Claims for Patent: 10,119,143

Summary for Patent: 10,119,143

Title:	Compositions and methods for inhibiting expression of the ALAS1 gene
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.
Inventor(s):	Bettencourt; Brian (Groton, MA), Fitzgerald; Kevin (Brookline, MA), Querbes; William (Cambridge, MA), Desnick; Robert J. (New York, NY), Yasuda; Makiko (New York, NY)
Assignee:	ALNYLAM PHARMACEUTICALS, INC. (Cambridge, MA) ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI (New York, NY)
Application Number:	15/027,176
Patent Claims:	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 ##STR00028## 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 .delta.-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: ##STR00029## or a pharmaceutically acceptable salt thereof, wherein Af, Cf, Gf, Uf=2'F ribonucleosides; Am, Cm, Gm, Um=2'-OMe ribonucleosides; ##STR00030## =phosphorothioate; ##STR00031## =phosphodiester, and wherein ##STR00032## 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: ##STR00033## pharmaceutically acceptable salt thereof, wherein Af, Cf, Gf, Uf=2'F ribonucleosides; Am, Cm, Gm, Um=2'-OMe ribonucleosides; ##STR00034## =phosphorothioate; ##STR00035## =phosphodiester, and wherein ##STR00036## 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: ##STR00037## pharmaceutically acceptable salt thereof, wherein Af, Cf, Gf, Uf=2'F ribonucleosides; Am, Cm, Gm, Um=2'-OMe ribonucleosides; ##STR00038## =phosphorothioate; ##STR00039## =phosphodiester, and wherein ##STR00040## 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 ##STR00041## 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.