Modified oligonucleotides targeting SNPs

Patent metadata

Field	Value
Publication number	US-12024706-B2
Application number	US-202016988391-A
Country	US
Kind code	B2
Filing date	Aug 7, 2020
Priority date	Aug 9, 2019
Publication date	Jul 2, 2024
Grant date	Jul 2, 2024

Abstract

Novel oligonucleotides that enhance silencing of the expression of a gene containing a single nucleotide polymorphism (SNP) relative to the expression of the corresponding wild-type gene are provided. Methods of using novel oligonucleotides that enhance silencing of the expression of a gene containing a SNP relative to the expression of the corresponding wild-type gene are provided.

First claim

The invention claimed is:

1. An siRNA molecule comprising:
a sense strand having complementarity to a target gene;
and an antisense strand having complementarity to the sense strand,
wherein the antisense strand comprises a nucleic acid comprising:
(a) a 5′ end and a 3′ end;
(b) a seed region that is complementary to a region of a gene comprising an allelic polymorphism;
(c) a single nucleotide polymorphism (SNP) position nucleotide at a position within the seed region, wherein the SNP position nucleotide is complementary to the allelic polymorphism;
(d) a mismatch (MM) position nucleotide that is a mismatch with a nucleotide in the gene; and
(e) at least one sugar-modified nucleotide (X) on either side of the SNP position nucleotide, wherein each X is located within four, three, or two nucleotides from the SNP position nucleotide; or
(f) at least one sugar-modified nucleotide (Y) on either side of the MM position nucleotide, wherein each Y is located within four, three or two nucleotides from the MM position nucleotide.

2. The siRNA molecule of claim 1 , wherein:
the sense strand has a length of from 13 nucleotides or nucleotide analogs to 17 nucleotides or nucleotide analogs.

3. The siRNA molecule of claim 1 , wherein the antisense strand has a length of from 18 nucleotides or nucleotide analogs to 22 nucleotides or nucleotide analogs.

4. The siRNA molecule of claim 1 , wherein the sense strand has a length of 15 nucleotides or nucleotide analogs and the antisense strand has a length of 20 nucleotides or nucleotide analogs.

5. The siRNA molecule of claim 1 , wherein the sense strand has a length of 16 nucleotides or nucleotide analogs and the antisense strand has a length of 20 nucleotides or nucleotide analogs.

6. A branched oligonucleotide comprising two or more siRNA molecules covalently bound to one another, wherein each siRNA molecule is, independently, an siRNA molecule of claim 1 .

7. The branched oligonucleotide of claim 6 , wherein the branched oligonucleotide comprises two siRNA molecules covalently bound to one another.

8. The branched oligonucleotide of claim 6 , wherein the siRNA molecules are covalently bound to one another by way of a linker.

9. A double-stranded nucleic acid comprising:
(a) a first strand of nucleotides comprising:
(i) a 5′ end and a 3′ end;
(ii) a seed region that is complementary to a region of a gene comprising an allelic polymorphism;
(iii) a single nucleotide polymorphism (SNP) position nucleotide at a position within the seed region, wherein the SNP position nucleotide is complementary to the allelic polymorphism;
(iv) a mismatch (MM) position nucleotide that is not complementary to a nucleotide in the gene; and
(v) at least one sugar-modified nucleotide located on either side of the SNP position nucleotide, on either side of the MM position nucleotide, or a combination thereof; wherein each sugar-modified nucleotide is located within four, three, or two nucleotides from the SNP position nucleotide or from the MM position nucleotide, respectively;

(b) a second strand of nucleotides that is complementary to the first strand of nucleotides.

10. The double-stranded nucleic acid of claim 9 , wherein:
the sugar-modified nucleotide comprises a modification selected from the group consisting of 2′-O-methyl(2′-OMe), 2′-fluoro (2′-F), 2′-ribo, 2′-deoxyribo, 2′-F-4′-thioarabino(2′-F-ANA), 2′-O-(2-methoxyethyl) (2′-MOE), 4′-S-RNA, locked nucleic acid (LNA), 4′-S-F-ANA, 2′-O-allyl, 2′-O-ethylamine, 2′-O-cyanoethyl-RNA(CNet-RNA), tricyclo-DNA, cyclohexenyl nucleic acid (CeNA), arabino nucleic acid (ANA), hexitol nucleic acid (HNA), and a combination thereof.

11. The double-stranded nucleic acid of claim 9 , wherein:
the sugar-modified nucleotide is positioned immediately 5′ to the SNP position nucleotide, immediately 3′ to the SNP position nucleotide, or a mixture thereof; or
the sugar-modified nucleotide is positioned immediately 5′ to the MM position nucleotide, immediately 3′ to the MM position nucleotide, or a mixture thereof.

12. The double-stranded nucleic acid of claim 9 , wherein the SNP position nucleotide is present from position 2 to position 6 from the 5′ end of the first strand of nucleotides.

13. The double-stranded nucleic acid of claim 9 , wherein:
the MM position nucleotide is located 2-11 nucleotides from the SNP position nucleotide of the first strand of nucleotides; or
the MM position nucleotide is located 2-6 nucleotides from the SNP position nucleotide of the first strand of nucleotides.

14. The double-stranded nucleic acid of claim 9 , wherein the sugar-modified nucleotides comprise identical nucleotide sugar modifications, different nucleotide sur modifications, or a mixture thereof.

15. The double-stranded nucleic acid of claim 9 , wherein:
the first strand has a length of from 13-17 nucleotides; or
the second strand has a length of from 18-22 nucleotides.

16. The double-stranded nucleic acid of claim 9 , wherein:
the first strand has a length of 15 nucleotides and the second strand has a length of 20 nucleotides; or
the first strand has a length of 16 nucleotides and the second strand has a length of 20 nucleotides.

17. The double-stranded nucleic acid of claim 9 , wherein the first strand has 3-7 more nucleotides than the second strand.

18. A branched oligonucleotide comprising two or more siRNA molecules covalently bound to one another, wherein each siRNA molecule comprises a double-stranded nucleic acid comprising:
(a) a first strand of nucleotides comprising:
(i) a 5′ end, a 3′ end;
(ii) a seed region that is complementary to a region of a gene comprising an allelic polymorphism;
(iii) a single nucleotide polymorphism (SNP) position nucleotide at a position within the seed region, wherein the SNP position nucleotide is complementary to the allelic polymorphism;
(iv) a mismatch (MM) position nucleotide that not complementary to a nucleotide in the gene; and
(v) at least one sur-modified nucleotide located on either side of the SNP position nucleotide, on either side of the MM position nucleotide, or a combination thereof; wherein each sugar-modified nucleotide is located within four, three, or two nucleotides from the SNP position nucleotide or from the MM position nucleotide, respectively;

(b) a second strand of nucleotides that is complementary to the first strand of nucleotides.

19. The branched oligonucleotide of claim 18 , wherein the branched oligonucleotide comprises two siRNA molecules covalently bound to one another.

20. The branched oligonucleotide of claim 18 , wherein the siRNA molecules are covalently bound to one another by way of a linker.

Assignees

• Univ Massachusetts

Inventors

• Khvorova Anastasia
• Alterman Julia
• Conroy Faith
• Pfister Edith
• Aronin Neil
• Yamada Ken

Classifications

• C12N2310/14

  interfering nucleic acids [NA] · CPC title

• C12N2310/52

  branched · CPC title

• C12N2310/343

  having patterns, e.g. ==--==--==-- · CPC title

• C12N2310/3515

  Lipophilic moiety, e.g. cholesterol · CPC title

• C12N2310/346

  having a combination of backbone and sugar modifications · CPC title