Patatin-like phospholipase domain containing 3 (PNPLA3) iRNA compositions and methods of use thereof
The present invention relates to RNAi agents, e.g., double stranded RNA (dsRNA) agents, targeting the Patatin-Like Phospholipase Domain Containing 3 (PNPLA3) gene. The invention also relates to methods of using such RNAi agents to inhibit expression of a PNPLA3 gene and to methods of preventing and treating an PNPLA3-associated disorder, e.g., Nonalcoholic Fatty Liver Disease (NAFLD).
1. A double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of Patatin-Like Phospholipase Domain Containing 3 (PNPLA3) in a cell, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of nucleotides 1214-1234 of SEQ ID NO:1, and the antisense strand comprises at least 15 contiguous nucleotides from the nucleotide sequence of nucleotides 1572-1592 of SEQ ID NO:2, wherein all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise a modification selected from the group consisting of a 2′-O-methyl modification and a 2′-fluoro modification, wherein both the sense strand and the antisense strand independently comprise at least one phosphorothioate or methylphosphonate internucleotide linkage, and wherein at least one strand is conjugated to a ligand.
2. The dsRNA agent of claim 1 , wherein the sense strand comprises 2-6 2′-fluoro modified nucleotides.
3. The dsRNA agent of claim 2 , wherein the sense strand comprises 4 2′-fluoro modified nucleotides.
4. The dsRNA agent of claim 1 , wherein the antisense strand comprises 2-8 2′-fluoro modified nucleotides.
5. The dsRNA agent of claim 4 , wherein the antisense strand comprises 6 2′-fluoro modified nucleotides.
6. The dsRNA agent of claim 1 , wherein the sense strand comprises 4 2′-fluoro modified nucleotides at nucleotides 7 and 9-11, counting from the 5′-end, and the antisense strand comprises 6 2′-fluoro modified nucleotides at nucleotides 2, 6, 8, 9, 14 and 16, counting from the 5′-end.
7. The dsRNA agent of claim 1 , wherein the sense strand comprises two phosphorothioate or methylphosphonate internucleotide linkages at the 5′-terminus.
8. The dsRNA agent of claim 1 , wherein the antisense strand comprises two phosphorothioate or methylphosphonate internucleotide linkages at both the 5′- and the 3′-terminus.
9. The dsRNA agent of claim 1 , wherein the sense strand comprises two phosphorothioate or methylphosphonate internucleotide linkages at the 5′-terminus and the antisense strand comprises two phosphorothioate or methylphosphonate internucleotide linkages at both the 5′- and the 3′-terminus.
10. The dsRNA agent of claim 1 , wherein the ligand is conjugated to the 3′-end of the sense strand.
11. The dsRNA agent of claim 1 , wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative.
12. The dsRNA agent of claim 11 , wherein the ligand is one or more GalNAc derivatives attached through a monovalent, bivalent, or trivalent branched linker.
13. The dsRNA agent of claim 12 , wherein the ligand is
14. The dsRNA agent of claim 13 , wherein the dsRNA agent is conjugated to the ligand as shown in the following schematic
and, wherein X is O.
15. A double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of Patatin-Like Phospholipase Domain Containing 3 (PNPLA3) in a cell, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand differs by no more than 3 modified nucleotides from the nucleotide sequence of 5′-csasuuagGfaUfAfAfugucuuaugu-3′ (SEQ ID NO:2648) and wherein the antisense strand differs by no more than 3 modified nucleotides from the nucleotide sequence of 5′-asCfsauaAfgAfCfauuaUfcCfuaaugsgsg-3′ (SEQ ID NO:2732), wherein a, g, c and u are 2′-O-methyl (2′-OMe) A, G, C, and U respectively; Af, Gf, Cf and Uf are 2′-fluoro A, G, C and U respectively; and s is a phosphorothioate linkage.
16. The dsRNA agent of claim 15 , wherein the sense strand differs by no more than 2 modified nucleotides from the nucleotide sequence of 5′-csasuuagGfaUfAfAfugucuuaugu-3′ (SEQ ID NO:2648) and wherein the antisense strand differs by no more than 2 modified nucleotides from the nucleotide sequence of 5′-asCfsauaAfgAfCfauuaUfcCfuaaugsgsg-3′ (SEQ ID NO:2732).
17. The dsRNA agent of claim 15 , wherein the sense strand differs by no more than 1 modified nucleotide from the nucleotide sequence of 5′-csasuuagGfaUfAfAfugucuuaugu-3′ (SEQ ID NO:2648) and wherein the antisense strand differs by no more than 1 modified nucleotides from the nucleotide sequence of 5′-asCfsauaAfgAfCfauuaUfcCfuaaugsgsg-3′ (SEQ ID NO:2732).
18. The dsRNA agent of claim 15 , wherein the dsRNA agent is conjugated to the ligand as shown in the following schematic
and, wherein X is O.
19. A double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of Patatin-Like Phospholipase Domain Containing 3 (PNPLA3) in a cell, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises the nucleotide sequence of 5′-csasuuagGfaUfAfAfugucuuaugu-3′ (SEQ ID NO:2648) and wherein the antisense strand comprises the nucleotide sequence of 5′-asCfsauaAfgAfCfauuaUfcCfuaaugsgsg-3′ (SEQ ID NO:2732), wherein a, g, c and u are 2′-O-methyl (2′-OMe) A, G, C, and U respectively; Af, Gf, Cf and Uf are 2′-fluoro A, G, C and U respectively; and s is a phosphorothioate linkage.
20. The dsRNA agent of claim 19 , wherein the sense strand comprises the nucleotide sequence of 5′-csasuuagGfaUfAfAfugucuuauguL96-3′ (SEQ ID NO:2648) and wherein the antisense strand comprises the nucleotide sequence of 5′-asCfsauaAfgAfCfauuaUfcCfuaaugsgsg-3′ (SEQ ID NO:2732), wherein a, g, c and u are 2′-O-methyl (2′-OMe) A, G, C, and U respectively; Af, Gf, Cf and Uf are 2′-fluoro A, G, C and U respectively; s is a phosphorothioate linkage, and L96 is N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol.
21. The dsRNA agent of claim 19 , wherein the 3′-end of the sense strand is conjugated to a ligand as shown in the following schematic:
wherein X is O.
22. A double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of Patatin-Like Phospholipase Domain Containing 3 (PNPLA3) in a cell, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand consists of the nucleotide sequence of 5′-csasuuagGfaUfAfAfugucuuaugu-3′ (SEQ ID NO:2648) and wherein the antisense strand consists of the nucleotide sequence of 5′-asCfsauaAfgAfCfauuaUfcCfuaaugsgsg-3′ (SEQ ID NO:2732), wherein a, g, c and u are 2′-O-methyl (2′-OMe) A, G, C, and U respectively; Af, Gf, Cf and Uf are 2′-fluoro A, G, C and U respectively; and s is a phosphorothioate linkage; and wherein the 3′-end of the sense strand is conjugated to a ligand as shown in the following schematic:
wherein X is O.
23. A pharmaceutical composition for inhibiting expression of a gene encoding Patatin-Like Phospholipase Domain Containing 3 (PNPLA3) comprising the dsRNA agent of claim 1 .
24. The pharmaceutical composition of claim 23 , wherein dsRNA agent is in an unbuffered solution.
25. The pharmaceutical composition of claim 24 , wherein the unbuffered solution is saline or water.
26. The pharmaceutical composition of claim 23 , wherein said dsRNA agent is in a buffer solution.
27. The pharmaceutical composition of claim 26 , wherein the buffer solution comprises acetate, citrate, prolamine, carbonate, or phosphate or any combination thereof.
28. The pharmaceutical composition of claim 27 , wherein the buffer solution is phosphate buffered saline (PBS).
29. A pharmaceutical composition for inhibiting expression of a gene encoding Patatin-Like Phospholipase Domain Containing 3 (PNPLA3) comprising the dsRNA agent of claim 21 .
30. A pharmaceutical composition for inhibiting expression of a gene encoding Patatin-Like Phospholipase Domain Containing 3 (PNPLA3) comprising the dsRNA agent of claim 22 .