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EP-4735603-A2 - GUIDE RNA COMPOSITIONS

EP4735603A2EP 4735603 A2EP4735603 A2EP 4735603A2EP-4735603-A2

Abstract

Disclosed herein are modified single guide RNAs having improved activity in gene editing methods.

Inventors

  • FRIEDLAND, ARI
  • MYER, Vic

Assignees

  • nChroma Bio, Inc.

Dates

Publication Date
20260506
Application Date
20240627

Claims (20)

  1. 1. A single guide RNA (sgRNA) comprising a tracr sequence and a spacer sequence, (i) wherein the tracr sequence comprises an upper stem, a hairpin region 1, and a hairpin region 2, and wherein (a) at least 50% of the nucleotides in the upper stem loop are 2’-0-Me modified, and at least one nucleotide in the upper stem region is not 2’-0-Me modified; and (b) at least 50% of the nucleotides in the hairpin region are 2’-0-Me modified, and at least one nucleotide in the hairpin region is not 2’-0-Me modified; or (ii) wherein the spacer sequence comprises a 5’ end, and wherein the spacer sequence comprises notmore than onePS bond within the first three seven nucleotides of the 5’ end.
  2. 2. The sgRNA of claim 1, wherein each nucleotide in the upper stem region is modified with 2’-O-Me except the first nucleotide of the upper stem region in the 5’ end to 3’ end direction.
  3. 3. The sgRNA of claim 1, wherein each nucleotide in the upper stem region is modified with 2’ -O-Me except the last nucleotide of the upper stem region in the 5’ end to 3’ end direction.
  4. 4. The sgRNA of claim 1, wherein the upper stem region further comprises nucleotides modified with 2'-deoxy (2'-H), 2-MOE, 2'-F, 2'-NH 2 , 2'-arabinosyl (2-arabino) nucleotide, 2- F-arabinosyl (2’-F-arabino) nucleotide, 2'- locked nucleic acid (LNA) nucleotide, 2'- unlocked nucleic acid (ULNA) nucleotide, a sugar in L form (L-sugar), 4'-thioribosyl nucleotide, or any combination thereof.
  5. 5. The sgRNA of claim 1, wherein at least 50%, 60%, 70%, 80%, or 90%, or 100% of nucleotides in the upper stem region are modified.
  6. 6. The sgRNA of claim 5, wherein at least 82.5% of the nucleotides in the upper stem region are modified.
  7. 7. The sgRNA of claim 1, wherein each nucleotide in the upper stem region is modified with 2’ -O-Me.
  8. 8. The sgRNA of claim 1, wherein the upper stem region comprises a linkage modification.
  9. 9. The sgRNA of claim 8, wherein the linkage modification comprises phosphorothioate, phosphonoacetate, thiophosphonoacetate, methylphosphonoate-P(CH 3 ), boranophosphonate, phosphorodithioate, or any combination thereof.
  10. 10. The sgRNA of claim 1, wherein the spacer sequence comprises not more than one PS bond within the first three nucleotides at the 5' end.
  11. 11 . The sgRNA of any one of claims 1-10, wherein each nucleotide in the hairpin region 2 is modified with 2’-0-Me except the first nucleotide in the 5' end to 3' end direction.
  12. 12. The sgRNA of any one of claims 1-10, wherein the hairpin region 2 further comprises nucleotides modified with 2'-deoxy (2'-H), 2-MOE, 2'-F, 2'-NH 2 , 2'-arabinosyl (2-arabino) nucleotide, 2-F-arabinosyl (2’-F-arabino) nucleotide, 2'- locked nucleic acid (LNA) nucleotide, 2'- unlocked nucleic acid (ULNA) nucleotide, a sugar in L form (L-sugar), 4'- thioribosyl nucleotide, or any combination thereof.
  13. 13. The sgRNA of any one of claims 1-10, wherein at least 50%, 60%, 70%, 80%, or 90%, or 100% of nucleotides in the hairpin region 2 are modified.
  14. 14. The sgRNA of any one of claims 1-10, wherein at least 82.5% of nucleotides in the hairpin region 2 are modified.
  15. 15. The sgRNA of any one of claims 1-10, wherein each nucleotide in the hairpin region 2 is modified with 2’-0-Me.
  16. 16. The sgRNA of claim 1-15, wherein the hairpin region 2 comprises a linkage modification.
  17. 17. The sgRNA of claim 16, wherein the linkage modification comprises phosphorothioate, phosphonoacetate, thiophosphonoacetate, methylphosphonoate-P(CH 3 ), boranophosphonate, phosphorodithioate, or any combination thereof.
  18. 18. The sgRNA of any one of claims 1-17, wherein the 5’ end of the 5’ terminus comprises the PS linkage between the first and second nucleotides.
  19. 19. The sgRNA of any one of claims 1-17, wherein the 5' terminus comprises nucleotides modified with 2'-0-Me, 2'-deoxy (2'-H), 2-MOE, 2'-F, 2'-NH 2 , 2'-arabinosyl (2-arabino) nucleotide, 2-F-arabinosyl (2’-F-arabino) nucleotide, 2'- locked nucleic acid (LNA) nucleotide, 2'- unlocked nucleic acid (ULNA) nucleotide, a sugar in L form (L-sugar), 4'- thioribosyl nucleotide, or any combination thereof.
  20. 20. The sgRNA of any one of claims 1-17, wherein at least 50%, 60%, 70%, 80%, or 90%, or 100% nucleotides of the 5' terminus are modified.

Description

GUIDE RNA COMPOSITIONS CROSS REFERENCE [0001] This application claims the benefit of U.S. Provisional Application No.63/511,624, filed June 30, 2023, of which is incorporated herein by reference in their entirety. BACKGROUND [0002] Oligonucleotides, such as guide RNAs (gRNAs) of CRISPR/Cas system for gene editing, can be subjected to degradation in cells by endonuclease or exonuclease cleavage. There is a need to prevent degradation of gRNAs and improve stability of gRNAs to enhance the gene editing efficiency. SUMMARY [0003] Described herein is a single guide RNA (sgRNA) comprising a tracr sequence and a spacer sequence, (i) wherein the tracr sequence comprises an upper stem, a hairpin region 1, and a hairpin region 2, and wherein (a) at least 50% of the nucleotides in the upper stem loop are 2’- OMe modified, and at least one nucleotide in the upper stem region is not 2’-O-Me modified; and (b) at least 50% of the nucleotides in the hairpin region are 2’-O-Me modified, and at least one nucleotide in the hairpin region is not 2’-O-Me modified; or (ii) wherein the spacer sequence comprises a 5’ end, and wherein the spacer sequence comprises not more than one PS bond within the first three seven nucleotides of the 5’ end. [0004] In some embodiments, each nucleotide in the upper stem region is modified with 2’-O- Me except the first nucleotide of the upper stem region in the 5’ end to 3’ end direction. In some embodiments, each nucleotide in the upper stem region is modified with 2’-O-Me except the last nucleotide of the upper stem region in the 5’ end to 3’ end direction. In some embodiments, the upper stem region further comprises nucleotides modified with 2'-deoxy (2'-H), 2-MOE, 2'-F, 2'- NH2, 2'-arabinosyl (2-arabino) nucleotide, 2-F-arabinosyl (2’-F-arabino) nucleotide, 2'- locked nucleic acid (LNA) nucleotide, 2'- unlocked nucleic acid (ULNA) nucleotide, a sugar in L form (L-sugar), 4'-thioribosyl nucleotide, or any combination thereof. In some embodiments, at least 50%, 60%, 70%, 80%, or 90%, or 100% of nucleotides in the upper stem region are modified. In some embodiments, at least 82.5% of the nucleotides in the upper stem region are modified. In some embodiments, each nucleotide in the upper stem region is modified with 2’-O-Me. [0005] In some embodiments, the upper stem region comprises a linkage modification. In some embodiments, the linkage modification comprises phosphorothioate, phosphonoacetate, thiophosphonoacetate, methylphosphonoate-P(CH3), boranophosphonate, phosphorodithioate, or any combination thereof. [0006] In some embodiments, the spacer sequence comprises not more than one PS bond within the first three nucleotides at the 5' end. [0007] In some embodiments, each nucleotide in the hairpin region 2 is modified with 2’-O-Me except the first nucleotide in the 5' end to 3' end direction. In some embodiments, the hairpin region 2 further comprises nucleotides modified with 2'-deoxy (2'-H), 2-MOE, 2'-F, 2'-NH2, 2'- arabinosyl (2-arabino) nucleotide, 2-F-arabinosyl (2’-F-arabino) nucleotide, 2'- locked nucleic acid (LNA) nucleotide, 2'- unlocked nucleic acid (ULNA) nucleotide, a sugar in L form (L- sugar), 4'-thioribosyl nucleotide, or any combination thereof. In some embodiments, at least 50%, 60%, 70%, 80%, or 90%, or 100% of nucleotides in the hairpin region 2 are modified. In some embodiments, atleast 82.5% of nucleotides in the hairpin region 2 are modified. In some embodiments, each nucleotide in the hairpin region 2 is modified with 2’-O-Me. [0008] In some embodiments, the hairpin region 2 comprises a linkage modification. In some embodiments, the linkage modification comprises phosphorothioate, phosphonoacetate, thiophosphonoacetate, methylphosphonoate-P(CH3), boranophosphonate, phosphorodithioate, or any combination thereof. [0009] In some embodiments, the 5’ end of the 5’ terminus comprises the PS linkage between the first and second nucleotides. In some embodiments, the 5' terminus comprises nucleotides modified with 2'-0-Me, 2'-deoxy (2'-H), 2-MOE, 2'-F, 2'-NH2, 2'-arabinosyl (2-arabino) nucleotide, 2-F-arabinosyl (2 ’-F-arabino) nucleotide, 2'- locked nucleic acid (LNA) nucleotide, 2'- unlocked nucleic acid (ULNA) nucleotide, a sugar in L form (L-sugar), 4'-thioribosyl nucleotide, or any combination thereof. In some embodiments, at least 50%, 60%, 70%, 80%, or 90%, or 100% nucleotides of the 5' terminus are modified. In some embodiments, the 5' end of the 5' terminus comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides modified with 2'-0-Me, 2'- deoxy (2'-H), 2-MOE, 2'-F, 2'-NH2, 2'-arabinosyl (2-arabino) nucleotide, 2-F-arabinosyl (2’-F- arabino) nucleotide, 2'- locked nucleic acid (LNA) nucleotide, 2'- unlocked nucleic acid (ULNA) nucleotide, a sugar in L form (L-sugar), 4 '-thioribosyl nucleotide, or any combination thereof. In some embodiments, nucleotides of the 5' terminus other than the first 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides are not modified. [0010] In som