Extrahepatic delivery

1. A double-stranded iRNA agent comprising:

an antisense strand which is complementary to a target gene;

a sense strand which is complementary to said antisense strand; and

one or more lipophilic moieties conjugated only to one or more internal positions on at least one strand, optionally via a linker or carrier;

wherein the one or more lipophilic moieties comprise a saturated or unsaturated C 4 -C 18 hydrocarbon chain, and an optional functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne;

wherein the one or more internal positions are selected from the positions consisting of: positions 4-8 and 13-18 on the sense strand, and positions 6-10 and 15-18 on the antisense strand, counting from the 5′end of each strand; and

wherein the double-stranded iRNA agent does not comprise a N-acetylgalactosamine (GalNAc) conjugate.

2. The double-stranded iRNA agent of claim 1 , wherein the one or more lipophilic moieties are conjugated only to one or more internal positions selected from the positions consisting of: positions 5, 6, 7, 15, and 17 on the sense strand, and positions 15 and 17 on the antisense strand, counting from the 5′-end of each strand.

3. The double-stranded iRNA agent of claim 1 , wherein said sense and antisense strands are each 15 to 30 nucleotides in length.

4. The double-stranded iRNA agent of claim 1 , wherein the one or more lipophilic moieties contain a saturated or unsaturated C 6 -C 18 hydrocarbon chain.

5. The double-stranded iRNA agent of claim 4 , wherein the one or more lipophilic moieties contain a saturated or unsaturated C 16 hydrocarbon chain.

6. The double-stranded iRNA agent of claim 1 , wherein the one or more lipophilic moieties are conjugated via a carrier that replaces one or more nucleotide(s) in the internal position(s).

7. The double-stranded iRNA agent of claim 6 , wherein the carrier is a cyclic group selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl; or is an acyclic moiety based on a serinol backbone or a diethanolamine backbone.

8. The double-stranded iRNA agent of claim 1 , wherein said iRNA agent comprises a single-stranded overhang on at least one of the termini.

9. The double-stranded iRNA agent of claim 1 , wherein the one or more lipophilic moieties are conjugated to a nucleobase, sugar moiety, or internucleosidic linkage.

10. The double-stranded iRNA agent of claim 1 , wherein the one or more lipophilic moieties are conjugated via a bio-cleavable linker selected from the group consisting of DNA, RNA, disulfide, amide, functionalized monosaccharides or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.

11. The double stranded iRNA agent of claim 1 , wherein the 3′ end of the sense strand is protected via an end cap which is a cyclic group having an amine, said cyclic group being selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl.

12. A method of reducing the expression of a target gene in a cell, comprising contacting said cell with the double-stranded iRNA agent according to claim 1 .

13. The method of claim 12 , wherein the cell is an extrahepatic cell.

14. The method of claim 12 , wherein the hydrophobicity of the double-stranded iRNA agent, measured by the unbound fraction in the plasma protein binding assay of the double-stranded iRNA agent, exceeds 0.2.

15. The method of claim 14 , wherein the plasma protein binding assay is an electrophoretic mobility shift assay using human serum albumin protein.

16. The method of claim 12 , wherein the one or more lipophilic moieties contain a saturated or unsaturated C 16 hydrocarbon chain.

17. A method of reducing the expression of a target gene in a subject, comprising administering to the subject the double-stranded iRNA agent according to claim 1 .

18. The method of claim 17 , wherein the double-stranded iRNA agent is administered extrahepatically.

19. The method of claim 17 , wherein the double-stranded iRNA agent is administered intrathecally.

20. The method of claim 19 , wherein the method reduces the expression of a target gene in a brain or spine tissue.

21. The method of claim 20 , wherein the brain or spine tissue is selected from the group consisting of cortex, cerebellum, cervical spine, lumbar spine, and thoracic spine.

22. The method of claim 17 , wherein the double-stranded iRNA agent is administered intravitreally.

23. The method of claim 22 , wherein the method reduces the expression of a target gene in an ocular tissue.

24. A method of treating a subject having a CNS disorder, comprising:

administering to the subject a therapeutically effective amount of the double-stranded RNAi agent of claim 1 , wherein the CNS disorder is selected from the group of alzheimer, amyotrophic lateral schlerosis (ALS), frontotemporal dementia, huntington, Parkinson, spinocerebellar, prion, and lafora.

25. The double-stranded iRNA agent of claim 1 , wherein the one or more lipophilic moieties are conjugated only to one or more internal positions selected from the group consisting of: positions 4-8 and 13-18 on the sense strand, counting from the 5′-end of the sense strand.

26. The double-stranded iRNA agent of claim 25 , wherein the double-stranded iRNA agent comprises one lipophilic moiety comprising a saturated or unsaturated C 6 -C 18 hydrocarbon chain, and an optional functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne, conjugated only to one internal position selected from the group consisting of: positions 4-8 and 13-18 on the sense strand, counting from the 5′ end of the sense strand, optionally via a linker or carrier.

27. The double-stranded iRNA agent of claim 26 , wherein the one lipophilic moiety consists of a saturated C 16 hydrocarbon chain, and an optional functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.

28. The double-stranded iRNA agent of claim 26 , wherein the one lipophilic moiety consists of a saturated C 16 hydrocarbon chain having a hydroxyl functional group.

29. The double-stranded iRNA agent of claim 26 , wherein the one lipophilic moiety consists of a saturated C 16 hydrocarbon chain.

30. The double-stranded iRNA agent of claim 26 , wherein the sense and antisense strands are each independently 21 to 23 nucleotides in length.

31. The double-stranded iRNA agent of claim 26 , wherein the internal position is position 5, counting from the 5′-end of the sense strand.

32. The double-stranded iRNA agent of claim 26 , wherein the internal position is position 6, counting from the 5′-end of the sense strand.

33. The double-stranded iRNA agent of claim 26 , wherein the internal position is position 7, counting from the 5′-end of the sense strand.

34. The double-stranded iRNA agent of claim 26 , wherein the internal position is position 13, counting from the 5′-end of the sense strand.

35. The double-stranded iRNA agent of claim 26 , wherein the sense strand and antisense strand are each 19 nucleotides in length, and the sense strand contains 2′-F modifications on the nucleotides at positions 7, 8, and 9 from the 5′-end of the sense strand.

36. The double-stranded iRNA agent of claim 35 , wherein the antisense strand contains 2′-O-methyl modifications on the nucleotides at positions 11, 12, and 13 from the 5′-end of the antisense strand.

37. The double-stranded iRNA agent of claim 26 , wherein the sense strand and antisense strand are each 20 nucleotides in length, and the sense strand contains 2′-F modifications on the nucleotides at positions 8, 9, and 10 from the 5′-end of the sense strand.

38. The double-stranded iRNA agent of claim 37 , wherein the antisense strand contains 2′-O-methyl modifications on the nucleotides at positions 11, 12, and 13 from the 5′-end of the antisense strand.

39. The double-stranded iRNA agent of claim 26 , wherein the sense strand and antisense strand are each 21 nucleotides in length, and the sense strand contains 2′-F modifications on the nucleotides at positions 9, 10, and 11 from the 5′-end of the sense strand.

40. The double-stranded iRNA agent of claim 39 , wherein the antisense strand contains 2′-O-methyl modifications on the nucleotides at positions 11, 12, and 13 from the 5′-end of the antisense strand.

41. The double-stranded iRNA agent of claim 40 , wherein:

the sense strand is 21 nucleotides in length, and the antisense strand is 23 nucleotides in length and has a two nucleotide overhang at the 3′-end of the antisense strand;

the sense strand contains 2′-F modifications on the nucleotides at positions 9, 10, and 11 from the 5′-end of the sense strand; and

the antisense strand contains 2′-O-methyl modifications on the nucleotides at positions 11, 12, and 13 from the 5′-end of the antisense strand.

42. The double-stranded iRNA agent of claim 26 , wherein the first base pair at the 5′-end of the antisense strand is an AU base pair.

43. The double-stranded iRNA agent of claim 26 , comprising at least one phosphorothioate or methylphosphonate internucleotide linkage.

44. The double-stranded iRNA agent of claim 43 , wherein the double-stranded iRNA agent comprises at least two phosphorothioate internucleotide linkages at the first five nucleotides on the antisense strand, counting from the 5′-end of the antisense strand.

45. The double-stranded iRNA agent of claim 44 , wherein the double-stranded iRNA agent comprises 8 phosphorothioate internucleotide linkages.

46. The double-stranded iRNA agent of claim 26 , wherein each of the sense and antisense strands contains at least two different modifications.

47. The double-stranded iRNA agent of claim 26 , wherein the double-stranded iRNA agent comprises a mismatch to the target RNA within the duplex region.

48. The double-stranded iRNA agent of claim 26 , wherein each of the sense and antisense strands is independently modified with one or more modifications selected from the group consisting of an acyclic nucleotide, LNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-deoxy, 2′-fluoro, 2′-O-N-methylacetamido (2′-O-NMA), a 2′-O-dimethylaminoethoxyethyl (2′-O-DMAEOE), 2′-O-aminopropyl (2′-O-AP), and 2′-ara-F.

49. The double-stranded iRNA agent of claim 48 , wherein one or more modification is acyclic nucleotide modification, and the acyclic nucleotide is an UNA or a GNA.

50. The double-stranded iRNA agent of claim 26 , wherein the double-stranded iRNA agent comprises a 2′-5′ internucleotide linkage.

51. The double-stranded iRNA agent of claim 26 , wherein the double-stranded iRNA agent comprises an overhang region containing two nucleotides at the 3′-end of the antisense strand and a phosphorothioate or methylphosphonate internucleotide linkage between the two overhang nucleotides.

52. The double-stranded iRNA agent of claim 26 , wherein the double-stranded iRNA agent comprises an overhang region at the 3′-end of the antisense strand and two phosphorothioate internucleotide linkages between the terminal three nucleotides at the 3′-end of the antisense strand, in which two of the three terminal nucleotides are the overhang nucleotides, and the third nucleotide is a paired nucleotide next to the overhang nucleotides.

53. The double-stranded iRNA agent of claim 26 , wherein the double-stranded iRNA agent comprises a phosphate at the 5′-end of the antisense strand.

54. The double-stranded iRNA agent of claim 26 , wherein the double-stranded iRNA agent comprises a phosphate mimic at the 5′-end of the antisense strand.

55. The double-stranded iRNA agent of claim 54 , wherein the phosphate mimic is a 5′-vinyl phosphonate (VP).

56. The double-stranded iRNA agent of claim 1 , wherein the double-stranded iRNA agent comprises a phosphate mimic at the 5′-end of the antisense strand.

57. The double-stranded iRNA agent of claim 56 , wherein the phosphate mimic is a 5′-vinyl phosphonate (VP).

58. The double-stranded iRNA agent of claim 25 , wherein the double-stranded iRNA agent comprises one lipophilic moiety conjugated only to one internal position selected from the group consisting of: positions 4-8 and 13-18 on the sense strand, counting from the 5′ end of the sense strand via a linker or carrier.

59. The double-stranded iRNA agent of claim 58 , wherein the lipophilic moiety is conjugated to the double-strand iRNA agent via a linker comprising an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide linkage, a triazole from the azide-alkyne cycloaddition, or carbamate.

60. The double-stranded iRNA agent of claim 59 , wherein the lipophilic moiety is conjugated to the double-strand iRNA agent via a linker comprising an amide.

61. The double-stranded iRNA agent of claim 59 , wherein the lipophilic moiety is conjugated to the double-strand iRNA agent via a linker comprising a disulfide.

62. The double-stranded iRNA agent of claim 58 , wherein the lipophilic moiety is conjugated to the double-strand iRNA agent via a linker comprising a formula of

TAP-(CH 2 ) n NH-;

TAP-(CH 2 ) n -C(O)-;

TAP-(CH 2 ) n -C(O)O-;

TAP-(CH 2 ) n -; or TAP-(CH 2 ) n -NH-C(O)-;

wherein TAP represents the attachment point to the double-stranded iRNA agent, and n is 1 - 20 .

63. The double-stranded iRNA agent of claim 62 , wherein the linker of the formula further comprises one or more additional linking groups selected from the group consisting of -O-(CH 2 ) n -,-(CH 2 ) n -SS-,-(CH 2 ) n -, and-(CH=CH)-.

64. The double-stranded iRNA agent of claim 63 , wherein the linker of the formula further comprises an additional linking group of-(CH 2 ) n -SS-.

65. The double-stranded iRNA agent of claim 60 , wherein the lipophilic moiety is TAP-(CH 2 ), n NH(LIGAND) or TAP-(CH 2 ) n -NH-C(O)(LIGAND), and wherein LIGAND consists of a saturated or unsaturated C 6 -C 18 hydrocarbon chain, and an optional functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.

66. The double-stranded iRNA agent of claim 59 , wherein the lipophilic moiety is conjugated to a sugar moiety of a nucleotide via a2′-O modification.