RNAi agents for inhibiting expression of PNPLA3, pharmaceutical compositions thereof, and methods of use
Described are RNAi agents, e.g., double stranded RNAi agents, for inhibiting patatin-like phospholipase domain-containing protein 3 (PNPLA3) gene expression. Also disclosed are pharmaceutical compositions that include PNPLA3 RNAi agents and methods of use thereof. The PNPLA3 RNAi agents disclosed herein may be conjugated to targeting ligands to facilitate the delivery to cells, including to hepatocytes. Delivery of the PNPLA3 RNAi agents in vivo provides for inhibition of PNPLA3 gene expression. The RNAi agents can be used in methods of treatment of PNPLA3-related diseases and disorders, including non-alcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), hepatic fibrosis, and alcoholic or non-alcoholic liver diseases, including cirrhosis.
1. An RNAi agent for inhibiting expression of a PNPLA3 gene, comprising:
an antisense strand comprising the nucleotide sequence usUfsasGfuUfaGfgUfgAfaAfaAfgGfuGfsu (SEQ ID NO: 95), wherein nucleotides 1-19 of the antisense strand are complementary to the PNPLA3 mRNA region comprising nucleotides 2180-2198 of SEQ ID NO: 1,
wherein a, g, and u represent 2′-O-methyl adenosine, 2′-O-methyl guanosine, and 2′-O-methyl uridine, respectively; Af, Gf, and Uf represent 2′-fluoro adenosine, 2′-fluoro guanosine, and 2′-fluoro uridine, respectively; and s represents a phosphorothioate linkage; and
a sense strand comprising a nucleotide sequence that is at least partially complementary to the antisense strand.
2. The RNAi agent of claim 1 , wherein at least one nucleotide of the sense strand of the RNAi agent is a modified nucleotide or includes a modified internucleoside linkage.
3. The RNAi agent of claim 1 , wherein all or substantially all of the nucleotides of the sense strand of the RNAi agent are modified nucleotides.
4. The RNAi agent of claim 3 , wherein the modified nucleotide is selected from the group consisting of: 2′-O-methyl nucleotide, 2′-fluoro nucleotide, 2′-deoxy nucleotide, 2′, 3′-seco nucleotide mimic, locked nucleotide, 2′-F-arabino nucleotide, 2′-methoxyethyl nucleotide, abasic nucleotide, ribitol, inverted nucleotide, inverted 2′-O-methyl nucleotide, inverted 2′-deoxy nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, vinyl phosphonate containing nucleotide, cyclopropyl phosphonate containing nucleotide, and 3′-O-methyl nucleotide.
5. The RNAi agent of claim 3 , wherein all or substantially all of the modified nucleotides are 2′-O-methyl nucleotides, 2′-fluoro nucleotides, or combinations thereof.
6. The RNAi agent of claim 1 , wherein the sense strand comprises the nucleotide sequence acaccuuuUfUfCfaccuaacuaa of SEQ ID NO: 136, wherein a, c, and u represent 2′-O-methyl adenosine, 2′-O-methyl cytidine, and 2′-O-methyl uridine, respectively; and Cf and Uf represent 2′-fluoro cytidine, and 2′-fluoro uridine, respectively.
7. The RNAi agent of claim 1 , wherein the RNAi agent is linked to a targeting ligand.
8. The RNAi agent of claim 7 , wherein the targeting ligand comprises N-acetyl-galactosamine.
9. The RNAi agent of claim 7 , wherein the targeting ligand comprises the structure of (NAG37) or (NAG37)s.
10. The RNAi agent of claim 7 , wherein the targeting ligand is linked to the sense strand.
11. The RNAi agent of claim 10 , wherein the targeting ligand is linked to the 5′ terminal end of the sense strand.
12. The RNAi agent of claim 1 , wherein the sense strand is between 18 and 30 nucleotides in length.
13. The RNAi agent of claim 12 , wherein the sense strand is between 18 and 27 nucleotides in length.
14. The RNAi agent of claim 13 , wherein the sense strand is between 18 and 24 nucleotides in length.
15. The RNAi agent of claim 14 , wherein the sense strand is 21 nucleotides in length.
16. The RNAi agent of claim 12 , wherein the RNAi agent has two blunt ends.
17. The RNAi agent of claim 1 , wherein the sense strand comprises one or two terminal caps.
18. The RNAi agent of claim 1 , wherein the sense strand comprises one or two inverted abasic residues.
19. The RNAi agent of claim 1 , wherein the RNAi agent is comprised of a sense strand and an antisense strand that form a duplex of SEQ ID NOs: (95 and 136).
20. The RNAi agent of claim 1 , wherein the sense strand further includes inverted abasic residues at the 3′ terminal end of the nucleotide sequence, at the 5′ end of the nucleotide sequence, or at both.
21. The RNAi agent of claim 1 , wherein the sense strand comprises a modified nucleotide sequence that differs by 0 or 1 nucleotides from the nucleotide sequence acaccuuuUfUfCfaccuaacuaa of SEQ ID NO: 136;
wherein a, c, g, and u represent 2′-O-methyl adenosine, 2′-O-methyl cytidine, 2′-O-methyl guanosine, and 2′-O-methyl uridine, respectively; Af, Cf, Gf, and Uf represent 2′-fluoro adenosine, 2′-fluoro cytidine, 2′-fluoro guanosine, and 2′-fluoro uridine, respectively; s represents a phosphorothioate linkage; and wherein all or substantially all of the nucleotides on the sense strand are modified nucleotides.
22. The RNAi agent of claim 6 , wherein the sense strand further includes inverted abasic residues at the 3′ terminal end of the nucleotide sequence, at the 5′ end of the nucleotide sequence, or at both.
23. The RNAi agent of claim 20 , wherein the sense strand of the RNAi agent is linked to a targeting ligand.
24. The RNAi agent of claim 23 , wherein the targeting ligand has affinity for the asialoglycoprotein receptor.
25. The RNAi agent of claim 24 , wherein the targeting ligand comprises N-acetyl-galactosamine.
26. The RNAi agent of claim 25 , wherein the targeting ligand comprises:
27. The RNAi agent of claim 1 , wherein the antisense strand consists of the modified nucleotide sequence usUfsasGfuUfaGfgUfgAfaAfaAfgGfuGfsu (SEQ ID NO: 95), and the sense strand consists of the modified nucleotide sequence (NAG37)s(invAb)sacaccuuuUfUfCfaccuaacuaas(invAb) (SEQ ID NO: 136);
wherein a, c, g, and u are 2′-O-methyl adenosine, 2′-O-methyl cytidine, 2′-O-methyl guanosine, and 2′-O-methyl uridine, respectively; Af, Cf, Gf, and Uf are 2′-fluoro adenosine, 2′-fluoro cytidine, 2′-fluoro guanosine, and 2′-fluoro uridine, respectively; s is a phosphorothioate linkage; (invAb) is an inverted abasic deoxyribose residue; and (NAG37)s has the following chemical structure:
28. A composition comprising the RNAi agent of claim 1 , wherein the composition further comprises a pharmaceutically acceptable excipient.
29. A method for inhibiting expression of a PNPLA3 gene in a cell, the method comprising introducing into a cell an effective amount of the RNAi agent of claim 1 .
30. The method of claim 29 , wherein the cell is within a subject.
31. The method of claim 30 , wherein the subject is a human subject.
32. The method of claim 29 , wherein the PNPLA3 gene expression is inhibited by at least 30%.
33. The method of claim 29 , wherein the cell is within a human subject and the RNAi agent is administered at a dose of 0.05 mg/kg to 5.0 mg/kg of body weight of the human subject.
34. The method of claim 31 , wherein the RNAi agent is administered in two or more doses.