EP4702145A2 - Arn guide modifié - Google Patents

Arn guide modifié

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Publication number
EP4702145A2
EP4702145A2 EP24730119.5A EP24730119A EP4702145A2 EP 4702145 A2 EP4702145 A2 EP 4702145A2 EP 24730119 A EP24730119 A EP 24730119A EP 4702145 A2 EP4702145 A2 EP 4702145A2
Authority
EP
European Patent Office
Prior art keywords
sgrna
nucleotides
sequence
modified
upper stem
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP24730119.5A
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German (de)
English (en)
Inventor
Brian CAFFERTY
Ho YAU
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Beam Therapeutics Inc
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Beam Therapeutics Inc
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Publication of EP4702145A2 publication Critical patent/EP4702145A2/fr
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C12N9/222—Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes
    • 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
    • C12N2310/10—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
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/344—Position-specific modifications, e.g. on every purine, at the 3'-end
    • 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
    • C12N2320/00—Applications; Uses
    • C12N2320/50—Methods for regulating/modulating their activity
    • C12N2320/52—Methods for regulating/modulating their activity modulating the physical stability, e.g. GC-content

Definitions

  • gRNA guide RNA
  • CRISPR-Cas9 activity is dependent on the sequence and structure of the gRNA.
  • a complete guide RNA comprises tracrRNA (trRNA) and crisprRNA (crRNA).
  • trRNA tracrRNA
  • crRNA crisprRNA
  • a crRNA comprising a guide region which can form a complete gRNA when being associated covalently or noncovalently with a trRNA.
  • the trRNA and crRNA may be contained within a single guide RNA (sgRNA) or in two separate RNA molecules.
  • Guide RNA either a single guide RNA (sgRNA) or as two separate crRNA and tracRNA molecules, forms common secondary structures, particularly the scaffold sequence of a guide RNA.
  • sgRNA single guide RNA
  • gRNA guide RNA
  • Modifications of guide RNAs that increase stability and on-target specificity would be useful.
  • SUMMARY OF THE INVENTION The present the application provides, among other things, modified gRNA molecules, compositions and methods for site specific gene editing and genomic modification, such as DNA cleavage, and gene activation or repression.
  • the present modified guide RNAs have modified secondary structure (e.g., long upper stem and a modified hairpin Attorney Docket No. BEM-020WO1 structure).
  • Modified gRNAs result in decreased of off-target activity, all while maintaining the ability to target a DNA sequence specifically.
  • the invention includes using modified single guide RNAs (sgRNAs) that enhance genome editing, e.g., editing of a target nucleic acid in a primary cell (e.g., cultured in vitro for use in ex vivo therapy) or in a cell in a subject such as a human.
  • sgRNAs modified single guide RNAs
  • the present invention also provides methods for treating a disease in a subject by enhancing precise genome editing to correct a mutation in a target gene associated with the disease.
  • the present invention can be used with any cell type and at any gene locus that is amenable to nuclease-mediated genome editing technology.
  • the present invention provides a modified guide RNA (gRNA) comprising a long (or extended) upper stem including more than 4 base pairs formed by complementary nucleotides, wherein one or more or all nucleotides of the upper stem are chemically modified nucleotides.
  • gRNA modified guide RNA
  • the long (or extended) upper stem includes 4-8 base pairs formed by complementary nucleotides, wherein one or more or all nucleotides of the upper stem are modified nucleotides. In one embodiment, the nucleotides of the extended upper stem are all chemically modified.
  • the modified gRNA comprises a long upper stem region comprising 5-15 base pairs formed by complementary nucleotides. In one embodiment, the long upper stem region comprises 5 base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides). In one embodiment, the long upper stem region comprises 6 base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides).
  • the long upper stem region comprises 7 base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides). In one embodiment, the long upper stem region comprises 8 base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides). In one embodiment, the long upper stem region comprises 9 base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides). In one embodiment, the long upper stem region comprises 10 base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides). In one embodiment, the long upper stem region comprises 11 base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides).
  • the long upper stem region comprises 12 base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides). In one embodiment, the long upper stem region comprises 13 base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides). In one embodiment, the Attorney Docket No. BEM-020WO1 long upper stem region comprises 14 base pairs formed by complementary nucleotides. In one embodiment, the long upper stem region comprises 15 base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides). In one embodiment, the long upper stem region comprises 15-20 base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides).
  • the long upper stem region comprises 20-200 base pairs formed by complementary nucleotides ((e.g., chemically modified nucleotides). In one embodiment, the long upper stem region comprises 20-40, 40- 80, 80-120, 120-160 or 160-200 base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides). [0010] In some embodiments, all the nucleotides of the upper stem of the modified gRNA described herein are chemically modified nucleotides. In other embodiments, at least 80%, at least 85%, at least 90%, or at least 95% of the nucleotides of the upper stem of the modified gRNA are chemically modified nucleotides.
  • the modified gRNA described herein further comprises one or more modified nucleotides within the hairpin 1 and hairpin 2 regions. In some embodiments, all the nucleotides within the hairpins 1 and 2 regions are modified nucleotides. In other embodiments, at least 80%, at least 85%, at least 90%, or at least 95% of the nucleotides within the hairpins 1 and 2 regions of the modified gRNA are modified nucleotides. [0012] In some embodiments, the modified gRNA described herein further comprises a modified stable hairpin 1 region, wherein the modified stable hairpin 1 region comprises an extended stem region comprising more than 4 base pairs and wherein the loop of hairpin 1 comprises locked nucleic acid.
  • the extended stem region of the modified stable hairpin comprises 8 base pairs.
  • the present invention provides a single guide RNA (sgRNA) comprising a sequence of GUUUUAGA N xn GAAA NynAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU CGGUGCUUUU (SEQ ID NO: 23), wherein N xn and N yn have the same number of nucleotides and are complementary nucleotides to form base pairs, and wherein the nucleotides of N xn and N yn are backbone modified nucleotides, and wherein n is an integral number from 5-15.
  • GAAA is the GNRA tetraloop that is used to lock the structure of the hairpin.
  • Other GNRA tetraloops include but are not limited to GUGA, Attorney Docket No. BEM-020WO1 GCAA, GAGA, GUAA, GGGA, GCGA, and GGAA.
  • another RNA tetraloop UNCG is incorporated to the sgRNA to lock the structure of the hairpin.
  • Exemplary UNCG tetraloops include but are not limited to UUCG, UACG, UCCG and UGCG.
  • the 5’ and 3’ end nucleotides of the sgRNA are backbone modified nucleotides.
  • the sgRNA comprises a sequence of GUUUUAGA m(Nxn)mGmAmAmA m(Nyn)AAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAm AmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUmUmUmUmU (SEQ ID NO: 24) , wherein “m” stands for 2’-OMe modification.
  • the Nx and Ny include 5 nucleotides respectively that are complementary nucleotides and that form the upper stem region of the sgRNA, and wherein the sgRNA comprises the sequence of GUUUUAGANxNxNxNxNxGAAANyNyNyNyNyAAGUUAAAAUAAGGCUAGUCCGUUA UCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 2).
  • each of the Nx and Ny nucleotides is backbone modified.
  • the sgRNA comprises a sequence of GUUUUAGAmNxmNxmNxmNxmNxmNxmGmAmAmAmNymNymNymNyAAGUUAAA AUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCm AmCmCmGmAmGmUmCmGmGmUmGmUmUmUmUmU (SEQ ID NO: 25), wherein “m” stands for 2’-OMe modification.
  • the Nx and Ny include 6 nucleotides respectively that are complementary nucleotides and that form the upper stem region of the sgRNA, and wherein the sgRNA comprises the sequence of GUUUUAGAN x N x N x N x N x N x GAAAN y N y N y N y N y N y AAGUUAAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO:3).
  • each of the Nx and Ny nucleotides is backbone modified.
  • the sgRNA comprises a sequence of GUUUUAGAmN x mN x mN x mN x mN x mN x mN x mGmAmAmN y mN y mN y mN y mN y mN y AAGUU AAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmAmGmUmGmG mCmAmCmGmAmGmUmCmGmGmUmGmCmUmUmUmUmU (SEQ ID NO: 26), wherein “m” stands for 2’-OMe modification.
  • the Nx and Ny include 7 nucleotides respectively that are complementary nucleotides and that form the upper stem region of the sgRNA, and wherein Attorney Docket No. BEM-020WO1 the sgRNA comprises the sequence of GUUUUAGANxNxNxNxNxNxNxGAAANyNyNyNyNyNyAAGUUAAAAUAAGGCUAGU CCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 4).
  • each of the Nx and Ny nucleotides is backbone modified.
  • the sgRNA comprises a sequence of GUUUUAGAmNxmNxmNxmNxmNxmNxmNxmNxmGmAmAmAmNymNymNymNymNymNymNymNymNymNy AAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGm UmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmGmUmGmCmUmUmUmUmUmU (SEQ ID NO: 27), wherein “m” stands for 2’-OMe modification.
  • the Nx and Ny include 8 nucleotides respectively that are complementary nucleotides and that form the upper stem region of the sgRNA, and wherein the sgRNA comprises the sequence of GUUUUAGANxNxNxNxNxNxNxNxNxGAAANyNyNyNyNyNyNyNyAAGUUAAAAUAAGGCU AGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 5).
  • each of the Nx and Ny nucleotides is backbone modified.
  • the sgRNA comprises a sequence of GUUUUAGAmN x mN x mN x mN x mN x mN x mN x mN x mG mAmAmN y mN y mN y mN y mNy mNy mNy mNy mNy mNy mNy mNy mNy mNy mNy mNy mNy mNy mNyAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmA mAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCUmUmUmU (SEQ ID NO: 28), wherein “m” stands for 2’-OMe modification.
  • the Nx and Ny include 9 nucleotides respectively that are complementary nucleotides and that form the upper stem region of the sgRNA, and wherein the sgRNA comprises the sequence of GUUUUAGAN x N x N x N x N x N x N x N x N x GAAAN y N y N y N y N y N y N y N y N y N y N y N y y y y y y y y y y y y y y y y y y y y y y y y y AAGUUAAAAUAAG GCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 6).
  • each of the Nx and Ny nucleotides is backbone modified.
  • the sgRNA comprises a sequence of GUUUUAGAmN x mN x mN x mN x mN x mN x mN x mN x mN x mG mAmAmN y mN y mN y mN y mNy mNy mNy mNyAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAm AmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmUmUmUmUm U (SEQ ID NO: 29), wherein “m” stands for 2’-OMe modification.
  • the Nx and Ny include 10 nucleotides respectively that are complementary nucleotides and that form the upper stem region of the sgRNA, and wherein Attorney Docket No. BEM-020WO1 the sgRNA comprises the sequence of GUUUUAGANxNxNxNxNxNxNxNxNxNxNxGAAANyNyNyNyNyNyNyNyNyNyNyNyNyAAGUUAAAAU AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 7).
  • each of the Nx and Ny nucleotides is backbone modified.
  • the sgRNA comprises a sequence of GUUUUAGAmNxmNxmNxmNxmNxmNxmNxmNxmNxmNxmNxmGmAmAmAmNymNymNy mN y mN y mN y mN y mN y mN y mN y mN y mN y mN y y AAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmU mGmAmAmAmAmGmUmGmGmCmAmCmCmGmGmUmGmGmUmGmUmUmUmU (SEQ ID NO: 30), wherein “m” stands for 2’-OMe modification.
  • the present invention provides a sgRNA comprising the sequence of GUUUUAGAGCGCGGAAACGCGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC UUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 8).
  • the present invention provides a sgRNA comprising the sequence of GUUUUAGAmGmCmGmCmGmGmGmAmAmAmCmGmCmGmCAAGUUAAAAUAAGGC UAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmAmGmUmGmGmCmAmCmCmG mAmGmUmCmGmAmGmUmCmG mAmGmUmGmUmUmUmUmUmUmU (SEQ ID NO: 31), wherein “m” stands for 2’-OMe modification.
  • the sgRNA of the present invention comprise the sequence of GUUUUAGAGCCGGCGGAAACGCCGGCAAGUUAAAAUAAGGCUAGUCCGUUAU CAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 9).
  • the sgRNA of the present invention comprise the sequence of GUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmCmCmGmGmCAAGUUAA AAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmC mAmCmGmAmGmUmCmGmGmUmGmUmUmUmUmUmU (SEQ ID NO: 32), wherein “m” stands for 2’-OMe modification.
  • one or more nucleotides within the hairpin 1 and hairpin 2 regions of the sgRNA described herein are modified nucleotides. In other embodiments, all the nucleotides within the hairpin 1 and hairpin 2 regions of the sgRNA described herein are modified nucleotides.
  • the loop of hairpin 1 of the sgRNA described herein comprises 2′-O-methyl modified nucleotides (e.g., 2′-O-methyl 3′-phosphorothioate (MS) nucleotide, 2′-O-methyl 3′-thioPACE (MSP) nucleotide), 2’-F modified nucleotides, Attorney Docket No.
  • the present invention provides a single guide RNA (sgRNA) comprising a sequence of GUUUUAGANxnGAAANynAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGGAC UUUGGUCCAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 10), wherein N xn and Nyn have the same number of nucleotides and are complementary nucleotides to form base pairs, and wherein the nucleotides of N xn and N yn are modified nucleotides and wherein n is an integral number from 5-15, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15.
  • the nucleotides of N xn and N yn are 2′-O-methyl modified nucleotides.
  • the sgRNA comprises a sequence of GUUUUAGAm(Nxn)mGmAmAmAm(Nyn)AAGUUAAAAUAAGGCUAGUCCGUUAUC mAmAmCmUmUmGmGmAmCmUmUmUmGmGmUmCmAmAmGmUmGmGmCmA mCmCmGmAmGmUmCmGmGmUmGmUmUmUmUmU (SEQ ID NO: 33), wherein “m” stands for 2’-OMe modification.
  • the Nx and Ny include 5 nucleotides respectively that are complementary nucleotides and that form the upper stem region of the sgRNA, and wherein the sgRNA comprises the sequence of GUUUUAGANxNxNxNxNxGAAANyNyNyNy AAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGGACUUUGGUCCAAGUGGCA CCGAGUCGGUGCUUUU (SEQ ID NO: 11).
  • each of the Nx and Ny nucleotides is backbone modified.
  • the sgRNA comprises a sequence of GUUUUAGAmN x mN x mN x mN x mN x mN x mGmAmAmN y mN y mN y mN y mN y AAGUUAAAAUAAGGCUAGUCCGUUAUCmAmAmCmUmUmGmGmAmCmUmUmU mU mGmGmUmCmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmAmGmUmCmGmGmUmGmGmC mUmGmGmC mUmUmUmU (SEQ ID NO: 34), wherein “m” stands for 2’-O-methyl modification.
  • the Nx and Ny include 6 nucleotides respectively that are complementary nucleotides and that form the upper stem region of the sgRNA, and wherein the sgRNA comprises the sequence of GUUUUAGANxNxNxNxNxNxGAAANyNyNyNyNy AAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGGACUUUGGUCCAAGUGGCA CCGAGUCGGUGCUUUU (SEQ ID NO.12).
  • each of the Nx and Ny nucleotides is backbone modified.
  • the sgRNA comprises a sequence of GUUUUAGAmNxmNxmNxmNxmNxmNxmNxmGmAmAmAmNymNymNymNymNymNymNymNymNymNymNymNymNymNylNylNylNylNylNylNylNylNylNylNy Attorney Docket No. BEM-020WO1 AAGUUAAAAUAAGGCUAGUCCGUUAUCmAmAmCmUmUmGmGmAmCmUmUmU mU mGmGmCmAmAmGmUmGmGmAmCmCmGmAmGmUmCmGmGmUmGmGmC mUmCmGmGmUmGmC mUmUmUmU (SEQ ID NO: 35), wherein “m” stands for 2’-OMe modification
  • the Nx and Ny include 7 nucleotides respectively that are complementary nucleotides and that form the upper stem region of the sgRNA, and wherein the sgRNA comprises the sequence of GUUUUAGANxNxNxNxNxNxNxNxGAAANyNyNyNyNyNyAAGUUAAAAUAAGGCUAGU CCGUUAUCAACUUGGACUUUGGUCCAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 13).
  • each of the Nx and Ny nucleotides is backbone modified.
  • the sgRNA comprises a sequence of GUUUUAGAmNxmNxmNxmNxmNxmNxmNxmNxmGmAmAmAmNymNymNymNymNymNymNymNymNy AAGUUAAAAUAAGGCUAGUCCGUUAUCmAmAmCmUmUmGmGmAmCmUmUmU mGmGmUmCmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmGmC mUmCmGmGmUmGmC mUmUmUmU (SEQ ID NO: 36), wherein “m” stands for 2’-OMe modification.
  • the Nx and Ny include 8 nucleotides respectively that are complementary nucleotides and that form the upper stem region of the sgRNA, wherein the sgRNA comprises the sequence of GUUUUAGANxNxNxNxNxNxNxNxNxGAAANyNyNyNyNyNyNyNyAAGUUAAAAUAAGGCU AGUCCGUUAUCAACUUGGACUUUGGUCCAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 14).
  • each of the Nx and Ny nucleotides is backbone modified.
  • the sgRNA comprises a sequence of GUUUUAGAmN x mN x mN x mN x mN x mN x mN x mN x mG x mGmG x mG x mG x mG x mG mG mG mG mG mG mG mG mG mGmNy mNy y mNy mNy mNy mNy mNy mNy mNy mNy mNy mNy mNy mNy mNy mNy mNy mNy mNy mNy mNy mNy mNy mNy mNy mNy mNy mNy mNy mNy mNy mNy mNy mNy mNy
  • the Nx and Ny include 9 nucleotides respectively that are complementary nucleotides and that form the upper stem region of the sgRNA, and wherein the sgRNA comprises the sequence of GUUUUAGAN x N x N x N x N x N x N x N x N x GAAAN y N y N y N y N y N y N y N y N y N y N y y y y y y y y y y y y y y y y y y y y y y y AAGUUAAAAUAAG GCUAGUCCGUUAUCAACUUGGACUUUGGUCCAAGUGGCACCGAGUCGGUGCUU UU (SEQ ID NO: 15).
  • each of the Nx and Ny nucleotides is backbone modified.
  • the sgRNA comprises a sequence of GUUUUAGAmNxmNxmNxmNxmNxmNxmNxmNxmNxmNxmNxmGmAmAmAmNymNymNymNymNymNymNyEffet No.
  • the Nx and Ny include 10 nucleotides respectively that are complementary nucleotides and that form the upper stem region of the sgRNA, and wherein the sgRNA comprises the sequence of GUUUUAGAN x N x N x N x N x N x N x N x N x N x GAAAN y N y N y N y N y NyNyNyNyAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGGACUUUGGUCCA AGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 16).
  • each of the Nx and Ny nucleotides is backbone modified.
  • the sgRNA comprises a sequence of GUUUUAGAmNxmNxmNxmNxmNxmNxmNxmNxmNxmNxmNxmNxmGmAmAmNymNymNy mNymNyAAGUUAAAAUAAGGCUAGUCCGUUAUCmAmmNyAmCmUm UmGmGmAmCmUmUmUmGmGmUmCmAmAmGmUmGmGmCmAmCmCmGmAm GmUmCmGmGmUmGmUmUmUmUmUmUmU (SEQ ID NO: 39), wherein “m” stands for 2’- OMe modification.
  • the sgRNA described herein comprises the sequence of GUUUUAGAGCCGGCGGAAACGCCGGCAAGUUAAAAUAAGGCUAGUCCGUUAU CAACUUGGACUUUGGUCCAAGUUUUU (SEQ ID NO: 17).
  • the sgRNA comprises backbone modified nucleotides.
  • the present invention provides a single guide RNA (sgRNA) comprising a sequence of GUUUUAGANxnGAAANynAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGGAC N z N z N z N z GUCCAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 41), wherein N xn and Nyn have the same number of nucleotides and are complementary nucleotides to form base pairs, and wherein the nucleotides of N xn and N yn are modified nucleotides and wherein n is an integral number from 5-15, and wherein the 4 nucleotides of the loop of hairpin 1 (NzNzNzNz) comprises a sequence of UUCG, CUUG or GCAA.
  • sgRNA single guide RNA
  • the sgRNA comprising a longer upper stem and a stable hair pin includes one or more modified nucleotides within the hairpin 1 and hairpin 2 regions.
  • the sgRNA comprising a longer upper stem and a stable hair pin includes the modified nucleotides within the hairpin 1 and hairpin 2 regions.
  • the modifications include 2′-O-methyl modified nucleotides (e.g., 2′-O-methyl 3′-phosphorothioate (MS) nucleotide, 2′-O-methyl 3′-thioPACE (MSP) nucleotide), 2’-F modified nucleotides and a combination thereof.
  • At least one modification comprises a 2’-O’methyl (2’-O-Me) modified nucleotide.
  • the loop of hairpin 1 comprises locked nucleic acid.
  • the gRNA described herein further comprises a spacer sequence at the 5′′′ end of the sgRNA; the spacer sequence comprises a sequence complementary to a target sequence of interest. In some embodiments, the spacer sequence comprises about 18-25, or 18-30, or 20-25, 20-30, 15-50, 20-50, or 20-40 nucleotides. As non-limiting examples, the spacer sequence comprises 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45 or 50 nucleotides.
  • the 3’ end of the sgRNA described herein is modified.
  • the 5′ end of the sgRNA described herein is modified.
  • the 3’ and 5′ ends of the sgRNA described herein are modified.
  • at least the first three nucleotides at the 5′ end of the sgRNA described herein are modified nucleotides.
  • the sgRNA described herein comprises a modification at the 5′ end of the sequence and a modification at the 3’ end of the sequence.
  • a gRNA described herein comprises a sequence as set forth in SEQ ID NO: 20. In one example, a gRNA described herein comprises a sequence as set forth in SEQ ID NO: 21. In one example, a gRNA described herein comprises a sequence as set Attorney Docket No. BEM-020WO1 forth in SEQ ID NO: 51. In one example, a gRNA described herein comprises a sequence as set forth in SEQ ID NO: 52.
  • the sgRNA described herein further comprises a nuclear localization sequence (NLS).
  • NLS nuclear localization sequence
  • the sgRNA described herein comprises about 102-150 nucleotides, about 102-120 nucleotides, or about 100-180 nucleotides.
  • the sgRNA of the present invention increases the gene modification efficacy about 2 to1000-fold, or about 2 to 100-fold, or about 2 to10-fold.
  • the sgRNA described herein increases the gene modification efficacy about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 550,, 600, 650, 700, 750, 800, 900 or 1000 fold.
  • a gene modification system comprising, (a) a CRISPR-associated protein (Cas) polypeptide, or variant thereof; and (b) a single guide RNA (sgRNA) described herein.
  • the Cas polypeptide is a Cas9 protein, including but not limited to an S. pyogenes Cas9 and an S.
  • the Cas9 polypeptide is a nickase or a dCas9.
  • provided also includes a composition comprising a single guide RNA described herein.
  • the composition may further comprise a Cas9 polypeptide, or variant thereof.
  • the Cas polypeptide is a Cas9 protein, dCas9 or a nickase.
  • the composition described herein is formulated in a lipid nanoparticle.
  • the present invention provides a method for modifying a target gene in a cell comprising introducing into the cell a gene editing system comprising a single guide RNA (sgRNA) of the present invention.
  • the method comprises introducing into the cell a modified single guide RNA (sgRNA) comprising a first nucleotide sequence that is complementary to the target nucleic acid and a second nucleotide sequence that interacts with a CRISPR- associated protein (Cas) polypeptide, wherein one or more of the nucleotides in the first nucleotide sequence and/or the second nucleotide sequence are modified nucleotides; and a Attorney Docket No.
  • sgRNA modified single guide RNA
  • FIG.1A shows the hairpin structures of exemplary end-modified sgRNA, standard heavily modified sgRNA modification and a LONGEST gRNA design including 5 base pair in the upper stem of the gRNA.
  • the chemical modified sgRNAs include only 4 base pairs in the upper stem.
  • FIG.1B shows an exemplary ALAS1 targeted editing in liver demonstrated that the editing potency decreased when the hairpin of the gRNA was extended without nucleotide modification.
  • the editing potency increased when extended in combination with nucleotide modification.
  • gRNA9 comprises an extended upper stem (LONGEST 1) without modifications internally including the upper stem of the extended hairpin.
  • sgRNA 10 comprises another extended upper stem (LONGEST 2) without modifications internally including the upper stem of the extended hairpin.
  • sgRNA3 is LONGEST1 including 2’OMe modified nucleotides (the sequence as set forth in SEQ ID NO: 51).
  • the ALAS1 editing was with base editor ABE8.8.
  • FIG.2A shows the hairpin structures of an ALAS1 targeting gRNA with end- modifications (gRNA 1, or standard-heavy modifications (gRNA 2) or LONGEST 1 with nucleotide modifications (gRNA 3).
  • FIG.2B shows the ALAS1 targeting gRNA editing potency in Lipid 1 at different doses .
  • FIG.2C shows the ALAS1 targeting gRNA editing potency in Lipid 2 at different dose.
  • FIG.3A shows the hairpin structures of a gRNA with end-modifications (EM), LONGEST-modifications, GOLD modifications, or LONGEST_GOLD combination modifications.
  • FIG.3B shows in vitro editing potencies of different gRNA modifications at three different target sites (TSBTx3228, TSBTx3215 and TSBTx3222).
  • FIG.4A shows the hairpin structures of gRNA 4 (standard end-modifications), gRNA 5 (GOLD modification) and gRNA 6(LONGEST-GOLD modifications). Attorney Docket No. BEM-020WO1
  • FIG.4B shows the editing potencies of gRNA 4, gRNA 5 and gRNA 6 after 5 days.
  • FIG.5 shows the editing potencies of three new hairpin extensions in ALAS1 targeting gRNAs: LONGEST 3, LONGEST 4 and unmodified LONGEST 4, as compared to the same gRNA with standard heavy modifications (Heavy-mod), end-modifications, or LONGEST modifications.
  • LONGEST 3, LONGEST 4 and unmodified LONGEST 4 as compared to the same gRNA with standard heavy modifications (Heavy-mod), end-modifications, or LONGEST modifications.
  • Administering includes oral administration, topical contact, administration as a suppository, intravenous, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal, or subcutaneous administration to a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial.
  • Binding region refers to the region within a nuclease target region that is recognized and bound by the nuclease, such as Cas9.
  • Complementary refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types.
  • a percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary).
  • Perfectly complementary means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.
  • “Substantially complementary” as used herein refers to a degree of complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%.97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions.
  • Effective amount refers to the amount of an agent (e.g., Cas nuclease, modified single guide RNA, etc.) that is sufficient to effect beneficial or desired results.
  • the therapeutically effective amount may vary depending upon one or more of: the subject and disease condition being treated, the weight and age of the Attorney Docket No. BEM-020WO1 subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art.
  • the specific amount may vary depending on one or more of: the particular agent chosen, the target cell type, the location of the target cell in the subject, the dosing regimen to be followed, whether it is administered in combination with other agents, timing of administration, and the physical delivery system in which it is carried.
  • Efficiency refers to the editing efficiency, or editing percentage; that is the total number of sequence reads with insertions or deletions of nucleotides into the target region of interest over the total number of sequence reads following cleavage by a gene editing system of the present disclosure.
  • Genome The term “genome” as used herein refers to the complete set of genes or genetic material present in a cell or organism. The genome includes DNA or RNA in RNA viruses. The genome includes both the genes, (the coding regions), the noncoding DNA and the genomes of the mitochondria and chloroplasts.
  • Genome editing The term “genome editing” as used herein refers to changing a gene.
  • Genome editing may include correcting or restoring a mutant gene. Genome editing may include knocking out a gene, such as a mutant gene or a normal gene. Genome editing may be used to treat disease or enhance muscle repair by changing the gene of interest.
  • Guide RNA or gRNA As used herein, the terms “guide RNA” and ‘gRNA” are used interchangeably.
  • guide RNA refers to a short synthetic RNA composed of a “scaffold” sequence necessary for Cas9-binding or Cpf1-binding and a user-defined “spacer” or “targeting sequence” (also referred to herein as a protospacer-targeting sequence or segment) which defines the genomic target to be modified.
  • “Modified gRNA” as used herein refers to a gRNA that has additional nucleotides or nucleotide modifications.
  • Hybridization means the pairing or annealing of complementary oligonucleotides and/or nucleic acids.
  • crRNA As used herein, the term“crRNA” refers to an RNA sequence comprising a sequence that is complementary to and recognizes a targeting nucleic acid sequence and a tracrRNA recognition sequence that is bound to or is capable of binding to tracrRNA.
  • the Attorney Docket No. BEM-020WO1 tracRNA recognition portion of crRNA may bind to tracrRNA via hybridization or covalent attachment.
  • tracRNA As used herein, the term “tracrRNA” means a nucleic acid sequence that can non-covalently bind to a Cas9 protein and that can bind to a crRNA sequence via hybridization or covalent attachment. In some embodiments, the tracrRNA and crRNA sequences can form a single guide RNA.
  • Hairpin The term “Hairpin” as used herein describes a duplex of nucleic acids that is created when a nucleic acid strand folds and forms base pairs with another section of the same strand. A hairpin may form a structure that comprises a loop or a U-shape. In some embodiments, a hairpin may be comprised of an RNA loop.
  • Hairpins can be formed with two complementary sequences in a single nucleic acid molecule bind together, with a folding or wrinkling of the molecule.
  • hairpins comprise stem or stem loop structures.
  • a “hairpin region” refers to hairpin 1 and hairpin 2 from the 5′ end to the 3’ end of the gRNA.
  • a conserved portion of a gRNA locates between hairpin 1 and hairpin 2 of the gRNA.
  • Stem loop As used herein, the term “stem loop” describes a secondary structure of nucleotides that form a base-paired “stem” that ends in a loop of unpaired nucleic acids.
  • a stem may be formed when two regions of the same nucleic acid strand are at least partially complementary in sequence when read in opposite directions.
  • Loop the term “loop” as used herein describes a region of nucleotides that do not base pair (i.e., are not complementary) that may cap a stem.
  • a “tetraloop” describes a loop of 4 nucleotides.
  • the upper stem of a modified gRNA may comprise a tetraloop.
  • GAAA is the GNRA tetraloop that is used to lock the structure of the hairpin.
  • the tetraloop is ANYA, CUYG, GNRA, UNAC, or UNCG.
  • Pharmaceutically acceptable carrier refers to a substance that aids the administration of an agent (e.g., Cas nuclease, modified single guide RNA, etc.) to a cell, an organism, or a subject.
  • “Pharmaceutically acceptable carrier” refers to a carrier or excipient that can be included in a composition or formulation and that causes no significant adverse toxicological effect on the patient.
  • Non- limiting examples of pharmaceutically acceptable carrier include water, NaCl, normal saline solutions, lactated Ringer's, normal sucrose, normal glucose, binders, fillers, disintegrants, Attorney Docket No.
  • Modified or modification refers to, in the context of guide RNA, various modifications, e.g., 2′-O-methyl modified nucleotides (e.g., 2′-O-methyl 3′-phosphorothioate (MS) nucleotide, 2′-O-methyl 3′- thioPACE (MSP) nucleotide), 2’-F modified nucleotides, locked nucleic acid, MOE (methoxyethyl), DNA nucleotides functionalized for conjugation, and a combination thereof.
  • 2′-O-methyl modified nucleotides e.g., 2′-O-methyl 3′-phosphorothioate (MS) nucleotide, 2′-O-methyl 3′- thioPACE (MSP) nucleotide
  • 2’-F modified nucleotides locked nucleic acid
  • MOE methoxyethyl
  • nucleic acid As used herein, the terms “nucleic acid” or “oligonucleotide” or “polynucleotide” as used herein means at least two nucleotides covalently linked together.
  • the depiction of a single strand also defines the sequence of the complementary strand.
  • a nucleic acid also encompasses the complementary strand of a depicted single strand.
  • Many variants of a nucleic acid may be used for the same purpose as a given nucleic acid.
  • a nucleic acid also encompasses substantially identical nucleic acids and complements thereof.
  • a single strand provides a probe that may hybridize to a target sequence under stringent hybridization conditions.
  • nucleic acid also encompasses a probe that hybridizes under stringent hybridization conditions.
  • Nucleic acids may be single stranded or double stranded, or may contain portions of both double stranded and single stranded sequence.
  • the nucleic acid may be DNA, both genomic and cDNA, RNA, or a hybrid, where the nucleic acid may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine.
  • Nucleic acids may be produced by chemical synthesis methods or by recombinant methods.
  • On-target site The term “on-target site” as used herein refers to the target region or sequence in a genome to which the gRNA is intended to target. Ideally, the on-target site has perfect homology (100% identity or homology) to the target DNA sequence with no homology elsewhere in the genome.
  • Off-target site The term “off-target site” as used herein refers to a region of the genome which has partial homology or partial identity to the on-target site or target region of the gRNA, but which the gRNA is not intended or designed to target.
  • Subject The terms “subject,” “patient,” and “individual” are used herein interchangeably to include a human or animal.
  • the animal subject may be a mammal, a primate (e.g., a monkey), a livestock animal (e.g., a horse, a cow, a sheep, a pig, or a goat), a companion animal (e.g., a dog, a cat), a laboratory test animal (e.g., a mouse, a Attorney Docket No. BEM-020WO1 rat, a guinea pig, a bird), an animal of veterinary significance, or an animal of economic significance.
  • a primate e.g., a monkey
  • livestock animal e.g., a horse, a cow, a sheep, a pig, or a goat
  • a companion animal e.g., a dog, a cat
  • laboratory test animal e.g., a mouse, a Attorney Docket No.
  • Treat refers to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit and/or a prophylactic benefit.
  • therapeutic benefit is meant any therapeutically relevant improvement in or effect on one or more diseases, conditions, or symptoms under treatment.
  • the compositions may be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject reporting one or more of the physiological symptoms of a disease, even though the disease, condition, or symptom may not have yet been manifested.
  • Modified guide RNA gRNA
  • Provided herein are modified guide RNAs (gRNAs) for use in gene editing methods.
  • modified gRNAs provided herein comprise a long or extended upper stem structure .
  • Modified guide RNAs provided herein may comprise an extended upper stem and a stable locked hairpin structure.
  • modified guide RNAs provided herein are single guide RNAs (sgRNAs). The modified sgRNAs of the present disclosure are more stable and show improved efficacy in gene editing as compared to the non-modified sgRNAs.
  • modified gRNAs are modified single guide RNAs (sgRNAs) comprising a long (or extended) upper stem including more than 4 base pairs formed by complementary nucleotides, wherein one or more nucleotides of the upper stem are chemically modified.
  • sgRNAs modified single guide RNAs
  • Exemplary modifications include 2’OMe modification, such as 2′- O-methyl (M) nucleotides, 2′-O-methyl 3′-phosphorothioate (MS) nucleotides, 2′-O-methyl 3′-thioPACE (MSP) nucleotides, or combinations thereof.
  • the modified sgRNA described herein comprises a long upper stem region comprising 5-15 base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides).
  • the long upper stem region comprises 5 base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides).
  • the long upper stem region comprises 6 base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides).
  • the Attorney Docket No. BEM-020WO1 long upper stem region comprises 7 base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides).
  • the long upper stem region comprises 8 base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides). In one embodiment, the long upper stem region comprises 9 base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides). In one embodiment, the long upper stem region comprises 10 base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides). In one embodiment, the long upper stem region comprises 11 base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides). In one embodiment, the long upper stem region comprises 12 base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides).
  • the long upper stem region comprises 13 base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides). In one embodiment, the long upper stem region comprises 14 base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides). In one embodiment, the long upper stem region comprises 15 base pairs formed by complementary nucleotides (e.g., chemically modified nucleotides). [0092] In some embodiments, the nucleotides of the upper stem of the modified sgRNA described herein are all modified nucleotides.
  • the sgRNA described herein comprises an upper stem modification, wherein the upper stem modification comprises a modification to any one or more of nucleotides in the upper stem region.
  • the sgRNA comprises an upper stem modification, wherein the upper stem modification comprises a modification of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in the upper stem region.
  • the gRNA comprises an upper stem modification, wherein the upper stem modification comprises a modification of about 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1- 10, 1-12, 1-14, 1-16, 2-4, 2-6, 2-8, 2-10, 2-12, 2-16, 2-18, 2-20, 4-8, 4-10, 6-12, 6-18, 6-20, 8-10, 8-18, 8-20, or 10-20 nucleotides in the upper stem region.
  • the modified nucleotides within the upper stem region of the sgRNA comprise the same chemical modifications.
  • the modified Attorney Docket No. BEM-020WO1 nucleotides within the upper stem region of the sgRNA comprise different chemical modifications.
  • the sgRNA comprises an upper stem modification, wherein the upper stem modification comprises a 2’-OMe modified nucleotide.
  • the upper stem modification comprises a 2’-O-MOE modified nucleotide.
  • the upper stem modification comprises a 2’-F modified nucleotide.
  • the upper stem modification comprises a 2’-Ome modified nucleotide, a 2’-O- MOE modified nucleotide, a 2’-F modified nucleotide, and/or combinations thereof.
  • Stable hairpins [0098]
  • the sgRNA described herein is further modified to comprise highly stable hairpin structures.
  • the modified sgRNA described herein further comprises one or more modified nucleotides within the hairpin 1 and hairpin 2 regions. In some embodiments, all the nucleotides within the hairpins 1 and 2 regions are modified nucleotides.
  • the modified sgRNA described herein further comprises a modified stable hairpin 1 region, wherein the modified stable hairpin 1 region comprises an extended stem region comprising more than 4 base pairs and wherein the loop of hairpin 1 comprises locked nucleic acids.
  • the extended stem region of the modified stable hairpin comprises 8 base pairs. In some embodiments, the extended stem region of the modified stable hairpin comprises 8 base pairs formed by chemically modified nucleotides. As a non-limiting example, all the nucleotides of the hairpin comprises comprise 2’-O-methyl modification.
  • the highly stable hairpin of a sgRNA refers to a “locked” hairpin.
  • the locked hairpin comprises a locked backbone, e.g., by incorporating one or more locked nucleic acid.
  • locked nucleic acid refers to a bicyclic RNA analogue in which the ribose is locked in a C3′-endo conformation by introduction of a 2′-O,4′-C methylene bridge.
  • Desirable LNA monomers and their method of synthesis also are disclosed in U.S. Pat. Nos.6,043,060, 6,268,490, PCT Publications WO 01/07455, WO 01/00641, WO Attorney Docket No. BEM-020WO1 98/39352, WO 00/56746, WO 00/56748 and WO 00/66604 as well as in the following papers: Morita et al., Bioorg. Med. Chem.
  • the hairpin region comprises locked nucleic acids or LNAs containing the 2′-O, 4′-C-methylene ribonucleoside (structure A) wherein the ribose sugar moiety is in a “locked” conformation.
  • the hairpin region comprises at least one 2′,4′-C- bridged 2′ deoxyribonucleoside (CDNA, structure B). See, e.g., U.S.
  • Locked nucleic acids are a class of high-affinity RNA analogs in which the ribose ring is “locked” in the ideal conformation for Watson-Crick binding.
  • LNATM oligonucleotides exhibit high thermal stability when hybridized to a complementary DNA or RNA strand.
  • LNATM oligonucleotides can be made shorter than traditional DNA or RNA oligonucleotides and still retain a high Tm.
  • LNATM oligonucleotides can consist of a mixture of LNATM and DNA or RNA. Incorporation of LNATM into oligonucleotides has been shown to improve sensitivity and specificity for many hybridization-based technologies including PCR, microarray and in situ hybridization.
  • Chemical modifications [0102]
  • the modified gRNA further comprises one or more chemically modified nucleotides.
  • such modified nucleosides comprise a modified sugar moiety or a modified nucleobase or both a modified sugar moiety and a modified nucleobase.
  • the modified sgRNA comprise one or more modified nucleosides comprising a modified sugar moiety.
  • modified sgRNA comprising one or more sugar-modified nucleosides may have desirable properties, such as enhanced nuclease stability or increased binding affinity with a target nucleic acid relative to guide RNA lacking such sugar-modified nucleosides.
  • modified sugar moieties are linearly modified sugar moieties.
  • modified sugar moieties are bicyclic or tricyclic sugar moieties.
  • modified sugar moieties are Attorney Docket No. BEM-020WO1 sugar surrogates. Such sugar surrogates may comprise one or more substitutions corresponding to those of substituted sugar moieties.
  • modified sugar moieties are linearly modified sugar moieties comprising a furanosyl ring with one or more acyclic substituent, including but not limited to substituents at the 2′ and/or 5′ positions.
  • 2′-substituent groups suitable for linearly modified sugar moieties include but are not limited to: 2′-F, 2′-OCH 3 (“Ome” or “O-methyl”), and 2′-O(CH2)2OCH3 (“MOE”).
  • 2′-substituent groups are selected from among: halo, allyl, amino, azido, SH, CN, OCN, CF 3 , OCF 3 , O—C 1 - C10 alkoxy, O—C1-C10 substituted alkoxy, O—C1-C10 alkyl, O—C1-C10 substituted alkyl, S- alkyl, N(R m )-alkyl, O-alkenyl, S-alkenyl, N(R m )-alkenyl, O-alkynyl, S-alkynyl, N(R m )- alkynyl, O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH2)2SCH3, O(CH2)2ON(Rm)(Rn) or OCH2C( ⁇ O)—N(Rm)
  • 2′-substituent groups can be further substituted with one or more substituent groups independently selected from among: hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy, thioalkyl, halogen, alkyl, aryl, alkenyl and alkynyl.
  • substituent groups independently selected from among: hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy, thioalkyl, halogen, alkyl, aryl, alkenyl and alkynyl.
  • substituent groups independently selected from among: hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy, thioalkyl, halogen, alkyl, aryl, alkenyl and al
  • linearly modified sugars comprise more than one non-bridging sugar substituent, for example, 2′-F-5′-methyl sugar moieties (see, e.g., PCT International Application WO 2008/101157, for additional 2′, 5′-bis substituted sugar moieties and nucleosides).
  • a 2′-substituted nucleoside or 2′-linearly modified nucleoside comprises a sugar moiety comprising a linear 2′-substituent group selected from: F, NH, N, OCF, OCH, O(CH)NH, CHCH ⁇ CH, OCHCH ⁇ CH, OCHCHOCH, O(CH)SCH, O(CH)ON(R )(R), O(CH)O(CH)N(CH), and N-substituted acetamide (OCHC( ⁇ O)—N(R )(R)), where each R and R is, independently, H, an amino protecting group, or substituted or unsubstituted C-C alkyl.
  • a linear 2′-substituent group selected from: F, NH, N, OCF, OCH, O(CH)NH, CHCH ⁇ CH, OCHCH ⁇ CH, OCHCHOCH, O(CH)SCH, O(CH)ON(R )(R), O(CH)O(CH)
  • a 2′-substituted nucleoside or 2′-linearly modified nucleoside comprises a sugar moiety comprising a linear 2′-substituent group selected from: F, OCF, OCH, OCHCHOCH, O(CH)SCH, O(CH) N(CH), O(CH)O(CH)N(CH), and OCHC( ⁇ O)—N(H)CH (“NMA”).
  • NMA OCHC( ⁇ O)—N(H)CH
  • a 2′-substituted nucleoside or 2′-linearly modified nucleoside comprises a sugar moiety comprising a linear 2′-substituent group selected from: F, OCH, and OCHCHOCH.
  • Certain modified sugar moieties comprise a bridging sugar substituent that forms a second ring resulting in a bicyclic sugar moiety.
  • the bicyclic sugar moiety comprises a bridge between the 4′ and the 2′ furanose ring atoms.
  • 4′ to 2′ bridging sugar substituents include but are not limited to: 4′-CH-2′, 4′-(CH)-2′, 4′-(CH)-2′, 4′-CH—O-2′ (“LNA”), 4′-CH—S-2′, 4′-(CH)—O-2′ (“ENA”), 4′-CH(CH)— O-2′ (referred to as “constrained ethyl” or “cEt” when in the S configuration), 4′-CH—O— CH-2′, 4′-CH—NI-2′, 4′-CH(CHOCH)—O-2′ (“constrained MOE” or “cMOE”) and analogs thereof (see, e.g., U.S. Pat.
  • such 4′ to 2′ bridges independently comprise from 1 to 4 linked groups independently selected from: —[C(R)(R)]—, —[C(R)(R)]—O—, — C(R) ⁇ C(R)—, —C(R) ⁇ N—, —C( ⁇ NR)—, —C( ⁇ O)—, —C( ⁇ S)—, —O—, —Si(R)—, —S( ⁇ O)—, and —N(R)—; wherein X is 0, 1 or 2 and n is 1, 2, 3 or 4.
  • bicyclic sugar moieties and nucleosides incorporating such bicyclic sugar moieties are further defined by isomeric configuration.
  • an LNA nucleoside (described above) may be in the ⁇ -L configuration or in the ⁇ -D configuration.
  • modified sugar moieties are sugar surrogates.
  • the oxygen atom of the sugar moiety is replaced, e.g., with a sulfur, carbon or nitrogen atom.
  • such modified sugar moieties also comprise bridging and/or non-bridging substituents as described above.
  • certain sugar surrogates comprise a 4′-sulfur atom and a substitution at the 2′-position (see, e.g., US2005/0130923) and/or the 5′ position.
  • Attorney Docket No. BEM-020WO1 [0111]
  • the modification in the sugar group can be a modification at the 2′ position of the ribose group.
  • the modification at the 2′ position of the ribose group is selected from the group consisting of 2′-O-methyl, 2′-fluoro, 2′-deoxy, and 2′-O-(2-methoxyethyl).
  • the one or more modifications in the modified gRNa comprises 2’OMe modification, such as 2′-O-methyl (M) nucleotides, 2′-O-methyl 3′- phosphorothioate (MS) nucleotides, 2′-O-methyl 3′-thioPACE (MSP) nucleotides, or combinations thereof.
  • the modified sgRNA comprise one or more nucleoside comprising a modified nucleobase.
  • the modified sgRNA comprise one or more nucleoside that does not comprise a nucleobase, referred to as an abasic nucleoside.
  • modified nucleobases are selected from: 5-substituted pyrimidines, 6-azapyrimidines, alkyl or alkynyl substituted pyrimidines, alkyl substituted purines, and N-2, N-6 and O-6 substituted purines.
  • modified nucleobases are selected from: 2-aminopropyladenine, 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-N-methylguanine, 6-N-methyladenine, 2-propyladenine, 2- thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl (—C ⁇ C—CH3) uracil, 5- propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-ribosyluracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, 8-aza and other 8-substituted purines, 5-halo, particularly 5-bromo, 5-trifluoromethyl, 5-halouracil, and 5-halocytosine, 7- methylguanine, 7-methyla
  • modified nucleobases include tricyclic pyrimidines, such as 1,3-diazaphenoxazine-2-one, 1,3-diazaphenothiazine-2-one and 9-(2-aminoethoxy)-1,3- diazaphenoxazine-2-one (G-clamp).
  • Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example, 7-deaza- adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.
  • nucleosides of modified sgRNAs described herein may be linked together using any internucleoside linkage.
  • the two main classes of internucleoside linking groups are defined by the presence or absence of a phosphorus atom.
  • Representative Attorney Docket No. BEM-020WO1 phosphorus-containing internucleoside linkages include but are not limited to phosphates, which contain a phosphodiester bond (“P ⁇ O”) (also referred to as unmodified or naturally occurring linkages), phosphotriesters, methylphosphonates, phosphoramidates, and phosphorothioates (“P ⁇ S”), and phosphorodithioates (“HS—P ⁇ S”).
  • P ⁇ O phosphodiester bond
  • P ⁇ S phosphotriesters
  • methylphosphonates methylphosphonates
  • phosphoramidates phosphoramidates
  • P ⁇ S phosphorothioates
  • HS—P ⁇ S phosphorodithioates
  • Non- phosphorus containing internucleoside linking groups include but are not limited to methylenemethylimino (—CH2—N(CH3)—O—CH2—), thiodiester (—O—C( ⁇ O)—S—), thionocarbamate (—O—C( ⁇ O)(NH)—S—); siloxane (—O—SiH 2 —O—); and N,N′- dimethylhydrazine (—CH2—N(CH3)—N(CH3)—).
  • Modified internucleoside linkages compared to naturally occurring phosphate linkages, can be used to alter, typically increase, nuclease resistance of the oligonucleotide.
  • internucleoside linkages having a chiral atom can be prepared as a racemic mixture, or as separate enantiomers.
  • Representative chiral internucleoside linkages include but are not limited to alkylphosphonates and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing internucleoside linkages are well known to those skilled in the art.
  • Neutral internucleoside linkages include, without limitation, phosphotriesters, methylphosphonates, MMI (3′-CH2—N(CH3)—O-5′), amide-3 (3′-CH2—C( ⁇ O)—N(H)-5′), amide-4 (3′-CH2—N(H)—C( ⁇ O)-5′), formacetal (3′-O—CH2—O-5′), methoxypropyl, and thioformacetal (3′-S—CH2—O-5′).
  • Further neutral internucleoside linkages include nonionic linkages comprising siloxane (dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonate ester and amides (See for example: Carbohydrate Modifications in Antisense Research; Y. S. Sanghvi and P. D. Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65). Further neutral internucleoside linkages include nonionic linkages comprising mixed N, O, S and CH2 component parts.
  • the one or more chemical modification in the phosphate group can be a phosphorothioate modification.
  • the modified sgRNA comprises one modified nucleotide at the 5′-end (e.g., the terminal nucleotide at the 5′-end) or near the 5′-end (e.g., within 1, 2, 3, 4, or 5 nucleotides of the terminal nucleotide at the 5′-end) of the modified gRNA nucleotide sequence and/or one modified nucleotide at the 3′-end (e.g., the terminal nucleotide at the 3′- end) or near the 3′-end (e.g., within 1, 2, 3, 4, or 5 nucleotides of the 3′-end) of the modified gRNA nucleotide sequence.
  • the modified sgRNA comprises two consecutive or non- consecutive modified nucleotides starting at the 5′-end (e.g., the terminal nucleotide at the 5′- end) or near the 5′-end (e.g., within 1, 2, 3, 4, or 5 nucleotides of the terminal nucleotide at the 5′-end) of the modified gRNA nucleotide sequence and/or two consecutive or non- consecutive modified nucleotides starting at the 3′-end (e.g., the terminal nucleotide at the 3′- end) or near the 3′-end (e.g., within 1, 2, 3, 4, or 5 nucleotides of the 3′-end) of the modified gRNA nucleotide sequence.
  • the modified gRNA comprises modified nucleotides at the 5′ terminus.
  • the 5′ terminus of the sgRNA comprises a spacer or guide region that functions to direct a Cas protein, e.g., a Cas9 protein, to a target nucleotide sequence.
  • the 5′ terminus does not comprise a guide region.
  • the 5′ terminus comprises a spacer and additional nucleotides that do not function to direct a Cas protein to a target nucleotide region.
  • the sgRNA has a 3’ end, which is the last nucleotide of the sgRNA.
  • the 3’ terminus region includes the last 1-7 nucleotides from the 3’ end.
  • the 3’ end is the end of hairpin 2.
  • the sgRNA comprises nucleotides after the hairpin region(s).
  • the sgRNA includes a 3’ tail region, in which case the last nucleotide of the 3’ tail is the 3’ terminus.
  • the 3’ tail comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 or more nucleotides, e.g., that are not associated with the secondary structure of a hairpin.
  • the 3’ tail region comprises 1, 2, 3, or 4 nucleotides that are not associated with the secondary structure of a hairpin.
  • the 3’ tail region comprises 4 nucleotides that are not associated with the secondary structure of a hairpin. In some embodiments, the 3’ tail region comprises 1, 2, or 3 nucleotides that are not associated with the secondary structure of a hairpin.
  • the modified gRNA further comprises a targeting nucleic acid sequence that is complementary to a sequence of a target nucleic acid. The complementary sequence is about 20 nucleotides in length. In some embodiments, at least two, three, four, five, six, seven, eight, nine, ten, or more of the nucleotides in the complementary nucleotide sequence are modified nucleotides.
  • nucleotides in the Attorney Docket No. BEM-020WO1 complementary nucleotide sequence are modified nucleotides.
  • all the nucleotides in the complementary nucleotide sequence are modified nucleotides.
  • the modified nucleotides are located at the 5′-end (e.g., the terminal nucleotide at the 5′-end) or near the 5′-end (e.g., within 1, 2, 3, 4, or 5 nucleotides of the terminal nucleotide at the 5′-end) of the complementary nucleotide sequence and/or at internal positions within the complementary nucleotide sequence. In other instances, from about 10% to about 30% of the nucleotides in the first nucleotide sequence are modified nucleotides.
  • Table 2 Exemplary guide RNA sequences Sequence (5′-3’) GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA m A : U A m C G Attorney Docket No.
  • a first RNA sequence that is a tran- activating RNA (tracrRNA) and a second RNA sequence that encompasses a clustered regularly interspersed short palindromic repeats (CRISPR) RNA (crRNA) comprising a sequence complementary to a target sequence are provided for synthesizing a modified sgRNA as described herein.
  • a first RNA sequence and a second RNA sequence are litigated together.
  • the ligation strategies described herein differ from previously reported chemical ligation strategies used to synthesize synthetic RNAs including guide RNAs.
  • the advantage of using the present segmented synthetic approach is that short sections of RNA can be produced with greater purity post-purification compared to full length gRNA.
  • the 5′ acceptor is the smallest RNA fragment (about 30-50 nts) and can thus be purified to a high level before ligation.
  • the 3′ donor is terminated with a phosphate that is required for synthesis and thus only the full-length fragment will be incorporated into the full-length product (i.e., truncations are not substrates.
  • the present modified gRNA is synthesized using a self- templating approach.
  • the method for synthesizing a modified gRNA comprises providing a first RNA sequence and a second RNA sequence having complementarity, wherein the complementarity allows for base pairing and the creation of a stem loop between the first and Attorney Docket No.
  • BEM-020WO1 the second RNA; ligating the first and second RNA with a ligating enzyme within the stem loop, thus producing a modified gRNA.
  • This allows for the use of a helix, or other structure, which is formed between the first RNA and the second RNA to template an enzymatic ligation of the two RNAs.
  • the length and sequence composition of the structure formed between the first and the second RNA is modified to promote non-covalent assembly and to create optimal ligation sites for enzymes compatible with RNA ligation.
  • the complementarity can either be partial or perfect among a stretch of nucleotides of the first and the second RNA sequences. The complementarity allows for base pairing between the complementary nucleotides.
  • the mismatched nucleotides would result in the formation of a bulge or a loop structure between the first and the second RNA sequences.
  • Various structures of the modified gRNA such the longest upper stem, a stem loop, a lower stem, hairpins, overhang, blunt end or a bulge can be formed between the first and the second RNA sequences based on hybridization between the two RNA sequences.
  • Various ligases can be used with the methods described herein.
  • T4 RNA ligase 1 is used to ligate the first RNA and the second RNA at the terminal loop.
  • T4 RNA ligase 2 is used for ligating the first RNA sequence and the second RNA sequence within the stem formed between the first RNA and the second RNA sequences.
  • Various kinds of ligation are possible using this approach, such as ligation within a terminal loop of a hairpin formed between the first RNA sequence and the second RNA sequence.
  • Various ligases are suitable for ligation at the terminal loop of a hairpin formed, such as T4 RNA ligase 1.
  • Another kind of ligation that is possible with this approach is ligation within the duplex formed between the first RNA and the second RNA.
  • a modified gRNA of the present disclosure may be synthesized using a splint templating approach.
  • a splint is used in the production of the synthetic RNAs. The use of a splint allows for one or more RNA Attorney Docket No. BEM-020WO1 molecules to be brought into physical proximity for the reaction using a splint as a template. When more than two RNAs are to be joined, the use of splints facilitates the production of the synthetic RNAs such as modified sgRNAs described herein.
  • the splints can be any suitable polymer that is capable of bringing the one or more RNA molecules in close proximity can be used.
  • the splint is an RNA molecule or a DNA molecule.
  • the splint has complementarity to sections of the first RNA sequence and the second RNA sequence. The complementarity can either be partial or perfect.
  • the method of producing a modified gRNA comprising providing a first RNA comprising a 5′ phophate; providing a second RNA comprising a free 3′-hydoxyl; providing an oligonucleotide that has partial complementarity to the first RNA and the second RNA, wherein the complementarity of the oligonucleotide allows for base pairing with the first and the second RNA; and providing a ligase to catalyze ligation between the first and the second RNA, thus producing a gRNA.
  • a non-templated approach is used to produce a modified sgRNA described herein.
  • a first RNA that has a 5′ phosphate (such as a 5′ monophosphate), and a second RNA is provided that comprises a blocked 3′ end (such as a blocked 3′ OH).
  • the purpose of blocking the 3′ OH of the second RNA is so that the second RNA cannot cyclize through an untemplated mechanism when ligation occurs.
  • RNA comprising 3′ hydroxyl of the 3′ terminal end of the donor molecule which is chemically blocked or removed (e.g., dideoxynucleotide) and an enzyme (particularly by T4 RNA Ligase 1) would catalyze proper ligation between the first RNA and the second RNA.
  • this ligation strategy is carried out at a high concentration.
  • the non-templating approach of producing a synthetic RNA comprises providing a first RNA comprising a 5′ –monophosphate; providing a second RNA comprising a blocked 3′ end; and providing a ligase to catalyze ligation between the first and the second RNA, thus producing a modified sgRNA described herein.
  • Gene editing system [0134] In accordance with the present disclosure, a gene editing system comprising one or more modified sgRNAs described herein is provided. In some embodiment, the system comprises a CRISPR-Cas9 nuclease or variant thereof. Attorney Docket No.
  • the present invention provides an gene editing system comprising one or modified sgRNAs described herein and a CRISPR-Cas9 nuclease or variant thereof.
  • the present invention provides an gene editing system comprising one or modified sgRNAs described herein and a base editor.
  • the base editor may comprise an adenosine deaminase domain or a cytidine deaminase domain.
  • the one or more modified guide RNAs target the base editor to effect an A»T to G*C alteration in a target polynucleotide (e.g., a target gene).
  • the base editor is a fusion protein comprising one or more domains having base editing activity.
  • the protein domains having base editing activity are linked to the guide RNA (e.g., via an RNA binding motif on the guide RNA and an RNA binding domain fused to the deaminase).
  • the domains having base editing activity are capable of deaminating a base within a nucleic acid molecule.
  • the base editor is capable of deaminating one or more bases within a DNA molecule.
  • the base editor is capable of deaminating a cytosine (C) or an adenosine (A) within DNA. In some embodiments, the base editor is capable of deaminating a cytosine (C) and an adenosine (A) within DNA. In some embodiments, the base editor is a cytidine base editor (CBE). In some embodiments, the base editor is an adenosine base editor (ABE). In some embodiments, the base editor is an adenosine base editor (ABE) or variant thereof.
  • the ABE includes but are not limited to ABE8.8, an editor variant ABEV1 (pNMG-B2000(ABE8.20 w/ F149Y “v1”) + S82T) or ABEV2 (pNMG-B2001(ABE8.20 w/ Y147D,F149Y,T166I,D167N “v2”) + S82T).
  • the base editor is a cytidine base editor (CBE).
  • the base editor is a nuclease-inactive Cas9 (dCas9) fused to an adenosine deaminase.
  • the base editor in some cases, may be fused to an inhibitor of base excision repair, for example, a UGI domain, or a dISN domain. In other embodiments the base editor is an abasic base editor.
  • a base editor Detailed descriptions of base editors can be found in PCT patent application publications WO2018/027078, WO2017/070632, WO 2022/204268 and WO2023/114953; the contents of each of which are incorporated herein by reference for all purpose.
  • Formulations and compositions Attorney Docket No. BEM-020WO1
  • Modified sgRNAs and gene editing systems of the present disclosure may be formulated in a carrier for delivery and administrations.
  • the pharmaceutical formulation comprises a lipid nanoparticle (LNP).
  • the pharmaceutical formulation comprises at least one modified gRNA of the present disclosure.
  • the pharmaceutical formulation comprises at least one modified gRNA and a Cas9 protein, a polynucleotide encoding a Cas9 protein, or an mRNA encoding a Cas9 protein.
  • Lipid nanoparticles are a well-known means for delivery of nucleotide and protein cargo, and may be used for delivery of the gRNAs, gene editing systems, compositions, or pharmaceutical formulations disclosed herein.
  • the LNPs deliver nucleic acid, protein, or nucleic acid together with protein.
  • the present disclosure provides a method for delivering any one of the gRNAs, gene editing systems, compositions, or pharmaceutical formulations disclosed herein to a subject, wherein the gRNA is associated with an LNP.
  • the gRNA/LNP is also associated with a Cas9 or an mRNA encoding Cas9.
  • the gRNA/LNP is also associated with a base editor or an mRNA encoding the base editor.
  • the invention comprises a composition comprising any one of the gRNAs disclosed and an LNP.
  • the composition further comprises a Cas9 protein or variant thereof, or an mRNA encoding Cas9 protein or variant thereof.
  • the LNP comprises one or more cationic lipids, one or more helper lipids, one or more PEGylated lipids and one or more cholesterol derived lipids.
  • compositions comprising modified gRNA, gene editing systems described herein are provided.
  • the composition is a pharmaceutical composition.
  • the pharmaceutical composition comprises one or more modified sgRNAs described herein.
  • Compositions comprising any of the gRNAs and/or gene editing systems described herein and a carrier, excipient, diluent, or the like are encompassed. [0146] In some embodiments.
  • Modified gRNAs, gene editing systems, compositions and formulations disclosed herein are for use in preparing a medicament for treating a disease or disorder.
  • Attorney Docket No. BEM-020WO1 Methods of use [0147]
  • the present disclosure further provides uses of the modified gRNAs described herein to alter the genome of a target nucleic acid in vitro (e.g., cells cultured in vitro for use in ex vivo therapy or other uses of genetically edited cells) or in a cell in a subject such as a human (e.g., for use in in vivo therapy).
  • the present invention comprises a method or use of modifying a target nucleic acid molecule comprising, administering or delivering any one or more of the gRNAs gene editing systems compositions, or pharmaceutical formulations described herein.
  • the present invention comprises a method or use for modulation of a target gene comprising, administering or delivering any one or more of the gRNAs, gene editing systems, compositions, or pharmaceutical formulations described herein.
  • the modulation is editing the target gene.
  • the modulation is a change in expression of the protein encoded by the target gene.
  • the method or use results in gene editing.
  • the method or use results in a double-stranded break within the target gene. In some embodiments, the method or use results in an insertion or deletion of nucleotides in a target gene. In some embodiments, the insertion or deletion of nucleotides in a target gene leads to a frameshift mutation or premature stop codon that results in a non-functional protein. In some embodiments, the insertion or deletion of nucleotides in a target gene leads to a knockdown or elimination of target gene expression. In some embodiments, the method or use further comprises delivering to the cell a template, wherein at least a part of the template incorporates into a target DNA at or near a double strand break site induced by the nuclease.
  • the method or use results in substitutions.
  • the gene modulation is an increase or decrease in gene expression, a change in methylation state of DNA, or modification of a histone subunit.
  • the method or use results in increased or decreased expression of the protein encoded by the target gene.
  • the efficacy of gRNAs, and/or gene editing systems can be tested in vitro and in vivo.
  • the invention comprises one or more of the gRNAs, gene editing systems, compositions, or pharmaceutical formulations described herein, wherein the gRNA Attorney Docket No. BEM-020WO1 results in gene modulation when provided to a cell together with Cas9 or mRNA encoding Cas9.
  • the efficacy of gRNA can be measured in vitro or in vivo.
  • the efficiency of a gRNA in increasing or decreasing target protein expression is determined by measuring the amount of target protein.
  • the efficiency of editing with specific gRNAs is determined by the editing present at the target location in the genome following delivery of Cas9 and the gRNA.
  • the efficiency of editing with specific gRNAs is measured by next-generation sequencing.
  • the editing percentage of the target region of interest is determined.
  • the total number of sequence reads with insertions or deletions of nucleotides into the target region of interest over the total number of sequence reads is measured following delivery of a modified gRNA, and/or a system or composition comprising a modified gRNA.
  • the activity of modified gRNAs is measured after in vivo dosing of LNPs comprising modified gRNAs.
  • in vivo efficacy of a gRNA or composition provided herein is determined by editing efficacy measured in DNA extracted from tissue (e.g., liver tissue) after administration of gRNA.
  • the method for modulating a target nucleic acid sequence in a cell comprises contacting the cell with a composition comprising a modified sgRNA described herein.
  • Methods of therapeutic uses [0157]
  • any one or more of the gRNAs, gene editing systems, compositions, or pharmaceutical formulations described herein is for use in preparing a medicament for treating or preventing a disease or disorder in a subject.
  • the present invention comprises a method of treating or preventing a disease or disorder in subject comprising administering any one or more of the gRNAs, gene editing systems, compositions, or pharmaceutical formulations described herein.
  • the gRNAs, gene editing systems, and compositions increases the editing potency at targeting sites.
  • the editing potency increases about 10% to 100% as compared to a gRNA without modification.
  • the Attorney Docket No. BEM-020WO1 editing potency increases 2 to 1000-fold as compared to a gRNA without modification.
  • the sgRNA of the present invention increases the gene modification efficacy about 2 to1000-fold, or about 2 to100-fold, or about 2 to 50- fold, or about 10 to 50- fold, or about 10 to 20- fold or about 2 to10-fold, or about 2 to 5- fold, or about 3 to 8 fold, or about 2 to 5 fold.
  • kits comprising one or more gRNAs, compositions, or pharmaceutical formulations described herein.
  • a kit further comprises one or more of a solvent, solution, buffer, each separate from the composition or pharmaceutical formulation, instructions, or desiccant.
  • Example 1 Editing potency of modified gRNAs
  • an ALAS1 Delta-aminolevulinate synthase 1 gene site was used to evaluate the influence of different hairpin designs on the editing potency of a guide RNA.
  • FIG.1A As measured for in vivo liver editing (FIG.1A), when the hairpin was extended without modification, the editing potency decreased. However, when the hairpin was extended with modifications, i.e., heavy modifications including all nucleotides, the editing potency increased.
  • gRNA1 end-modifications
  • gRNA2 standard heavy modifications
  • gRNA3 a heavily modified LONGEST hairpin design
  • the editing potency in liver of each gRNA was measured in in both Lipid 1 (FIG.2B) and Lipid 2 (FIG.2C).
  • gRNA3 with the LONGEST design was found to be 2 to 5-fold more potent at sub-saturating dose (8% editing at 0.005 mpk) over gRNA2 with standard heavy modification design for ALAS1.
  • the targeting sequence for ALAS1 CAGGATCCGCACAGACTCCAGGG (SEQ ID NO: 59), and the protospacer sequence: CAGGAUCCGCACAGACUCCA (SEQ ID NO: 60) were used in the study.
  • Attorney Docket No. BEM-020WO1 Table 3 Examples of gRNA designs used in the study Na Sequence me A G m s A m U N [0165]
  • the end modified gRNA is standard design for gRNA used in the prior art (Hendel, Ayal, et al. Nature biotechnology 33.9 (2015):- 3290).
  • the standard heavily modified gRNA without extension of the upper stem region is also used for comparison (Finn, Jonathan D., et al. Cell reports 22.9 (2016): 2227-2235).
  • Both the end modified and heavily modified gRNAs comprises 100 nucleotides in length.
  • the hairpin was extended with an additional 3 base pairs compared to end modified gRNA.
  • the unmodified LONGEST1 has the same sequence as LONGEST 1 (SEQ ID NO: 51) but is not modified with 2’OMe internally. It has 106 nts in length.
  • Riesenberg, et al Riesenberg, et al, Improved gRNA secondary structures allow editing of target sites resistant to CRISPR-Cas9 cleavage.” Nature Communications 13.1 (2022): 1-8) recently reported an engineered gRNA with “locked” hairpins can increase the Attorney Docket No.
  • FIG.3B shows that both the LONGEST design and the GOLD design outperformed the gRNA with end-modifications (EM shown in FIG.3A). About 7-fold increase was observed for the GOLD design as compared to the end-modification design. About 3-fold increase was observed for the LONGEST design as compared to the end-modification design. [0167] A gRNA with both the LONGEST and GOLD designs (LONGEST-GOLD),was synthesized and evaluated for its editing potency (gRNA 6 in FIG.4A).
  • ABEV1 is an editor variant pNMG-B2000(ABE8.20 w/ F149Y “v1”) + S82T.
  • ABEV2 is an editor variant pNMG- B2001(ABE8.20 w/ Y147D,F149Y,T166I,D167N “v2”) + S82T.
  • the editing efficacy after 5 days shows that the hairpin extensions increase overall editing of gRNAs at saturating conditions (FIG.4B).
  • Example 2 New extension designs [0169] This study further tested different hairpin extensions in gRNA. Three new hairpin extensions that have less GC content than LONGEST modifications promote the stability of the hairpin. The sequences and modifications of the three gRNA designs are shown in Table 4. LONGEST 3 has 3 additional base pairs with full modification of the upper stem loop. LONGEST 4 has 5 additional base pairs with full modification. unmodified LONGEST 4 has the same extension as LONGEST 4 but is unmodified. Table 4 Name Sequence A A Attorney Docket No.
  • LONGEST 4 mNsmNsmNsNNNNNNNNNNNNNGUUUUAGAmGmCmUmAmU mGmCmUmGmGmAmAmAmCmAmGmCmAmUmAmGmCAAGUUAAA m U
  • the results show that LONGEST 4 showed higher potency than the standard heavy modification and end modification designs (FIG.5).
  • the unmodified LONGEST 4 showed lower potency, which demonstrates that extension alone is enough and modification is needed for increasing potency. Similar findings are also observed for LONGEST design.

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Abstract

La présente invention concerne des molécules d'ARNg modifiées, des compositions et des procédés pour l'édition génique et la modification génomique spécifiques à un site, telles que le clivage d'ADN, et l'activation ou la répression de gènes. Les présents ARN guides modifiés ont une structure secondaire modifiée (par exemple, une longue tige supérieure et une structure en épingle à cheveux modifiée), qui cible la séquence d'ADN cible spécifiquement avec une activité hors cible réduite.
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CN121079412A (zh) 2025-12-05

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