WO2025111452A2 - Modifications chimiques dans pegarn et ngarn - Google Patents

Modifications chimiques dans pegarn et ngarn Download PDF

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WO2025111452A2
WO2025111452A2 PCT/US2024/056877 US2024056877W WO2025111452A2 WO 2025111452 A2 WO2025111452 A2 WO 2025111452A2 US 2024056877 W US2024056877 W US 2024056877W WO 2025111452 A2 WO2025111452 A2 WO 2025111452A2
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modified
pegrna
nucleotide
forgoing
modification
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WO2025111452A9 (fr
WO2025111452A3 (fr
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Ekambareswara Rao KANDIMALLA
Mallikarjuna Putta
Seth Alexander
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Prime Medicine Inc
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Prime Medicine Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/317—Chemical structure of the backbone with an inverted bond, e.g. a cap structure
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/322—2'-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • C12N2310/335—Modified T or U

Definitions

  • the present disclosure describes modified PEgRNA and ngRNA sequences and compositions comprising the same, as well as using the same in methods for gene editing.
  • RNA molecules used for prime editing such as prime editing guide RNA (PEgRNA) and nicking guide RNA (ngRNA) are intrinsically unstable and prone to degradation, although some chemical modifications have been reported to improve their stability.
  • PEgRNA prime editing guide RNA
  • ngRNA nicking guide RNA
  • PEgRNA prime editing guide RNA
  • ngRNA prime editor nicking guide RNA
  • the disclosure provides a modified PEgRNA comprising, 5 ? to 3 ? :
  • gRNA guide RNA
  • the gRNA core comprises, in 5’ to 3’ order, a tetraloop, a first stem loop (SL1). a second stem loop (SL2), and a third stem loop (SL3);
  • an extension arm comprising:
  • a primer binding site that comprises a region of complementarity to a region upstream of a nick site in a non-target strand of the double- stranded target DNA
  • the PEgRNA comprises 5 or more nucleotide modifications.
  • the disclosure provides a modified ngRNA comprising, 5’ to 3’:
  • gRNA guide RNA
  • the gRNA core comprises, in 5’ to 3’ order, a tetraloop, a first stem loop (SL1). a second stem loop (SL2), and a third stem loop (SL3); and
  • ngRNA comprises 5 or more nucleotide modifications.
  • a nucleotide modification is located in the 3’ end.
  • a nucleotide modification is located in the 5’ end.
  • nucleotide modifications are located in the 3’ end and the 5' end.
  • a nucleotide modification is located in the spacer.
  • a nucleotide modification is located in the gRNA core.
  • a nucleotide modification is located in the extension arm.
  • a nucleotide modification is located in the tetraloop.
  • a nucleotide modification is located in the SL1.
  • a nucleotide modification is located in the SL2.
  • a nucleotide modification is located in the SL3.
  • At least 2 or more nucleotide modifications are located in the 5’ end, 3’ end, tetraloop, SL2, or SL3.
  • nucleotide modifications are located in the tetraloop, SL1, SL2, and SL3.
  • nucleotide modifications are located in the tetraloop, SL2, and SL3.
  • nucleotide modifications are located in the 5’ end, 3’ end, tetraloop, SL2, and SL3.
  • At least 2 or more nucleotide modifications occur within the gRNA.
  • the gRNA core comprises a nucleotide modification at nucleotide 25, 37, 38, 46, 64, 65, 67, or 68.
  • the gRNA core comprises a nucleotide modification at nucleotide 25.
  • the gRNA core comprises a nucleotide modification at nucleotide 37 and/or 38.
  • the gRNA core comprises a nucleotide modification at nucleotide 65.
  • the gRNA core comprises a nucleotide modification at nucleotide 68.
  • the PEgRNA or ngRNA comprises 10 or more nucleotide modifications.
  • the PEgRNA or ngRNA comprises 20 or more nucleotide modifications.
  • the PEgRNA or ngRNA comprises 30 or more nucleotide modifications.
  • the PEgRNA or ngRNA comprises 40 or more nucleotide modifications.
  • the PEgRNA or ngRNA comprises 50 or more nucleotide modifications.
  • the PEgRNA or ngRNA comprises 60 or more nucleotide modifications. [0036] In some embodiments, the PEgRNA or ngRNA comprises 70 or more nucleotide modifications.
  • the PEgRNA or ngRNA comprises 80 or more nucleotide modifications.
  • the PEgRNA or ngRNA comprises 90 or more nucleotide modifications.
  • the PEgRNA or ngRNA comprises 100 or more modified nucleotides.
  • a base modification is selected from the group consisting of N 6 -methyladenosine (m 6 A), N 6 -methyl-2'-O-methyladenosine (2’0Me-m 6 A), xanthine, allyaminouracil, allyaminothymidine, hypoxanthine, digoxigeninated adenine, digoxigeninated cytosine, digoxigeninated guanine, digoxigeninated uracil, 6- chloropurineriboside, N6-methyladenine, methylpseudouracil, 2-thiocytosine, 2-thiouracil, 5- methyluracil.
  • 2,6-daminoguanine 5 -carboxamide- uracil, 5-ethynyluracil, N 6 -isopentenyladenine (i 6 A), 2- methyl-thio- N 6 -isopentenyladenine (ms2i 6 A), 2-methylthio- N 6 -methyladenine (ms2m 6 A), N6-(cis-hydroxyisopentenyl)adenine (io 6 A).
  • 2-methylthio- N 6 -(cis- hydroxyisopentenyl)adenine (ms2io 6 A), N 6 -glycinyl carbamoyl adenine (g 6 A), N 6 - threonylcarbamoyladenine (t 6 A), 2-methylthio- N 6 -threonyl carbamoyladenine (ms2t 6 A), N 6 - methyl- N 6 -threonylcarbamoyladenine (m 6 t 6 A), N 6 -hy droxynorvalylcarbamoyladenine (hn 6 A).
  • the modification comprises an inverted nucleotide located in the 3’ end.
  • the inverted nucleotide comprises
  • a nucleotide modification is a sugar modification.
  • the sugar modification comprises [0045] In some embodiments, the sugar modification is selected from the group consisting of 2’ -thioribose, 2’. 3 ’ -di deoxy ribose, 2’-amino-2’-deoxyribose. 2’ deoxyribose, 2’-azido-2’- deoxyribose, 2’ -fl uoro-2’ -deoxyribose, 2’-O-methoxyethyl (MOE).
  • the sugar modification is selected from the group consisting of 2’ -thioribose, 2’. 3 ’ -di deoxy ribose, 2’-amino-2’-deoxyribose. 2’ deoxyribose, 2’-azido-2’- deoxyribose, 2’ -fl uoro-2’ -deoxyribose, 2’-O-methoxyethyl (MOE).
  • a nucleotide modification is a phosphate modification.
  • a phosphate modification comprises phosphorothioate, a stereospecific phosphorothioate, or a combination thereof.
  • the phosphate modification is selected from the group consisting of phosphorothioate (PS), a stereospecific phosphorothioate, phosphorodithioate, thiophosphate, 5’-O-methylphosphonate, 3’-O- methylphosphonate, 5’-hydroxyphosphonate, hydroxyphosphanate, phosphoroselenoate, selenophosphate, phosphoramidate, carbophosphonate.
  • PS phosphorothioate
  • a stereospecific phosphorothioate phosphorodithioate
  • thiophosphate 5’-O-methylphosphonate
  • 3’-O- methylphosphonate 3’-O- methylphosphonate
  • 5’-hydroxyphosphonate hydroxyphosphanate
  • phosphoroselenoate selenophosphate
  • phosphoramidate carbophosphonate
  • methylphosphonate methylphosphonate, phenylphosphonate, ethylphosphonate, H- phosphonate, guanidinium ring, triazole ring, boranophosphate (BP), methylphosphonate, and guanidinopropyl phosphoramidate.
  • the nucleotide modification comprises a phosphate modification, a base modiication, a sugar modification, or a combination thereof.
  • a modified PEgRNA sequence is selected from the group consisting of Tables 5 or 6.
  • the nucleotide modification comprises a non-nucleotide modification comprises a hairpin a C2-C12 linker, an ethylene glycol linker, 2’-5‘ linkages, or a combination thereof.
  • Another aspect of the disclosure provides a method for producing a modified PEgRNA or modified ngRNA, the method comprising ligating a coding nucleotide sequence with the modified nucleic acid sequence disclosed herein.
  • Another aspect of the disclosure provides a method for producing a modified PEgRNA or modified ngRNA, the method comprising ligating a sequence encoding a prime editor with the modified nucleic acid sequence disclosed herein.
  • the ligation comprises a self-templated enzy matic ligation.
  • the self-templated enzymatic ligation is splint-mediated.
  • the ligation comprises a templated enzymatic ligation.
  • the enzymatic ligation is catalyzed by a nucleic acid ligase to generate a natural phosphodiester linkage between the coding nucleotide sequence and the modified nucleic acid sequence of any one of the foregoing claims, or between the sequence encoding a prime editor with the modified nucleic acid sequence of any one of the foregoing claims, or between two fragments of the nucleotide sequences containing the chemical modifications of any one of the foregoing claims.
  • the nucleic acid ligase is T4 RNA Ligase I, T4 RNA Ligase II, or T4 DNA Ligase.
  • Another aspect of the disclosure provises a prime editing system comprising a modified PEgRNA or modified ngRNA disclosed herein or one or more polynucleotides encoding the modified PEgRNA or modified ngRNA.
  • Another aspect of the disclosure provides a lipid nanoparticle comprising the modified PEgRNA or modified ngRNA disclosed herein.
  • Another aspect of the disclosure provides a lipid nanoparticle comprising the prime editing system disclosed herein.
  • Another aspect of the disclosure provides a method for editing a gene, the method comprising contacting the gene with the modified PEgRNA, modified ngRNA, prime editing system, lipid nanoparticle, or as disclosed herein.
  • FIG. 1 shows exemplified modifications in a PEgRNA or ngRNA (nucleotide and non-nucleotide modifications).
  • FIG. 2 depicts a PEgRNA architectural overview in an exemplar ⁇ ' schematic of PEgRNA designed for a prime editor.
  • FIG. 3 is a schematic showing the spacer and gRNA core part of an exemplary guide RNA, in two separate molecules (crRNA/tracrRNA). The rest of the PEgRNA structure is not shown.
  • FIG. 4 is a schematic showing modification sites in exemplified PEgRNAs.
  • FIG. 5 shows prime editing efficiency for targeted end modifications and gRNA chemical modifications of the exemplified PEgRNAs.
  • FIG. 6 shows prime editing efficiency and Indel qualifications for exemplified PEgRNA No. 1 -PEgRNA No. 6 molecules and visual representation of the modifications within the tested PEgRNAs is also shown.
  • FIG. 7A shows metabolite cleavage or nuclease degradation profiles of exemplary PEgRNA No. 1 with specific site modifications, and the respective liquid chromatography mass spectrometry (LCMS) profiles after incubation in human S9 fractions.
  • LCMS liquid chromatography mass spectrometry
  • FIG. 7B shows metabolite cleavage or nuclease degradation profiles of exemplary PEgRNA No. 2 with specific site modifications, and the respective liquid chromatography mass spectrometry (LCMS) profiles after incubation in human S9 fractions.
  • LCMS liquid chromatography mass spectrometry
  • FIG. 7C shows metabolite cleavage or nuclease degradation profiles of exemplary PEgRNA No. 3 with specific site modifications, and the respective liquid chromatography mass spectrometry (LCMS) profiles after incubation in human S9 fractions.
  • LCMS liquid chromatography mass spectrometry
  • FIG. 7D shows metabolite cleavage or nuclease degradation profiles of exemplary PEgRNA No. 6 with specific site modifications, and the respective liquid chromatography mass spectrometry (LCMS) profiles after incubation in human S9 fractions.
  • LCMS liquid chromatography mass spectrometry
  • FIG. 8 shows exemplified site-specific modifications in a PEgRNA or ngRNA (nucleotide and non-nucleotide modifications)
  • FIG. 9 shows in-vitro half-life stability in human liver S9 for modifications present in the PEgRNAs No. 1, 2, 6.
  • FIG. 10A depicts exemplified modifications (i.e. sugar modifications) in a PEgRNA or ngRNA.
  • FIG. 10A depicts exemplified modifications (i.e. phosphate modifications) in a PEgRNA or ngRNA.
  • FIG. 10B depicts exemplified modifications (i.e. base modifications and inverted nucleotide) in a PEgRNA or ngRNA.
  • FIG. 11 shows results for shows a UV-Vis melt (Tm), curves fur RNP structure formation using (Differential Scanning Fluorimetry, DSF) and in-vitro half-life stability related to melting temperatures for RNP stability (TM - melting temperature) for exemplary modifications to PEgRNA No. 2, 4, 5, and 6.
  • FIG. 12 shows in vivo prime editing efficiency of PEgRNAs P2, P31, P32, P33, P34, P35, P36, P37, P38, and Pl.
  • FIG. 13 shows a detection method and assay to identify metabolites using in vitro assays human liver cells, fractions S9 and detected with LCMS techniques.
  • modified prime editing guide RNA PEgRNA
  • modified prime editor nicking guide RNA ngRNA
  • PEgRNA modified prime editing guide RNA
  • ngRNA modified prime editor nicking guide RNA
  • 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. For example, “about” can mean within 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” meaning within an acceptable error range for the particular value should be assumed.
  • the term “substantially” as used herein may refer to a value approaching 100% of a given value. In some embodiments, the term may refer to an amount that may be at least about 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% of a total amount. In some embodiments, the term may refer to an amount that may be about 100% of a total amount.
  • protein and “polypeptide” can be used interchangeably to refer to a polymer of two or more amino acids joined by covalent bonds (e.g., an amide bond) that can adopt a three-dimensional conformation.
  • a protein or polypeptide comprises at least 10 amino acids, 15 amino acids, 20 amino acids, 30 amino acids or 50 amino acids joined by covalent bonds (e.g., amide bonds).
  • a protein comprises at least tw o amide bonds.
  • a protein comprises multiple amide bonds.
  • a protein comprises an enzyme, enzyme precursor proteins, regulator ⁇ ' protein, structural protein, receptor, nucleic acid binding protein, a biomarker, a member of a specific binding pair (e.g., a ligand or aptamer), or an antibody.
  • a protein may be a full-length protein (e.g, a fully processed protein having certain biological function).
  • a protein may be a variant or a fragment of a full-length protein.
  • a Cas9 protein domain comprises an H840A amino acid substitution compared to a naturally occurring A pyogenes Cas9 protein.
  • a variant of a protein or enzyme for example a variant reverse transcriptase, comprises a polypeptide having an amino acid sequence that is about 60% identical, about 70% identical, about 80% identical, about 90% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, about 99% identical, about 99.5% identical, or about 99.9% identical to the amino acid sequence of a reference protein.
  • a protein comprises one or more protein domains or subdomains.
  • polypeptide domain' refers to a polypeptide chain that has one or more biological functions, e.g., a catalytic function, a protein-protein binding function, or a protein-DNA function.
  • a protein comprises multiple protein domains.
  • a protein comprises multiple protein domains that are naturally occurring.
  • a protein comprises multiple protein domains from different naturally occurring proteins.
  • a prime editor may be a fusion protein comprising a Cas9 protein domain of S pyogenes and a reverse transcriptase protein domain of Moloney murine leukemia virus.
  • a protein that comprises amino acid sequences from different origins or naturally occurring proteins may be referred to as a fusion, or chimeric protein.
  • polynucleotide can be any polymeric form of nucleotides, including DNA, RNA, a hybridization thereof, or RNA-DNA chimeric molecules.
  • a polynucleotide comprises cDNA, genomic DNA, mRNA, tRNA, rRNA, or microRNA.
  • a polynucleotide is double stranded, e.g, a double-stranded DNA in a gene.
  • a polynucleotide is single-stranded or substantially single-stranded, e g., single-stranded DNA or an mRNA. In some embodiments, a polynucleotide is a cell-free nucleic acid molecule. In some embodiments, a polynucleotide circulates in blood. In some embodiments, a polynucleotide is a cellular nucleic acid molecule. In some embodiments, a polynucleotide is a cellular nucleic acid molecule in a cell circulating in blood.
  • Polynucleotides can have any three-dimensional structure.
  • a polynucleotide comprises deoxyribonucleotides, ribonucleotides or analogs thereof.
  • a polynucleotide comprises modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide.
  • the sequence of nucleotides can be interrupted by non-nucleotide components.
  • a polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component.
  • a polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA.
  • the polynucleotide may comprise one or more other nucleotide bases, such as inosine (1), which is read by the translation machinery as guanine (G).
  • the terms “modified” or “modification” refers to chemical modification with respect to the A, C, G, T and U nucleotides.
  • modifications may be on the nucleoside base and/or sugar portion of the nucleosides that comprise the polynucleotide.
  • the modification may be on the intemucleoside linkage (e.g., phosphate backbone).
  • multiple modifications are included in the modified nucleic acid molecule.
  • a single modification is included in the modified nucleic acid molecule.
  • the terms “modified” or “modification” refers to RNA modifications with respect incorporation of inverted nucleotides in the PEgRNA or ngRNA sequences, or non-nucleotide modifications wherein PEgRNA or ngRNA sequences of nucleotides can be interrupted by non-nucleotide components, or further modified after polymerization, such as by conjugation with a labeling component.
  • complement refers to the ability of two polynucleotide molecules to base pair with each other.
  • Complementary' polynucleotides may base pair via hydrogen bonding, which may be Watson Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding.
  • hydrogen bonding may be Watson Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding.
  • an adenine on one polynucleotide molecule will base pair to a thymine or uracil on a second polynucleotide molecule and a cytosine on one polynucleotide molecule will base pair to a guanine on a second polynucleotide molecule.
  • Two polynucleotide molecules are complementary 7 to each other when a first polynucleotide molecule comprising a first nucleotide sequence can base pair with a second polynucleotide molecule comprising a second nucleotide sequence.
  • the two DNA molecules 5'-ATGC-3' and 5'-GCAT-3' are complementary, and the complement of the DNA molecule 5'-ATGC-3' is 5 -GCAT-3'.
  • a percentage of complementarity indicates the percentage of nucleotides in a polynucleotide molecule which can base pair with a second polynucleotide molecule (e.g, 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary, respectively).
  • Perfectly complementary means that all the contiguous nucleotides of a polynucleotide molecule will base pair with the same number of contiguous nucleotides in a second polynucleotide molecule. “Substantially complementary” as used herein refers to a degree of complementarity that can be 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% over all or a portion of two polynucleotide molecules. In some embodiments, the portion of complementarity may be a region of 10, 15, 20, 25, 30, 35, 40, 45, 50, or more nucleotides.
  • “Substantial complementary” can also refer to a 100% complementarity over a portion of two polynucleotide molecules.
  • the portion of complementarity' between the two polynucleotide molecules is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% of the length of at least one of the two polynucleotide molecules or a functional or defined portion thereof.
  • subject and its grammatical equivalents as used herein may refer to a human or a non-human.
  • a subject may be a mammal.
  • a human subject may be male or female.
  • a human subject may be of any age.
  • a subject may be a human embryo.
  • a human subject may be a newborn, an infant, a child, an adolescent, or an adult.
  • a human subject may be up to about 100 years of age.
  • a human subject may be in need of treatment for a genetic disease or disorder.
  • treatment may refer to the medical management of a subject with an intent to cure, ameliorate, or ameliorate a symptom of, a disease, condition, or disorder.
  • Treatment may include active treatment, that is, treatment directed specifically toward the improvement of a disease, condition, or disorder.
  • Treatment may include causal treatment, that is, treatment directed toward removal of the cause of the associated disease, condition, or disorder.
  • this treatment may include palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, condition, or disorder.
  • Treatment may include supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the disease, condition, or disorder.
  • a condition may be pathological.
  • a treatment may not completely cure or prevent a disease, condition, or disorder. In some embodiments, a treatment ameliorates, but does not completely cure or prevent a disease, condition, or disorder. In some embodiments, a subject may be treated for 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months. 3 months, 4 months. 5 months, 6 months, 1 year, 2 years. 3 years, 4 years, 5 years, 6 years, indefinitely, or life of the subject.
  • ameliorate and its grammatical equivalents means to decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.
  • the terms “prevent” or “preventing” means delaying, forestalling, or avoiding the onset or development of a disease, condition, or disorder for a period of time. Prevent also means reducing risk of developing a disease, disorder, or condition. Prevention includes minimizing or partially or completely inhibiting the development of a disease, condition, or disorder.
  • a composition e.g, a pharmaceutical composition, prevents a disorder by delaying the onset of the disorder for 12 hours, 24 hours, 2 days, 3 days, 4 days,
  • an effective amount or “therapeutically effective amount” may refer to a quantity of a composition, for example a composition comprising a construct, that can be sufficient to result in a desired activity upon introduction into a subject as disclosed herein.
  • An effective amount of the prime editing compositions can be provided to the target gene or cell, whether the cell is ex vivo or in vivo.
  • An effective amount can be the amount to induce, for example, at least about a 2- fold change (increase or decrease) or more in the amount of target nucleic acid modulation (e.g. , expression of a gene to produce functional a protein) observed relative to a negative control.
  • target nucleic acid modulation e.g. , expression of a gene to produce functional a protein
  • An effective amount or dose can induce, for example, about 2-fold increase, about 3- fold increase, about 4-fold increase, about 5-fold increase, about 6-fold increase, about 7-fold increase, about 8-fold increase, about 9-fold increase, about 10-fold increase, about 25-fold increase, about 50-fold increase, about 100-fold increase, about 200-fold increase, about 500- fold increase, about 700-fold increase, about 1000-fold increase, about 5000-fold increase, or about 10,000-fold increase in target gene modulation (e.g., expression of a target gene to produce a functional protein).
  • target gene modulation e.g., expression of a target gene to produce a functional protein.
  • the amount of target gene modulation may be measured by any suitable method known in the art.
  • the “effective amount” or “therapeutically effective amount” is the amount of a composition that is required to ameliorate the symptoms of a disease relative to an untreated patient.
  • an effective amount is the amount of a composition sufficient to introduce an alteration in a gene of interest in a cell (e.g., a cell in vitro or in vivo).
  • the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 th Ed.
  • An aspect of the disclosure provides a modified prime editing guide RNA (PEgRNA) comprising, 5’ to 3’:
  • PEgRNA prime editing guide RNA
  • gRNA guide RNA
  • the gRNA core comprises, in 5’ to 3’ order, a tetraloop, a first stem loop (SL1). a second stem loop (SL2), and a third stem loop (SL3);
  • an extension arm comprising:
  • a primer binding site (li) a primer binding site (PBS) that comprises a region of complementarity to a region upstream of a nick site in a non-target strand of the double- stranded target DNA;
  • the PEgRNA comprises 5 or more nucleotide modifications.
  • ngRNA modified prime editor nicking guide RNA
  • gRNA guide RNA
  • the gRNA core comprises, in 5’ to 3’ order, a tetraloop, a first stem loop (SL1), a second stem loop (SL2), and a third stem loop (SL3);
  • ngRNA comprises 5 or more nucleotide modifications.
  • the nucleotide modification is located in the 3' end.
  • nucleotide modification is located in the 5’ end.
  • nucleotide modifications are located in the 3’ end and the 5‘ end.
  • the nucleotide modification is located in the spacer.
  • the nucleotide modification is located in the gRNA core.
  • the nucleotide modification is located in the extension arm.
  • the nucleotide modification is located in the tetraloop.
  • the nucleotide modification is located in the SL1.
  • the nucleotide modification is located in the SL2.
  • the nucleotide modification is located in the SL3.
  • At least 2 or more nucleotide modifications are located in the 5’ end, 3’ end, tetraloop, SL2, or SL3.
  • the nucleotide modifications are located in the tetraloop, SL1. SL2, and SL3.
  • nucleotide modifications are located in the tetraloop, SL2, and SL3.
  • the nucleotide modifications are located in the 5’ end, 3’ end, tetraloop. SL2, and SL3.
  • At least 2 or more nucleotide modifications occur within the gRNA.
  • the gRNA core comprises a nucleotide modification at nucleotide 25, 37, 38, 46, 64, 65, 67, or 68.
  • the gRNA core comprises a nucleotide modification at nucleotide 25.
  • the gRNA core comprises a nucleotide modification at nucleotide 37 and/or 38.
  • the gRNA core comprises a nucleotide modification at nucleotide 65.
  • the gRNA core comprises a nucleotide modification at nucleotide 68.
  • the PEgRNA comprises 10 or more nucleotide modifications. [00130] In some embodiments, the PEgRNA comprises 20 or more nucleotide modifications.
  • the PEgRNA comprises 30 or more nucleotide modifications.
  • the PEgRNA comprises 40 or more nucleotide modifications.
  • the PEgRNA comprises 50 or more nucleotide modifications.
  • the PEgRNA comprises 60 or more nucleotide modifications.
  • the PEgRNA comprises 70 or more nucleotide modifications.
  • the PEgRNA comprises 80 or more nucleotide modifications.
  • the PEgRNA comprises 90 or more nucleotide modifications.
  • the PEgRNA comprises 100 or more modified nucleotides.
  • a nucleotide modification comprises a phosphate modification, a base modification, a sugar modification, or a combination thereof.
  • a nucleotide modification comprises a base modification.
  • the base modification is selected from the group consisting of N 6 -methyladenosine (m 6 A), N 6 -methyl-2’-O-methyladenosine (2’0Me-m 6 A), xanthine, allyaminouracil, allyaminothymidine, hypoxanthine, digoxigeninated adenine, digoxigeninated cytosine, digoxigeninated guanine, digoxigeninated uracil, 6- chloropurineriboside, N6-methyladenine, methylpseudouracil, 2-thiocytosine, 2-thiouracil. 5- methyluracil. 4-thiothymidine.
  • N'-methoxymethylpseudouracil N 1 -methyl adenine, N 1 - methylpseudouracil, N 1 -propyl pseudouraci 1, N 2 -methylguanme, N 4 -biotin-OBEA-cytosine, N 4 -methylcytosine, N 6 - methyladenine, O 6 -methylguanine, pseudoisocytosine, pseudouracil, thienocytosine, thienoguanine, thienouracil, xanthosine, 3 -deazaadenine, 2,6-diaminoadenine, 2,6-daminoguanine, 5 -carboxamide- uracil, 5-ethynyluracil, N 6 -isopentenyladenine (i 6 A), 2- methyl-thio- N 6 -isopentenyladenine (ms2i 6 A), 2-methylthio- N 6 -methyladenine (m
  • the modification comprises an inverted nucleotide located in the 3’ end.
  • the inverted nucleotide comprises
  • the nucleotide modification comprises a sugar modification.
  • the sugar modification comprises
  • the sugar modification is selected from the structures above with substituted bases (e.g. guanine, cytosine, thymine, and uracil is used in place of adenine).
  • substituted bases e.g. guanine, cytosine, thymine, and uracil is used in place of adenine.
  • the sugar modification is selected from the group consisting of 2’- thioribose, 2’, 3 ‘-di deoxyribose, 2’-amino-2’-deoxyribose, 2’ deoxyribose, 2’-azido-2’- deoxyribose, 2’ -fluoro-2’ -deoxyribose, 2’-O-methoxy ethyl (MOE), 2’-O- methylribose, 2’-O-methyldeoxyribose, 3'-amino- 2’,3’-dideoxyribose, 3 ‘-azido-2’, 3 ‘- dideoxyribose, 3 ‘-deoxyribose, 3'-O-(2-nitrobenzyl)-2'-deoxyribose, 3’-0-methylnbose.
  • MOE methoxy ethyl
  • the nucleotide modification comprises a phosphate modification.
  • the phosphate modification comprises phosphorothioate, a stereospecific phosphorothioate, phosphorodithioate, thiophosphate, 5’- O-methylphosphonate, 3’-O-methylphosphonate, 5’-hydroxyphosphonate, hydroxyphosphanate, phosphoroselenoate, selenophosphate, phosphoramidate, carbophosphonate. methylphosphonate, phenylphosphonate, ethylphosphonate, H- phosphonate. guanidinium ring, triazole ring, boranophosphate (BP), methylphosphonate, guanidinopropyl phosphoramidate, or a combination thereof.
  • BP boranophosphate
  • the non-nucleotide modification comprises a hairpin, a C2-C12 linker, an ethylene glycol linker, 2‘-5’ linkages, or a combination thereof. In some embodiments, the non-nucleotide modification comprises a hairpin, , Tetra ethylene glycol, 2’-5’ linkages, or a combination thereof. In some embodiments, the non-nucleotide modification comprises a hairpin. In some embodiments, the non-nucleotide modification comprises a C2- Ce linker.
  • the non-nucleotide modification comprises a C2 linker, a C3 linker, a C4 linker, a C5 linker, a Cr> linker, a C7 linker, a Cs linker, a C9 linker, a C10 linker, a C11 linker, or a C12 linker.
  • the non-nucleotide modification comprises
  • the non-nucleotide modification comprises an ethylene glycol linker.
  • the ethylene glycol linker comprises from one to twelve ethylene glycol moieties.
  • the ethylene glycol linker comprises a single ethylene glycol moiety' (e.g., -OCH2CH2-).
  • the ethylene glycol linker comprises a di-, tri-, tetra-, penta-, hexa-, hepta-, octa-, nona-, or decaethylene glycol linker.
  • the ethylene glycol linker comprises 1, 2, 3, 4. 5, 6, 7, 8, 9, 10, 11, or 12 ethylene glycol moieties.
  • the non-nucleotide modification comprises ⁇ EG j n some embodiments, the non- nucleotide modification comprises tetra-ethylene glycol.
  • the non-nucleotide modification comprises an abasic deoxyribonucleotide, an abasic ribonucleotide, an abasic 2’ -substituted ribonucleotide or a combination thereof.
  • the modification comprises an inverted nucleotide
  • the prime editor comprises a Cas protein and a DNA polymerase.
  • the modified nucleic acid sequence comprises about 15 nucleotides to about 180 nucleotides. In some embodiments, the modified nucleic acid sequence comprises about 15 nucleotides to about 150 nucleotides. In some embodiments, the modified nucleic acid sequence comprises about 15 nucleotides to about 120 nucleotides. In some embodiments, the modified nucleic acid sequence comprises about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, or 180 nucleotides.
  • the modified nucleic acid sequence comprises about 5 to about 180 unmodified nucleotides. In some embodiments, the modified nucleic acid sequence comprises about 5 to about 150 unmodified nucleotides. In some embodiments, the modified nucleic acid sequence comprises about 5 to about 120 unmodified nucleotides. In some embodiments, the modified nucleic acid sequence comprises about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110. 115, 120. 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, or 180 unmodified nucleotides.
  • the modified nucleic acid sequence comprises from 1 to about 25 modifications. In some embodiments, the modified nucleic acid sequence comprises from 1 to about 20 modifications. In some embodiments, the modified nucleic acid sequence comprises from about 5 to about 25 modifications. In some embodiments, the modified nucleic acid sequence comprises from about 5 to about 25 modifications. In some embodiments the modified nucleic acid sequence comprises from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23. 24, or 25 modifications.
  • the modified nucleic acid sequence further comprises a deoxyribose sugar at the 3' end.
  • the modifications are nucleotide modifications.
  • the nucleotide modifications comprise a phosphate modification, a base modification, a sugar modification, or a combination thereof.
  • nucleotide modifications are the same.
  • nucleotide modifications are different.
  • the non-nucleotide modifications are the same.
  • the non-nucleotide modifications are different.
  • the modifications are a combination of nucleotide and non-nucleotide modifications.
  • the nucleotide modifications comprise a base modification.
  • the base modification is selected from the group consisting of N 6 -methyladenosine (m 6 A), N 6 -methyl-2’-O-methyladenosine (2’0Me-m 6 A), xanthine, allyaminouracil, allyaminothymidine, hypoxanthine, digoxigeninated adenine, digoxigeninated cytosine, digoxigeninated guanine, digoxigeninated uracil, 6- chloropurineriboside, N6-methyladenine, methylpseudouracil, 2-thiocytosine, 2-thiouracil, 5- methyluracil. 4-thiothymidine.
  • 5-aminoallyluracil 5- bromouracil, 5- bromocytosine, 5-carboxycytosine, 5-carboxymethylesteruracil, 5- carboxyuracil, 5 -fluorouracil, 5- formylcytosine, 5-formyluracil, 5-hydroxycytosine, 5- hydroxymethylcytosine, 5- hydroxymethyluracil, 5-hydroxyuracil, 5 -iodocytosine, 5- iodouracil, 5-methoxy cytosine, 5- methoxyuracil, 5 -methylcytosine, 5 -methyluracil, 5- propargylaminocytosine, 5- propargylaminouracil, 5-propynylcytosine, 5-propynyluracil, 6- azacytosine, 6-azauracil, 6- chloropurine, 6-thioguanine, 7-deazaadenine, 7-deazaguanine, 7- deaza-7-propargylaminoadenine
  • the nucleotide modifications comprise a sugar modification.
  • the sugar modification comprises
  • the nucleotide modifications comprise a phosphate modification.
  • the phosphate modification comprises phosphorothioate, a stereospecific phosphorothioate, phosphorodi thioate, thiophosphate, 5 ? -
  • the nucleotide modifications comprise a hairpin, a C2- C12 linker, an ethylene glycol linker. 2'-5' linkages, or a combination thereof.
  • the inverted nucleotide comprises
  • Another aspect of the disclosure provides a method for producing a modified PEgRNA or ngRNA, the method comprising ligating one or more modified nucleotide sequences with one or more modified nucleic acid sequences disclosed herein.
  • Another aspect of the disclosure provides a method for producing a modified PEgRNA or ngRNA, the method comprising ligating a nucleotide sequence encoding a prime editor with one or more modified nucleic acid sequences disclosed herein.
  • the ligation comprises a self-tempi ated enzymatic ligation.
  • the self-templated enzymatic ligation is splint-mediated.
  • the ligation comprises a templated enzy matic ligation.
  • the enzymatic ligation is catalyzed by a nucleic acid ligase to generate a natural phosphodiester linkage between the coding nucleotide sequence and the modified nucleic acid sequence of any one of the foregoing claims, or between the sequence encoding a prime editor with the modified nucleic acid sequence of any one of the foregoing claims, or between two fragments of the nucleotide sequences containing the chemical modifications of any one of the foregoing claims.
  • RNA fragments are brought into proximity 7 by way of sequence complementarity resulting in base pairing hybridization either between the RNA fragments directly (self-templated) or via an additional complementary DNA splint (splinted).
  • Subsequent enzymatic catalysis by a nucleic acid ligase can generate a natural phosphodiester linkage between fragments, yielding the full-length PEgRNA or ngRNA.
  • a nucleic acid ligase is T4 RNA Ligase I, T4 RNA Ligase II, or T4 DNA Ligase.
  • RNA fragments with complementary' base pairs are hybridized for enzy atic ligation (e.g.. ligase-mediated conjugation).
  • the fragments take natural advantage of the sequence complementarity driven structure of any region within the RNA to help facilitate a very specific ligation reaction between the fragments.
  • the RNA duplex forms a native secondary' structure mimic to the full length RNA, resulting in a net increase in the effective local concentration of the terminal 3 ’-OH and 5 ’-monophosphate functional ends required for ligase activity.
  • Subsequent ligase catalysis forms a phosphodiester bond between the RNA fragments which covalently seals the nick and generates a full-length RNA.
  • Template Ligation In some embodiments, two functionalized, singlestranded RNA fragments are conjugated with ligase, similarly to self-templated ligation.
  • a short, complementary’ nucleic acid splint facilitates a trimeric secondary structure formation during templated ligation.
  • the nucleic acid splint is designed such that it bears proximal sequence complementarity of both the 3’ end of the upstream RNA fragment and 5’ end of the downstream RNA fragment to facilitate a splinted trimeric structure.
  • the RNA fragments may or may not require complementarity at the ligation site, and so the advantage of templated ligation for RNA synthesis is that the splinted approach enables ligation at RNA locations which lack secondary structure and anywhere in the sequence without extensions.
  • the splint is a DNA strand.
  • the splint is a modified nucleic acid strand.
  • sgRNA single guide RNA
  • ngRNA nicking guide RNA
  • sgRNA or ngRNA are designed to have (i) a guide RNA sequence domain (designated as a nucleotide spacer sequence as disclosed) at the 5' end, which is complementary' to a target DNA sequence and (ii) guide core RNA that’s binds to DNA nickase enzyme or other polypeptides for form ribonucleoproteins (RNP).
  • ngRNAs are used to guide the DNA nickase enzyme (e.g. Cas9 protein) to specific sites in the genome for targeted single stranded cleavage of DNA.
  • PEgRNA refers to a guide polynucleotide that comprises one or more intended nucleotide edits for incorporation into the target double stranded DNA.
  • the PEgRNA associates with and directs a prime editor to incorporate the one or more intended nucleotide edits into the target gene via prime editing.
  • a PEgRNA comprises a spacer that is complementary or substantially complementary to a search target sequence on a target strand of the target gene.
  • the PEgRNA comprises a gRNA core that associates with a DNA binding domain, e.g., a CRISPR-Cas protein domain, of a prime editor.
  • the PEgRNA comprises an editing template.
  • the PEgRNA comprises a primer binding site (PBS)
  • a PEgRNA comprises an extension arm that comprises an editing template and a PBS.
  • a PEgRNA sequence or fragments thereof such as a spacer, PBS. or RTT sequence, unless indicated otherwise, it should be appreciated that the letter “T” or “thymine” indicates a nucleobase in a DNA sequence that encodes the PEgRNA or guide RNA sequence, and is intended to refer to a uracil (U) nucleobase of the PEgRNA or guide RNA or any chemically modified uracil nucleobase known in the art, such as 5- methoxy uracil.
  • U uracil
  • the PEgRNA or naRNA may comprise a first polynucleotide comprising the spacer and a first portion of a gRNA core referred to as a crRNA.
  • the PEgRNA comprise a second polynucleotide comprising a second portion of the gRNA core and the extension arm, wherein the second portion of the gRNA core may also be referred to as a trans-activating crRNA, or tracr RNA.
  • the crRNA portion and the tracr RNA portion of the gRNA core are at least partially complementary to each other.
  • the partially complementary' portions of the crRNA and the tracr RNA form a lower stem, a bulge, and an upper stem.
  • the partially complementary portions of the crRNA and the tracr RNA form a lower stem, a bulge, and an upper stem, as exemplified in Fig. 3.
  • the crRNArtracrRNA duplex forms the lower stem, bulge, and upper stem modules.
  • the crRNA contains the spacer module, and the tracrRNA contains the nexus and terminal hairpins.
  • PEgRNAs comprises a spacer complementary or substantially complementary' to a separate search target sequence.
  • a PEgRNA anneals with a separate search target sequence through its spacer.
  • a PEgRNA comprises a spacer complementary to a search target sequence on a strand of a double stranded target DNA. e.g., a double stranded target gene.
  • the PEgRNA complexes with and directs a prime editor to bind the double stranded target DNA at the position corresponding to the search target sequence.
  • a spacer comprises a region that has substantial complementarity to a search target sequence on a target strand, or strand, of a double stranded target DNA.
  • the first spacer is substantially complementary to the search target sequence.
  • a spacer sequence may further comprise additional nucleotides beside a region of complementarity to genomic search target sequence.
  • a spacer sequence (as well as the full PEgRNA sequence) may comprise an additional G at the 5’ end, for example, wherein the 5’ most nucleotide of the spacer (or the PEgRNA) is not a G.
  • the gRNA core comprises the sequence GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU GAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO.: 19), or GUUUGAGAGCUAGAAAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUU GAAAAAGUGGGACCGAGUCGGUCC (SEQ ID NO.: 20), or GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO.: 21).
  • the gRNA core comprises the sequence GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU GAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO.: 19). In some embodiments, the gRNA core comprises the sequence AGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAA
  • a guide RNA core (also referred to herein as the gRNA core, gRNA scaffold, or gRNA backbone sequence) of a PEgRNA may contain a polynucleotide sequence that binds to a DNA binding domain (e.g., Cas9) of a prime editor.
  • the gRNA core may interact with a prime editor as described herein, for example, by association with a DNA binding domain, such as a Cas9 nickase of the prime editor.
  • a prime editor such as a Cas9 nickase of the prime editor.
  • the gRNA core is capable of binding to a Cas9-based prime editor.
  • the gRNA core is capable of binding to a Cpfl -based prime editor. In some embodiments, the gRNA core is capable of binding to a Casl2b-based prime editor. [00193] In some embodiments, the gRNA core comprises regions and secondary structures involved in binding with specific CRISPR Cas proteins. For example, in a Cas9 based prime editing system, the gRNA core of a PEgRNA may comprise one or more regions of a base paired “lower stem” adjacent to the spacer and a base paired “upper stem” following the lower stem, where the lower stem and upper stem may be connected by a “bulge” comprising unpaired RNAs.
  • the gRNA core may further comprise a “nexus” distal from the spacer, followed by a hairpin structure, e.g., at the 3’ end.
  • the gRNA core comprises modified nucleotides as compared to a wild type gRNA core in the lower stem, upper stem, and/or the hairpin.
  • nucleotides in the lower stem, upper stem, and/or the hairpin regions may be modified, deleted, or replaced.
  • RNA nucleotides in the lower stem, upper stem, and/or the hairpin regions may be replaced with one or more DNA sequences.
  • the gRNA core comprises unmodified or wild type RNA sequences in the nexus and/or the bulge regions.
  • the gRNA core does not include long stretches of A-T pairs, for example, a GUUUU-AAAAC (SEQ ID NO.: 23) pairing element.
  • a guide RNA core (also referred to herein as the gRNA core, gRNA scaffold, or gRNA backbone sequence) of a PEgRNA or ngRNA may contain a polynucleotide sequence that binds to a DNA binding domain (e.g., Cas9) of a prime editor.
  • the gRNA core may interact with a prime editor as described herein, for example, by association with a DNA binding domain, such as a Cas9 nickase of the prime editor.
  • a prime editor such as a Cas9 nickase of the prime editor.
  • the gRNA core is capable of binding to a Cas9-based prime editor. In some embodiments, the gRNA core is capable of binding to a Cpfl -based prime editor. In some embodiments, the gRNA core is capable of binding to a Casl2b-based prime editor.
  • the gRNA core comprises regions and secondary structures involved in binding with specific CRISPR Cas proteins.
  • the gRNA core of a PEgRNA may comprise one or more regions of a base paired "‘lower stem” adjacent to the spacer and a base paired “upper stem” following the lower stem, where the lower stem and upper stem may be connected by a “bulge” comprising unpaired RNAs.
  • the gRNA core may further comprise a “nexus” distal from the spacer, followed by a hairpin structure, e.g., at the 3’ end.
  • the gRNA core comprises modified nucleotides as compared to a wild ty pe gRNA core in the lower stem, upper stem, and/or the hairpin.
  • nucleotides in the lower stem, upper stem, and/or the hairpin regions may be modified, deleted, or replaced.
  • RNA nucleotides in the lower stem, upper stem, and/or the hairpin regions may be replaced with one or more DNA sequences.
  • the gRNA core comprises unmodified or wild type RNA sequences in the nexus and/or the bulge regions.
  • the gRNA core does not include long stretches of A-T pairs, for example, a GUUUU-AAAAC (SEQ ID NO.: 23) pairing element.
  • the gRNA core comprises the sequence, GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU GAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO.: 19), or GUUUGAGAGCUAGAAAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUU GAAAAAGUGGGACCGAGUCGGUCC (SEQ ID NO.: 20), or GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO.: 21).
  • the gRNA core comprises the sequence, GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU GAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO.: 19). In some embodiments, the gRNA core comprises the sequence, AGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAA AGUGGCACCGAGUCG (SEQ ID NO.: 22). [00198] Any gRNA core sequences known in the art are also contemplated in the prime editing compositions described herein. In some embodiments, one or more nucleotides in the gRNA core is DNA.
  • Prime editor refers to the polypeptide or polypeptide components involved in prime editing, or any polynucleotide(s) encoding the polypeptide or polypeptide components.
  • a prime editor includes a polypeptide domain having DNA binding activity and a polypeptide domain having DNA polymerase activity.
  • the polypeptide domain having DNA binding activity' is a polypeptide domain having programmable DNA binding activity.
  • the prime editor further comprises a polypeptide domain having nuclease activity’.
  • the polypeptide domain having DNA binding activity comprises a nuclease domain or nuclease activity 7 .
  • the polypeptide domain having nuclease activity comprises a nickase, or a fully active nuclease.
  • the polypeptide domain having DNA polymerase activity comprises a template-dependent DNA polymerase, for example, a DNA-dependent DNA polymerase or an RNA-dependent DNA polymerase.
  • the DNA polymerase is a reverse transcriptase.
  • the prime editor comprises additional polypeptides or polypeptide domains involved in prime editing, for example, a polypeptide domain having 5’ endonuclease activity, e.g., a 5‘ endogenous DNA flap endonucleases (e.g., FEN1), for helping to drive the prime editing process towards the edited product formation.
  • a polypeptide domain having 5’ endonuclease activity e.g., a 5‘ endogenous DNA flap endonucleases (e.g., FEN1)
  • the prime editor further comprises an RNA-protein recruitment polypeptide, for example, a MS2 coat protein.
  • a prime editor may be engineered.
  • the polypeptide components of a prime editor do not naturally occur in the same organism or cellular environment.
  • the polypeptide components of a prime editor may be of different origins or from different organisms.
  • a prime editor comprises a DNA binding domain and a DNA polymerase domain that are derived from different species.
  • a prime editor comprises a Cas polypeptide and a reverse transcriptase polypeptide that are derived from different species.
  • a prime editor may comprise a S. pyogenes Cas9 polypeptide and a Moloney murine leukemia virus (M- MLV) reverse transcriptase polypeptide.
  • M- MLV Moloney murine leukemia virus
  • polypeptide domains of a prime editor may be fused or linked by a peptide linker to form a fusion protein.
  • the term “prime editor complex'’ is used interchangeably with the term “prime editing complex” and refers to a complex comprising one or more prime editor components (e.g., a polypeptide domain having DNA binding activity and a polypeptide domain having DNA polymerase activity) complexed with a PEgRNA.
  • a prime editor comprises a M-MLV RT variant, wherein the M-MLV RT variant consists of the following amino acid sequence: TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTP VSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLR EVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWR DPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAAT SELDCQQGTRALLQTLGNLGYRASAKKAQ1CQKQVKYLGYLLKEGQRWLTEARKE TVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKA YQEIKQALLTAPALGL
  • a prime editor comprises a eukaryotic RT, for example, a yeast, drosophila, rodent, or primate RT.
  • the prime editor comprises a Group II intron RT, for example, a. Geobacillus stearothermophilus Group II Intron (Gsl- IIC) RT or a Eubacterium rectale group II intron (Eu.re.I2) RT.
  • the prime editor comprises a retron RT.
  • the DNA-binding domain of a prime editor is a programmable DNA binding domain.
  • a programmable DNA binding domain refers to a protein domain that is designed to bind a specific nucleic acid sequence, e.g., a target DNA or a target RNA.
  • the DNA-binding domain is a polynucleotide programmable DNA-binding domain that can associate with a guide polynucleotide (e.g., a PEgRNA) that guides the DNA-binding domain to a specific DNA sequence, e.g., a search target sequence in a target gene.
  • a guide polynucleotide e.g., a PEgRNA
  • the DNA-binding domain comprises a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Associated (Cas) protein.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • Cas Clustered Regularly Interspaced Short Palindromic Repeats
  • a Cas protein may comprise any Cas protein described herein or a functional fragment or functional variant thereof.
  • a DNA-binding domain may also comprise a zine-finger protein domain.
  • a DNA-binding domain comprises a transcription activator-like effector domain (TALE).
  • TALE transcription activator-like effector domain
  • the DNA- binding domain comprises a DNA nuclease.
  • the DNA-binding domain of a prime editor may comprise an RNA-guided DNA endonuclease, e.g, a Cas protein.
  • the DNA-binding domain comprises a zinc finger nuclease (ZFN) or a transcription activator like effector domain nuclease (TALEN), where one or more zinc finger motifs or TALE motifs are associated with one or more nucleases, e.g., a Fok I nuclease domain.
  • ZFN zinc finger nuclease
  • TALEN transcription activator like effector domain nuclease
  • the DNA-binding domain comprise a nuclease activity .
  • the DNA-binding domain of a prime editor comprises an endonuclease domain having single strand DNA cleavage activity.
  • the endonuclease domain may comprise a FokI nuclease domain.
  • the DNA-binding domain of a prime editor comprises a nuclease having full nuclease activity.
  • the DNA-binding domain of a prime editor comprises a nuclease having modified or reduced nuclease activity as compared to a wild type endonuclease domain.
  • the endonuclease domain may comprise one or more amino acid substitutions as compared to a wild type endonuclease domain.
  • the DNA-binding domain of a prime editor has nickase activity.
  • the DNA-binding domain of a prime editor comprises a Cas protein domain that is a nickase with single stranded DNA nicking activities.
  • the Cas nickase comprises one or more amino acid substitutions in a nuclease domain that reduces or abolishes its double strand nuclease activity but retains DNA binding activity.
  • the Cas nickase comprises an amino acid substitution in a HNH domain.
  • the Cas nickase comprises an amino acid substitution in a RuvC domain.
  • the DNA-binding domain comprises a CRISPR associated protein (Cas protein) domain.
  • Cas protein CRISPR associated protein
  • a Cas protein may be a Class 1 or a Class 2 Cas protein.
  • a Cas protein can be a type I, type II, type III, type IV, type V Cas protein, or a ty pe VI Cas protein.
  • Non-limiting examples of Cas proteins include Cas9, Casl2a (Cpfl), Casl2e (CasX), Casl2d (CasY), Cast 2b 1 (C2cl), Casl2b2, Cas 12c (C2c3), C2c4, C2c8, C2c5, C2cl0, C2c9, Cas 14a, Casl4b, Casl4c, Casl4d, Casl4e, Casl4f, Casl4g, Casl4h, Casl4u, Cns2, Cas ⁇ D, and homologs, functional fragments, or modified versions thereof.
  • a Cas protein can be a chimeric Cas protein that is fused to other proteins or polypeptides.
  • a Cas protein can be a chimera of various Cas proteins, for example, comprising domains of Cas proteins from different organisms.
  • a Cas protein, e.g., Cas9 can be from any suitable organism.
  • the organism is Streptococcus pyogenes (S. pyogenes).
  • the organism is Staphylococcus aureus (S. aureus).
  • the organism is Streptococcus thermophilus (S. thermophilus).
  • the organism is Staphylococcus lugdunensis.
  • a Cas protein may comprise one or more domains.
  • Cas domains include, guide nucleic acid recognition and/or binding domain, nuclease domains (e.g., DNase or RNase domains, RuvC, HNH), DNA binding domain, RNA binding domain, helicase domains, protein-protein interaction domains, and dimerization domains.
  • a Cas protein comprises a guide nucleic acid recognition and/or binding domain that can interact with a guide nucleic acid, and one or more nuclease domains that comprise catalytic activity for nucleic acid cleavage.
  • a prime editor comprises a Cas nickase that can bind to the target gene in a sequence-specific manner and generate a single-strand break at a protospacer within double-stranded DNA in the target gene, but not a double-strand break.
  • the Cas nickase can cleave the edit strand (i.e., the PAM strand) or the non-edit strand of the target gene, but may not cleave both.
  • a prime editor comprises a Cas nickase comprising two nuclease domains (e.g., Cas9), with one of the two nuclease domains modified to lack catalytic activity or deleted.
  • the Cas nickase of a prime editor comprises a nuclease inactive RuvC domain and a nuclease active HNH domain. In some embodiments, the Cas nickase of a prime editor comprises a nuclease inactive HNH domain and a nuclease active RuvC domain. In some embodiments, a prime editor comprises a Cas9 nickase having an amino acid substitution in the RuvC domain. In some embodiments, the Cas9 nickase comprises a DlOX amino acid substitution compared to a wild type S. pyogenes Cas9, wherein X is any amino acid other than D.
  • a prime editor comprises a Cas9 nickase having an amino acid substitution in the HNH domain.
  • the Cas9 nickase comprises a H840X amino acid substitution compared to a wild type S. pyogenes Cas9, wherein X is any amino acid other than H.
  • a prime editor comprises a Cas protein, e.g., Cas9, containing modifications that allow altered PAM recognition.
  • a “protospacer adjacent motif (PAM)”, PAM sequence, or P AM- like motif may be used to refer to a short DNA sequence immediately following the protospacer on the PAM strand of the target gene.
  • the PAM is recognized by the Cas nuclease in the prime editor during prime editing.
  • the PAM is required for target binding of the Cas protein.
  • the specific PAM sequence required for Cas protein recognition may depend on the specific type of the Cas protein.
  • a PAM can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides in length. In some embodiments, a PAM is between 2-6 nucleotides in length. 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). In some embodiments, the Cas protein of a prime editor recognizes a canonical PAM, for example, a SpCas9 recognizes 5’-NGG-3’ PAM. In some embodiments, the Cas protein of a prime editor has altered or non-canonical PAM specificities.
  • Prime editing system comprising: (a) a prime editing guide RNA (PEgRNA), single guide RNA (sgRNA), nicking guide RNA (ngRNA) or one or more polynucleotides encoding the PEgRNA; and (b) a modified mRNA disclosed herein or the modified mRNA produced by the methods described herein.
  • the modified mRNA encodes a prime editor.
  • the prime editor comprises a DNA binding domain and a DNA polymerase domain.
  • the DNA binding domain is a CRISPR associated (Cas) protein domain.
  • Prime editing refers to programmable editing of a target DNA using a prime editor complexed with a PEgRNA to incorporate an intended nucleotide edit into the target DNA through target-primed DNA synthesis.
  • a target polynucleotide e.g., a target gene of prime editing may comprise a double stranded DNA molecule having two complementary strands: a first strand that may be referred to as a “target strand” or a “nonedit strand”, and a second strand that may be referred to as a “non-target strand,” or an “edit strand.”
  • a spacer sequence is complementary or substantially complementary to a specific sequence on the target strand, which may be referred to as a “search target sequence”.
  • the spacer sequence anneals with the target strand at the search target sequence.
  • the target strand may also be referred to as the “non-Protospacer Adjacent Motif (non-PAM strand).”
  • the non-target strand may also be referred to as the “PAM strand”.
  • the PAM strand comprises a protospacer sequence and optionally a protospacer adjacent motif (PAM) sequence.
  • PAM sequence refers to a short DNA sequence immediately adjacent to the protospacer sequence on the PAM strand of the target gene.
  • a PAM sequence may be specifically recognized by a programmable DNA binding protein, e.g., a Cas nickase or a Cas nuclease
  • a specific PAM is characteristic of a specific programmable DNA binding protein, e.g., a Cas nickase or a Cas nuclease.
  • a protospacer sequence refers to a specific sequence in the PAM strand of the target gene that is complementary to the search target sequence.
  • a spacer sequence may have a substantially identical sequence as the protospacer sequence on the edit strand of a target gene, except that the spacer sequence may comprise Uracil (U) and the protospacer sequence may comprise Thymine (T).
  • the double stranded target DNA comprises a nick site on the PAM strand (or non-target strand).
  • a “nick site” refers to a specific position in between two nucleotides or two base pairs of the double stranded target DNA.
  • the position of a nick site is determined relative to the position of a specific PAM sequence.
  • the nick site is the particular position where a nick will occur when the double stranded target DNA is contacted with a nickase, for example, a Cas nickase, that recognizes a specific PAM sequence.
  • the nick site is upstream of a specific PAM sequence on the PAM strand of the double stranded target DNA. In some embodiments, the nick site is downstream of a specific PAM sequence on the PAM strand of the double stranded target DNA. In some embodiments, the nick site is 3 base pairs upstream of the PAM sequence, and the PAM sequence is recognized by a Streptococcus pyogenes Cas9 nickase, aP. lavamentivorans Cas9 nickase, a C. diphtheriae Cas9 nickase, a A. cinereci Cas9, a S.
  • the nick site is 3 base pairs upstream of the PAM sequence, and the PAM sequence is recognized by a Cas9 nickase, wherein the Cas9 nickase comprises a nuclease active HNH domain and a nuclease inactive RuvC domain.
  • the nick site is 2 base pairs upstream of the PAM sequence, and the PAM sequence is recognized by a S. thermophilus Cas9 nickase.
  • a PEgRNA complexes with and directs a prime editor to bind to the search target sequence of the target gene.
  • the bound prime editor generates a nick on the edit strand (PAM strand) of the target gene at the nick site.
  • a primer binding site (PBS) of the PEgRNA anneals with a free 3' end formed at the nick site, and the prime editor initiates DNA synthesis from the nick site, using the free 3' end as a primer. Subsequently, a single-stranded DNA encoded by the editing template of the PEgRNA is synthesized.
  • the newly synthesized single-stranded DNA comprises one or more intended nucleotide edits compared to the endogenous target gene sequence.
  • the editing template of a PEgRNA is complementary to a sequence in the edit strand except for one or more mismatches at the intended nucleotide edit positions in the editing template partially complementary to the editing template may be referred to as an “editing target sequence”.
  • the newly synthesized single stranded DNA has identity or substantial identity to a sequence in the editing target sequence, except for one or more insertions, deletions, or substitutions at the intended nucleotide edit positions.
  • the newly synthesized single-stranded DNA equilibrates with the editing target on the edit strand of the target gene for pairing with the target strand of the target gene.
  • the editing target sequence of the target gene is excised by a flap endonuclease (FEN), for example, FEN1.
  • the FEN is an endogenous FEN, for example, in a cell comprising the target gene.
  • the FEN is provided as part of the prime editor, either linked to other components of the prime editor or provided in trans.
  • the newly synthesized single stranded DNA which comprises the intended nucleotide edit, replaces the endogenous single stranded editing target sequence on the edit strand of the target gene.
  • the newly synthesized single stranded DNA and the endogenous DNA on the target strand form a heteroduplex DNA structure at the region corresponding to the editing target sequence of the target gene.
  • the newly synthesized single-stranded DNA comprising the nucleotide edit is paired in the heteroduplex with the target strand of the target DNA that does not comprise the nucleotide edit, thereby creating a mismatch between the two otherwise complementary strands.
  • the mismatch is recognized by DNA repair machinery, e.g.. an endogenous DNA repair machinery.
  • the intended nucleotide edit is incorporated into the target gene.
  • the Cas protein domain has nickase activity.
  • the Cas protein domain is a Cas9.
  • the Cas9 comprises a mutation in an HNH domain.
  • the Cas9 comprises a H840A mutation in the HNH domain.
  • the Cas protein domain is a Cas 12b.
  • the Cas protein domain is a Casl2a, Casl2b, Casl2c, Casl2d, Casl2e, Casl4a, Casl4b, Casl4c, Casl4d, Casl4e, Casl4f, Casl4g, Casl4h, Casl4u, or a Cascp.
  • the DNA polymerase domain is a reverse transcriptase.
  • the reverse transcriptase is a retrovirus reverse transcriptase.
  • the reverse transcriptase is a Moloney murine leukemia virus (M-MLV) reverse transcriptase.
  • M-MLV Moloney murine leukemia virus
  • the DNA polymerase and the DNA binding domain are fused or linked to form a fusion protein.
  • a PEgRNA comprises a gRNA core that comprises a modified direct repeat compared to the sequence of a naturally occurring CRISPR-Cas guide RNA scaffold, for example, a Cas9 gRNA scaffold.
  • the PEgRNA comprises a ‘'flip and extension (F+E)” gRNA core, wherein one or more base pairs in a direct repeat is modified.
  • the PEgRNA comprises a first direct repeat (the first paring element or the lower stem), wherein a Uracil is changed to a Adenine (such that in the stem region, a U-A base pair is changed to a A-U base pair).
  • the PEgRNA comprises a first direct repeat wherein the fourth U-A base pair in the stem is changed to a A-U base pair. In some embodiments, the PEgRNA comprises a first direct repeat wherein one or more U-A base pair is changed to a G-C or C-G base pair. For example, in some embodiments, the PEgRNA comprises a first direct repeat comprising a modification to a GUUUU-AAAAC (SEQ ID NO.: 23) pairing element, wherein one or more of the U-A base pairs is changed to a A-U base pair, a G-C base pair, or a C-G base pair. In some embodiments, the PEgRNA comprises an extended first direct repeat.
  • polynucleotides encoding polypeptide components of a prime editing composition are codon optimized by replacing at least one codon (e.g, about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence.
  • a polynucleotide encoding a polypeptide component of a prime editing composition are operably linked to one or more expression regulatory elements, for example, a promoter, a 3’ UTR, a 5’ UTR, or any combination thereof.
  • a polynucleotide encoding a prime editing composition component is a messenger RNA (mRNA).
  • mRNA messenger RNA
  • the mRNA comprises a Cap at the 5’ end and/or a poly A tail at the 3’ end.
  • a prime editing system comprises a PEgRNA, and a nuclease that recognizes the PAM sequence “NG.”
  • a PAM motif on the edit strand comprises an “NG” motif, wherein N is any nucleotide.
  • a prime editing system comprises a PEgRNA, and a nuclease that recognizes the PAM sequence “NAG.”
  • a PAM motif on the edit strand comprises an “NAG” motif, wherein N is any nucleotide.
  • a prime editing system comprises a PEgRNA, and a nuclease that recognizes the PAM sequence “NGA.”
  • a PAM motif on the edit strand comprises an “NGA” motif, wherein N is any nucleotide.
  • a prime editing system comprises a PEgRNA, and a nuclease that recognizes the PAM sequence “NNGG.’'
  • a PAM motif on the edit strand comprises an "NNGG" motif, wherein N is any nucleotide.
  • a prime editing system comprises a PEgRNA, and a nuclease that recognizes the PAM sequence “NNGRRT.”
  • a PAM motif on the edit strand comprises an “NNGRRT” motif, wherein N is any nucleotide and R is A or G.
  • N is any nucleotide
  • R is A or G.
  • a PAM motif on the edit strand comprises an “NGG” motif, wherein N is any nucleotide.
  • a prime editing system comprises a PEgRNA, and a nuclease recognizes the PAM motif NGG, wherein the comprising a spacer, a PBS sequence, an RTT sequence, and a gRNA core sequence.
  • the PEgRNA is part of a prime editing system that recognizes the PAM motif NGG and comprises, contiguously from 5’ to 3’: a spacer sequence, a gRNA core sequence, a RTT sequence, and a PBS sequence.
  • one or more of the components (e.g., spacer, PBS or RTT) of a 5' PEgRNA can operate as part of a single or stand-alone PEgRNA.
  • one or more of the components of a 3’ PEgRNA can operate as part of a single or stand-alone PEgRNA.
  • the prime editing compositions provided herein are capable of incorporating one or more intended nucleotide edits without generating a significant proportion of indels.
  • the term "indel(s)" refers to the insertion or deletion of a nucleotide base within a polynucleotide, for example, a target gene. Such insertions or deletions can lead to frame shift mutations within a coding region of a gene.
  • the prime editing systems comprising the modified mRNA described herein can be delivered to a cellular environment with any approach known in the art.
  • Components of a prime editing composition can be delivered to a cell by the same mode or different modes.
  • a prime editor can be delivered as a polypeptide or a polynucleotide (DNA or RNA) encoding the polypeptide.
  • a PEgRNA can be delivered directly as an RNA or as a DNA encoding the PEgRNA.
  • the polynucleotide encoding one or more prime editing composition components is a part of, or is encoded by, a vector.
  • the vector is a viral vector.
  • the vector is a non-viral vector. Exemplary 7 delivery methods are shown in Table 2 below.
  • a prime editing system for example, prime editor polypeptide components and PEgRNA/ngRNA are introduced to a target cell by nanoparticles.
  • the prime editor polypeptide components and the PEgRNA and/or ngRNA form a complex in the nanoparticle.
  • Any suitable nanoparticle design can be used to deliver genome editing system components or nucleic acids encoding such components.
  • the nanoparticle is inorganic.
  • the nanoparticle is organic.
  • a prime editing composition is delivered to a target cell, e.g., a hepatocyte, in an organic nanoparticle, e.g. a lipid nanoparticle (LNP) or polymer nanoparticle.
  • LNP lipid nanoparticle
  • LNPs are formulated from cationic, anionic, neutral lipids, or combinations thereof.
  • neutral lipids such as the fusogenic phospholipid DOPE or the membrane component cholesterol, are included to enhance transfection activity and nanoparticle stability.
  • LNPs are formulated with hydrophobic lipids, hydrophilic lipids, or combinations thereof. Lipids may be formulated in a wide range of molar ratios to produce an LNP. Any lipid or combination of lipids that are known in the art can be used to produce an LNP. Exemplary lipids used to produce LNPs are provided in Table 3 below.
  • components of a prime editing system form a complex prior to delivery to a target cell.
  • a PEgRNA can form a complex prior to delivery to the target cell.
  • a prime editing polypeptide e.g.. a prime editor fusion protein
  • a guide polynucleotide e.g., a PEgRNA or ngRNA
  • RNP ribonucleoprotein
  • the RNP comprises a prime editor fusion protein in complex with a PEgRNA.
  • RNPs may be delivered to cells using known methods, such as electroporation, nucleofection, or cationic lipid- mediated methods, or any other approaches known in the art.
  • delivery of a prime editing composition or complex to the target cell does not require the delivery of foreign DNA into the cell.
  • the RNP comprising the prime editing complex is degraded over time in the target cell.
  • Exemplary lipids for use in nanoparticle formulations and/or gene transfer are shown in Table 3 below.
  • Table 3 Exemplary lipids for nanoparticle formulation or gene transfer
  • Table 4 Exemplary lipids for nanoparticle formulation or gene transfer
  • compositions comprising any of the prime editing composition components, for example, prime editors, fusion proteins, polynucleotides encoding prime editor polypeptides, PEgRNAs, and/or prime editing complexes described herein.
  • prime editing composition components for example, prime editors, fusion proteins, polynucleotides encoding prime editor polypeptides, PEgRNAs, and/or prime editing complexes described herein.
  • composition refers to a composition formulated for pharmaceutical use.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
  • the pharmaceutical composition comprises additional agents, e.g., for specific delivery, increasing half-life, or other therapeutic compounds.
  • a pharmaceutically-acceptable carrier comprises any vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the compound from one site (e.g. the delivery site) of the body, to another site (e.g., organ, tissue or portion of the body).
  • a pharmaceutically acceptable carrier is “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the tissue of the subject (e.g., physiologically compatible, sterile, physiologic pH, etc.)
  • compositions comprising the modified mRNA, the prime editing system, or the LNP or RNP described herein.
  • composition refers to a composition formulated for pharmaceutical use.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
  • the pharmaceutical composition comprises additional agents, e.g., for specific delivery, increasing half-life, or other therapeutic compounds.
  • a pharmaceutically-acceptable carrier comprises any vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carry ing or transporting the compound from one site (e.g., the delivery 7 site) of the body, to another site (e.g, organ, tissue or portion of the body).
  • a pharmaceutically acceptable carrier is “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the tissue of the subject (e.g., physiologically compatible, sterile, physiologic pH, etc.)
  • Formulations of the pharmaceutical compositions described herein can be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient(s) into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping and/or packaging the product into a desired single- or multi-dose unit.
  • compositions can additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.
  • a pharmaceutically acceptable excipient includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.
  • compositions disclosed herein can be used to edit a target gene or sequence of interest by editing.
  • contacting the target gene with the prime editing composition results in binding of the PEgRNA to a strand of the target gene. In some embodiments, contacting the target gene with the prime editing composition results in binding of the PEgRNA to a search target sequence on the strand of the target gene upon contacting with the PEgRNA. In some embodiments, contacting the target gene with the prime editing composition results in binding of the prime editor to the target gene, upon the contacting of the PE composition with the target gene. In some embodiments, components of a prime editing composition form a complex prior to deliver ⁇ 7 to a target cell. For example, a prime editor fusion protein and a PEgRNA can form a complex prior to delivery to the target cell.
  • a prime editing polypeptide e.g.. a prime editor fusion protein
  • a guide polynucleotide e.g, a PEgRNA
  • the RNP comprises a prime editor fusion protein in complex with a PEgRNA.
  • RNPs may be delivered to cells using known methods, such as electroporation, nucleofection. or cationic lipid-mediated methods, or any other approaches known in the art.
  • delivery of a prime editing composition or complex to the target cell does not require the delivery of foreign DNA into the cell.
  • the RNP comprising the prime editing complex is degraded over time in the target cell.
  • compositions disclosed herein can be used to edit a target gene of interest by prime editing.
  • the prime editing method comprises contacting a target gene, with the prime editing system or lipid nanoparticle described herein.
  • a method for editing a gene comprises contacting a PEgRNA and a prime editor (PE) comprising a DNA binding domain and a DNA polymerase domain, wherein the PEgRNA directs the prime editor to incorporate the intended nucleotide edit in the gene, thereby editing the gene.
  • PE prime editor
  • a method for editing a gene comprises contacting the gene with the prime editing system described herein, wherein the PEgRNA directs the prime editor to incorporate the intended nucleotide edit in the gene, thereby editing the gene.
  • the editing efficiency is at least 10%, at least 15%. at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, 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%, at least 99%, at least 99.5%, at least 99.9% equivalent as compared to editing with an unmodified control PEgRNA.
  • the prime editor synthesizes a single stranded DNA encoded by the editing template, wherein the single stranded DNA replaces the editing target sequence and results in incorporation of the intended nucleotide edit into a region corresponding to the editing target sequence.
  • the subject is a human.
  • the method further comprising administering the cell to the subj ect after incorporation of the intended nucleotide edit.
  • EXAMPLE 1 Examples of Chemically Modified PEgRNA and ngRNA Sequences
  • FIG. 1 discloses possible PEgRNA or ngRNA modifications in nucleotides for both nucleotide and non-nucleotide modifications.
  • the modified PEgRNAs or ngRNAs synthesis can by achieved by in-vitro transcription (IVT) or solid phase synthesis on automated synthesizers. These methods utilize synthesis of short oligonucleotides of about 10-120 nucleotides with desired chemical modifications.
  • RNAs > 100-nt because just several nucleotides shorter or longer becomes a trivial difference for long oligonucleotides (> 100-nt length) compared to much shorter oligonucleotides (20-nt length).
  • Applicant has developed alternate synthesis and purification methods for long-RNA synthesis to overcome these synthesis challenges.
  • the methods developed included synthesizing long RNAs, such as PEgRNAs or ngRNAs, via split synthesis using functionalized fragments of RNA.
  • the functionalized fragments can be joined together via enzymatic ligation or chemical conjugation.
  • RNA split synthesis methods An important factor to consider in long RNA split synthesis methods is choosing the appropriate split site for chemical ligations.
  • PEgRNAs due to the additional chemical structure formed after chemical ligation, it is important the chemical structure avoids interfering with the (a) Cas9 nuclease interaction and (b) its activity, by disturbing the gRNA/Cas9 complex.
  • a crystal structure analysis of gRNA/Cas9 complex has showed that the TL and SL2 in the gRNA core have minimal interactions with the Cas9 protein. Therefore, TL and SL2 are considered as the potential split sites for chemical ligations.
  • Other potential split sites include the junction between the gRNA core and the RTT and the end of the PBS and extensions of the 3’ end.
  • the PEgRNAs or ngRNAs may be enzymatically ligated between two or more oligoribonucleotides synthesized by solid phase synthesis which may or may not contain chemically modifications of the sugar, phosphodiester backbone, or nucleobase canonical structures.
  • the PEgRNAs or ngRNAs can be generated from one 5’ fragment, and one or more 3’ fragments which are 5' terminally modified with a free monophosphate to facilitate the ligation by way of a nucleotide ligase enzyme.
  • Free 3‘ ends and phosphoroylated 5’ ends are brought into proximity by way of sequence complementarity resulting in base pairing hybridization either between the RNA fragments directly (self- templated) or via an additional complementary DNA splint (splinted).
  • a nucleic acid ligase such as, but not limited to, T4 RNA Ligase I, T4 RNA Ligase II, or T4 DNA Ligase can generate a natural phosphodiester linkage between fragments, yielding the full-length PEgRNA.
  • Other ligase variants can also be used to ligate the fragments.
  • Scheme A shows a potential fragment functionalization for enzymatic split synthesis, where “Upstream” or 5’ end RNA fragments have a free 3’ OH available for ligation, while “Downstream” or 3’ end RNA fragments are terminally functionalized with a 5 ’ -monophosphate.
  • Scheme A Schematic of upstream and downstream fragments are shown.
  • the light shaded segments are complementary and facilitate self-tempi ated ligation.
  • RNA fragments with complementary base pairs are hybridized for ligase-mediated conjugation.
  • the full-length PEgRNA is split into two or more fragments that take natural advantage of the sequence complementarity driven structure of either the Tetraloop or Stem-loop 2 features within the scaffold of a PEgRNA to help facilitate a very specific ligation reaction between the fragments.
  • the RNA duplex can form a native secondary structure mimic to the full length PEgRNA, resulting in a net increase in the effective local concentration of the terminal 3 -OH and 5 ’-monophosphate functional ends required for ligase activity.
  • Subsequent ligase catalysis forms a phosphodiester bond between the RNA fragments which covalently seals the nick and generates a full-length PEgRNA. Accordingly, during self-templated ligation two RNA fragments with significant complementarity are enzymatically conjugated.
  • the first step is to anneal the fragments, bringing the reactive functional groups together and creating a nicked double-stranded substrate for ligase catalysis. In the presence of ligase, a phosphodi ester bond is formed between the 5 ’-monophosphate and native 3’-hydroxyl of the fragments, yielding a full- length PEgRNA.
  • Editing efficiency of the modified PEgRNA or ngRNA compositions and methods described herein can be measured by calculating the percentage of edited target genes in a population of cells introduced with the prime editing composition.
  • the editing efficiency is determined after 1 hour, 2 hours, 6 hours, 12 hours. 24 hours, 36 hours, 48 hours, 72, hours, 3 days, 4 days, 5 days, 7 days, 10 days, or 14 days of exposing a target gene to a prime editing composition.
  • the population of cells introduced with the prime editing composition is ex vivo.
  • the population of cells introduced with the prime editing composition is in vitro.
  • PEgRNAs plasmids encoding a prime editor fusion protein and PEgRNA were transfected into primary hepatocyte cells with Lipofectamine transfection reagents.
  • Next generation sequencing-based methods or ddPCR- based methods may be developed to determine editing efficiency at 72 hours after transfection.
  • PEgRNAs Nos. 1, 2, 4, 5, and 6 successfully tested with top editing efficiency reaching over 50% (FIG. 5).
  • the chemical modifications of these PEgRNAs are shown in FIG. 8. Chemical modifications to PEgRNA and ngRNA were evaluated and compared to control sequences for editing efficiency as measured by percent editing of the target DNA. Five PEgRNAs and their results are displayed in FIG. 5.
  • PEgRNAl showed reproducible editing efficiency, and exemplary PEgRNA6 with novel 3 '-end modifications showed better editing than the PEgRNA2.
  • Tested PEgRNAs with modifications as indicated in pink, orange, and blue are site specific chemical modifications that were incorporated into the PEgRNAs (FIG. 4).
  • the novel end modifications in the 3" end in exemplary' PEgRNAs 4, 5, 6 increased prime editing compared with PEgRNA3, all of which contain the chemically modified gRNAs, crRNAs and tracrRNAs.
  • These PEgRNAs were screened in primary hepatocytes, suggesting that novel end modifications to 3’ could contribute to improved prime editing.
  • PEgRNAs were prioritized to further evaluation.
  • PEgRNAs were identified of metabolites using in vitro assays human liver cells, fractions S9 and detected with LCMS techniques. The detection method and assay are disclosed in FIG. 13.
  • PEgRNA No.3 stabilized and eliminated cleavage sites 5 ? -U37, A38-3', A65-3, A68-3’within that region that were found for example in PEgRNA No. l. This was observed by comparing metabolite peaks in the LCMS profiles found in FIG. 7A and FIG. 7C. Newly identified metabolites (i.e., cleavage sites) were visible in the PEgRNA No.3 LCMS profile (A46 and A26).
  • UV-Vis melting (T m ) experiments were performed by monitoring PEgRNAs formation of RNPs by absorbance at 260nm in phosphate-buffered saline (PBS) against a 0.2°C/min rise in temperature (FIG. 11). Selected PEgRNAs were used to study the impact and RNP formation maintenance with substituting single ‘U’ residues with 2’-0-Me-U modifications in the gRNA and scaffold.
  • the melting temperature T m -2 (the melting of the potential duplex between protospacer and RTT-PBS) was not affected much by a single 2’-O- Me-U substitution. Slight stabilization of the secondary' structures in the scaffold region was observed in the PEgRNAS. Additionally, the data is supports the notion that the 3’ sequence modifications in the PEgRNAs does not greatly alter the specificity of the RNP and formation.
  • FIG. 12 shows evaluation of Pcsk9 editing efficiency with PEgRNAs P2, P31, P32, P33, P34. P35, P36, P37. P38, and Pl.
  • Embodiment 1 A modified PEgRNA comprising, 5’ to 3’:
  • gRNA guide RNA
  • the gRNA core comprises, in 5’ to 3’ order, a tetraloop, a first stem loop (SL1), a second stem loop (SL2), and a third stem loop (SL3);
  • an extension arm comprising:
  • a primer binding site that comprises a region of complementarity to a region upstream of a nick site in a non-target strand of the double- stranded target DNA
  • the PEgRNA comprises 5 or more nucleotide modifications.
  • Embodiment 2 In another aspect, the disclosure provides a modified ngRNA comprising, 5’ to 3’:
  • gRNA guide RNA
  • the gRNA core comprises, in 5’ to 3 ? order, a tetraloop, a first stem loop (SL1), a second stem loop (SL2), and a third stem loop (SL3);
  • ngRNA comprises 5 or more nucleotide modifications.
  • Embodiment 3 a nucleotide modification is located in the 3’ end.
  • Embodiment 4 a nucleotide modification is located in the 5’ end.
  • Embodiment s nucleotide modifications are located in the 3’ end and the 5' end.
  • Embodiment 6 a nucleotide modification is located in the spacer.
  • Embodiment 7 a nucleotide modification is located in the gRNA core.
  • Embodiment 8 a nucleotide modification is located in the extension arm.
  • Embodiment 9 a nucleotide modification is located in the tetraloop.
  • Embodiment 10 a nucleotide modification is located in the SL1.
  • Embodiment 11 a nucleotide modification is located in the SL2.
  • Embodiment 12 a nucleotide modification is located in the SL3.
  • Embodiment 13 at least 2 or more nucleotide modifications are located in the 5’ end. 3’ end, tetraloop, SL2, or SL3.
  • Embodiment 14 nucleotide modifications are located in the tetraloop. SL1, SL2, and SL3.
  • Embodiment 15 nucleotide modifications are located in the tetraloop
  • Embodiment 16 nucleotide modifications are located in the 5’ end, 3’ end, tetraloop, SL2, and SL3.
  • Embodiment 17 at least 2 or more nucleotide modifications occur within the gRNA.
  • Embodiment 18 the gRNA core comprises a nucleotide modification at nucleotide 25, 37, 38, 46, 64, 65, 67, or 68.
  • Embodiment 19 the gRNA core comprises a nucleotide modification at nucleotide 25.
  • Embodiment 20 the gRNA core comprises a nucleotide modification at nucleotide 37 and/or 38.
  • Embodiment 21 the gRNA core comprises a nucleotide modification at nucleotide 65.
  • Embodiment 22 the gRNA core comprises a nucleotide modification at nucleotide 68.
  • Embodiment 23 the PEgRNA or ngRNA comprises 10 or more nucleotide modifications.
  • Embodiment 24 the PEgRNA or ngRNA comprises 20 or more nucleotide modifications.
  • Embodiment 25 the PEgRNA or ngRNA comprises 30 or more nucleotide modifications.
  • Embodiment 26 the PEgRNA or ngRNA comprises 40 or more nucleotide modifications.
  • Embodiment 27 the PEgRNA or ngRNA comprises 50 or more nucleotide modifications.
  • Embodiment 28 the PEgRNA or ngRNA comprises 60 or more nucleotide modifications.
  • Embodiment 29 the PEgRNA or ngRNA comprises 70 or more nucleotide modifications.
  • Embodiment 30 the PEgRNA or ngRNA comprises 80 or more nucleotide modifications.
  • Embodiment 31 the PEgRNA or ngRNA comprises 90 or more nucleotide modifications.
  • Embodiment 32 the PEgRNA or ngRNA comprises 100 or more modified nucleotides.
  • Embodiment 33 a base modification is selected from the group consisting of N 6 -methyladenosine (m 6 A), N 6 -methyl-2’-O-methyladenosine (2’0Me-m 6 A), xanthine, allyaminouracil, allyaminothymidine, hypoxanthine, digoxigeninated adenine, digoxigeninated cytosine, digoxigeninated guanine, digoxigeninated uracil, 6- chloropurineriboside, N6-methyladenine, methylpseudouracil, 2-thiocytosine, 2-thiouracil.
  • 5- iodouracil 5-methoxy cytosine, 5- methoxyuracil, 5 -methyl cytosine, 5 -methyluracil, 5- propargylaminocytosine, 5- propargylaminouracil.
  • cyanine 5- aminoallylcytosine cyanine 5-aminoallyluracil, cyanine 7-aminoallyluracil, dabcyl-5-3 - aminoallyluracil, desthiobiotin- 16-aminoallyl-uracil, desthiobiotin-6-aminoallylcytosine, isoguanine, N 1 -ethylpseudouracil. N'-methoxymeth ⁇ lpseudouracil.
  • N 6 -hydroxynorvalylcarbamoyladenine (hn 6 A), 2- methylthio- N 6 -hydroxynorvalyl carbamoyladenine (ms2hn 6 A), N 6 , N 6 - dimethyladenine (m 6 2A), and ⁇ 6 -acetyladenine (ac 6 A).
  • Embodiment 34 the modification comprises an inverted nucleotide located in the 3‘ end.
  • Embodiment 35 the inverted nucleotide comprises
  • Embodiment 36 a nucleotide modification is a sugar modification.
  • Embodiment 37 the sugar modification comprises .or a combination thereof.
  • Embodiment 38 the sugar modification is selected from the group consisting of 2 ’-thioribose, 2’, 3 ’-dideoxyribose, 2’-amino-2'-deoxyribose, 2’ deoxyribose, 2’-azido-2’- deoxyribose, 2’-fluoro-2’-deoxyribose.
  • Embodiment 39 a nucleotide modification is a phosphate modification.
  • Embodiment 40 a phosphate modification comprises phosphorothioate, a stereospecific phosphorothioate, or a combination thereof.
  • the phosphate modification is selected from the group consisting of phosphorothioate (PS), a stereospecific phosphorothioate. phosphorodithioate, thiophosphate, 5’-O-methylphosphonate, 3’-O- methylphosphonate, 5’-hydroxyphosphonate, hydroxyphosphanate, phosphoroselenoate, selenophosphate, phosphoramidate, carbophosphonate, methylphosphonate, phenylphosphonate, ethylphosphonate. H- phosphonate. guanidinium ring, triazole ring, boranophosphate (BP), methylphosphonate, and guanidinopropyl phosphoramidate.
  • PS phosphorothioate
  • a stereospecific phosphorothioate phosphorodithioate, thiophosphate, 5’-O-methylphosphonate, 3’-O- methylphosphonate, 5’-hydroxyphosphonate, hydroxypho
  • Embodiment 42 the nucleotide modification comprises a phosphate modification, a base modiication, a sugar modification, or a combination thereof.
  • Embodiment 43 a modified PEgRNA sequence is selected from the group consisting of Tables 5 or 6.
  • Embodiment 44 the non-nucleotide modification comprises a hairpin, a
  • Embodiment 45 a method for producing a modified PEgRNA or modified ngRNA, the method comprising ligating a coding nucleotide sequence with the modified nucleic acid sequence of any of the claims disclosed herein.
  • Embodiment 46 a method for producing a modified PEgRNA or modified ngRNA, the method comprising ligating a sequence encoding a prime editor with the modified nucleic acid sequence of any of the claims disclosed herein.
  • Embodiment 47 the ligation comprises a self-templated enzymatic ligation, optionally the self-templated enzy matic ligation is splint-mediated.
  • Embodiment 48 the ligation comprises a templated enzymatic ligation.
  • Embodiment 49 the enzymatic ligation is catalyzed by a nucleic acid ligase to generate a natural phosphodiester linkage between the coding nucleotide sequence and the modified nucleic acid sequence of any one of the foregoing claims, or between the sequence encoding a prime editor with the modified nucleic acid sequence of any one of the foregoing claims, or between two fragments of the nucleotide sequences containing the chemical modifications of any one of the foregoing claims.
  • Embodiment 50 the nucleic acid ligase is T4 RNA Ligase I, T4 RNA
  • Embodiment 51 a prime editing system comprising a modified PEgRNA or modified ngRNA of any of the claims disclosed herein or one or more polynucleotides encoding the modified PEgRNA or modified ngRNA.
  • Embodiment 52 a lipid nanoparticle comprising the modified PEgRNA or modified ngRNA of any one of the claims disclosed herein.
  • Embodiment 53 a lipid nanoparticle comprising the prime editing system disclosed herein.
  • Embodiment 54 a method for editing a gene, the method comprising contacting the gene with the modified PEgRNA, modified ngRNA, prime editing system, lipid nanoparticle, or any of the disclosed claims described herein.

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Abstract

L'invention concerne des séquences modifiées d'ARN guide d'édition primaire (PEgARN) et d'ARN guide de coupure (ngARN) et des compositions les comprenant, ainsi que l'utilisation de celles-ci dans des procédés d'édition de gènes.
PCT/US2024/056877 2023-11-21 2024-11-21 Modifications chimiques dans pegarn et ngarn Pending WO2025111452A2 (fr)

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EP4352230A4 (fr) * 2021-06-03 2025-06-11 Prime Medicine, Inc. Compositions d'édition de génome et méthodes de traitement de la maladie de wilson
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Publication number Priority date Publication date Assignee Title
WO2025215514A1 (fr) * 2024-04-08 2025-10-16 Crispr Therapeutics Ag Synthèse d'arn médiée par ponts

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