RNA INTERFERENCE MEDIATED INHIBITION OF GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACIDS (siNA)

• US 20160264964A1
• Filed: 12/30/2015
• Published: 09/15/2016
• Est. Priority Date: 10/29/2010
• Status: Active Grant

• Current Assignee: Sirna Therapeutics, Inc. (Alnylam Pharmaceuticals Incorporated)
• Original Assignee: Sirna Therapeutics, Inc. (Alnylam Pharmaceuticals Incorporated)
• Inventors: Cancilla, Mark, Cunningham, James John, Flanagan, Michael William, Haringsma, Henry J., Kenski, Denise M., Stanton, Matthew G., Stirdivant, Steven M., Willingham, Aarron T.
• Granted Patent: US 9,970,005 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61K 31/713 Double-stranded nucleic acids or oligonucleotides
• CPC Class Codes: C07H 21/02 with ribosyl as saccharide radical
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/322 2'-R Modification
• CPC Class Codes: C12N 2310/3231 having an additional ring, e.g. LNA, ENA
• CPC Class Codes: C12N 2310/331 Universal or degenerate base
• CPC Class Codes: C12N 2310/344 Position-specific modifications, e.g. on every purine, at the 3'-end
• CPC Class Codes: C12N 2310/346 having a combination of backbone and sugar modifications
• CPC Class Codes: C12N 2310/351 Conjugate
• CPC Class Codes: C12N 2310/3521 Methyl
• CPC Class Codes: C12N 2310/3527 Other alkyl chain
• CPC Class Codes: C12N 2310/3533 Halogen
• CPC Class Codes: C12N 2320/32 Special delivery means, e.g. tissue-specific
• CPC Class Codes: C12N 2320/51 modulating the chemical stability, e.g. nuclease-resistance

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.

Claims

1. A composition comprising:

I) 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);

2. The composition 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 composition 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 composition according to claim 1, wherein X5 is 3.

5. The composition 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 ribonucletide;

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 composition 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 composition 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 composition 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 composition 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 composition according to claim 1, wherein X1 is 2 and X2 is 2.

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

12. The composition according to claim 1, wherein said double-stranded short interfering nucleic acid (siNA) molecule includes one or more universal base substitutions.

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

14. The composition according to claim 1, wherein the nucleotide at position 14 from the 5′

-end of the antisense strand of said double-stranded short interfering nucleic acid (siNA) molecule is a 2′

-deoxy-2′

-fluoro nucleotide.

15. The composition according to claim 1, wherein one or more overhang nucleotides of said double-stranded short interfering nucleic acid (siNA) molecule is a 2′

-O-methyl nucleotide.

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

17. The composition 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.

18. The composition according to claim 1, wherein X5=3;

each X1 and X2=2;

X3=19, and X4=16.

19. 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):

20. A polymer comprising the double-stranded short interfering nucleic acid (siNA) molecule according to claim 19.

21. A compound comprising the double-stranded short interfering nucleic acid (siNA) molecule according to claim 19 covalently attached to a ligand.

22. A lipid nanoparticle composition comprising the double-stranded short interfering nucleic acid (siNA) molecule according to claim 19.

23. The lipid nanoparticle composition according to claim 22, further comprising:

(a) (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine;

(b) cholesterol;

(c) DSPC; and

(d) PEG-DMG.

24. The lipid nanoparticle composition according to claim 23, wherein the (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine, cholesterol, DSPC, and PEG-DMG have a molar ratio of 50:

30;

10;

2 respectively

25. A composition comprising the double-stranded short interfering nucleic acid (siNA) molecule according to claim 19 and a pharmaceutically acceptable carrier or diluent.