Login| Sign Up| Help| Contact|

Patent Searching and Data


Title:
COMPOSITIONS AND METHODS FOR INHIBITING SNCA EXPRESSION
Document Type and Number:
WIPO Patent Application WO/2023/220351
Kind Code:
A1
Abstract:
Oligonucleotides (e.g., RNAi oligonucleotides) are provided herein that inhibit SNCA gene expression, including oligonucleotides conjugated to a targeting ligand (e.g., GalNAC moiety or lipid moiety). Also provided are compositions including the same and uses thereof, particularly uses relating to treating diseases, disorders, and/or conditions associated with SNCA gene expression.

Inventors:
BROWN BOB (US)
DUDEK HENRYK (US)
CHEONG SEONGMOON (US)
WANG SHIYU (US)
GRIM TRAVIS (US)
COSTALES MATTHEW GUESE (US)
JUNG MAIRE (US)
Application Number:
PCT/US2023/022007
Publication Date:
November 16, 2023
Filing Date:
May 12, 2023
Export Citation:
Click for automatic bibliography generation   Help
Assignee:
DICERNA PHARMACEUTICALS INC (US)
International Classes:
C12N15/113
Domestic Patent References:
WO2022072447A12022-04-07
WO2009079399A22009-06-25
WO2022031847A22022-02-10
WO2010033225A22010-03-25
WO2018045317A12018-03-08
WO2016100401A12016-06-23
WO2011133871A22011-10-27
Foreign References:
US20050137155A12005-06-23
US8372968B22013-02-12
US8883996B22014-11-11
US8513207B22013-08-20
US8927705B22015-01-06
US9012138B22015-04-21
US9012621B22015-04-21
US9193753B22015-11-24
US20090099115A12009-04-16
US9567587B22017-02-14
US10131912B22018-11-20
US20080274462A12008-11-06
US20070254362A12007-11-01
US201662383207P2016-09-02
US201662393401P2016-09-12
US8927513B22015-01-06
US5804683A1998-09-08
US5831071A1998-11-03
US5998203A1999-12-07
US6008400A1999-12-28
US6111086A2000-08-29
US6117657A2000-09-12
US6353098B12002-03-05
US6362323B12002-03-26
US6437117B12002-08-20
US6469158B12002-10-22
Other References:
KRAYNACKBAKER, RNA, vol. 12, 2006, pages 163 - 76
MOORE, METHODS MOL. BIOL, vol. 629, 2010, pages 141 - 58
SUN, NAT. BIOTECHNOL., vol. 26, 2008, pages 1379 - 82
CHANG, MOL. THER, vol. 17, 2009, pages 725 - 732
HOHJOH, FEBSLETT, vol. 557, 2004, pages 193 - 98
ELSNER, NAT. BIOTECHNOL., vol. 30, 2012, pages 1063
ABE ET AL., J. AM. CHEM. SOC., vol. 129, 2007, pages 15108 - 09
VAN AERSCHOT ET AL., NUCLEIC ACIDS RES., vol. 22, 1994, pages 4039 - 4370
HAMILTON ET AL., EMBO J., vol. 21, 2002, pages 4671 - 79
MATSUI ET AL., MOL. THER, vol. 24, 2016, pages 946 - 955
BENNETT ET AL., ANNU. REV. PHARMACOL, vol. 57, 2017, pages 81 - 105
KOSHKIN ET AL., TETRAHEDON, vol. 54, 1998, pages 3607 - 30
SNEAD ET AL., MOL. THER-NUCL. ACIDS, vol. 2, 2013, pages e103
IMANISHIOBIKA, CHEM COMMUN. (CAMB, vol. 21, 2002, pages 1653 - 59
LOAKES ET AL., NUCLEIC ACIDS RES, vol. 23, 1995, pages 2361 - 66
"Remington: THE SCIENCE AND PRACTICE OF PHARMACY", 2013, PHARMACEUTICAL PRESS
PRAKASH ET AL., NUCLEIC ACIDS RES., vol. 43, 2015, pages 2993 - 3011
CHEONG ET AL., NATURE, vol. 346, 1990, pages 680 - 682
HEUSPARDI, SCIENCE, vol. 253, 1991, pages 191 - 94
CORNISH-BOWDEN, NUCLEIC ACIDS RES., vol. 13, 1985, pages 3021 - 30
WOESE ET AL., PROC. NATL. ACAD. SCI. USA, vol. 87, 1990, pages 8467 - 71
ANTAO ET AL., NUCLEIC ACIDS RES., vol. 19, 1991, pages 5901 - 05
NAKANO ET AL., BIOCHEM., vol. 41, 2002, pages 4281 - 92
SHINJI, NIPPON KAGAKKAI KOEN YOKOSHU, vol. 78, 2000, pages 731
SCARINGE ET AL., NUCLEIC ACIDS RES., vol. 18, 1990, pages 5433 - 5441
USMAN ET AL., J. AM. CHEM. SOC., vol. 109, 1987, pages 7845 - 45
HUGHESELLINGTON, COLD SPRING HARB PERSPECT, vol. 9, no. 1, 2017, pages a023812
BEAUCAGE SLCARUTHERS MH: "Deoxynucleoside phosphoramidites-A new class of key intermediates for deoxypolynucleotides", TETRAHEDRON LETT., vol. 22, no. 20, 1981, pages 1859 - 1862, XP002007271, DOI: 10.1016/S0040-4039(01)90461-7
DAMHAOLGIVIE, METHODS MOL. BIOL, vol. 20, 1993, pages 81 - 114
WINCOTT ET AL., NUCLEIC ACIDS RES., vol. 23, 1995, pages 2677 - 84
MATTEUCCI MDCARUTHERS MH: "The synthesis of oligodeoxypyrimidines on a polymer su", TETRAHEDRON LETT., vol. 21, no. 8, 1980, pages 719 - 722
Attorney, Agent or Firm:
REID, Andrea L.C. et al. (US)
Download PDF:
Claims:
CLAIMS

The invention claimed is:

1. A RNAi oligonucleotide for reducing SNCA gene expression, the oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a duplex region, wherein the antisense strand comprises a region of complementarity to a SNCA mRNA target sequence of any one of SEQ ID NOs: 1683-2066, and wherein the region of complementarity is at least 15 contiguous nucleotides in length.

2. The RNAi oligonucleotide of claim 1, wherein the sense strand is 15 to 50 nucleotides in length.

3. The RNAi oligonucleotide of claims 1 or 2, wherein the sense strand is 18 to 36 nucleotides in length.

4. The RNAi oligonucleotide of any one of claims 1 to 3, wherein the antisense strand is 15 to 30 nucleotides in length.

5. The RNAi oligonucleotide of any one of claims 1 to 4, wherein the antisense strand is 22 nucleotides in length and wherein antisense strand and the sense strand form a duplex region of at least 19 nucleotides in length, optionally at least 20 nucleotides in length.

6. The RNAi oligonucleotide of any one of claims 1 to 5, wherein the region of complementarity is at least 19 contiguous nucleotides in length.

7. The RNAi oligonucleotide of any one of claims 1 to 6, wherein the region of complementarity is at least 20 contiguous nucleotides in length.

8. A double-stranded RNAi oligonucleotide for reducing SNCA gene expression, the oligonucleotide comprising:

(i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand comprises a nucleotide sequence comprising a region of complementarity to a SNCA mRNA target sequence, wherein the region of complementarity is selected from SEQ ID NOs: 2067-2450, and

(ii) a sense strand of 19-50 nucleotides in length comprising a region of complementarity to the antisense strand, wherein the antisense and sense strands are separate strands which form an asymmetric duplex region having an overhang of 1 -4 nucleotides at the 3’ terminus of the antisense strand.

9. The RNAi oligonucleotide of any one of claims 1-8, wherein the 3’ end of the sense strand comprises a stem-loop set forth as S1-L-S2, wherein SI is complementary to S2, and wherein L forms a loop between S I and S2 of 3-5 nucleotides in length.

10. The RNAi oligonucleotide of claim 9, wherein L is a triloop or a tetraloop.

11. The RNAi oligonucleotide of claim 10, wherein L is a tetraloop.

12. The RNAi oligonucleotide of claim 11, wherein the tetraloop comprises the sequence 5’- GAAA-3’.

13. The RNAi oligonucleotide of any one of claims 9-12, wherein the SI and S2 are 1-10 nucleotides in length and have the same length.

14. The RNAi oligonucleotide of claim 13, wherein SI and S2 are 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, or 10 nucleotides in length.

15. The RNAi oligonucleotide of claim 14, wherein SI and S2 are 6 nucleotides in length.

16. The RNAi oligonucleotide of any one of claims 9-15, wherein the stem-loop comprises the sequence 5’-GCAGCCGAAAGGCUGC-3’ (SEQ ID NO: 1680).

17. The RNAi oligonucleotide of any one of claims 1-8, wherein the oligonucleotide comprises a blunt end.

18. The RNAi oligonucleotide of claim 17, wherein the blunt end comprises the 3’ end of the sense strand.

19. The RNAi oligonucleotide of any one of claims 17-18, wherein the sense strand is 20-22 nucleotides.

20. The RNAi oligonucleotide of claim 19, wherein the sense strand is 20 nucleotides.

21. The RNAi oligonucleotide of any one of claims 1 to 20, wherein the antisense strand comprises a 3’ overhang sequence of one or more nucleotides in length.

22. The RNAi oligonucleotide of claim 21, wherein the overhang comprises purine nucleotides.

23. The RNAi oligonucleotide of claim 21 or 22, wherein the 3’ overhang sequence is 2 nucleotides in length.

24. The RNAi oligonucleotide of claim 23, wherein the 3’ overhang is selected from AA, GG, AG, and GA.

25. The RNAi oligonucleotide of claim 24, wherein the overhang is GG or AA.

26. The RNAi oligonucleotide of claim 25, wherein the overhang is GG.

27. The RNAi oligonucleotide of any one of the preceding claims, wherein the oligonucleotide comprises at least one modified nucleotide.

28. The RNAi oligonucleotide of claim 27, wherein the modified nucleotide comprises a 2'- modifi cation.

29. The RNAi oligonucleotide of claim 28, wherein the 2'-modification is a modification selected from 2'-aminoethyl, 2 -fluoro, 2'-O-methyl, 2'-O-methoxyethyl, and 2'-deoxy-2'-fluoro- P-d-arabinonucleic acid.

30. The RNAi oligonucleotide of any one of claims 28-29, wherein the modification is a 2’- modifi cation selected from 2’-fluoro and 2’-O-methyl.

31. The RNAi oligonucleotide of any one of claims 29-30, wherein about 18% to about 23%, or 18%, 19%, 20%, 21%, 22% or 23% of the nucleotides of the sense strand comprise the 2’-fluoro modification.

32. The RNAi oligonucleotide of any one of claims 29-30, wherein about 38% to about 43%, or 38%, 39%, 40%, 41%, 42% or 43% of the nucleotides of the sense strand comprise the 2’-fluoro modification.

33. The RNAi oligonucleotide of any one of claims 29-32, wherein about 25% to about 35%, or 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34% or 35% of the nucleotides of the antisense strand comprise the 2’-fluoro modification.

34. The RNAi oligonucleotide of any one of claims 29-33, wherein about 25% to about 35%, or 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34% or 35% of the nucleotides of the oligonucleotide comprise the 2’-fluoro modification.

35. The RNAi oligonucleotide of any one of claims 29-33, wherein about 35-45%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44% or 45% of the nucleotides of the oligonucleotide comprise the 2’ -fluoro modification.

36. The RNAi oligonucleotide of any one of claims 29-35, wherein the sense strand comprises 36 nucleotides with positions 1-36 from 5’ to 3’, wherein each of positions 3, 5, 8, 10, 12, 13, 15, and 17 comprise the 2’ -fluoro modification.

37. The RNAi oligonucleotide of any one of claims 29-35, wherein the sense strand comprises 20 nucleotides with positions 1-20 from 5’ to 3’, wherein each of positions 3, 5, 8, 10, 12, 13, 15, and 17 comprise the 2’ -fluoro modification.

38. The RNAi oligonucleotide of any one of claims 29-37, wherein the antisense strand comprises 22 nucleotides with positions 1-22 from 5’ to 3’, and wherein each of positions 2, 3, 4, 5, 7, 10 14, 16 and 19 comprise the 2’-fluoro modification.

39. The RNAi oligonucleotide of any one of claims 31-38, wherein the remaining nucleotides comprise the 2’-O-methyl modification.

40. The RNAi oligonucleotide of any one of the preceding claims, wherein the oligonucleotide comprises at least one modified intemucleotide linkage.

41. The RNAi oligonucleotide of claim 40, wherein the at least one modified intemucleotide linkage is a phosphorothioate linkage.

42. The RNAi oligonucleotide of claim 41, wherein the antisense strand comprises the phosphorothioate linkage (i) between positions 1 and 2, and between positions 2 and 3; or (ii) between positions 1 and 2, between positions 2 and 3, and between positions 3 and 4, and wherein positions are numbered 1-4 from 5’ to 3’.

43. The RNAi oligonucleotide of claim 41 or 42, wherein the antisense strand is 22 nucleotides in length, and wherein the antisense strand comprises the phosphorothioate linkage between positions 20 and 21 and between positions 21 and 22, and wherein positions are numbered 1 -22 from 5 ’ to 3 ’ .

44. The RNAi oligonucleotide of any one of claims 41-43, wherein the sense strand comprises the phosphorothioate linkage between positions 1 and 2, and wherein positions are numbered 1-2 from 5’ to 3’.

45. The RNAi oligonucleotide of any one of claims 41-43, wherein the sense strand is 20 nucleotides in length, wherein the sense strand comprises the phosphorothioate linkage between positions between positions 1 and 2, between positions 18 and 19 and between positions 19 and 20, and wherein positions are numbered 1-20 from 5’ to 3’.

46. The RNAi oligonucleotide of any one of the preceding claims, wherein the 4'-carbon of the sugar of the 5 '-nucleotide of the antisense strand comprises a phosphate analog.

47. The RNAi oligonucleotide of claim 46, wherein the phosphate analog is oxymethyl phosphonate, vinyl phosphonate or malonyl phosphonate, optionally wherein the phosphate analog is a 4’-phosphate analog comprising 4’-oxymethylphosphonate.

48. The RNAi oligonucleotide of any one of the preceding claims, wherein at least one nucleotide of the oligonucleotide is conjugated to one or more targeting ligands.

49. The RNAi oligonucleotide of claim 48, wherein each targeting ligand comprises a carbohydrate, amino sugar, lipid, cholesterol, or polypeptide.

50. The RNAi oligonucleotide of any one of claims 9-16 and 21-49, wherein the stem-loop comprises one or more targeting ligands conjugated to one or more nucleotides of the stem-loop.

51. The RNAi oligonucleotide of claim 50, wherein the one or more targeting ligands is conjugated to one or more nucleotides of the loop (L).

52. The RNAi oligonucleotide of claim 51, wherein the loop (L) comprises 4 nucleotides numbered 1-4 from 5’ to 3’, wherein nucleotides at positions 2, 3, and 4 each comprise one or more targeting ligands, and wherein the targeting ligands are the same or different.

53. The RNAi oligonucleotide of claim 52, wherein each targeting ligand comprises a N- acetylgalactosamine (GalNAc) moiety.

54. The RNAi oligonucleotide of claim 53, wherein the GalNac moiety is a monovalent GalNAc moiety, a bivalent GalNAc moiety, a trivalent GalNAc moiety, or a tetravalent GalNAc moiety.

55. The RNAi oligonucleotide of any one of claims 9-16 and 21-54, wherein up to 4 nucleotides of loop (L) of the stem-loop are each conjugated to a monovalent GalNAc moiety.

56. The RNAi oligonucleotide of any one of claims 17-48, wherein the one or more targeting ligands is a lipid moiety.

57. The RNAi oligonucleotide of claim 56, wherein the lipid moiety is conjugated to the 5 ’terminal nucleotide of the sense strand.

58. The RNAi oligonucleotide of claim 56 or 57, wherein the lipid moiety is a hydrocarbon chain.

59. The RNAi oligonucleotide of claim 58, wherein the hydrocarbon chain is a Cs-Cso hydrocarbon chain.

60. The RNAi oligonucleotide of claim 58 or 59, wherein the hydrocarbon chain is a Ci6 hydrocarbon chain.

61. The RNAi oligonucleotide of claim 60, wherein the Ci6 hydrocarbon chain is represented by:

62. The RNAi oligonucleotide of any one of claims 56-61, wherein the lipid moiety is conjugated to the 2’ carbon of the ribose ring of the 5’ terminal nucleotide.

63. The RNAi oligonucleotide of any one of claims 1-62, wherein the region of complementarity is fully complementary to the SNCA mRNA target sequence at nucleotide positions 2-8 of the antisense strand, and wherein nucleotide positions are numbered 5’ to 3’.

64. The RNAi oligonucleotide of any one of claims 1-62, wherein the region of complementarity is fully complementary to the SNCA mRNA target sequence at nucleotide positions 2-11 of the antisense strand, and wherein nucleotide positions are numbered 5’ to 3’.

65. The RNAi oligonucleotide of any one of claims 1 to 64, wherein the sense strand comprises a nucleotide sequence of any one of SEQ ID NOs: 1537-1571 and 1681.

66. The RNAi oligonucleotide of any one of claims 1 to 65, wherein the antisense strand comprises a nucleotide sequence of any one of SEQ ID NOs: 1572-1606.

67. The RNAi oligonucleotide of any one of claims 1 to 66, wherein the sense strand and the antisense strand comprise nucleotide sequences selected from the group consisting of: a) SEQ ID NOs: 1537 and 1572, respectively; b) SEQ ID NOs: 1538 and 1573, respectively; c) SEQ ID NOs: 1539 and 1574, respectively; d) SEQ ID NOs: 1540 and 1575, respectively; e) SEQ ID NOs: 1541 and 1576, respectively; f) SEQ ID NOs: 1542 and 1577, respectively; g) SEQ ID NOs: 1543 and 1578, respectively; h) SEQ ID NOs: 1544 and 1579, respectively; i) SEQ ID NOs: 1545 and 1580, respectively; j) SEQ ID NOs: 1546 and 1581, respectively; k) SEQ ID NOs: 1547 and 1582, respectively; l) SEQ ID NOs: 1548 and 1583, respectively; m) SEQ ID NOs: 1549 and 1584, respectively; n) SEQ ID NOs: 1550 and 1585, respectively; o) SEQ ID NOs: 1551 and 1586, respectively; p) SEQ ID NOs: 1552 and 1587, respectively; q) SEQ ID NOs: 1553 and 1588, respectively; r) SEQ ID NOs: 1554 and 1589, respectively; s) SEQ ID NOs: 1555 and 1590, respectively; t) SEQ ID NOs: 1556 and 1591, respectively; u) SEQ ID NOs: 1557 and 1592, respectively; v) SEQ ID NOs: 1558 and 1593, respectively; w) SEQ ID NOs: 1559 and 1594, respectively; x) SEQ ID NOs: 1560 and 1595, respectively; y) SEQ ID NOs: 1561 and 1596, respectively; z) SEQ ID NOs: 1562 and 1597, respectively; aa) SEQ ID NOs: 1563 and 1598, respectively; bb) SEQ ID NOs: 1564 and 1599, respectively; cc) SEQ ID NOs: 1565 and 1600, respectively; dd) SEQ ID NOs: 1566 and 1601, respectively; ee) SEQ ID NOs: 1567 and 1602, respectively; ff) SEQ ID NOs: 1568 and 1603, respectively; gg) SEQ ID NOs: 1569 and 1604, respectively; hh) SEQ ID NOs: 1570 and 1605, respectively; ii) SEQ ID NOs: 1571 and 1606, respectively; and jj) SEQ ID NOs: 1681 and 1586, respectively.

68. The RNAi oligonucleotide of any one of claims 1 to 66, wherein the sense strand and the antisense strand comprise nucleotide sequences selected from the group consisting of: a) SEQ ID NOs: 1540 and 1575, respectively; b) SEQ ID NOs: 1544 and 1579, respectively; c) SEQ ID NOs: 1546 and 1581, respectively; d) SEQ ID NOs: 1551and 1586, respectively; e) SEQ ID NOs: 1552 and 1587, respectively; f) SEQ ID NOs: 1553 and 1588, respectively; g) SEQ ID NOs: 1558 and 1594, respectively; h) SEQ ID NOs: 1560 and 1595, respectively; i) SEQ ID NOs: 1564 and 1599, respectively; j) SEQ ID NOs: 1565 and 1600, respectively; k) SEQ ID NOs: 1566 and 1601, respectively; l) SEQ ID NOs: 1570 and 1605, respectively; and m) SEQ ID NOs: 1681 and 1586, respectively.

69. The RNAi oligonucleotide of any one of claims 1 to 66, wherein the sense strand and antisense strands comprise nucleotide sequences selected from the group consisting of: a) SEQ ID NOs: 1553 and 1588, respectively; b) SEQ ID NOs: 1560 and 1595, respectively; c) SEQ ID NOs: 1564 and 1599, respectively; d) SEQ ID NOs: 1551 and 1586, respectively; e) SEQ ID NOs: 1570 and 1605, respectively; and, f) SEQ ID NOs: 1681 and 1586, respectively.

70. TheRNAi oligonucleotide of any one of claims 1 to 66, wherein the sense strand comprises the nucleotide sequence as set forth in SEQ ID NO: 1553, and wherein the antisense strand comprises the nucleotide sequence as set forth in SEQ ID NO: 1588.

71. The RNAi oligonucleotide of any one of claims 1 to 66, wherein the sense strand comprises the nucleotide sequence as set forth in SEQ ID NO: 1560, and wherein the antisense strand comprises the nucleotide sequence as set forth in SEQ ID NO: 1595.

72. The RNAi oligonucleotide of any one of claims 1 to 66, wherein the sense strand comprises the nucleotide sequence as set forth in SEQ ID NO: 1564, and wherein the antisense strand comprises the nucleotide sequence as set forth in SEQ ID NO: 1599.

73. The RNAi oligonucleotide of any one of claims 1 to 66, wherein the sense strand comprises the nucleotide sequence as set forth in SEQ ID NO: 1551, and wherein the antisense strand comprises the nucleotide sequence as set forth in SEQ ID NO: 1586.

74. The RNAi oligonucleotide of any one of claims 1 to 66, wherein the sense strand comprises the nucleotide sequence as set forth in SEQ ID NO: 1570, and wherein the antisense strand comprises the nucleotide sequence as set forth in SEQ ID NO: 1605.

75. The RNAi oligonucleotide of any one of claims 1 to 66, wherein the sense strand comprises the nucleotide sequence as set forth in SEQ ID NO: 1681, and wherein the antisense strand comprises the nucleotide sequence as set forth in SEQ ID NO: 1586.

76. The RNAi oligonucleotide of any one of claims 1-64, wherein the antisense strand is 22 nucleotides in length.

77. The RNAi oligonucleotide of claim 76, wherein the antisense strand comprises a nucleotide sequence selected from SEQ ID NOs: 1588, 1595, 1599, 1586, and 1605.

78. The RNAi oligonucleotide of any one of claims 1-64 and 76-77, wherein the sense strand comprises a nucleotide sequence selected from SEQ ID NOs: 1865, 1721, 1847, 1846, and 1955.

79. The RNAi oligonucleotide of any one of claims 1-64 and 76-78, wherein the sense strand is 36 nucleotides in length.

80. The RNAi oligonucleotide of claim 79, wherein the sense strand comprises a nucleotide sequence selected from SEQ ID NOs: 1553, 1560, 1564, 1551, and 1570.

81. The RNAi oligonucleotide of any one of claims 1-64, wherein the sense strand comprises the nucleotide sequence of any one of SEQ ID NOs: 1607-1641, and 1682.

82. The RNAi oligonucleotide of any one of claims 1 to 64 and 81 , wherein the antisense strand comprises a nucleotide sequence of any one of SEQ ID NOs: 1642-1676.

83. The RNAi oligonucleotide of any one of claims 1-64, wherein the sense strand and the antisense strand comprise nucleotide sequences selected from the group consisting of: a) SEQ ID NOs: 1607 and 1642, respectively; b) SEQ ID NOs: 1608 and 1643, respectively; c) SEQ ID NOs: 1609 and 1644, respectively; d) SEQ ID NOs: 1610 and 1645, respectively; e) SEQ ID NOs: 1611 and 1646, respectively; f) SEQ ID NOs: 1612 and 1647, respectively; g) SEQ ID NOs: 1613 and 1648, respectively; h) SEQ ID NOs: 1614 and 1649, respectively; i) SEQ ID NOs: 1615 and 1650, respectively; j) SEQ ID NOs: 1616 and 1651, respectively; k) SEQ ID NOs: 1617 and 1652, respectively; l) SEQ ID NOs: 1618 and 1653, respectively; m) SEQ ID NOs: 1619 and 1654, respectively; n) SEQ ID NOs: 1620 and 1655, respectively; o) SEQ ID NOs: 1621 and 1656, respectively; p) SEQ ID NOs: 1622 and 1657, respectively; q) SEQ ID NOs: 1623 and 1658, respectively; r) SEQ ID NOs: 1624 and 1659, respectively; s) SEQ ID NOs: 1625 and 1660, respectively; t) SEQ ID NOs: 1626 and 1661, respectively; u) SEQ ID NOs: 1627 and 1662, respectively; v) SEQ ID NOs: 1628 and 1663, respectively; w) SEQ ID NOs: 1629 and 1664, respectively; x) SEQ ID NOs: 1630 and 1665, respectively; y) SEQ ID NOs: 1631 and 1666, respectively; z) SEQ ID NOs: 1632 and 1667, respectively; aa) SEQ ID NOs: 1633 and 1668, respectively; bb) SEQ ID NOs: 1634 and 1669, respectively; cc) SEQ ID NOs: 1635 and 1670, respectively; dd) SEQ ID NOs: 1636 and 1671, respectively; ee) SEQ ID NOs: 1637 and 1672, respectively; ff) SEQ ID NOs: 1638 and 1673, respectively; gg) SEQ ID NOs: 1639 and 1674, respectively; hh) SEQ ID NOs: 1640 and 1675, respectively; ii) SEQ ID NOs: 1641 and 1676, respectively; and jj) SEQ ID NOs: 1682 and 1656, respectively.

84. The RNAi oligonucleotide of any one of claims 1-64, wherein the sense strand and the antisense strand comprise nucleotide sequences selected from the group consisting of: a) SEQ ID NOs: 1610 and 1645, respectively; b) SEQ ID NOs: 1614 and 1649, respectively; c) SEQ ID NOs: 1616 and 1651, respectively; d) SEQ ID NOs: 1621 and 1656, respectively; e) SEQ ID NOs: 1622 and 1657, respectively; f) SEQ ID NOs: 1623 and 1658, respectively; g) SEQ ID NOs: 1629 and 1664, respectively; h) SEQ ID NOs: 1630 and 1665, respectively; i) SEQ ID NOs: 1634 and 1669, respectively; j) SEQ ID NOs: 1635 and 1670, respectively; k) SEQ ID NOs: 1636 and 1671, respectively; l) SEQ ID NOs: 1640 and 1675, respectively; and m) SEQ ID NOs: 1682 and 1656, respectively.

85. The RNAi oligonucleotide of any one of claims 1-64, wherein the sense strand and the antisense strand comprise nucleotide sequences selected from the group consisting of: a) SEQ ID NOs: 1623 and 1658, respectively; b) SEQ ID NOs: 1630 and 1665, respectively; c) SEQ ID NOs: 1634 and 1669, respectively; d) SEQ ID NOs: 1621 and 1656, respectively; e) SEQ ID NOs: 1640 and 1675, respectively; and f) SEQ ID NOs: 1682 and 1656, respectively.

86. The RNAi oligonucleotide of any one of claims 1 to 64, wherein the sense strand comprises the nucleotide sequence as set forth in SEQ ID NO: 1623, and wherein the antisense strand comprises the nucleotide sequence as set forth in SEQ ID NO: 1658.

87. The RNAi oligonucleotide of any one of claims 1 to 64, wherein the sense strand comprises the nucleotide sequence as set forth in SEQ ID NO: 1630, and wherein the antisense strand comprises the nucleotide sequence as set forth in SEQ ID NO: 1665.

88. The RNAi oligonucleotide of any one of claims 1 to 64, wherein the sense strand comprises the nucleotide sequence as set forth in SEQ ID NO: 1634, and wherein the antisense strand comprises the nucleotide sequence as set forth in SEQ ID NO: 1669.

89. The RNAi oligonucleotide of any one of claims 1 to 64, wherein the sense strand comprises the nucleotide sequence as set forth in SEQ ID NO: 1621, and wherein the antisense strand comprises the nucleotide sequence as set forth in SEQ ID NO: 1656.

90. The RNAi oligonucleotide of any one of claims 1 to 64, wherein the sense strand comprises the nucleotide sequence as set forth in SEQ ID NO: 1640, and wherein the antisense strand comprises the nucleotide sequence as set forth in SEQ ID NO: 1676.

91. The RNAi oligonucleotide of any one of claims 1 to 64, wherein the sense strand comprises the nucleotide sequence as set forth in SEQ ID NO: 1682, and wherein the antisense strand comprises the nucleotide sequence as set forth in SEQ ID NO: 1656.

92. The RNAi oligonucleotide of claim 1, wherein the sense strand comprises the sequence 5'- [mC s] [mA] [fG] [mC] [f A] [mG] [mU] [fG] [mA] [fU] [mU]

[fG] [f A] [mA] [fG] [mU] [f A] [mU] [mC] [mA] [mG] [mC] [mA] [mG] [mC] [mC ] [mG] [adem A- GalNAc] [ademA-GalNAc] [ademA-GalNAc] [mG][mG][mC][mU][mG][mC]-3’ (SEQ ID NO: 1623), wherein the antisense strand comprises the sequence 5'-[MePhosphonate-4O- mUs] [fGs] [fA] [fU] [fA] [mC] [fU] [mU] [mC] [f A] [mA] [mU] [mC] [fA] [mC][fU][mG][mC][fU][mGs][mGs][mG]-3' (SEQ ID NO: 1658), and wherein mC, mA, mG, mU = 2'-0Me ribonucleosides; fA, fC, fG, fU = 2'-F ribonucleosides; s = phosphorothioate, and wherein ademA-GalNAc =

93. The RNAi oligonucleotide of claim 1, wherein the sense strand comprises the sequence 5'- [mAs] [mG] [fA] [mG] [fC] [mA] [mA] [fG] [mU] [fG] [mA] [fC] [fA][m A] [fA] [mU] [fG] [mU] [mU] [mA] [mG] [mC] [mA] [mG] [mC] [mC] [mG] [adem A-GalNAc] [ademA-GalNAc] [ademA- GalNAc][mG][mG][mC][mU][mG][mC]-3’ (SEQ ID NO: 1630), wherein the antisense strand comprises the sequence 5'-[MePhosphonate-4O-mUs][fAs][fA][fC][fA][mU][fU][mU][mG] [fU][mC][mA][mC][fU][mU][fG][mC][mU][fC][mUs][mGs][mG]-3' (SEQ ID NO: 1665), and wherein mC, mA, mG, mU = 2'-0Me ribonucleosides; fA, fC, fG, fU = 2'-F ribonucleosides; s = phosphorothioate, and wherein ademA-GalNAc =

94. The RNAi oligonucleotide of claim 1, wherein the sense strand comprises the sequence 5'- [mAs] [mG] [fU] [mC] [fA] [mU] [mG] [fA] [mC][f A] [mU] [fU] [fU][mC][fU] [mC] [fA] [mA] [mA] [mA] [mG] [mC] [mA] [mG] [mC] [mC] [mG] [adem A-GalNAc] [adem A-GalNAc] [adem A- GalNAc][mG][mG][mC][mU][mG][mC]-3’ (SEQ ID NO: 1634), wherein the antisense strand comprises the sequence 5'-[MePhosphonate-4O-mUs][fUs][fU][fU][fG][mA][fG][mA][mA] [fA][mU][mG][mU][fC][mA][fU][mG][mA][fC][mUs][mGs][mG]-3' (SEQ ID NO: 1669), and wherein mC, mA, mG, mU = 2'-0Me ribonucleosides; fA, fC, fG, fU = 2'-F ribonucleosides; s = phosphorothioate, and wherein ademA-GalNAc =

95. The RNAi oligonucleotide of claim 1, wherein the sense strand comprises the sequence 5'- [mC s] [mA] [fG] [mU] [fC] [mA] [mU] [fG] [mA] [fC] [mA] [fU] [fU] [mU] [fC] [mU] [fC] [mA] [mA] [mA] [mG] [mC] [mA] [mG] [mC] [mC] [mG] [ademA-GalNAc] [adem A-GalNAc] [ademA- GalNAc] [mG][mG][mC][mU][mG][mC]- 3’ (SEQ ID NO: 1621), wherein the antisense strand comprises the sequence 5'-[MePhosphonate-4O-mUs][fUs][fU][fG][fA][mG][fA][mA] [mA][fU][mG][mU][mC][fA][mU][fG][mA][mC][fU][mGs][mGs][mG]-3' (SEQ ID NO: 1656), and wherein mC, mA, mG, mU = 2'-0Me ribonucleosides; fA, fC, fG, fU = 2'-F ribonucleosides; s = phosphorothioate, and wherein ademA-GalNAc =

96. The RNAi oligonucleotide of claim 1, wherein the sense strand comprises the sequence 5'-

[mAs] [mG] [fU] [mU] [fG] [mU] [mU] [f A] [mG] [fU] [mG] [f ] [fU] [mU] [fU] [mG] [fC] [mU] [mA] [mA] [mG] [mC] [mA] [mG] [mC] [mC] [mG] [adem A-GalNAc] [adem A-GalNAc] [adem A- GalNAc][mG][mG][mC][mU][mG][mC]-3’ (SEQ ID NO: 1640), wherein the antisense strand comprises the sequence 5'-[MePhosphonate-4O-mUs][fUs][fA][fG][fC][mA][fA][mA][mU][fC] [mA][mC][mU][fA][mA][fC][mA][mA][fC][mUs][mGs][mG]-3' (SEQ ID NO: 1675), and wherein mC, mA, mG, mU = 2'-0Me ribonucleosides; fA, fC, fG, fU = 2'-F ribonucleosides; s = phosphorothioate, and wherein ademA-GalNAc =

97. The RNAi oligonucleotide of claim 1, wherein the sense strand comprises the sequence 5'- [ademCs-Cie] [mA] [fG] [mU] [fC][mA] [mU] [fG] [mA] [fC] [mA] [fU] [fU] [mU] [fC] [mU] [fC][mAs][mAs][mA]-3’ (SEQ ID NO: 1682), wherein the antisense strand comprises the sequence 5 [MePhosphonate-4O-mU s] [fU s] [fU] [fG] [fA] [mG] [fA] [mA] [mA] [fU] [mG] [mU] [mC][fA][mU][fG][mA][mC][fU][mGs][mGs][mG]-3' (SEQ ID NO: 1656), and wherein mC, mA, mG, mU = 2'-OMe ribonucleosides; fA, fC, fG, fU = 2'-F ribonucleosides; s = phosphorothioate, and [ademCs-Cis]=

98. A pharmaceutical composition comprising the RNAi oligonucleotide of any one of claims

1 to 97, and a pharmaceutically acceptable carrier, delivery agent, or excipient.

99. A method for treating a subject having a disease, disorder, or condition associated with SNCA gene expression, the method comprising administering to the subject a therapeutically effective amount of the RNAi oligonucleotide of any one of claim 1-97, or pharmaceutical composition of claim 98, thereby treating the subject.

100. A method of delivering an oligonucleotide to a subject, the method comprising administering to the subject the pharmaceutical composition of claim 98.

101. A method for reducing SNCA gene expression in a cell, a population of cells, or a subject, the method comprising the steps of: i. contacting the cell or the population of cells with the RNAi oligonucleotide of any one of claims 1 to 97, or the pharmaceutical composition of claim 98; or ii. administering to the subject the RNAi oligonucleotide of any one of claims 1 to 97, or the pharmaceutical composition of claim 98.

102. The method of claim 101, wherein reducing SNCA gene expression comprises reducing an amount or level of SNCA mRNA, an amount or level of SNCA protein, a SNCA activity/function, or a combination thereof.

103. The method of claim 101 or 102, wherein the subject has a disease, disorder, or condition associated with SNCA gene expression.

104. The method of claim 99 or 103, wherein the disease, disorder, or condition associated with SNCA gene expression is multiple system atrophy, dementia with Lewy bodies, or Parkinson disease.

105. The method of any one of claims 99-104, wherein SNCA gene expression is reduced in tissue of one or more regions of the central nervous system (CNS), and wherein the tissue is associated with Parkinson disease.

106. The method of claim 105, wherein tissue associated with Parkinson disease is selected from: putamen, midbrain tegmentum, substantia nigra, pons, and medulla.

107. The method of any one of claims 99-104, wherein SNCA gene expression is reduced in tissue of one or more regions of the central nervous system (CNS), and wherein the tissue is associated with multiple system atrophy.

108. The method of claim 107, wherein tissue associated with multiple system atrophy is selected from: caudate nucleus, putamen, midbrain tegmentum, substantia nigra, pons, cerebellar cortex, cerebellar white matter, medulla, cervical spinal cord, thoracic spinal cord, and lumbar spinal cord.

109. The method of any one of claims 99-104, wherein SNCA gene expression is reduced in one or more regions of the central nervous system (CNS) selected from: cervical spinal cord, thoracic spinal cord, lumbar spinal cord, frontal cortex, temporal cortex, cerebellum, midbrain, occipital cortex, parietal cortex, hippocampus, caudate nucleus, thalamus, brainstem, motor cortex, globus pallidus, midbrain tegmentum, substantia nigra, pons, cerebellar white matter, and cerebellar dentate nucleus.

110. The method of any one of claims 99-109, wherein the RNAi oligonucleotide, or pharmaceutical composition, is administered in combination with a second composition or therapeutic agent.

111. Use of the RNAi oligonucleotide of any one of claims 1 to 97, or the pharmaceutical composition of claim 98, in the manufacture of a medicament for the treatment of a disease, disorder, or condition associated with SNCA gene expression.

112. The RNAi oligonucleotide of any one of claims 1 to 97, or the pharmaceutical composition of claim 98, for use, or adaptable for use, in the treatment of a disease, disorder, or condition associated with SNCA gene expression.

113. A kit comprising the RNAi oligonucleotide of any one of claims 1 to 97, an optional pharmaceutically acceptable carrier, and a package insert comprising instructions for administration to a subject having a disease, disorder, or condition associated with SNCA gene expression.

114. The use of claim 111, the RNAi oligonucleotide or pharmaceutical composition for use, or adaptable for use, of claim 112, or the kit of claim 113, wherein the disease, disorder, or condition associated with SNCA gene expression is multiple system atrophy, dementia with Lewy bodies, and Parkinson disease.

Description:
COMPOSITIONS AND METHODS FOR INHIBITING SNCA EXPRESSION

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority under 35 U.S.C. § 119(e) from U.S. Provisional Application No. 63/364,639, filed May 13, 2022, which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

[0002] The disclosure relates generally to biology and medicine, and more particularly it relates to oligonucleotides and compositions including the same for inhibiting or reducing (i.e., modulating) alpha-synuclein (SNCA) gene expression, as well as their use for treating diseases and disorders associated with SNCA gene expression.

BACKGROUND

[0003] Synapses are functional compartment between cells where information is passed from one cell to another in the brain. SNCA is a protein primarily found in the brain which regulates synaptic vesicle trafficking and release of neurotransmitters. Among other functions, SNCA acts as a molecular chaperone to assist in folding synaptic fusion components e.g., SNAREs). Mutations (e.g., insertions and mismatches) in SNCA that alter SNCA function and expression or general aberrant expression of SNCA are known causes of several diseases impacting the CNS (e.g, Parkinson’s disease and multiple system atrophy). Strategies for targeting the SNCA gene to prevent such diseases are needed.

[0004] The mammalian CNS is a complex system of tissues, including cells, fluids and chemicals that interact in concert to enable a wide variety of functions, including movement, navigation, cognition, speech, vision, and emotion. Unfortunately, a variety of diseases and disorders of the CNS are known e.g., neurological disorders) and affect or disrupt some or all of these functions. Typically, treatments for diseases and disorders of the CNS have been limited to small molecule drugs, antibodies and/or to adaptive or behavioral therapies. There exists an ongoing need to develop treatment of diseases and disorders of the CNS associated with inappropriate gene expression.

SUMMARY OF DISCLOSURE

[0005J To address this need, the disclosure describes compositions for and methods of treating a disease, disorder, or condition associated with SNCA gene expression. The present disclosure is based, at least in part, on discovering RNAi oligonucleotides that effectively target and reduce SNCA gene expression in tissues of the CNS. Specifically, target sequences within SNCA mRNA were identified and oligonucleotides that bind to these target sequences and inhibiting SNCA mRNA expression were generated. As demonstrated herein, the oligonucleotides inhibited human and non-human primate (NHP) SNCA gene expression in CNS tissue. Further, SNCA mRNA expression was reduced in CNS tissue associated with Parkinson’s disease or multiple system atrophy lipid-conjugated SNCA -targeting oligonucleotides. Without being bound by theory, the oligonucleotides described herein are useful for treating a disease, disorder, or condition associated with SNCA gene expression.

[0006] Accordingly, in some aspects, the disclosure provides a RNAi oligonucleotide for reducing SNCA gene expression, the oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a duplex region, wherein the antisense strand comprises a region of complementarity to a SNCA mRNA target sequence of any one of SEQ ID NOs: 1683-2066, and wherein the region of complementarity is at least about 15 contiguous nucleotides in length.

[0007] In any of the foregoing or related aspects, the sense strand is about 15 to about 50 nucleotides in length. In some aspects, the sense strand is about 18 to about 36 nucleotides in length. In some aspects, the antisense strand is about 15 to about 30 nucleotides in length. In some aspects, the antisense strand is 22 nucleotides in length, and the antisense strand and the sense strand form a duplex region of at least 19 nucleotides in length, optionally at least 20 nucleotides in length. In some aspects, the region of complementarity is at least 19 contiguous nucleotides in length. In some aspects, the region of complementarity is at least 20 contiguous nucleotides in length. [0008] In other aspects, the disclosure provides a double-stranded (ds) RNAi oligonucleotide for reducing SNCA gene expression, the oligonucleotide comprising:

(i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand comprises a nucleotide sequence comprising a region of complementarity to a SNCA mRNA target sequence, wherein the region of complementarity is selected from SEQ ID NOs: 2067-2450, and

(ii) a sense strand of 19-50 nucleotides in length comprising a region of complementarity to the antisense strand, wherein the antisense and sense strands are separate strands which form an asymmetric duplex region having an overhang of 1 -4 nucleotides at the 3’ terminus of the antisense strand.

[0009] In some aspects, the 3’ end of the sense strand comprises a stem-loop set forth as Sl-L- S2, wherein SI is complementary to S2, and wherein L forms a loop between SI and S2 of 3-5 nucleotides in length. In some aspects, L is a triloop or a tetraloop. In some aspects, L is a tetraloop. In some aspects, the tetraloop comprises the sequence 5’-GAAA-3’. In some aspects, the S I and S2 are 1-10 nucleotides in length and have the same length. In some aspects, SI and S2 are 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, or 10 nucleotides in length. In some aspects, SI and S2 are 6 nucleotides in length. In some aspects, the stem-loop comprises the sequence 5’- GCAGCCGAAAGGCUGC-3’ (SEQ ID NO: 1680).

[0010] In other aspects, the oligonucleotides comprise a blunt end. In some aspects, the blunt end comprises the 3’ end of the sense strand. In some aspects, the sense strand is 20 to 22 nucleotides. In some aspects, the sense strand is 20 nucleotides.

[0011] In any of the foregoing or related aspects, the antisense strand comprises a 3’ overhang sequence of one or more nucleotides in length. In some aspects, the overhang comprises purine nucleotides. In some aspects, the 3’ overhang sequence is 2 nucleotides in length. In some aspects, the 3’ overhang is selected from AA, GG, AG, and GA. In some aspects, the overhang is GG or AA. In some aspects, the overhang is GG.

[0012] In any of the foregoing or related aspects, the oligonucleotide comprises at least one modified nucleotide. In some aspects, the modified nucleotide comprises a 2'-modification. In some aspects, the 2'-modification is a modification selected from 2 '-aminoethyl, 2'-fluoro (2’-F), 2'-0-methyl (2’-0Me), 2 '-0-m ethoxy ethyl, and 2'-deoxy-2'-fluoro-P-d-arabinonucleic acid. In some aspects, the modification is a 2’ -modification selected from 2’-F and 2’-0Me. In some aspects, about 18% to about 23%, or 18%, 19%, 20%, 21%, 22% or 23%, of the nucleotides of the sense strand comprise a 2’-F modification. In some aspects, about 38-43%, 38%, 39%, 40%, 41%, 42% or 43% of the nucleotides of the sense strand comprise a 2’-F modification. In some aspects, about 25% to about 35%, or 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34% or 35%, of the nucleotides of the antisense strand comprise a 2’-F modification. In some aspects, about 25% to about 35%, or 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34% or 35%, of the nucleotides of the oligonucleotide comprise a 2’-F modification. In some aspects, about 35% to about 45%, or 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44% or 45%, of the nucleotides of the oligonucleotide comprise a 2’-F modification. In some aspects, the sense strand comprises 36 nucleotides with positions 1-36 from 5’ to 3’, wherein each of positions 3, 5, 8, 10, 12, 13, 15, and 17 comprise a 2’-F modification. In some aspects, the sense strand comprises 20 nucleotides with positions 1-20 from 5’ to 3’, wherein each of positions 3, 5, 8, 10, 12, 13, 15, and 17 comprise a 2’-F modification. In some aspects, the antisense strand comprises 22 nucleotides with positions 1-22 from 5’ to 3’, and wherein each of positions 2, 3, 4, 5, 7, 10 14, 16 and 19 comprise a 2’-F modification. In some aspects, the remaining nucleotides comprise a 2’-0Me modification.

[0013] In any of the foregoing or related aspects, the oligonucleotide comprises at least one modified internucleotide linkage. In some aspects, the at least one modified intemucleotide linkage is a phosphor othioate linkage. In some aspects, the antisense strand comprises a phosphorothioate linkage (i) between positions 1 and 2, and between positions 2 and 3; or (ii) between positions 1 and 2, between positions 2 and 3, and between positions 3 and 4, wherein positions are numbered 1 -4 from 5 ’ to 3 ’ . In some aspects, the antisense strand is 22 nucleotides in length, and the antisense strand comprises a phosphorothioate linkage between positions 20 and 21 and between positions 21 and 22, wherein positions are numbered 1-22 from 5’ to 3’. In some aspects, the sense strand comprises a phosphorothioate linkage between positions 1 and 2, wherein positions are numbered 1-2 from 5’ to 3’. In some aspects, the sense strand is 20 nucleotides in length, and the sense strand comprises a phosphorothioate linkage between positions between positions 1 and 2, between positions 18 and 19 and between positions 19 and 20, wherein positions are numbered 1-20 from 5’ to 3’.

[0014] In any of the foregoing or related aspects, the 4'-carbon of the sugar of the 5 '-nucleotide of the antisense strand comprises a phosphate analog. In some aspects, the phosphate analog is oxymethyl phosphonate, vinyl phosphonate or malonyl phosphonate, optionally wherein the phosphate analog is a 4’-phosphate analog comprising 4’-oxymethylphosphonate.

[0015] In any of the foregoing or related aspects, at least one nucleotide of the oligonucleotide is conjugated to one or more targeting ligands. Tn some aspects, each targeting ligand comprises a carbohydrate, amino sugar, lipid, cholesterol, or polypeptide. In some aspects, the stem-loop comprises one or more targeting ligands conjugated to one or more nucleotides of the stem-loop. In some aspects, the one or more targeting ligands is conjugated to one or more nucleotides of the loop. In some aspects, the loop comprises 4 nucleotides numbered 1-4 from 5’ to 3’, wherein nucleotides at positions 2, 3, and 4 each comprise one or more targeting ligands, wherein the targeting ligands are the same or different. In some aspects, each targeting ligand comprises a N- acetylgalactosamine (GalNAc) moiety. In some aspects, the GalNAc moiety is a monovalent GalNAc moiety, a bivalent GalNAc moiety, a trivalent GalNAc moiety or a tetravalent GalNAc moiety. In some aspects, up to 4 nucleotides of L of the stem-loop are each conjugated to a monovalent GalNAc moiety.

[0016] In other aspects, the one or more targeting ligands is a lipid moiety. In some aspects, the lipid moiety is conjugated to the 5’terminal nucleotide of the sense strand. In some aspects, the lipid moiety is a hydrocarbon chain. In some aspects, the hydrocarbon chain is a Cs-Cso hydrocarbon chain. In some aspects, the hydrocarbon chain is a Ci6 hydrocarbon chain. In some aspects, the Ci6 hydrocarbon chain is represented by:

. In some aspects, the lipid moiety is conjugated to the 2’ carbon of the ribose ring of the 5’ terminal nucleotide.

[0017] In any of the foregoing or related aspects, the region of complementarity is fully complementary to the SNCA mRNA target sequence at nucleotide positions 2-8 of the antisense strand, wherein nucleotide positions are numbered 5’ to 3’. In some aspects, the region of complementarity is fully complementary to the SNCA mRNA target sequence at nucleotide positions 2-11 of the antisense strand, wherein nucleotide positions are numbered 5’ to 3’.

[0018] In any of the foregoing or related aspects, the sense strand comprises a nucleotide sequence of any one of SEQ ID NOs: 1537-1571 and 1681. In some aspects, the antisense strand comprises a nucleotide sequence of any one of SEQ ID NOs: 1572-1606. In some aspects, the sense strand and antisense strands comprise nucleotide sequences selected from the group consisting of: a) SEQ ID NOs: 1537 and 1572, respectively; b) SEQ ID NOs: 1538 and 1573, respectively; c) SEQ ID NOs: 1539 and 1574, respectively; d) SEQ ID NOs: 1540 and 1575, respectively; e) SEQ ID NOs: 1541 and 1576, respectively; f) SEQ ID NOs: 1542 and 1577, respectively; g) SEQ ID NOs: 1543 and 1578, respectively; h) SEQ ID NOs: 1544 and 1579, respectively; i) SEQ ID NOs: 1545 and 1580, respectively; j) SEQ ID NOs: 1546 and 1581, respectively; k) SEQ ID NOs: 1547 and 1582, respectively; l) SEQ ID NOs: 1548 and 1583, respectively; m) SEQ ID NOs: 1549 and 1584, respectively; n) SEQ ID NOs: 1550 and 1585, respectively; o) SEQ ID NOs: 1551 and 1586, respectively; p) SEQ ID NOs: 1552 and 1587, respectively; q) SEQ ID NOs: 1553 and 1588, respectively; r) SEQ ID NOs: 1554 and 1589, respectively; s) SEQ ID NOs: 1555 and 1590, respectively; t) SEQ ID NOs: 1556 and 1591, respectively; u) SEQ ID NOs: 1557 and 1592, respectively; v) SEQ ID NOs: 1558 and 1593, respectively; w) SEQ ID NOs: 1559 and 1594, respectively; x) SEQ ID NOs: 1560 and 1595, respectively; y) SEQ ID NOs: 1561 and 1596, respectively; z) SEQ ID NOs: 1562 and 1597, respectively; aa) SEQ ID NOs: 1563 and 1598, respectively; bb) SEQ ID NOs: 1564 and 1599, respectively; cc) SEQ ID NOs: 1565 and 1600, respectively; dd) SEQ ID NOs: 1566 and 1601, respectively; ee) SEQ ID NOs: 1567 and 1602, respectively; ff) SEQ ID NOs: 1568 and 1603, respectively; gg) SEQ ID NOs: 1569 and 1604, respectively; hh) SEQ ID NOs: 1570 and 1605, respectively; ii) SEQ ID NOs: 1571 and 1606, respectively; and jj) SEQ ID NOs: 1681 and 1586, respectively.

[0019] In some aspects, the sense strand and antisense strands comprise nucleotide sequences selected from the group consisting of: a) SEQ ID NOs: 1540 and 1575, respectively; b) SEQ ID NOs: 1544 and 1579, respectively; c) SEQ ID NOs: 1546 and 1581, respectively; d) SEQ ID NOs: 1551and 1586, respectively; e) SEQ ID NOs: 1552 and 1587, respectively; f) SEQ ID NOs: 1553 and 1588, respectively; g) SEQ ID NOs: 1558 and 1594, respectively; h) SEQ ID NOs: 1560 and 1595, respectively; i) SEQ ID NOs: 1564 and 1599, respectively; j) SEQ ID NOs: 1565 and 1600, respectively; k) SEQ ID NOs: 1566 and 1601, respectively; l) SEQ ID NOs: 1570 and 1605, respectively; and m) SEQ ID NOs: 1681 and 1586, respectively. [0020] In some aspects, the sense strand and antisense strands comprise nucleotide sequences selected from the group consisting of: a) SEQ ID NOs: 1553 and 1588, respectively; b) SEQ ID NOs: 1560 and 1595, respectively; c) SEQ ID NOs: 1564 and 1599, respectively; d) SEQ ID NOs: 1551 and 1586, respectively; e) SEQ ID NOs: 1570 and 1605, respectively; and, f) SEQ ID NOs: 1681 and 1586, respectively.

[0021] In some aspects, the sense strand comprises the nucleotide sequence as set forth in SEQ ID NO: 1553, and the antisense strand comprises the nucleotide sequence as set forth in SEQ ID NO: 1588. In some aspects, the sense strand comprises the nucleotide sequence as set forth in SEQ ID NO: 1560, and the antisense strand comprises the nucleotide sequence as set forth in SEQ ID NO: 1595. In some aspects, the sense strand comprises the nucleotide sequence as set forth in SEQ ID NO: 1564, and the antisense strand comprises the nucleotide sequence as set forth in SEQ ID NO: 1599. In some aspects, the sense strand comprises the nucleotide sequence as set forth in SEQ ID NO: 1551, and the antisense strand comprises the nucleotide sequence as set forth in SEQ ID NO: 1586. In some aspects, the sense strand comprises the nucleotide sequence as set forth in SEQ ID NO: 1570, and the antisense strand comprises the nucleotide sequence as set forth in SEQ ID NO: 1605. In some aspects, the sense strand comprises the nucleotide sequence as set forth in SEQ ID NO: 1681, and the antisense strand comprises the nucleotide sequence as set forth in SEQ ID NO: 1586.

[0022] In any of the foregoing or related aspects, the antisense strand is 22 nucleotides in length. In some aspects, the antisense strand comprises a nucleotide sequence comprising the nucleotide sequence selected from SEQ ID NOs: 1588, 1595, 1599, 1586, and 1605. In some aspects, the sense strand is 36 nucleotides in length. In some aspects, the sense strand comprises a nucleotide sequence comprising the nucleotide sequence selected from SEQ ID NOs: 1865, 1721, 1847, 1846, and 1955. In some aspects, the sense strand comprises a nucleotide sequence selected from SEQ ID NOs: 1553, 1560, 1564, 1551, and 1570. [0023] In any of the foregoing or related aspects, the sense strand comprises the nucleotide sequence of any one of SEQ ID NOs: 1607-1641, and 1682. In some aspects, the antisense strand comprises a nucleotide sequence of any one of SEQ ID NOs: 1642-1676.

[0024] In some aspects, the sense strand and antisense strands comprise nucleotide sequences selected from the group consisting of: a) SEQ ID NOs: 1607 and 1642, respectively; b) SEQ ID NOs: 1608 and 1643, respectively; c) SEQ ID NOs: 1609 and 1644, respectively; d) SEQ ID NOs: 1610 and 1645, respectively; e) SEQ ID NOs: 1611 and 1646, respectively; f) SEQ ID NOs: 1612 and 1647, respectively; g) SEQ ID NOs: 1613 and 1648, respectively; h) SEQ ID NOs: 1614 and 1649, respectively; i) SEQ ID NOs: 1615 and 1650, respectively; j) SEQ ID NOs: 1616 and 1651, respectively; k) SEQ ID NOs: 1617 and 1652, respectively; l) SEQ ID NOs: 1618 and 1653, respectively; m) SEQ ID NOs: 1619 and 1654, respectively; n) SEQ ID NOs: 1620 and 1655, respectively; o) SEQ ID NOs: 1621 and 1656, respectively; p) SEQ ID NOs: 1622 and 1657, respectively; q) SEQ ID NOs: 1623 and 1658, respectively; r) SEQ ID NOs: 1624 and 1659, respectively; s) SEQ ID NOs: 1625 and 1660, respectively; t) SEQ ID NOs: 1626 and 1661, respectively; u) SEQ ID NOs: 1627 and 1662, respectively; v) SEQ ID NOs: 1628 and 1663, respectively; w) SEQ ID NOs: 1629 and 1664, respectively; x) SEQ ID NOs: 1630 and 1665, respectively; y) SEQ ID NOs: 1631 and 1666, respectively; z) SEQ ID NOs: 1632 and 1667, respectively; aa) SEQ ID NOs: 1633 and 1668, respectively; bb) SEQ ID NOs: 1634 and 1669, respectively; cc) SEQ ID NOs: 1635 and 1670, respectively; dd) SEQ ID NOs: 1636 and 1671, respectively; ee) SEQ ID NOs: 1637 and 1672, respectively; ff) SEQ ID NOs: 1638 and 1673, respectively; gg) SEQ ID NOs: 1639 and 1674, respectively; hh) SEQ ID NOs: 1640 and 1675, respectively; ii) SEQ ID NOs: 1641 and 1676, respectively; and, jj) SEQ ID NOs: 1682 and 1656, respectively.

[0025] In some aspects, the sense strand and antisense strands comprise nucleotide sequences selected from the group consisting of: a) SEQ ID NOs: 1610 and 1645, respectively; b) SEQ ID NOs: 1614 and 1649, respectively; c) SEQ ID NOs: 1616 and 1651, respectively; d) SEQ ID NOs: 1621 and 1656, respectively; e) SEQ ID NOs: 1622 and 1657, respectively; f) SEQ ID NOs: 1623 and 1658, respectively; g) SEQ ID NOs: 1629 and 1664, respectively; h) SEQ ID NOs: 1630 and 1665, respectively; i) SEQ ID NOs: 1634 and 1669, respectively; j) SEQ ID NOs: 1635 and 1670, respectively; k) SEQ ID NOs: 1636 and 1671, respectively; l) SEQ ID NOs: 1640 and 1675, respectively; and, m) SEQ ID NOs: 1682 and 1656, respectively.

[0026] In some aspects, the sense strand and antisense strands comprise nucleotide sequences selected from the group consisting of: a) SEQ ID NOs: 1623 and 1658, respectively; b) SEQ ID NOs: 1630 and 1665, respectively; c) SEQ ID NOs: 1634 and 1669, respectively; d) SEQ ID NOs: 1621 and 1656, respectively; e) SEQ ID NOs: 1640 and 1675, respectively; and, f) SEQ ID NOs: 1682 and 1656, respectively.

[0027] In some aspects, the sense strand comprises the nucleotide sequence as set forth in SEQ ID NO: 1623, and the antisense strand comprises the nucleotide sequence as set forth in SEQ ID NO: 1658. In some aspects, the sense strand comprises the nucleotide sequence as set forth in SEQ ID NO: 1630, and the antisense strand comprises the nucleotide sequence as set forth in SEQ ID NO: 1665. In some aspects, the sense strand comprises the nucleotide sequence as set forth in SEQ ID NO: 1634, and the antisense strand comprises the nucleotide sequence as set forth in SEQ ID NO: 1669. In some aspects, the sense strand comprises the nucleotide sequence as set forth in SEQ ID NO: 1621, and the antisense strand comprises the nucleotide sequence as set forth in SEQ ID NO: 1656. In some aspects, the sense strand comprises the nucleotide sequence as set forth in SEQ ID NO: 1640, and the antisense strand comprises the nucleotide sequence as set forth in SEQ ID NO: 1676. In some aspects, the sense strand comprises the nucleotide sequence as set forth in SEQ ID NO: 1682, and the antisense strand comprises the nucleotide sequence as set forth in SEQ ID NO: 1656.

[0028] In some aspects, the sense strand comprises the sequence and all of the modifications of 5 [mC s] [mA] [fG] [mC] [f A] [mG] [mU] [fG] [mA] [fU] [mU] [fG] [fA] [mA] [fG] [mU] [f A] [mU] [mC] [mA] [mG] [mC] [mA] [mG] [mC] [mC] [mG] [adem A-GalNAc] [adem A-GalNAc] [adem A- GalNAc][mG][mG][mC][mU][mG][mC]-3’ (SEQ ID NO: 1623), and wherein the antisense strand comprises the sequence and all of the modifications of 5'-[MePhosphonate-4O- mUs] [fGs] [fA] [fU] [fA] [mC] [fU] [mU] [mC] [f A] [mA] [mU] [mC] [fA] [mC] [fU] [mG] [mC] [fU] [m Gs][mGs][mG]-3' (SEQ ID NO: 1658), wherein mC, mA, mG, mU = 2'-OMe ribonucleosides; fA, fC, fG, fU = 2'-F ribonucleosides; s = phosphorothioate, and wherein ademA-GalNAc =

[0029] In some aspects, the sense strand comprises the sequence and all of the modifications of 5 [m As] [mG] [f A] [mG] [fC] [mA] [mA] [fG] [mU] [fG] [mA] [fC] [f A] [mA] [f A] [mU] [fG] [mU] [mU]

[mA] [mG] [mC] [mA] [mG] [mC] [mC] [mG] [adem A-GalNAc] [adem A-GalNAc] [adem A-

GalNAc][mG][mG][mC][mU][mG][mC]-3’ (SEQ ID NO: 1630), and wherein the antisense strand comprises the sequence and all of the modifications of 5'-[MePhosphonate-4O- mUs] [f As] [fA] [fC] [f A] [mU] [fU] [mU] [mG] [fU] [mC] [m A] [mC] [fU] [mU] [fG] [mC] [mU] [fC] [m Us][mGs][mG]-3' (SEQ ID NO: 1665), wherein mC, mA, mG, mU = 2'-0Me ribonucleosides; f , fC, fG, fU = 2'-F ribonucleosides; s = phosphorothioate, and wherein ademA-GalNAc =

[0030] In some aspects, the sense strand comprises the sequence and all of the modifications of 5 [m As] [mG] [fU] [mC] [fA] [mU] [mG] [f A] [mC] [f A] [mU] [fU] [fU] [mC] [fU] [mC] [f A] [mA] [mA] [mA] [mG] [mC] [mA] [mG] [mC] [mC] [mG] [adem A-GalNAc] [adem A-GalNAc] [adem A- GalNAc][mG][mG][mC][mU][mG][mC]-3’ (SEQ ID NO: 1634), and wherein the antisense strand comprises the sequence and all of the modifications of 5'-[MePhosphonate-4O- mUs] [fUs] [fU] [fU] [fG] [mA] [fG] [mA] [mA] [f A] [mU] [mG] [mU] [fC ] [mA] [fU] [mG] [mA] [fC] [m Us][mGs][mG]-3' (SEQ ID NO: 1669), wherein mC, mA, mG, mU = 2'-OMe ribonucleosides; fA, fC, fG, fU = 2'-F ribonucleosides; s = phosphorothioate, and wherein ademA-GalNAc =

[0031] In some aspects, the sense strand comprises the sequence and all of the modifications of 5 '-[mCs] [mA] [fG] [mU] [fC] [mA] [mU] [fG] [mA] [fC] [mA] [fU] [fU] [mU] [fC] [mU] [fC] [mA] [mA] [mA] [mG] [mC] [mA] [mG] [mC] [mC] [mG] [adem A-GalNAc] [adem A-GalNAc] [adem A- GalNAc][mG][mG][mC][mU][mG][mC]-3’ (SEQ ID NO: 1621), and wherein the antisense strand comprises the sequence and all of the modifications of 5'-[MePhosphonate-4O- mUs] [fUs] [fU] [fG] [fA] [mG] [f A] [mA] [mA] [fU] [mG] [mU] [mC] [f A] [mU] [fG] [mA] [mC] [fU] [m Gs][mGs][mG]-3' (SEQ ID NO: 1656), wherein mC, mA, mG, mU = 2'-OMe ribonucleosides; fA, fC, fG, fU = 2'-F ribonucleosides; s = phosphorothioate, and wherein ademA-GalNAc =

[0032] In some aspects, the sense strand comprises the sequence and all of the modifications of 5 [m As] [mG] [fU] [mU] [fG] [mU] [mU] [f A] [mG] [fU] [mG] [f A] [fU] [mU] [fU] [mG] [fC] [mU] [mA]

[mA] [mG] [mC] [mA] [mG] [mC] [mC] [mG] [adem A-GalNAc] [adem A-GalNAc] [adem A-

GalNAc][mG][mG][mC][mU][mG][mC]-3’ (SEQ ID NO: 1640), and wherein the antisense strand comprises the sequence and all of the modifications of 5'-[MePhosphonate-4O- mUs] [fUs] [fA] [fG] [fC] [mA] [fA] [mA] [mU][fC] [mA] [mC] [mU] [fA] [mA] [fC] [mA] [mA] [fC] [m Us][mGs][mG]-3' (SEQ ID NO: 1675), wherein mC, mA, mG, mU = 2'-0Me ribonucleosides; f , fC, fG, fU = 2'-F ribonucleosides; s = phosphorothioate, and wherein ademA-GalNAc =

In some aspects, the sense strand comprises the sequence and all of the modifications of 5'- [ademCs-Ci 6 ] [mA] [fG] [mU] [fC][mA] [mU] [fG] [mA] [fC] [mA] [fU] [fU] [mU] [fC] [mU] [fC] [mAs] [mAs][mA]-3’ (SEQ ID NO: 1682), and wherein the antisense strand comprises the sequence and all of the modifications of 5'-[MePhosphonate-4O- mUs] [fUs] [fU] [fG] [fA] [mG] [f A] [mA] [mA] [fU] [mG] [mU] [mC] [f A] [mU] [fG] [mA] [mC] [fU] [m Gs][mGs][mG]-3' (SEQ ID NO: 1656), wherein mC, mA, mG, mU = 2'-OMe ribonucleosides; f , fC, fG, fU = 2'-F ribonucleosides; s = phosphorothioate, and [ademCs-Cifi]=

[0033] In some aspects, the disclosure provides a pharmaceutical composition comprising a RNAi oligonucleotide described herein, and a pharmaceutically acceptable carrier, delivery agent or excipient.

[0034] In other aspects, the disclosure provides a method for treating a subject having a disease, disorder, or condition associated with SNCA gene expression, the method comprising administering to the subject a therapeutically effective amount of a RNAi oligonucleotide described herein, or pharmaceutical composition thereof, thereby treating the subject.

[0035] In further aspects, the disclosure provides a method of delivering an oligonucleotide to a subject, the method comprising administering a pharmaceutical composition described herein to the subject.

[0036] In yet further aspects, the disclosure provides, a method for reducing SNCA gene expression in a cell, a population of cells or a subject, the method comprising the step of: i. contacting the cell or the population of cells with a RNAi oligonucleotide or pharmaceutical composition described herein; or ii. administering to the subject a RNAi oligonucleotide or pharmaceutical composition described herein. [0037] In some aspects, reducing SNCA gene expression comprises reducing an amount or level of SNCA mRNA, an amount or level of SNCA protein, or both. In some aspects, the subject has a disease, disorder, or condition associated with SNCA gene expression. In some aspects, the disease, disorder, or condition associated with SNCA expression is multiple system atrophy, dementia with Lewy bodies, or Parkinson disease.

[0038] In any of the foregoing or related aspects, a RNAi oligonucleotide or pharmaceutical composition described herein SNCA gene expression is reduced in tissue of one or more regions of the CNS, wherein the tissue is associated with Parkinson disease. Tn some aspects, the tissue associated with Parkinson disease is selected from: putamen, midbrain tegmentum, substantia nigra, pons, and medulla. In some aspects, SNCA gene expression is reduced in tissue of one or more regions of the CNS, wherein the tissue is associated with multiple system atrophy. In some aspects, tissue associated with multiple system atrophy is selected from: caudate nuclease, putamen, midbrain tegmentum, substantia nigra, pons, cerebellar cortex, cerebellar white matter, medulla, cervical spinal cord, thoracic spinal cord, and lumbar spinal cord. In some aspects, SNCA gene expression is reduced in one or more regions of the CNS selected from: cervical spinal cord, thoracic spinal cord, lumbar spinal cord, frontal cortex, temporal cortex, cerebellum, midbrain, occipital cortex, parietal cortex, hippocampus, caudate nucleus, thalamus, brainstem, motor cortex, globus pallidus, midbrain tegmentum, substantia nigra, pons, cerebellar white matter, and cerebellar dentate nucleus. In some aspects, SNCA gene expression is reduced in one or more regions of the CNS selected from: cervical spinal cord, thoracic spinal cord, lumbar spinal cord, frontal cortex, temporal cortex, cerebellum, midbrain, occipital cortex, parietal cortex, hippocampus, caudate nucleus, thalamus, brainstem, motor cortex, globus pallidus, midbrain tegmentum, substantia nigra, pons, cerebellar white matter, cerebellar dentate nucleus, LI dorsal root ganglion (DRG), L2 DRG, L3 DRG, L4 DRG, L5 DRG, L6 DRG, putamen, midbrain tegmentum, substantia nigra, pons, medulla, cerebellar cortex, and cerebellar white matter.

[0039] In any of the foregoing or related aspects, the RNAi oligonucleotide, or pharmaceutical composition, is administered in combination with a second composition or therapeutic agent. [0040] In other aspects, the disclosure provides use of aRNAi oligonucleotide or pharmaceutical composition described herein, in the manufacture of a medicament for the treatment of a disease, disorder, or condition associated with SNCA gene expression.

[0041] In further aspects, the disclosure provides a RNAi oligonucleotide or pharmaceutical composition described herein for use, or adaptable for use, in the treatment of a disease, disorder, or condition associated with SNCA expression.

[0042] In some aspects, the disclosure provides a kit comprising an RNAi oligonucleotide described herein, an optional pharmaceutically acceptable carrier, and a package insert comprising instructions for administration to a subject having a disease, disorder or condition associated with SNCA expression.

[0043] In any of the foregoing or related aspects, the disease, disorder, or condition associated with SNCA gene expression is multiple system atrophy, dementia with Lewy bodies, and Parkinson disease.

BRIEF DESCRIPTION OF THE FIGURES

[0044] FIGs. 1A and IB provide graphs depicting the percent (%) of human SNCA mRNA remaining in the liver of mice exogenously expressing human SNCA (hydrodynamic injection model) after treatment with GalN Ac-conjugated SNCA-targeting oligonucleotides. CD-I mice were dosed subcutaneously with 3 mg/kg of the indicated GalNAc-conjugated SNCA-targeting oligonucleotide formulated in PBS. Three days post-dose mice were hydrodynamically injected (HD I) with a DNA plasmid encoding human SNCA. The level of human SNCA mRNA was determined from livers collected 24 hours later. Hs-Mf = construct is human and monkey SNCA specific. Hs-Mf-Mm = construct is human, monkey, and mouse SNCA specific. SNCA-291 was used as a benchmark control.

[0045] FIGs. 2A and 2B provide graphs depicting the dose response of GalNAc-conjugated SNCA oligonucleotides selected based on inhibitory efficacy shown in FIGs. 1A-1B. The percent (%) of SNCA mRNA remaining in liver tissue was measured in CD-I HDI mice as described in FIGs. 1A-1B. Following injection with 0.3mg/kg, 1.0mg/kg, or 3mg/kg of the indicated GalNAc- conjugated SNCA oligonucleotide, percent (%) mRNA remaining was determined in two cohorts, FIG. 2A and FIG. 2B. Hs-Mf = construct is human and monkey SNCA specific. Hs-Mf-Mm= construct is human, monkey, and mouse SNCA specific.

[0046] FIGs. 3A-3S provide graphs depicting the percent (%) of non-human primate (NHP; Mf) SNCA mRNA remaining in the CNS of NHPs after treatment with GalNAc-conjugated SNCA- targeting oligonucleotides. NHPs were dosed by intra cistema magna (i.c.m) injection with 50 mg of the indicated GalNAc-conjugated SNCA-targeting oligonucleotide formulated in artificial cerebrospinal fluid (aCSF) on study days 0 and 7. The level of SNCA mRNA was determined relative to the percent (%) of SNCA mRNA remaining in aCSF treated animals. Central nervous tissues measured included frontal cortex (FIG. 3A), caudate nucleus (FIG. 3B), hippocampus (FIG. 3C), mid brain (FIG. 3D), parietal cortex (FIG. 3E), occipital cortex (FIG. 3F), thalamus (FIG. 3G), temporal cortex (FIG. 3H), cerebellum (FIG. 31), brainstem (FIG. 3J), cervical spinal cord (FIG. 3K), thoracic spinal cord (FIG. 3L), lumbar spinal cord (FIG. 3M), LI dorsal root ganglion (DRG) (FIG. 3N), L2 DRG (FIG. 30), L3 DRG (FIG. 3P), L4 DRG (FIG. 3Q), L5 DRG (FIG. 3R), and L6 DRG (FIG. 3S). GaLXC = GalNAc-conjugated SNCA-targeting oligonucleotide.

[0047] FIGs. 4A-4B provide graphs depicting the percent (%) of non-human primate (NHP; Mf) SNCA mRNA remaining (FIG. 4A) and concentration of oligonucleotide (FIG. 4B) in NHP CNS tissue associated with Parkinson’s disease. NHPs were intrathecally administered aCSF or SNCA- B15 conjugated to a Ci6 lipid. Tissue was collected and analyzed 28 days after administration of the oligonucleotide.

[0048] FIGs. 5A-5B provide graphs depicting the percent (%) of non-human primate (NHP; Mf) SNCA mRNA remaining (FIG. 5A) and concentration of oligonucleotide (FIG. 5B) in NHP CNS tissue associated with Multiple Systems Atrophy. NHPs were intrathecally administered aCSF or SNCA-B15 conjugated to a Ci6 lipid. Tissue was collected and analyzed 28 days after administration of the oligonucleotide.

DETAILED DESCRIPTION

[0049] According to some aspects, the disclosure provides oligonucleotides that reduce SNCA gene expression in the CNS. In some embodiments, the oligonucleotides provided herein are designed to treat diseases associated with SNCA expression in the CNS. In other embodiments, the disclosure provides methods of treating a disease associated with SNCA expression by reducing SNCA gene expression in cells (e.g., cells of the CNS).

Oligonucleotide Inhibitors of SNCA Expression

[0050] The disclosure provides, inter alia, oligonucleotides that inhibit SNCA gene expression e.g., RNAi oligonucleotides). In some embodiments, an oligonucleotide that inhibits SNCA gene expression is targeted to a SNCA mRNA.

SNCA Target Sequences

[0051] In some embodiments, an oligonucleotide herein (e.g., a RNAi oligonucleotide) is targeted to a target sequence comprising a SNCA mRNA. In some embodiments, the oligonucleotide described herein is targeted to a target sequence within a SNCA mRNA sequence. [0052] In some embodiments, the oligonucleotide described herein corresponds to a target sequence within a SNCA mRNA sequence. In some embodiments, the oligonucleotide, or a portion, fragment, or strand thereof (e.g., an antisense strand or a guide strand of a ds RNAi oligonucleotide) binds or anneals to a target sequence comprising SNCA mRNA, thereby inhibiting SNCA gene expression.

[0053] In some embodiments, the oligonucleotide is targeted to a SNCA target sequence for the purpose of inhibiting SNCA gene expression in vivo. In some embodiments, the amount or extent of inhibition of SNCA gene expression by an oligonucleotide targeted to a SNCA target sequence correlates with the potency of the oligonucleotide. In some embodiments, the amount or extent of inhibition of SNCA gene expression by an oligonucleotide targeted to a SNCA target sequence correlates with the amount or extent of therapeutic benefit in a subject or patient having a disease, disorder, or condition associated with SNCA gene expression treated with the oligonucleotide.

[0054] Through examination of the nucleotide sequence of mRNAs encoding SNCA, including mRNAs of multiple different species (e.g., human, cynomolgus monkey, and mouse; see, e.g., Example 1) and as a result of in vitro and in vivo testing (see, e.g., Examples 2-5), it has been discovered that certain nucleotide sequences of SNCA mRNA are more amenable than others to oligonucleotide-based inhibition and are thus useful as target sequences for the oligonucleotides herein. In some embodiments, a sense strand of an oligonucleotide (e.g., a RNAi oligonucleotide) described herein comprises a SNCA target sequence. In some embodiments, a portion or region of the sense strand of a ds oligonucleotide described herein comprises a SNCA target sequence. In some embodiments, the SNCA target sequence comprises, or consists of, a nucleotide sequence of any one of SEQ ID NOs: 1683-2066. In some embodiments, the SNCA target sequence comprises, or consists of, a nucleotide sequence of any one of SEQ ID Nos: 1781, 1782, 1796, 1798, 1802, 1808, 1814, 1817, 1713, 1718, 1726, 1830, 1839, 1742, 1846, 1852, 1865, 1784, 1804, 1721 , 1822, 1840, 1735, 1847, 1855, 1864, 1901, 1902, 1938, 1947, 1955, 1964, 1973, and 1978. In some embodiments, the SNCA target sequence comprises, or consists of, a nucleotide sequence of any one of SEQ ID Nos: 1798, 1817, 1718, 1846, 1852, 1865, 1804, 1721, 1847, 1855, 1864, and 1955. In some embodiments, a SNCA target sequence comprises, or consists of, a nucleotide sequence of any one of SEQ ID NOs: 1865, 1721, 1847, 1846, and 1955. In some embodiments, the SNCA target sequence comprises the nucleotide sequence set forth in SEQ ID NO: 1865. In some embodiments, the SNCA target sequence comprises the nucleotide sequence set forth in SEQ ID NO: 1721. In some embodiments, the SNCA target sequence comprises the nucleotide sequence set forth in SEQ ID NO: 1847. In some embodiments, the SNCA target sequence comprises the nucleotide sequence set forth in SEQ ID NO: 1846. In some embodiments, the SNCA target sequence comprises the nucleotide sequence set forth in SEQ ID NO: 1955.

SNCA-Targeting Sequences

[0055] In some embodiments, an oligonucleotide herein has a region of complementarity to SNCA mRNA (e.g., within a target sequence of SNCA mRNA) for purposes of targeting the mRNA in cells and inhibiting its expression. In some embodiments, the oligonucleotide comprises a SNCA targeting sequence (e.g., an antisense strand or a guide strand of a ds oligonucleotide) having a region of complementarity that binds or anneals to the SNCA target sequence by complementary (Watson-Crick) base pairing. The targeting sequence or region of complementarity is generally of suitable length and base content to enable binding or annealing of the oligonucleotide (or a strand thereof) to a SNCA mRNA for purposes of inhibiting its expression. In some embodiments, the targeting sequence or region of complementarity is at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, or at least about 30 nucleotides in length. In some embodiments, the targeting sequence or region of complementarity is at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 nucleotides. In some embodiments, the targeting sequence or region of complementarity is about 12 to about 30 (e.g., 12 to 30, 12 to 22, 15 to 25, 17 to 21 , 18 to 27, 19 to 27, or 15 to 30) nucleotides in length. In some embodiments, the targeting sequence or region of complementarity is about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, the targeting sequence or region of complementarity is 18 nucleotides in length. In some embodiments, the targeting sequence or region of complementarity is 19 nucleotides in length. In some embodiments, the targeting sequence or region of complementarity is 20 nucleotides in length. In some embodiments, the targeting sequence or region of complementarity is 21 nucleotides in length. In some embodiments, the targeting sequence or region of complementarity is 22 nucleotides in length. In some embodiments, the targeting sequence or region of complementarity is 23 nucleotides in length. In some embodiments, the targeting sequence or region of complementarity is 24 nucleotides in length. In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity complementary to a sequence of any one of SEQ ID NOs: 1683-2066, and the targeting sequence or region of complementarity is 18 nucleotides in length. In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity complementary to a sequence of any one of SEQ ID NOs: 1683-2066, and the targeting sequence or region of complementarity is 19 nucleotides in length. In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity complementary to a sequence of any one of SEQ ID NOs: 1-384, and the targeting sequence or region of complementarity is 20 nucleotides in length. In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity complementary to a sequence of any one of SEQ ID NOs: 1-384, and the targeting sequence or region of complementarity is 21 nucleotides in length. In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity complementary to a sequence of any one of SEQ ID NOs: 1-384, and the targeting sequence or region of complementarity is 22 nucleotides in length. In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity complementary to a sequence of any one of SEQ ID NOs: 1-384, and the targeting sequence or region of complementarity is 23 nucleotides in length. In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity complementary to a sequence of any one of SEQ ID NOs: 1-384 and the targeting sequence or region of complementarity is 24 nucleotides in length.

[0056] In some embodiments, the oligonucleotide comprises a targeting sequence or a region of complementarity (e.g., an antisense strand or a guide strand of a ds oligonucleotide) that is fully complementary to a SNCA target sequence. In some embodiments, the targeting sequence or region of complementarity is partially complementary to a5 L4 target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity that is fully complementary to a sequence of any one of SEQ ID NOs: 1683-2066. In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity that is partially complementary to a sequence of any one of SEQ ID NOs: 1683-2066. In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity that is fully complementary to a sequence of any one of SEQ ID NOs: 1781, 1782, 1796, 1798, 1802, 1808, 1814, 1817, 1713, 1718, 1726, 1830, 1839, 1742, 1846, 1852, 1865, 1784, 1804, 1721, 1822, 1840, 1735, 1847, 1855, 1864, 1901, 1902, 1938, 1947, 1955, 1964, 1973, and 1978. In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity that is partially complementary to a sequence of any one of SEQ ID NOs: 1781, 1782, 1796, 1798, 1802, 1808, 1814, 1817, 1713, 1718, 1726, 1830, 1839, 1742, 1846, 1852, 1865, 1784, 1804, 1721, 1822, 1840, 1735, 1847, 1855, 1864, 1901, 1902, 1938, 1947, 1955, 1964, 1973, and 1978. In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity that is fully complementary to a sequence of any one of SEQ ID NOs: 1798, 1817, 1718, 1846, 1852, 1865, 1804, 1721, 1847, 1855, 1864, and 1955. In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity that is partially complementary to a sequence of any one of SEQ ID NOs: 1798, 1817, 1718, 1846, 1852, 1865, 1804, 1721, 1847, 1855, 1864, and 1955. In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity that is fully complementary to a sequence of any one of SEQ ID NOs: 1865, 1721, 1847, 1846, and 1955. In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity that is partially complementary to a sequence of any one of SEQ ID NOs: 1865, 1721, 1847, 1846, and 1955. In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity that is fully complementary to the sequence set forth in SEQ ID NO: 1865. In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity that is fully complementary to the sequence set forth in SEQ ID NO: 1721. In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity that is fully complementary to the sequence set forth in SEQ ID NO: 1847. In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity that is fully complementary to the sequence set forth in SEQ ID NO: 1846. In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity that is fully complementary to the sequence set forth in SEQ ID NO: 1955. In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity that is partially complementary to a sequence of any one of SEQ ID NOs: 1865. In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity that is partially complementary to the sequence of SEQ ID NO: 1721. In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity that is partially complementary to the sequence of SEQ ID NO: 1847. In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity that is partially complementary to the sequence of SEQ ID NO: 1846. In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity that is partially complementary to the sequence of SEQ ID NO: 1955.

[0057] In some embodiments, the oligonucleotide herein comprises a targeting sequence or region of complementarity that is complementary to a contiguous sequence of nucleotides comprising a SNCA mRNA, wherein the contiguous sequence of nucleotides is about 12 to about 30 nucleotides in length (e.g., 12 to 30, 12 to 28, 12 to 26, 12 to 24, 12 to 20, 12 to 18, 12 to 16, 14 to 22, 16 to 20, 18 to 20, or 18 to 19 nucleotides in length). In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity that is complementary to a contiguous sequence of nucleotides comprising a SNCA mRNA, wherein the contiguous sequence of nucleotides is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity that is complementary to a contiguous sequence of nucleotides comprising a SNCA mRNA, wherein the contiguous sequence of nucleotides is 19 nucleotides in length. In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity that is complementary to a contiguous sequence of nucleotides comprising a SNCA mRNA, wherein the contiguous sequence of nucleotides is 20 nucleotides in length.

[0058] In some embodiments, the oligonucleotide comprises a targeting sequence or a region of complementarity that is complementary to a contiguous sequence of nucleotides of any one of SEQ ID NOs: 1683-2066, optionally wherein the contiguous sequence of nucleotides is 19 nucleotides in length. In some embodiments, the oligonucleotide comprises a targeting sequence or a region of complementarity that is complementary to a contiguous sequence of nucleotides of any one of SEQ ID NOs: 1781, 1782, 1796, 1798, 1802, 1808, 1814, 1817, 1713, 1718, 1726, 1830, 1839, 1742, 1846, 1852, 1865, 1784, 1804, 1721, 1822, 1840, 1735, 1847, 1855, 1864, 1901, 1902, 1938, 1947, 1955, 1964, 1973, and 1978, optionally wherein the contiguous sequence of nucleotides is 19 nucleotides in length. In some embodiments, the oligonucleotide comprises a targeting sequence or a region of complementarity that is complementary to a contiguous sequence of nucleotides of any one of SEQ ID NOs: 1798, 1817, 1718, 1846, 1852, 1865, 1804, 1721, 1847, 1855, 1864, and 1955, optionally wherein the contiguous sequence of nucleotides is 19 nucleotides in length. In some embodiments, the oligonucleotide comprises a targeting sequence or a region of complementarity that is complementary to a contiguous sequence of nucleotides of SEQ ID NO: 1865, 1721, 1847, 1846, and 1955, optionally wherein the contiguous sequence of nucleotides is 19 nucleotides in length.

[0059] In some embodiments, the targeting sequence or region of complementarity of the oligonucleotide (e.g, a RNAi oligonucleotide) is complementary to contiguous nucleotides of a sequence as set forth in any one of SEQ ID NOs: 1683-2066 and spans the entire length of an antisense strand. In some embodiments, the targeting sequence or region of complementarity of the oligonucleotide is complementary to contiguous nucleotides of a sequence as set forth in any one of SEQ ID NOs: 1683-2066 and spans a portion of the entire length of an antisense strand. In some embodiments, the oligonucleotide comprises a region of complementarity (e.g., on an antisense strand of a ds oligonucleotide) that is at least partially (e.g., fully) complementary to a contiguous stretch of nucleotides spanning nucleotides 1-20 of a sequence as set forth in any one of SEQ ID NOs: 1683-2066. In some embodiments, the targeting sequence or region of complementarity of the oligonucleotide is complementary to contiguous nucleotides of a sequence as set forth in any one of SEQ ID NOs: 1-384 and spans the entire length of an antisense strand. In some embodiments, the region of complementarity of the oligonucleotide is complementary to contiguous nucleotides of a sequence as set forth in any one of SEQ ID NOs: 1-384 and spans a portion of the entire length of an antisense strand. In some embodiments, the oligonucleotide comprises a region of complementarity (e.g., on an antisense strand of a ds oligonucleotide) that is at least partially (e.g., fully) complementary to a contiguous stretch of nucleotides spanning nucleotides 1 to 19 of a sequence as set forth in any one of SEQ ID NOs: 1-384.

[0060] In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity having one or more base pair (bp) mismatches with the corresponding SNCA target sequence. In some embodiments, the targeting sequence or region of complementarity may have up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, etc. mismatches with the corresponding SNCA target sequence provided that the ability of the targeting sequence or region of complementarity to bind or anneal to the SNCA mRNA under appropriate hybridization conditions and/or the ability of the oligonucleotide to inhibit SNCA gene expression is maintained. Alternatively, in some embodiments, the targeting sequence or region of complementarity comprises no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 mismatches with the corresponding SNCA target sequence provided that the ability of the targeting sequence or region of complementarity to bind or anneal to the SNCA mRNA under appropriate hybridization conditions and/or the ability of the oligonucleotide to inhibit SNCA gene expression is maintained. In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity having 1 mismatch with the corresponding target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity having 2 mismatches with the corresponding target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity having 3 mismatches with the corresponding target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity having 4 mismatches with the corresponding target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity having 5 mismatches with the corresponding target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity more than one mismatch (e.g., 2, 3, 4, 5, or more mismatches) with the corresponding target sequence, wherein at least 2 (e.g., all) of the mismatches are positioned consecutively (e.g., 2, 3, 4, 5, or more mismatches in a row), or wherein the mismatches are interspersed in any position throughout the targeting sequence or region of complementarity. In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity more than one mismatch (e.g., 2, 3, 4, 5, or more mismatches) with the corresponding target sequence, wherein at least 2 (e.g., all) of the mismatches are positioned consecutively (e.g., 2, 3, 4, 5, or more mismatches in a row), or wherein at least one or more non-mismatched base pair is located between the mismatches, or a combination thereof.

[0061] In some embodiments, the oligonucleotide comprises a targeting sequence or a region of complementary that is complementary to a contiguous sequence of nucleotides of any one of SEQ ID NOs: 1683-2066, wherein the targeting sequence or region of complementarity may have up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, etc. mismatches with the corresponding SNCA target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or a region of complementary that is complementary to a contiguous sequence of nucleotides of any one of SEQ ID NOs: 1683-2066, wherein the targeting sequence or region of complementarity may have no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 mismatches with the corresponding SNCA target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or a region of complementary that is complementary to a contiguous sequence of nucleotides of any one of SEQ ID NOs: 1781, 1782, 1796, 1798, 1802, 1808, 1814, 1817, 1713, 1718, 1726, 1830, 1839, 1742, 1846, 1852, 1865, 1784, 1804, 1721, 1822, 1840, 1735, 1847, 1855, 1864, 1901, 1902, 1938, 1947, 1955, 1964, 1973, and 1978, wherein the targeting sequence or region of complementarity may have up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, etc. mismatches with the corresponding SNCA target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or a region of complementary that is complementary to a contiguous sequence of nucleotides of any one of SEQ ID NOs: 1781, 1782, 1796, 1798, 1802, 1808, 1814, 1817, 1713, 1718, 1726, 1830, 1839, 1742, 1846, 1852, 1865, 1784, 1804, 1721, 1822, 1840, 1735, 1847, 1855, 1864, 1901, 1902, 1938, 1947, 1955, 1964, 1973, and 1978, wherein the targeting sequence or region of complementarity may have no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 mismatches with the corresponding SNCA target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or a region of complementary that is complementary to a contiguous sequence of nucleotides of any one of SEQ ID NOs: 1798, 1817, 1718, 1846, 1852, 1865, 1804, 1721, 1847, 1855, 1864, and 1955, wherein the targeting sequence or region of complementarity may have up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, etc. mismatches with the corresponding SNCA target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or a region of complementary that is complementary to a contiguous sequence of nucleotides of any one of SEQ ID NOs: 1798, 1817, 1718, 1846, 1852, 1865, 1804, 1721, 1847, 1855, 1864, and 1955, wherein the targeting sequence or region of complementarity may have no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 mismatches with the corresponding SNCA target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or a region of complementary that is complementary to a contiguous sequence of nucleotides of any one of SEQ ID NOs: 1865, 1721, 1847, 1846, and 1955, wherein the targeting sequence or region of complementarity may have up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, etc. mismatches with the corresponding SNCA target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or a region of complementary that is complementary to a contiguous sequence of nucleotides of any one of SEQ ID NOs: 1865, 1721, 1847, 1846, and 1955, wherein the targeting sequence or region of complementarity may have no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 mismatches with the corresponding SNCA target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or a region of complementary that is complementary to a contiguous sequence of nucleotides of SEQ ID NO: 1865, wherein the targeting sequence or region of complementarity may have up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, etc. mismatches with the corresponding SNCA target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or a region of complementary that is complementary to a contiguous sequence of nucleotides of SEQ ID NO : 1865, wherein the targeting sequence or region of complementarity may have no more than 1 , no more than 2, no more than 3, no more than 4, or no more than 5 mismatches with the corresponding SNCA target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or a region of complementary that is complementary to a contiguous sequence of nucleotides of SEQ ID NO: 1721, wherein the targeting sequence or region of complementarity may have up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, etc. mismatches with the corresponding SNCA target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or a region of complementary that is complementary to a contiguous sequence of nucleotides of SEQ ID NO: 1721, wherein the targeting sequence or region of complementarity may have no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 mismatches with the corresponding SNCA target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or a region of complementary that is complementary to a contiguous sequence of nucleotides of SEQ ID NO: 1847, wherein the targeting sequence or region of complementarity may have up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, etc. mismatches with the corresponding SNCA target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or a region of complementary that is complementary to a contiguous sequence of nucleotides of SEQ ID NO: 1847, wherein the targeting sequence or region of complementarity may have no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 mismatches with the corresponding SNCA target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or a region of complementary that is complementary to a contiguous sequence of nucleotides of SEQ ID NO: 1846, wherein the targeting sequence or region of complementarity may have up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, etc. mismatches with the corresponding SNCA target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or a region of complementary that is complementary to a contiguous sequence of nucleotides of SEQ ID NO: 1846, wherein the targeting sequence or region of complementarity may have no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 mismatches with the corresponding SNCA target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or a region of complementary that is complementary to a contiguous sequence of nucleotides of SEQ TD NO: 1955, wherein the targeting sequence or region of complementarity may have up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, etc. mismatches with the corresponding SNCA target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or a region of complementary that is complementary to a contiguous sequence of nucleotides of SEQ ID NO: 1955, wherein the targeting sequence or region of complementarity may have no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 mismatches with the corresponding SNCA target sequence.

Types of Oligonucleotides

[0062] A variety of oligonucleotide types and/or structures are useful for targeting SNCA mRNA in the methods herein including, but not limited to, RNAi oligonucleotides, antisense oligonucleotides, miRNAs, etc. Any of the oligonucleotide types described herein or elsewhere are contemplated for use as a framework to incorporate a SNCA mRNA targeting sequence herein for the purposes of inhibiting SNCA gene expression.

[0063] In some embodiments, the oligonucleotides herein inhibit SNCA gene expression by engaging with RNA interference (RNAi) pathways upstream or downstream of Dicer involvement (e.g., a RNAi oligonucleotide). For example, RNAi oligonucleotides have been developed with each strand having sizes of about 19 to about 25 nucleotides with at least one 3' overhang of about 1 to about 5 nucleotides (see, e.g., US Patent No. 8,372,968). Longer oligonucleotides also have been developed that are processed by Dicer to generate active RNAi products (see, e.g., US Patent No. 8,883,996). Further work produced extended ds oligonucleotides where at least one end of at least one strand is extended beyond a duplex targeting region, including structures where one of the strands includes a thermodynamically stabilizing tetraloop structure (see, e.g., US Patent Nos. 8,513,207 and 8,927,705, as well as Inti. Patent Application Publication No. WO 2010/033225). Such structures may include single-stranded (ss) extensions (on one or both sides of the molecule) as well as ds extensions.

[0064J In some embodiments, the oligonucleotides engage with the RNAi pathway downstream of the involvement of Dicer (e.g., Dicer cleavage). In some embodiments, the oligonucleotide has an overhang (e.g., of 1, 2, or 3 nucleotides in length) in the 3' end of the sense strand. In some embodiments, the oligonucleotide (e.g., siRNA) comprises a 21 -nucleotide antisense strand that is antisense to a target mRNA (e.g., SNCA mRNA) and a complementary passenger sense strand, in which both strands anneal to form a 19-bp duplex and 2 nucleotide overhangs at either or both 3' ends. Longer oligonucleotide designs also are contemplated including oligonucleotides having a antisense strand of 23 nucleotides and a passenger strand of 21 nucleotides, where there is a blunt end on the right side of the molecule (3' end of sense strand/5' end of antisense strand) and a two nucleotide 3' antisense strand overhang on the left side of the molecule (5' end of the sense strand/3' end of the antisense strand). In such molecules, there is a 21-bp duplex region. See, e.g., US Patent Nos. 9,012,138; 9,012,621 and 9,193,753.

[0065] In some embodiments, the oligonucleotide herein comprises sense and antisense strands that are both in the range of about 17 to about 36 (e.g., 17 to 26, 20 to 25, or 21-23) nucleotides in length. In some embodiments, the oligonucleotide comprises an antisense strand of 19-30 nucleotides in length and a sense strand of 19-50 nucleotides in length, wherein the antisense and sense strands are separate strands that form an asymmetric duplex region having an overhang of 1-4 nucleotides at the 3’ terminus of the antisense strand. In some embodiments, the oligonucleotide comprises sense and antisense strands that are both in the range of about 19 to about 22 nucleotides in length. In some embodiments, the sense and antisense strands are of equal length. In some embodiments, the oligonucleotide comprises sense and antisense strands, such that there is a 3' overhang on either the sense strand or the antisense strand, or both the sense and antisense strand. In some embodiments, for oligonucleotides that have sense and antisense strands that are both in the range of about 21 -23 nucleotides in length, a 3 ' overhang on the sense, antisense, or both strands is 1 or 2 nucleotides in length. In some embodiments, the oligonucleotide has an antisense strand of 22 nucleotides and a sense strand of 20 nucleotides, where there is a blunt end on the right side of the molecule (3' end of sense strand/5' end of antisense strand) and a 2 nucleotide 3' antisense strand overhang on the left side of the molecule (5' end of the sense strand/3' end of the antisense strand). In such molecules, there is a 20-bp duplex region.

[0066J Other oligonucleotide designs for use with the compositions and methods herein include: 16-mer siRNAs (see, e.g, NUCLEIC ACIDS IN CHEMISTRY AND BIOLOGY, Blackbum (ed.), Royal Society of Chemistry, 2006), shRNAs (e.g., having 19 bp or shorter stems; see, e.g., Moore et al. (2010) METHODS MOL. BIOL. 629: 141 -58), blunt siRNAs (e.g., of 19 bps in length; see, e.g., Kraynack & Baker (2006) RNA 12: 163-76), asymmetrical siRNAs (aiRNA; see, e.g., Sun et al. (2008) Nat. Biotechnol. 26:1379-82), asymmetric shorter-duplex siRNA (see, e.g., Chang et al. (2009) Mol. Ther. 17:725-732), fork siRNAs (see, e.g., Hohjoh (2004) FEBS Lett. 557: 193-98), single-stranded siRNAs (Elsner (2012) Nat. Biotechnol. 30: 1063), dumbbell-shaped circular siRNAs (see, e.g., Abe et al. (2007) J. Am. Chem. Soc. 129: 15108-09), and small internally segmented interfering RNA (siRNA; see, e.g., Bramsen et al. (2007) Nucleic Acids Res. 35:5886- 97). Further non-limiting examples of an oligonucleotide designs that may be used in some embodiments to reduce or inhibit SNCA gene expression are microRNA (miRNA), short hairpin RNA (shRNA) and short siRNA (see, e.g., Hamilton et l. (2002) EMBO J. 21:4671-79; see also, US Patent Application Publication No. 2009/0099115).

[0067] Still, in some embodiments, an oligonucleotide for reducing or inhibiting SNCA gene expression herein is ss. Such structures may include but are not limited to ss RNAi molecules. Recent efforts have demonstrated the activity of ss RNAi molecules (see, e.g., Matsui et al. (2016) Mol. Ther. 24:946-955). However, in some embodiments, the oligonucleotide is an antisense oligonucleotide (ASO). An antisense oligonucleotide is a ss oligonucleotide that has a nucleobase sequence and that, when written or depicted in the 5' to 3' direction, comprises the reverse complement of a targeted segment of a particular nucleic acid and is suitably modified (e.g., as a gapmer) so as to induce RNaseH-mediated cleavage of its target RNA in cells or (e.g., as a mixmer) so as to inhibit translation of the target mRNA in cells. ASOs for use herein may be modified in any suitable manner known in the art including, for example, as shown in US Patent No. 9,567,587 (including, e.g., length, sugar moi eties of the nucleobase (pyrimidine, purine), and alterations of the heterocyclic portion of the nucleobase). Further, ASOs have been used for decades to reduce expression of specific target genes (see, e.g., Bennett el al. (2017) Annu. Rev. Pharmacol. 57:81- 105).

[0068] In some embodiments, the antisense oligonucleotide (ASO) shares a region of complementarity with SNCA mRNA. In some embodiments, the ASO targets various areas of the human SNCA identified as NM_000345.3. In some embodiments, the ASO is about 15 to about 50 nucleotides in length. In some embodiments, the ASO is 15-25 nucleotides in length. In some embodiments, the ASO is 22 nucleotides in length. Tn some embodiments, the ASO is complementary to any one of SEQ ID NOs: 1683-2066. In some embodiments, the ASO is at least 15 contiguous nucleotides in length. In some embodiments, the ASO is at least 19 contiguous nucleotides in length. In some embodiments, the ASO is at least 20 contiguous nucleotides in length. In some embodiments, the ASO differs by 1, 2, or 3 nucleotides from the target sequence.

Double-Stranded RNAi Oligonucleotides

[0069] In some aspects, the disclosure provides ds RNAi oligonucleotides for targeting SNCA mRNA and inhibiting SNCA gene expression (e.g., via the RNAi pathway) comprising a sense strand (also referred to herein as a passenger strand) and an antisense strand (also referred to herein as a guide strand). In some embodiments, the sense strand and antisense strand are separate strands and are not covalently linked. In some embodiments, the sense strand and the antisense strand are covalently linked. In some embodiments, the sense strand and the antisense strand form a duplex region, wherein the sense strand and the antisense strand, or a portion thereof, binds with one another in a complementary fashion (e.g., by Watson-Crick base pairing).

[0070] In some embodiments, the sense strand has a first region (Rl) and a second region (R2), wherein R2 comprises a first subregion (S 1), a loop (L) such as a tetraloop (tetraL) or triloop (triL), and a second subregion (S2), wherein L is located between SI and S2, and wherein SI and S2 form a second duplex (D2). D2 may have various lengths. In some embodiments, D2 is about 1 to about 6 bp in length. In some embodiments, D2 is 2-6, 3-6, 4-6, 5-6, 1-5, 2-5, 3-5, or 4-5 bp in length. In some embodiments, D2 is 1, 2, 3, 4, 5, or 6 bp in length. In some embodiments, D2 is 6 bp in length. [0071] In some embodiments, R1 of the sense strand and the antisense strand form a first duplex (DI). In some embodiments, DI is at least about 15 (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21) nucleotides in length. In some embodiments, DI is in the range of about 12 to about 30 nucleotides in length (e.g., 12 to 30, 12 to 27, 15 to 22, 18 to 22, 18 to 25, 18 to 27, 18 to 30 or 21 to 30 nucleotides in length). In some embodiments, DI is at least 12 nucleotides in length (e.g., at least 12, at least 15, at least 20, at least 25, or at least 30 nucleotides in length). In some embodiments, DI is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, DI is 19 nucleotides in length. Tn some embodiments, DI is 20 nucleotides in length. In some embodiments, DI comprising the sense strand and the antisense strand does not span the entire length of the sense strand and/or the antisense strand. In some embodiments, DI comprising the sense strand and the antisense strand spans the entire length of either the sense strand or the antisense strand or both. In certain embodiments, DI comprising the sense strand and the antisense strand spans the entire length of both the sense strand and the antisense strand.

[0072] In some embodiments, the sense strand is 36 nucleotides in length and positions are numbered l-36 from 5’ to 3’. In some embodiments, the antisense strand is 22 nucleotides in length and positions are numbered 1-22 from 5’ to 3’. In some embodiments, position numbers described herein adhere to this numbering format.

[0073] In some embodiments, the oligonucleotide comprises a sense strand having a sequence of any one of SEQ ID NOs: 1-384 and an antisense strand comprising a complementary sequence of any one of SEQ ID NOs: 385-768. In some embodiments, the oligonucleotide comprises a sense strand having a sequence of SEQ ID NOs: 1683-2066 and an antisense strand comprising a complementary sequence of any one of SEQ ID NOs: 2067-2450.

[0074] In some embodiments, the oligonucleotide comprises a sense strand having a sequence of any one of SEQ ID NOs: 1537-1571 and an antisense strand comprising a complementary sequence of any one of SEQ ID NOs: 1572-1606. In some embodiments, the oligonucleotide comprises a sense strand having a sequence of any one of SEQ ID NOs: 1537-1571 and 1681 and an antisense strand comprising a complementary sequence of any one of SEQ ID NOs: 1572-1606. [0075] In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand comprising nucleotide sequences selected from: a) SEQ ID NOs: 1537 and 1572, respectively; b) SEQ ID NOs: 1538 and 1573, respectively; c) SEQ ID NOs: 1539 and 1574, respectively; d) SEQ ID NOs: 1540 and 1575, respectively; e) SEQ ID NOs: 1541 and 1576, respectively; f) SEQ ID NOs: 1542 and 1577, respectively; g) SEQ ID NOs: 1543 and 1578, respectively; h) SEQ ID NOs: 1544 and 1579, respectively; i) SEQ ID NOs: 1545 and 1580, respectively; j) SEQ ID NOs: 1546 and 1581, respectively; k) SEQ ID NOs: 1547 and 1582, respectively; l) SEQ ID NOs: 1548 and 1583, respectively; m) SEQ ID NOs: 1549 and 1584, respectively; n) SEQ ID NOs: 1550 and 1585, respectively; o) SEQ ID NOs: 1551 and 1586, respectively; p) SEQ ID NOs: 1552 and 1587, respectively; q) SEQ ID NOs: 1553 and 1588, respectively; r) SEQ ID NOs: 1554 and 1589, respectively; s) SEQ ID NOs: 1555 and 1590, respectively; t) SEQ ID NOs: 1556 and 1591, respectively; u) SEQ ID NOs: 1557 and 1592, respectively; v) SEQ ID NOs: 1558 and 1593, respectively; w) SEQ ID NOs: 1559 and 1594, respectively; x) SEQ ID NOs: 1560 and 1595, respectively; y) SEQ ID NOs: 1561 and 1596, respectively; z) SEQ ID NOs: 1562 and 1597, respectively; aa) SEQ ID NOs: 1563 and 1598, respectively; bb) SEQ ID NOs: 1564 and 1599, respectively; cc) SEQ ID NOs: 1565 and 1600, respectively; dd) SEQ ID NOs: 1566 and 1601, respectively; ee) SEQ ID NOs: 1567 and 1602, respectively; ff) SEQ ID NOs: 1568 and 1603, respectively; gg) SEQ ID NOs: 1569 and 1604, respectively; hh) SEQ ID NOs: 1570 and 1605, respectively; ii) SEQ ID NOs: 1571 and 1606, respectively; and jj) SEQ ID NOs: 1681 and 1586, respectively.

[0076] In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand comprising nucleotide sequences selected from: a) SEQ ID NOs: 1540 and 1575, respectively; b) SEQ ID NOs: 1544 and 1579, respectively; c) SEQ ID NOs: 1546 and 1581, respectively; d) SEQ ID NOs: 1551and 1586, respectively; e) SEQ ID NOs: 1552 and 1587, respectively; f) SEQ ID NOs: 1553 and 1588, respectively; g) SEQ ID NOs: 1558 and 1594, respectively; h) SEQ ID NOs: 1560 and 1595, respectively; i) SEQ ID NOs: 1564 and 1599, respectively; j) SEQ ID NOs: 1565 and 1600, respectively; k) SEQ ID NOs: 1566 and 1601, respectively; l) SEQ ID NOs: 1570 and 1605, respectively; and m) SEQ ID NOs: 1681 and 1586, respectively.

[0077] In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand comprising nucleotide sequences selected from: a) SEQ ID NOs: 1553 and 1588, respectively; b) SEQ ID NOs: 1560 and 1595, respectively; c) SEQ ID NOs: 1564 and 1599, respectively; d) SEQ ID NOs: 1551 and 1586, respectively; e) SEQ ID NOs: 1570 and 1605, respectively; and f) SEQ ID NOs: 1681 and 1586, respectively.

[0078] In some embodiments, the sense strand comprises the sequence of SEQ ID NO: 1553 and the antisense strand comprises the sequence of SEQ ID NO: 1588. In some embodiments, the sense strand comprises the sequence of SEQ ID NO: 1560 and the antisense strand comprises the sequence of SEQ ID NO: 1595. In some embodiments, the sense strand comprises the sequence of SEQ ID NO: 1564 and the antisense strand comprises the sequence of SEQ ID NO: 1599. In some embodiments, the sense strand comprises the sequence of SEQ ID NO: 1551 and the antisense strand comprises the sequence of SEQ ID NO: 1586. In some embodiments, the sense strand comprises the sequence of SEQ ID NO: 1570 and the antisense strand comprises the sequence of SEQ ID NO: 1605. In some embodiments, the sense strand comprises the sequence of SEQ ID NO: 1681 and the antisense strand comprises the sequence of SEQ ID NO: 1586.

[0079] It should be appreciated that, in some embodiments, sequences presented in the Sequence Listing may be referred to in describing the structure of the oligonucleotide (e.g., a RNAi oligonucleotide) or other nucleic acid. In such embodiments, the actual oligonucleotide or other nucleic acid may have one or more alternative nucleotides (e.g., a RNA counterpart of a DNA nucleotide or a DNA counterpart of a RNA nucleotide) and/or one or more modified nucleotides and/or one or more modified intemucleotide linkages and/or one or more other modification when compared with the specified sequence while retaining essentially same or similar complementary properties as the specified sequence.

[0080] In some embodiments, a RNAi oligonucleotide herein comprises a 25-nucleotide sense strand and a 27-nucleotide antisense strand that when acted upon by a Dicer enzyme results in an antisense strand that is incorporated into the mature RNA-induced silencing complex (RISC). In some embodiments, the 25-nucleotide sense strand comprises a sequence selected from SEQ ID NOs: 1-384. In some embodiments, the 27-nucleotide antisense strand comprises a sequence selected from SEQ ID NOs: 385-768. In some embodiments, the sense strand is longer than 27 nucleotides (e.g., 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides). In some embodiments, the sense strand is longer than 25 nucleotides (e.g., 26, 27, 28, 29, or 30 nucleotides). In some embodiments, the sense strand comprises a nucleotide sequence selected from SEQ ID NOs: 1683-2066, wherein the nucleotide sequence is longer than 27 nucleotides (e.g, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides). In some embodiments, the sense strand comprises a nucleotide sequence selected from SEQ ID NOs: 1683-2066, wherein the nucleotide sequence is longer than 25 nucleotides (e.g., 26, 27, 28, 29, or 30 nucleotides).

[0081] In some embodiments, the oligonucleotide has one 5' end that is thermodynamically less stable when compared to the other 5' end. Tn some embodiments, an asymmetric RNAi oligonucleotide is provided that comprises a blunt end at the 3' end of a sense strand and a 3' overhang at the 3' end of an antisense strand. In some embodiments, the 3' overhang on the antisense strand is about 1 to about 8 nucleotides in length (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides in length). Typically, the oligonucleotide has a two-nucleotide overhang on the 3' end of the antisense strand. However, other overhangs are possible. In some embodiments, the overhang is a 3' overhang comprising a length of between 1 and 6 nucleotides, optionally 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 6, 3 to 5, 3 to 4, 4 to 6, 4 to 5, 5 to 6 nucleotides, or 1, 2, 3, 4, 5, or 6 nucleotides. In other embodiments, the overhang is a 5' overhang comprising a length of between 1 and 6 nucleotides, optionally 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 6, 3 to 5, 3 to 4, 4 to 6, 4 to 5, 5 to 6 nucleotides, or 1, 2, 3, 4, 5, or 6 nucleotides.

[0082] In some embodiments, two terminal nucleotides on the 3' end of the antisense strand are modified. In some embodiments, the two terminal nucleotides on the 3' end of the antisense strand are complementary with the target mRNA (e.g., SNCA mRNA). In some embodiments, the two terminal nucleotides on the 3' end of the antisense strand are not complementary with the target mRNA. In some embodiments, the two terminal nucleotides on the 3’ end of the antisense strand of the oligonucleotide are unpaired. In some embodiments, the two terminal nucleotides on the 3’ end of the antisense strand of the oligonucleotide comprise an unpaired GG. In some embodiments, the two terminal nucleotides on the 3’ end of the antisense strand of the oligonucleotide are not complementary to the target mRNA. In some embodiments, two terminal nucleotides on each 3' end of the oligonucleotide are GG. Typically, one or both of the two terminal GG nucleotides on each 3' end of a ds oligonucleotide are not complementary with the target mRNA. In some embodiments, two terminal nucleotides on each 3' end of the oligonucleotide are GG. In some embodiments, one or both of the two terminal GG nucleotides on each 3 ' end of the oligonucleotide are not complementary with the target mRNA. In some embodiments, the oligonucleotide comprises a targeting sequence or a region of complementary that is complementary to a contiguous sequence of nucleotides of any one of SEQ ID NOs: 2067-2045, wherein the two terminal nucleotides on the 3' end of the antisense strand of the oligonucleotide comprise an unpaired GG. In some embodiments, the oligonucleotide comprises an antisense strand comprising a nucleotide sequence selected from SEQ ID NOs: 2067-2450, wherein the two terminal nucleotides on the 3' end of the antisense strand of the oligonucleotide comprise an unpaired GG. In some embodiments, the oligonucleotide comprises a sense strand comprising a nucleotide sequence selected from SEQ ID NOs: 1683-2066 and antisense strand comprising a nucleotide sequence selected from SEQ ID NOs: 2067-2450, wherein the two terminal nucleotides on the 3' end of the antisense strand of the oligonucleotide comprise an unpaired GG.

[0083] In some embodiments, there is one or more (e.g., 1, 2, 3, 4, or 5) mismatch(es) between the sense and antisense strands comprising an oligonucleotide herein. If there is more than one mismatch between the sense and antisense strands, they may be positioned consecutively (e.g., 2, 3, or more in a row) or may be interspersed throughout the region of complementarity. In some embodiments, the 3' end of the sense strand contains one or more mismatches. In one embodiment, two mismatches are incorporated at the 3' end of the sense strand. In some embodiments, base mismatches or destabilization of segments at the 3' end of the sense strand of the oligonucleotide improves or increases the potency of the oligonucleotide.

[0084] In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand comprising nucleotide sequences selected from: a) SEQ ID NOs: 1537 and 1572, respectively; b) SEQ ID NOs: 1538 and 1573, respectively; c) SEQ ID NOs: 1539 and 1574, respectively; d) SEQ ID NOs: 1540 and 1575, respectively; e) SEQ ID NOs: 1541 and 1576, respectively; f) SEQ ID NOs: 1542 and 1577, respectively; g) SEQ ID NOs: 1543 and 1578, respectively; h) SEQ ID NOs: 1544 and 1579, respectively; i) SEQ ID NOs: 1545 and 1580, respectively; j) SEQ ID NOs: 1546 and 1581, respectively; k) SEQ ID NOs: 1547 and 1582, respectively; l) SEQ ID NOs: 1548 and 1583, respectively; m) SEQ ID NOs: 1549 and 1584, respectively; n) SEQ ID NOs: 1550 and 1585, respectively; o) SEQ ID NOs: 1551 and 1586, respectively; p) SEQ ID NOs: 1552 and 1587, respectively; q) SEQ ID NOs: 1553 and 1588, respectively; r) SEQ ID NOs: 1554 and 1589, respectively; s) SEQ ID NOs: 1555 and 1590, respectively; t) SEQ ID NOs: 1556 and 1591, respectively; u) SEQ ID NOs: 1557 and 1592, respectively; v) SEQ ID NOs: 1558 and 1593, respectively; w) SEQ ID NOs: 1559 and 1594, respectively; x) SEQ ID NOs: 1560 and 1595, respectively; y) SEQ ID NOs: 1561 and 1596, respectively; z) SEQ ID NOs: 1562 and 1597, respectively; aa) SEQ ID NOs: 1563 and 1598, respectively; bb) SEQ ID NOs: 1564 and 1599, respectively; cc) SEQ ID NOs: 1565 and 1600, respectively; dd) SEQ ID NOs: 1566 and 1601, respectively; ee) SEQ ID NOs: 1567 and 1602, respectively; ff) SEQ ID NOs: 1568 and 1603, respectively; gg) SEQ ID NOs: 1569 and 1604, respectively; hh) SEQ ID NOs: 1570 and 1605, respectively; ii) SEQ ID NOs: 1571 and 1606, respectively; and jj) SEQ ID NOs: 1681 and 1586, respectively, wherein there is one or more (e.g., 1, 2, 3, 4, or 5) mismatch(s) between the sense and antisense strands.

[0085] In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand comprising nucleotide sequences selected from: a) SEQ ID NOs: 1540 and 1575, respectively; b) SEQ ID NOs: 1544 and 1579, respectively; c) SEQ ID NOs: 1546 and 1581 , respectively; d) SEQ ID NOs: 1551and 1586, respectively; e) SEQ ID NOs: 1552 and 1587, respectively; f) SEQ ID NOs: 1553 and 1588, respectively; g) SEQ ID NOs: 1558 and 1594, respectively; h) SEQ ID NOs: 1560 and 1595, respectively; i) SEQ ID NOs: 1564 and 1599, respectively; j) SEQ ID NOs: 1565 and 1600, respectively; k) SEQ ID NOs: 1566 and 1601, respectively; l) SEQ ID NOs: 1570 and 1605, respectively; and m) SEQ ID NOs: 1681 and 1586, respectively, wherein there is one or more (e.g, 1, 2, 3, 4, or 5) mismatch(s) between the sense and antisense strands.

[0086] In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand comprising sequence selected from: a) SEQ ID NOs: 1553 and 1588, respectively; b) SEQ ID NOs: 1560 and 1595, respectively; c) SEQ ID NOs: 1564 and 1599, respectively; d) SEQ ID NOs: 1551 and 1586, respectively; e) SEQ ID NOs: 1570 and 1605, respectively; and, f) SEQ ID NOs: 1681 and 1586, respectively, wherein there is one or more (e.g., 1, 2, 3, 4 or 5) mismatch(s) between the sense and antisense strands.

Antisense Strands

[0087J In some embodiments, an antisense strand of an oligonucleotide is referred to as a “guide strand,” which engages with RISC and binds to an Argonaute protein such as Ago2, or engages with or binds to one or more similar factors, and directs silencing of a target gene. In some embodiments, a sense strand complementary to the antisense strand is referred to as a “passenger strand.”

[0088] In some embodiments, the oligonucleotide comprises an antisense strand of up to about 50 nucleotides in length (e. ., up to 50, up to 40, up to 35, up to 30, up to 27, up to 25, up to 21, up to 19, up to 17, up to 15, or up to 12 nucleotides in length). In some embodiments, the oligonucleotide comprises an antisense strand of at least about 12 nucleotides in length (e.g., at least 12, at least 15, at least 19, at least 21, at least 22, at least 25, at least 27, at least 30, at least 35, or at least 38 nucleotides in length). In some embodiments, the antisense strand is in a range of about 12 to about 40 (e.g., 12 to 40, 12 to 36, 12 to 32, 12 to 28, 15 to 40, 15 to 36, 15 to 32, 15 to 30, 15 to 28, 17 to 22, 17 to 25, 19 to 27, 19 to 30, 20 to 40, 22 to 40, 25 to 40, or 32 to 40) nucleotides in length. In some embodiments, the oligonucleotide comprises an antisense of 15 to 30 nucleotides in length. In some embodiments, the antisense strand of any one of the oligonucleotides disclosed herein is of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length. In some embodiments, the antisense strand is 22 nucleotides in length.

[0089] In some embodiments, the antisense strand comprises or consists of a sequence as set forth in any one of SEQ ID NOs: 1683-2066. In some embodiments, the antisense strand comprises at least about 12 (e.g., at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) contiguous nucleotides of a sequence as set forth in any one of SEQ ID NOs: 2067-2450. In some embodiments, the antisense strand comprises or consists of a sequence as set forth in any one of SEQ ID NOs: 385-768. In some embodiments, the antisense strand comprises at least about 12 (e.g., at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) contiguous nucleotides of a sequence as set forth in any one of SEQ ID NOs: 385-768. In some embodiments, the antisense strand comprises or consists of a sequence as set forth in any one of SEQ ID NOs: 1572-1606. In some embodiments, the antisense strand comprises at least about 12 (e.g., at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least

18, at least 19, at least 20, at least 21, at least 22, or at least 23) contiguous nucleotides of a sequence as set forth in any one of SEQ ID NOs: 1572-1606. In some embodiments, the antisense strand comprises or consists of a sequence as set forth in any one of SEQ ID NOs: 2067-2450. Tn some embodiments, the antisense strand comprises at least about 12 (e.g., at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) contiguous nucleotides of a sequence as set forth in any one of SEQ ID NOs: 2067-2450. In some embodiments, the antisense strand comprises or consists of a sequence as set forth in any one of SEQ ID NOs: 1575, 1579, 1581, 1586, 1587, 1588, 1594, 1595, 1599, 1600, 1601, 1605, and 1586. In some embodiments, the antisense strand comprises at least about 12 (e.g., at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) contiguous nucleotides of a sequence as set forth in any one of SEQ ID NOs: 1575, 1579, 1581, 1586, 1587, 1588, 1594, 1595, 1599, 1600, 1601, 1605, and 1586. In some embodiments, the antisense strand comprises or consists of a sequence as set forth in any one of SEQ ID NOs: 1588, 1595, 1599, 1586, and 1605 In some embodiments, the antisense strand comprises at least about 12 (e.g., at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) contiguous nucleotides of a sequence as set forth in any one of SEQ ID NOs: 1588, 1595, 1599, 1586, and 1605.

Sense Strands

[0090] In some embodiments, the oligonucleotide comprises a sense strand sequence as set forth in in any one of SEQ ID NOs: 1683-2066. In some embodiments, the sense strand comprises at least about 12 (e.g., at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least

19, at least 20, at least 21, at least 22, or at least 23) contiguous nucleotides of a sequence as set forth in in any one of SEQ ID NOs: 1683-2066. In some embodiments, the sense strand comprises a sequence a set forth in any one of SEQ ID NOs: 1-384. In some embodiments, the sense strand comprises at least about 12 (e.g., at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) contiguous nucleotides of a sequence as set forth in in any one of SEQ ID NOs: 1-384. In some embodiments, the sense strand comprises a sequence as set forth in any one of SEQ ID NOs: 1537-1571. In some embodiments, the sense strand comprises a sequence as set forth in any one of SEQ ID NOs: 1537-1571 and 1681 . Tn some embodiments, the sense strand is SEQ ID NO: 1681 . Tn some embodiments, the sense strand comprises at least about 12 e.g., at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) contiguous nucleotides of a sequence as set forth in any one of SEQ ID NOs: 1537-1571. In some embodiments, the sense strand comprises a sequence as set forth in any one of SEQ ID NOs: 1781, 1782, 1796, 1798, 1802, 1808, 1814, 1817, 1713, 1718, 1726, 1830, 1839, 1742, 1846, 1852, 1865, 1784, 1804, 1721, 1822, 1840, 1735, 1847, 1855, 1864, 1901, 1902, 1938, 1947, 1955, 1964, 1973, and 1978. In some embodiments, the sense strand comprises at least about 12 (e.g., at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) contiguous nucleotides of a sequence as set forth in any one of SEQ ID NOs: 1781, 1782, 1796, 1798, 1802, 1808, 1814, 1817, 1713, 1718, 1726, 1830, 1839, 1742, 1846, 1852, 1865, 1784, 1804, 1721, 1822, 1840, 1735, 1847, 1855, 1864, 1901, 1902, 1938, 1947, 1955, 1964, 1973, and 1978. In some embodiments, the sense strand comprises a sequence as set forth in any one of SEQ ID NOs: 1540, 1544, 1546, 1551, 1552, 1553, 1558, 1560, 1564, 1565, 1566, and 1570. In some embodiments, the sense strand comprises at least about 12 (e.g., at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22 or at least 23) contiguous nucleotides of a sequence as set forth in any one of SEQ ID NOs: 1540, 1544, 1546, 1551, 1552, 1553, 1558, 1560, 1564, 1565, 1566, and 1570. In some embodiments, the sense strand comprises a sequence as set forth in any one of SEQ ID NOs: 1553, 1560, 1564, 1551, and 1570. In some embodiments, the sense strand that comprises at least about 12 (e.g., at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) contiguous nucleotides of a sequence as set forth in any one of SEQ ID NOs: 1553, 1560, 1564, 1551, and 1570.

[0091] In some embodiments, the oligonucleotide comprises a sense strand sequence as set forth in any one of SEQ ID NOs: 1540, 1544, 1546, 1551, 1552, 1553, 1558, 1560, 1564, 1565, 1566, 1570, and 1681. In some embodiments, the sense strand comprises at least about 12 (e.g., at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) contiguous nucleotides of a sequence as set forth in any one of SEQ ID NOs: 1540, 1544, 1546, 1551 , 1552, 1553, 1558, 1560, 1564, 1565, 1566, 1570, and 1681. In some embodiments, the sense strand comprises a sequence as set forth in any one of SEQ ID NOs: 1553, 1560, 1564, 1551, 1570, and 1681. In some embodiments, the sense strand comprises at least about 12 e.g., at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) contiguous nucleotides of a sequence as set forth in any one of SEQ ID NOs: 1553, 1560, 1564, 1551, 1570, and 1681.

[0092] In some embodiments, the sense strand comprises up to about 50 nucleotides in length (e.g., up to 50, up to 40, up to 36, up to 30, up to 27, up to 25, up to 21, up to 19, up to 17, or up to 12 nucleotides in length). In some embodiments, the sense strand comprises at least about 12 nucleotides in length (e.g., at least 12, at least 15, at least 19, at least 21, at least 25, at least 27, at least 30, at least 36, or at least 38 nucleotides in length). In some embodiments, the sense strand is in a range of about 12 to about 50 (e.g., 12 to 50, 12 to 40, 12 to 36, 12 to 32, 12 to 28, 15 to 40, 15 to 36, 15 to 32, 15 to 28, 17 to 21, 17 to 25, 19 to 27, 19 to 30, 20 to 40, 22 to 40, 25 to 40, or 32 to 40) nucleotides in length. In some embodiments, the sense strand comprises 15 to 50 nucleotides in length. In some embodiments, the sense strand comprises 18 to 36 nucleotides in length. In some embodiments, the sense strand comprises 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In some embodiments, the sense strand is 36 nucleotides in length. [0093] In some embodiments, the sense strand comprises a stem-loop structure at its 3' end. In some embodiments, the stem-loop is formed by intrastrand base pairing. In some embodiments, the sense strand comprises a stem-loop structure at its 5' end. In some embodiments, the stem of the stem-loop comprises a duplex of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 2 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 3 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 4 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 5 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 6 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 7 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 8 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 9 nucleotides in length. Tn some embodiments, the stem of the stem-loop comprises a duplex of 10 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 11 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 12 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 13 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 14 nucleotides in length. [0094] In some embodiments, a stem-loop provides the oligonucleotide protection against degradation (e.g., enzymatic degradation), facilitates or improves targeting and/or delivery to a target cell, tissue, or organ (e.g., the liver or brain), or both. For example, in some embodiments, the loop of the stem-loop provides nucleotides comprising one or more modifications that facilitate, improve, or increase targeting to a target mRNA (e.g., a SNCA mRNA), inhibition of target gene expression (e.g., SNCA gene expression), and/or delivery to a target cell, tissue, or organ (e.g., the CNS), or a combination thereof. In some embodiments, the stem -loop itself or modification(s) to the stem-loop do not substantially affect the inherent gene expression inhibition activity of the oligonucleotide, but facilitates, improves, or increases stability e.g., provides protection against degradation) and/or delivery of the oligonucleotide to a target cell, tissue, or organ (e.g., the CNS). In certain embodiments, the oligonucleotide comprises a sense strand comprising (e.g., at its 3' end) a stem-loop set forth as: S1-L-S2, in which SI is complementary to S2, and in which L forms a single-stranded loop between S 1 and S2 of up to about 10 nucleotides in length (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length). In some embodiments, the loop (L) is 3 nucleotides in length (e.g., triloop ortriL). In some embodiments, the loop (L) is 4 nucleotides in length (e.g., tetraloop or tetraL). In some embodiments, the loop (L) is 5 nucleotides in length (e.g., pentaloop or pentaL). In some embodiments, the loop (L) is 6 nucleotides in length (e.g., hexaloop or hexaL). In some embodiments, the loop (L) is 7 nucleotides in length (e.g., heptaloop or heptaL). In some embodiments, the loop (L) is 8 nucleotides in length e.g., octaloop or octaL). In some embodiments, the loop (L) is 9 nucleotides in length (e.g., nonaloop or nonaL). In some embodiments, the loop (L) is 10 nucleotides in length (e.g., decaloop or decaL).

[0095] In some embodiments, the oligonucleotide comprises a targeting sequence or a region of complementary that is complementary to a contiguous sequence of nucleotides of any one of SEQ ID NOs: 1683-2066, and the oligonucleotide comprises a sense strand comprising (e.g., at its 3' end) a stem-loop set forth as: S1-L-S2, in which SI is complementary to S2, and in which L forms a ss loop between SI and S2 of up to about 10 nucleotides in length (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length). In some embodiments, the oligonucleotide comprises a targeting sequence or a region of complementary that is complementary to a contiguous sequence of nucleotides of any one of SEQ ID NOs: 1683-2066, and the oligonucleotide comprises a sense strand comprising (e.g., at its 3' end) a stem-loop set forth as: S1-L-S2, in which SI is complementary to S2, and in which L forms a ss loop between SI and S2 of 4 nucleotides in length. In some embodiments, a loop (L) of a stem-loop having the structure S1-L-S2 as described above is a tetraL as described in US Patent No. 10,131,912, incorporated herein by reference (e.g., within a nicked tetraloop structure). In some embodiments, the oligonucleotide comprises a targeting sequence or a region of complementary that is complementary to a contiguous sequence of nucleotides of any one of SEQ ID NOs: 1683-2066 and a tetraL. In some embodiments, the tetraloop comprises the sequence 5’-GAAA-3’. In some embodiments, the stem loop comprises the sequence 5’- GCAGCCGAAAGGCUGC-3’ (SEQ ID NO: 1680).

[0096] In other embodiments, the loop (L) is a triL. In some embodiments, the oligonucleotide comprises a targeting sequence or a region of complementary that is complementary to a contiguous sequence of nucleotides of any one of SEQ ID NOs: 1683-2066 and a triL. In some embodiments, the triL comprises ribonucleotides, deoxyribonucleotides, modified nucleotides, delivery ligands, and combinations thereof.

Duplex Length [0097] In some embodiments, a duplex formed between the sense and antisense strands is at least about 12 (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21) nucleotides in length. In some embodiments, the duplex is in the range of about 12 to about 30 nucleotides in length (e.g., 12 to 30, 12 to 27, 12 to 22, 15 to 25, 18 to 30, 18 to 22, 18 to 25, 18 to 27, 18 to 30, 19 to 30, or 21 to 30 nucleotides in length). In some embodiments, the duplex is 12, 13, 14, 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, the duplex is 12 nucleotides in length. In some embodiments, the duplex is 13 nucleotides in length. Tn some embodiments, the duplex is 14 nucleotides in length. Tn some embodiments, the duplex is 15 nucleotides in length. In some embodiments, the duplex is 16 nucleotides in length. In some embodiments, the duplex is 17 nucleotides in length. In some embodiments, the duplex is 18 nucleotides in length. In some embodiments, the duplex is 19 nucleotides in length. In some embodiments, the duplex is 20 nucleotides in length. In some embodiments, the duplex is 21 nucleotides in length. In some embodiments, the duplex is 22 nucleotides in length. In some embodiments, the duplex is 23 nucleotides in length. In some embodiments, the duplex formed is 24 nucleotides in length. In some embodiments, the duplex is 25 nucleotides in length. In some embodiments, the duplex is 26 nucleotides in length. In some embodiments, the duplex is 27 nucleotides in length. In some embodiments, the duplex is 28 nucleotides in length. In some embodiments, the duplex is 29 nucleotides in length. In some embodiments, the duplex is 30 nucleotides in length. In some embodiments, the duplex does not span the entire length of the sense strand and/or the antisense strand. In some embodiments, the duplex spans the entire length of either the sense or antisense strands. In some embodiments, the duplex spans the entire length of both the sense strand and the antisense strand.

[0098] In some embodiments, the sense and antisense strands comprise nucleotide sequences selected from the group consisting of: a) SEQ ID NOs: 1537 and 1572, respectively; b) SEQ ID NOs: 1538 and 1573, respectively; c) SEQ ID NOs: 1539 and 1574, respectively; d) SEQ ID NOs: 1540 and 1575, respectively; e) SEQ ID NOs: 1541 and 1576, respectively; f) SEQ ID NOs: 1542 and 1577, respectively; g) SEQ ID NOs: 1543 and 1578, respectively; h) SEQ ID NOs: 1544 and 1579, respectively; i) SEQ ID NOs: 1545 and 1580, respectively; j) SEQ ID NOs: 1546 and 1581, respectively; k) SEQ ID NOs: 1547 and 1582, respectively; l) SEQ ID NOs: 1548 and 1583, respectively; m) SEQ ID NOs: 1549 and 1584, respectively; n) SEQ ID NOs: 1550 and 1585, respectively; o) SEQ ID NOs: 1551 and 1586, respectively; p) SEQ ID NOs: 1552 and 1587, respectively; q) SEQ ID NOs: 1553 and 1588, respectively; r) SEQ ID NOs: 1554 and 1589, respectively; s) SEQ ID NOs: 1555 and 1590, respectively; t) SEQ ID NOs: 1556 and 1591, respectively; u) SEQ ID NOs: 1557 and 1592, respectively; v) SEQ ID NOs: 1558 and 1593, respectively; w) SEQ ID NOs: 1559 and 1594, respectively; x) SEQ ID NOs: 1560 and 1595, respectively; y) SEQ ID NOs: 1561 and 1596, respectively; z) SEQ ID NOs: 1562 and 1597, respectively; aa) SEQ ID NOs: 1563 and 1598, respectively; bb) SEQ ID NOs: 1564 and 1599, respectively; cc) SEQ ID NOs: 1565 and 1600, respectively; dd) SEQ ID NOs: 1566 and 1601, respectively; ee) SEQ ID NOs: 1567 and 1602, respectively; ff) SEQ ID NOs: 1568 and 1603, respectively; gg) SEQ ID NOs: 1569 and 1604, respectively; hh) SEQ ID NOs: 1570 and 1605, respectively; ii) SEQ ID NOs: 1571 and 1606, respectively; and jj) SEQ ID NOs: 1681 and 1586, respectively, wherein a duplex formed between the sense and antisense strands is in the range of about 12 to about 30 nucleotides in length (e.g, 12 to 30, 12 to 27, 12 to 22, 15 to 25, 18 to 30, 18 to 22, 18 to 25, 18 to 27, 18 to 30, 19 to 30, or 21 to 30 nucleotides in length).

[0099] In some embodiments, the sense and antisense strands comprise nucleotide sequences selected from the group consisting of: a) SEQ ID NOs: 1540 and 1575, respectively; b) SEQ ID NOs: 1544 and 1579, respectively; c) SEQ ID NOs: 1546 and 1581, respectively; d) SEQ ID NOs: 1551and 1586, respectively; e) SEQ ID NOs: 1552 and 1587, respectively; f) SEQ ID NOs: 1553 and 1588, respectively; g) SEQ ID NOs: 1558 and 1594, respectively; h) SEQ ID NOs: 1560 and 1595, respectively; i) SEQ ID NOs: 1564 and 1599, respectively; j) SEQ ID NOs: 1565 and 1600, respectively; k) SEQ ID NOs: 1566 and 1601, respectively; l) SEQ ID NOs: 1570 and 1605, respectively; and m) SEQ ID NOs: 1681 and 1586, respectively, wherein a duplex formed between the sense and antisense strands is in the range of about 12 to about 30 nucleotides in length (e.g., 12 to 30, 12 to 27, 12 to 22, 15 to 25, 18 to 30, 18 to 22, 18 to 25, 18 to 27, 18 to 30, 19 to 30, or 21 to 30 nucleotides in length).

[0100] In some embodiments, the sense and antisense strands comprise nucleotide sequences selected from the group consisting of: a) SEQ ID NOs: 1553 and 1588, respectively; b) SEQ ID NOs: 1560 and 1595, respectively; c) SEQ ID NOs: 1564 and 1599, respectively; d) SEQ ID NOs: 1551 and 1586, respectively; e) SEQ ID NOs: 1570 and 1605, respectively; and, f) SEQ ID NOs: 1681 and 1586, respectively, wherein a duplex formed between the sense and antisense strands is in the range of about 12 to about 30 nucleotides in length (e.g, 12 to 30, 12 to 27, 12 to 22, 15 to 25, 18 to 30, 18 to 22, 18 to 25, 18 to 27, 18 to 30, 19 to 30, or 21 to 30 nucleotides in length).

Oligonucleotide Termini

[0101] In some embodiments, the oligonucleotide (e.g, a RNAi oligonucleotide) comprises a sense strand and an antisense strand, wherein the termini of either or both strands comprise a blunt end. In some embodiments, the oligonucleotide comprises sense and antisense strands that are separate strands that form an asymmetric duplex region having an overhang at the 3’ terminus of the antisense strand. In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand, wherein the termini of either or both strands comprise an overhang comprising one or more nucleotides. In some embodiments, the one or more nucleotides comprising the overhang are unpaired nucleotides. In some embodiments, the oligonucleotide herein comprises a sense strand and an antisense strand, wherein the 3’ termini of the sense strand and the 5’ termini of the antisense strand comprise a blunt end. In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand, wherein the 5’ termini of the sense strand and the 3 ’ termini of the antisense strand comprise a blunt end.

[0102] In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand, wherein a 3’ terminus of either or both strands comprises a 3’ overhang comprising one or more nucleotides. In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand, wherein the sense strand comprises a 3’ overhang comprising one or more nucleotides. In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand, wherein the antisense strand comprises a 3’ overhang comprising one or more nucleotides. In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand, wherein both the sense strand and the antisense strand comprise a 3’ overhang comprising one or more nucleotides. [0103] In some embodiments, the 3’ overhang is about 1 to about 20 nucleotides in length (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or about 20 nucleotides in length). In some embodiments, the 3’ overhang is about 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 nucleotides in length. In some embodiments, the 3’ overhang is 1 nucleotide in length. In some embodiments, the 3’ overhang is 2 nucleotides in length. In some embodiments, the 3’ overhang is 3 nucleotides in length. In some embodiments, the 3 ’-overhang is 4 nucleotides in length. In some embodiments, the 3’ overhang is 5 nucleotides in length. Tn some embodiments, the 3’ overhang is 6 nucleotides in length. In some embodiments, the 3’ overhang is 7 nucleotides in length. In some embodiments, the 3’ overhang is 8 nucleotides in length. In some embodiments, the 3’ overhang is 9 nucleotides in length. In some embodiments, the 3’ overhang is 10 nucleotides in length. In some embodiments, the 3’ overhang is 11 nucleotides in length. In some embodiments, the 3’ overhang is 12 nucleotides in length. In some embodiments, the 3’ overhang is 13 nucleotides in length. In some embodiments, the 3’ overhang is 14 nucleotides in length. In some embodiments, the 3’ overhang is 15 nucleotides in length. In some embodiments, the 3’ overhang is 16 nucleotides in length. In some embodiments, the 3’ overhang is 17 nucleotides in length. In some embodiments, the 3’ overhang is 18 nucleotides in length. In some embodiments, the 3’ overhang is 19 nucleotides in length. In some embodiments, the 3’ overhang is 20 nucleotides in length.

[0104] In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand, wherein the sense and antisense strands comprise nucleotide sequences selected from the group consisting of: a) SEQ ID NOs: 1537 and 1572, respectively; b) SEQ ID NOs: 1538 and 1573, respectively; c) SEQ ID NOs: 1539 and 1574, respectively; d) SEQ ID NOs: 1540 and 1575, respectively; e) SEQ ID NOs: 1541 and 1576, respectively; f) SEQ ID NOs: 1542 and 1577, respectively; g) SEQ ID NOs: 1543 and 1578, respectively; h) SEQ ID NOs: 1544 and 1579, respectively; i) SEQ ID NOs: 1545 and 1580, respectively; j) SEQ ID NOs: 1546 and 1581, respectively; k) SEQ ID NOs: 1547 and 1582, respectively; l) SEQ ID NOs: 1548 and 1583, respectively; m) SEQ ID NOs: 1549 and 1584, respectively; n) SEQ ID NOs: 1550 and 1585, respectively; o) SEQ ID NOs: 1551 and 1586, respectively; p) SEQ ID NOs: 1552 and 1587, respectively; q) SEQ ID NOs: 1553 and 1588, respectively; r) SEQ ID NOs: 1554 and 1589, respectively; s) SEQ ID NOs: 1555 and 1590, respectively; t) SEQ ID NOs: 1556 and 1591, respectively; u) SEQ ID NOs: 1557 and 1592, respectively; v) SEQ ID NOs: 1558 and 1593, respectively; w) SEQ ID NOs: 1559 and 1594, respectively; x) SEQ ID NOs: 1560 and 1595, respectively; y) SEQ ID NOs: 1561 and 1596, respectively; z) SEQ ID NOs: 1562 and 1597, respectively; aa) SEQ ID NOs: 1563 and 1598, respectively; bb) SEQ ID NOs: 1564 and 1599, respectively; cc) SEQ ID NOs: 1565 and 1600, respectively; dd) SEQ ID NOs: 1566 and 1601, respectively; ee) SEQ ID NOs: 1567 and 1602, respectively; ff) SEQ ID NOs: 1568 and 1603, respectively; gg) SEQ ID NOs: 1569 and 1604, respectively; hh) SEQ ID NOs: 1570 and 1605, respectively; ii) SEQ ID NOs: 1571 and 1606, respectively; and jj) SEQ ID NOs: 1681 and 1586, respectively, wherein the antisense strand comprises a 3’ overhang about 1 to about 20 nucleotides in length (e.g, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or about 20 nucleotides in length), optionally wherein the 3’ overhang is 2 nucleotides in length.

[0105] In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand, wherein the sense and antisense strands comprise nucleotide sequences selected from the group consisting of: a) SEQ ID NOs: 1540 and 1575, respectively; b) SEQ ID NOs: 1544 and 1579, respectively; c) SEQ ID NOs: 1546 and 1581, respectively; d) SEQ ID NOs: 1551and 1586, respectively; e) SEQ ID NOs: 1552 and 1587, respectively; f) SEQ ID NOs: 1553 and 1588, respectively; g) SEQ ID NOs: 1558 and 1594, respectively; h) SEQ ID NOs: 1560 and 1595, respectively; i) SEQ ID NOs: 1564 and 1599, respectively; j) SEQ ID NOs: 1565 and 1600, respectively; k) SEQ ID NOs: 1566 and 1601, respectively; l) SEQ ID NOs: 1570 and 1605, respectively; and m) SEQ ID NOs: 1681 and 1586, respectively, and wherein the antisense strand comprises a 3 ’-overhang about 1 to about 20 nucleotides in length (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or about 20 nucleotides in length), optionally wherein the 3’ overhang is 2 nucleotides in length.

[0106] In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand, wherein the sense and antisense strands comprise nucleotides sequences selected from the group consisting of: a) SEQ ID NOs: 1553 and 1588, respectively; b) SEQ ID NOs: 1560 and 1595, respectively; c) SEQ ID NOs: 1564 and 1599, respectively; d) SEQ ID NOs: 1551 and 1586, respectively; e) SEQ ID NOs: 1570 and 1605, respectively; and f) SEQ ID NOs: 1681 and 1586, respectively, wherein the antisense strand comprises a 3’ overhang about 1 to about 20 nucleotides in length (e.g, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or about 20 nucleotides in length), optionally wherein the 3’ overhang is 2 nucleotides in length.

[0107] In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand, wherein the sense strand comprises a 5’ overhang comprising one or more nucleotides.

[0108] In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand, wherein the sense and antisense strands comprise nucleotide sequences selected from the group consisting of: a) SEQ ID NOs: 1540 and 1575, respectively; b) SEQ ID NOs: 1544 and 1579, respectively; c) SEQ ID NOs: 1546 and 1581, respectively; d) SEQ ID NOs: 1551and 1586, respectively; e) SEQ ID NOs: 1552 and 1587, respectively; f) SEQ ID NOs: 1553 and 1588, respectively; g) SEQ ID NOs: 1558 and 1594, respectively; h) SEQ ID NOs: 1560 and 1595, respectively; i) SEQ ID NOs: 1564 and 1599, respectively; j) SEQ ID NOs: 1565 and 1600, respectively; k) SEQ ID NOs: 1566 and 1601, respectively; l) SEQ ID NOs: 1570 and 1605, respectively; and m) SEQ ID NOs: 1681 and 1586, respectively, wherein the antisense strand comprises a 5’ overhang about 1 to about 20 nucleotides in length (e.g, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or about 20 nucleotides in length), optionally wherein the 5’ overhang is 2 nucleotides in length.

[0109] In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand, wherein the sense and antisense strands comprise nucleotide sequences selected from the group consisting of: a) SEQ ID NOs: 1553 and 1588, respectively; b) SEQ ID NOs: 1560 and 1595, respectively; c) SEQ ID NOs: 1564 and 1599, respectively; d) SEQ ID NOs: 1551 and 1586, respectively; e) SEQ ID NOs: 1570 and 1605, respectively; and f) SEQ ID NOs: 1681 and 1586, respectively, wherein the antisense strand comprises a 5’ overhang about 1 to about 20 nucleotides in length (e.g, about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or about 20 nucleotides in length), optionally wherein the 5’ overhang is 2 nucleotides in length.

[0110] In some embodiments, one or more (e.g, 2, 3, 4, 5, or more) nucleotides comprising the 3’ terminus or 5’ terminus of the sense and/or antisense strand are modified. For example, in some embodiments, one or two terminal nucleotides of the 3’ terminus of the antisense strand are modified. In some embodiments, the last nucleotide at the 3’ terminus of the antisense strand is modified (e.g, comprises a 2’ modification, e.g., a 2’-O-methoxyethyl). In some embodiments, the last one or two terminal nucleotides at the 3’ terminus of an antisense strand are complementary with the target. In some embodiments, the last one or two nucleotides at the 3’ terminus of the antisense strand are not complementary with the target.

[OHl] In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand, wherein the 3’ terminus of the sense strand comprises a stem-loop described herein, and the 3’ terminus of the antisense strand comprises a 3’ overhang. In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand that form a nicked tetraL structure, wherein the 3’ terminus of the sense strand comprises a stem-loop, wherein the loop (L) is a tetraL described herein, and wherein the 3’ terminus of the antisense strand comprises a 3’ overhang described herein. In some embodiments, the 3’ overhang is 2 nucleotides in length. In some embodiments, the 2 nucleotides comprising the 3’ overhang both comprise guanine (G) nucleobases. Typically, one or both of the nucleotides comprising the 3’ overhang of the antisense strand are not complementary with the target mRNA.

Oligonucleotide Modifications [0112] In some embodiments, an oligonucleotide comprises a modification. Oligonucleotides (e.g. a RNAi oligonucleotide) may be modified in various ways to improve or control specificity, stability, delivery, bioavailability, resistance from nuclease degradation, immunogenicity, basepairing properties, RNA distribution and cellular uptake, and other features relevant to therapeutic research use.

[0113] In some embodiments, the modification is a modified sugar. In some embodiments, the modification is a 5 ’-terminal phosphate group. In some embodiments, the modification is a modified internucleoside linkage. Tn some embodiments, the modification is a modified base. Tn some embodiments, the modification is a reversible modification. In some embodiments, the oligonucleotide can comprise any one of the modifications described herein or any combination thereof. For example, in some embodiments, the oligonucleotide comprises at least one modified sugar, a 5’ terminal phosphate group, at least one modified internucleoside linkage, at least one modified base, and at least one reversible modification.

[0114] In some embodiments, the oligonucleotide comprises at least one modified sugar, a 5’ terminal phosphate group, at least one modified intemucleotide linkage, and at least one modified base. In some embodiments, the sense and antisense strands of the oligonucleotide comprise nucleotide sequences selected from the group consisting of: a) SEQ ID NOs: 1537 and 1572, respectively; b) SEQ ID NOs: 1538 and 1573, respectively; c) SEQ ID NOs: 1539 and 1574, respectively; d) SEQ ID NOs: 1540 and 1575, respectively; e) SEQ ID NOs: 1541 and 1576, respectively; f) SEQ ID NOs: 1542 and 1577, respectively; g) SEQ ID NOs: 1543 and 1578, respectively; h) SEQ ID NOs: 1544 and 1579, respectively; i) SEQ ID NOs: 1545 and 1580, respectively; j) SEQ ID NOs: 1546 and 1581, respectively; k) SEQ ID NOs: 1547 and 1582, respectively; l) SEQ ID NOs: 1548 and 1583, respectively; m) SEQ ID NOs: 1549 and 1584, respectively; n) SEQ ID NOs: 1550 and 1585, respectively; o) SEQ ID NOs: 1551 and 1586, respectively; p) SEQ ID NOs: 1552 and 1587, respectively; q) SEQ ID NOs: 1553 and 1588, respectively; r) SEQ ID NOs: 1554 and 1589, respectively; s) SEQ ID NOs: 1555 and 1590, respectively; t) SEQ ID NOs: 1556 and 1591, respectively; u) SEQ ID NOs: 1557 and 1592, respectively; v) SEQ ID NOs: 1558 and 1593, respectively; w) SEQ ID NOs: 1559 and 1594, respectively; x) SEQ ID NOs: 1560 and 1595, respectively; y) SEQ ID NOs: 1561 and 1596, respectively; z) SEQ ID NOs: 1562 and 1597, respectively; aa) SEQ ID NOs: 1563 and 1598, respectively; bb) SEQ ID NOs: 1564 and 1599, respectively; cc) SEQ ID NOs: 1565 and 1600, respectively; dd) SEQ ID NOs: 1566 and 1601, respectively; ee) SEQ ID NOs: 1567 and 1602, respectively; ff) SEQ ID NOs: 1568 and 1603, respectively; gg) SEQ ID NOs: 1569 and 1604, respectively; hh) SEQ ID NOs: 1570 and 1605, respectively; ii) SEQ ID NOs: 1571 and 1606, respectively; and jj) SEQ ID NOs: 1681 and 1586, respectively, wherein the oligonucleotide comprises at least one modified sugar, a 5’ terminal phosphate group, at least one modified intemucleotide linkage, and at least one modified base.

[0115] In some embodiments, the sense and antisense strands comprise nucleotides sequences selected from the group consisting of: a) SEQ ID NOs: 1540 and 1575, respectively; b) SEQ ID NOs: 1544 and 1579, respectively; c) SEQ ID NOs: 1546 and 1581, respectively; d) SEQ ID NOs: 1551and 1586, respectively; e) SEQ ID NOs: 1552 and 1587, respectively; f) SEQ ID NOs: 1553 and 1588, respectively; g) SEQ ID NOs: 1558 and 1594, respectively; h) SEQ ID NOs: 1560 and 1595, respectively; i) SEQ ID NOs: 1564 and 1599, respectively; j) SEQ ID NOs: 1565 and 1600, respectively; k) SEQ ID NOs: 1566 and 1601, respectively; l) SEQ ID NOs: 1570 and 1605, respectively; and m) SEQ ID NOs: 1681 and 1586, respectively, wherein the oligonucleotide comprises at least one modified sugar, a 5’ terminal phosphate group, at least one modified intemucleotide linkage, and at least one modified base.

[0116] In some embodiments, the sense and antisense strands comprise nucleotides sequences selected from the group consisting of: a) SEQ ID NOs: 1553 and 1588, respectively; b) SEQ ID NOs: 1560 and 1595, respectively; c) SEQ ID NOs: 1564 and 1599, respectively; d) SEQ ID NOs: 1551 and 1586, respectively; e) SEQ ID NOs: 1570 and 1605, respectively; and f) SEQ ID NOs: 1681 and 1586, respectively, wherein the oligonucleotide comprises at least one modified sugar, a 5’ terminal phosphate group, at least one modified intemucleotide linkage, and at least one modified base.

[0117] The number of modifications on the oligonucleotide and the position of those nucleotide modifications may influence the properties of the oligonucleotide. For example, the oligonucleotide may be delivered in vivo by encompassing them in a lipid nanoparticle (LNP) or similar carrier. However, when the oligonucleotide is not protected by an LNP or similar carrier, it may be advantageous for at least some of the nucleotides to be modified. Accordingly, in some embodiments, all or substantially all of the nucleotides of the oligonucleotide are modified. In some embodiments, more than half of the nucleotides are modified. In some embodiments, less than half of the nucleotides are modified. In some embodiments, the sugar moiety of all nucleotides comprising the oligonucleotide is modified at the 2’ position. The modifications may be reversible or irreversible. In some embodiments, the oligonucleotide has a number and type of modified nucleotides sufficient to cause the desired characteristics (e.g., protection from enzymatic degradation, capacity to target a desired cell after in vivo administration, and/or thermodynamic stability).

Sugar Modifications

[0118] In some embodiments, the oligonucleotide comprises a modified sugar. In some embodiments, the modified sugar (also referred herein to a sugar analog) includes a modified deoxyribose or ribose moiety in which, for example, one or more modifications occur at the 2', 3', 4' and/or 5' carbon position of the sugar. In some embodiments, the modified sugar may also include non-natural alternative carbon structures such as those present in locked nucleic acids (“LNA”; see, e.g., Koshkin etal. (1998) Tetrahedon 54:3607-30), unlocked nucleic acids (“UNA”; see, e.g., Snead et al. (2013) Afo/. Ther-Nucl. Acids 2:el03) and bridged nucleic acids (“BNA”; see, e.g., Imanishi & Obika (2002) Chem Commun. (Camb) 21 : 1653-59).

[0119] In some embodiments, a nucleotide modification in the sugar comprises a 2'- modification. In some embodiments, the 2 '-modification may be 2'-O-propargyl, 2'-O-propylamin, 2'-amino, 2'-ethyl, 2'-F, 2'-aminoethyl (EA), 2'-OMe, 2'-O-methoxyethyl (2'-M0E), 2'-O-[2- (methylamino)-2-oxoethyl] (2'-0-NMA) or 2'-deoxy-2'-fluoro-P-d-arabinonucleic acid (2'- FANA). In some embodiments, the modification is 2'-F, 2'-0Me or 2'-MOE. In some embodiments, the modified sugar comprises a modification of the sugar ring, which may comprise modification of one or more carbons of the sugar ring. For example, a modification of a sugar of a nucleotide may comprise a 2'-oxygen of a sugar is linked to a 1 '-carbon or 4'-carbon of the sugar, or a 2'-oxygen is linked to the l'-carbon or 4'-carbon via an ethylene or methylene bridge. In some embodiments, the modified nucleotide has an acyclic sugar that lacks a 2'-carbon to 3 '-carbon bond. In some embodiments, the modified nucleotide has a thiol group (e.g., in the 4' position of the sugar).

[0120] In some embodiments, the oligonucleotide comprises at least about 1 modified nucleotide (e.g., at least 1, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, or more). In some embodiments, the sense strand of the oligonucleotide comprises at least about 1 modified nucleotide (e.g., at least 1, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, or more). In some embodiments, the antisense strand of the oligonucleotide comprises at least about 1 modified nucleotide (e.g., at least 1, at least 5, at least 10, at least 15, at least 20, or more).

[0121] In some embodiments, all the nucleotides of the sense strand are modified. In some embodiments, all the nucleotides of the antisense strand are modified. In some embodiments, all the nucleotides (i.e., both the sense strand and the antisense strand) are modified. In some embodiments, the modified nucleotide comprises a 2 '-modification (e.g., a 2'-F or 2'-OMe, 2'- MOE, and 2'-deoxy-2'-fluoro-P-d-arabinonucleic acid). In some embodiments, the modified nucleotide comprises a 2'-modification (e.g., a 2'-F or 2'-OMe)

[0122] In some embodiments, the disclosure provides oligonucleotides having different modification patterns. In some embodiments, the oligonucleotides comprise a sense strand sequence having a modification pattern as set forth in the Examples and Sequence Listing and an antisense strand having a modification pattern as set forth in the Examples and Sequence Listing. [0123] In some embodiments, the oligonucleotide comprises an antisense strand having nucleotides that are modified with 2'-F. In other embodiments, the oligonucleotide comprises an antisense strand having nucleotides that are modified with 2'-F and 2'-OMe. In some embodiments, the oligonucleotide comprises a sense strand having nucleotides that are modified with 2'-F. In other embodiments, the oligonucleotide comprises a sense strand having nucleotides that are modified with 2'-F and 2'-0Me.

[0124] In some embodiments, the oligonucleotide comprises a sense strand with about 10% to about 15%, or 10%, 11%, 12%, 13%, 14%, or 15% of the nucleotides of the sense strand comprising a 2’-F modification. In some embodiments, the oligonucleotide comprises a sense strand with about 18% to about 23%, or 18%, 19%, 20%, 21%, 22%, or 23% of the nucleotides comprising a 2’-F modification. In some embodiments, the oligonucleotide comprises a sense strand with about 38% to about 43%, or 38%, 39%, 40%, 41%, 42%, or 43% of the nucleotides comprising a 2’-F modification. In some embodiments, about 11% of the nucleotides of the sense strand comprise a 2-F modification. In some embodiments, about 22% of the nucleotides of the sense strand comprise a 2-F modification. In some embodiments, about 40% of the nucleotides of the sense strand comprise a 2-F modification.

[0125] In some embodiments, the oligonucleotide comprises an antisense strand with about 25% to about 35%, or 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35% of the nucleotides comprising a 2’-F modification. In some embodiments, about 32% of the nucleotides of the antisense strand comprise a 2’-F modification. In some embodiments, the oligonucleotide has about 15% to about 25%, or 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% of the nucleotides comprising a 2’-F modification. In some embodiments, the oligonucleotide has about 35% to about 45%, or 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, or 45% of the nucleotides comprising a 2’-F modification. In some embodiments, about 19% of the nucleotides comprise a 2’-F modification. In some embodiments, about 29% of the nucleotides comprise a 2’-F modification. In some embodiments, about 40% of the nucleotides comprise a 2’- F modification.

[0126] In some embodiments, one or more of positions 8, 9, 10, or 11 of a 36-nucleotide sense strand are modified with a 2'-F group. In some embodiments, one or more of positions 8, 9, 10, or 11 of a sense strand comprising a stem-loop are modified with a 2’-F group. In some embodiments, the sugar moiety at each of nucleotides at positions 1-7 and 12-20 of a 36-nucleotide sense strand is modified with a 2'-0Me. In some embodiments, the sugar moiety at each of nucleotides at positions 1-7 and 12-20 of a sense strand comprising a stem-loop is modified with a 2’-0Me. In some embodiments, the sugar moiety at each of nucleotides at positions 1-7 and 12-36 in the sense strand is modified with a 2'-0Me.

[0127] In some embodiments, one or more of positions 3, 5, 8, 10, 12, 13, 15, and 17 of the sense strand are modified with a 2'-F group.

[0128] In some embodiments, the antisense strand has 3 nucleotides that are modified at the 2'- position of the sugar moiety with a 2'-F. In some embodiments, the sugar moiety at positions 2, 5, and 14 and optionally up to 3 of the nucleotides at positions 1, 3, 7 and 10 of the antisense strand are modified with a 2'-F. In some embodiments, the sugar moiety at positions 2, 5, and 14 and optionally up to 3 of the nucleotides at positions 3, 4, 7 and 10 of the antisense strand are modified with a 2'-F. In other embodiments, the sugar moiety at each of the positions at positions 2, 5, and 14 of the antisense strand is modified with the 2'-F. In other embodiments, the sugar moiety at each of the positions at positions 1, 2, 5, and 14 of the antisense strand is modified with the 2'-F. In other embodiments, the sugar moiety at each of the positions at positions 2, 4, 5, and 14 of the antisense strand is modified with the 2'-F. Tn still other embodiments, the sugar moiety at each of the positions at positions 1, 2, 3, 5, 7, and 14 of the antisense strand is modified with the 2'-F. In other embodiments, the sugar moiety at each of the positions at positions 2, 3, 4, 5, 7, and 14 of the antisense strand is modified with the 2'-F. In yet another embodiment, the sugar moiety at each of the positions at positions 1, 2, 3, 5, 10, and 14 of the antisense strand is modified with the 2'-F. In other embodiments, the sugar moiety at each of the positions at positions 2, 3, 4, 5, 10, and 14 of the antisense strand is modified with the 2'-F. In another embodiment, the sugar moiety at each of the positions at positions 2, 3, 5, 7, 10, and 14 of the antisense strand is modified with the 2'-F. In other embodiments, the sugar moiety at each of the positions at positions 2, 3, 4, 5, 7, 10, and 14 of an antisense strand duplexed with a 36-nucleotide sense strand is modified with the 2'-F. In some embodiments, the sugar moiety at each of the positions at positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand duplexed with a sense strand comprising a stem-loop is modified with the 2’-F.

[0129] In some embodiments, the oligonucleotide comprises an antisense strand having the sugar moiety at positions 2 and 14 modified with 2'-F. In some embodiments, the oligonucleotide comprises an antisense strand having the sugar moiety at positions 2, 5, and 14 modified with 2'- F. In some embodiments, the oligonucleotide comprises an antisense strand having the sugar moiety at positions 1, 2, 5, and 14 modified with 2'-F. In some embodiments, the oligonucleotide comprises an antisense strand having the sugar moiety at positions 2, 4, 5, and 14 modified with 2'-F. In some embodiments, the oligonucleotide comprises an antisense strand having the sugar moiety at positions 1, 2, 3, 5, 7, and 14 modified with 2'-F. In some embodiments, the oligonucleotide comprises an antisense strand having the sugar moiety at positions 2, 3, 4, 5, 7, and 14 modified with 2'-F. In some embodiments, the oligonucleotide comprises an antisense strand having the sugar moiety at positions 1, 2, 3, 5, 10, and 14 modified with 2'-F. In some embodiments, the oligonucleotide comprises an antisense strand having the sugar moiety at positions 2, 3, 4, 5, 10, and 14 modified with 2'-F. In some embodiments, the oligonucleotide comprises a 36-nucleotide sense strand and an antisense strand, wherein the antisense strand comprises a sugar moiety at positions 2, 3, 4, 5, 7, 10, and 14 modified with 2'-F. In some embodiments, the oligonucleotide comprises a sense strand comprising a stem-loop and an antisense strand, wherein the antisense strand comprises a sugar moiety at positions 2, 3, 4, 5, 7, 10, and 14 modified with 2'-F. In some embodiments, the oligonucleotide comprises an antisense strand having the sugar moiety at positions 2, 3, 4, 5, 7, 10, 14, 16, and 19 modified with 2'-F.

[0130] In some embodiments, the oligonucleotide comprises an antisense strand having the sugar moiety of each of the nucleotides at positions 2, 5, and 14 modified with 2'-F and the sugar moiety of each of the remaining nucleotides of the antisense strand modified with a modification selected from the group consisting of 2'-O-propargyl, 2 -O-propylamin, 2'-amino, 2'-ethyl, EA, 2'- OMe, 2'-M0E, 2'-0-NMA, and 2'-FANA.

[0131] In some embodiments, the oligonucleotide comprises an antisense strand having the sugar moiety of each of the nucleotides at positions 1, 2, 5, and 14 modified with 2'-F and the sugar moiety of each of the remaining nucleotides of the antisense strand modified with a modification selected from the group consisting of 2'-O-propargyl, 2 -O-propylamin, 2'-amino, 2'-ethyl, EA, 2'- OMe, 2'-M0E, 2'-0-NMA, and 2'-FANA.

[0132] In some embodiments, the oligonucleotide comprises an antisense strand having the sugar moiety of each of the nucleotides at positions 2, 4, 5, and 14 modified with 2'-F and the sugar moiety of each of the remaining nucleotides of the antisense strand modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamin, 2'-amino, 2'-ethyl, EA, 2'- OMe, 2'-M0E, 2'-0-NMA, and 2'-FANA.

[0133] In some embodiments, the oligonucleotide comprises an antisense strand having the sugar moiety of each of the nucleotides at positions 1, 2, 3, 5, 7, and 14 modified with 2'-F and the sugar moiety of each of the remaining nucleotides of the antisense strand modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamin, 2'-amino, 2'- ethyl, EA, 2'-0Me, 2'-M0E, 2'-0-NMA, and 2'-FANA.

[0134] In some embodiments, the oligonucleotide comprises an antisense strand having the sugar moiety of each of the nucleotides at positions 2, 3, 4, 5, 7, and 14 modified with 2'-F and the sugar moiety of each of the remaining nucleotides of the antisense strand modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamin, 2'-amino, 2'- ethyl, EA, 2'-0Me, 2'-M0E, 2'-0-NMA, and 2'-FANA.

[0135] In some embodiments, the oligonucleotide comprises an antisense strand having the sugar moiety of each of the nucleotides at positions 1, 2, 3, 5, 10, and 14 modified with 2'-F and the sugar moiety of each of the remaining nucleotides of the antisense strand modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamin, 2'-amino, 2'- ethyl, EA, 2'-0Me, 2'-M0E, 2'-0-NMA, and 2'-FANA.

[0136] In some embodiments, the oligonucleotide comprises an antisense strand having the sugar moiety of each of the nucleotides at positions 2, 3, 4, 5, 10, and 14 modified with 2'-F and the sugar moiety of each of the remaining nucleotides of the antisense strand modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamin, 2'-amino, 2'- ethyl, EA, 2'-0Me, 2'-M0E, 2'-0-NMA, and 2'-FANA.

[0137] In some embodiments, the oligonucleotide comprises an antisense strand having the sugar moiety of each of the nucleotides at positions 2, 3, 5, 7, 10, and 14 modified with 2'-F and the sugar moiety of each of the remaining nucleotides of the antisense strand modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamin, 2'-amino, 2'- ethyl, EA, 2'-0Me, 2'-M0E, 2'-0-NMA, and 2'-FANA.

[0138] In some embodiments, the oligonucleotide comprises a 36-nucleotide sense strand and an antisense strand, wherein the antisense strand comprises a sugar moiety at positions 2, 3, 4, 5, 7, 10, and 14 modified with 2'-F and the sugar moiety of each of the remaining nucleotides of the antisense strand modified with a modification selected from the group consisting of 2'-O- propargyl, 2'-O-propylamin, 2'-amino, 2'-ethyl, EA, 2'-OMe, 2'-M0E, 2'-0-NMA, and 2'-FANA. In some embodiments, the oligonucleotide comprises a sense strand comprising a stem-loop and an antisense strand, wherein the antisense strand comprises a sugar moiety at positions 2, 3, 4, 5, 7, 10, and 14 modified with 2'-F and the sugar moiety of each of the remaining nucleotides of the antisense strand modified with a modification selected from the group consisting of 2'-O- propargyl, 2'-O-propylamin, 2'-amino, 2'-ethyl, EA, 2'-OMe, 2'-M0E, 2'-0-NMA, and 2'-FANA. [0139] In some embodiments, the oligonucleotide comprises an antisense strand having the sugar moiety of each of the nucleotides at positions 2, 3, 4, 5, 7, 10, 14, 16, and 19 modified with 2'-F and the sugar moiety of each of the remaining nucleotides of the antisense strand modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamin, 2'- amino, 2'-ethyl, EA, 2'-0Me, 2'-M0E, 2'-0-NMA, and 2'-FANA.

[0140] In some embodiments, the oligonucleotide comprises an antisense strand having the sugar moiety at position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, or position 22 modified with 2'-F.

[0141] In some embodiments, the oligonucleotide comprises an antisense strand having the sugar moiety at position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, or position 22 modified with 2'-OMe.

[0142] In some embodiments, the oligonucleotide comprises an antisense strand having the sugar moiety at position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, or position 22 modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamin, 2'- amino, 2'-ethyl, EA, 2'-0Me, 2'-M0E, 2'-0-NMA, and 2'-FANA.

[0143] In some embodiments, the oligonucleotide comprises a 36-nucleotide sense strand having the sugar moiety at positions 8-11 modified with 2'-F. In some embodiments, the oligonucleotide comprises a sense strand comprising a stem-loop and the sugar moiety at positions 8-11 modified with 2’-F. In some embodiments, the oligonucleotide comprises a 36-nucleotide sense strand having the sugar moiety at positions 1-7 and 12-17 or 12-20 modified with 2’-OMe. In some embodiments, the oligonucleotide comprises a sense strand comprising a stem-loop and the sugar moiety at positions 1-7 and 12-17 or 12-20 modified with 2’-OMe. In some embodiments, the oligonucleotide comprises a 36-nucleotide sense strand having the sugar moiety at positions 1-7 and 12-17, 12-20 or 12-22 modified with 2’-OMe. In some embodiments, the oligonucleotide comprises a sense strand comprising a stem-loop and the sugar moiety at positions 1-7 and 12-17, 12-20 or 12-22 modified with 2’-OMe. In some embodiments, the oligonucleotide comprises a 36- nucleotide sense strand having the sugar moiety of each of the nucleotides at positions 1-7 and 12- 17 or 12-20 of the sense strand modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamin, 2'-amino, 2'-ethyl, EA, 2'-0Me, 2'-M0E, 2'-0-NMA, and 2'- FANA. In some embodiments, the oligonucleotide comprises a sense strand comprising a stemloop and having the sugar moiety of each of the nucleotides at positions 1-7 and 12-17 or 12-20 of the sense strand modified with a modification selected from the group consisting of 2'-O- propargyl, 2'-O-propylamin, 2'-amino, 2'-ethyl, EA, 2'-OMe, 2'-M0E, 2'-0-NMA, and 2'-FANA. In some embodiments, the oligonucleotide comprises a 36-nucleotde sense strand having the sugar moiety of each of the nucleotides at positions 1-7 and 12-17, 12-20 or 12-22 of the sense strand modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O- propylamin, 2'-amino, 2'-ethyl, EA, 2'-0Me, 2'-M0E, 2 -0-NMA, and 2’-FANA. In some embodiments, the oligonucleotide comprises a sense strand comprising a stem-loop and the sugar moiety of each of the nucleotides at positions 1-7 and 12-17, 12-20 or 12-22 of the sense strand modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O- propylamin, 2'-amino, 2'-ethyl, EA, 2'-0Me, 2'-M0E, 2'-0-NMA, and 2'-FANA.

[0144] In some embodiments, the oligonucleotide comprises a sense strand having the sugar moiety at positions 3, 5, 8, 10, 12, 13, 15, and 17 modified with 2'-F. In some embodiments, the oligonucleotide comprises a sense strand having the sugar moiety at positions 1, 2, 4, 6, 7, 9, 11, 14, 16, and 18-20 modified with 2’-OMe. In some embodiments, the oligonucleotide comprises a sense strand having the sugar moiety of each of the nucleotides at positions 1, 2, 4, 6, 7, 9, 11, 14, 16, and 18-20 of the sense strand modified with a modification selected from the group consisting of 2 -O-propargyl, 2'-O-propylamin, 2'-amino, 2'-ethyl, EA, 2'-0Me, 2'-M0E, 2'-0-NMA, and 2'- FANA. In some embodiments, the oligonucleotide comprises a sense strand having the sugar moiety of each of the nucleotides at positions 1-7 and 12-17, 12-20 or 12-22 of the sense strand modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O- propylamin, 2'-amino, 2'-ethyl, EA, 2'-0Me, 2'-M0E, 2'-0-NMA, and 2'-FANA.

[0145] In some embodiments, the oligonucleotide comprises a sense strand having the sugar moiety at position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, position 22, position 23, position 24, position 25, position 26, position 27, position 28, position 29, position 30, position 31, position 32, position 33, position 34, position 35, or position 36 modified with 2'-F.

[0146] In some embodiments, the oligonucleotide comprises a sense strand having the sugar moiety at position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, position 22, position 23, position 24, position 25, position 26, position 27, position 28, position 29, position 30, position 31, position 32, position 33, position 34, position 35, or position 36 modified with 2'-0Me.

[0147] In some embodiments, the oligonucleotide comprises a sense strand having the sugar moiety at position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, position 22, position 23, position 24, position 25, position 26, position 27, position 28, position 29, position 30, position 31, position 32, position 33, position 34, position 35, or position 36 modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamin, 2'-amino, 2'-ethyl, EA, 2'-0Me, 2'- MOE, 2'-0-NMA, and 2'-FANA.

[0148] In some embodiments, the oligonucleotide comprises an antisense strand having the sugar moiety of each of the nucleotides at positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand modified with 2'-F and the sugar moiety of each of the remaining nucleotides of the antisense strand modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O- propylamin, 2'-amino, 2'-ethyl, EA, 2'-0Me, 2'-M0E, 2 -0-NMA, and 2'-FANA; and a sense strand having the sugar moiety at each of the nucleotides at positions 8-11 of the sense strand modified with 2’-F and the sugar moiety of each of the remaining nucleotides of the sense strand modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O- propylamin, 2'-amino, 2'-ethyl, EA, 2'-0Me, 2'-M0E, 2 -0-NMA, and 2’-FANA. In some embodiments, the oligonucleotide comprises an antisense strand having the sugar moiety of each of the nucleotides at positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand modified with 2 -F and the sugar moiety of each of the remaining nucleotides of the antisense strand modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamin, 2'-amino, 2'- ethyl, EA, 2'-0Me, 2'-M0E, 2'-0-NMA, and 2'-FANA; and a sense strand having and stem-loop and the sugar moiety at each of the nucleotides at positions 8-11 of the sense strand modified with 2’-F and the sugar moiety of each of the remaining nucleotides of the sense strand modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamin, 2'-amino, 2'-ethyl, EA, 2'-0Me, 2'-M0E, 2'-0-NMA, and 2'-FANA.

[0149] In some embodiments, the oligonucleotide comprises an antisense strand having the sugar moiety of each of the nucleotides at positions 2, 3, 4, 5, 7, 10, 14, 16, and 19 of the antisense strand modified with 2'-F and the sugar moiety of each of the remaining nucleotides of the antisense strand modified with a modification selected from the group consisting of 2'-O- propargyl, 2'-O-propylamin, 2'-amino, 2'-ethyl, EA, 2'-0Me, 2'-M0E, 2'-0-NMA, and 2'-FANA; and a sense strand having the sugar moiety at each of the nucleotides at positions 3, 5, 8, 10, 12, 13, 15, and 17 of the sense strand modified with 2’-F and the sugar moiety of each of the remaining nucleotides of the sense strand modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamin, 2'-amino, 2'-ethyl, EA, 2'-0Me, 2'-M0E, 2'-0-NMA, and 2'- FANA.

[0150] In some embodiments, the sense and antisense strands of an oligonucleotide comprise nucleotides sequences selected from the group consisting of: a) SEQ ID NOs: 1537 and 1572, respectively; b) SEQ ID NOs: 1538 and 1573, respectively; c) SEQ ID NOs: 1539 and 1574, respectively; d) SEQ ID NOs: 1540 and 1575, respectively; e) SEQ ID NOs: 1541 and 1576, respectively; f) SEQ ID NOs: 1542 and 1577, respectively; g) SEQ ID NOs: 1543 and 1578, respectively; h) SEQ ID NOs: 1544 and 1579, respectively; i) SEQ ID NOs: 1545 and 1580, respectively; j) SEQ ID NOs: 1546 and 1581, respectively; k) SEQ ID NOs: 1547 and 1582, respectively; l) SEQ ID NOs: 1548 and 1583, respectively; m) SEQ ID NOs: 1549 and 1584, respectively; n) SEQ ID NOs: 1550 and 1585, respectively; o) SEQ ID NOs: 1551 and 1586, respectively; p) SEQ ID NOs: 1552 and 1587, respectively; q) SEQ ID NOs: 1553 and 1588, respectively; r) SEQ ID NOs: 1554 and 1589, respectively; s) SEQ ID NOs: 1555 and 1590, respectively; t) SEQ ID NOs: 1556 and 1591, respectively; u) SEQ ID NOs: 1557 and 1592, respectively; v) SEQ ID NOs: 1558 and 1593, respectively; w) SEQ ID NOs: 1559 and 1594, respectively; x) SEQ ID NOs: 1560 and 1595, respectively; y) SEQ ID NOs: 1561 and 1596, respectively; z) SEQ ID NOs: 1562 and 1597, respectively; aa) SEQ ID NOs: 1563 and 1598, respectively; bb) SEQ ID NOs: 1564 and 1599, respectively; cc) SEQ ID NOs: 1565 and 1600, respectively; dd) SEQ ID NOs: 1566 and 1601, respectively; ee) SEQ ID NOs: 1567 and 1602, respectively; ff) SEQ ID NOs: 1568 and 1603, respectively; gg) SEQ ID NOs: 1569 and 1604, respectively; hh) SEQ ID NOs: 1570 and 1605, respectively; ii) SEQ ID NOs: 1571 and 1606, respectively; and jj) SEQ ID NOs: 1681 and 1586, respectively, wherein one or more of positions 3, 5, 8, 10, 12, 13, 15, or 17 of the sense strand are modified with a 2'-F group.

[0151J In some embodiments, the sense and antisense strands of the oligonucleotide comprise nucleotides sequences selected from the group consisting of: a) SEQ ID NOs: 1540 and 1575, respectively; b) SEQ ID NOs: 1544 and 1579, respectively; c) SEQ ID NOs: 1546 and 1581, respectively; d) SEQ ID NOs: 155 land 1586, respectively; e) SEQ ID NOs: 1552 and 1587, respectively; f) SEQ ID NOs: 1553 and 1588, respectively; g) SEQ ID NOs: 1558 and 1594, respectively; h) SEQ ID NOs: 1560 and 1595, respectively; i) SEQ ID NOs: 1564 and 1599, respectively; j) SEQ ID NOs: 1565 and 1600, respectively; k) SEQ ID NOs: 1566 and 1601, respectively; l) SEQ ID NOs: 1570 and 1605, respectively; and m) SEQ ID NOs: 1681 and 1586, respectively, wherein one or more of positions 3, 5, 8, 10, 12, 13, 15, or 17 of the sense strand are modified with a 2'-F group.

[0152] In some embodiments, the sense and antisense strands of the oligonucleotide comprise nucleotides sequences selected from the group consisting of: a) SEQ ID NOs: 1553 and 1588, respectively; b) SEQ ID NOs: 1560 and 1595, respectively; c) SEQ ID NOs: 1564 and 1599, respectively; d) SEQ ID NOs: 1551 and 1586, respectively; e) SEQ ID NOs: 1570 and 1605, respectively; and f) SEQ ID NOs: 1681 and 1586, respectively, wherein one or more of positions 3, 5, 8, 10, 12, 13, 15, or 17 of the sense strand are modified with a 2'-F group.

5 ’-Terminal Phosphate

[0153] In some embodiments, an oligonucleotide described herein (e.g., an RNAi oligonucleotide) comprises a sense strand and an antisense strand, wherein the antisense strand comprises a 5’ terminal phosphate. In some embodiments, 5' terminal phosphate groups enhance the interaction with Ago2. However, oligonucleotides comprising a 5' phosphate group may be susceptible to degradation via phosphatases or other enzymes, which can limit their bioavailability in vivo. In some embodiments, oligonucleotides include analogs of 5' phosphates that are resistant to such degradation. In some embodiments, the phosphate analog is oxymethyl phosphonate, vinyl phosphonate or malonyl phosphonate, or a combination thereof. In certain embodiments, the 5' end of an oligonucleotide strand is attached to chemical moiety that mimics the electrostatic and steric properties of a natural 5' phosphate group (“phosphate mimic”).

[0154] In some embodiments, the oligonucleotide has a phosphate analog at a 4'-carbon position of the sugar (referred to as a “4'-phosphate analog”). See, e.g., Inti. Patent Application Publication No. WO 2018/045317. In some embodiments, the oligonucleotide comprises a 4'-phosphate analog at a 5' terminal nucleotide. In some embodiments, the phosphate analog is an oxymethyl phosphonate, in which the oxygen atom of the oxymethyl group is bound to the sugar moiety (e.g., at its 4'-carbon) or analog thereof. In other embodiments, the 4'-phosphate analog is a thiomethylphosphonate or an aminomethylphosphonate, in which the sulfur atom of the thiomethyl group or the nitrogen atom of the amino methyl group is bound to the 4'-carbon of the sugar moiety or analog thereof. In certain embodiments, the 4'-phosphate analog is an oxymethyl phosphonate. In some embodiments, the oxymethyl phosphonate is represented by the formula -O-CH2- PO(OH)2,-O-CH2-PO(OR)2, or -O-CH2-POOH(R), in which R is independently selected from H, CH3, an alkyl group, CH2CH2CN, CH2OCOC(CH3)3, CthOCthCHjSi (CH3)3 or a protecting group. In certain embodiments, the alkyl group is CH2CH3. More typically, R is independently selected from H, CH3, or CH2CH3. In some embodiment, R is CH3. In some embodiments, the 4’- phosphate analog is 4’-oxymethylphosphonate. In some embodiments, the modified nucleotide having the 4 ’-phosphonate analog is a uridine. In some embodiments, the modified nucleotide is 4’-O-monomethylphosphonate-2’-O-methyl uridine.

[0155] In some embodiments, the sense and antisense strands of the oligonucleotide comprise nucleotides sequences selected from the group consisting of: a) SEQ ID NOs: 1537 and 1572, respectively; b) SEQ ID NOs: 1538 and 1573, respectively; c) SEQ ID NOs: 1539 and 1574, respectively; d) SEQ ID NOs: 1540 and 1575, respectively; e) SEQ ID NOs: 1541 and 1576, respectively; f) SEQ ID NOs: 1542 and 1577, respectively; g) SEQ ID NOs: 1543 and 1578, respectively; h) SEQ ID NOs: 1544 and 1579, respectively; i) SEQ ID NOs: 1545 and 1580, respectively; j) SEQ ID NOs: 1546 and 1581, respectively; k) SEQ ID NOs: 1547 and 1582, respectively; l) SEQ ID NOs: 1548 and 1583, respectively; m) SEQ ID NOs: 1549 and 1584, respectively; n) SEQ ID NOs: 1550 and 1585, respectively; o) SEQ ID NOs: 1551 and 1586, respectively; p) SEQ ID NOs: 1552 and 1587, respectively; q) SEQ ID NOs: 1553 and 1588, respectively; r) SEQ ID NOs: 1554 and 1589, respectively; s) SEQ ID NOs: 1555 and 1590, respectively; t) SEQ ID NOs: 1556 and 1591, respectively; u) SEQ ID NOs: 1557 and 1592, respectively; v) SEQ ID NOs: 1558 and 1593, respectively; w) SEQ ID NOs: 1559 and 1594, respectively; x) SEQ ID NOs: 1560 and 1595, respectively; y) SEQ ID NOs: 1561 and 1596, respectively; z) SEQ ID NOs: 1562 and 1597, respectively; aa) SEQ ID NOs: 1563 and 1598, respectively; bb) SEQ ID NOs: 1564 and 1599, respectively; cc) SEQ ID NOs: 1565 and 1600, respectively; dd) SEQ ID NOs: 1566 and 1601, respectively; ee) SEQ ID NOs: 1567 and 1602, respectively; ff) SEQ ID NOs: 1568 and 1603, respectively; gg) SEQ ID NOs: 1569 and 1604, respectively; hh) SEQ ID NOs: 1570 and 1605, respectively; ii) SEQ ID NOs: 1571 and 1606, respectively; and jj) SEQ ID NOs: 1681 and 1586, respectively, wherein the oligonucleotide comprises a 5’-terminal phosphate, optionally a 5’-terminal phosphate analog.

[0156] In some embodiments, the sense and antisense strands of an oligonucleotide comprise nucleotides sequences selected from the group consisting of: a) SEQ ID NOs: 1540 and 1575, respectively; b) SEQ ID NOs: 1544 and 1579, respectively; c) SEQ ID NOs: 1546 and 1581, respectively; d) SEQ ID NOs: 1551and 1586, respectively; e) SEQ ID NOs: 1552 and 1587, respectively; f) SEQ ID NOs: 1553 and 1588, respectively; g) SEQ ID NOs: 1558 and 1594, respectively; h) SEQ ID NOs: 1560 and 1595, respectively; i) SEQ ID NOs: 1564 and 1599, respectively; j) SEQ ID NOs: 1565 and 1600, respectively; k) SEQ ID NOs: 1566 and 1601, respectively; l) SEQ ID NOs: 1570 and 1605, respectively; and m) SEQ ID NOs: 1681 and 1586, respectively,

13 wherein the oligonucleotide comprises a 5’-terminal phosphate, optionally a 5’-terminal phosphate analog.

[0157] In some embodiments, the sense and antisense strands of the oligonucleotide comprise nucleotides sequences selected from the group consisting of: a) SEQ ID NOs: 1553 and 1588, respectively; b) SEQ ID NOs: 1560 and 1595, respectively; c) SEQ ID NOs: 1564 and 1599, respectively; d) SEQ ID NOs: 1551 and 1586, respectively; e) SEQ ID NOs: 1570 and 1605, respectively; and f) SEQ ID NOs: 1681 and 1586, respectively, wherein the oligonucleotide comprises a 5’-terminal phosphate, optionally a 5’-terminal phosphate analog.

[0158] In some embodiments, the oligonucleotide comprises an antisense strand comprising a 4'-phosphate analog at the 5' terminal nucleotide, wherein 5’ terminal nucleotide comprises the following structure: 4’-O-monomethylphosphonate-2’-O-methyl uridine phosphorothioate [MePhosphonate-4O- mUs],

Modified Internucleotide Linkage

[0159J In some embodiments, an oligonucleotide e.g., a RNAi oligonucleotide) comprises a modified internucleotide linkage. In some embodiments, phosphate modifications or substitutions result in an oligonucleotide that comprises at least about 1 (e.g., at least 1, at least 2, at least 3, at least 4, or at least 5) modified intemucleotide linkage. Tn some embodiments, the oligonucleotide comprises about 1 to about 10 (e.g., 1 to 10, 2 to 8, 4 to 6, 3 to 10, 5 to 10, 1 to 5, 1 to 3 or 1 to 2) modified intemucleotide linkages. In some embodiments, the oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modified internucleotide linkages.

[0160] A modified intemucleotide linkage may be a phosphorodithioate linkage, a phosphorothioate linkage, a phosphotriester linkage, a thionoalkylphosphonate linkage, a thionalkylphosphotriester linkage, a phosphoramidite linkage, a phosphonate linkage, or a boranophosphate linkage. In some embodiments, at least one modified intemucleotide linkage is a phosphorothioate linkage.

[0161] In some embodiments, the oligonucleotide has a phosphorothioate linkage between one or more of positions 1 and 2 of the sense strand, positions 1 and 2 of the antisense strand, positions 2 and 3 of the antisense strand, positions 3 and 4 of the antisense strand, positions 20 and 21 of the antisense strand, and positions 21 and 22 of the antisense strand. In some embodiments, the oligonucleotide has a phosphorothioate linkage between each of positions 1 and 2 of the sense strand, positions 1 and 2 of the antisense strand, positions 2 and 3 of the antisense strand, positions 20 and 21 of the antisense strand, and positions 21 and 22 of the antisense strand. In some embodiments, the oligonucleotide has a phosphorothioate linkage between each of (i) positions 1 and 2 of the sense strand; and (ii) positions 1 and 2, positions 2 and 3, positions 3 and 4, positions 20 and 21, and positions 21 and 22 of the antisense strand.

[0162] In some embodiments, the sense and antisense strands of the oligonucleotide comprise nucleotides sequences selected from the group consisting of: a) SEQ ID NOs: 1537 and 1572, respectively; b) SEQ ID NOs: 1538 and 1573, respectively; c) SEQ ID NOs: 1539 and 1574, respectively; d) SEQ ID NOs: 1540 and 1575, respectively; e) SEQ ID NOs: 1541 and 1576, respectively; f) SEQ ID NOs: 1542 and 1577, respectively; g) SEQ ID NOs: 1543 and 1578, respectively; h) SEQ ID NOs: 1544 and 1579, respectively; i) SEQ ID NOs: 1545 and 1580, respectively; j) SEQ ID NOs: 1546 and 1581, respectively; k) SEQ ID NOs: 1547 and 1582, respectively; l) SEQ ID NOs: 1548 and 1583, respectively; m) SEQ ID NOs: 1549 and 1584, respectively; n) SEQ ID NOs: 1550 and 1585, respectively; o) SEQ ID NOs: 1551 and 1586, respectively; p) SEQ ID NOs: 1552 and 1587, respectively; q) SEQ ID NOs: 1553 and 1588, respectively; r) SEQ ID NOs: 1554 and 1589, respectively; s) SEQ ID NOs: 1555 and 1590, respectively; t) SEQ ID NOs: 1556 and 1591, respectively; u) SEQ ID NOs: 1557 and 1592, respectively; v) SEQ ID NOs: 1558 and 1593, respectively; w) SEQ ID NOs: 1559 and 1594, respectively; x) SEQ ID NOs: 1560 and 1595, respectively; y) SEQ ID NOs: 1561 and 1596, respectively; z) SEQ ID NOs: 1562 and 1597, respectively; aa) SEQ ID NOs: 1563 and 1598, respectively; bb) SEQ ID NOs: 1564 and 1599, respectively; cc) SEQ ID NOs: 1565 and 1600, respectively; dd) SEQ ID NOs: 1566 and 1601, respectively; ee) SEQ ID NOs: 1567 and 1602, respectively; ff) SEQ ID NOs: 1568 and 1603, respectively; gg) SEQ ID NOs: 1569 and 1604, respectively; hh) SEQ ID NOs: 1570 and 1605, respectively; ii) SEQ ID NOs: 1571 and 1606, respectively; and jj) SEQ ID NOs: 1681 and 1586, respectively, wherein the oligonucleotide comprises a modified intemucleotide linkage.

[0163] In some embodiments, the sense and antisense strands of the oligonucleotide comprise nucleotides sequences selected from the group consisting of: a) SEQ ID NOs: 1540 and 1575, respectively; b) SEQ ID NOs: 1544 and 1579, respectively; c) SEQ ID NOs: 1546 and 1581, respectively; d) SEQ ID NOs: 1551and 1586, respectively; e) SEQ ID NOs: 1552 and 1587, respectively; f) SEQ ID NOs: 1553 and 1588, respectively; g) SEQ ID NOs: 1558 and 1594, respectively; h) SEQ ID NOs: 1560 and 1595, respectively; i) SEQ ID NOs: 1564 and 1599, respectively; j) SEQ ID NOs: 1565 and 1600, respectively; k) SEQ ID NOs: 1566 and 1601, respectively; l) SEQ ID NOs: 1570 and 1605, respectively; and m) SEQ ID NOs: 1681 and 1586, respectively, wherein the oligonucleotide comprises a modified intemucleotide linkage.

[0164] In some embodiments, the sense and antisense strands of the oligonucleotide comprise nucleotides sequences selected from the group consisting of: a) SEQ ID NOs: 1553 and 1588, respectively; b) SEQ ID NOs: 1560 and 1595, respectively; c) SEQ ID NOs: 1564 and 1599, respectively; d) SEQ ID NOs: 1551 and 1586, respectively; e) SEQ ID NOs: 1570 and 1605, respectively; and f) SEQ ID NOs: 1681 and 1586, respectively, wherein the oligonucleotide comprises a modified intemucleotide linkage.

Base Modifications

[0165] In some embodiments, an oligonucleotide herein (e.g., a RNAi oligonucleotide) has one or more modified nucleobases. In some embodiments, modified nucleobases (also referred to herein as base analogs) are linked at the 1 ' position of a nucleotide sugar moiety. In certain embodiments, a modified nucleobase is a nitrogenous base. In certain embodiments, a modified nucleobase does not contain nitrogen atom. See, e.g., US Patent Application Publication No. 2008/0274462. In some embodiments, a modified nucleotide comprises a universal base. In some embodiments, a modified nucleotide does not contain a nucleobase (abasic).

[0166] In some embodiments, the sense and antisense strands of the oligonucleotide comprise nucleotides sequences selected from the group consisting of: a) SEQ ID NOs: 1537 and 1572, respectively; b) SEQ ID NOs: 1538 and 1573, respectively; c) SEQ ID NOs: 1539 and 1574, respectively; d) SEQ ID NOs: 1540 and 1575, respectively; e) SEQ ID NOs: 1541 and 1576, respectively; f) SEQ ID NOs: 1542 and 1577, respectively; g) SEQ ID NOs: 1543 and 1578, respectively; h) SEQ ID NOs: 1544 and 1579, respectively; i) SEQ ID NOs: 1545 and 1580, respectively; j) SEQ ID NOs: 1546 and 1581, respectively; k) SEQ ID NOs: 1547 and 1582, respectively; l) SEQ ID NOs: 1548 and 1583, respectively; m) SEQ ID NOs: 1549 and 1584, respectively; n) SEQ ID NOs: 1550 and 1585, respectively; o) SEQ ID NOs: 1551 and 1586, respectively; p) SEQ ID NOs: 1552 and 1587, respectively; q) SEQ ID NOs: 1553 and 1588, respectively; r) SEQ ID NOs: 1554 and 1589, respectively; s) SEQ ID NOs: 1555 and 1590, respectively; t) SEQ ID NOs: 1556 and 1591, respectively; u) SEQ ID NOs: 1557 and 1592, respectively; v) SEQ ID NOs: 1558 and 1593, respectively; w) SEQ ID NOs: 1559 and 1594, respectively; x) SEQ ID NOs: 1560 and 1595, respectively; y) SEQ ID NOs: 1561 and 1596, respectively; z) SEQ ID NOs: 1562 and 1597, respectively; aa) SEQ ID NOs: 1563 and 1598, respectively; bb) SEQ ID NOs: 1564 and 1599, respectively; cc) SEQ ID NOs: 1565 and 1600, respectively; dd) SEQ ID NOs: 1566 and 1601, respectively; ee) SEQ ID NOs: 1567 and 1602, respectively; ff) SEQ ID NOs: 1568 and 1603, respectively; gg) SEQ ID NOs: 1569 and 1604, respectively; hh) SEQ ID NOs: 1570 and 1605, respectively; ii) SEQ ID NOs: 1571 and 1606, respectively; and jj) SEQ ID NOs: 1681 and 1586, respectively, wherein the oligonucleotide comprises one or more modified nucleobases.

[0167] In some embodiments, the sense and antisense strands of an oligonucleotide comprise nucleotides sequences selected from the group consisting of: a) SEQ ID NOs: 1540 and 1575, respectively; b) SEQ ID NOs: 1544 and 1579, respectively; c) SEQ ID NOs: 1546 and 1581, respectively; d) SEQ ID NOs: 155 land 1586, respectively; e) SEQ ID NOs: 1552 and 1587, respectively; f) SEQ ID NOs: 1553 and 1588, respectively; g) SEQ ID NOs: 1558 and 1594, respectively; h) SEQ ID NOs: 1560 and 1595, respectively; i) SEQ ID NOs: 1564 and 1599, respectively; j) SEQ ID NOs: 1565 and 1600, respectively; k) SEQ ID NOs: 1566 and 1601, respectively; l) SEQ ID NOs: 1570 and 1605, respectively; and m) SEQ ID NOs: 1681 and 1586, respectively, wherein the oligonucleotide comprises one or more modified nucleobases.

[0168] In some embodiments, the sense and antisense strands of an oligonucleotide comprise nucleotides sequences selected from the group consisting of: a) SEQ ID NOs: 1553 and 1588, respectively; b) SEQ ID NOs: 1560 and 1595, respectively; c) SEQ ID NOs: 1564 and 1599, respectively; d) SEQ ID NOs: 1551 and 1586, respectively; e) SEQ ID NOs: 1570 and 1605, respectively; and f) SEQ ID NOs: 1681 and 1586, respectively, wherein the oligonucleotide comprises one or more modified nucleobases.

[0169] In some embodiments, a universal base is a heterocyclic moiety located at the 1 ' position of a nucleotide sugar moiety in a modified nucleotide, or the equivalent position in a nucleotide sugar moiety substitution, that, when present in a duplex, can be positioned opposite more than one type of base without substantially altering structure of the duplex. In some embodiments, compared to a reference ss nucleic acid (e.g., oligonucleotide) that is fully complementary to a target nucleic acid, a ss nucleic acid containing a universal base forms a duplex with the target nucleic acid that has a lower T m than a duplex formed with the complementary nucleic acid. In some embodiments, when compared to a reference ss nucleic acid in which the universal base has been replaced with a base to generate a single mismatch, the ss nucleic acid containing the universal base forms a duplex with the target nucleic acid that has a higher T m than a duplex formed with the nucleic acid comprising the mismatched base. [0170] Non-limiting examples of universal-binding nucleotides include, but are not limited to, inosine, l-P-D-ribofuranosyl-5-nitroindole and/or l-P-D-ribofuranosyl-3 -nitropyrrole (see, US Patent Application Publication No. 2007/0254362; Van Aerschot et al. (1995) Nucleic Acids Res. 23:4363-4370; Loakes et al. (1995) Nucleic Acids Res. 23:2361-66; and Loakes & Brown (1994) Nucleic Acids Res. 22:4039-43).

Targeting Ligands

[0171] In some embodiments, it is desirable to target the oligonucleotide (e.g., a RNAi oligonucleotide) to one or more cells or one or more organs. Such a strategy can help to avoid undesirable effects in other organs or avoid undue loss of the oligonucleotide to cells, tissue, or organs that would not benefit from the oligonucleotide. Accordingly, in some embodiments, the oligonucleotide is modified to facilitate targeting and/or delivery to a particular tissue, cell, or organ (e.g., to facilitate delivery of the oligonucleotide to the CNS). In some embodiments, the oligonucleotide comprises at least one nucleotide (e.g., 1, 2, 3, 4, 5, 6 or more nucleotides) conjugated to one or more targeting ligand(s). In some embodiments, the sense and antisense strands of the oligonucleotide comprise nucleotides sequences selected from the group consisting of: a) SEQ ID NOs: 1537 and 1572, respectively; b) SEQ ID NOs: 1538 and 1573, respectively; c) SEQ ID NOs: 1539 and 1574, respectively; d) SEQ ID NOs: 1540 and 1575, respectively; e) SEQ ID NOs: 1541 and 1576, respectively; f) SEQ ID NOs: 1542 and 1577, respectively; g) SEQ ID NOs: 1543 and 1578, respectively; h) SEQ ID NOs: 1544 and 1579, respectively; i) SEQ ID NOs: 1545 and 1580, respectively; j) SEQ ID NOs: 1546 and 1581, respectively; k) SEQ ID NOs: 1547 and 1582, respectively; l) SEQ ID NOs: 1548 and 1583, respectively; m) SEQ ID NOs: 1549 and 1584, respectively; n) SEQ ID NOs: 1550 and 1585, respectively; o) SEQ ID NOs: 1551 and 1586, respectively; p) SEQ ID NOs: 1552 and 1587, respectively; q) SEQ ID NOs: 1553 and 1588, respectively; r) SEQ ID NOs: 1554 and 1589, respectively; s) SEQ ID NOs: 1555 and 1590, respectively; t) SEQ ID NOs: 1556 and 1591, respectively; u) SEQ ID NOs: 1557 and 1592, respectively; v) SEQ ID NOs: 1558 and 1593, respectively; w) SEQ ID NOs: 1559 and 1594, respectively; x) SEQ ID NOs: 1560 and 1595, respectively; y) SEQ ID NOs: 1561 and 1596, respectively; z) SEQ ID NOs: 1562 and 1597, respectively; aa) SEQ ID NOs: 1563 and 1598, respectively; bb) SEQ ID NOs: 1564 and 1599, respectively; cc) SEQ ID NOs: 1565 and 1600, respectively; dd) SEQ ID NOs: 1566 and 1601, respectively; ee) SEQ ID NOs: 1567 and 1602, respectively; ff) SEQ ID NOs: 1568 and 1603, respectively; gg) SEQ ID NOs: 1569 and 1604, respectively; hh) SEQ ID NOs: 1570 and 1605, respectively; ii) SEQ ID NOs: 1571 and 1606, respectively; and jj) SEQ ID NOs: 1681 and 1586, respectively, wherein the oligonucleotide comprises a targeting ligand conjugated to at least one nucleotide.

[0172] In some embodiments, the oligonucleotide comprises at least one nucleotide (e.g, I, 2, 3, 4, 5, 6 or more nucleotides) conjugated to one or more targeting ligand(s). In some embodiments, the sense and antisense strands of the oligonucleotide comprise nucleotides sequences selected from the group consisting of: a) SEQ ID NOs: 1540 and 1575, respectively; b) SEQ ID NOs: 1544 and 1579, respectively; c) SEQ ID NOs: 1546 and 1581, respectively; d) SEQ ID NOs: 155 land 1586, respectively; e) SEQ ID NOs: 1552 and 1587, respectively; f) SEQ ID NOs: 1553 and 1588, respectively; g) SEQ ID NOs: 1558 and 1594, respectively; h) SEQ ID NOs: 1560 and 1595, respectively; i) SEQ ID NOs: 1564 and 1599, respectively; j) SEQ ID NOs: 1565 and 1600, respectively; k) SEQ ID NOs: 1566 and 1601, respectively; l) SEQ ID NOs: 1570 and 1605, respectively; and m) SEQ ID NOs: 1681 and 1586, respectively, wherein the oligonucleotide comprises a targeting ligand conjugated to at least one nucleotide. [0173] In some embodiments, the oligonucleotide comprises at least one nucleotide (e.g, 1, 2, 3, 4, 5, 6 or more nucleotides) conjugated to one or more targeting ligand(s). In some embodiments, the sense and antisense strands of the oligonucleotide comprise nucleotides sequences selected from the group consisting of: a) SEQ ID NOs: 1553 and 1588, respectively; b) SEQ ID NOs: 1560 and 1595, respectively; c) SEQ ID NOs: 1564 and 1599, respectively; d) SEQ ID NOs: 1551 and 1586, respectively; e) SEQ ID NOs: 1570 and 1605, respectively; and, f) SEQ ID NOs: 1681 and 1586, respectively, wherein the oligonucleotide comprises a targeting ligand conjugated to at least one nucleotide. [0174] In some embodiments, the targeting ligand comprises a carbohydrate, amino sugar, cholesterol, peptide, polypeptide, or protein or part of a protein (e.g., an antibody or antibody fragment). In some embodiments, the targeting ligand is an aptamer. For example, a targeting ligand may be a RGD peptide that is used to target tumor vasculature or glioma cells, CREKA peptide to target tumor vasculature or stoma, transferring, lactoferrin, or an aptamer to target transferrin receptors expressed on CNS vasculature, or an anti-EGFR antibody to target EGFR on glioma cells. In certain embodiments, the targeting ligand is one or more GalNAc moieties. In some embodiments, the targeting ligand is one or more lipid moieties.

10175] In some embodiments, 1 or more (e.g., 1, 2, 3, 4, 5, or 6) nucleotides of the oligonucleotide are each conjugated to a separate targeting ligand. In some embodiments, 2 to 4 nucleotides of the oligonucleotide are each conjugated to a separate targeting ligand. In some embodiments, targeting ligands are conjugated to 2 to 4 nucleotides at either ends of the sense or antisense strand e.g., targeting ligands are conjugated to a 2 to 4 nucleotide overhang or extension on the 5' or 3' end of the sense or antisense strand) such that the targeting ligands resemble bristles of a toothbrush, and the oligonucleotide resembles a toothbrush. For example, the oligonucleotide may comprise a stem-loop at either the 5' or 3' end of the sense strand and 1, 2, 3, or 4 nucleotides of the loop of the stem may be individually conjugated to a targeting ligand. In some embodiments, the oligonucleotide comprises a stem-loop at the 3' end of the sense strand, wherein the loop of the stem-loop comprises a triL or a tetraL, and wherein the 3 or 4 nucleotides comprising the triL or tetraL, respectfully, are individually conjugated to a targeting ligand. In some embodiments, the oligonucleotide comprises a blunt end at its 3’ end and one or more targeting ligands conjugated to at least one nucleotide. In some embodiments, the oligonucleotide comprises a blunt end at its 3’ end and one or more targeting ligands conjugated to the 5’ terminal nucleotide of the sense strand.

GalNAc Conjugation

[0176] GalNAc is a high affinity ligand for the ASGPR, which is primarily expressed on the sinusoidal surface of hepatocyte cells and has a major role in binding, internalizing and subsequent clearing circulating glycoproteins that contain terminal galactose or GalNAc residues (asialoglycoproteins). Conjugation (either indirect or direct) of GalNAc moieties to an oligonucleotide herein (e.g., a RNAi oligonucleotide) can be used to target the oligonucleotide to the ASGPR expressed on cells. In some embodiments, the oligonucleotide is conjugated to at least one or more GalNAc moieties, wherein the GalNAc moieties target the oligonucleotide to an ASGPR expressed on human liver cells (e.g., human hepatocytes). In some embodiments, the GalNAc moiety targets the oligonucleotide to the liver.

[0177] In some embodiments, the oligonucleotide is conjugated directly or indirectly to a monovalent GalNAc. In some embodiments, the oligonucleotide is conjugated directly or indirectly to more than one monovalent GalNAc (i.e., is conjugated to 2, 3, or 4 monovalent GalNAc moieties, and is typically conjugated to 3 or 4 monovalent GalNAc moieties). In some embodiments, the oligonucleotide is conjugated to one or more bivalent GalNAc, trivalent GalNAc, or tetravalent GalNAc moieties. In some embodiments, the bivalent, trivalent, or tetravalent GalNAc moiety is conjugated to the oligonucleotide via a branched linker. In some embodiments, the monovalent GalNAc moiety is conjugated to a first nucleotide and the bivalent, trivalent, or tetravalent GalNAc moiety is conjugated to a second nucleotide via a branched linker. [0178] In some embodiments, 1 or more (e.g., 1, 2, 3, 4, 5, or 6) nucleotides of the oligonucleotide are each conjugated to a GalNAc moiety. In some embodiments, 2 to 4 nucleotides of a tetraL are each conjugated to a separate GalNAc. In some embodiments, 1 to 3 nucleotides of a triL are each conjugated to a separate GalNAc. In some embodiments, targeting ligands are conjugated to 2 to 4 nucleotides at either ends of the sense or antisense strand (e.g., ligands are conjugated to a 2 to 4 nucleotide overhang or extension on the 5' or 3' end of the sense or antisense strand) such that the GalNAc moieties resemble bristles of a toothbrush, and the oligonucleotide resembles a toothbrush. In some embodiments, GalNAc moieties are conjugated to a nucleotide of the sense strand. For example, 4 GalNAc moieties can be conjugated to nucleotides in the tetraL of the sense strand where each GalNAc moiety is conjugated to 1 nucleotide.

[0179] In some embodiments, the oligonucleotide comprises a tetraL, wherein the tetraL is any combination of adenine (A) and guanine (G) nucleotides. In some embodiments, the tetraL comprises a monovalent GalNAc moiety attached to any one or more guanine (G) nucleotides of the tetraloop via any linker described herein, as depicted below (X=heteroatom):

[0180] In some embodiments, the tetraL has a monovalent GalNAc attached to any one or more adenine nucleotides of the tetraL via any linker described herein, as depicted below (X=heteroatom):

[0181] In some embodiments, the oligonucleotide comprises a monovalent GalNAc attached to a guanine (G) nucleotide referred to as [ademG-GalNAc] or 2'-aminodiethoxymethanol-Guanine- GalNAc, as depicted below:

[0182] In some embodiments, the oligonucleotide comprises a monovalent GalNAc attached to an adenine (A) nucleotide, referred to as [ademA-GalNAc] or 2'-aminodiethoxymethanol-

Adenine-GalNAc, as depicted below:

[0183] An example of such conjugation is shown below for a loop comprising from 5' to 3' the nucleotide sequence GAAA (L = linker, X = heteroatom) stem attachment points are shown. Such a loop may be present, for example, at positions 27-30 of the sense strand of any one of the sense strands listed in Tables 4 and 5. In the chemical formula, is used to describe an attachment point to the oligonucleotide strand.

[0184] Appropriate methods or chemistry (e.g., click chemistry) can be used to link the targeting ligand to a nucleotide. In some embodiments, the targeting ligand is conjugated to a nucleotide using a click linker. In some embodiments, an acetal-based linker is used to conjugate a targeting ligand to a nucleotide of any one of the oligonucleotides described herein. Acetal-based linkers are disclosed, for example, in Inti. Patent Application Publication No. WO 2016/100401. In some embodiments, the linker is a labile linker. However, in other embodiments, the linker is stable. Examples are shown below for a loop comprising from 5' to 3' the nucleotides GAAA, in which GalNAc moieties are attached to 3 or 4 nucleotides of the loop using an acetal linker. Such a loop may be present, for example, at positions 27-30 of the any one of the sense strands listed in Tables

4 and 5 In the chemical formula, is an attachment point to the oligonucleotide strand:

[0185] As mentioned, various appropriate methods or chemistry synthetic techniques (e.g., click chemistry) can be used to link the targeting ligand to a nucleotide. In some embodiments, the targeting ligand is conjugated to a nucleotide using a click linker. In some embodiments, an acetal- based linker is used to conjugate the targeting ligand to a nucleotide of any one of the oligonucleotides described herein. Acetal-based linkers are disclosed, for example, in Inti. Patent Application Publication No. WO 2016/100401 . In some embodiments, the linker is a labile linker. However, in other embodiments, the linker is a stable linker.

[0186] In some embodiments, a duplex extension (e.g., of up to 3, 4, 5, or 6 bp in length) is provided between the targeting ligand (e.g., a GalNAc moiety) and the oligonucleotide. In some embodiments, the oligonucleotide does not have a GalNAc conjugated thereto. [0187] In some embodiments, the sense and antisense strands of an oligonucleotide comprise nucleotides sequences selected from the group consisting of: a) SEQ ID NOs: 1537 and 1572, respectively; b) SEQ ID NOs: 1538 and 1573, respectively; c) SEQ ID NOs: 1539 and 1574, respectively; d) SEQ ID NOs: 1540 and 1575, respectively; e) SEQ ID NOs: 1541 and 1576, respectively; f) SEQ ID NOs: 1542 and 1577, respectively; g) SEQ ID NOs: 1543 and 1578, respectively; h) SEQ ID NOs: 1544 and 1579, respectively; i) SEQ ID NOs: 1545 and 1580, respectively; j) SEQ ID NOs: 1546 and 1581, respectively; k) SEQ ID NOs: 1547 and 1582, respectively; l) SEQ ID NOs: 1548 and 1583, respectively; m) SEQ ID NOs: 1549 and 1584, respectively; n) SEQ ID NOs: 1550 and 1585, respectively; o) SEQ ID NOs: 1551 and 1586, respectively; p) SEQ ID NOs: 1552 and 1587, respectively; q) SEQ ID NOs: 1553 and 1588, respectively; r) SEQ ID NOs: 1554 and 1589, respectively; s) SEQ ID NOs: 1555 and 1590, respectively; t) SEQ ID NOs: 1556 and 1591, respectively; u) SEQ ID NOs: 1557 and 1592, respectively; v) SEQ ID NOs: 1558 and 1593, respectively; w) SEQ ID NOs: 1559 and 1594, respectively; x) SEQ ID NOs: 1560 and 1595, respectively; y) SEQ ID NOs: 1561 and 1596, respectively; z) SEQ ID NOs: 1562 and 1597, respectively; aa) SEQ ID NOs: 1563 and 1598, respectively; bb) SEQ ID NOs: 1564 and 1599, respectively; cc) SEQ ID NOs: 1565 and 1600, respectively; dd) SEQ ID NOs: 1566 and 1601, respectively; ee) SEQ ID NOs: 1567 and 1602, respectively; ff) SEQ ID NOs: 1568 and 1603, respectively; gg) SEQ ID NOs: 1569 and 1604, respectively; hh) SEQ ID NOs: 1570 and 1605, respectively; ii) SEQ ID NOs: 1571 and 1606, respectively; and jj) SEQ ID NOs: 1681 and 1586, respectively, wherein the oligonucleotide comprises at least one GalNAc moiety conjugated to a nucleotide.

[0188] In some embodiments, the sense and antisense strands of an oligonucleotide comprise nucleotides sequences selected from the group consisting of: a) SEQ ID NOs: 1540 and 1575, respectively; b) SEQ ID NOs: 1544 and 1579, respectively; c) SEQ ID NOs: 1546 and 1581, respectively; d) SEQ ID NOs: 155 land 1586, respectively; e) SEQ ID NOs: 1552 and 1587, respectively; f) SEQ ID NOs: 1553 and 1588, respectively; g) SEQ ID NOs: 1558 and 1594, respectively; h) SEQ ID NOs: 1560 and 1595, respectively; i) SEQ ID NOs: 1564 and 1599, respectively; j) SEQ ID NOs: 1565 and 1600, respectively; k) SEQ ID NOs: 1566 and 1601, respectively; l) SEQ ID NOs: 1570 and 1605, respectively; and m) SEQ ID NOs: 1681 and 1586, respectively, wherein the oligonucleotide comprises at least one GalNAc moiety conjugated to a nucleotide.

[0189] In some embodiments, the sense and antisense strands of an oligonucleotide comprise nucleotides sequences selected from the group consisting of: a) SEQ ID NOs: 1553 and 1588, respectively; b) SEQ ID NOs: 1560 and 1595, respectively; c) SEQ ID NOs: 1564 and 1599, respectively; d) SEQ ID NOs: 1551 and 1586, respectively; e) SEQ ID NOs: 1570 and 1605, respectively; and f) SEQ ID NOs: 1681 and 1586, respectively, wherein the oligonucleotide comprises at least one GalNAc moiety conjugated to a nucleotide.

Lipid Conjugation

[0190] In some embodiments, one or more lipid moi eties are conjugated to a 5’ terminal nucleotide of a sense strand. In some embodiments, one or more lipid moieties are conjugated to an adenine nucleotide. In some embodiments, one or more lipid moieties are conjugated to a guanine nucleotide. In some embodiments, one or more lipid moieties are conjugated toa cytosine nucleotide. In some embodiments, one or more lipid moieties are conjugated to a thymine (T) nucleotide. In some embodiments, one or more lipid moieties are conjugated to a uracil (U) nucleotide.

[0191] In some embodiments, the lipid moiety is a hydrocarbon chain. In some embodiments, the hydrocarbon chain is saturated. In other embodiments, the hydrocarbon chain is unsaturated. In some embodiments, the hydrocarbon chain is branched. In other embodiments, the hydrocarbon chain is straight. In some embodiments, the lipid moiety is a Cs-Cso hydrocarbon chain. In certain embodiments, the lipid moiety is a Cs:0, Cio:O, Cn:0, Ci2:0, Ci4:0, Cie:0, Ci?:0, Cis:0, Cis: 1, Cis:2, C22A, C22:0, C24:0, C26:0, €22:6, C24:l, diacyl Cie:0 or diacyl Cis:l. In some embodiments, the lipid moiety is a Ci6 hydrocarbon chain. In some embodiments, the Ci6 hydrocarbon chain is represented as:

[0192] In some embodiments, the sense strand is 20-22 nucleotides in length, and a lipid moiety is conjugated to the 5’ terminal nucleotide of the sense strand. In some embodiments, the sense strand is 20-22 nucleotides in length, and a hydrocarbon chain is conjugated to the 5’ terminal nucleotide of the sense strand. In some embodiments, the sense strand is 20-22 nucleotides in length, and a C14-C22 hydrocarbon chain is conjugated to the 5’ terminal nucleotide of the sense strand. In some embodiments, the sense strand is 20-22 nucleotides in length and a Ci6 hydrocarbon chain is conjugated to the 5’ terminal nucleotide of the sense strand. In some embodiments, the sense strand is 20 nucleotides in length, and a lipid moiety is conjugated to the 5’ terminal nucleotide of the sense strand. In some embodiments, the sense strand is 20 nucleotides in length, and a hydrocarbon chain is conjugated to the 5’ terminal nucleotide of the sense strand. In some embodiments, the sense strand is 20 nucleotides in length, and a C14-C22 hydrocarbon chain is conjugated to the 5’ terminal nucleotide of the sense strand. In some embodiments, the sense strand is 20 nucleotides in length ,and a Ci6 hydrocarbon chain is conjugated to the 5’ terminal nucleotide of the sense strand.

[0193] In some embodiments, the oligonucleotide comprises (i) a sense strand of 20-22 nucleotides in length; (ii) an antisense strand comprising a 3’ overhang sequence of one or more nucleotides in length; (iii) a blunt end comprising the 3’ end of the sense strand; and (iv) a lipid moiety conjugated to the 5’ terminal nucleotide of the sense strand. In some embodiments, the oligonucleotide comprises (i) a sense strand of 20-22 nucleotides in length; (ii) an antisense strand comprising a 3’ overhang sequence of one or more nucleotides in length; (iii) a blunt end comprising the 3’ end of the sense strand; and (iv) a hydrocarbon chain conjugated to the 5’ terminal nucleotide of the sense strand. In some embodiments, the oligonucleotide comprises (i) a sense strand of 20-22 nucleotides in length; (ii) an antisense strand comprising a 3’ overhang sequence of one or more nucleotides in length; (iii) a blunt end comprising the 3’ end of the sense strand; and (iv) a C14-C22 hydrocarbon chain conjugated to the 5’ terminal nucleotide of the sense strand. In some embodiments, the oligonucleotide comprises (i) a sense strand of 20-22 nucleotides in length; (ii) an antisense strand comprising a 3’ overhang sequence of one or more nucleotides in length; (iii) a blunt end comprising the 3’ end of the sense strand; and (iv) a Ci6 hydrocarbon chain conjugated to the 5’ terminal nucleotide of the sense strand.

[0194] In some embodiments, the oligonucleotide comprises (i) a sense strand of 20 nucleotides in length; (ii) an antisense strand of 22 nucleotides in length comprising a 3’ overhang sequence of two nucleotides in length; (iii) a blunt end comprising the 3’ end of the sense strand; and (iv) a lipid moiety conjugated to the 5’ terminal nucleotide of the sense strand. In some embodiments, the oligonucleotide comprises (i) a sense strand of 20 nucleotides in length; (ii) an antisense strand of 22 nucleotides in length comprising a 3’ overhang sequence of two nucleotides in length; (iii) a blunt end comprising the 3’ end of the sense strand; and (iv) a hydrocarbon chain conjugated to the 5’ terminal nucleotide of the sense strand. In some embodiments, the oligonucleotide comprises (i) a sense strand of 20 nucleotides in length; (ii) an antisense strand of 22 nucleotides in length comprising a 3’ overhang sequence of 2 nucleotides in length; (iii) a blunt end comprising the 3’ end of the sense strand; and (iv) a C14-C22 hydrocarbon chain conjugated to the 5’ terminal nucleotide of the sense strand. In some embodiments, the oligonucleotide comprises (i) a sense strand of 20 nucleotides in length; (ii) an antisense strand of 22 nucleotides in length comprising a 3’ overhang sequence of 2 nucleotides in length; (iii) a blunt end comprising the 3’ end of the sense strand; and (iv) a Ci6 hydrocarbon chain conjugated to the 5’ terminal nucleotide of the sense strand.

[0195] In some embodiments, the oligonucleotide comprises (i) an antisense strand of 19-30 nucleotides comprising a region of complementarity to a SNCA mRNA target sequence selected from SEQ ID NOs: 1781, 1782, 1796, 1798, 1802, 1808, 1814, 1817, 1713, 1718, 1726, 1830, 1839, 1742, 1846, 1852, 1865, 1784, 1804, 1721, 1822, 1840, 1735, 1847, 1855, 1864, 1901, 1902, 1938, 1947, 1955, 1964, 1973, and 1978; (ii) a sense strand of 19-25 nucleotides that forms a duplex region with the antisense strand; and (iii) a lipid moiety conjugated to the 5’ terminal nucleotide of the sense strand. In some embodiments, the oligonucleotide comprises (i) an antisense strand of 19-30 nucleotides comprising a region of complementarity to a SNCA mRNA target sequence selected from SEQ ID NOs: 1798, 1817, 1718, 1846, 1852, 1865, 1804, 1721, 1847, 1855, 1864, and 1955; (ii) a sense strand of 19-25 nucleotides that forms a duplex region with the antisense strand; and (iii) a lipid moiety conjugated to the 5’ terminal nucleotide of the sense strand. In some embodiments, the oligonucleotide comprises (i) an antisense strand of 19- 30 nucleotides comprising a region of complementarity to a SNCA mRNA target sequence selected from SEQ ID NOs: 1865, 1721, 1847, 1846, and 1955; (ii) a sense strand of 19-25 nucleotides that forms a duplex region with the antisense strand; and (iii) a lipid moiety conjugated to the 5’ terminal nucleotide of the sense strand.

[0196] In some embodiments, the oligonucleotide comprises (i) an antisense strand of 19-30 nucleotides comprising a region of complementarity to a SNCA mRNA target sequence selected from SEQ ID NOs: 1781, 1782, 1796, 1798, 1802, 1808, 1814, 1817, 1713, 1718, 1726, 1830, 1839, 1742, 1846, 1852, 1865, 1784, 1804, 1721, 1822, 1840, 1735, 1847, 1855, 1864, 1901, 1902, 1938, 1947, 1955, 1964, 1973, and 1978; (ii) a sense strand of 19-25 nucleotides that forms a duplex region with the antisense strand; and (iii) a hydrocarbon chain conjugated to the 5’ terminal nucleotide of the sense strand. In some embodiments, the oligonucleotide comprises (i) an antisense strand of 19-30 nucleotides comprising a region of complementarity to a SNCA mRNA target sequence selected from SEQ ID NOs: 1798, 1817, 1718, 1846, 1852, 1865, 1804, 1721, 1847, 1855, 1864, and 1955; (ii) a sense strand of 19-25 nucleotides that forms a duplex region with the antisense strand; and (iii) a hydrocarbon chain conjugated to the 5’ terminal nucleotide of the sense strand. In some embodiments, the oligonucleotide comprises (i) an antisense strand of 19-30 nucleotides comprising a region of complementarity to a SNCA mRNA target sequence selected from SEQ ID NOs: 1865, 1721, 1847, 1846, and 1955; (ii) a sense strand of 19-25 nucleotides that forms a duplex region with the antisense strand; and (iii) a hydrocarbon chain conjugated to the 5’ terminal nucleotide of the sense strand.

[0197] In some embodiments, the oligonucleotide comprises (i) an antisense strand of 19-30 nucleotides comprising a region of complementarity to a SNCA mRNA target sequence selected from SEQ ID NOs: 1781, 1782, 1796, 1798, 1802, 1808, 1814, 1817, 1713, 1718, 1726, 1830, 1839, 1742, 1846, 1852, 1865, 1784, 1804, 1721, 1822, 1840, 1735, 1847, 1855, 1864, 1901, 1902, 1938, 1947, 1955, 1964, 1973, and 1978; (ii) a sense strand of 19-25 nucleotides that forms a duplex region with the antisense strand; and (iii) a C14-C22 hydrocarbon chain conjugated to the 5’ terminal nucleotide of the sense strand. In some embodiments, the oligonucleotide comprises (i) an antisense strand of 19-30 nucleotides comprising a region of complementarity to a SNCA mRNA target sequence selected from SEQ ID NOs: 1798, 1817, 1718, 1846, 1852, 1865, 1804, 1721, 1847, 1855, 1864, and 1955; (ii) a sense strand of 19-25 nucleotides that forms a duplex region with the antisense strand; and (iii) a C14-C22 hydrocarbon chain conjugated to the 5’ terminal nucleotide of the sense strand. In some embodiments, the oligonucleotide comprises (i) an antisense strand of 19-30 nucleotides comprising a region of complementarity to a SNCA mRNA target sequence selected from SEQ ID NOs: 1865, 1721, 1847, 1846, and 1955; (ii) a sense strand of 19-25 nucleotides that forms a duplex region with the antisense strand; and (iii) a C14-C22 hydrocarbon chain conjugated to the 5’ terminal nucleotide of the sense strand.

[0198] In some embodiments, the oligonucleotide comprises (i) an antisense strand of 19-30 nucleotides comprising a region of complementarity to a SNCA mRNA target sequence selected from SEQ ID NOs:1781, 1782, 1796, 1798, 1802, 1808, 1814, 1817, 1713, 1718, 1726, 1830, 1839, 1742, 1846, 1852, 1865, 1784, 1804, 1721, 1822, 1840, 1735, 1847, 1855, 1864, 1901, 1902, 1938, 1947, 1955, 1964, 1973, and 1978; (ii) a sense strand of 19-25 nucleotides that forms a duplex region with the antisense strand; and (iii) a Ci6 hydrocarbon chain conjugated to the 5’ terminal nucleotide of the sense strand. In some embodiments, the oligonucleotide comprises (i) an antisense strand of 19-30 nucleotides comprising a region of complementarity to a SNCA mRNA target sequence selected from SEQ ID NOs: 1798, 1817, 1718, 1846, 1852, 1865, 1804, 1721, 1847, 1855, 1864, and 1955; (ii) a sense strand of 19-25 nucleotides that forms a duplex region with the antisense strand; and (iii) a Ci6 hydrocarbon chain conjugated to the 5’ terminal nucleotide of the sense strand. In some embodiments, the oligonucleotide comprises (i) an antisense strand of 19-30 nucleotides comprising a region of complementarity to a SNCA mRNA target sequence selected from SEQ ID NOs: 1865, 1721, 1847, 1846, and 1955; (ii) a sense strand of 19-25 nucleotides that forms a duplex region with the antisense strand; and (iii) a Ci6 hydrocarbon chain conjugated to the 5’ terminal nucleotide of the sense strand.

[0199] In some embodiments, the oligonucleotide comprises a sense strand comprising the nucleotide sequence of SEQ ID NO: 1681 and an antisense strand comprising the nucleotide sequence of SEQ ID NO: 1586, wherein the sense strand comprises a lipid moiety conjugated to the 5’ terminal nucleotide of the sense strand. In some embodiments, the oligonucleotide comprises a sense strand comprising the nucleotide sequence of SEQ ID NO: 1681 and an antisense strand comprising the nucleotide sequence of SEQ ID NO: 1586, wherein the sense strand comprises a hydrocarbon chain conjugated to the 5’ terminal nucleotide of the sense strand. In some embodiments, the oligonucleotide comprises a sense strand comprising the nucleotide sequence of SEQ ID NO: 1681 and an antisense strand comprising the nucleotide sequence of SEQ ID NO: 1586, wherein the sense strand comprises a C14-C22 hydrocarbon chain conjugated to the 5’ terminal nucleotide of the sense strand. In some embodiments, the oligonucleotide comprises a sense strand comprising the nucleotide sequence of SEQ ID NO: 1681 and an antisense strand comprising the nucleotide sequence of SEQ ID NO: 1586, wherein the sense strand comprises a Ci6 hydrocarbon chain conjugated to the 5’ terminal nucleotide of the sense strand.

Exemplary SNCA-Targeting RNAi Oligonucleotides

[0200] In some embodiments, the SNCA -targeting RNAi oligonucleotide for reducing SNCA gene expression comprise a sense strand and an antisense strand, wherein all nucleotides comprising the sense strand and the antisense strand are modified, wherein the antisense strand comprises a region of complementarity to a SNCA mRNA target sequence of any one of SEQ ID NOs: 1683-2066, and wherein the region of complementarity is at least 15 contiguous nucleotides in length. In some embodiments, the 5’ terminal nucleotide of the antisense strand comprises 4’- O-monomethylphosphonate-2’-O-methyluridine [MePhosphonate-4O-mU], as described herein. In some embodiments, the 5’ terminal nucleotide of the antisense strand comprises a phosphorothioate linkage. In some embodiments, the antisense strand and the sense strand comprise one or more 2'-F and 2'-OMe modified nucleotides and at least one phosphorothioate linkage. In some embodiments, the antisense strand comprises 4 phosphorothioate linkages and the sense strand comprises 1 phosphorothioate linkage. In some embodiments, the antisense strand comprises 5 phosphorothioate linkages and the sense strand comprises 1 phosphorothioate linkage. [0201] In some embodiments, the oligonucleotide comprises a sense strand having a sequence of any one of SEQ ID NOs: 1683-2066 and an antisense strand comprising a complementary sequence selected from any one of SEQ ID NOs: 2067-2450.

[0202] In some embodiments, the oligonucleotide comprises a sense strand having a sequence of any one of SEQ ID NOs: 1-384 and an antisense strand comprising a complementary sequence selected from any one of SEQ ID NOs: 385-768.

[0203] In some embodiments, the oligonucleotide comprises a sense strand having a sequence of any one of SEQ ID NOs: 1537-1571 and an antisense strand comprising a complementary sequence selected from any one of SEQ ID NOs: 1572-1606. [0204] In some embodiments, the oligonucleotide comprises a sense strand having a sequence of any one of SEQ ID NOs: 1537-1571 and 1681 and an antisense strand comprising a complementary sequence selected from any one of SEQ ID NOs: 1572-1606.

[0205] In some embodiments, the oligonucleotide consists a sense strand having a sequence of any one of SEQ ID NOs: 1537-1571 and an antisense strand comprising a complementary sequence selected from any one of SEQ ID NOs: 1572-1606.

[0206] In some embodiments, the oligonucleotide consists a sense strand having a sequence of any one of SEQ ID NOs: 1537-1571 and 1681 and an antisense strand comprising a complementary sequence selected from any one of SEQ ID NOs: 1572-1606.

[0207] In some embodiments, the oligonucleotide is for reducing SNCA gene expression and comprises: a sense strand of 36 nucleotides comprising a 2'-F modified nucleotide at positions 3, 5, 8, 10, 12, 13, 15, and 17; a 2'-OMe modified nucleotide at positions I, 2, 4, 6, 7, 9, I I, 14, 16, 18- 27, and 31-36; a GalN Ac-conjugated nucleotide at position 28, 29, and 30; and a phosphorothioate linkage between positions 1 and 2; and an antisense strand of 22 nucleotides comprising a 2'-F modified nucleotide at positions 2, 3, 4, 5, 7, 10, 14, 16, and 19; a 2'-0Me at positions 1, 6, 8, 9, 11, 12, 13, 15, 17, 18, and 20-22; a phosphorothioate linkage between positions 1 and 2, positions 2 and 3, positions 20 and 21, and positions 21 and 22; and a 5’ terminal nucleotide at position 1 comprising a 4’ -phosphate analog, optionally wherein the 5’ terminal nucleotide comprises 4’-O-monomethylphosphonate-2’-O- methyluridine [MePhosphonate-4O-mU]; wherein positions 1-20 of the antisense strand form a duplex region with positions 1-20 of the sense strand, wherein positions 21-36 of the sense strand form a stem-loop, wherein positions 27-30 form the loop of the stem-loop, optionally wherein positions 27-30 comprise a tetraL, wherein positions 21 and 22 of the antisense strand comprise an overhang, and wherein the sense strand and antisense strands comprise nucleotide sequences selected from the group consisting of: a) SEQ ID NOs: 1537 and 1572, respectively; b) SEQ ID NOs: 1538 and 1573, respectively; c) SEQ ID NOs: 1539 and 1574, respectively; d) SEQ ID NOs: 1540 and 1575, respectively; e) SEQ ID NOs: 1541 and 1576, respectively; f) SEQ ID NOs: 1542 and 1577, respectively; g) SEQ ID NOs: 1543 and 1578, respectively; h) SEQ ID NOs: 1544 and 1579, respectively; i) SEQ ID NOs: 1545 and 1580, respectively; j) SEQ ID NOs: 1546 and 1581, respectively; k) SEQ ID NOs: 1547 and 1582, respectively; l) SEQ ID NOs: 1548 and 1583, respectively; m) SEQ ID NOs: 1549 and 1584, respectively; n) SEQ ID NOs: 1550 and 1585, respectively; o) SEQ ID NOs: 1551 and 1586, respectively; p) SEQ ID NOs: 1552 and 1587, respectively; q) SEQ ID NOs: 1553 and 1588, respectively; r) SEQ ID NOs: 1554 and 1589, respectively; s) SEQ ID NOs: 1555 and 1590, respectively; t) SEQ ID NOs: 1556 and 1591, respectively; u) SEQ ID NOs: 1557 and 1592, respectively; v) SEQ ID NOs: 1558 and 1593, respectively; w) SEQ ID NOs: 1559 and 1594, respectively; x) SEQ ID NOs: 1560 and 1595, respectively; y) SEQ ID NOs: 1561 and 1596, respectively; z) SEQ ID NOs: 1562 and 1597, respectively; aa) SEQ ID NOs: 1563 and 1598, respectively; bb) SEQ ID NOs: 1564 and 1599, respectively; cc) SEQ ID NOs: 1565 and 1600, respectively; dd) SEQ ID NOs: 1566 and 1601, respectively; ee) SEQ ID NOs: 1567 and 1602, respectively; ff) SEQ ID NOs: 1568 and 1603, respectively; gg) SEQ ID NOs: 1569 and 1604, respectively; hh) SEQ ID NOs: 1570 and 1605, respectively; and ii) SEQ ID NOs: 1571 and 1606, respectively.

[0208] In some embodiments, the oligonucleotide is for reducing SNCA gene expression and comprises: a sense strand of 36 nucleotides comprising a 2'-F modified nucleotide at positions 3, 5, 8, 10, 12, 13, 15, and 17; a 2'-OMe modified nucleotide at positions 1, 2, 4, 6, 7, 9, 11, 14, 16, 18- 27, and 31 -36; a GalNAc-conjugated nucleotide at position 28, 29, and 30; and a phosphorothioate linkage between positions 1 and 2; and an antisense strand of 22 nucleotides comprising a 2'-F modified nucleotide at positions 2, 3, 4, 5, 7, 10, 14, 16, and 19; a 2'-0Me at positions 1, 6, 8, 9, 11, 12, 13, 15, 17, 18, and 20-22; a phosphorothioate linkage between positions 1 and 2, positions 2 and 3, positions 20 and 21, and positions 21 and 22; and a 5’ terminal nucleotide at position 1 comprising a 4’ -phosphate analog, optionally wherein the 5’ terminal nucleotide comprises 4’-O-monomethylphosphonate-2’-O- methyluridine [MePhosphonate-4O-mU]; wherein positions 1-20 of the antisense strand form a duplex region with positions 1-20 of the sense strand, wherein positions 21-36 of the sense strand form a stem-loop, wherein positions 27-30 form the loop of the stem-loop, optionally wherein positions 27-30 comprise a tetraL, wherein positions 21 and 22 of the antisense strand comprise an overhang, and wherein the sense strand and antisense strands comprise nucleotide sequences selected from the group consisting of: a) SEQ ID NOs: 1540 and 1575, respectively; b) SEQ ID NOs: 1544 and 1579, respectively; c) SEQ ID NOs: 1546 and 1581, respectively; d) SEQ ID NOs: 155 land 1586, respectively; e) SEQ ID NOs: 1552 and 1587, respectively; f) SEQ ID NOs: 1553 and 1588, respectively; g) SEQ ID NOs: 1558 and 1594, respectively; h) SEQ ID NOs: 1560 and 1595, respectively; i) SEQ ID NOs: 1564 and 1599, respectively; j) SEQ ID NOs: 1565 and 1600, respectively; k) SEQ ID NOs: 1566 and 1601, respectively; and l) SEQ ID NOs: 1570 and 1605, respectively.

[0209] In some embodiments, the oligonucleotide is for reducing SNCA gene expression and comprises: a sense strand of 36 nucleotides comprising a 2'-F modified nucleotide at positions 3, 5, 8, 10, 12, 13, 15, and 17; a 2'-OMe modified nucleotide at positions 1, 2, 4, 6, 7, 9, 11, 14, 16, 18- 27, and 31 -36; a GalNAc-conjugated nucleotide at position 28, 29 and 30; and a phosphorothioate linkage between positions 1 and 2; and an antisense strand of 22 nucleotides comprising a 2'-F modified nucleotide at positions 2, 3, 4, 5, 7, 10, 14, 16, and 19; a 2'-0Me at positions 1, 6, 8, 9, 11, 12, 13, 15, 17, 18, and 20-22; a phosphorothioate linkage between positions 1 and 2, positions 2 and 3, positions 20 and 21, and positions 21 and 22; and a 5’ terminal nucleotide at position 1 comprising a 4’ -phosphate analog, optionally wherein the 5’-terminal nucleotide comprises 4’-O-monomethylphosphonate-2’-O- methyluridine [MePhosphonate-4O-mU]; wherein positions 1-20 of the antisense strand form a duplex region with positions 1-20 of the sense strand, wherein positions 21-36 of the sense strand form a stem-loop, wherein positions 27-30 form the loop of the stem-loop, optionally wherein positions 27-30 comprise a tetraL, wherein positions 21 and 22 of the antisense strand comprise an overhang, and wherein the sense strand and antisense strands comprise nucleotide sequences selected from the group consisting of: a) SEQ ID NOs: 1553 and 1588, respectively; b) SEQ ID NOs: 1560 and 1595, respectively; c) SEQ ID NOs: 1564 and 1599, respectively; d) SEQ ID NOs: 1551 and 1586, respectively; and e) SEQ ID NOs: 1570 and 1605, respectively.

[0210] In some embodiments, the oligonucleotide is for reducing SNCA gene expression and comprises: a sense strand of 20 nucleotides comprising a 2'-F modified nucleotide at positions 3, 5, 8, 10, 12, 13, 15, and 17; a 2'-0Me modified nucleotide at positions 2, 4, 6, 7, 9, 11, 14, 16, and 18-20; a Ci6 hydrocarbon chain conjugated to a nucleotide at position 1; and a phosphorothioate linkage between positions 1 and 2, between positions 18 and 19, and between positions 19 and 20; and an antisense strand of 22 nucleotides comprising a 2'-F modified nucleotide at positions 2, 3, 4, 5, 7, 10, 14, 16, and 19; a 2'-0Me at positions 1, 6, 8, 9, 11, 12, 13, 15, 17, 18, and 20-22; a phosphorothioate linkage between positions 1 and 2, positions 2 and 3, positions 20 and 21, and positions 21 and 22; and a 5’ terminal nucleotide at position 1 comprising a 4’ -phosphate analog, optionally wherein the 5’ terminal nucleotide comprises 4’-O-monomethylphosphonate-2’-O- methyluridine [MePhosphonate-4O-mU]; wherein positions 1-20 of the antisense strand form a duplex region with positions 1-20 of the sense strand, wherein positions 21 and 22 of the antisense strand comprise an overhang, and wherein the sense strand and antisense strands comprise nucleotide sequences of SEQ ID NOs: 1681 and 1586, respectively.

[0211] In some embodiments, the oligonucleotide is for reducing SNCA gene expression and comprises a sense strand comprising the nucleotide sequence as set forth in SEQ ID NO: 1553 and an antisense strand comprising the nucleotide sequence as set forth in SEQ ID NO: 1588. In some embodiments, the oligonucleotide is for reducing SNCA gene expression and comprises a sense strand comprising the nucleotide sequence as set forth in SEQ ID NO: 1560 and an antisense strand comprising the nucleotide sequence as set forth in SEQ ID NO: 1595. In some embodiments, the oligonucleotide is for reducing SNCA gene expression and comprises a sense strand comprising the nucleotide sequence as set forth in SEQ ID NO: 1564 and an antisense strand comprising the nucleotide sequence as set forth in SEQ ID NO: 1599. In some embodiments, the oligonucleotide is for reducing SNCA gene expression comprises a sense strand comprising the nucleotide sequence as set forth in SEQ ID NO: 1551 and an antisense strand comprising the nucleotide sequence as set forth in SEQ ID NO: 1586. In some embodiments, the oligonucleotide is for reducing SNCA gene expression and comprises a sense strand comprising the nucleotide sequence as set forth in SEQ ID NO: 1570 and an antisense strand comprising the nucleotide sequence as set forth in SEQ ID NO: 1605. In some embodiments, the oligonucleotide is for reducing SNCA gene expression and comprises a sense strand comprising the nucleotide sequence as set forth in SEQ ID NO: 1681 and an antisense strand comprising the nucleotide sequence as set forth in SEQ ID NO: 1586.

[0212] In some embodiments, the oligonucleotide is for reducing SNCA gene expression and comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand comprises a nucleotide sequence comprising a region of complementarity to a SNCA mRN A target sequence, wherein the region of complementarity is set forth in SEQ ID NO: 1865; and (ii) a sense strand of 19-50 nucleotides in length comprising a region of complementarity to the antisense strand, wherein the antisense and sense strands are separate strands that form an asymmetric duplex region having an overhang of 1-4 nucleotides at the 3’ terminus of the antisense strand.

[0213] In some embodiments, the oligonucleotide is for reducing SNCA gene expression and comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand comprises a nucleotide sequence comprising a region of complementarity to a SNCA mRNA target sequence, wherein the region of complementarity is set forth in SEQ ID NO: 1721; and (ii) a sense strand of 19-50 nucleotides in length comprising a region of complementarity to the antisense strand, wherein the antisense and sense strands are separate strands that form an asymmetric duplex region having an overhang of 1-4 nucleotides at the 3’ terminus of the antisense strand.

[0214] In some embodiments, the oligonucleotide is for reducing SNCA expression and comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand comprises a nucleotide sequence comprising a region of complementarity to a SNCA mRNA target sequence, wherein the region of complementarity is set forth in SEQ ID NO: 1847; and (ii) a sense strand of 19-50 nucleotides in length comprising a region of complementarity to the antisense strand, wherein the antisense and sense strands are separate strands that form an asymmetric duplex region having an overhang of 1-4 nucleotides at the 3’ terminus of the antisense strand.

[0215] In some embodiments, the oligonucleotide is for reducing SNCA gene expression and comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand comprises a nucleotide sequence comprising a region of complementarity to a SNCA mRNA target sequence, wherein the region of complementarity is set forth in SEQ ID NO: 1846; and (ii) a sense strand of 19-50 nucleotides in length comprising a region of complementarity to the antisense strand, wherein the antisense and sense strands are separate strands that form an asymmetric duplex region having an overhang of 1-4 nucleotides at the 3’ terminus of the antisense strand.

[0216] In some embodiments, the oligonucleotide is for reducing SNCA gene expression and comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand comprises a nucleotide sequence comprising a region of complementarity to a SNCA mRN A target sequence, wherein the region of complementarity is set forth in SEQ ID NO: 1955; and (ii) a sense strand of 19-50 nucleotides in length comprising a region of complementarity to the antisense strand, wherein the antisense and sense strands are separate strands that form an asymmetric duplex region having an overhang of 1-4 nucleotides at the 3’ terminus of the antisense strand.

[0217] In some embodiments, the oligonucleotide is for reducing SNCA gene expression and comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand comprises a nucleotide sequence comprising a region of complementarity to a SNCA mRNA target sequence, wherein the region of complementarity is set forth in SEQ ID NO: 1865; and (ii) a sense strand of 19-50 nucleotides in length comprising a region of complementarity to the antisense strand and a stem-loop at the 3’ terminus, wherein the stem-loop is set forth as S1-L-S2, wherein SI is complementary to S2, and wherein L forms a loop between SI and S2 of 3 to 5 nucleotides in length, wherein the antisense and sense strands are separate strands that form an asymmetric duplex region having an overhang of 1-4 nucleotides at the 3’ terminus of the antisense strand.

[0218] In some embodiments, the oligonucleotide is for reducing SNCA gene expression and comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand comprises a nucleotide sequence comprising a region of complementarity to a SNCA mRNA target sequence, wherein the region of complementarity is set forth in SEQ ID NO: 1721; and (ii) a sense strand of 19-50 nucleotides in length comprising a region of complementarity to the antisense strand and a stem-loop at the 3’ terminus, wherein the stem-loop is set forth as S1-L-S2, wherein SI is complementary to S2, and wherein L forms a loop between SI and S2 of 3 to 5 nucleotides in length, wherein the antisense and sense strands are separate strands that form an asymmetric duplex region having an overhang of 1-4 nucleotides at the 3’ terminus of the antisense strand.

[0219] In some embodiments, the oligonucleotide is for reducing SNCA gene expression and comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand comprises a nucleotide sequence comprising a region of complementarity to a SNCA mRNA target sequence, wherein the region of complementarity is set forth in SEQ ID NO: 1847; and (ii) a sense strand of 19-50 nucleotides in length comprising a region of complementarity to the antisense strand and a stem-loop at the 3’ terminus, wherein the stem-loop is set forth as S1-L-S2, wherein SI is complementary to S2, and wherein L forms a loop between SI and S2 of 3 to 5 nucleotides in length, wherein the antisense and sense strands are separate strands that form an asymmetric duplex region having an overhang of 1-4 nucleotides at the 3’ terminus of the antisense strand.

[0220] In some embodiments, the oligonucleotide is for reducing SNCA gene expression and comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand comprises a nucleotide sequence comprising a region of complementarity to a SNCA mRNA target sequence, wherein the region of complementarity is set forth in SEQ ID NO: 1846; and (ii) a sense strand of 19-50 nucleotides in length comprising a region of complementarity to the antisense strand and a stem-loop at the 3’ terminus, wherein the stem-loop is set forth as S1-L-S2, wherein SI is complementary to S2, and wherein L forms a loop between SI and S2 of 3 to 5 nucleotides in length, wherein the antisense and sense strands are separate strands that form an asymmetric duplex region having an overhang of 1-4 nucleotides at the 3’ terminus of the antisense strand.

[0221] In some embodiments, the oligonucleotide is for reducing SNCA gene expression and comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand comprises a nucleotide sequence comprising a region of complementarity to a SNCA mRNA target sequence, wherein the region of complementarity is set forth in SEQ ID NO: 1955; and (ii) a sense strand of 19-50 nucleotides in length comprising a region of complementarity to the antisense strand and a stem-loop at the 3’ terminus, wherein the stem-loop is set forth as S1-L-S2, wherein SI is complementary to S2, and wherein L forms a loop between SI and S2 of 3 to 5 nucleotides in length, wherein the antisense and sense strands are separate strands that form an asymmetric duplex region having an overhang of 1-4 nucleotides at the 3’ terminus of the antisense strand.

[0222] In some embodiments, the oligonucleotide is for reducing SNCA gene expression and comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand comprises a nucleotide sequence comprising a region of complementarity to a SNCA mRNA target sequence, wherein the region of complementarity is set forth in SEQ ID NO: 1846; and (ii) a sense strand of 19-25 nucleotides in length comprising a region of complementarity to the antisense strand, wherein the oligonucleotide comprises a blunt end comprising the 3’ end of the sense strand, and wherein the antisense and sense strands are separate strands that form an asymmetric duplex region having an overhang of 1-4 nucleotides at the 3’ terminus of the antisense strand.

[0223] In some embodiments, the oligonucleotide is for reducing SNCA gene expression and comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand comprises a nucleotide sequence comprising a region of complementarity to a SNCA mRNA target sequence, wherein the region of complementarity is set forth in SEQ ID NO: 1865; and (ii) a sense strand of 19-50 nucleotides in length comprising a region of complementarity to the antisense strand, wherein the region of complementarity to the antisense strand is set forth in SEQ ID NO: 2249, and wherein the antisense and sense strands are separate strands that form an asymmetric duplex region having an overhang of 1-4 nucleotides at the 3’ terminus of the antisense strand.

[0224] In some embodiments, the oligonucleotide is for reducing SNCA gene expression and comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand comprises a nucleotide sequence comprising a region of complementarity to a SNCA mRNA target sequence, wherein the region of complementarity is set forth in SEQ ID NO: 1721; and (ii) a sense strand of 19-50 nucleotides in length comprising a region of complementarity to the antisense strand, wherein the region of complementarity to the antisense strand is set forth in SEQ ID NO: 2105, and wherein the antisense and sense strands are separate strands that form an asymmetric duplex region having an overhang of 1-4 nucleotides at the 3’ terminus of the antisense strand.

[0225] In some embodiments, the oligonucleotide is for reducing SNCA gene expression and comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand comprises a nucleotide sequence comprising a region of complementarity to a SNCA mRNA target sequence, wherein the region of complementarity is set forth in SEQ ID NO: 1847; and (ii) a sense strand of 19-50 nucleotides in length comprising a region of complementarity to the antisense strand, wherein the region of complementarity to the antisense strand is set forth in SEQ ID NO: 2231, and wherein the antisense and sense strands are separate strands that form an asymmetric duplex region having an overhang of 1-4 nucleotides at the 3’ terminus of the antisense strand. [0226] In some embodiments, the oligonucleotide is for reducing SNCA gene expression and comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand comprises a nucleotide sequence comprising a region of complementarity to a SNCA mRNA target sequence, wherein the region of complementarity is set forth in SEQ ID NO: 1846; and (ii) a sense strand of 19-50 nucleotides in length comprising a region of complementarity to the antisense strand, wherein the region of complementarity to the antisense strand is set forth in SEQ ID NO: 2230, and wherein the antisense and sense strands are separate strands that form an asymmetric duplex region having an overhang of 1-4 nucleotides at the 3’ terminus of the antisense strand.

[0227] In some embodiments, the oligonucleotide is for reducing SNCA gene expression and comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand comprises a nucleotide sequence comprising a region of complementarity to a SNCA mRNA target sequence, wherein the region of complementarity is set forth in SEQ ID NO: 1955; and (ii) a sense strand of 19-50 nucleotides in length comprising a region of complementarity to the antisense strand, wherein the region of complementarity to the antisense strand is set forth in SEQ ID NO: 2339, and wherein the antisense and sense strands are separate strands that form an asymmetric duplex region having an overhang of 1-4 nucleotides at the 3’ terminus of the antisense strand.

[0228] In some embodiments, the oligonucleotide is for reducing SNCA gene expression and comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand comprises a nucleotide sequence comprising a region of complementarity to a SNCA mRNA target sequence, wherein the region of complementarity is set forth in SEQ ID NO: 1865; and (ii) a sense strand of 19-50 nucleotides in length comprising a region of complementarity to the antisense strand and a stem-loop at the 3’ terminus, wherein the region of complementarity to the antisense strand is set forth in SEQ ID NO: 2249, wherein the stem-loop is set forth as S1-L-S2, wherein SI is complementary to S2, and wherein L forms a loop between SI and S2 of 3 to 5 nucleotides in length, wherein the antisense and sense strands are separate strands that form an asymmetric duplex region having an overhang of 1-4 nucleotides at the 3’ terminus of the antisense strand.

[0229] In some embodiments, the oligonucleotide is for reducing SNCA gene expression and comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand comprises a nucleotide sequence comprising a region of complementarity to a SNCA mRNA target sequence, wherein the region of complementarity is set forth in SEQ ID NO: 1721; and (ii) a sense strand of 19-50 nucleotides in length comprising a region of complementarity to the antisense strand and a stem-loop at the 3’ terminus, wherein the region of complementarity to the antisense strand is set forth in SEQ ID NO: 2105, wherein the stem-loop is set forth as S1-L-S2, wherein SI is complementary to S2, and wherein L forms a loop between SI and S2 of 3 to 5 nucleotides in length, wherein the antisense and sense strands are separate strands that form an asymmetric duplex region having an overhang of 1-4 nucleotides at the 3’ terminus of the antisense strand.

[0230] In some embodiments, the oligonucleotide is for reducing SNCA gene expression and comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand comprises a nucleotide sequence comprising a region of complementarity to a SNCA mRNA target sequence, wherein the region of complementarity is set forth in SEQ ID NO: 1847; and (ii) a sense strand of 19-50 nucleotides in length comprising a region of complementarity to the antisense strand and a stem-loop at the 3’ terminus, wherein the region of complementarity to the antisense strand is set forth in SEQ ID NO: 2231, wherein the stem-loop is set forth as S1-L-S2, wherein SI is complementary to S2, and wherein L forms a loop between SI and S2 of 3 to 5 nucleotides in length, wherein the antisense and sense strands are separate strands that form an asymmetric duplex region having an overhang of 1-4 nucleotides at the 3’ terminus of the antisense strand.

[0231] In some embodiments, the oligonucleotide is for reducing SNCA gene expression and comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand comprises a nucleotide sequence comprising a region of complementarity to a SNCA mRNA target sequence, wherein the region of complementarity is set forth in SEQ ID NO: 1846; and (ii) a sense strand of 19-50 nucleotides in length comprising a region of complementarity to the antisense strand and a stem-loop at the 3’ terminus, wherein the region of complementarity to the antisense strand is set forth in SEQ ID NO: 2230, wherein the stem-loop is set forth as S1-L-S2, wherein SI is complementary to S2, and wherein L forms a loop between SI and S2 of 3 to 5 nucleotides in length, wherein the antisense and sense strands are separate strands that form an asymmetric duplex region having an overhang of 1-4 nucleotides at the 3’ terminus of the antisense strand.

[0232] In some embodiments, the oligonucleotide is for reducing SNCA gene expression and comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand comprises a nucleotide sequence comprising a region of complementarity to a SNCA mRNA target sequence, wherein the region of complementarity is set forth in SEQ ID NO: 1955; and (ii) a sense strand of 19-50 nucleotides in length comprising a region of complementarity to the antisense strand and a stem-loop at the 3’ terminus, wherein the region of complementarity to the antisense strand is set forth in SEQ ID NO: 2339, wherein the stem-loop is set forth as S1-L-S2, wherein SI is complementary to S2, and wherein L forms a loop between SI and S2 of 3 to 5 nucleotides in length, wherein the antisense and sense strands are separate strands that form an asymmetric duplex region having an overhang of 1-4 nucleotides at the 3’ terminus of the antisense strand.

[0233] In some embodiments, the oligonucleotide is for reducing SNCA gene expression and comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand comprises a nucleotide sequence comprising a region of complementarity to a SNCA mRNA target sequence, wherein the region of complementarity is set forth in SEQ ID NO: 1846; and (ii) a sense strand of 19-25 nucleotides in length comprising a region of complementarity to the antisense strand, wherein the region of complementarity to the antisense strand is set forth in SEQ ID NO: 2230, wherein the oligonucleotide comprises a blunt end comprising the 3’ end of the sense strand, and wherein the antisense and sense strands are separate strands that form an asymmetric duplex region having an overhang of 1-4 nucleotides at the 3’ terminus of the antisense strand.

[0234] In some embodiments, the oligonucleotide is for reducing SNCA gene expression and comprises a sense strand and an antisense strand according to:

Sense Strand: 5 ’-mX-X-mX-fX-mX-fX-mX-rnX-fX- X-fX-rnX-fX-fX-rnX-fX-rnX-fX-rnX- mX-mX-mX-mX-mX-mX-mX-mX-mX- [ademX-GalNAc] [ademX-GalNAc] [ademX-GalNAc]- mX-mX-mX-mX-mX-mX-3 ’ ; hybridized to:

Antisense Strand: 5 ’-[MePhosphonate-dO-mXJ-X-fX-X-fX-fX-fX-mX-fX-mX-mX-fX-mX- m X-m X-fX-m X-fX-m X-m X-fX-m X-,S'-m X-,S'-m X-3 ’ ; wherein mX = 2’-0Me modified nucleotide, fX = 2’-F modified nucleotide, -S- = phosphorothioate linkage, - = phosphodiester linkage, [MePhosphonate-4O-mX] = 4’-O- monomethylphosphonate-2’-O-methyl modified nucleotide, and ademX-GalNAc = GalNAc attached to a nucleotide. [0235] In some embodiments, the oligonucleotide is for reducing SNCA gene expression and comprises a sense strand and an antisense strand according to:

Sense Strand: 5 ’ -[AdemX-L]-X-mX-fX-mX-fX-mX-mX-fX-mX-fX-mX-fX-fX-mX-fX-mX- fX-mX-5-mX-5-mX-3 ’ ; hybridized to:

Antisense Strand: 5’-[MePhosphonate-4O-mX]-5-fX-5-fX-fX-fX-mX-fX-mX-mX-fX-mX - mX-mX-fX-mX-fX-mX-mX-fX-mX-5-mX-5-mX-3 ’ ; wherein mX = 2’-0Me modified nucleotide, fX = 2’-F modified nucleotide, -S- = phosphorothioate linkage, - = phosphodi ester linkage, [MePhosphonate-4O-mX] = 4’-O- monomethylphosphonate-2’-O-methyl modified nucleotide, and ademX-L = lipid moiety attached to a nucleotide.

[0236] In some embodiments, the oligonucleotide is for reducing SNCA gene expression and comprises a sense strand and an antisense strand according to:

Sense Strand: 5 ’ -[AdemX-Ci6]-5-mX-fX-mX-fX-mX-mX-fX-mX-fX-mX-fX-fX-mX-fX-mX- fX-mX-X-mX-,S'-mX-3’; hybridized to:

Antisense Strand: 5’-[MePhosphonate-4O-mX]-5'-fX-5'-fX-fX-fX-mX-fX-mX-mX-fX- mX- m X-m X-fX-m X-fX-m X-m X-fX-m X-X-m X-X-m X-3 ’ ; wherein mX = 2’-0Me modified nucleotide, fX = 2’-F modified nucleotide, -S- = phosphorothioate linkage, - = phosphodiester linkage, [MePhosphonate-4O-mX] = 4’-O- monomethylphosphonate-2’-O-methyl modified nucleotide, and ademX-Ci6 = Ci6 hydrocarbon chain attached to a nucleotide.

[0237] In some embodiments, the oligonucleotide is for reducing SNCA gene expression and comprises a sense strand and an antisense strand comprising nucleotide sequences selected from the group consisting of: a) SEQ ID NOs: 1607 and 1642, respectively; b) SEQ ID NOs: 1608 and 1643, respectively; c) SEQ ID NOs: 1609 and 1644, respectively; d) SEQ ID NOs: 1610 and 1645, respectively; e) SEQ ID NOs: 1611 and 1646, respectively; f) SEQ ID NOs: 1612 and 1647, respectively; g) SEQ ID NOs: 1613 and 1648, respectively; h) SEQ ID NOs: 1614 and 1649, respectively; i) SEQ ID NOs: 1615 and 1650, respectively; j) SEQ ID NOs: 1616 and 1651, respectively; k) SEQ ID NOs: 1617 and 1652, respectively; l) SEQ ID NOs: 1618 and 1653, respectively; m) SEQ ID NOs: 1619 and 1654, respectively; n) SEQ ID NOs: 1620 and 1655, respectively; o) SEQ ID NOs: 1621 and 1656, respectively; p) SEQ ID NOs: 1622 and 1657, respectively; q) SEQ ID NOs: 1623 and 1658, respectively; r) SEQ ID NOs: 1624 and 1659, respectively; s) SEQ ID NOs: 1625 and 1660, respectively; t) SEQ ID NOs: 1626 and 1661, respectively; u) SEQ ID NOs: 1627 and 1662, respectively; v) SEQ ID NOs: 1628 and 1663, respectively; w) SEQ ID NOs: 1629 and 1664, respectively; x) SEQ ID NOs: 1630 and 1665, respectively; y) SEQ ID NOs: 16 1 and 1666, respectively; z) SEQ ID NOs: 1632 and 1667, respectively; aa) SEQ ID NOs: 1633 and 1668, respectively; bb) SEQ ID NOs: 1634 and 1669, respectively; cc) SEQ ID NOs: 1635 and 1670, respectively; dd) SEQ ID NOs: 1636 and 1671, respectively; ee) SEQ ID NOs: 1637 and 1672, respectively; ff) SEQ ID NOs: 1638 and 1673, respectively; gg) SEQ ID NOs: 1639 and 1674, respectively; hh) SEQ ID NOs: 1640 and 1675, respectively; ii) SEQ ID NOs: 1641 and 1676, respectively; and jj) SEQ ID NOs: 1682 and 1656, respectively.

[0238] In some embodiments, the oligonucleotide is for reducing SNCA gene expression and comprises a sense strand and an antisense strand comprising nucleotide sequences selected from the group consisting of: a) SEQ ID NOs: 1610 and 1645, respectively; b) SEQ ID NOs: 1614 and 1649, respectively; c) SEQ ID NOs: 1616 and 1651, respectively; d) SEQ ID NOs: 1621 and 1656, respectively; e) SEQ ID NOs: 1622 and 1657, respectively; f) SEQ ID NOs: 1623 and 1658, respectively; g) SEQ ID NOs: 1629 and 1664, respectively; h) SEQ ID NOs: 1630 and 1665, respectively; i) SEQ ID NOs: 1634 and 1669, respectively; j) SEQ ID NOs: 1635 and 1670, respectively; k) SEQ ID NOs: 1636 and 1671, respectively; l) SEQ ID NOs: 1640 and 1675, respectively; and m) SEQ ID NOs: 1682 and 1656, respectively.

[0239] In some embodiments, the oligonucleotide is for reducing SNCA gene expression and comprises a sense strand and an antisense strand comprising nucleotide sequences selected from the group consisting of: a) SEQ ID NOs: 1623 and 1658, respectively; b) SEQ ID NOs: 1630 and 1665, respectively; c) SEQ ID NOs: 1634 and 1669, respectively; d) SEQ ID NOs: 1621 and 1656, respectively; e) SEQ ID NOs: 1640 and 1675, respectively; and, f) SEQ ID NOs: 1682 and 1656, respectively.

[0240] In some embodiments, the oligonucleotide is for reducing SNCA gene expression and comprises a sense strand comprising the nucleotide sequence as set forth in SEQ ID NO: 1623 and an antisense strand comprising the nucleotide sequence as set forth in SEQ ID NO: 1658. In some embodiments, the oligonucleotide is for reducing SNCA gene expression and comprises a sense strand comprising the nucleotide sequence as set forth in SEQ ID NO: 1630 and an antisense strand comprising the nucleotide sequence as set forth in SEQ ID NO: 1665. In some embodiments, the oligonucleotide is for reducing SNCA gene expression and comprises a sense strand comprising the nucleotide sequence as set forth in SEQ ID NO: 1634 and an antisense strand comprising the nucleotide sequence as set forth in SEQ ID NO: 1669. In some embodiments, the oligonucleotide is for reducing SNCA gene expression and comprises a sense strand comprising the nucleotide sequence as set forth in SEQ ID NO: 1621 and an antisense strand comprising the nucleotide sequence as set forth in SEQ ID NO: 1656. In some embodiments, the oligonucleotide is for reducing SNCA gene expression and comprises a sense strand comprising the nucleotide sequence as set forth in SEQ ID NO: 1640 and an antisense strand comprising the nucleotide sequence as set forth in SEQ ID NO: 1675. In some embodiments, the oligonucleotide is for reducing SNCA gene expression and comprises a sense strand comprising the nucleotide sequence as set forth in SEQ ID NO: 1682 and an antisense strand comprising the nucleotide sequence as set forth in SEQ ID NO: 1656.

Formulations

[0241] Various formulations have been developed to facilitate oligonucleotide use. For example, oligonucleotides (e.g, RNAi oligonucleotides) can be delivered to a subject or a cellular environment using a formulation that minimizes degradation, facilitates delivery and/or uptake, or provides another beneficial property to the oligonucleotide in the formulation. In some embodiments, the formulation is a composition comprising oligonucleotides that reduce SNCA gene expression. Such a composition can be suitably formulated such that when administered to a subject, either into the immediate environment of a target cell or systemically, a sufficient portion of the oligonucleotides enter the cell to reduce SNCA gene expression. Any variety of suitable oligonucleotide formulations can be used to deliver oligonucleotides for the reduction of SNCA gene expression. In some embodiments, the oligonucleotides are formulated in buffer solutions such as phosphate buffered saline solutions, liposomes, micellar structures, and capsids. In other embodiments, the oligonucleotides are formulated in buffer solutions such as phosphate buffered saline solutions.

[0242] Formulations of oligonucleotides with cationic lipids can be used to facilitate transfection of the oligonucleotides into cells. For example, cationic lipids, such as lipofectin, cationic glycerol derivatives, and polycationic molecules (e.g, polylysine) can be used. Suitable lipids include Oligofectamine, Lipofectamine (Life Technologies), NC388 (Ribozyme Pharmaceuticals, Inc.; Boulder, CO), or FuGene 6 (Roche), all of which can be used according to the manufacturer's instructions. Tn some embodiments, the oligonucleotide is not formulated with a component to facilitate transfection into cells.

[0243] Accordingly, in some embodiments, the formulation comprises a lipid nanoparticle. In some embodiments, the lipid nanoparticle comprises a liposome, a lipid, a lipid complex, a microsphere, a microparticle, a nanosphere or a nanoparticle, or may be otherwise formulated for administration to the cells, tissues, organs, or body of a subject in need thereof (see, e.g., Remington: THE SCIENCE AND PRACTICE OF PHARMACY, 22nd ed, Pharmaceutical Press, 2013).

[0244] In some embodiments, the formulation comprises an excipient, which confers to a composition improved stability, improved absorption, improved solubility, and/or therapeutic enhancement of the active ingredient. In some embodiments, the excipient is a buffering agent (e.g., sodium citrate, sodium phosphate, a tris base, or sodium hydroxide) or a vehicle (e.g, a buffered solution, petrolatum, dimethyl sulfoxide, or mineral oil). In some embodiments, the oligonucleotide is lyophilized for extending its shelf-life and then made into a solution before use (e.g, administration to a subject). Accordingly, the excipient may be a lyoprotectant (e.g., mannitol, lactose, polyethylene glycol, or polyvinylpyrrolidone) or a collapse temperature modifier (e.g., dextran, Ficoll™ or gelatin).

[0245] In some embodiments, the formulation is a pharmaceutical composition compatible with its intended route of administration. Examples of routes of administration include, but are not limited to, parenteral (e.g, intravenous, intramuscular, intraperitoneal, intradermal, subcutaneous), oral (e.g., inhalation), transdermal (e.g, topical), transmucosal, and rectal administration.

[0246] In some embodiments, the formulation is formulated for administration into the CNS. In some embodiments, the formulation is formulated for administration into the cerebral spinal fluid. In some embodiments, the formulation is formulated for administration to the spinal cord. In some embodiments, the formulation is formulated for intrathecal administration. In some embodiments, the formulation is formulated for administration to the brain. In some embodiments, the formulation is formulated for intracerebroventricular administration. In some embodiments, the formulation is formulated for the brain stem. In some embodiments, the formulation is formulated for intraci sternal magna administration.

[0247] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF; Parsippany, NJ), or phosphate buffered saline (PBS). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Sterile injectable solutions can be prepared by incorporating the oligonucleotides in a required amount in a selected solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.

[0248] In some embodiments, the formulation may contain at least about 0.1% of the oligonucleotide or more, although the percentage of the active ingredient(s) may be between about 1% to about 80% or more of the weight or volume of the total composition. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.

Methods of Use

Reducing SNCA Expression

[0249] In some embodiments, methods of contacting or delivering to a cell or population of cells comprise administering an effective amount of an oligonucleotide (e.g., a RNAi oligonucleotide) to reduce SNCA gene expression. In some embodiments, a reduction of SNCA gene expression is determined by measuring a reduction in the amount or level of SNCA mRNA, SNCA protein, SNCA activity in a cell, or a combination thereof. The methods include those described herein and known to one of ordinary skill in the art.

[0250J In some embodiments, methods of reducing SNCA gene expression in the CNS comprise administering an effective amount of an oligonucleotide (e.g., a RNAi oligonucleotide) to reduce SNCA gene expression. In some embodiments, the CNS comprises the brain and spinal cord. In some embodiments, SNCA gene expression is reduced in at least one region of the brain, which includes, but is not limited to, the cervical spinal cord, thoracic spinal cord, lumbar spinal cord, frontal cortex, temporal cortex, cerebellum, midbrain, occipital cortex, parietal cortex, hippocampus, caudate nucleus, thalamus, and brainstem. In some embodiments, SNCA gene expression is reduced in at least one region of the spinal cord, which includes, but is not limited to, the cervical spinal cord, thoracic spinal cord, and lumbar spinal cord. In some embodiments, SNCA gene expression is reduced in at least one region of the brain and at least one region of the spinal cord. In some embodiments, SNCA gene expression is reduced in at least one of the cervical spinal cord, thoracic spinal cord, lumbar spinal cord, frontal cortex, temporal cortex, cerebellum, midbrain, occipital cortex, parietal cortex, hippocampus, caudate nucleus, thalamus, brainstem, motor cortex, globus pallidus, midbrain tegmentum, substantia nigra, pons, cerebellar white matter, and cerebellar dentate nucleus. In some embodiments, SNCA gene expression is reduced in at least one of the lumbar spinal cord, thoracic spinal cord, and cervical spinal cord. In some embodiments, SNCA gene expression is reduced in tissue of the brain and/or spinal cord associated with Parkinson’s disease. In some embodiments, tissue associated with Parkinson’s disease includes, but is not limited to, putamen, midbrain tegmentum, substantia nigra, pons, and medulla. In some embodiments, SNCA gene expression is reduced in tissue of the brain and/or spinal cord associated with multiple systems atrophy. In some embodiments, tissue associated with Parkinson’ s disease includes, but is not limited to, caudate nucleus, putamen, midbrain tegmentum, substantia nigra, pons, cerebellar cortex, cerebellar white matter, medulla, cervical spinal cord, thoracic spinal cord, and lumbar spinal cord. [0251] In some embodiments, SNCA gene expression is reduced for about 1 week to about 12 weeks after administration of an oligonucleotide or a formulation including the same. In some embodiments, SNCA gene expression is reduced for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks after administration of the oligonucleotide or the formulation. In some embodiments, SNCA gene expression is reduced for about 1 month to about 4 months after administration of the oligonucleotide or the formulation. In some embodiments, SNCA gene expression is reduced for about 1 month to about 6 months after administration of the oligonucleotide or the formulation. In some embodiments, SNCA gene expression is reduced for 1 , 2, 3, or 4 months after administration of the oligonucleotide or the formulation. In some embodiments, SNCA gene expression is reduced for 1, 2, 3 4, 5, or 6 months after administration of the oligonucleotide or the formulation. In some embodiments, SNCA gene expression is reduced for about 7days to about 91 days after administration of the oligonucleotide or the formulation. In some embodiments, SNCA gene expression is reduced for 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, or 91 days after administration of the oligonucleotide or the formulation.

[0252] In some embodiments, SNCA gene expression is reduced in at least one region of the brain and/or at least one region of the spinal cord for about 1 to about 12 weeks after administration of the oligonucleotide or the formulation. In some embodiments, SNCA gene expression is reduced in at least one region of the brain and/or at least one region of the spinal cord for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks after administration of the oligonucleotide or the formulation. In some embodiments, SNCA gene expression is reduced in at least one region of the brain and/or at least one region of the spinal cord for about 1 month to about 4 months after administration of the oligonucleotide or the formulation. In some embodiments, SNCA gene expression is reduced in at least one region of the brain and/or at least one region of the spinal cord for about 1 month to about 6 months after administration of the oligonucleotide or the formulation. In some embodiments, SNCA gene expression is reduced in at least one region of the brain and/or at least one region of the spinal cord for 1, 2, 3, or 4 months after administration of the oligonucleotide or the formulation. In some embodiments, SNCA gene expression is reduced in at least one region of the brain and/or at least one region of the spinal cord for 1, 2, 3 4, 5, or 6 months after administration of the oligonucleotide or the formulation. In some embodiments, SNCA gene expression is reduced in at least one region of the brain and/or at least one region of the spinal cord for about 7 days to about 91 days after administration of the oligonucleotide or the formulation. In some embodiments, SNCA gene expression is reduced in at least one region of the brain and/or at least one region of the spinal cord for 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, or 91 days after administration of the oligonucleotide or the formulation.

[0253] The methods herein are useful in any appropriate cell type. In some embodiments, the cell type is any cell that expresses SNCA mRNA (e.g., oligodendrocyte). In some embodiments, the cell type is a primary cell obtained from a subject. In some embodiments, the primary cell has undergone a limited number of passages such that the cell substantially maintains is natural phenotypic properties. In some embodiments, a cell to which the oligonucleotide is delivered is ex vivo or in vitro (i.e., can be delivered to a cell in culture or to an organism in which the cell resides). [0254] In some embodiments, the oligonucleotide is delivered to a cell or population of cells using a nucleic acid delivery method known in the art including, but not limited to, injection of a solution or pharmaceutical composition containing the oligonucleotide (i.e., a formulation), bombardment by particles covered by the oligonucleotide, exposing the cell or population of cells to a solution containing the oligonucleotide, or electroporation of cell membranes in the presence of the oligonucleotide. Other methods known in the art for delivering oligonucleotides to cells may be used, such as lipid-mediated carrier transport, chemical-mediated transport, and cationic liposome transfection such as calcium phosphate, and others.

[0255] In some embodiments, reduction of SNCA gene expression is determined by an assay or technique that evaluates one or more molecules, properties or characteristics of a cell or population of cells associated with SNCA gene expression, or by an assay or technique that evaluates molecules that are directly indicative of SNCA gene expression in a cell or population of cells (e.g. , SNCA mRNA or SNCA protein). In some embodiments, the extent to which the oligonucleotide reduces SNCA gene expression is evaluated by comparing SNCA gene expression in a cell or population of cells contacted with the oligonucleotide to a control cell or population of cells (e.g., a cell or population of cells not contacted with the oligonucleotide or contacted with a control oligonucleotide). In some embodiments, a control amount or level of SNCA gene expression in a control cell or population of cells is predetermined, such that the control amount or level need not be measured in every instance the assay or technique is performed. The predetermined level or value can take a variety of forms. In some embodiments, a predetermined level or value can be single cut-off value, such as a median or mean.

[0256] In some embodiments, contacting or delivering the oligonucleotide to a cell or a population of cells results in a reduction in SNCA gene expression. In some embodiments, the reduction in SNCA gene expression is relative to a control amount or level of SNCA gene expression in cell or population of cells not contacted with the oligonucleotide or contacted with a control oligonucleotide. Tn some embodiments, the reduction in SNCA gene expression is about 1% or lower, about 5% or lower, about 10% or lower, about 15% or lower, about 20% or lower, about 25% or lower, about 30% or lower, about 35% or lower, about 40% or lower, about 45% or lower, about 50% or lower, about 55% or lower, about 60% or lower, about 70% or lower, about 80% or lower, or about 90% or lower relative to a control amount or level of SNCA gene expression. In some embodiments, the control amount or level of SNCA gene expression is an amount or level of SNCA mRNA and/or SNCA protein and/or SNCA activity/function in a cell or population of cells that has not been contacted with the oligonucleotide. In some embodiments, the effect of delivery of the oligonucleotide to a cell or population of cells according to a method herein is assessed after any finite period or amount of time (e.g, minutes, hours, days, weeks, months). For example, in some embodiments, SNCA gene expression is determined in a cell or population of cells at least about 4 hours, about 8 hours, about 12 hours, about 18 hours, about 24 hours; or at least about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 21 days, about 28 days, about 35 days, about 42 days, about 49 days, about 56 days, about 63 days, about 70 days, about 77 days, or about 84 days, or more after contacting or delivering the oligonucleotide to the cell or population of cells. In some embodiments, SNCA gene expression is determined in a cell or population of cells at least about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months, or more after contacting or delivering the oligonucleotide to the cell or population of cells.

[0257] In some embodiments, the oligonucleotide is delivered in the form of a transgene that is engineered to express in a cell the oligonucleotide or strands comprising the oligonucleotide (e.g., its sense and antisense strands). In some embodiments, the oligonucleotide is delivered using a transgene engineered to express any oligonucleotide. Transgenes may be delivered using viral vectors (e.g., adenovirus, retrovirus, vaccinia virus, poxvirus, adeno-associated virus, or herpes simplex virus) or non-viral vectors (e.g., plasmids or synthetic mRNAs). In some embodiments, transgenes can be injected directly to a subject.

Treatment Methods

[0258] Oligonucleotides (e.g., RNAi oligonucleotides) also are provided for use, or adaptable for use, to treat a subject (e.g., a human having a disease, disorder, or condition associated with SNCA expression) that would benefit from reducing SNCA gene expression. In some aspects, the disclosure provides oligonucleotides for use, or adapted for use, to treat a subject having a disease, disorder or condition associated with expression of SNCA. Oligonucleotides also are provided for use, or adaptable for use, in the manufacture of a medicament or formulation/pharmaceutical composition for treating a disease, disorder, or condition associated with SNCA gene expression. In some embodiments, the oligonucleotides for use, or adaptable for use, target SNCA mRNA and reduce SNCA gene expression (e.g., via the RNAi pathway). In some embodiments, the oligonucleotides for use, or adaptable for use, target SNCA mRNA and reduce the amount or level of SNCA mRNA, SNCA protein and/or SNCA activity/function.

[0259] In addition, in some embodiments of the methods herein, a subject having a disease, disorder, or condition associated with SNCA expression or is predisposed to the same is selected for treatment with the oligonucleotide or the formulation. In some embodiments, the method comprises selecting an individual having a marker (e.g., a biomarker) for a disease, disorder, or condition associated with SNCA gene expression, or predisposed to the same, such as, but not limited to, SNCA mRNA, SNCA protein, SNCA activity/function, or a combination thereof. Likewise, and as detailed below, some embodiments of the methods include steps such as measuring or obtaining a baseline value for a marker of SNCA gene expression, and then comparing such obtained value to one or more other baseline values or values obtained after the subject is administered the oligonucleotide to assess the effectiveness of treatment.

[0260] Methods of treating a subject having, suspected of having, or at risk of developing a disease, disorder, or condition associated with SNCA gene expression with the oligonucleotide or formulation are provided herein. In some aspects, methods of treating or attenuating the onset or progression of a disease, disorder, or condition associated with SNCA gene expression using the oligonucleotide or formulation are provided herein. In other aspects, methods to achieve one or more therapeutic benefits in a subject having a disease, disorder, or condition associated with SNCA gene expression using the oligonucleotide or formulation are provided herein. In some embodiments, the subject is treated by administering a therapeutically effective amount of any one or more of the oligonucleotides provided herein. Tn some embodiments, treatment comprises reducing SNCA gene expression. In some embodiments, the subject is treated therapeutically. In other embodiments, the subject is treated prophylactically.

[0261] In some embodiments of the methods herein, the oligonucleotide, or a pharmaceutical composition comprising the oligonucleotide, is administered to a subject having a disease, disorder, or condition associated with SNCA expression such that SNCA gene expression is reduced in the subject, thereby treating the subject. In some embodiments, an amount or level of SNCA mRNA is reduced in the subject. In some embodiments, an amount or level of SNCA protein is reduced in the subject. In other embodiments, an amount or level of SNCA activity/function is reduced in the subject.

[0262] In some embodiments, the oligonucleotide or pharmaceutical composition/formulation comprising the oligonucleotide, is administered to a subj ect having a disease, disorder, or condition associated with SNCA gene expression such that SNCA expression is reduced in the subject by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or greater than 99% when compared to SNCA expression prior to administration of the oligonucleotide or pharmaceutical composition. In some embodiments of the methods herein, the oligonucleotide or the pharmaceutical composition is administered to a subject having a disease, disorder, or condition associated with SNCA gene expression such that SNCA expression is reduced in the subject by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or greater than 99% for about 1 week to about 12 weeks, about 1 month to about 6 months, or about 7 days to about 91 days when compared to SNCA expression prior to administration of the oligonucleotide or pharmaceutical composition. In some embodiments, SNCA expression is reduced in the subject by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or greater than 99% when compared to SNCA expression in a subject (e.g., a reference or control subject) not receiving the oligonucleotide or pharmaceutical composition or receiving a control oligonucleotide, pharmaceutical composition, or treatment. In some embodiments, SNCA expression is reduced in the subject by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or greater than 99% for about 1 week to about 12 weeks, about 1 month to about 6 months, or about 7 days to about 91 days when compared to SNCA expression in a subject e.g., a reference or control subject) not receiving the oligonucleotide or pharmaceutical composition or receiving a control oligonucleotide, pharmaceutical composition or treatment.

[0263] In some embodiments of the methods herein, the oligonucleotide or the pharmaceutical composition is administered to a subject having a disease, disorder, or condition associated with SNCA gene expression such that an amount or level of SNCA mRNA is reduced in the subject by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or greater than 99% when compared to the amount or level of SNCA mRNA prior to administration of the oligonucleotide or pharmaceutical composition. In some embodiments of the methods herein, the oligonucleotide or the pharmaceutical composition is administered to a subject having a disease, disorder, or condition associated with SNCA gene expression such that an amount or level of SNCA mRNA is reduced in the subject by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or greater than 99% for about 1 week to about 12 weeks, about 1 month to about 6 months, or about 7 days to about 91 days when compared to the amount or level of SNCA mRNA prior to administration of the oligonucleotide or pharmaceutical composition. In some embodiments, an amount or level of SNCA mRNA is reduced in the subject by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or greater than 99% when compared to an amount or level of SNCA mRNA in a subject (e.g., a reference or control subject) not receiving the oligonucleotide or pharmaceutical composition or receiving a control oligonucleotide, pharmaceutical composition, or treatment. In some embodiments, an amount or level of SNCA mRNA is reduced in the subject by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or greater than 99% for about 1 week to about 12 weeks, about 1 month to about 6 months, or about 7 days to about 91 days when compared to an amount or level of SNCA mRNA in a subject (e.g., a reference or control subject) not receiving the oligonucleotide or pharmaceutical composition or receiving a control oligonucleotide, pharmaceutical composition, or treatment.

[0264] In some embodiments of the methods herein, the oligonucleotide or the pharmaceutical composition is administered to a subject having a disease, disorder, or condition associated with SNCA gene expression such that an amount or level of SNCA protein is reduced in the subject by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or greater than 99% when compared to the amount or level of SNCA protein prior to administration of the oligonucleotide or pharmaceutical composition. In some embodiments of the methods herein, the oligonucleotide or the pharmaceutical composition is administered to a subject having a disease, disorder, or condition associated with SNCA gene expression such that an amount or level of SNCA protein is reduced in the subject by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or greater than 99% for about 1 week to about 12 weeks, about 1 month to about 6 months, or about 7 days to about 91 days when compared to the amount or level of SNCA protein prior to administration of the oligonucleotide or pharmaceutical composition. In some embodiments, an amount or level of SNCA protein is reduced in the subject by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or greater than 99% when compared to an amount or level of SNCA protein in a subject (e.g., a reference or control subject) not receiving the oligonucleotide or pharmaceutical composition or receiving a control oligonucleotide, pharmaceutical composition, or treatment. In some embodiments, an amount or level of SNCA protein is reduced in the subject by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or greater than 99% for about 1 week to about 12 weeks, about 1 month to about 6 months, or about 7 days to about 91 days when compared to an amount or level of SNCA protein in a subject (e.g., a reference or control subject) not receiving the oligonucleotide or pharmaceutical composition or receiving a control oligonucleotide, pharmaceutical composition, or treatment.

[0265] In some embodiments of the methods herein, the oligonucleotide or the pharmaceutical composition is administered to a subject having a disease, disorder, or condition associated with SNCA gene expression such that an amount or level of SNCA activity /function is reduced in the subject by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or greater than 99% when compared to the amount or level of SNCA activity/function prior to administration of the oligonucleotide or pharmaceutical composition. In some embodiments of the methods herein, the oligonucleotide or the pharmaceutical composition is administered to a subject having a disease, disorder, or condition associated with SNCA gene expression such that an amount or level of SNCA activity/function is reduced in the subject by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% ,or greater than 99% for about 1 week to about 12 weeks, about 1 month to about 6 months, or about 7 days to about 91 days when compared to the amount or level of SNCA activity/function prior to administration of the oligonucleotide or pharmaceutical composition. In some embodiments, an amount or level of SNCA activity/function is reduced in the subject by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or greater than 99% when compared to an amount or level of SNCA activity/function in a subject (e.g., a reference or control subject) not receiving the oligonucleotide or pharmaceutical composition or receiving a control oligonucleotide, pharmaceutical composition or treatment. In some embodiments, an amount or level of SNCA activity /function is reduced in the subject by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or greater than 99% for about 1 week to about 12 weeks, about 1 month to about 6 months, or about 7 days to about 91 days when compared to an amount or level of SNCA activity/function in a subject (e.g., a reference or control subject) not receiving the oligonucleotide or pharmaceutical composition or receiving a control oligonucleotide, pharmaceutical composition, or treatment.

[0266] Suitable methods for determining SNCA gene expression such as SNCA expression, an amount or level of SNCA mRNA, an amount or level of SNCA protein, and/or an amount or level of SNCA activity/function in the subject, or in a sample from the subject, are known in the art. Further, the Examples set forth herein illustrate exemplary methods for determining SNCA gene expression.

[0267] In some embodiments, SNCA gene expression such as SNCA expression, an amount or level of SNCA mRNA, an amount or level of SNCA protein, an amount or level of SNCA activity/function, or any combination thereof, is reduced in a cell (e.g., an oligodendrocyte), a population or a group of cells (e.g., an organoid), an organ (e.g., frontal cortex), blood or a fraction thereof (e.g., plasma), a tissue (e.g., brain tissue), a sample (e.g., a brain biopsy sample), or any other biological material obtained or isolated from the subject. In some embodiments, SNCA expression, an amount or level of SNCA mRNA, an amount or level of SNCA protein, an amount or level of SNCA activity/function, or any combination thereof, is reduced in more than one type of cell (e.g., an oligodendrocyte and one or more other type(s) of cell), more than one groups of cells, more than one organ (e.g., brain and one or more other organ(s)), more than one fraction of blood (e.g., plasma and one or more other blood fraction(s)), more than one type of tissue (e.g., brain tissue and one or more other type(s) of tissue), more than one type of sample (e.g., a brain biopsy sample and one or more other type(s) of biopsy sample) obtained or isolated from the subject. In some embodiments, SNCA expression, an amount or level of SNCA mRNA, an amount or level of SNCA protein, an amount or level of SNCA activity/function, or any combination thereof is reduced in one or more of the cervical spinal cord, thoracic spinal cord, lumbar spinal cord, frontal cortex, temporal cortex, cerebellum, midbrain, occipital cortex, parietal cortex, hippocampus, caudate nucleus, thalamus, brainstem, motor cortex, globus pallidus, midbrain tegmentum, substantia nigra, pons, cerebellar white matter, and cerebellar dentate nucleus. In some embodiments, SNCA expression, an amount or level of SNCA mRNA, an amount or level of SNCA protein, an amount or level of SNCA activity/function, or any combination thereof is reduced in tissue of the brain and/or spinal cord associated with Parkinson’s disease. In some embodiments, tissue associated with Parkinson’s disease includes, but is not limited to, putamen, midbrain tegmentum, substantia nigra, pons, and medulla. In some embodiments, SNCA expression, an amount or level of SNCA mRNA, an amount or level of SNCA protein, an amount or level of SNCA activity/function, or any combination thereof is reduced in tissue of the brain and/or spinal cord associated with multiple system atrophy. In some embodiments, tissue associated with multiple system atrophy includes, but is not limited to caudate nucleus, putamen, midbrain tegmentum, substantia nigra, pons, cerebellar cortex, cerebellar white matter, medulla, cervical spinal cord, thoracic spinal cord, and lumbar spinal cord.

[0268] Examples of a disease, disorder or condition associated with SNCA gene expression include, multiple system atrophy, dementia with Lewy bodies, and Parkinson disease.

[0269] Because of their high specificity, the oligonucleotide herein specifically targets SNCA mRNA of target genes of cells, tissue(s), or organ(s) (e.g, brain). In preventing disease, the target gene may be one which is required for initiation or maintenance of the disease or which has been identified as being associated with a higher risk of contracting the disease. In treating disease, the oligonucleotide can be brought into contact with the cells, tissue(s), or organ(s) (e.g, brain) exhibiting or responsible for mediating the disease. For example, an oligonucleotide substantially identical to all or part of a wild-type (i.e., native) or mutated gene associated with a disease, disorder, or condition associated with SNCA gene expression may be brought into contact with or introduced into a cell or tissue type of interest such as an oligodendrocyte or other brain cell.

[0270] In some embodiments, SNCA may be from any mammal, such as a human and may be silenced according to the method described herein.

[0271] The methods herein typically involve administering to a subject a therapeutically effective amount of the oligonucleotide, that is, an amount capable of producing a desirable therapeutic result. A therapeutically acceptable amount may be an amount that can therapeutically treat a disease, disorder, or condition. The appropriate amount/dosage for any one subject will depend on certain factors, including the subject's size, body surface area, age, the particular composition to be administered, the active ingredient(s) in the composition, time and route of administration, general health, and other drugs being administered concurrently.

[0272] In some embodiments, the subject is administered any one of the oligonucleotides or compositions herein either enterally (e.g., orally, by gastric feeding tube, by duodenal feeding tube, via gastrostomy or rectally), parenterally (e.g., subcutaneous injection, intravenous injection or infusion, intra-arterial injection or infusion, intraosseous infusion, intramuscular injection, intracerebral injection, intracerebroventricular injection, intrathecal), topically e.g., epi cutaneous, inhalational, via eye drops, or through a mucous membrane), or by direct injection into a target organ (e.g., the brain of a subject). Typically, the oligonucleotide or composition is administered intravenously or subcutaneously. In some embodiments, the oligonucleotide or composition is administered to the cerebral spinal fluid. In some embodiments, the oligonucleotide or composition is administered intrathecally. In some embodiments, the oligonucleotide or composition is administered intracerebroventricularly. In some embodiments, the oligonucleotide or composition is administered by intraci sternal magna injection.

[0273] As a non-limiting set of examples, the oligonucleotide would typically be administered quarterly (once every three months), bi-monthly (once every two months), monthly, or weekly. For example, the oligonucleotide may be administered every week or at intervals of two, or three weeks. Alternatively, the oligonucleotide may be administered daily. In some embodiments, a subject is administered one or more loading doses of the oligonucleotide followed by one or more maintenance doses of the oligonucleotide.

[0274] In some embodiments, the subject to be treated is a human or non-human primate or other mammalian subject. Other exemplary subjects include domesticated animals such as dogs and cats; livestock such as horses, cattle, pigs, sheep, goats, and chickens; and animals such as mice, rats, guinea pigs, and hamsters. Kits

[0275] In some embodiments, a kit is provided comprising an oligonucleotide described herein (e.g., a RNAi oligonucleotide), and instructions for its use. In some embodiments, the kit comprises the oligonucleotide, and a package insert containing instructions for use of the kit and/or any component thereof In some embodiments, the kit comprises, in a suitable container, the oligonucleotide, one or more controls, and various buffers, reagents, enzymes, and other standard ingredients well known in the art. In some embodiments, the container comprises at least one vial, well, test tube, flask, bottle, syringe, or other container means, into which the oligonucleotide is placed, and in some instances, suitably aliquoted. In some embodiments where an additional component is provided, the kit contains additional containers into which this component is placed. The kits can also include a means for containing the oligonucleotide and any other reagent in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained. Containers and/or kits can include labeling with instructions for use and/or warnings.

[0276] In some embodiments, the kit comprises the oligonucleotide and a pharmaceutically acceptable carrier, or the pharmaceutical composition, and instructions for treating or delaying progression of a disease, disorder, or condition associated with SNCA gene expression in a subject in need thereof.

[0277] In some embodiments, the kit comprises the oligonucleotide and a pharmaceutically acceptable carrier, or the pharmaceutical composition, and instructions for administering the oligonucleotide or pharmaceutical composition to the cerebral spinal fluid to reduce SNCA gene expression in at least one region of the brain and/or at least one region of the spinal cord in a subject in need thereof.

Definitions

[0278] As used herein, “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0279] As used herein, “administer,” “administering,” “administration” and the like refer to providing a substance (e.g., an oligonucleotide) to a subject in a manner that is pharmacologically useful (e.g., to treat a disease, disorder, or condition in the subject).

[0280] As used herein, “asialoglycoprotein receptor” or “ASGPR” refers to a bipartite C-type lectin formed by a major 48 kDa subunit (ASGPR- 1) and minor 40 kDa subunit (ASGPR-2). ASGPR is primarily expressed on the sinusoidal surface of hepatocyte cells and has a major role in binding, internalizing, and subsequent clearing of circulating glycoproteins that contain terminal galactose or GalNAc residues (asialoglycoproteins).

[0281] As used herein, “attenuate,” “attenuating,” “attenuation” and the like refer to reducing or effectively halting. As a non-limiting example, one or more of the treatments herein may reduce or effectively halt the onset or progression of a disease, disorder, or condition associated with SNCA gene expression in a subject. This attenuation may be exemplified by, for example, a decrease in one or more aspects (e.g., symptoms, tissue characteristics, and cellular, inflammatory, or immunological activity, etc.) of a disease associated with SNCA gene expression, no detectable progression (worsening) of one or more aspects of the disease, disorder, or condition, or no detectable aspects of the disease in a subject when they might otherwise be expected.

[0282] As used herein, “complementary” refers to a structural relationship between two nucleotides (e.g., on two opposing nucleic acids or on opposing regions of a single nucleic acid strand) that permits the two nucleotides to form base pairs with one another. For example, a purine nucleotide of one nucleic acid that is complementary to a pyrimidine nucleotide of an opposing nucleic acid may base pair together by forming hydrogen bonds with one another. In some embodiments, complementary nucleotides can base pair in the Watson-Crick manner or in any other manner that allows for the formation of stable duplexes. In some embodiments, two nucleic acids may have regions of multiple nucleotides that are complementary with each other to form regions of complementarity, as described herein.

[0283] As used herein, “deoxyribonucleotide” refers to a nucleotide having a hydrogen in place of a hydroxyl at the 2' position of its pentose sugar when compared with a ribonucleotide. A modified deoxyribonucleotide is a deoxyribonucleotide having one or more modifications or substitutions of atoms other than at the 2' position, including modifications or substitutions in or of the sugar, phosphate group or base.

[0284] As used herein, “double-stranded oligonucleotide” or “ds oligonucleotide” refers to an oligonucleotide that is substantially in a duplex form. In some embodiments, the complementary base-pairing of duplex region(s) of a ds oligonucleotide is formed between antiparallel sequences of nucleotides of covalently separate nucleic acid strands. In some embodiments, complementary base-pairing of duplex region(s) of a ds oligonucleotide is formed between antiparallel sequences of nucleotides of nucleic acid strands that are covalently linked. In some embodiments, complementary base-pairing of duplex region(s) of a ds oligonucleotide is formed from single nucleic acid strand that is folded (e.g, via a hairpin) to provide complementary antiparallel sequences of nucleotides that base pair together. In some embodiments, a ds oligonucleotide comprises two covalently separate nucleic acid strands that are fully duplexed with one another. However, in other embodiments, a ds oligonucleotide comprises two covalently separate nucleic acid strands that are partially duplexed (e.g, having overhangs at one or both ends). In some embodiments, a ds oligonucleotide comprises antiparallel sequence of nucleotides that are partially complementary, and thus, may have one or more mismatches, which may include internal mismatches or end mismatches.

[0285] As used herein, “duplex,” in reference to nucleic acids (e.g, oligonucleotides), refers to a structure formed through complementary base pairing of two antiparallel sequences of nucleotides.

[0286] As used herein, “excipient” refers to a non-therapeutic agent that may be included in a composition, for example, to provide or contribute to a desired consistency or stabilizing effect. [0287] As used herein, “labile linker” refers to a linker that can be cleaved (e.g., by acidic pH). A “fairly stable linker” refers to a linker that cannot be cleaved.

[0288] As used herein, “loop” refers to a unpaired region of a nucleic acid (e.g., oligonucleotide) that is flanked by two antiparallel regions of the nucleic acid that are sufficiently complementary to one another, such that under appropriate hybridization conditions (e.g., in a phosphate buffer, in a cells), the two antiparallel regions, which flank the unpaired region, hybridize to form a duplex (referred to as a “stem”).

[0289] As used herein, “modified internucleotide linkage” refers to an internucleotide linkage having one or more chemical modifications when compared with a reference internucleotide linkage comprising a phosphodiester bond. In some embodiments, a modified nucleotide is a non- naturally occurring linkage. Typically, a modified internucleotide linkage confers one or more desirable properties to a nucleic acid in which the modified internucleotide linkage is present. For example, a modified nucleotide may improve thermal stability, resistance to degradation, nuclease resistance, solubility, bioavailability, bioactivity, reduced immunogenicity, etc.

[0290] As used herein, “modified nucleotide” refers to a nucleotide having one or more chemical modifications when compared with a corresponding reference nucleotide selected from: adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, adenine deoxyribonucleotide, guanine deoxyribonucleotide, cytosine deoxyribonucleotide, and thymidine deoxyribonucleotide. In some embodiments, a modified nucleotide is a non-naturally occurring nucleotide. In some embodiments, a modified nucleotide has one or more chemical modification in its sugar, nucleobase, and/or phosphate group. In some embodiments, a modified nucleotide has one or more chemical moieties conjugated to a corresponding reference nucleotide. Typically, a modified nucleotide confers one or more desirable properties to a nucleic acid in which the modified nucleotide is present. For example, a modified nucleotide may improve thermal stability, resistance to degradation, nuclease resistance, solubility, bioavailability, bioactivity, reduced immunogenicity, etc.

[0291] As used herein, “nicked tetraloop structure” or “nicked tetraL structure” refers to a structure of an oligonucleotide (e.g., a RNAi oligonucleotide) that is characterized by separate sense (passenger) and antisense (guide) strands, in which the sense strand has a region of complementarity with the antisense strand, and in which at least one of the strands, generally the sense strand, has a tetraL configured to stabilize an adjacent stem region formed within the at least one strand.

[0292] As used herein, “oligonucleotide” refers to a short nucleic acid (e.g., less than about 100 nucleotides in length). An oligonucleotide may be ss or ds. An oligonucleotide may or may not have duplex regions. As a set of non-limiting examples, an oligonucleotide may be, but is not limited to, a small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), dicer substrate interfering RNA (dsiRNA), antisense oligonucleotide (ASO), short siRNA, or ss siRNA. In some embodiments, the oligonucleotide is a ds oligonucleotide and is an RNAi oligonucleotide.

[0293] As used herein, “overhang” refers to terminal non-base pairing nucleotide(s) resulting from one strand or region extending beyond the terminus of a complementary strand with which the one strand or region forms a duplex. Tn some embodiments, an overhang comprises one or more unpaired nucleotides extending from a duplex region at the 5' terminus or 3' terminus of a ds oligonucleotide. In certain embodiments, the overhang is a 3' or 5' overhang on the antisense strand or sense strand of a ds oligonucleotides.

[0294] As used herein, “phosphate analog” refers to a chemical moiety that mimics the electrostatic and/or steric properties of a phosphate group. In some embodiments, a phosphate analog is positioned at the 5' terminal nucleotide of an oligonucleotide in place of a 5'-phosphate, which is often susceptible to enzymatic removal. In some embodiments, a 5' phosphate analog contains a phosphatase-resistant linkage Examples of phosphate analogs include, but are not limited to, 5' phosphonates, such as 5' methylenephosphonate (5'-MP) and 5 '-(E)- vinylphosphonate (5'-VP). In some embodiments, an oligonucleotide has a phosphate analog at a 4'-carbon position of the sugar (referred to as a “4'-phosphate analog”) at a 5 '-terminal nucleotide. An example of a 4'-phosphate analog is oxymethylphosphonate, in which the oxygen atom of the oxymethyl group is bound to the sugar moiety (e.g, at its 4'-carbon) or analog thereof. See, e.g., US Provisional Patent Application Nos. 62/383,207 (fded on 2 September 2016) and 62/393,401 (fded on 12 September 2016). Other modifications have been developed for the 5' end of oligonucleotides (see, e.g., Inti. Patent Application No. WO 2011/133871; US Patent No. 8,927,513; and Prakash et al. (2015) Nucleic Acids Res. 43:2993-3011).

[0295] As used herein, “SNCA” refers to Synyclein Alpha. SNCA is found abundantly in the brain, and inhibits phospholipase D2. It functions in resynaptic signaling and membrane trafficking. The mRNA encoding wild-type human SNCA is set forth in SEQ ID NO: 1677. The mRNA encoding mouse SNCA is set forth in SEQ ID NO: 1678. The mRNA encoding monkey SNCA is set forth in SEQ ID NO: 1679.

[0296] As used herein, “reduced expression” of a gene (e.g, SNCA) refers to a decrease in the amount or level of RNA transcript (e.g, SNCA mRNA) or protein encoded by the gene and/or a decrease in the amount or level of activity/function of the gene and/or protein in a cell, a population of cells, a sample, or a subject, when compared to an appropriate reference (e.g., a reference cell, population of cells, sample, or subject). For example, the act of contacting a cell with an oligonucleotide (e.g, an oligonucleotide such as a RNAi oligonucleotide comprising an antisense strand having a nucleotide sequence that is complementary to a nucleotide sequence comprising SNCA mRNA) may result in a decrease in the amount or level of SNCA mRNA, SNCA protein and/or SNCA activity/function (e.g, via inactivation and/or degradation of SNCA mRNA by the RNAi pathway) when compared to a cell that is not treated with the oligonucleotide. Similarly, and as used herein, “reducing expression” refers to an act that results in reduced expression of a gene (e.g, SNCA).

[0297] As used herein, “reduction of SNCA gene expression” refers to a decrease in the amount or level of SNCA mRNA, SNCA protein and/or SNCA activity/function in a cell, a population of cells, a sample, or a subject when compared to an appropriate reference (e.g., a reference cell, population of cells, sample, or subject).

[0298] As used herein, “region of complementarity” refers to a sequence of nucleotides of a nucleic acid (e.g, a ds oligonucleotide) that is sufficiently complementary to an antiparallel sequence of nucleotides to permit hybridization between the two sequences of nucleotides under appropriate hybridization conditions (e.g, in a phosphate buffer, in a cell, etc.). In some embodiments, the oligonucleotide comprises a targeting sequence having a region of complementary to a mRNA target sequence.

[0299] As used herein, “ribonucleotide” refers to a nucleotide having a ribose as its pentose sugar, which contains a hydroxyl group at its 2' position. A “modified ribonucleotide” refers to a ribonucleotide having one or more modifications or substitutions of atoms other than at the 2' position, including modifications or substitutions in or of the ribose, phosphate group, or base. [0300] As used herein, “RNAi oligonucleotide” refers to either (a) a ds oligonucleotide having a sense strand and an antisense strand in which the antisense strand or part of the antisense strand is used by the Argonaute 2 (Ago2) endonuclease in the cleavage of a target mRNA (e.g., SNCA mRNA) or (b) a ss oligonucleotide having a single antisense strand, where that antisense strand (or part of that antisense strand) is used by the Ago2 endonuclease in the cleavage of a target mRNA (e.g., SNCA mRNA).

[0301] As used herein, “strand” refers to a single, contiguous sequence of nucleotides linked together through intemucleotide linkages (e.g., phosphodi ester linkages or phosphorothioate linkages). In some embodiments, a strand has two free ends (e.g., a 5' end and a 3' end).

[0302] As used herein, “subject” means any mammal, including mice, rabbits, and humans. In one embodiment, the subject is a human orNHP. Moreover, “individual” or “patient” may be used interchangeably with “subject.”

[0303] As used herein, “synthetic” refers to a nucleic acid or other molecule that is artificially synthesized (e.g., using a machine such as, for example, a solid-state nucleic acid synthesizer) or that is otherwise not derived from a natural source (e.g. , a cell or organism) that normally produces the molecule.

[0304] As used herein, “targeting ligand” refers to a molecule (e.g. , a carbohydrate, amino sugar, cholesterol, or polypeptide) that selectively binds to a cognate molecule (e.g. , a receptor) of a tissue or cell of interest and that is conjugatable to another substance for purposes of targeting the other substance to the tissue or cell of interest. For example, in some embodiments, a targeting ligand may be conjugated to an oligonucleotide for purposes of targeting the oligonucleotide to a specific tissue or cell of interest. In some embodiments, a targeting ligand selectively binds to a cell surface receptor. Accordingly, in some embodiments, a targeting ligand when conjugated to an oligonucleotide facilitates delivery of the oligonucleotide into a particular cell through selective binding to a receptor expressed on the surface of the cell and endosomal internalization by the cell of the complex comprising the oligonucleotide, targeting ligand, and receptor. In some embodiments, a targeting ligand is conjugated to an oligonucleotide via a linker that is cleaved following or during cellular internalization such that the oligonucleotide is released from the targeting ligand in the cell. [0305] As used herein, “tetraloop” or “tetraL” refers to a loop that increases stability of an adjacent duplex formed by hybridization of flanking sequences of nucleotides. The increase in stability is detectable as an increase in melting temperature (T m ) of an adjacent stem duplex that is higher than the T m of the adjacent stem duplex expected, on average, from a set of loops of comparable length consisting of randomly selected sequences of nucleotides. For example, a tetraL can confer a T m of at least about 50°C, at least about 55°C, at least about 56°C, at least about 58°C, at least about 60°C, at least about 65°C, or at least about 75°C in 10 mM NaHPCL to a hairpin comprising a duplex of at least 2 base pairs (bp) in length. Tn some embodiments, a tetraL may stabilize a bp in an adjacent stem duplex by stacking interactions. In addition, interactions among the nucleotides in a tetraL include, but are not limited to, non-Watson-Crick base pairing, stacking interactions, hydrogen bonding, and contact interactions (Cheong et al. (1990) Nature 346:680- 682; and Heus & Pardi (1991) Science 253: 191-94). In some embodiments, a tetraL comprises or consists of 3 to 6 nucleotides and is typically 4 to 5 nucleotides. In certain embodiments, a tetraL comprises or consists of 3, 4, 5, or 6 nucleotides, which may or may not be modified (e.g., which may or may not be conjugated to a targeting moiety). In certain embodiments, a tetraL comprises or consists of 3, 4, 5, or 6 nucleotides, which may or may not be modified (e.g., which may or may not be conjugated to a targeting ligand). In one embodiment, a tetraL consists of 4 nucleotides. Any nucleotide may be used in the tetraL and standard IUPAC-IUB symbols for such nucleotides may be used as described in Cornish-Bowden (1985) Nucleic Acids Res. 13:3021-30. For example, the letter “N” may be used to mean that any base may be in that position, the letter “R” may be used to show that A (adenine) or G (guanine) may be in that position, and “B” may be used to show that C (cytosine), G (guanine), T (thymine) or U (uracil) may be in that position. Examples of tetraloops include the UNCG family of tetraloops (e.g., UUCG), the GNRA family of tetraloops (e.g., GAAA), and the CUUG tetraloop (Woese et al. (1990) Proc. Natl. Acad. Sci. USA 87:8467- 71; and Antao etal. (1991) Nucleic Acids Res. 19:5901-05). Examples of DNA tetraloops include the d(GNNA) family of tetraloops (e.g., d(GTTA), the d(GNRA)) family of tetraloops, the d(GNAB) family of tetraloops, the d(CNNG) family of tetraloops, and the d(TNCG) family of tetraloops (e.g., d(TTCG)). See, e.g., Nakano et al. (2002) Biochem. 41 :4281-92; Shinji et al. (2000) Nippon Kagakkai Koen Yokoshu 78:731. In some embodiments, the tetraL is contained within a nicked tetraL structure.

[0306] As used herein, “treat” or “treating” refers to the act of providing care to a subject in need thereof, for example, by administering a therapeutic agent (e.g., an oligonucleotide herein such as a RNAi oligonucletoide) to the subject, for purposes of improving the health and/or well-being of the subject with respect to an existing condition (e.g., a disease, disorder) or to prevent or decrease the likelihood of the occurrence of a condition. In some embodiments, treatment involves reducing the frequency or severity of at least one sign, symptom, or contributing factor of a condition (e.g., disease, disorder) experienced by the subject.

EXAMPLES

Example 1: Preparation of RNAi Oligonucleotides

Oligonucleotide Synthesis and Purification

[0307] The oligonucleotides (i.e., RNAi oligonucleotides) described in the foregoing Examples are chemically synthesized using methods described herein. Generally, RNAi oligonucleotides are synthesized using solid phase oligonucleotide synthesis methods as described for 19-23mer siRNAs (see, e.g. , Scaringe etal. (1990) Nucleic Acids Res. 18:5433-5441 and Usman etal. (1987) J. Am. Chem. Soc. 109:7845-45; see also, US Patent Nos. 5,804,683; 5,831,071; 5,998,203; 6,008,400; 6,111,086; 6,117,657; 6,353,098; 6,362,323; 6,437,117 and 6,469,158) in addition to using known phosphoramidite synthesis (see, e.g., Hughes & Ellington (2017) Cold Spring Harb Perspect Biol. 9(l):a023812; Beaucage & Caruthers (1981) Tetrahedron Lett. 22:1859-62). dsRNAi oligonucleotides with a 19mer core sequence were formatted into constructs having a 25mer sense strand and a 27mer antisense strand to allow for processing by the RNAi machinery. The 19mer core sequence is complementary to a region in the SNCA mRNA.

[0308] Individual RNA strands were synthesized and HPLC purified according to standard methods (Integrated DNA Technologies; Coralville, IA). For example, RNA oligonucleotides were synthesized using solid phase phosphoramidite chemistry, deprotected, and desalted on NAP - 5 columns (Amersham Pharmacia Biotech; Piscataway, NJ) using standard techniques (Damha & Olgivie (1993) Methods Mol. Biol. 20:81-114; Wincott et al. (1995) Nucleic Acids Res. 23'.26T1- 84). The oligomers were purified using ion-exchange high performance liquid chromatography (IE-HPLC) on an Amersham Source 15Q column (1.0 cm><25 cm; Amersham Pharmacia Biotech) using a 15 min step-linear gradient. The gradient varied from 90: 10 Buffers A:B to 52:48 Buffers A:B, where Buffer A is 100 mM Tris pH 8.5 and Buffer B is 100 mM Tris pH 8.5, 1 M NaCl. Samples were monitored at 260 nm, and peaks corresponding to the full-length oligonucleotide species were collected, pooled, desalted on NAP-5 columns, and lyophilized.

[0309] The purity of each oligomer was determined by capillary electrophoresis (CE) on a Beckman PACE 5000 (Beckman Coulter, Inc.; Fullerton, CA). The CE capillaries have a 100 pm inner diameter and contain ssDNA 100R Gel (Beckman-Coulter). Typically, about 0.6 nmole of oligonucleotide was injected into a capillary, run in an electric field of 444 V/cm, and detected by UV absorbance at 260 nm. Denaturing Tris-Borate-7 M-urea running buffer was purchased from Beckman-Coulter. Oligoribonucleotides were obtained that were at least 90% pure as assessed by CE for use in experiments described below. Compound identity was verified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectroscopy on a Voyager DE™ Biospectometry Work Station (Applied Biosystems; Foster City, CA) following the manufacturer's recommended protocol. Relative molecular masses of all oligomers were obtained, often within 0.2% of expected molecular mass.

Preparation of Duplexes

[0310] ss RNA oligomers were resuspended ( .g., at 100 pM concentration) in duplex buffer consisting of 100 mM potassium acetate, 30 mM HEPES, pH 7.5. Complementary sense and antisense strands were mixed in equal molar amounts to yield a final solution of, for example, 50 pM duplex. Samples were heated to 100°C for 5 min in RNA buffer (IDT) and were allowed to cool to room temperature before use. The RNAi oligonucleotides were stored at -20° C. ss RNA oligomers were stored lyophilized or in nuclease-free water at -80° C. Example 2: Generation of SAC l-Targeting RNAi Oligonucleotides

[0311] SNCA encodes SNCA, a neuronal protein that inhibits phospholipase D2. SNCA participates in the regulation of synaptic vesicle trafficking and neurotransmitter release. Abnormal expression of SNCA can lead to various diseases of the brain, including but not limited to Parkinson disease and multiple system atrophy. Oligonucleotides capable of inhibiting SNCA mRNA expression were identified and generated.

Identification of SNCA mRNA Target Sequences

To generate SNCA -targeting RNAi oligonucleotides, a computer-based algorithm was used to computationally identify SNCA mRNA target sequences suitable for assaying inhibition of SNCA expression by the RNAi pathway. The algorithm provided RNAi oligonucleotide antisense strand sequences each having a region of complementarity to a suitable SNCA mRNA target sequence of human (Hs) or murine (Mm) mRNA (e.g., SEQ ID NOs: 1677 and 1678, respectively; Table 1). Due to sequence conservation across species, some of the SNCA mRNA target sequences identified for human SNCA mRNA are homologous to the corresponding SNCA mRNA target sequence of murine (mM) SNCA mRNA (SEQ ID NO: 1678; Table 1; i.e., double-common) and/or monkey (Mf) SNCA mRNA (SEQ ID NO: 1679; Table 1; i.e., triple-common). SNCA- targeting RNAi oligonucleotides comprising a region of complementarity to homologous SNCA mRNA target sequences with nucleotide sequence similarity are predicted to have the ability to target homologous SNCA mRNAs (e.g., human SNCA and monkey SNCA mRNAs).

[0312] Table 1: Exemplary Human SNCA, Monkey SNCA, and Mouse SNCA mRNA Sequences.

[0313] RNAi oligonucleotides (formatted as DsiRNA oligonucleotides) were generated as described in Example 1 for evaluation in vitro. Each DsiRNA was generated with the same modification pattern, and each with a unique guide strand having a region of complementarity to a SNCA target sequence identified by the algorithm. Modifications for the sense and anti- sense DsiRNA included the following (X- any nucleotide; m- 2’-0Me modified nucleotide; r- ribosyl modified nucleotide):

Sense Strand:

5’ rXmXrXmXrXrXrXrXrXrXrXrXrXmXrXmXrXrXrXrXrXrXrXXX 3’

Anti-sense Strand:

3’ mXmXmXrXmXrXrXrXrXrXrXrXrXrXmXrXmXrXrXrXrXrXrXmXmXmXmX 5’

In Vitro Cell-Based Assays

[0314] The ability of each of the modified DsiRNA in Table 2 to reduce SNCA mRNA was measured using in vitro cell-based assays. Briefly, human Huh7 cells (hepatocyte cell line) expressing endogenous human SNCA were transfected with each of the DsiRNAs listed in Table 2 at 0.5 nM (Phase 1) or select DsiRNAs at 0.5 nM, 0.1 nM, and 0.02 nM (Phase 2) in separate wells of a multi-well cell-culture plate. Cells were maintained for 24 hours following transfection with the modified DsiRNA, and then the amount of remaining SNCA mRNA from the transfected cells was determined using a TAQMAN®-based qPCR assay using the following primers: Forward: AGG GTG TTC TCT ATG TAG GCT (SEQ ID NO: 2451); Reverse: ACT GCT CCT CCA ACA TTT GTC (SEQ ID NO: 2452); Probe:

TGCTCTTTG/ZEN/GTCTTCTCAGCCACTG (SEQ ID NO:2453). Primer pairs were assayed for % remaining mRNA as shown in Table 2. DsiRNAs resulting in less than or equal to 13% SNCA mRNA remaining in DsiRNA-transfected cells when compared to mock-transfected cells were considered DsiRNA “hits”. The Huh7 cell-based assay evaluating the ability of the DsiRNAs listed in Table 2 to inhibit SNCA gene expression identified several candidate DsiRNAs.

[0315] Taken together, these results show that DsiRNAs designed to target human SNCA mRNA inhibit SNCA gene expression in cells, as determined by a reduced amount of SNCA mRNA in DsiRNA-transfected cells relative to control cells. These results demonstrate that the nucleotide sequences comprising the DsiRNA are useful for generating RNAi oligonucleotides to inhibit SNCA gene expression. Further, these results demonstrate that multiple SNCA mRNA target sequences are suitable for the RNAi-mediated inhibition of SNCA gene expression.

[0316] Table 2: In Vitro Screening Results.

Example 3: GalNAc-Conjugated SAC l- I argeting RNAi Oligonucleotides Inhibit Human SNCA mRNA Expression In Vivo

[0317] The in vitro screening assays in Example 2 validated the ability of NVC.4 -targeting RNAi oligonucleotides to knock-down target SNCA mRNA. To further evaluate the ability of SNCA- targeting RNAi oligonucleotides to inhibit SNCA mRNA expression, GalN Ac-conjugated SNCA- targeting RNAi oligonucleotides were generated to confirm knockdown in vivo.

[0318] Specifically, a subset of the DsiRNAs identified in Example 2 were used to generate corresponding ds RNAi oligonucleotides comprising a nicked tetraL GalN Ac-conjugated structure (referred to herein as “GalNAc-conjugated SNCA oligonucleotides” or “GalNAc-SVCN oligonucleotides”) having a 36-mer passenger strand and a 22-mer guide strand (Tables 4 and 5). Further, the nucleotide sequences comprising the sense strand and the antisense strand have a distinct pattern of modified nucleotides and phosphorothioate linkages. Three of the nucleotides comprising the tetraL were each conjugated to a GalNAc moiety (CAS#14131-60-3). The modification patterns are illustrated below:

Sense Strand: 5’-mX-X-mX-fX-mX-fX-mX-mX-fX-mX-fX-mX-fX-fX-mX-fX-mX- fX-mX-mX-mX-mX-mX-mX-mX-mX-mX-mX-[ademX-GalNAc][ademX-GalNAc ][ademX- GalNAc]-mX-mX-mX-mX-mX-mX-3 ’ hybridized to:

Antisense Strand: 5 ’-MePhosphonate-4O-mX-5-fX-5-fX-fX-fX-mX-fX-mX-mX-fX- mX-mX-mX-fX-mX-fX-mX-mX-fX-mX-5-mX-X-mX-3’ (Modification key: Table 3).

Or, represented as:

Sense Strand: 5'-[mXs][mX][fX][mX][fX][mX][mX][fX][mX][fX][mX]

[fX] [fX] [mX] [IX] [mX] [fX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [ademX- GalNAc] [ademX-GalNAc] [ademX-GalNAc] [mX] [mX] [mX] [mX] [mX] [mX]-3 ’ hybridized to:

Antisense Strand : 5 ’ -[MePhosphonate-4O-mXs] [fXs] [fX] [fX] [fX] [mX] [fX] [mX] [mX] [fX] [mX] [mX][mX] [fX] [mX] [fX] [mX] [mX] [fX] [mXs] [mXs] [mX]-3 ’ (Modification key : Table 3)

[0319] Table 3: Key for Modification Patterns.

[0320] The GalN Ac-conjugated SNCA -targeting oligonucleotides were used in a hydrodynamic injection (HDI) mouse model to confirm the ability of the RNAi oligonucleotides to knockdown SNCA gene expression in vivo. The GalNAc-conjugated SNCA -targeting oligonucleotides listed in Tables 4 and 5 were evaluated in mice engineered to transiently express human SNCA mRNA in hepatocytes of the mouse liver. Briefly, 6-8-week-old female CD-I mice were subcutaneously administered the indicated GalNAc-conjugated SNCA -targeting oligonucleotides at a dose of 3 mg/kg formulated in PBS. A control group of mice (n = 5) were administered only PBS. Three days later (72 hours), the mice were HDI with a DNA plasmid encoding the full human SNCA (SEQ ID NO: 1677) (10 pg) under control of a ubiquitous cytomegalovirus (CMV) promoter sequence. One day after introduction of the DNA plasmid, liver samples from HDI mice were collected. Total RNA derived from these HDI mice were subjected to qRT-PCR analysis to determine human SNCA mRNA levels as described in Example 2. The values were normalized for transfection efficiency using the NeoR gene included on the DNA plasmid. Benchmark control SNCA-291 was used to confirm successful knock-down.

[0321] Table 4: GalNAc-Conjugated Human SNCA RNAi Oligonucleotides for HDI screen.

[0322] Table 5: GalNAc-Conjugated Human SNCA RNAi Oligonucleotides for HDI screen.

[0323] The results in FIGs. 1A and IB demonstrate that GalNAc-conjugated SNCA oligonucleotides (as shown in Tables 4 and 5, respectively) designed to target human SNCA mRNA successfully inhibited human SNCA mRNA expression in HDI mice, as determined by a reduction in the amount of human SNCA mRNA expression in liver samples from HDI mice treated with GalNAc-conjugated SNCA oligonucleotides relative to control HDI mice treated with only PBS.

Example 4: GalNAc-Conjugated SNCA RNAi Oligonucleotides Inhibit Human SNCA Gene Expression in A Dose-Dependent Manner

[0324] To further evaluate the ability of GalNAc-conjugated SNCA RNAi oligonucleotides to inhibit SNCA expression a dose response study was carried out. Specifically, in separate treatment groups, selected GalNAc-conjugated SNCA RNAi oligonucleotides (Tables 6 and 7) were formulated in PBS and administered to CD-I mice at doses of 0.3 mg/kg, 1 mg/kg, or 3 mg/kg subcutaneously. As described in Example 3, a human SNCA DNA expression plasmid was administered to the mice 3 days post-oligonucleotide dosing, and livers were collected 24 hours later for qRT-PCR analysis. As shown in FTGs. 2A and 2B, all of the GalNAc-conjugated SNCA RNAi oligonucleotides tested inhibited human SNCA gene expression in a dose-dependent manner. Potent GalNAc-conjugated SNCA oligonucleotides (i.e., SNCA-244, SNCA-429, SNCA- 751, SNCA-752, SNCA-800, SNCA-801, and SNCA1003) reduced SNCA mRNA by about 50% or more at 1 mg/kg and even further at 3 mg/kg. Select constructs were chosen for further studies in non-human primates.

[0325] Table 6: GalNAc-Conjugated Human SNCA RNAi Oligonucleotides for Dose Screen.

[0326] Table 7: GalNAc-Conjugated Human SNCA RNAi Oligonucleotides for Dose Screen. Example 5: RNAi Oligonucleotide Inhibition of SNCA Gene Expression in NHP CNS

[0327] Effective GalN Ac-conjugated SNC IA -targeting oligonucleotides identified in the HDI mouse studies were assayed for inhibition in NHPs. Specifically, GalN Ac-conjugated SNCA- targeting oligonucleotides listed in Table 8 were evaluated in non-naive cynomolgus monkeys (Macaca fascicular is). Each cohort contained 2 male and 2 female subjects. The GalNAc- conjugated SNCA -targeting oligonucleotides were administered at a dose of 50 mg in 1.6 mL of artificial cerebrospinal fluid (aCSF) on study days 0 and 7 via intra cisterna magna (i.c.m.) injection.

[0328] Table 8. GalN Ac-Conjugated SNCA RNAi Oligonucleotides for NHP Study.

[0329] On study day 14, CNS tissue was collected and subjected to qRT-PCR analysis to measure SNCA mRNA in oligonucleotide-treated monkeys relative to those treated with a comparable volume of aCSF. To normalize the data, the measurements were made relative to 2 reference genes, RPL23 and GAPDH (the geometric mean between the two was used as the set point for on target KD). The following SYBR assays purchased from Integrated DNA Technologies were used to evaluate gene expressions: Forward: ACAGTGG

CTGAGAAGACCAA (SEQ ID NO: 2454), Reverse: CTCCCTCCACTGTCTTCTGG (SEQ ID NO: 2455); and Probe: ACCCGTCACCACCGCTCCTCC (SEQ ID NO: 2456).

[0330] As shown in FIGs. 3A-3S (Day 14), treating NHPs with the GalNAc-conjugated SNCA- targeting oligonucleotides inhibited SNCA gene expression in several regions of the CNS, as determined by a reduced amount of SNCA mRNA in brain samples from oligonucleotide-treated NHPs relative to NHPs treated with aCSF. Several GalNAc-conjugated SNCA -targeting oligonucleotides reduced SNCA gene expression throughout the CNS. SNCA-801 and SNCA-751 were particularly potent and reduced SNCA mRNA by at least 50% in the frontal cortex, hippocampus, parietal cortex, occipital cortex, temporal cortex, brain stem, cervical spinal cord, thoracic spinal cord, and lumbar spinal cord. These results demonstrate that treating NHPs with the GalNAc-conjugated SNCA -targeting oligonucleotides reduces the amount of SNCA mRNA in the CNS.

Example 6: Lipid-Conjugated RNAi Oligonucleotide Targeting SNCA Reduces Gene Expression in NHP CNS

[0331] To further investigate the efficacy of oligonucleotides targeting SNCA, a lipid-conjugated oligonucleotide was assessed in NHP. Specifically, SNCA-0751 was selected based on the above studies, and the sense strand was formatted as a 20mer sense strand with a lipid conjugated to the 5’ terminal nucleotide. This construct is referred to as SNCA-B15, having a 20mer sense strand and 22mer antisense strand (SEQ ID NOs: 1682 and 1656, respectively). The chemical modification pattern of the lipid-conjugated oligonucleotide is provided below:

Sense Strand: 5’-[ademX-Ci6]-5-mX-fX-mX-fX-mX-mX-fX-mX-fX-mX-fX-fX-mX- fX-mX-fX-mX-5-mX-5-mX-3 ’ hybridized to:

Antisense Strand: 5’-[MePhosphonate-4O-mX]-5-fX-S-fX-fX-fX-mX-fX-mX-mX- fX-mX- X-mX-fX- X-fX- X- X-fX-rriX-S'-rriX-S'-rriX 3 ’ (Modification key: Table 3).

Or, represented as:

Sense Strand : 5 ’ - [ademXs-C 16 ] [mX] [fX] [mX] [fX] [mX] [mX] [fX] [mX] [fX] [mX] [fX] [fX] [mX] [fX] [mX] [fX] [mXs] [mXs] [mX]-3 ’ hybridized to:

Antisense Strand: 5 ’ -[MePhosphonate-4O-mXs] [fXs] [fX] [fX] [fX] [mX] [fX] [mX] [mX] [fX] [mX] [mX][mX] [IX] [mX] [fX] [mX] [mX] [fX] [mXs] [mXs] [mX]-3 ’ (Modification key : Table 3)

Lipid Conjugation [0332] Lipid-conjugated blunt-ended oligonucleotides were synthesized using a standard procedure known in the literature for oligo synthesis on a synthesizer using amidite chemistry (see, Matteucci & Caruthers (1981) Tetrahedron Lett. 2C719-22 1 ; Beaucage & Caruthers (1981) Tetrahedron Lett. 22: 1859-62 2 ).

'Matteucci MD, Caruthers MH. The synthesis of oligodeoxypyrimidines on a polymer support. Tetrahedron Lett. 1980;21(8):719-722.

2 Beaucage SL, Caruthers MH. Deoxynucleoside phosphoramidites-A new class of key intermediates for deoxypolynucleotides. Tetrahedron Lett. 1981;22(20): 1859-1862

[0333] Conjugation of a lipid moiety to the SNCA -targeting oligonucleotide was carried out using phosphoramidite synthesis as shown below:

[0334] Synthesis of 2-(2-((((6aR,8R9R,9aR)-8-(6-benzamido-9H-purin-9-yl)-2,2,4,4 - tetraisopropyltetrahydro-6H-furo[3, 2-f][L 3, 5, 2, 4]trioxadisilocin-9-yl)oxy)methoxy)ethoxy) [0335] A solution of compound 1-1 (25.00 g, 67.38 mmol) in 20 mL of DMF was treated with pyridine (11 mL, 134.67 mmol) and tetraisopropyldisiloxane dichloride (22.63 mL, 70.75 mmol) at 10 °C. The resulting mixture was stirred at 25 °C for 3 hr and quenched with 20% citric acid (50 mL). The aqueous layer was extracted with EtOAc (3X50 mL), and the combined organic layers were concentrated in vacuo. The crude residue was recrystallized from a mixture of MTBE and n- heptane (1 :15, 320 mL) to afford compound 1-2 (37.20 g, 90%) as a white oily solid.

[0336] A solution of compound 1-2 (37.00 g, 60.33 mmol) in 20 mL of DMSO was treated with AcOH (20 mL, 317.20 mmol) and Ac O (15 mL, 156.68 mmol). The mixture was stirred at 25 °C for 15 hr. The reaction was diluted with EtOAc (100 mL) and quenched with sat. K2CO3 (50 mL). The aqueous layer was extracted with EtOAc (3X50 mL). The combined organic layers were concentrated and recrystallized with ACN (30 mL) to afford compound 1-3 (15.65 g, 38.4%) as a white solid.

[0337] A solution of compound 1-3 (20.00 g, 29.72 mmol) in 120 mL of DCM was treated with Fmoc-amino-ethoxy ethanol (11.67 g, 35.66 mmol) at 25 °C. The mixture was stirred to afford a clear solution and then treated with 4A molecular sieves (20.0 g), A-iodosuccinimide (8.02 g, 35.66 mmol), and TfOH (5.25 mL, 59.44 mmol). The mixture was stirred at 30 °C until the HPLC analysis indicated >95% consumption of compound 1-3. The reaction was quenched with TEA (6 mL) and filtered. The filtrate was diluted with EtOAc, washed with sat. NaHCCh (2X100 mL), sat. Na2SO3 (2X100 mL), and water (2X100 mL) and concentrated in vacuo to afford crude compound 1-4 (26.34 g, 93.9%) as a yellow solid, which was used directly for the next step without further purification.

[0338] A solution of compound 1-4 (26.34 g, 27.62 mmol) in a mixture of DCM/water (10:7, 170 mL) was treated with DBU (7.00 mL, 45.08 mmol) at 5 °C. The mixture was stirred at 5-25 °C for 1 hr. The organic layer was then separated, washed with water (100 mL), and diluted with DCM (130 mL). The solution was treated with fumaric acid (7.05 g, 60.76 mmol) and 4A molecular sieves (26.34 g) in 4 portions. The mixture was stirred for 1 hr, concentrated, and recrystallized from a mixture of MTBE and DCM (5:1) to afford compound 1-6 (14.74 g, 62.9%) as a white solid: 'H NMR (400 MHz, tL-DMSO) 8.73 (s, 1H), 8.58 (s, 1H), 8.15-8.02 (m, 2H), 7.65-7.60 (m, 1H), 7.59-7.51 (m, 2H), 6.52 (s, 2H), 6.15(s, 1H), 5.08-4.90 (m, 3H), 4.83-4.78 (m, 1H), 4.15-3.90 (m, 3H), 3.79-3.65 (m, 2H), 2.98-2.85 (m, 6H), 1.20-0.95 (m, 28H).

[0339] Synthesis of ( 2R, 3R, 4R, 5R)-5-( 6-benzamido-9H-purin-9-yl)-2-( t'bisf 4- methoxyphenyl)(phenyl)methoxy)methyl)-4-((2-(2-[lipid]-amido ethoxy)ethoxy)methoxy)

[0340] A solution of compound 1-6 (50.00 g, 59.01 mmol) in 150 mL of 2- methyltetrahydrofuran was washed with ice cold aqueous K2HPO4 (6%, 100 mL) and brine (20%, 2X100 mL). The organic layer was separated and treated with hexanoic acid (10.33 mL, 82.61 mmol), HATU (33.66 g, 88.52 mmol), and DMAP (10.81 g, 147.52 mmol) at 0 °C. The resulting mixture was warmed to 25 °C and stirred for 1 hr. The solution was washed with water (2X100 mL), brine (100 mL), and concentrated in vacuo to afford a crude residue. Flash chromatography on silica gel (1 :1 hexanes/acetone) gave compound 2-la (34.95 g, 71.5%) as a white solid. [0341] A mixture of compound 2-la (34.95 g, 42.19 mmol) and TEA (9.28 mL, 126.58 mmol) in 80 mL of THF was treated with triethylamine trihydrofluoride (20.61 mL, 126.58 mmol) dropwise at 10 °C. The mixture was warmed to 25 °C and stirred for 2 hr. The reaction was concentrated, dissolved in DCM (100 mL), and washed with sat. NaHCOs (5X20 mL) and brine (50 mL). The organic layer was concentrated in vacuo to afford crude compound 2-2a (24.72 g, 99%), which was used directly for the next step without further purification.

[0342] A solution of compound 2-2a (24.72 g, 42.18 mmol) in 50 mL of DCM was treated with N-m ethyl morpholine (18.54 mL, 168.67 mmol) andDMTr-Cl (15.69 g, 46.38 mmol). The mixture was stirred at 25 °C for 2 hr and quenched with sat. NaHCCL (50 mL). The organic layer was separated, washed with water, and concentrated to afford a slurry crude. Flash chromatography on silica gel (1: 1 hexanes/acetone) gave compound 2-3a (30.05 g, 33.8 mmol, 79.9%) as a white solid. [0343] A solution of compound 2-3a (25.00 g, 28.17 mmol) in 50 mL of DCM was treated with A-methylmorpholine (3.10 mL, 28.17 mmol) and tetrazole (0.67 mL, 14.09 mmol) under nitrogen atmosphere. Bis(diisopropylamino) chlorophosphine (9.02 g, 33.80 mmol) was added to the solution dropwise and the resulting mixture was stirred at 25 °C for 4 hr. The reaction was quenched with water (15 mL), and the aqueous layer was extracted with DCM (3X50 mL). The combined organic layers were washed with sat. NaHCCh (50 mL), concentrated to afford a crude solid that was recrystallized from a mixture of DCM/MTBE/n-hexane (1 :4:40) to afford compound 2-4a (25.52 g, 83.4%) as a white solid: 'H NMR (400 MHz, tL-DMSO) 11.25 (s, 1H), 8.65-8.60 (m, 2 H), 8.09-8.02 (m, 2H), 7.71 (s, 1H), 7.67-7.60 (m, 1H), 7.59-7.51 (m, 2H), 7.38-7.34 (m, 2H), 7.30-7.25 (m, 7H), 6.85-6.79 (m, 4H), 6.23-6.20 (m, 1H), 5.23-5.14 (m, 1H), 4. 80-4.69 (m, 3H), 4.33-4.23 (m, 2H), 3.90-3.78 (m, 1H), 3.75 (s, 6H), 3.74-3.52 (m, 3H), 3.50-3.20 (m, 6H), 3.14-3.09 (m, 2H), 3.09 (s, 1H), 2.82-2.80 (m, 1H), 2.65-2.60 (m, 1H), 2.05-1.96 (m, 2H), 1.50- 1.39 (m, 2H), 1.31-1.10 (m, 14H), 1.08-1.05 (m, 2 H), 0.85-0.79 (m, 3H); 31 P NMR (162 MHz, MSO) 149.43, 149.18.

[0344] Compound 2-4b, 2-4c, 2-4d, and 2-4e were prepared using similar procedures described above for compound 2-4a. Compound 2-4b was obtained (25.50 g, 85.4%) as a white solid: 'H NMR (400 MHz, tL-DMSO) 11.23 (s, 1H), 8.65-8.60 (m, 2 H), 8.05-8.02 (m, 2H), 7.73-7.70 (m, 1H), 7.67-7.60 (m, 1H), 7.59-7.51 (m, 2H), 7.38-7.34 (m, 2H), 7.30-7.25 (m, 7H), 6.89-6.80 (m, 4H), 6.21-6.15 (m, 1H), 5.23-5.17 (m, 1H), 4. 80-4.69 (m, 3H), 4.40-4.21 (m, 2H), 3.91-3.80 (m, 1H), 3.74 (s, 6H), 3.74-3.52 (m, 3H), 3.50-3.20 (m, 6H), 3.14-3.09 (m, 2H), 3.09 (s, 1H), 2.83- 2.79 (m, 1H), 2.68-2.62 (m, 1H), 2.05-1.97 (m, 2H), 1.50-1.38 (m, 2H), 1.31-1.10 (m, 18H), 1.08- 1.05 (m, 2H), 0.85-0.78 (m, 3H); 31 P NMR (162 MHz, t/g-DMSO) 149.43, 149.19.

[0345J Compound 2-4c was obtained (36.60 g, 66.3%) as an off-white solid: 'H NMR (400 MHz, ^-DMSO) 11.22 (s, 1H), 8.64-8.59 (m, 2H), 8.05-8.00 (m, 2H), 7.73-7.70 (m, 1H), 7.67- 7.60 (m, 1H), 7.59-7.51 (m, 2H), 7.38-7.34 (m, 2H), 7.30-7.25 (m, 7H), 6.89-6.80 (m, 4H), 6.21- 6.15 (m, 1H), 5.25-5.17 (m, 1H), 4.80-4.69 (m, 3H), 4.40-4.21 (m, 2H), 3.91 -3.80 (m, 1H), 3.74 (s, 6H), 3.74-3.50 (m, 3H), 3.50-3.20 (m, 6H), 3.14-3.09 (m, 2H), 3.09 (s, 1H), 2.83-2.79 (m, 1H), 2.68-2.62 (m, 1H), 2.05-1.99 (m, 2H), 1.50-1.38 (m, 2H), 1.33-1.12 (m, 38H), 1.08-1.05 (m, 2 H), 0.86-0.80 (m, 3H); 31 P NMR (162 MHz, ^-DMSO) 149.42, 149.17.

[0346] Compound 2-4d was obtained (26.60 g, 72.9%) as an off-white solid: 'll NMR (400 MHz, tfc-DMSO) 11.22 (s, 1H), 8.64-8.59 (m, 2H), 8.05-8.00 (m, 2H), 7.73-7.70 (m, 1H), 7.67- 7.60 (m, 1H), 7.59-7.51 (m, 2H), 7.38-7.33 (m, 2H), 7.30-7.25 (m, 7H), 6.89-6.80 (m, 4H), 6.21- 6.15 (m, 1H), 5.22-5.17 (m, 1H), 4.80-4.69 (m, 3H), 4.40-4.21 (m, 2H), 3.91-3.80 (m, 1H), 3.74 (s, 6H), 3.74-3.52 (m, 3H), 3.50-3.20 (m, 6H), 3.14-3.09 (m, 2H), 3.09 (s, 1H), 2.83-2.79 (m, 1H), 2.68-2.62 (m, 1H), 2.05-1.99 (m, 2H), 1.50-1.38 (m, 2H), 1.35-1.08 (m, 38H), 1.08-1.05 (m, 2 H), 0.85-0.79 (m, 3H); 31 P NMR (162 MHz, t/ 6 -DMSO) 149.47, 149.22.

[0347] Compound 2-4e was obtained (38.10 g, 54.0%) as a white solid: 'H NMR (400 MHz, OMSO) 11.21 (s, 1H), 8.64-8.59 (m, 2H), 8.05-8.00 (m, 2H), 7.73-7.70 (m, 1H), 7.67-7.60 (m, 1H), 7.59-7.51 (m, 2H), 7.38-7.34 (m, 2H), 7.30-7.25 (m, 7H), 6.89-6.80 (m, 4H), 6.21-6.15 (m, 1H), 5.23-5.17 (m, 1H), 4.80-4.69 (m, 3H), 4.40-4.21 (m, 2H), 3.91-3.80 (m, 1H), 3.73 (s, 6H), 3.74-3.52 (m, 3H), 3.47-3.22 (m, 6H), 3.14-3.09 (m, 2H), 3.09 (s, 1H), 2.83-2.79 (m, 1H), 2.68- 2.62 (m, 1H), 2.05-1.99 (m, 2H), 1.50-1.38 (m, 2H), 1.35-1.06 (m, 46H), 1.08-1.06 (m, 2 H), 0.85- 0.77 (m, 3H); 31 P NMR (162 MHz, tfc-DMSO) 149.41, 149.15.

NHP study

[0348] NHPs (n=4) were intrathecally administered 37.5 mg lipid-conjugated SNCA-B15 via lumbar infusion at LI (see Table 9). aCSF was used as a control. [0349] Table 9. Lipid-Conjugated SNCA -Targeting RNAi Oligonucleotide for NHP Study

[0350] 28 days after administration, CNS tissue was collected to determine the concentration of the oligonucleotide and the SCNA gene expression level. Parkinson’s Disease (PD) is a movement disorder characterized by tremors, slowness of movement, stiff muscles, unsteady walk, and/or loss of balance, whereas Multiple Systems Atrophy (MSA) is a rare disorder affecting autonomic functions such as blood pressure, breathing and bladder control, and motor function. Accordingly, CNS tissues associated with PD or MSA were analyzed separately.

[0351] As shown in FIG. 4A, SNCA gene expression was reduced in tissues associated with AD, including the putamen, midbrain tegmentum, substantia nigra, pons, and medulla, with the lipid- conjugated SNCA-B15. SNCA gene expression was determined as described in the above Examples. FIG. 4B shows concentration of lipid-conjugated SNCA-B15 in the tissues after 28 days. These results indicate lipid-conjugated SNCA -targeting oligonucleotides have enhanced potency across tissues associated with PD.

[0352] Likewise, and as shown in FIG. 5A, SNCA gene expression was reduced in tissues associated with MSA, including the putamen, midbrain tegmentum, substantia nigra, pons, cerebellar white matter, medulla, cervical spinal cord, thoracic spinal cord, and lumbar spinal cord with lipid-conjugated SNCA- Bl 5. SNCA gene expression was determined as described in the above Examples. FIG. 5B shows the concentration of lipid-conjugated SNCA-B15 in the tissues after 28 days. These results indicate lipid-conjugated SNCA -targeting oligonucleotides have enhanced potency across tissues associated with MSA.

SEQUENCE LISTING

[0353] The following nucleic and/or amino acid sequences are referred to in the disclosure and are provided below for reference.