wherein “N” indicates A, C, G, or T, “R” indicates A or G, “Y” indicates C or T, and “H” indicates A, C, or T.

19. The LNP of claim 18, wherein the napDNAbp is a nickase.

20. The LNP of claim 8 or claim 9, wherein the base editor polypeptide comprises an amino acid sequence with at least about 90% identity to a sequence listed in Table 2.

21. A lipid nanoparticle (LNP) comprising:

A) a guide RNA having a nucleotide sequence selected from the group consisting of: mUsmUsmAsCCCGGCAGUGGGGGUGGGUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmCmC mGmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmC mAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 746); and mUsmUsmAsCCCGGCAGUGGGGGUGGUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmCmCm GmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCm AmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 791), wherein “mN” indicates a 2'-0Me modification of the nucleotide “N”, and “Ns” indicates that the nucleotide “N” is linked to the following nucleotide by a phosphorothioate (PS);

B) a base editor polypeptide, or a polynucleotide encoding the base editor polypeptide, wherein the base editor polypeptide comprises a nucleic acid programmable DNA binding protein (napDNAbp) domain and a deaminase domain comprising a deaminase domain comprising a deaminase selected from the group consisting of 6b, RrA3f, and T1.52; and ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

C) an ionizable lipid having the following structure, or a pharmaceutically acceptable salt

thereof:

22. The lipid nanoparticle of claim 21, wherein the base editor polypeptide comprises an amino acid sequence with at least about 90% identity to a sequence listed in Table 2.

23. A base editor system comprising a guide RNA, or a polynucleotide encoding the guide RNA, and a base editor comprising a deaminase domain capable of deaminating cytidine in DNA and a nucleic acid programmable DNA binding protein (napDNAbp) domain, or one or more polynucleotides encoding the base editor, wherein said guide RNA targets said base editor to effect an alteration to a nucleobase in codon 188 of a GALT polynucleotide encoding an R amino acid, wherein the alteration to the nucleobase changes codon 188 to a codon encoding a Q amino acid, and wherein:

UUACCCGGCAGUGGGGGUGG (SEQ ID NO: 705), UUACCCGGCAGUGGGGGUG (SEQ ID NO: 793), CCCUUACCCGGCAGUGGGGG (SEQ ID NO: 699), CCUUACCCGGCAGUGGGGGU (SEQ ID NO: 701), CUUACCCGGCAGUGGGGGUG (SEQ ID NO: 703), UACCCGGCAGUGGGGGUGGG (SEQ ID NO: 707), CUGACACCCUUACCCGGCAG (SEQ ID NO: 709), UGACACCCUUACCCGGCAGU (SEQ ID NO: 711), GACACCCUUACCCGGCAGUG (SEQ ID NO: 713), ACACCCUUACCCGGCAGUGG (SEQ ID NO: 715), CCCUUACCCGGCAGUGGGGGU (SEQ ID NO: 717), and ACCCGGCAGUGGGGGUGGGG (SEQ ID NO: 719).

24. A base editor system comprising a guide RNA and mRNA encoding a base editor comprising a deaminase domain comprising a deaminase selected from the group consisting of 6b, RrA3f, and T1.52, and a nucleic acid programmable DNA binding protein (napDNAbp) domain, wherein the napDNAbp is an SpCas9, an SpRY, an SpRYc, an saCas9, or an spCas9 variant having specificity for a protospacer-adjacent motif (PAM) selected from the group ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

consisting of: NG, NRRH, and NRTH, wherein “N” indicates A, C, G, or T, “R” indicates A or G, “Y” indicates C or T, and “H” indicates A, C, or T, wherein said guide RNA targets said base editor to effect an alteration to a nucleobase in codon 188 of the GALT polynucleotide encoding an R amino acid, wherein the alteration to the nucleobase changes codon 188 to a codon encoding a Q amino acid, and wherein the guide RNA comprises a spacer, wherein the spacer consists of a sequence selected from the group consisting of: UUACCCGGCAGUGGGGGUGG (SEQ ID NO: 705), UUACCCGGCAGUGGGGGUG (SEQ ID NO: 793), CCCUUACCCGGCAGUGGGGG (SEQ ID NO: 699), CCUUACCCGGCAGUGGGGGU (SEQ ID NO: 701), CUUACCCGGCAGUGGGGGUG (SEQ ID NO: 703), UACCCGGCAGUGGGGGUGGG (SEQ ID NO: 707), CUGACACCCUUACCCGGCAG (SEQ ID NO: 709), UGACACCCUUACCCGGCAGU (SEQ ID NO: 711), GACACCCUUACCCGGCAGUG (SEQ ID NO: 713), ACACCCUUACCCGGCAGUGG (SEQ ID NO: 715), CCCUUACCCGGCAGUGGGGGU (SEQ ID NO: 717), and ACCCGGCAGUGGGGGUGGGG (SEQ ID NO: 719).

25. A polynucleotide or set of polynucleotides encoding the base editor system of claim 23 or claim 24, or a component thereof.

26. A lipid nanoparticle comprising the base editor system of claim 23 or claim 24, or the polynucleotide or set of polynucleotides of claim 25.

27. A method for modifying a target nucleobase in a galactose- 1 -phosphate uridylyltransferase (GALT) polynucleotide in a cell, the method comprising contacting the cell with the lipid nanoparticle of any one of claims 8-22 or claim 26, the base editor system of claim 23 or claim 24, or the polynucleotide or set of polynucleotides of claim 25, thereby modifying the target nucleobase in the GALT polynucleotide.

28. The method of claim 27, wherein the cell is in a subject.

29. A method of treating a disease in a subject in need thereof, wherein the disease is associated with a pathogenic mutation in a galactose- 1 -phosphate uridylyltransferase (GALT) polynucleotide in the subject, the method comprising administering to the subject the lipid nanoparticle of any one of claims 8-22 or claim 26, the base editor system of claim 23 or claim 24, or the polynucleotide or set of polynucleotides of claim 25, thereby altering a target nucleobase in the GALT polynucleotide in the subject. ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

30. The method of claim 29, wherein the disease is galactosemia.

31. A composition comprising: a guide RNA and mRNA encoding a base editor comprising a deaminase domain comprising a deaminase selected from the group consisting of 6b, RrA3f, and T1.52, and a nucleic acid programmable DNA binding protein (napDNAbp) domain, wherein the napDNAbp is an spCas9, SpRY, an SpRYc, an saCas9, or an spCas9 variant having specificity for a protospacer-adjacent motif (PAM) selected from the group consisting of: NG, NRRH, and NRTH, wherein “N” indicates A, C, G, or T, “R” indicates A or G, “Y” indicates C or T, and “H” indicates A, C, or T, wherein said guide RNA targets said base editor to effect an alteration to a nucleobase in codon 188 of the GALT polynucleotide encoding an R amino acid, wherein the alteration to the nucleobase changes codon 188 to a codon encoding a Q amino acid, and wherein the guide RNA comprises a spacer, wherein the spacer consists of a sequence selected from the group consisting of: UUACCCGGCAGUGGGGGUGG (SEQ ID NO: 705);

UUACCCGGCAGUGGGGGUG (SEQ ID NO: 793), CCCUUACCCGGCAGUGGGGG (SEQ ID NO: 699), CCUUACCCGGCAGUGGGGGU (SEQ ID NO: 701), CUUACCCGGCAGUGGGGGUG (SEQ ID NO: 703), UACCCGGCAGUGGGGGUGGG (SEQ ID NO: 707), CUGACACCCUUACCCGGCAG (SEQ ID NO: 709), UGACACCCUUACCCGGCAGU (SEQ ID NO: 711), GACACCCUUACCCGGCAGUG (SEQ ID NO: 713), ACACCCUUACCCGGCAGUGG (SEQ ID NO: 715), CCCUUACCCGGCAGUGGGGGU (SEQ ID NO: 717), and ACCCGGCAGUGGGGGUGGGG (SEQ ID NO: 719).

32. A lipid nanoparticle (LNP) composition comprising: a guide RNA and mRNA encoding a base editor comprising a deaminase domain comprising a deaminase selected from the group consisting of 6b, RrA3f, and T1.52, and a nucleic acid programmable DNA binding protein (napDNAbp) domain, wherein the napDNAbp is an spCas9, SpRY, an SpRYc, an saCas9, or an spCas9 variant having specificity for a protospacer-adjacent motif (PAM) selected from the group consisting of: NG, NRRH, and NRTH, wherein “N” indicates A, C, G, or T, “R” indicates A or G, “Y” indicates C or T, and “H” indicates A, C, or T, wherein said guide RNA targets said base editor to effect an alteration to a nucleobase in codon 188 of the GALT polynucleotide encoding an R amino acid, wherein the alteration to the nucleobase changes codon 188 to a codon encoding a Q amino acid, and wherein the guide RNA comprises a spacer, wherein the spacer consists of a sequence selected from the group consisting of:

UUACCCGGCAGUGGGGGUGG (SEQ ID NO: 705); UUACCCGGCAGUGGGGGUG (SEQ ID NO: ATTORNEY DOCKET NO. 180802-47402/PCT

ELECTRONIC DEPOSIT DATE: December 10, 2025

793), CCCUUACCCGGCAGUGGGGG (SEQ ID NO: 699), CCUUACCCGGCAGUGGGGGU (SEQ ID NO: 701), CUUACCCGGCAGUGGGGGUG (SEQ ID NO: 703), UACCCGGCAGUGGGGGUGGG (SEQ ID NO: 707), CUGACACCCUUACCCGGCAG (SEQ ID NO: 709), UGACACCCUUACCCGGCAGU (SEQ ID NO: 711), GACACCCUUACCCGGCAGUG (SEQ ID NO: 713), ACACCCUUACCCGGCAGUGG (SEQ ID NO: 715), CCCUUACCCGGCAGUGGGGGU (SEQ ID NO: 717), and ACCCGGCAGUGGGGGUGGGG (SEQ ID NO: 719).

33. A guide polynucleotide comprising a spacer or gRNA nucleotide sequence listed in Table 1

34. A pharmaceutical composition comprising an effective amount of the lipid nanoparticle of any one of claims 8-22 or claim 26, the base editor system of claim 23 or claim 24, the polynucleotide or set of polynucleotides of claim 25, the composition of claim 31 or claim 32, or the guide polynucleotide of claim 33, and a pharmaceutically acceptable excipient.

35. A kit comprising the lipid nanoparticle of any one of claims 8-22 or claim 26, the base editor system of claim 23 or claim 24, the polynucleotide or set of polynucleotides of claim 25, the composition of claim 31 or claim 32, the guide polynucleotide of claim 33, or the pharmaceutical composition of claim 34, disposed within a container.