Compositions and methods for inhibiting expression of the ALAS1 gene

Patent metadata

Field	Value
Publication number	US-10119143-B2
Application number	US-201415027176-A
Country	US
Kind code	B2
Filing date	Oct 3, 2014
Priority date	Oct 4, 2013
Publication date	Nov 6, 2018
Grant date	Nov 6, 2018

Abstract

The invention relates to double-stranded ribonucleic acid (dsRNA) compositions targeting the ALAS1 gene, and methods of using such dsRNA compositions to alter (e.g., inhibit) expression of ALAS1.

First claim

We claim:

1. A double-stranded ribonucleic acid (dsRNA) for inhibiting expression of ALAS1, wherein said dsRNA comprises a sense strand and an antisense strand, the antisense strand comprising a region of complementarity to an ALAS1 RNA transcript,
wherein the sense strand comprises the sequence and all of the modifications of csasgaaaGfaGfuGfuCfuCfaucuuaL96 (SEQ ID NO: 4160), and wherein the antisense strand comprises the sequence and all of the modifications of usAfsAfGfaUfgAfgAfcAfcUfcUfuUfcUfgsgsu (SEQ ID NO: 4161),
wherein c, a, g, u=2′-OMe ribonucleosides; Af, Cf, Gf, Uf=2′F ribonucleosides; s=phosphorothioate, and
wherein

2. The dsRNA of claim 1 , wherein the dsRNA comprises a duplex region which is 21-23 nucleotide pairs in length.

3. The dsRNA of claim 1 , wherein at least one strand comprises a 3′ overhang of at least 2 nucleotides.

4. The dsRNA of claim 1 , wherein each strand is no more than 26 nucleotides in length.

5. The dsRNA of claim 1 , wherein:
(i) the antisense strand consists of the sequence of usAfsAfGfaUfgAfqAfcAfcUfcUfuUfcUfqsqsu (SEQ ID NO: 4161);
(ii) the sense strand consists of the sequence of csasqaaaGfaGfuGfuCfuCfaucuuaL96 (SEQ ID NO: 4160); or
(iii) the sense strand consists of the sequence of csasqaaaGfaGfuGfuCfuCfaucuuaL96 (SEQ ID NO: 4160), and the antisense strand consists of the sequence of usAfsAfGfaUfgAfqAfcAfcUfcUfufcUfqsqsu (SEQ ID NO: 4161).

6. An isolated cell comprising the dsRNA of claim 1 .

7. A pharmaceutical composition for inhibiting expression of an ALAS1 gene, the composition comprising the dsRNA of claim 1 .

8. A method of inhibiting ALAS1expression in a liver cell, the method comprising:
(a) introducing into the cell the dsRNA of claim 1 , and
(b) maintaining the cell of step (a) for a time sufficient to obtain degradation of the mRNA transcript of an ALAS1 gene, thereby inhibiting expression of the ALAS1 gene in the cell.

9. A method for decreasing a level of a porphyrin or a porphyrin precursor in a cell, comprising contacting the cell with the dsRNA of claim 1 , in an amount effective to decrease the level of the porphyrin or the porphyrin precursor in the cell.

10. A method of treating a porphyria, the method comprising administering to a subject in need of such treatment a therapeutically effective amount of the dsRNA of claim 1 , thereby treating the porphyria.

11. The method of claim 10 , wherein the porphyria is acute intermittent porphyria or ALA-dehydratase deficiency porphyria.

12. The method of claim 10 , wherein the dsRNA is administered after an acute attack of porphyria.

13. The method of claim 10 , wherein the dsRNA is administered at a dose of 0.05 mg/kg to 50 mg/kg, 0.01 mg/kg to 5 mg/kg, 1 mg/kg to 2.5 mg/kg bodyweight of the subject, or at a dose of 1 mg/kg, 2.5 mg/kg, or 5 mg/kg bodyweight of the subject.

14. The method of claim 10 , wherein the method decreases a level of a porphyrin or a porphyrin precursor in the subject, wherein the porphyrin precursor is δ-aminolevulinic acid (ALA) or porphopilinogen (PBG).

15. The method of claim 10 , wherein said method ameliorates a symptom associated with an ALAS1 related disorder.

16. The method of claim 10 , wherein the dsRNA is administered weekly, biweekly, or monthly.

17. The method of claim 10 , wherein the subject has an elevated level of ALA and/or PBG.

18. The pharmaceutical composition of claim 7 , comprising about 200 mg/mL of the dsRNA.

19. The pharmaceutical composition of claim 7 , wherein the pharmaceutical composition has a pH of 6.0-7.5.

20. A double-stranded ribonucleic acid (dsRNA) for inhibiting expression of ALAS1, wherein said dsRNA comprises a sense strand comprising SEQ ID NO. 4160 and an antisense strand comprising SEQ ID NO: 4161, the antisense strand comprising a region of complementarity to an ALAS1 RNA transcript,
wherein said dsRNA is in the form of a conjugate having the structure of:

or a pharmaceutically acceptable salt thereof,
wherein Af, Cf, Gf, Uf=2′F ribonucleosides; Am, Cm, Gm, Um=2′-OMe ribonucleosides;

=phosphorothioate;

=phosphodiester, and
wherein

21. The dsRNA of claim 1 wherein the dsRNA comprises a duplex region which is 21 nucleotide pairs in length.

22. The dsRNA of claim 1 , wherein the antisense strand comprises a 3′ overhang of two nucleotides.

23. An isolated cell comprising the dsRNA of claim 2 .

24. An isolated cell comprising the dsRNA of claim 3 .

25. An isolated cell comprising the dsRNA of claim 4 .

26. An isolated cell comprising the dsRNA of claim 5 .

27. An isolated cell comprising the dsRNA of claim 20 .

28. An isolated cell comprising the dsRNA of claim 21 .

29. An isolated cell comprising the dsRNA of claim 22 .

30. A pharmaceutical composition for inhibiting expression of an ALAS1 gene, the composition comprising the dsRNA of claim 2 .

31. A pharmaceutical composition for inhibiting expression of an ALAS1 gene, the composition comprising the dsRNA of claim 3 .

32. A pharmaceutical composition for inhibiting expression of an ALAS1 gene, the composition comprising the dsRNA of claim 4 .

33. A pharmaceutical composition for inhibiting expression of an ALAS1 gene, the composition comprising the dsRNA of claim 5 .

34. A pharmaceutical composition for inhibiting expression of an ALAS1 gene, the composition comprising a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of ALAS1, wherein said dsRNA comprises a sense strand comprising SEQ ID NO: 4160 and an antisense strand comprising SEQ ID NO: 4161, the antisense strand comprising a region of complementarity to an ALAS1 RNA transcript, wherein said dsRNA is in the form of a conjugate having the structure of:

pharmaceutically acceptable salt thereof,
wherein Af, Cf, Gf, Uf=2′F ribonucleosides; Am, Cm, Gm, Um=2′-OMe ribonucleosides;

=phosphorothioate;

=phosphodiester, and
wherein

35. A pharmaceutical composition for inhibiting expression of an ALAS1 gene, the composition comprising the dsRNA of claim 21 .

36. A pharmaceutical composition for inhibiting expression of an ALAS1 gene, the composition comprising the dsRNA of claim 22 .

37. The method of claim 8 , wherein the expression of ALAS1 is inhibited by at least 80% at 10 nM of the dsRNA as measured by b

Assignees

• Alnylam Pharmaceuticals Inc
• Icahn School Med Mount Sinai

Inventors

• Bettencourt Brian
• Fitzgerald Kevin
• Querbes William
• Desnick Robert J
• Yasuda Makiko

Classifications

• A61P43/00

  Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 · CPC title

• A61P7/00

  Drugs for disorders of the blood or the extracellular fluid · CPC title

• A61P3/00

  Drugs for disorders of the metabolism (of the blood or the extracellular fluid A61P7/00) · CPC title

• A61P25/04

  Centrally acting analgesics, e.g. opioids · CPC title

• A61P1/16

  for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics · CPC title