Compositions and methods for inhibiting expression of the ALAS1 gene

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 branched DNA (bDNA) assay at 24 hours post-transfection.

38. The method of claim 10 , wherein the subject is at risk for developing, or is diagnosed with, a porphyria.

39. The method of claim 10 , wherein the porphyria is an acute hepatic porphyria.

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

41. The method of claim 10 , wherein the dsRNA is administered prophylactically to prevent an acute attack of porphyria.

42. The method of claim 14 , wherein the level is decreased by at least 40%.

43. The method of claim 10 , wherein the method inhibits ALAS1 expression by at least 40% in the subject.

44. The method of claim 10 , wherein said method decreases frequency of acute attacks of symptoms associated with a porphyria in the subject.

45. The method of claim 10 , wherein said method decreases incidence of acute attacks of symptoms associated with a porphyria in the subject when the subject is exposed to a precipitating factor.

46. The method of claim 17 , wherein the subject suffers from chronic pain.

47. The method of claim 10 , wherein the method decreases or prevents pain, neuropathy, and/or nerve damage.

48. 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 2 , thereby treating the porphyria.

49. 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 3 , thereby treating the porphyria.

50. 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 4 , thereby treating the porphyria.

51. 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 5 , thereby treating the porphyria.

52. A method of treating a porphyria, the method comprising administering to a subject in need of such treatment a therapeutically effective amount of a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of ALAS1, wherein said dsRNA comprises a sense strand comprising SEQ ID NO: 4060 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

thereby treating the porphyria.

53. 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 21 , thereby treating the porphyria.

54. 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 22 , thereby treating the porphyria.

55. The method of claim 48 , wherein the porphyria is an acute hepatic porphyria.

56. The method of claim 49 , wherein the porphyria is an acute hepatic porphyria.

57. The method of claim 50 , wherein the porphyria is an acute hepatic porphyria.

58. The method of claim 51 , wherein the porphyria is an acute hepatic porphyria.

59. The method of claim 52 , wherein the porphyria is an acute hepatic porphyria.

60. The method of claim 53 , wherein the porphyria is an acute hepatic porphyria.

61. The method of claim 54 , wherein the porphyria is an acute hepatic porphyria.

62. A method of treating a porphyria, the method comprising administering to a subject in need of such treatment a therapeutically effective amount of the pharmaceutical composition of claim 7 , thereby treating the porphyria.

63. The method of claim 62 , wherein the porphyria is an acute hepatic porphyria.

64. A method of inhibiting ALAS1 expression in a liver cell, the method comprising:

(a) introducing into the cell the dsRNA of claim 20 , 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.

65. 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 20 , in an amount effective to decrease the level of the porphyrin or the porphyrin precursor in the cell.

66. A method of treating a porphyria, the method comprising administering to a subject in need of such treatment a therapeutically effective amount of the pharmaceutical composition of claim 34 , thereby treating the porphyria.

67. The method of claim 66 , wherein the porphyria is an acute hepatic porphyria.

68. A pharmaceutically acceptable salt of 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

69. The pharmaceutically acceptable salt of claim 68 , wherein the pharmaceutically acceptable salt is a sodium salt.

70. A pharmaceutical composition for inhibiting expression of an ALAS1 gene, the composition comprising the pharmaceutically acceptable salt of claim 68 .

71. A method of treating a porphyria, the method comprising administering to a subject in need of such treatment a therapeutically effective amount of the pharmaceutically acceptable salt of claim 68 , thereby treating the porphyria.

72. The method of claim 71 , wherein the porphyria is an acute hepatic porphyria.

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

74. The method of claim 10 , wherein the dsRNA is administered monthly at a dose of 2.5 mg/kg bodyweight of the subject.

75. The method of claim 74 , wherein the porphyria is an acute hepatic porphyria.

76. The method of claim 52 , wherein the dsRNA is administered monthly at a dose of 0.01 mg/kg to 5 mg/kg or 1 mg/kg to 2.5 mg/kg bodyweight of the subject.

77. The method of claim 52 , wherein the dsRNA is administered monthly at a dose of 2.5 mg/kg bodyweight of the subject.

78. The method of claim 77 , wherein the porphyria is an acute hepatic porphyria.