Claims for Patent: 10,240,152

Summary for Patent: 10,240,152

Title:	Compositions and methods for inhibiting expression of transthyretin
Abstract:	The invention relates to a double-stranded ribonucleic acid (dsRNA) targeting a transthyretin (TTR) gene, and methods of using the dsRNA to inhibit expression of TTR.
Inventor(s):	Sah; Dinah Wen-Yee (Boston, MA), Hinkle; Gregory (Plymouth, MA), Alvarez; Rene (Boxborough, MA), Milstein; Stuart (Cambridge, MA), Chen; Qingmin (Lincoln, MA)
Assignee:	Alnylam Pharmaceuticals, Inc. (Cambridge, MA)
Application Number:	15/380,571
Patent Claims:	1. A pharmaceutical composition comprising a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of transthyretin (TTR), wherein the dsRNA comprises a sense strand and an antisense strand, wherein the antisense strand is 19, 20, 21, 22, 23, or 24 nucleotides in length and comprises AUGGAAUACUCUUGGUUACNN (SEQ ID NO:450) and the sense strand of the dsRNA is 19, 20, 21, 22, 23, or 24 nucleotides in length and comprises a region complementary to the antisense strand, wherein the dsRNA comprises at least one chemically modified nucleotide and administration of 10 nM of the dsRNA to a HepG2 cell results in at least 80% inhibition of TTR mRNA expression as measured by a real time PCR assay. 2. A pharmaceutical composition comprising a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of transthyretin (TTR), wherein the dsRNA comprises a sense strand and an antisense strand, wherein the antisense strand is 19, 20, 21, 22, 23, or 24 nucleotides in length and comprises AUGGAAUACUCUUGGUUACNN (SEQ ID NO:450) and the sense strand of the dsRNA is 19, 20, 21, 22, 23, or 24 nucleotides in length, and comprises a region complementary to the antisense strand, wherein the dsRNA comprises at least one chemically modified nucleotide and wherein the pharmaceutical composition has an ED50 in humans of less than 0.03 mg dsRNA/kg. 3. A pharmaceutical composition comprising a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of transthyretin (TTR), wherein the dsRNA comprises a sense strand and an antisense strand, wherein the antisense strand comprises AUGGAAUACUCUUGGUUACNN (SEQ ID NO:450) and the sense strand of the dsRNA is 19, 20, 21, 22, 23, or 24 nucleotides in length, and comprises a region complementary to the antisense strand, wherein the dsRNA comprises at least one chemically modified nucleotide and wherein the pharmaceutical composition reduces human serum TTR protein by at least 50%, as compared prior to treatment, at a dose of less than 0.03 mg dsRNA/kg. 4. A pharmaceutical composition comprising a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of transthyretin (TTR), wherein the dsRNA comprises a sense strand and an antisense strand, wherein the antisense strand comprises AUGGAAUACUCUUGGUUACNN (SEQ ID NO:450) and the sense strand of the dsRNA is 19, 20, 21, 22, 23, or 24 nucleotides in length, wherein the dsRNA comprises at least one chemically modified nucleotide and wherein the pharmaceutical composition reduces human serum TTR protein by at least 70%, as compared to prior to treatment, at a dose of 0.3 mg dsRNA/kg. 5. The pharmaceutical composition of claim 1, wherein the at least one chemically modified nucleotide is selected from the group of a 2'-O-methyl modified nucleotide, a nucleotide comprising a 5'-phosphorothioate group. 6. The pharmaceutical composition of claim 1, wherein the nucleotide sequence of the sense strand comprises GUAACCAAGAGUAUUCCAUNN (SEQ ID NO:449). 7. The pharmaceutical composition of claim 1, wherein the nucleotide sequence of the antisense strand comprises AUGGAAUACUCUUGGUUACdTdT (SEQ ID NO:730). 8. The pharmaceutical composition of claim 1, wherein the nucleotide sequence of the sense strand comprises GUAACCAAGAGUAUUCCAUdTdT (SEQ ID NO: 729). 9. The pharmaceutical composition of claim 7, wherein the nucleotide sequence of the sense strand comprises GUAACCAAGAGUAUUCCAUdTdT (SEQ ID NO: 729). 10. The pharmaceutical composition of claim 1, wherein the antisense strand consists of AUGGAAuACUCUUGGUuACdTdT (SEQ ID NO:1010), wherein A is adenosine, C is cytidine, G is guanosine, U is uridine, a is 2'-O-methyladenosine, c is 2'-O-methylcytidine, g is 2'-O-methylguanosine, u is 2'-O-methyluridine and dT is 2'-deoxythymidine. 11. The pharmaceutical composition of claim 1, wherein the sense strand consists of GuAAccAAGAGuAuuccAudTdT (SEQ ID NO: 1009), wherein A is adenosine, C is cytidine, G is guanosine, U is uridine, a is 2'-O-methyladenosine, c is 2'-O-methylcytidine, g is 2'-O-methylguanosine, u is 2'-O-methyluridine and dT is 2'-deoxythymidine. 12. The pharmaceutical composition of claim 1, wherein the sense strand is 21 nucleotides in length. 13. The pharmaceutical composition of claim 2, wherein the sense strand is 21 nucleotides in length. 14. The pharmaceutical composition of claim 3, wherein the sense strand is 21 nucleotides in length. 15. The pharmaceutical composition of claim 4, wherein the sense strand is 21 nucleotides in length. 16. The pharmaceutical composition of claim 1, wherein the antisense strand comprises 4 modified ribonucleotides. 17. The pharmaceutical composition of claim 1, wherein the sense strand comprises 11 modified ribonucleotides. 18. The pharmaceutical composition of claim 1, wherein administration of the dsRNA to a cell results in at least 90% inhibition of TTR mRNA expression as measured by a real time PCR assay, wherein the cell is a HepG2 cell, and wherein the concentration of the dsRNA is 10 nM. 19. The pharmaceutical composition of claim 1, wherein the dsRNA is formulated in a lipid formulation. 20. The pharmaceutical composition of claim 19 wherein the dsRNA is formulated in a LNP formulation. 21. The pharmaceutical composition of claim 20, wherein the dsRNA is formulated in a LNP11 formulation comprising MC3/DSPC/Chol/PEG.sub.2000-C14 in a ratio of about 50/10/38.5/1.5 mol %. 22. The pharmaceutical composition of claim 21, wherein the dsRNA is formulated in a LNP11 formulation comprising MC3/DSPC/Chol/PEG.sub.2000-C14 in a ratio of about 50/10/38.5/1.5 mol % and a lipid:siRNA ratio of about 11:1. 23. A method of inhibiting TTR expression in a mammalian cell, the method comprising: (a) contacting the cell with the pharmaceutical composition of claim 1 and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of a TTR gene, thereby inhibiting expression of the TTR gene in the cell. 24. A method of treating a transthyretin amyloidosis comprising administering to a human subject with transthyretin amyloidosis the pharmaceutical composition of claim 1. 25. The method of claim 24, wherein the dsRNA is administered at not more than a 2 week interval or not more than a 3 week interval or not more than a 4 week interval. 26. The method of claim 24, wherein the dsRNA is administered every 3 to 4 weeks. 27. The method of claim 24, wherein the dsRNA is administered at a dose of 0.3 mg/kg. 28. The method of claim 24, wherein the subject suffers from at least one symptom of TTR amyloidosis selected from the group consisting of polyneuropathy, cardiomyopathy, gastrointestinal dysfunction and weight loss. 29. The method of claim 24, wherein the method reduces serum TTR protein by at least 70% as compared to prior to treatment or reduces serum TTR protein by at least 80% as compared to prior to treatment. 30. A method of treating a subject with hereditary transthyretin mediated amyloidosis comprising administering via intravenous infusion to a human in need of such treatment 0.3 mg dsRNA/kg every three weeks, wherein the dsRNA comprises a sense strand and an antisense strand, the sense strand consisting of the nucleotide sequence GuAAccAAGAGuAuuccAudTdT (SEQ ID NO: 1009) and the antisense strand consisting of the nucleotide sequence AUGGAAuACUCUUGGUuACdTdT (SEQ ID NO: 1010), wherein A is adenosine, C is cytidine, G is guanosine, U is uridine, a is 2'-O-methyladenosine, c is 2'-O-methylcytidine, g is 2'-O-methylguanosine, u is 2'-O-methyluridine and dT is 2'-deoxythymidine, and wherein the dsRNA is in a lipid nanoparticle formulation comprising MC3/DSPC/cholesterol/PEG-DMG at a ratio of about 50/10/38.5/1.5 mol %. 31. A pharmaceutical composition comprising a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of transthyretin (TTR), wherein the dsRNA comprises a sense strand and an antisense strand, wherein the antisense strand consists of the nucleotide sequence AUGGAAuACUCUUGGUuACdTdT (SEQ ID NO:1010), wherein A is adenosine, C is cytidine, G is guanosine, U is uridine, a is 2'-O-methyladenosine, c is 2'-O-methylcytidine, g is 2'-O-methylguanosine, u is 2'-O-methyluridine and dT is 2'-deoxythymidine, and the sense strand consists of GuAAccAAGAGuAuuccAudTdT (SEQ ID NO: 1009), wherein A is adenosine, C is cytidine, G is guanosine, U is uridine, a is 2'-O-methyladenosine, c is 2'-O-methylcytidine, g is 2'-O-methylguanosine, u is 2'-O-methyluridine and dT is 2'-deoxythymidine. 32. The pharmaceutical composition of claim 31, wherein at least one nucleotide comprises a 5'-phosphorothioate group. 33. The pharmaceutical composition of claim 31, wherein administration of the dsRNA to a cell results in at least 90% inhibition of TTR mRNA expression as measured by a real time PCR assay, wherein the cell is a HepG2 cell, and wherein the concentration of the dsRNA is 10 nM. 34. The pharmaceutical composition of claim 32, wherein administration of the dsRNA to a cell results in at least 90% inhibition of TTR mRNA expression as measured by a real time PCR assay, wherein the cell is a HepG2 cell, and wherein the concentration of the dsRNA is 10 nM. 35. The pharmaceutical composition of claim 31, wherein the dsRNA is formulated in a lipid formulation. 36. The pharmaceutical composition of claim 32, wherein the dsRNA is formulated in a lipid formulation. 37. The pharmaceutical composition of claim 33, wherein the dsRNA is formulated in a lipid formulation. 38. The pharmaceutical composition of claim 34, wherein the dsRNA is formulated in a lipid formulation. 39. The pharmaceutical composition of claim 35, wherein the dsRNA is formulated in a LNP formulation. 40. The pharmaceutical composition of claim 36, wherein the dsRNA is formulated in a LNP formulation. 41. The pharmaceutical composition of claim 37, wherein the dsRNA is formulated in a LNP formulation. 42. The pharmaceutical composition of claim 38, wherein the dsRNA is formulated in a LNP formulation. 43. The pharmaceutical composition of claim 39, wherein the dsRNA is formulated in a LNP11 formulation comprising MC3/DSPC/Chol/PEG.sub.2000-C14 in a ratio of about 50/10/38.5/1.5 mol %. 44. The pharmaceutical composition of claim 40, wherein the dsRNA is formulated in a LNP11 formulation comprising MC3/DSPC/Chol/PEG.sub.2000-C14 in a ratio of about 50/10/38.5/1.5 mol %. 45. The pharmaceutical composition of claim 41, wherein the dsRNA is formulated in a LNP11 formulation comprising MC3/DSPC/Chol/PEG.sub.2000-C14 in a ratio of about 50/10/38.5/1.5 mol %. 46. The pharmaceutical composition of claim 42, wherein the dsRNA is formulated in a LNP11 formulation comprising MC3/DSPC/Chol/PEG.sub.2000-C14 in a ratio of about 50/10/38.5/1.5 mol %. 47. A method of inhibiting TTR expression in a mammalian cell, the method comprising: (a) contacting the cell with the pharmaceutical composition of claim 31 and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of a TTR gene, thereby inhibiting expression of the TTR gene in the cell. 48. A method of treating a transthyretin amyloidosis comprising administering to a human subject with transthyretin amyloidosis the pharmaceutical composition of claim 31. 49. The method of claim 48, wherein the dsRNA is administered at not more than a 2 week interval or not more than a 3 week interval or not more than a 4 week interval. 50. The method of claim 48, wherein the dsRNA is administered every 3 to 4 weeks. 51. The method of claim 48, wherein the dsRNA is administered at a dose of 0.3 mg/kg. 52. The method of claim 48, wherein the subject suffers from at least one symptom of TTR amyloidosis selected from the group consisting of polyneuropathy, cardiomyopathy, gastrointestinal dysfunction and weight loss. 53. The method of claim 48, wherein the method reduces serum TTR protein by at least 70% as compared to prior to treatment or by at least 80% as compared to prior to treatment.