IP Library › Granted Patent US 12,649,921
Granted Patent B2
US 12,649,921 · App. 17/918,090 · Granted Jun 9, 2026

Chemically modified guide RNAs for genome editing with Cas12b

Inventors: Kallanthottathil G. Rajeev (Wayland, MA); Alexandra Chadwick (Somerville, MA); Caroline Reiss (Somerville, MA)
Assignee: Verve Therapeutics, Inc.
C12N15/11C12N9/22C12N15/907C12N2310/20C12N2310/315C12N2310/321C12N2310/331C12N2800/80
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Quick Facts
Patent No.
US 12,649,921
App. No.
17/918,090
Granted
Jun 9, 2026
Kind
B2
Abstract

Provided herein are compositions related to Cas12b guide RNAs. Also provided herein are methods for modifying target polynucleotide sequences and methods of treating or preventing a condition in a subject in need thereof, such as conditions resultant from the expression of ANGPTL3 or mutations thereto.

Claims (41)

1 . A chemically modified single guide RNA for a Cas12b protein, comprising:

(i) a spacer sequence complimentary to a polynucleotide sequence located on a strand of a human ANGPTL3 gene, and wherein the spacer sequence is designed to hybridize to the polynucleotide sequence located on the human ANGPTL3 gene, and

(ii) a scaffold sequence that serves as a binding scaffold for the Cas12b protein,

wherein the spacer sequence comprises about 10 to about 40 nucleotides in length,

wherein the scaffold sequence has a nucleotide base sequence, wherein the nucleotide base sequence has at least 90% identity to positions 1 to 97 of SEQ ID NO: 1,

wherein the scaffold sequence comprises a plurality of different chemical modifications,

wherein the plurality of different chemical modifications are at two or more nucleotide positions selected from positions 1-7, 11, 16-21, 25-31, 39, 42, 45-52, 57-62, 64, 65, and 70-87 as numbered in SEQ ID NO: 1 or a corresponding position or combination of corresponding positions thereof, and

wherein the one or more chemical modifications comprise a 2′-O-methyladenosine, a 2′-O-methylcytidine, a 2′-O-methylguanosine, a 2′-O-methyluridine, a nebularine, a 2′-O-methylnebularine, or a phosphorothioate linkage.

2 . The chemically modified single guide RNA of claim 1 , wherein the plurality of different chemical modifications are at one or more nucleotide positions selected from positions 1-7, 11, 16-21, 25-31, 45-52, 57-62, 64, 65, and 70-87 as numbered in SEQ ID NO: 1 or a corresponding position or combination of corresponding positions thereof.

3 . The chemically modified single guide RNA of claim 1 , wherein the plurality of different chemical modifications are at two or more nucleotide positions selected from positions 1-3, 5, 16, 21, 25-26, 29, 30, 33, 35, 37, 39, 44, 45, 47, 49-51, 62, 63, 65, 67-69, 72, 75, 76, 79, 82-84, 86, 87, 89, 92, and 95 as numbered in SEQ ID NO: 1 or a corresponding position or combination of corresponding positions thereof.

4 . The chemically modified single guide RNA of claim 1 , wherein the plurality of different chemical modifications are at two or more nucleotide positions selected from positions 1-3, 5, 16, 21, 25-26, 29, 30, 44, 45, 49, 50, 62, 63, 67-69, 72, 75, 76, 79, and 82-84 as numbered in SEQ ID NO: 1 or a corresponding position or combination of corresponding positions thereof.

5 . The chemically modified single guide RNA of claim 1 , wherein the plurality of different chemical modifications are at two or more nucleotide positions selected from positions 16, 21, 25, 29, 30, 44, 45, 49, 50, 62, 63, 67-69, 72, 75, 76, 79, and 82-84 as numbered in SEQ ID NO: 1 or a corresponding position or combination of corresponding positions thereof.

6 . The chemically modified single guide RNA of claim 1 , wherein the scaffold sequence comprises the plurality of different chemical modifications

(i) at positions 1-7, 11, 16-21, 25-31, 39, 42, 45-52, 57-62, 64, 65, and 70-87,

(ii) at positions 1-7, 11, 16-21, 25-31, 45-52, 57-62, 64, 65, and 70-87,

(iii) at positions 1-3, 5, 16, 21, 25-26, 29, 30, 44, 45, 49, 50, 62, 63, 67-69, 72, 75, 76, 79, and 82-84, or

(iv) at positions 1-3, 5, 16, 21, 25-26, 29, 30, 44, 45, 49, 50, 62, 63, 67-69, 72, 75, 76, 79, and 82-84,

as numbered in SEQ ID NO: 1 or a corresponding position or combination of corresponding positions thereof.

7 . The chemically modified single guide RNA of claim 1 , wherein the chemically modified single guide RNA comprises one or more unmodified nucleotides.

8 . The chemically modified single guide RNA of claim 7 , wherein the one or more unmodified nucleotides are at one or more nucleotide positions selected from positions 4, 6-15, 17-20, 22-24, 27, 28, 31-43, 46-48, 51-61, 64-66, 70, 71, 73, 74, 77, 78, 80, 81, and 85-116 as numbered in SEQ ID NO: 1 or a corresponding position or combination of corresponding positions thereof.

9 . The chemically modified single guide RNA of claim 7 , wherein the one or more unmodified nucleotides comprise a 2′-hydroxyl group,

wherein the 2′-hydroxyl group is in a region of the scaffold sequence that serves as a binding scaffold for the Cas12b protein, and/or

wherein the 2′-hydroxyl group is in close proximity to the Cas12b protein when the Cas12b protein is bound to the scaffold sequence.

10 . The chemically modified single guide RNA of claim 1 , wherein the one or more chemical modifications comprise the phosphorothioate linkage, and wherein the chemically modified single guide RNA comprises two and no more than two contiguous phosphorothioate linkages at the 5′ end and/or at the 3′ end of the chemically modified single guide RNA.

11 . The chemically modified single guide RNA of claim 1 , wherein the chemically modified single guide RNA comprises the phosphorothioate linkage, and wherein the chemically modified single guide RNA comprises three and no more than three contiguous phosphorothioate linkages at the 5′ end and/or the 3′ end of the chemically modified single guide RNA.

12 . The chemically modified single guide RNA of claim 1 , wherein the chemically modified single guide RNA comprises a sequence of 5′-NsNsN-3′, 5′-NsNsNsN-3′, 5′-nsnsnsn-3′, or 5′-nsnsn-3′ at the 3′ end of the chemically modified single guide RNA, wherein N independently is an unmodified nucleotide adenosine, cytidine, guanosine or uridine; and n independently is a modified nucleotide adenosine, cytidine, guanosine or uridine, wherein the modification of the nucleotide is a 2′-H, 2′-OMe or nucleobase modification; and each s is a phosphorothioate linkage.

13 . The chemically modified single guide RNA of claim 1 , wherein the chemically modified single guide RNA comprises a sequence of 5′-UsUsU-3′, 5′-UsUsUsU-3′, 5′-usususu-3′, or 5′-ususu-3′ at the 3′ end of the chemically modified single guide RNA, wherein U is an unmodified uridine; and u is a modified uridine, wherein the modification of the uridine is a 2′-H, 2′-OMe or nucleobase modification; and each s is a phosphorothioate linkage.

14 . The chemically modified single guide RNA of claim 1 , wherein each one of the last four nucleotides at the 3′ end of the chemically modified single guide RNA comprises a 2′-OMe modification.

15 . The chemically modified single guide RNA of claim 1 , wherein the human ANGPTL3 gene comprises a sequence of SEQ ID NO: 108.

16 . The chemically modified single guide RNA of claim 1 , wherein the human ANGPTL3 gene comprises a protospacer sequence of any one of SEQ ID NOs: 90-101.

17 . The chemically modified single guide RNA of claim 1 , wherein the chemically modified single guide RNA comprises a sequence with at least 90% 18 identity to a sequence selected from any one of SEQ ID NOs: 2-89.

18 . The chemically modified single guide RNA of claim 1 , wherein the chemically modified single guide RNA comprises a nucleotide sequence of gsususCuGUCUUIUUGGUcAGGAcAACcgUCuaGCUAUAAGUGCUGcaGGGugUGA GAAACUCCuaUUGcugGAcGAugUCuCUuacGAGGCAUUAGCACAACCAACAGC AUAGUCAAAusasasa, wherein uppercase A, U, G, and C represent unmodified adenosine, uridine, guanosine and cytidine respectively, lowercase a, u, g, and c represent 2′-O-methyl modified adenosine, uridine, guanosine and cytidine respectively, and the lowercase s represents a phosphorothioate linkage.

19 . The chemically modified single guide RNA of claim 1 , wherein the chemically modified single guide RNA is about 90 to about 135 nucleotides in length.

20 . The chemically modified single guide RNA of claim 1 , wherein the chemically modified single guide RNA comprises a nucleotide sequence of gsususCuGUCUUUUGGUcAGGAcAACcgUCuaGCuAuAaGuGCUGcaGgGuguGAGA AACUCCuaUuGcugGAcGAugUCuCUuacGagGcAUuAGcACAACCAACAGCAUAG UCAAAusasasa, wherein uppercase A, U, G, and C represent unmodified adenosine, uridine, guanosine and cytidine respectively, lowercase a, u, g, and c represent 2′-O-methyl modified adenosine, uridine, guanosine and cytidine respectively, and the lowercase s represents a phosphorothioate linkage.

21 . The chemically modified single guide RNA of claim 1 , wherein the chemically modified single guide RNA comprises a nucleotide sequence of gsususcuguCUUuUGGUcaggacAACcgucuagCUAUAAGuGCuGCagggugugAGAAacuc cuAuuGCUGgacgaugucucuuacgagGCAUUAGCACAACCAACAGCAUAGUCAAAusa sasa, wherein uppercase A, U, G, and C represent unmodified adenosine, uridine, guanosine and cytidine respectively, lowercase a, u, g, and c represent 2′-O-methyl modified adenosine, uridine, guanosine and cytidine respectively, and the lowercase s represents a phosphorothioate linkage.

22 . The chemically modified single guide RNA of claim 1 , wherein the chemically modified single guide RNA comprises a nucleotide sequence of gsususcuguCUUuUGGUcaggacAACcgucuagCUAUAAGUGCUGCagggugugAGAAacu ccuAuuGCUGgacgaugucucuuacgagGCAUUAGCACAACCAACAGCAUAGUCAAAus asasa, wherein uppercase A, U, G, and C represent unmodified adenosine, uridine, guanosine and cytidine respectively, lowercase a, u, g, and c represent 2′-O-methyl modified adenosine, uridine, guanosine and cytidine respectively, and the lowercase s represents a phosphorothioate linkage.

23 . The chemically modified single guide RNA of claim 1 , wherein the chemically modified single guide RNA comprises a nucleotide sequence of gsususCuGUCUUUUGGUcAGGAcAACcgUCuaGCUAUAAGUGCUGcaGGGugUGA GAAACUCCuaUUGcugGAcGAugUCuCUuacGAGGCAUUAGCACAACCAACAGC AuAGUcAAAusasasa, wherein uppercase A, U, G, and C represent unmodified adenosine, uridine, guanosine and cytidine respectively, lowercase a, u, g, and c represent 2′-O-methyl modified adenosine, uridine, guanosine and cytidine respectively, and the lowercase s represents a phosphorothioate linkage.

24 . The chemically modified single guide RNA of claim 1 , wherein the chemically modified single guide RNA comprises a nucleotide sequence of gsususCuGUCUUUUGGUcAGGAcAACcgUCuaGCuAuAaGuGCUGcaGgGuguGAGA AACUCCuaUuGcugGAcGAugUCuCUuacGagGcAUuAGcACAACCAACAGCAuAGU cAAAusasasa, wherein uppercase A, U, G, and C represent unmodified adenosine, uridine, guanosine and cytidine respectively, lowercase a, u, g, and c represent 2′-O-methyl modified adenosine, uridine, guanosine and cytidine respectively, and the lowercase s represents a phosphorothioate linkage.

25 . The chemically modified single guide RNA of claim 1 , wherein the chemically modified single guide RNA comprises a nucleotide sequence of gsususCUGUCUUUUGGUCAGGACAACcgucuagCUAUAAGuGCuGCagggugugAGA AacuccuAuuGCUGgacgaugucucuuacgagGCAUUAGCACAACCAACAGCAUAGUCA AAusasasa, wherein uppercase A, U, G, and C represent unmodified adenosine, uridine, guanosine and cytidine respectively, lowercase a, u, g, and c represent 2′-O-methyl modified adenosine, uridine, guanosine and cytidine respectively, and the lowercase s represents a phosphorothioate linkage.

26 . The chemically modified single guide RNA of claim 1 , wherein the chemically modified single guide RNA comprises a nucleotide sequence of gsususcuguCUUuUGGUcaggacAACcgucuagCUAUAAGuGCuGCAGGGUGUGAGAA ACUCCUAuuGCUGgacgaugucucuuacgagGCAUUAGCACAACCAACAGCAUAGUC AAAusasasa, wherein uppercase A, U, G, and C represent unmodified adenosine, uridine, guanosine and cytidine respectively, lowercase a, u, g, and c represent 2′-O-methyl modified adenosine, uridine, guanosine and cytidine respectively, and the lowercase s represents a phosphorothioate linkage.

27 . The chemically modified single guide RNA of claim 1 , wherein the chemically modified single guide RNA comprises a nucleotide sequence of gsususcuguCUUuUGGUcaggacAACcgucuagCUAUAAGuGCuGCagggugugAGAAacuc cuAuuGCUGgacgaugucucuuacgagGCAUUAGCACaACCAAcaGCAuaGUCAAAusasas a, wherein uppercase A, U, G, and C represent unmodified adenosine, uridine, guanosine and cytidine respectively, lowercase a, u, g, and c represent 2′-O-methyl modified adenosine, uridine, guanosine and cytidine respectively, and the lowercase s represents a phosphorothioate linkage.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Oct 11, 2022
From: RAJEEV, KALLANTHOTTATHIL G.; CHADWICK, ALEXANDRA; REISS, CAROLINE
To: VERVE THERAPEUTICS, INC.
Reel/Frame 061381/0107 →
Continuity (2)
Provisional Application 63007814 · Apr 9, 2020
Related Publication 20230134582A1 · May 4, 2023
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