Fully stabilized asymmetric siRNA

Patent metadata

Field	Value
Publication number	US-12173286-B2
Application number	US-202016927543-A
Country	US
Kind code	B2
Filing date	Jul 13, 2020
Priority date	Apr 3, 2015
Publication date	Dec 24, 2024
Grant date	Dec 24, 2024

Abstract

Provided herein are self-delivering oligonucleotides that are characterized by efficient RISC entry, minimum immune response and off-target effects, efficient cellular uptake without formulation, and efficient and specific tissue distribution.

First claim

The invention claimed is:

1. A double-stranded small interfering RNA (siRNA) molecule comprising an antisense strand and a sense strand, the sense strand having complementarity to the antisense strand and the antisense strand comprising an oligonucleotide comprising a 5′ end, a 3′ end, and complementarity to a target mRNA molecule, wherein:
(1) the oligonucleotide comprises alternating 2′-methoxy-ribonucleotides and 2′-fluoro-ribonucleotides;
(2) the nucleotides at positions 2 and 14 from the 5′ end of the oligonucleotide are 2′-fluoro-ribonucleotides;
(3) the nucleotides are connected to one another by way of phosphodiester or phosphorothioate linkages;
(4) the nucleotides at positions 1-6 from the 3′ end of the oligonucleotide, or positions 1-7 from the 3′ end of the oligonucleotide, are connected to one another by way of phosphorothioate linkages; and
(5) the oligonucleotide comprises from 6-17 phosphorothioated bonds,
wherein the oligonucleotide has a chemical structure represented by formula (Ia), in the 5′-to-3′ direction:
X(-K-B-K-A) j (-S-B-S-A) r -OR  (Ia)

wherein X is

each A is, independently, a 2′-methoxy-ribonucleotide;
each B is, independently, a 2′-fluoro-ribonucleotide;
each K is, independently, a phosphodiester or phosphorothioate linkage;
each S is a phosphorothioate linkage;
R is a capping group;
j is an integer selected from 4, 5, 6, or 7; and
r is an integer selected from 2 and 3; and
wherein the siRNA molecule comprises a single-stranded tail of 5-8 bases.

2. The double-stranded siRNA molecule of claim 1 , wherein
X is

3. The double-stranded siRNA molecule of claim 1 , wherein j is 7.

4. The double-stranded siRNA molecule of claim 2 , wherein j is 7.

5. The double-stranded siRNA molecule of claim 1 , wherein r is 3.

6. The double-stranded siRNA molecule of claim 2 , wherein r is 3.

7. The double-stranded siRNA molecule of claim 3 , wherein r is 3.

8. The double-stranded siRNA molecule of claim 1 , wherein the oligonucleotide has a chemical structure represented by formula X(-K-B-K-A) 7 (-S-B-S-A) 3 -OR, in the 5′-to-3′ direction, and wherein X is

9. The double-stranded siRNA molecule of claim 1 , wherein the target mRNA corresponds to a portion of a mutant Huntingtin allele.

10. The double-stranded siRNA molecule of claim 8 , wherein the target mRNA corresponds to a portion of a mutant Huntingtin allele.

11. The double-stranded siRNA molecule of claim 1 , wherein the antisense strand has a length of from 16 to 30 nucleotides.

12. The double-stranded siRNA molecule of claim 11 , wherein the antisense strand has a length of 19, 20, 21, 22, or 23 nucleotides.

13. The double-stranded siRNA molecule of claim 12 , wherein the antisense strand has a length of 20, 21, or 22 nucleotides.

14. The double-stranded siRNA molecule of claim 13 , wherein the antisense strand has a length of 21 nucleotides.

15. The double-stranded siRNA molecule of claim 11 , wherein the sense strand has a length of 16, 17, 18, 19, 20, or 21 nucleotides.

16. The double-stranded siRNA molecule of claim 15 , wherein the sense strand has a length of 16, 17, or 18 nucleotides.

17. The double-stranded siRNA molecule of claim 16 , wherein the sense strand has a length of 16 nucleotides.

18. The double-stranded siRNA molecule of claim 1 , wherein the double-stranded siRNA molecule comprises a duplex region having a length of from 11 to 16 base pairs.

19. The double-stranded siRNA molecule of claim 18 , wherein the double-stranded siRNA molecule comprises a duplex region having a length of 16 base pairs.

20. The double-stranded siRNA of claim 1 , wherein the 3′ end of the antisense strand comprises the tail.

21. The double-stranded siRNA of claim 20 , wherein the tail is fully phosphorothioated.

22. A double-stranded siRNA molecule comprising an antisense strand and a sense strand, wherein the antisense strand comprises an oligonucleotide comprising a 5′ end, a 3′ end, and complementarity to a target mRNA molecule, wherein:
(1) the sense strand has complementarity to the antisense strand;
(2) the oligonucleotide comprises alternating 2′-methoxy-ribonucleotides and 2′-fluoro-ribonucleotides;
(3) the nucleotides at positions 2 and 14 from the 5′ end of the oligonucleotide are 2′-fluoro-ribonucleotides;
(4) the nucleotides are connected to one another by way of phosphodiester or phosphorothioate linkages;
(5) the nucleotides at positions 1-6 from the 3′ end of the oligonucleotide, or positions 1-7 from the 3′ end of the oligonucleotide, are connected to one another by way of phosphorothioate linkages;
(6) the oligonucleotide comprises from 6-17 phosphorothioated bonds;
(7) each of the antisense strand and the sense strand is, independently, from 16 to 30 nucleotides in length, with the proviso that the antisense strand is 5-8 nucleotides longer than the sense strand; and
(8) the oligonucleotide has a chemical structure represented by formula (Ia), in the 5′-to-3′ direction:
X(-K-B-K-A) j (-S-B-S-A) r -OR  (Ia)

wherein X is

each A is, independently, a 2′-methoxy-ribonucleotide;
each B is, independently, a 2′-fluoro-ribonucleotide;
each K is, independently, a phosphodiester or phosphorothioate linkage;
each S is a phosphorothioate linkage;
R is a capping group;
j is an integer selected from 4, 5, 6, or 7; and
r is an integer selected from 2 and 3.

23. The double-stranded siRNA molecule of claim 22 , wherein X is

j is 7, and r is 3.

24. The double-stranded siRNA molecule of claim 23 , wherein the antisense strand has a length of 19, 20, 21, 22, or 23 nucleotides.

25. The double-stranded siRNA molecule of claim 24 , wherein the antisense strand has a length of 20, 21, or 22 nucleotides.

26. The double-stranded siRNA molecule of claim 24 , wherein the antisense strand has a length of 21 nucleotides.

27. The double-stranded siRNA molecule of claim 23 , wherein the sense strand has a length of 16, 17, 18, 19, 20, or 21 nucleotides.

28. The double-stranded siRNA molecule of claim 27 , wherein the sense strand has a length of 16, 17, or 18 nucleotides.

29. The double-stranded siRNA molecule of claim 28 , wherein the sense strand has a length of 16 nucleotides.

30. The double-stranded siRNA molecule of claim 23 , wherein the double-stranded siRNA molecule comprises a duplex region having a length of from 11 to 16 base pairs.

31. The double-stranded siRNA molecule of claim 30 , wherein the double-stranded siRNA molecule comprises a duplex region having a length of 16

Assignees

• Univ Massachusetts

Inventors

• Khvorova Anastasia
• Aronin Neil
• Alterman Julia
• Hassler Matthew

Classifications

• C12N2310/343

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

• C12N2310/321

  2'-O-R Modification · CPC title

• C12N2320/53

  reducing unwanted side-effects · CPC title

• C12N2320/51

  modulating the chemical stability, e.g. nuclease-resistance · CPC title

• C12N2310/3515

  Lipophilic moiety, e.g. cholesterol · CPC title