COMPOSITIONS AND METHODS FOR INHIBITING EXPRESSION OF THE ALAS1 GENE

• US 20180037886A1
• Filed: 03/13/2017
• Published: 02/08/2018
• Est. Priority Date: 04/10/2012
• Status: Active Grant

• Current Assignee: Alnylam Pharmaceuticals Incorporated, Icahn School of Medicine at Mount Sinai
• Original Assignee: Alnylam Pharmaceuticals Incorporated, Icahn School of Medicine at Mount Sinai
• Inventors: Bettencourt, Brian, Fitzgerald, Kevin, Querbes, William, Desnick, Robert J., Yasuda, Makiko
• Granted Patent: US 10,400,239 B2
• Time in Patent Office: Days
• CPC Class Codes: A61K 31/713 Double-stranded nucleic acids or oligonucleotides
• CPC Class Codes: A61K 47/60 the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/04 Centrally acting analgesics, e.g. opioids
• CPC Class Codes: A61P 25/20 Hypnotics; Sedatives
• CPC Class Codes: A61P 25/22 Anxiolytics
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 29/02 without antiinflammatory effect
• CPC Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/00 Drugs for disorders of the blood or the extracellular fluid
• 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 15/1137 against enzymes viral enzymes C12N15/1131; receptors C12N15/1138
• 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 2310/343 : having patterns, e.g. ==--==--==--
• CPC Class Codes: C12N 2310/344 : Position-specific modifications, e.g. on every purine, at the 3'-end
• CPC Class Codes: C12N 2310/351 : Conjugate
• CPC Class Codes: C12N 2310/3521 : Methyl
• CPC Class Codes: C12N 2310/3533 : Halogen
• CPC Class Codes: C12N 2310/50 : Physical structure
• CPC Class Codes: C12N 2310/533 : having a mismatch or nick in at least one of the strands
• CPC Class Codes: C12N 2320/30 : Special therapeutic applications
• CPC Class Codes: C12Y 203/01037 : 5-Aminolevulinate synthase (2.3.1.37)

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.

Claims

1. (canceled)

2. 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.

3. The dsRNA of claim 2, wherein said dsRNA comprises at least one modified nucleotide.

7. The dsRNA of claim 2, wherein the region of complementarity is 19 to 24 nucleotides in length.

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

overhang of at least 1 nucleotide or 2 nucleotides.

12. The dsRNA of claim 2, further comprising a ligand.

13. The dsRNA of claim 12, wherein said ligand is a GalNAc ligand.

18. A cell containing the dsRNA of claim 2.

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

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

(a) introducing into the cell the dsRNA of claim 2, 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.

42. 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 2.

51. The method of claim 42, wherein the dsRNA is administered at a dose of 0.05-50 mg/kg or 0.01-5 mg/kg bodyweight of the subject.

62. A vector encoding at least one strand of a dsRNA of claim 2.

66. A cell comprising the vector of claim 62.

85. The dsRNA of claim 2, wherein the duplex region is 15-30, 17-23, 19-21, or 21-23 nucleotide pairs in length.

4-6. -6. (canceled)

8-9. -9. (canceled)

11. (canceled)

14-17. -17. (canceled)

20-31. -31. (canceled)

33-41. -41. (canceled)

43. 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.

44. The method of claim 43, wherein the subject is at risk for developing, or is diagnosed with, a porphyria.

46. The method of claim 43, 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.

50. The method of claim 43, wherein the dsRNA is in the form of a GalNAc conjugate.

55. The method of claim 43, wherein the method decreases a level of a porphyrin or a porphyrin precursor in the subject.

57. The method of claim 55, wherein the porphyrin precursor is δ

-aminolevulinic acid (ALA) or porphopilinogen (PBG).

58. The method of claim 43, wherein the dsRNA has an IC50 in the range of 0.01-1 nM.

73. The method of claim 43, 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.

89. The method of claim 43, 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.

90. The method of claim 89, wherein the modifications on the nucleotides are 2′

-O-methyl, 2′

-fluoro or both.

91. The method of claim 43, wherein the ligand comprises a carbohydrate ligand.

92. The method of claim 91, wherein the ligand is attached via a linker.

93. The method of claim 92, wherein the linker is a bivalent or trivalent branched linker.

94. The method of claim 91, wherein the ligand is

95. The method of claim 92, wherein the ligand and linker are as shown in Formula XXIV:

96. The method of claim 95, wherein the ligand is attached to the 3′

end of the sense strand.

112. The method of claim 43, 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.

118. The method of claim 43, wherein the dsRNA or composition comprising dsRNA is administered during a prodrome.

124. The method of claim 43, wherein the subject has an elevated level of ALA, PBG, or both.

126. The method of claim 124, wherein the subject suffers from chronic pain.

155. The method of claim 91, wherein the ligand comprises one or more N-acetylgalactosamine (GalNAc) derivatives.

156. The method of claim 43, wherein the dsRNA comprises one or more phosphorothioate linkages.

157. The method of claim 89, wherein the dsRNA comprises at least 20 modified nucleotides.

158. The method of claim 157, wherein the dsRNA comprises nucleotide modifications on the entire length of the sense and antisense strands.

159. The method of claim 43, wherein the dsRNA comprises at least one blunt end.

160. The method of claim 43, wherein the dsRNA comprises a 3′

overhang of at least one nucleotide on the antisense strand or the sense strand.

161. The method of claim 160, wherein the 3′

overhang is two nucleotides in length.

162. The method of claim 160, wherein the 3′

overhang is present on the 3′

ends of both the antisense strand and the sense strand.

163. The method of claim 160, wherein the 3′

overhang is present on the antisense strand.

164. The method of claim 163, wherein one or more of the nucleotides in the overhang is a nucleoside thiophosphate.

165. The method of claim 43, wherein the region of complementarity is 19 to 24 nucleotides in length.

166. The method of claim 43, wherein the dsRNA comprises a duplex region that is 17-23 nucleotide pairs in length.

167. The method of claim 43, wherein each strand of the dsRNA is 19-24 nucleotides in length.

45. (canceled)

47-49. -49. (canceled)

52-54. -54. (canceled)

56. (canceled)

59-61. -61. (canceled)

63-65. -65. (canceled)

67-72. -72. (canceled)

74-84. -84. (canceled)

86-88. -88. (canceled)

97-111. -111. (canceled)

113-117. -117. (canceled)

119-123. -123. (canceled)

125. (canceled)

127-150. -150. (canceled)

151. 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.

153. The method of claim 151, wherein the method is effective to decrease the level of ALA, PBG, or both ALA and PBG.

152. (canceled)

154. (canceled)

168. 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

169. The method of claim 168, wherein the dsRNA comprises one or more phosphorothioate linkages.

170. The method of claim 168, wherein the ligand is attached via a linker as shown in Formula XXIV: