RNA interference mediated inhibition of gene expression using short interfering nucleic acids (siNA)

Patent metadata

Field	Value
Publication number	US-9970005-B2
Application number	US-201514984065-A
Country	US
Kind code	B2
Filing date	Dec 30, 2015
Priority date	Oct 29, 2010
Publication date	May 15, 2018
Grant date	May 15, 2018

Abstract

The present invention relates to compounds, compositions, and methods for the study, diagnosis, and treatment of traits, diseases and conditions that respond to the modulation of gene expression and/or activity, and/or modulate a gene expression pathway. Specifically, the invention relates to double-stranded nucleic acid molecules including small nucleic acid molecules, such as short interfering nucleic acid (siNA) molecules that are capable of mediating or that mediate RNA interference (RNAi) against target gene expression.

First claim

What we claim is:

1. A compound comprising:
I) a double-stranded short interfering nucleic acid (siNA) molecule that inhibits the expression of a target gene via RNA interference, comprising a sense strand and an antisense strand, said siNA molecule comprising formula (A):

wherein, the upper strand is the sense strand and the lower strand is the antisense strand of the siNA molecule; wherein the antisense strand comprises a sequence having at least 15 nucleotides that are complementary to a target RNA sequence encoded by the target gene and the sense strand comprises a sequence that is complementary to the antisense strand; each N is independently a nucleotide which is unmodified or chemically modified, or is a non-nucleotide; each B is independently a terminal cap that is present or absent; (N) represents overhanging nucleotides, each of which is independently unmodified or chemically modified; [N] represents nucleotides at the 5′-terminus of the antisense strand; X1 and X2 are independently integers from 0 to 4; X3 is an integer from 15 to 30; X4 is an integer from 12 to 27; and X5 is an integer from 1-6, provided that the sum of X4 and X5 is an integer from 15-30; and wherein
(a) five or more pyrimidine nucleotides in N X4 positions are a combination of two or more of independently 2′-deoxy-2′-fluoro nucleotides, 2′-O-alkyl nucleotides, 2′deoxy nucleotides, ribonucleotides, or any combination thereof;
(b) five or more purine nucleotides in N X4 positions are independently 2′-deoxy-2′-fluoro nucleotides, 2′-O-alkyl nucleotides, 2′deoxy nucleotides, ribonucleotides, or any combination thereof;
(c) five or more pyrimidine nucleotides in N X3 positions are independently 2′-deoxy-2′-fluoro nucleotides, 2′-O-alkyl nucleotides, 2′deoxy nucleotides, ribonucleotides, or any combination thereof;
(d) five or more purine nucleotides in N X3 positions are independently 2′-deoxy-2′-fluoro nucleotides, 2′-O-alkyl nucleotides, 2′deoxy nucleotides, ribonucleotides;
(e) [N] position nucleotide(s) are ribonucleotides, deoxyribonucleotides, 2′-O-alkyl nucleotides, or 2′-halo nucleotides, or any combination thereof irrespective of purine or pyrimidine content; and
(f) the nucleotide at position 14 from the 5′-end of the antisense strand is a 2′-deoxy-2′-fluoro nucleotide regardless of whether it is a purine or pyrimidine;
and
II) a ligand.

2. The compound according to claim 1 , wherein:
(a) five or more pyrimidine nucleotides in N X4 positions are 2′-O-methyl nucleotides;
(b) five or more purine nucleotides in N X4 positions are 2′-deoxy-2′-fluoro nucleotides;
(c) five or more pyrimidine nucleotides in N X3 positions are 2′-O-methyl nucleotides;
(d) five or more purine nucleotides in N X3 positions are 2′-deoxy-2′-fluoro nucleotides; and
(e) [N] position nucleotide(s) are any combination of ribonucleotides, deoxyribonucleotides, 2′-O-alkyl nucleotides, or 2′-halo nucleotides.

3. The compound according to claim 1 , wherein:
(a) 5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides in N X4 positions are 2′-O-methyl nucleotides;
(b) 5, 6, 7, 8, 9, 10 or more purine nucleotides in N X4 positions are 2′-deoxy-2′-fluoro nucleotides;
(c) 5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides in N X3 positions are 2′-O-methyl nucleotides;
(d) 5, 6, 7, 8, 9, 10 or more purine nucleotides in N X3 positions are 2′-deoxy-2′-fluoro nucleotides; and
(e) [N] position nucleotide(s) are any combination of ribonucleotides, deoxyribonucleotides, 2′-O-alkyl nucleotides, or 2′-halo nucleotides.

4. The compound according to claim 1 , wherein X5 is 3.

5. The compound according to claim 4 , wherein the three [N] nucleotides of formula (A) are represented as 5′-[N1, N2, N3]-3′, wherein:
a. each N1, N2, and N3 is a ribonucleotide; or
b. each N1, N2, and N3 is a 2′-deoxy-2′-fluoro nucleotide; or
c. each N1, N2, and N3 is a 2′-deoxy nucleotide; or
d. each N1, N2, and N3 is a 2′-O-alkyl nucleotide; and
e. any of N1, N2, or N3 optionally comprises a phosphorothioate internucleotide linkage.

6. The compound according to claim 4 , wherein the three [N] nucleotides of formula (A) are represented as 5′-[N1, N2, N3]-3′, wherein:
a. N1 is a 2′-deoxy-2′-fluoro nucleotide, N2 is 2′-deoxy-2′-fluoro nucleotide, and N3 is a 2′-deoxynucleotide; and
b. any of N1, N2, or N3 optionally comprises a phosphorothioate internucleotide linkage.

7. The compound according to claim 4 , wherein the three [N] nucleotides of formula (A) are represented as 5′-[N1, N2, N3]-3′, wherein:
a. N1 is a 2′-deoxy nucleotide, N2 is 2′-deoxy-2′-fluoro nucleotide, and N3 is a 2′-O-methyl nucleotide; and
b. any of N1, N2, or N3 optionally comprises a phosphorothioate internucleotide linkage.

8. The compound according to claim 4 , wherein the three [N] nucleotides of formula (A) are represented as 5′-[N1, N2, N3]-3′, wherein:
a. N1 is a 2′-deoxy nucleotide, N2 is 2′-deoxy-2′-fluoro nucleotide, and N3 is a 2′-deoxy nucleotide; and
b. any of N1, N2, or N3 optionally comprises a phosphorothioate internucleotide linkage.

9. The compound according to claim 4 , wherein the three [N] nucleotides of formula (A) are represented as 5′-[N1, N2, N3]-3′, wherein:
a. N1 is a 2′-deoxy-2′-fluoro nucleotide, N2 is 2′-deoxy-2′-fluoro nucleotide, and N3 is a 2′-deoxy-2′-fluoro nucleotide; and
b. any of N1, N2, or N3 optionally comprises a phosphorothioate internucleotide linkage.

10. The compound according to claim 1 , wherein X1 is 2.

11. The compound according to claim 1 , wherein X5 is 3, X1 is 2 and X2 is 2.

12. The compound according to claim 1 , wherein said siNA molecule comprises one or more universal base substitutions.

13. The compound according to claim 1 , wherein said double-stranded short interfering nucleic acid (siNA) molecule includes one or more locked nucleic acid (LNA) substitutions.

14. The compound according to claim 1 , wherein one or more overhanging nucleotides of said siNA molecule is a 2′-O-methyl nucleotide.

15. The compound according to claim 1 , wherein said double-stranded short interfering nucleic acid (siNA) molecule comprises at least one phosphorothioate internucleotide linkage.

16. The compound according to claim 1 , wherein X5=3; each X1 and X2=1 or 2; X3=18, 19, 20, 21, 22, or 24, and X4=17, 18, 19, 20, 21, 22, or 23.

17. The compound according to claim 1 , wherein X5=3; each X1 and X2=2; X3=19, and X4=16.

18. A double-stranded short interfering nucleic acid (siNA) molecule that inhibits the expression of a target gene via RNA interference, having a sense strand and an antisense strand and comprising formula (A):

wherein, the upper strand is the sense strand and the lower strand is the antisense strand of the siNA molecule; wherein the antisense strand comprises a sequence having at least 15 nucleotides that are complementary to a target RNA sequence encoded by the target gene and the sense strand comprises a sequence that is complementary to the antisense strand; each N is independently a nucleotide which is unmodified or chemically modified, or is a non-nucleotide; each B is independently a terminal cap that is present or absent; (N) represents overhanging nucleotides, each of which is independently unmodified or chemically modified; [N] represents nucleotides at the 5′-terminus of the antisense strand;

Assignees

• Sirna Therapeutics Inc

Inventors

• Cancilla Mark
• Cunningham James John
• Flanagan William Michael
• Haringsma Henry J
• Kenski Denise M
• Stanton Matthew G
• Stirdivant Steven M
• Willingham Aarron T

Classifications

• C12N2310/321

  2'-O-R Modification · CPC title

• C12N2310/346

  having a combination of backbone and sugar modifications · CPC title

• C12N2320/51

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

• C12N2310/3527

  Other alkyl chain · CPC title

• C12N2310/3533

  Halogen · CPC title