Compositions and methods for inhibiting expression of the ALAS1 gene
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.
1. A double-stranded ribonucleic acid (dsRNA) for inhibiting expression of ALAS1, wherein said dsRNA comprises a sense strand and an antisense strand each of which is 15-30 nucleotides in length, the antisense strand comprising a region of complementarity to an ALAS1 RNA transcript, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from SEQ ID NO: 3686, SEQ ID NO: 3688, or SEQ ID NO: 3690, and wherein the region of complementarity is 19 to 24 nucleotides in length.
2. The dsRNA of claim 1 , wherein said dsRNA comprises at least one modified nucleotide.
3. The dsRNA of claim 1 , wherein at least one strand comprises a 3′ overhang of at least 1 nucleotide or 2 nucleotides.
4. The dsRNA of claim 1 , further comprising a ligand.
5. The dsRNA of claim 4 , wherein said ligand is a GalNAc ligand.
6. An isolated cell containing 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 ALAS1 expression in a 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 of treating a disorder related to ALAS1 expression comprising administering to a subject in need of such treatment a therapeutically effective amount of the dsRNA of claim 1 .
10. A method of treating a porphyria comprising administering to a subject in need of such treatment a double-stranded ribonucleic acid (dsRNA), wherein said dsRNA comprises a sense strand and an antisense strand each of which is 15-30 nucleotides in length, the antisense strand comprising a region of complementarity to an ALAS1 RNA transcript, and wherein the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from SEQ ID NO: 3686, SEQ ID NO: 3688, or SEQ ID NO: 3690.
11. The method of claim 10 , wherein the subject is at risk for developing, or is diagnosed with, a porphyria.
12. The method of claim 10 , wherein the dsRNA is administered before, during, or after an acute attack of porphyria, or is administered prophylactically to prevent an acute attack of porphyria.
13. The method of claim 10 , wherein the dsRNA is in the form of a GalNAc conjugate.
14. The method of claim 9 , wherein the dsRNA is administered at a dose of 0.05-50 mg/kg or 0.01-5 mg/kg bodyweight of the subject.
15. The method of claim 10 , wherein the method decreases a level of a porphyrin or a porphyrin precursor in the subject.
16. The method of claim 15 , wherein the porphyrin precursor is δ-aminolevulinic acid (ALA) or porphopilinogen (PBG).
17. The method of claim 10 , wherein the dsRNA has an IC50 in the range of 0.01-1 nM.
18. A vector encoding at least one strand of a dsRNA of claim 1 .
19. An isolated cell comprising the vector of claim 18 .
20. The method of claim 10 , wherein said method
(i) ameliorates a symptom associated with a porphyria,
(ii) inhibits ALAS1 expression in the subject,
(iii) decreases a level of a porphyrin precursor or a porphyrin in the subject,
(iv) decreases frequency of acute attacks of symptoms associated with a porphyria in the subject, or
(v) decreases incidence of acute attacks of symptoms associated with a porphyria in the subject when the subject is exposed to a precipitating factor.
21. The dsRNA of claim 1 , comprising a duplex region that is 15-30, 17-23, 19-21, or 21-23 nucleotide pairs in length.
22. The dsRNA of claim 1 , wherein the dsRNA comprises modifications on the nucleotides and the modifications are selected from the group consisting of LNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-alkyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro, 2′-deoxy, 2′-hydroxyl, and combinations thereof.
23. The dsRNA of claim 22 , wherein the modifications on the nucleotides are 2′-O-methyl, 2′-fluoro or both.
24. The dsRNA of claim 4 , wherein the ligand comprises a carbohydrate ligand.
25. The dsRNA of claim 24 , wherein the ligand is attached via a linker.
26. The dsRNA of claim 25 , wherein the linker is a bivalent or trivalent branched linker.
27. The dsRNA of claim 24 , wherein the ligand is
28. The dsRNA of claim 25 , wherein the ligand and linker are as shown in Formula XXIV:
29. The dsRNA of claim 28 , wherein the ligand is attached to the 3′ end of the sense strand.
30. The method of claim 10 , wherein the porphyria is a hepatic porphyria selected from the group consisting of acute intermittent porphyria (AIP) hereditary coproporphyria (HCP), variegate porphyria (VP), ALA deyhdratase deficiency porphyria (ADP), and hepatoerythropoietic porphyria.
31. The method of claim 10 , wherein the dsRNA or composition comprising dsRNA is administered during a prodrome.
32. The method of claim 10 , wherein the subject has an elevated level of ALA, PBG, or both.
33. The method of claim 32 , wherein the subject suffers from chronic pain.
34. A method of treating a subject with an elevated level of ALA, PBG, or both ALA and PBG, the method comprising administering to the subject a double-stranded ribonucleic acid (dsRNA), wherein said dsRNA comprises a sense strand and an antisense strand each of which is 15-30 nucleotides in length, the antisense strand comprising a region of complementarity to an ALAS1 RNA transcript, and wherein the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from SEQ ID NO: 3686, SEQ ID NO: 3688, or SEQ ID NO: 3690.
35. The method of claim 34 , wherein the method is effective to decrease the level of ALA, PBG, or both ALA and PBG.
36. The dsRNA of claim 24 , wherein the ligand comprises one or more N-acetylgalactosamine (GalNAc) derivatives.
37. The dsRNA of claim 1 , wherein the dsRNA comprises one or more phosphorothioate linkages.
38. The dsRNA of claim 22 , wherein the dsRNA comprises at least 20 modified nucleotides.
39. The dsRNA of claim 38 , wherein the dsRNA comprises nucleotide modifications on the entire length of the sense and antisense strands.
40. The dsRNA of claim 1 , wherein the dsRNA comprises at least one blunt end.
41. The dsRNA of claim 1 , wherein the dsRNA comprises a 3′ overhang of at least one nucleotide on the antisense strand or the sense strand.
42. The dsRNA of claim 41 , wherein the 3′ overhang is two nucleotides in length.
43. The method of claim 41 , wherein the 3′ overhang is present on the 3′ ends of both the antisense strand and the sense strand.
44. The dsRNA of claim 41 , wherein the 3′ overhang is present on the antisense strand.
45. The dsRNA of claim 44 , wherein one or more of the nucleotides in the overhang is a nucleoside thiophosphate.
46. The method of claim 10 , wherein the region of complementarity is 19 to 24 nucleotides in length.
47. The dsRNA of claim 1 , wherein the dsRNA comprises a duplex region that is 17-23 nucleotide pairs in length.
48. The dsRNA of claim 1 , wherein each strand of the dsRNA is 19-24 nucleotides in length.
49. A method of treating a subject having a porphyria, the method comprising administering to the subject a double-stranded ribonucleic acid (dsRNA), wherein said dsRNA comprises a sense strand and an antisense strand each of which are 19-24 nucleotides in length, wherein
(i) the antisense strand comprises at least 15 contiguous nucleotides of SEQ ID NO: 3686, SEQ ID NO: 3688, or SEQ ID NO: 3690,
(ii) the sense strand comprises at least 15 contiguous nucleotides of SEQ ID NO: 3685, SEQ ID NO: 3687, or SEQ ID NO: 3689,
(iii) the dsRNA comprises a ligand attached to the 3′ end of the sense strand and having the structure
(iv) the nucleotides of the dsRNA are modified on the entire length of the sense strand and the antisense strand with 2′-O-methyl and 2′-fluoro modifications, and
(v) the antisense strand comprises a 3′-overhang of two nucleotides.
50. The method of claim 49 , wherein the dsRNA comprises one or more phosphorothioate linkages.
51. The method of claim 49 , wherein the ligand is attached via a linker as shown
in Formula XXIV: