Compositions and methods for inhibiting SNCA expression

Abstract

Provided herein are oligonucleotides (e.g., RNAi oligonucleotides, including oligonucleotides conjugated to targeting ligands (e.g., GalNAC moieties or lipid moieties) that inhibit expression of SNCA genes. Compositions comprising the oligonucleotides and uses thereof are also provided, in particular to uses in treating diseases, disorders and / or conditions associated with the expression of SNCA genes.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63 / 364,639, filed on May 13, 2022, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure relates generally to biology and medicine, and more particularly to oligonucleotides and compositions comprising the oligonucleotides for inhibiting or reducing (i.e., regulating) α-synuclein (SNCA) gene expression, and their use for treating diseases and disorders associated with SNCA gene expression. Background Art

[0004] Synapses are functional compartments between cells where information is transmitted from one cell in the brain to another. SNCA is a protein found primarily in the brain that regulates synaptic vesicle trafficking and the release of neurotransmitters. Among other functions, SNCA acts as a molecular chaperone to help fold synaptic fusion components (e.g., SNAREs). Mutations in SNCA that alter SNCA function and expression or general abnormal expression of SNCA (e.g., insertions and mispairings) are known causes of several diseases that affect the CNS (e.g., Parkinson's disease and multiple system atrophy). Strategies that target the SNCA gene to prevent such diseases are needed.

[0005] The mammalian CNS is a complex system of tissues (including cells), fluids and chemicals that synergistically interact to achieve multiple functions, including motion, navigation, cognition, language, vision and emotion. Unfortunately, it is known that there are multiple CNS diseases and disorders (e.g., neurological disorders) and some or all of these functions are affected or destroyed. Typically, the treatment of CNS diseases and disorders is limited to small molecule drugs, antibodies and / or adaptive therapy or behavioral therapy. There is a continued need to develop the treatment of CNS diseases and disorders related to inappropriate gene expression. Summary of the invention

[0006] To meet this need, the present disclosure describes compositions and methods for treating diseases, disorders or conditions associated with SNCA gene expression. The present disclosure is based, at least in part, on the discovery of RNAi oligonucleotides that effectively target and reduce SNCA gene expression in CNS tissues. Specifically, target sequences within SNCA mRNA are identified, and oligonucleotides are generated that bind to these target sequences and inhibit SNCA mRNA expression. As demonstrated herein, the oligonucleotides inhibit human and non-human primate (NHP) SNCA gene expression in CNS tissues. In addition, SNCA mRNA expression is reduced using lipid-conjugated SNCA targeting oligonucleotides in CNS tissues associated with Parkinson's disease or multiple system atrophy. Without being bound by theory, the oligonucleotides described herein can be used to treat diseases, disorders or conditions associated with SNCA gene expression.

[0007] Thus, in some aspects, the present disclosure provides an 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 complementary region to the SNCA mRNA target sequence of any one of SEQ ID NOs: 1683-2066, and wherein the length of the complementary region is at least about 15 consecutive nucleotides.

[0008] In any of the foregoing or related aspects, the length of the sense strand is about 15 to about 50 nucleotides. In some aspects, the length of the sense strand is about 18 to about 36 nucleotides. In some aspects, the length of the antisense strand is about 15 to about 30 nucleotides. In some aspects, the length of the antisense strand is 22 nucleotides, and the antisense strand and the sense strand form a duplex region, the length of the duplex region is at least 19 nucleotides, optionally at least 20 nucleotides in length. In some aspects, the length of the complementary region is at least 19 consecutive nucleotides. In some aspects, the length of the complementary region is at least 20 consecutive nucleotides.

[0009] In other aspects, the present disclosure provides a double-stranded (ds) RNAi oligonucleotide for reducing SNCA gene expression, the oligonucleotide comprising:

[0010] (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand comprises a nucleotide sequence comprising a complementary region to a SNCA mRNA target sequence, wherein the complementary region is selected from the group consisting of SEQ ID NOs: 2067-2450, and

[0011] (ii) a sense strand of 19-50 nucleotides in length, the sense strand comprising a complementary region to the antisense strand, wherein the antisense strand and the sense strand are independent strands forming an asymmetric duplex region having an overhang of 1-4 nucleotides at the 3' end of the antisense strand.

[0012] In some aspects, the 3' end of the sense strand comprises a stem loop as shown in S1-L-S2, wherein S1 is complementary to S2, and wherein L forms a loop of 3-5 nucleotides in length between S1 and S2. In some aspects, L is a tricyclic or tetracyclic ring. In some aspects, L is a tetracyclic ring. In some aspects, the tetracyclic ring comprises the sequence 5'-GAAA-3'. In some aspects, the length of S1 and S2 is 1-10 nucleotides and has the same length. In some aspects, the length of S1 and S2 is 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides or 10 nucleotides. In some aspects, the length of S1 and S2 is 6 nucleotides. In some aspects, the stem loop comprises the sequence 5'-GCAGCCGAAAGGCUGC-3' (SEQID NO: 1680).

[0013] In other aspects, the oligonucleotide comprises 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.

[0014] 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 a purine nucleotide. 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.

[0015] 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 selected from 2'-aminoethyl, 2'-fluoro (2'-F), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl and 2'-deoxy-2'-fluoro-β-d-arabinose nucleic acid. In some aspects, the modification is a 2'-modification selected from 2'-F and 2'-OMe. 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 from 5' to 3' at positions 1-36, wherein each of positions 3, 5, 8, 10, 12, 13, 15, and 17 comprises a 2'-F modification. In some aspects, the sense strand comprises 20 nucleotides from 5' to 3' at positions 1-20, wherein each of positions 3, 5, 8, 10, 12, 13, 15, and 17 comprises a 2'-F modification. In some aspects, the antisense strand comprises 22 nucleotides from 5' to 3' at positions 1-22, and wherein each of positions 2, 3, 4, 5, 7, 10, 14, 16, and 19 comprises a 2'-F modification. In some aspects, the remaining nucleotides comprise a 2'-OMe modification.

[0016] In any of the foregoing or related aspects, the oligonucleotide comprises at least one modified internucleotide bond. In some aspects, at least one modified internucleotide bond is a phosphorothioate bond. In some aspects, the antisense strand comprises a phosphorothioate bond at the following positions: (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 bond 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 bond 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 phosphorothioate linkages 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'.

[0017] 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 an oxymethylphosphonate, a vinylphosphonate, or a malonylphosphonate, optionally wherein the phosphate analog is a 4'-phosphate analog comprising a 4'-oxymethylphosphonate.

[0018] In any one of the foregoing or related aspects, at least one nucleotide of the oligonucleotide is conjugated to one or more targeting ligands. In some aspects, each targeting ligand comprises a carbohydrate, an amino sugar, a lipid, cholesterol or a 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, one or more targeting ligands are conjugated to one or more nucleotides of the ring. In some aspects, the ring comprises 4 nucleotides numbered 1-4 from 5' to 3', wherein the nucleotides at positions 2, 3 and 4 each comprise one or more targeting ligands, wherein the targeting ligands are identical or different. In some aspects, each targeting ligand comprises N-acetylgalactosamine (GalNAc) moiety. In some aspects, the GalNAc moiety is a monovalent GalNAc moiety, a divalent GalNAc moiety, a trivalent GalNAc moiety or a tetravalent GalNAc moiety. In some aspects, up to 4 nucleotides of the L of the stem loop are each conjugated to a monovalent GalNAc moiety.

[0019] In other aspects, one or more targeting ligands are lipid moieties. 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 C8-C30 hydrocarbon chain. In some aspects, the hydrocarbon chain is a C16 hydrocarbon chain. In some aspects, the C16 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.

[0020] 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 the nucleotide positions are numbered from 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 the nucleotide positions are numbered from 5' to 3'.

[0021] In any of the foregoing or related aspects, the sense strand comprises the nucleotide sequence of any one of SEQ ID NOs: 1537-1571 and 1681. In some aspects, the antisense strand comprises the nucleotide sequence of any one of SEQ ID NOs: 1572-1606. In some aspects, the sense strand and the antisense strand comprise a nucleotide sequence selected from the group consisting of:

[0022] a) SEQ ID NO: 1537 and 1572, respectively;

[0023] b) SEQ ID NOs: 1538 and 1573, respectively;

[0024] c) SEQ ID NOs: 1539 and 1574, respectively;

[0025] d) SEQ ID NOs: 1540 and 1575, respectively;

[0026] e) SEQ ID NOs: 1541 and 1576, respectively;

[0027] f) SEQ ID NO: 1542 and 1577, respectively;

[0028] g) SEQ ID NOs: 1543 and 1578, respectively;

[0029] h) SEQ ID NOs: 1544 and 1579, respectively;

[0030] i) SEQ ID NOs: 1545 and 1580, respectively;

[0031] j) SEQ ID NOs: 1546 and 1581, respectively;

[0032] k) SEQ ID NOs: 1547 and 1582, respectively;

[0033] 1) are SEQ ID NOs: 1548 and 1583, respectively;

[0034] m) are SEQ ID NOs: 1549 and 1584, respectively;

[0035] n) are SEQ ID NOs: 1550 and 1585, respectively;

[0036] o) are SEQ ID NOs: 1551 and 1586, respectively;

[0037] p) are SEQ ID NOs: 1552 and 1587, respectively;

[0038] q) are SEQ ID NOs: 1553 and 1588, respectively;

[0039] r) are SEQ ID NOs: 1554 and 1589, respectively;

[0040] s) are SEQ ID NOs: 1555 and 1590, respectively;

[0041] t) are SEQ ID NOs: 1556 and 1591, respectively;

[0042] u) are SEQ ID NOs: 1557 and 1592, respectively;

[0043] v) SEQ ID NOs: 1558 and 1593, respectively;

[0044] w) are SEQ ID NOs: 1559 and 1594, respectively;

[0045] x) are SEQ ID NOs: 1560 and 1595, respectively;

[0046] y) are SEQ ID NOs: 1561 and 1596, respectively;

[0047] z) are SEQ ID NOs: 1562 and 1597, respectively;

[0048] aa) are SEQ ID NOs: 1563 and 1598, respectively;

[0049] bb) are SEQ ID NOs: 1564 and 1599, respectively;

[0050] cc) are SEQ ID NOs: 1565 and 1600, respectively;

[0051] dd) are SEQ ID NOs: 1566 and 1601, respectively;

[0052] ee) are SEQ ID NOs: 1567 and 1602, respectively;

[0053] ff) are SEQ ID NOs: 1568 and 1603, respectively;

[0054] gg) are SEQ ID NOs: 1569 and 1604, respectively;

[0055] hh) are SEQ ID NOs: 1570 and 1605, respectively;

[0056] ii) SEQ ID NO: 1571 and 1606, respectively; and

[0057] jj) are SEQ ID NOs: 1681 and 1586, respectively.

[0058] In some aspects, the sense strand and the antisense strand comprise a nucleotide sequence selected from the group consisting of:

[0059] a) SEQ ID NO: 1540 and 1575, respectively;

[0060] b) SEQ ID NOs: 1544 and 1579, respectively;

[0061] c) SEQ ID NOs: 1546 and 1581, respectively;

[0062] d) SEQ ID NOs: 1551 and 1586, respectively;

[0063] e) SEQ ID NOs: 1552 and 1587, respectively;

[0064] f) SEQ ID NOs: 1553 and 1588, respectively;

[0065] g) SEQ ID NOs: 1558 and 1594, respectively;

[0066] h) SEQ ID NOs: 1560 and 1595, respectively;

[0067] i) SEQ ID NOs: 1564 and 1599, respectively;

[0068] j) are SEQ ID NOs: 1565 and 1600, respectively;

[0069] k) are SEQ ID NOs: 1566 and 1601, respectively;

[0070] 1) are SEQ ID NOs: 1570 and 1605, respectively; and

[0071] m) are SEQ ID NOs: 1681 and 1586, respectively.

[0072] In some aspects, the sense strand and the antisense strand comprise a nucleotide sequence selected from the group consisting of:

[0073] a) SEQ ID NO: 1553 and 1588, respectively;

[0074] b) SEQ ID NOs: 1560 and 1595, respectively;

[0075] c) SEQ ID NOs: 1564 and 1599, respectively;

[0076] d) SEQ ID NOs: 1551 and 1586, respectively;

[0077] e) SEQ ID NOs: 1570 and 1605, respectively; and

[0078] f) are SEQ ID NOs: 1681 and 1586, respectively.

[0079] In some aspects, the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 1553, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 1588. In some aspects, the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 1560, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 1595. In some aspects, the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 1564, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 1599. In some aspects, the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 1551, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 1586. In some aspects, the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 1570, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 1605. In some aspects, the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 1681, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 1586.

[0080] 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 a nucleotide sequence selected from the group consisting of 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 a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1865, 1721, 1847, 1846, and 1955. In some aspects, the sense strand comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1553, 1560, 1564, 1551, and 1570.

[0081] 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 the nucleotide sequence of any one of SEQ ID NOs: 1642-1676.

[0082] In some aspects, the sense strand and the antisense strand comprise a nucleotide sequence selected from the group consisting of:

[0083] a) SEQ ID NO: 1607 and 1642, respectively;

[0084] b) SEQ ID NO: 1608 and 1643, respectively;

[0085] c) SEQ ID NOs: 1609 and 1644, respectively;

[0086] d) SEQ ID NO: 1610 and 1645, respectively;

[0087] e) SEQ ID NOs: 1611 and 1646, respectively;

[0088] f) SEQ ID NO: 1612 and 1647, respectively;

[0089] g) SEQ ID NO: 1613 and 1648, respectively;

[0090] h) SEQ ID NOs: 1614 and 1649, respectively;

[0091] i) SEQ ID NOs: 1615 and 1650, respectively;

[0092] j) SEQ ID NOs: 1616 and 1651, respectively;

[0093] k) are SEQ ID NOs: 1617 and 1652, respectively;

[0094] 1) are SEQ ID NOs: 1618 and 1653, respectively;

[0095] m) are SEQ ID NOs: 1619 and 1654, respectively;

[0096] n) are SEQ ID NOs: 1620 and 1655, respectively;

[0097] o) are SEQ ID NOs: 1621 and 1656, respectively;

[0098] p) are SEQ ID NOs: 1622 and 1657, respectively;

[0099] q) are SEQ ID NOs: 1623 and 1658, respectively;

[0100] r) are SEQ ID NOs: 1624 and 1659, respectively;

[0101] s) are SEQ ID NOs: 1625 and 1660, respectively;

[0102] t) are SEQ ID NOs: 1626 and 1661, respectively;

[0103] u) are SEQ ID NOs: 1627 and 1662, respectively;

[0104] v) SEQ ID NOs: 1628 and 1663, respectively;

[0105] w) are SEQ ID NOs: 1629 and 1664, respectively;

[0106] x) are SEQ ID NOs: 1630 and 1665, respectively;

[0107] y) are SEQ ID NOs: 1631 and 1666, respectively;

[0108] z) are SEQ ID NOs: 1632 and 1667, respectively;

[0109] aa) are SEQ ID NOs: 1633 and 1668, respectively;

[0110] bb) are SEQ ID NOs: 1634 and 1669, respectively;

[0111] cc) are SEQ ID NOs: 1635 and 1670, respectively;

[0112] dd) are SEQ ID NOs: 1636 and 1671, respectively;

[0113] ee) are SEQ ID NOs: 1637 and 1672, respectively;

[0114] ff) are SEQ ID NOs: 1638 and 1673, respectively;

[0115] gg) are SEQ ID NOs: 1639 and 1674, respectively;

[0116] hh) are SEQ ID NOs: 1640 and 1675, respectively;

[0117] ii) SEQ ID NO: 1641 and 1676, respectively; and,

[0118] jj) are SEQ ID NOs: 1682 and 1656, respectively.

[0119] In some aspects, the sense strand and the antisense strand comprise a nucleotide sequence selected from the group consisting of:

[0120] a) SEQ ID NO: 1610 and 1645, respectively;

[0121] b) SEQ ID NOs: 1614 and 1649, respectively;

[0122] c) SEQ ID NOs: 1616 and 1651, respectively;

[0123] d) SEQ ID NO: 1621 and 1656, respectively;

[0124] e) SEQ ID NOs: 1622 and 1657, respectively;

[0125] f) SEQ ID NOs: 1623 and 1658, respectively;

[0126] g) SEQ ID NOs: 1629 and 1664, respectively;

[0127] h) SEQ ID NOs: 1630 and 1665, respectively;

[0128] i) SEQ ID NOs: 1634 and 1669, respectively;

[0129] j) SEQ ID NOs: 1635 and 1670, respectively;

[0130] k) are SEQ ID NOs: 1636 and 1671, respectively;

[0131] 1) are SEQ ID NOs: 1640 and 1675, respectively; and

[0132] m) are SEQ ID NOs: 1682 and 1656, respectively.

[0133] In some aspects, the sense strand and the antisense strand comprise a nucleotide sequence selected from the group consisting of:

[0134] a) SEQ ID NO: 1623 and 1658, respectively;

[0135] b) SEQ ID NOs: 1630 and 1665, respectively;

[0136] c) SEQ ID NOs: 1634 and 1669, respectively;

[0137] d) SEQ ID NO: 1621 and 1656, respectively;

[0138] e) SEQ ID NOs: 1640 and 1675, respectively; and

[0139] f) are SEQ ID NOs: 1682 and 1656, respectively.

[0140] In some aspects, the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 1623, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 1658. In some aspects, the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 1630, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 1665. In some aspects, the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 1634, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 1669. In some aspects, the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 1621, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 1656. In some aspects, the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 1640, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 1676. In some aspects, the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 1682, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 1656.

[0141] In some aspects, the sense strand comprises 5'-[mCs][mA][fG][mC][fA][mG][mU][fG][mA][fU][mU][fG][fA][mA][fG][mU][fA][mU][mC][mA][mG][mC][mC][mG][ademA-GalNAc][ademA-GalNAc][ademA-GalNAc][mG][mC][mU][mG][mC]-3' (SEQ ID NO:1623) and all modifications, and wherein the antisense strand comprises the sequence of 5'-[Me phosphonate-4O-mUs][fGs][fA][fU][fA][mC][fU][mU][mC][fA][mA][mU][mC][fA][mC][fU][mG][mC][fU][mGs][mGs][mG]-3' (SEQ ID NO:1658) and all modifications, wherein mC, mA, mG, mU=2'-OMe ribonucleoside; fA, fC, fG, fU=2'-F ribonucleoside; s=thiophosphate, and wherein ademA-GalNAc=

[0142]

[0143] In some aspects, the sense strand comprises 5'-[mAs][mG][fA][mG][fC][mA][mA][fG][mU][fG][mA][fC][fA][mA][fA][mU][fG][mU][mU][mA][mG][mC][mA][mG][mC][mC][mG][ademA-GalNAc][ademA-GalNAc][mG][mG][mC][mU][mG][mC]-3' (SEQ ID NO:1630) and all modifications, and wherein the antisense strand comprises the sequence of 5'-[Me phosphonate-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 all modifications, wherein mC, mA, mG, mU=2'-OMe ribonucleoside; fA, fC, fG, fU=2'-F ribonucleoside; s=thiophosphate, and wherein ademA-GalNAc=

[0144]

[0145] In some aspects, the sense strand comprises 5'-[mAs][mG][fU][mC][fA][mU][mG][fA][mC][fA][mU][fU][fU][mC][fU][mC][fA][mA][mA][mG][mC][mA][mG][mC][mC][mG][ademA-GalNAc][ademA-GalNAc][ademA-GalNAc][mG][mC][mU][mG][mC]-3' (SEQ ID NO:1634) and all modifications, and wherein the antisense strand comprises the sequence of 5'-[Me phosphonate-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 all modifications, wherein mC, mA, mG, mU=2'-OMe ribonucleoside; fA, fC, fG, fU=2'-F ribonucleoside; s=thiophosphate, and wherein ademA-GalNAc=

[0146]

[0147] In some aspects, the sense strand comprises 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][ademA-GalNAc][ademA-GalNAc][ademA-GalNAc][mG][mC][mU][mG][mC]-3' (SEQ ID NO:1621) and all modifications, and wherein the antisense strand comprises the sequence of 5'-[Me phosphonate-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 all modifications, wherein mC, mA, mG, mU=2'-OMe ribonucleoside; fA, fC, fG, fU=2'-F ribonucleoside; s=thiophosphate, and wherein ademA-GalNAc=

[0148]

[0149] In some aspects, the sense strand comprises 5'-[mAs][mG][fU][mU][fG][mU][mU][fA][mG][fU][mG][fA][fU][mU][fU][mG][fC][mU][mA][mA][mG][mC][mA][mG][mC][mC][mG][ademA-GalNAc][ademA-GalNAc][ademA-GalNAc][mG][mG][mC][mU][mG][mC]-3' (SEQ ID NO:1640) and all modifications, and wherein the antisense strand comprises the sequence of 5'-[Me phosphonate-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 all modifications, wherein mC, mA, mG, mU=2'-OMe ribonucleoside; fA, fC, fG, fU=2'-F ribonucleoside; s=thiophosphate, and wherein ademA-GalNAc=

[0150]

[0151] In some aspects, the sense strand comprises the sequence of 5'-[ademCs-C16][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 all modifications, and wherein the antisense strand comprises 5'-[Me phosphonate-4O-mUs][fUs][fU][fG][fA][mG][fA][mA][fU][mG][mU][mC][fA][mU][fG][mA][mC][fU][mGs][mGs][mG]-3' (SEQ ID NO: 1684). NO:1656) and all modifications, wherein mC, mA, mG, mU = 2′-OMe ribonucleoside; fA, fC, fG, fU = 2′-F ribonucleoside; s = phosphorothioate, and [ademC s-C16] =

[0152]

[0153] In some aspects, the present disclosure provides a pharmaceutical composition comprising an RNAi oligonucleotide described herein and a pharmaceutically acceptable carrier, delivery agent, or excipient.

[0154] In other aspects, the present 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 an RNAi oligonucleotide described herein or a pharmaceutical composition thereof, thereby treating the subject.

[0155] In other aspects, the present disclosure provides a method of delivering an oligonucleotide to a subject, the method comprising administering to the subject a pharmaceutical composition described herein.

[0156] In other aspects, the present disclosure provides a method for reducing SNCA gene expression in a cell, a cell population, or a subject, the method comprising the steps of:

[0157] i. contacting the cell or the cell population with an RNAi oligonucleotide or a pharmaceutical composition described herein; or

[0158] ii. administering to the subject an RNAi oligonucleotide or pharmaceutical composition described herein.

[0159] In some aspects, reducing SNCA gene expression comprises reducing the amount or level of SNCA mRNA, the amount or level of SNCA protein, or both. In some aspects, the subject suffers from 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 Lewybodies, or Parkinson's disease.

[0160] In any of the foregoing or related aspects, using the RNAi oligonucleotides or pharmaceutical compositions described herein, SNCA gene expression is reduced in tissues of one or more regions of the CNS, wherein the tissues are associated with Parkinson's disease. In some aspects, the tissues associated with Parkinson's disease are selected from the group consisting of putamen, midbrain tegmentum, substantia nigra, pons, and medulla. In some aspects, SNCA gene expression is reduced in tissues of one or more regions of the CNS, wherein the tissues are associated with multiple system atrophy. In some aspects, the tissues associated with multiple system atrophy are selected from the group consisting of caudate nucleus, 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 decreased in one or more regions of the CNS selected from the group consisting of: 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 decreased in one or more regions of the CNS selected from the group consisting of 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, tegmentum, substantia nigra, pons, cerebellar white matter, cerebellar dentate nucleus, L1 dorsal root ganglion (DRG), L2 DRG, L3 DRG, L4 DRG, L5 DRG, L6 DRG, putamen, tegmentum, substantia nigra, pons, medulla, cerebellar cortex, and cerebellar white matter.

[0161] 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.

[0162] In other aspects, the disclosure provides uses of the RNAi oligonucleotides or pharmaceutical compositions described herein for the manufacture of a medicament for treating a disease, disorder or condition associated with SNCA gene expression.

[0163] In other aspects, the disclosure provides an RNAi oligonucleotide or a pharmaceutical composition described herein for use or application in treating a disease, disorder or condition associated with SNCA expression.

[0164] In some aspects, the disclosure provides a kit comprising an RNAi oligonucleotide described herein, optionally a pharmaceutically acceptable carrier, and a package insert comprising instructions for administration to a subject having a disease, disorder, or condition associated with SNCA expression.

[0165] 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's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0166] Figure 1A and Figure 1B A graph depicting the percentage (%) of human SNCA mRNA remaining in the liver of mice exogenously expressing human SNCA (hydrodynamic injection model) after treatment with GalNAc-conjugated SNCA targeting oligonucleotides is provided. CD-1 mice were subcutaneously administered 3 mg / kg of the indicated GalNAc-conjugated SNCA targeting oligonucleotides formulated in PBS. Three days after dosing, mice were hydrodynamically injected (HDI) with a DNA plasmid encoding human SNCA. The level of human SNCA mRNA was determined from the harvested liver 24 hours later. Hs-Mf = constructs are specific for human and monkey SNCA. Hs-Mf-Mm = constructs are specific for human, monkey and mouse SNCA. SNCA-291 was used as a benchmark control.

[0167] Figure 2A and Figure 2B Provides a description based on Figure 1A-1B The inhibitory efficacy of selected GalNAc-conjugated SNCA oligonucleotides is shown in the dose-response graph. Figure 1A-1B The percentage (%) of SNCA mRNA remaining in liver tissue was measured in CD-1 HDI mice as described in . The percentage (%) of mRNA remaining was determined in both groups after injection of 0.3 mg / kg, 1.0 mg / kg, or 3 mg / kg of the indicated GalNAc-conjugated SNCA oligonucleotides, Figure 2A and Figure 2B Hs-Mf = The construct is specific for human and monkey SNCA. Hs-Mf-Mm = The construct is specific for human, monkey and mouse SNCA.

[0168] Figures 3A-3S A graph depicting the percentage (%) of non-human primate (NHP; Mf) SNCA mRNA remaining in the CNS of NHPs after treatment with GalNAc-conjugated SNCA targeting oligonucleotides is provided. On study days 0 and 7, NHPs were dosed by intracisternal (icm) injection of 50 mg of the indicated GalNAc-conjugated SNCA targeting oligonucleotides formulated in artificial cerebrospinal fluid (aCSF). The level of SNCA mRNA was determined relative to the percentage (%) of SNCA mRNA remaining in aCSF-treated animals. CNS tissues measured included the frontal cortex ( Figure 3A ), caudate nucleus ( Figure 3B ), hippocampus( Figure 3C)、midbrain( Figure 3D ), parietal cortex ( Figure 3E ), occipital cortex( Figure 3F ),thalamus( Figure 3G ), temporal cortex ( Figure 3H ), cerebellum( Fig. 3I )、brain stem( Figure 3J )、cervical spinal cord( Figure 3K )、thoracic spinal cord( Figure 3L )、lumbar spinal cord( Figure 3M )、L1 dorsal root ganglion (DRG) ( Figure 3N )、L2 DRG( Fig.3O )、L3 DRG( Figure 3P )、L4 DRG( Figure 3Q )、L5 DRG( Figure 3R ) and L6 DRG( Figure 3S ). GaLXC = GalNAc-conjugated SNCA targeting oligonucleotide.

[0169] Figures 4A-4B Provided are graphs depicting the percentage (%) of non-human primate (NHP; Mf) SNCA mRNA remaining in NHP CNS tissues associated with Parkinson's disease ( Figure 4A ) and the concentration of oligonucleotides ( Figure 4B ). Intrathecal administration of C-conjugated to NHP 16 aCSF or SNCA-B15 lipids. Tissues were collected and analyzed 28 days after oligonucleotide administration.

[0170] Figure 5A-5B Provided are graphs depicting the percentage (%) of non-human primate (NHP; Mf) SNCA mRNA remaining in NHP CNS tissue associated with multiple system atrophy ( Figure 5A ) and the concentration of oligonucleotides ( Figure 5B ). Intrathecal administration of C-conjugated to NHP 16 aCSF or SNCA-B15 lipids. Tissues were collected and analyzed 28 days after oligonucleotide administration. DETAILED DESCRIPTION

[0171] According to some aspects, the present 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 present disclosure provides methods for treating diseases associated with SNCA expression by reducing SNCA gene expression in cells (e.g., cells of the CNS).

[0172] Oligonucleotide inhibitors of SNCA expression

[0173] The present disclosure provides, inter alia, oligonucleotides (eg, RNAi oligonucleotides) that inhibit SNCA gene expression. In some embodiments, the oligonucleotides that inhibit SNCA gene expression target SNCA mRNA.

[0174] SNCA target sequence

[0175] In some embodiments, the oligonucleotides herein (eg, RNAi oligonucleotides) target a target sequence comprising SNCA mRNA. In some embodiments, the oligonucleotides described herein target a target sequence within the SNCA mRNA sequence.

[0176] In some embodiments, the oligonucleotides described herein correspond to a target sequence within the SNCA mRNA sequence. In some embodiments, an oligonucleotide or a portion, fragment or strand thereof (e.g., the antisense strand or guide strand of a ds RNAi oligonucleotide) binds or affixes to a target sequence comprising SNCA mRNA, thereby inhibiting SNCA gene expression.

[0177] In some embodiments, the oligonucleotide targets the SNCA target sequence for the purpose of inhibiting SNCA gene expression in vivo. In some embodiments, the amount or degree to which an oligonucleotide targeting the SNCA target sequence inhibits SNCA gene expression correlates with the efficacy of the oligonucleotide. In some embodiments, the amount or degree to which an oligonucleotide targeting the SNCA target sequence inhibits SNCA gene expression correlates with the amount or degree of therapeutic benefit in a subject or patient suffering from a disease, disorder or condition associated with SNCA gene expression treated with the oligonucleotide.

[0178] By examining the nucleotide sequences of mRNA encoding SNCA, including mRNAs of a number of different species (e.g., humans, cynomolgus monkeys, and mice; 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 susceptible to oligonucleotide-based inhibition than others and can therefore be used as target sequences for the oligonucleotides herein. In some embodiments, the sense strand of an oligonucleotide (e.g., 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 the nucleotide sequence of any one of SEQ ID NOs: 1683-2066. In some embodiments, the SNCA target sequence comprises or consists of the 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 the 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, the SNCA target sequence comprises or consists of the 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.

[0179] SNCA targeting sequence

[0180] In some embodiments, the oligonucleotides herein have a complementary region to SNCA mRNA (e.g., within a target sequence of SNCA mRNA) for the purpose of targeting mRNA in a cell and inhibiting its expression. In some embodiments, the oligonucleotide comprises a SNCA targeting sequence (e.g., the antisense strand or guide strand of a ds oligonucleotide) having a complementary region that binds or affixes to the SNCA target sequence by complementary (Watson-Crick) base pairing. The targeting sequence or complementary region generally has a suitable length and base content to enable the oligonucleotide (or its strand) to bind or affix to SNCA mRNA for the purpose of inhibiting its expression. In some embodiments, the length of the targeting sequence or complementary region 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 some embodiments, the targeting sequence or complementary region 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 length of the targeting sequence or complementary region 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 some embodiments, the length of the targeting sequence or complementary region is about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the length of the targeting sequence or complementary region is 18 nucleotides. In some embodiments, the length of the targeting sequence or complementary region is 19 nucleotides. In some embodiments, the length of the targeting sequence or complementary region is 20 nucleotides. In some embodiments, the length of the targeting sequence or complementary region is 21 nucleotides. In some embodiments, the length of the targeting sequence or complementary region is 22 nucleotides. In some embodiments, the length of the targeting sequence or complementary region is 23 nucleotides. In some embodiments, the length of the targeting sequence or complementary region is 24 nucleotides. In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region complementary to a sequence of any one of SEQ ID NOs: 1683-2066, and the length of the targeting sequence or complementary region is 18 nucleotides.In some embodiments, the oligonucleotide comprises a targeting sequence or a complementary region complementary to a sequence of any one of SEQ ID NO: 1683-2066, and the length of the targeting sequence or the complementary region is 19 nucleotides. In some embodiments, the oligonucleotide comprises a targeting sequence or a complementary region complementary to a sequence of any one of SEQ ID NO: 1-384, and the length of the targeting sequence or the complementary region is 20 nucleotides. In some embodiments, the oligonucleotide comprises a targeting sequence or a complementary region complementary to a sequence of any one of SEQ ID NO: 1-384, and the length of the targeting sequence or the complementary region is 21 nucleotides. In some embodiments, the oligonucleotide comprises a targeting sequence or a complementary region complementary to a sequence of any one of SEQ ID NO: 1-384, and the length of the targeting sequence or the complementary region is 22 nucleotides. In some embodiments, the oligonucleotide comprises a targeting sequence or a complementary region complementary to a sequence of any one of SEQ ID NO: 1-384, and the length of the targeting sequence or the complementary region is 23 nucleotides. In some embodiments, the oligonucleotide comprises a targeting sequence or a complementary region that is complementary to the sequence of any one of SEQ ID NOs: 1-384, and the targeting sequence or the complementary region is 24 nucleotides in length.

[0181] In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region that is fully complementary to a SNCA target sequence (e.g., the antisense strand or guide strand of a ds oligonucleotide). In some embodiments, the targeting sequence or complementary region is partially complementary to a SNCA target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region 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 complementary region 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 a complementary region that is fully complementary to the 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 a complementary region that is complementary to a portion of the 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 a complementary region that is fully complementary to the 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 a complementary region that is partially complementary to the 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 a complementary region that is fully complementary to the sequence of any one of SEQ ID NOs: 1865, 1721, 1847, 1846, and 1955.In some embodiments, the oligonucleotide comprises a targeting sequence or a complementary region 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 a complementary region that is fully complementary to a sequence set forth in SEQ ID NO: 1865. In some embodiments, the oligonucleotide comprises a targeting sequence or a complementary region that is fully complementary to a sequence set forth in SEQ ID NO: 1721. In some embodiments, the oligonucleotide comprises a targeting sequence or a complementary region that is fully complementary to a sequence set forth in SEQ ID NO: 1847. In some embodiments, the oligonucleotide comprises a targeting sequence or a complementary region that is fully complementary to a sequence set forth in SEQ ID NO: 1846. In some embodiments, the oligonucleotide comprises a targeting sequence or a complementary region that is fully complementary to a sequence set forth in SEQ ID NO: 1955. In some embodiments, the oligonucleotide comprises a targeting sequence or a complementary region 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 a complementary region that is partially complementary to a sequence of SEQ ID NO: 1721. In some embodiments, the oligonucleotide comprises a targeting sequence or a complementary region that is partially complementary to the sequence of SEQ ID NO: 1847. In some embodiments, the oligonucleotide comprises a targeting sequence or a complementary region that is partially complementary to the sequence of SEQ ID NO: 1846. In some embodiments, the oligonucleotide comprises a targeting sequence or a complementary region that is partially complementary to the sequence of SEQ ID NO: 1955.

[0182] In some embodiments, the oligonucleotides herein comprise a targeting sequence or a complementary region that is complementary to a contiguous nucleotide sequence comprising SNCA mRNA, wherein the contiguous nucleotide sequence 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 oligonucleotides comprise a targeting sequence or a complementary region that is complementary to a contiguous nucleotide sequence comprising SNCA mRNA, wherein the contiguous nucleotide sequence is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In some embodiments, the oligonucleotides comprise a targeting sequence or a complementary region that is complementary to a contiguous nucleotide sequence comprising SNCA mRNA, wherein the contiguous nucleotide sequence is 19 nucleotides in length. In some embodiments, the oligonucleotide comprises a targeting sequence or a complementary region that is complementary to a contiguous nucleotide sequence comprising SNCA mRNA, wherein the contiguous nucleotide sequence is 20 nucleotides in length.

[0183] In some embodiments, the oligonucleotide comprises a targeting sequence or a complementary region that is complementary to a contiguous nucleotide sequence of any one of SEQ ID NOs: 1683-2066, optionally wherein the contiguous nucleotide sequence is 19 nucleotides in length. In some embodiments, the oligonucleotide comprises a targeting sequence or a complementary region that is complementary to a contiguous 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, optionally wherein the contiguous nucleotide sequence is 19 nucleotides in length. In some embodiments, the oligonucleotide comprises a targeting sequence or a complementary region that is complementary to a contiguous nucleotide sequence of any one of SEQ ID NOs: 1798, 1817, 1718, 1846, 1852, 1865, 1804, 1721, 1847, 1855, 1864, and 1955, optionally wherein the contiguous nucleotide sequence is 19 nucleotides in length. In some embodiments, the oligonucleotide comprises a targeting sequence or a complementary region that is complementary to a contiguous nucleotide sequence of SEQ ID NOs: 1865, 1721, 1847, 1846, and 1955, optionally wherein the contiguous nucleotide sequence is 19 nucleotides in length.

[0184] In some embodiments, the targeting sequence or complementary region of an oligonucleotide (e.g., an RNAi oligonucleotide) is complementary to a continuous nucleotide sequence as shown in any one of SEQ ID NOs: 1683-2066 and spans the entire length of the antisense strand. In some embodiments, the targeting sequence or complementary region of an oligonucleotide is complementary to a continuous nucleotide sequence as shown in any one of SEQ ID NOs: 1683-2066 and spans a portion of the entire length of the antisense strand. In some embodiments, an oligonucleotide comprises a complementary region (e.g., completely) complementary to a continuous nucleotide segment of nucleotides 1-20 spanning a sequence as shown in any one of SEQ ID NOs: 1683-2066 (e.g., on the antisense strand of a ds oligonucleotide). In some embodiments, the targeting sequence or complementary region of an oligonucleotide is complementary to a continuous nucleotide sequence as shown in any one of SEQ ID NOs: 1-384 and spans the entire length of the antisense strand. In some embodiments, the complementary region of an oligonucleotide is complementary to a continuous nucleotide sequence as shown in any one of SEQ ID NOs: 1-384 and spans a portion of the entire length of the antisense strand. In some embodiments, the oligonucleotide comprises a complementary region (eg, on the antisense strand of the ds oligonucleotide) that is at least partially (eg, 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.

[0185] In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region that has one or more base pair (bp) mismatches with the corresponding SNCA target sequence. In some embodiments, the targeting sequence or complementary region 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 complementary region to bind or adhere to 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 complementary region 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 complementary region to bind or adhere to 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 complementary region that has 1 mismatch with the corresponding target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region that has 2 mismatches with the corresponding target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region with 3 mismatches with the corresponding target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region with 4 mismatches with the corresponding target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region with 5 mismatches with the corresponding target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region with 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) mismatches are continuously positioned (e.g., 2, 3, 4, 5 or more mismatches in a row), or wherein mismatches are scattered throughout any position of the entire targeting sequence or complementary region. In some embodiments, an oligonucleotide comprises a targeting sequence or complementary region that has more than one mismatch (e.g., 2, 3, 4, 5 or more mismatches) with a 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 pairs are located between the mismatches, or a combination thereof.

[0186] In some embodiments, the oligonucleotide comprises a targeting sequence or a complementary region that is complementary to a contiguous nucleotide sequence of any one of SEQ ID NOs: 1683-2066, wherein the targeting sequence or the complementary region may have at most about 1, at most about 2, at most about 3, at most about 4, at most about 5, etc. mismatches with the corresponding SNCA target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or a complementary region that is complementary to a contiguous nucleotide sequence of any one of SEQ ID NOs: 1683-2066, wherein the targeting sequence or the complementary region 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 complementary region that is complementary to a contiguous 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, wherein the targeting sequence or complementary region may have at most about 1, at most about 2, at most about 3, at most about 4, at most about 5, etc. mismatches with the corresponding SNCA target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region that is complementary to a contiguous 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, wherein the targeting sequence or complementary region 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 complementary region that is complementary to a contiguous nucleotide sequence 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 complementary region may have at most about 1, at most about 2, at most about 3, at most about 4, at most about 5, etc. mismatches with the corresponding SNCA target sequence.In some embodiments, the oligonucleotide comprises a targeting sequence or a complementary region that is complementary to a contiguous nucleotide sequence 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 the complementary region 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 complementary region that is complementary to a contiguous nucleotide sequence of any one of SEQ ID NOs: 1865, 1721, 1847, 1846, and 1955, wherein the targeting sequence or the complementary region may have at most about 1, at most about 2, at most about 3, at most about 4, at most about 5, etc. mismatches with the corresponding SNCA target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or a complementary region that is complementary to a contiguous nucleotide sequence of any one of SEQ ID NOs: 1865, 1721, 1847, 1846, and 1955, wherein the targeting sequence or complementary region 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 complementary region that is complementary to a contiguous nucleotide sequence of SEQ ID NO: 1865, wherein the targeting sequence or complementary region may have no more than about 1, no more than about 2, no more than about 3, no more than about 4, no more than about 5, etc. mismatches with the corresponding SNCA target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or a complementary region that is complementary to a contiguous nucleotide sequence of SEQ ID NO: 1865, wherein the targeting sequence or complementary region 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 complementary region that is complementary to a contiguous nucleotide sequence of SEQ ID NO: 1721, wherein the targeting sequence or the complementary region may have at most about 1, at most about 2, at most about 3, at most about 4, at most about 5, etc. mismatches with the corresponding SNCA target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or a complementary region that is complementary to a contiguous nucleotide sequence of SEQ ID NO: 1721, wherein the targeting sequence or the complementary region 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 complementary region that is complementary to a contiguous nucleotide sequence of SEQ ID NO: 1847, wherein the targeting sequence or the complementary region may have at most about 1, at most about 2, at most about 3, at most about 4, at most about 5, etc. mismatches with the corresponding SNCA target sequence.In some embodiments, the oligonucleotide comprises a targeting sequence or a complementary region that is complementary to a contiguous nucleotide sequence of SEQ ID NO: 1847, wherein the targeting sequence or the complementary region 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 complementary region that is complementary to a contiguous nucleotide sequence of SEQ ID NO: 1846, wherein the targeting sequence or the complementary region may have no more than about 1, no more than about 2, no more than about 3, no more than about 4, no more than about 5, etc. mismatches with the corresponding SNCA target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or a complementary region that is complementary to a contiguous nucleotide sequence of SEQ ID NO: 1846, wherein the targeting sequence or the complementary region 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 complementary region that is complementary to a contiguous nucleotide sequence of SEQ ID NO: 1955, wherein the targeting sequence or the complementary region may have at most about 1, at most about 2, at most about 3, at most about 4, at most about 5, etc. mismatches with the corresponding SNCA target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or a complementary region that is complementary to a contiguous nucleotide sequence of SEQ ID NO: 1955, wherein the targeting sequence or the complementary region 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.

[0187] Types of Oligonucleotides

[0188] A variety of oligonucleotide types and / or structures can be used to target SNCA mRNA in the methods herein, including but not limited to RNAi oligonucleotides, antisense oligonucleotides, miRNA, etc. Any oligonucleotide type described herein or elsewhere is contemplated for use as a framework to incorporate the SNCA mRNA targeting sequences herein for the purpose of inhibiting SNCA gene expression.

[0189] In some embodiments, the oligonucleotides herein inhibit SNCA gene expression by participating in an RNA interference (RNAi) pathway (e.g., RNAi oligonucleotides) upstream or downstream of Dicer involvement. For example, RNAi oligonucleotides have been developed in which each strand has a size of about 19 to about 25 nucleotides with at least one 3' overhang of about 1 to about 5 nucleotides (see, e.g., U.S. Pat. No. 8,372,968). Longer oligonucleotides have also been developed that are processed by Dicer to produce active RNAi products (see, e.g., U.S. Pat. No. 8,883,996). Further work has produced extended ds oligonucleotides in which at least one end of at least one strand extends beyond the duplex targeting region, including structures in which one strand includes a thermodynamically stable tetraloop structure (see, e.g., U.S. Pat. Nos. 8,513,207 and 8,927,705, and International 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.

[0190] In some embodiments, the oligonucleotide participates in the RNAi pathway downstream of Dicer involvement (e.g., Dicer cleavage). In some embodiments, the oligonucleotide has an overhang (e.g., 1, 2, or 3 nucleotides in length) at 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 the target mRNA (e.g., SNCA mRNA) and a complementary passenger sense strand, wherein the two strands are bonded to form a 19-bp duplex and an overhang of 2 nucleotides at either or both 3' ends. Longer oligonucleotide designs are also contemplated, including oligonucleotides having an antisense strand of 23 nucleotides and a passenger strand of 21 nucleotides, wherein 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 sense strand / 3' end of antisense strand). In such molecules, there is a 21-bp duplex region. See, e.g., U.S. Patent Nos. 9,012,138, 9,012,621, and 9,193,753.

[0191] In some embodiments, the oligonucleotides herein include sense strands and antisense strands with a length of about 17 to about 36 (e.g., 17 to 26, 20 to 25, or 21-23) nucleotides. In some embodiments, the oligonucleotides include antisense strands with a length of 19-30 nucleotides and sense strands with a length of 19-50 nucleotides, wherein the antisense strand and the sense strand are independent strands forming an asymmetric duplex region, and the duplex region has an overhang of 1-4 nucleotides at the 3′ end of the antisense strand. In some embodiments, the oligonucleotides include sense strands and antisense strands with a length of about 19 to about 22 nucleotides. In some embodiments, the lengths of the sense strand and the antisense strand are equal. In some embodiments, the oligonucleotides include sense strands and antisense strands, so that there is a 3' overhang on either the sense strand or the antisense strand or on both the sense strand and the antisense strand. In some embodiments, for oligonucleotides having sense and antisense strands each ranging from about 21-23 nucleotides in length, the length of the 3' overhang on the sense strand, antisense strand, or both strands is 1 or 2 nucleotides. In some embodiments, the oligonucleotide has a sense strand of 22 nucleotides and an antisense strand of 20 nucleotides, with a blunt end on the right side of the molecule (3' end of sense strand / 5' end of antisense strand) and a 3' antisense strand overhang of 2 nucleotides on the left side of the molecule (5' end of sense strand / 3' end of antisense strand). In such molecules, there is a 20-bp duplex region.

[0192] Other oligonucleotide designs for use in the compositions and methods herein include: 16-mer siRNA (see, e.g., NUCLEIC ACIDS IN CHEMISTRY AND BIOLOGY, Blackburn (ed.), Royal Society of Chemistry, 2006), shRNA (e.g., having a stem of 19 bp or less; see, e.g., Moore et al. (2010) METHODS MOL. BIOL. 629: 141-58), blunt siRNA (e.g., 19 bp in length; see, e.g., Kraynack and Baker (2006) RNA 12: 163-76), asymmetric siRNA (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), forked siRNA (see, e.g., Hohjoh (2004) FEBS Lett. 557: 193-98), single-stranded siRNA (Elsner (2012) Nat. Biotechnol. 30: 1063), dumbbell-shaped circular siRNA (see, e.g., Abe et al. (2007) J. Am. Chem. Soc. 129: 15108-09), and small internal segmented interfering RNA (siRNA; see, e.g., Bramsen et al. (2007) Nucleic Acids Res. 35: 5886-97). Other non-limiting examples of oligonucleotide designs that can be used to reduce or inhibit SNCA gene expression in some embodiments are microRNA (miRNA), short hairpin RNA (shRNA), and short siRNA (see, e.g., Hamilton et al. (2002) EMBO J. 21: 4671-79; see also U.S. Patent Application Publication No. 2009 / 0099115).

[0193] In addition, in some embodiments, the oligonucleotide used herein to reduce or inhibit SNCA gene expression 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, for example, Matsui et al. (2016) Mol. Ther. 24: 946-955). However, in some embodiments, the oligonucleotide is an antisense oligonucleotide (ASO). An antisense oligonucleotide is an ss oligonucleotide having a nucleobase sequence, and when written or depicted in the 5′ to 3′ direction, the nucleobase sequence comprises the reverse complement of the targeted segment of a specific nucleic acid and is appropriately modified (e.g., as a gapmer) to induce RNaseH-mediated cleavage of its target RNA in a cell or (e.g., as a mixmer) to inhibit translation of a target mRNA in a cell. The ASOs used herein can be modified in any suitable manner known in the art, including, for example, as shown in U.S. Pat. No. 9,567,587 (including, for example, changes in length, sugar moieties of nucleobases (pyrimidines, purines), and heterocyclic moieties of nucleobases). In addition, ASOs have been used for decades to reduce the expression of specific target genes (see, for example, Bennett et al. (2017) Annu. Rev. Pharmacol. 57: 81-105).

[0194] In some embodiments, the antisense oligonucleotide (ASO) shares a complementary region with the SNCA mRNA. In some embodiments, the ASO targets various regions of 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. In 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 from the target sequence by 1, 2, or 3 nucleotides.

[0195] Double-stranded RNAi oligonucleotides

[0196] In some aspects, the present disclosure provides ds RNAi oligonucleotides for targeting SNCA mRNA and inhibiting SNCA gene expression (e.g., via an RNAi pathway), the oligonucleotides 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 the antisense strand are independent strands and are not covalently linked. In some embodiments, the sense strand is covalently linked to the antisense strand. 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, bind to each other in a complementary manner (e.g., by Watson-Crick base pairing).

[0197] In some embodiments, the sense strand has a first region (R1) and a second region (R2), wherein R2 comprises a first subregion (S1), a loop (L) (such as a tetraloop (tetraL) or a triloop (triL)) and a second subregion (S2), wherein L is located between S1 and S2, and wherein S1 and S2 form a second duplex (D2). D2 can have various lengths. In some embodiments, the length of D2 is about 1 to about 6 bp. In some embodiments, the length of D2 is 2-6, 3-6, 4-6, 5-6, 1-5, 2-5, 3-5 or 4-5 bp. In some embodiments, the length of D2 is 1, 2, 3, 4, 5 or 6 bp. In some embodiments, the length of D2 is 6 bp.

[0198] In some embodiments, the R1 of the sense strand and the antisense strand form a first duplex (D1). In some embodiments, the length of D1 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 some embodiments, the length of D1 is in the range of about 12 to about 30 nucleotides (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, the length of D1 is at least 12 nucleotides (e.g., at least 12, at least 15, at least 20, at least 25, or at least 30 nucleotides in length). In some embodiments, D1 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, D1 is 19 nucleotides in length. In some embodiments, D1 is 20 nucleotides in length. In some embodiments, D1 comprising a sense strand and an antisense strand does not span the entire length of the sense strand and / or antisense strand. In some embodiments, D1 comprising a sense strand and an antisense strand spans the entire length of either or both of the sense strand or antisense strand. In certain embodiments, D1 comprising a sense strand and an antisense strand spans the entire length of both the sense strand or the antisense strand.

[0199] In some embodiments, the sense strand is 36 nucleotides in length and positions are numbered 1-36 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, the position numbering described herein follows this numbering format.

[0200] 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.

[0201] 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.

[0202] In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand, the sense strand and the antisense strand comprising a nucleotide sequence selected from the group consisting of:

[0203] a) SEQ ID NO: 1537 and 1572, respectively;

[0204] b) SEQ ID NOs: 1538 and 1573, respectively;

[0205] c) SEQ ID NOs: 1539 and 1574, respectively;

[0206] d) SEQ ID NOs: 1540 and 1575, respectively;

[0207] e) SEQ ID NOs: 1541 and 1576, respectively;

[0208] f) SEQ ID NO: 1542 and 1577, respectively;

[0209] g) SEQ ID NOs: 1543 and 1578, respectively;

[0210] h) SEQ ID NOs: 1544 and 1579, respectively;

[0211] i) SEQ ID NOs: 1545 and 1580, respectively;

[0212] j) SEQ ID NOs: 1546 and 1581, respectively;

[0213] k) SEQ ID NOs: 1547 and 1582, respectively;

[0214] 1) are SEQ ID NOs: 1548 and 1583, respectively;

[0215] m) are SEQ ID NOs: 1549 and 1584, respectively;

[0216] n) are SEQ ID NOs: 1550 and 1585, respectively;

[0217] o) are SEQ ID NOs: 1551 and 1586, respectively;

[0218] p) are SEQ ID NOs: 1552 and 1587, respectively;

[0219] q) are SEQ ID NOs: 1553 and 1588, respectively;

[0220] r) are SEQ ID NOs: 1554 and 1589, respectively;

[0221] s) are SEQ ID NOs: 1555 and 1590, respectively;

[0222] t) are SEQ ID NOs: 1556 and 1591, respectively;

[0223] u) are SEQ ID NOs: 1557 and 1592, respectively;

[0224] v) SEQ ID NOs: 1558 and 1593, respectively;

[0225] w) are SEQ ID NOs: 1559 and 1594, respectively;

[0226] x) are SEQ ID NOs: 1560 and 1595, respectively;

[0227] y) are SEQ ID NOs: 1561 and 1596, respectively;

[0228] z) are SEQ ID NOs: 1562 and 1597, respectively;

[0229] aa) are SEQ ID NOs: 1563 and 1598, respectively;

[0230] bb) are SEQ ID NOs: 1564 and 1599, respectively;

[0231] cc) are SEQ ID NOs: 1565 and 1600, respectively;

[0232] dd) are SEQ ID NOs: 1566 and 1601, respectively;

[0233] ee) are SEQ ID NOs: 1567 and 1602, respectively;

[0234] ff) are SEQ ID NOs: 1568 and 1603, respectively;

[0235] gg) are SEQ ID NOs: 1569 and 1604, respectively;

[0236] hh) are SEQ ID NOs: 1570 and 1605, respectively;

[0237] ii) SEQ ID NO: 1571 and 1606, respectively; and

[0238] jj) are SEQ ID NOs: 1681 and 1586, respectively.

[0239] In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand, the sense strand and the antisense strand comprising a nucleotide sequence selected from the group consisting of:

[0240] a) SEQ ID NO: 1540 and 1575, respectively;

[0241] b) SEQ ID NOs: 1544 and 1579, respectively;

[0242] c) SEQ ID NOs: 1546 and 1581, respectively;

[0243] d) SEQ ID NOs: 1551 and 1586, respectively;

[0244] e) SEQ ID NOs: 1552 and 1587, respectively;

[0245] f) SEQ ID NOs: 1553 and 1588, respectively;

[0246] g) SEQ ID NOs: 1558 and 1594, respectively;

[0247] h) SEQ ID NOs: 1560 and 1595, respectively;

[0248] i) SEQ ID NOs: 1564 and 1599, respectively;

[0249] j) are SEQ ID NOs: 1565 and 1600, respectively;

[0250] k) are SEQ ID NOs: 1566 and 1601, respectively;

[0251] 1) are SEQ ID NOs: 1570 and 1605, respectively; and

[0252] m) are SEQ ID NOs: 1681 and 1586, respectively.

[0253] In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand, the sense strand and the antisense strand comprising a nucleotide sequence selected from the group consisting of:

[0254] a) SEQ ID NO: 1553 and 1588, respectively;

[0255] b) SEQ ID NOs: 1560 and 1595, respectively;

[0256] c) SEQ ID NOs: 1564 and 1599, respectively;

[0257] d) SEQ ID NOs: 1551 and 1586, respectively;

[0258] e) SEQ ID NOs: 1570 and 1605, respectively; and

[0259] f) are SEQ ID NOs: 1681 and 1586, respectively.

[0260] 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.

[0261] It will be appreciated that in some embodiments, reference may be made to the sequences presented in the sequence listing when describing the structure of an oligonucleotide (e.g., an 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., an RNA counterpart of a DNA nucleotide or a DNA counterpart of an RNA nucleotide) and / or one or more modified nucleotides and / or one or more modified internucleotide bonds and / or one or more other modifications when compared to a specified sequence while retaining a complementary property that is substantially identical or similar to the specified sequence.

[0262] In some embodiments, the RNAi oligonucleotide herein comprises a sense strand of 25 nucleotides and an antisense strand of 27 nucleotides, which, when acted upon by a dicer, produces an antisense strand that is incorporated into a mature RNA-induced silencing complex (RISC). In some embodiments, the sense strand of 25 nucleotides comprises a sequence selected from SEQ ID NO: 1-384. In some embodiments, the antisense strand of 27 nucleotides comprises a sequence selected from SEQ ID NO: 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).

[0263] In some embodiments, the oligonucleotide has a 5' end that is thermodynamically less stable when compared to another 5' end. In some embodiments, an asymmetric RNAi oligonucleotide is provided, the oligonucleotide comprising a flat end at the 3' end of the sense strand and a 3' overhang at the 3' end of the antisense strand. In some embodiments, the length of the 3' overhang on the antisense strand is about 1 to about 8 nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides in length). Typically, the oligonucleotide has an overhang of two nucleotides at the 3' end of the antisense strand. However, there may be other overhangs. In some embodiments, the overhang is a 3' overhang comprising a length 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 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.

[0264] In some embodiments, the 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 to 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 to 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 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, the 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 the ds oligonucleotide are not complementary to the target mRNA. In some embodiments, the 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 to the target mRNA. In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region complementary to a contiguous nucleotide sequence 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 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 unpaired GG. In some embodiments, the oligonucleotide comprises a sense strand comprising a nucleotide sequence selected from SEQ ID NOs: 1683-2066; and 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 unpaired GG.

[0265] In some embodiments, there are one or more (e.g., 1, 2, 3, 4, or 5) mismatches between the sense strand and the antisense strand that constitute the oligonucleotide herein. If there are more than one mismatch between the sense strand and the antisense strand, they may be located consecutively (e.g., 2, 3, or more in a row) or may be dispersed throughout the complementary region. 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 a segment at the 3' end of the sense strand of an oligonucleotide improves or increases the efficacy of the oligonucleotide.

[0266] In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand, the sense strand and the antisense strand comprising a nucleotide sequence selected from the group consisting of:

[0267] a) SEQ ID NO: 1537 and 1572, respectively;

[0268] b) SEQ ID NOs: 1538 and 1573, respectively;

[0269] c) SEQ ID NOs: 1539 and 1574, respectively;

[0270] d) SEQ ID NOs: 1540 and 1575, respectively;

[0271] e) SEQ ID NOs: 1541 and 1576, respectively;

[0272] f) SEQ ID NO: 1542 and 1577, respectively;

[0273] g) SEQ ID NOs: 1543 and 1578, respectively;

[0274] h) SEQ ID NOs: 1544 and 1579, respectively;

[0275] i) SEQ ID NOs: 1545 and 1580, respectively;

[0276] j) SEQ ID NOs: 1546 and 1581, respectively;

[0277] k) SEQ ID NOs: 1547 and 1582, respectively;

[0278] 1) are SEQ ID NOs: 1548 and 1583, respectively;

[0279] m) are SEQ ID NOs: 1549 and 1584, respectively;

[0280] n) are SEQ ID NOs: 1550 and 1585, respectively;

[0281] o) are SEQ ID NOs: 1551 and 1586, respectively;

[0282] p) are SEQ ID NOs: 1552 and 1587, respectively;

[0283] q) are SEQ ID NOs: 1553 and 1588, respectively;

[0284] r) are SEQ ID NOs: 1554 and 1589, respectively;

[0285] s) are SEQ ID NOs: 1555 and 1590, respectively;

[0286] t) are SEQ ID NOs: 1556 and 1591, respectively;

[0287] u) are SEQ ID NOs: 1557 and 1592, respectively;

[0288] v) SEQ ID NOs: 1558 and 1593, respectively;

[0289] w) are SEQ ID NOs: 1559 and 1594, respectively;

[0290] x) are SEQ ID NOs: 1560 and 1595, respectively;

[0291] y) are SEQ ID NOs: 1561 and 1596, respectively;

[0292] z) are SEQ ID NOs: 1562 and 1597, respectively;

[0293] aa) are SEQ ID NOs: 1563 and 1598, respectively;

[0294] bb) are SEQ ID NOs: 1564 and 1599, respectively;

[0295] cc) are SEQ ID NOs: 1565 and 1600, respectively;

[0296] dd) are SEQ ID NOs: 1566 and 1601, respectively;

[0297] ee) are SEQ ID NOs: 1567 and 1602, respectively;

[0298] ff) are SEQ ID NOs: 1568 and 1603, respectively;

[0299] gg) are SEQ ID NOs: 1569 and 1604, respectively;

[0300] hh) are SEQ ID NOs: 1570 and 1605, respectively;

[0301] ii) SEQ ID NO: 1571 and 1606, respectively; and

[0302] jj) are SEQ ID NOs: 1681 and 1586,

[0303] wherein there are one or more (eg, 1, 2, 3, 4, or 5) mismatches between the sense strand and the antisense strand.

[0304] In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand, the sense strand and the antisense strand comprising a nucleotide sequence selected from the group consisting of:

[0305] a) SEQ ID NO: 1540 and 1575, respectively;

[0306] b) SEQ ID NOs: 1544 and 1579, respectively;

[0307] c) SEQ ID NOs: 1546 and 1581, respectively;

[0308] d) SEQ ID NOs: 1551 and 1586, respectively;

[0309] e) SEQ ID NOs: 1552 and 1587, respectively;

[0310] f) SEQ ID NOs: 1553 and 1588, respectively;

[0311] g) SEQ ID NOs: 1558 and 1594, respectively;

[0312] h) SEQ ID NOs: 1560 and 1595, respectively;

[0313] i) SEQ ID NOs: 1564 and 1599, respectively;

[0314] j) are SEQ ID NOs: 1565 and 1600, respectively;

[0315] k) are SEQ ID NOs: 1566 and 1601, respectively;

[0316] 1) are SEQ ID NOs: 1570 and 1605, respectively; and

[0317] m) are SEQ ID NOs: 1681 and 1586, respectively,

[0318] wherein there are one or more (eg, 1, 2, 3, 4, or 5) mismatches between the sense strand and the antisense strand.

[0319] In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand, the sense strand and the antisense strand comprising a nucleotide sequence selected from the group consisting of:

[0320] a) SEQ ID NO: 1553 and 1588, respectively;

[0321] b) SEQ ID NOs: 1560 and 1595, respectively;

[0322] c) SEQ ID NOs: 1564 and 1599, respectively;

[0323] d) SEQ ID NOs: 1551 and 1586, respectively;

[0324] e) SEQ ID NOs: 1570 and 1605, respectively; and

[0325] f) are SEQ ID NOs: 1681 and 1586, respectively,

[0326] wherein there are one or more (eg, 1, 2, 3, 4, or 5) mismatches between the sense strand and the antisense strand.

[0327] Antisense strand

[0328] In some embodiments, the antisense strand of the oligonucleotide is referred to as the "guide strand", which engages with RISC and binds to an Argonaute protein, such as Ago2, or engages or binds to one or more similar factors, and directs the silencing of the target gene. In some embodiments, the sense strand complementary to the antisense strand is referred to as the "passenger strand".

[0329] In some embodiments, the oligonucleotide comprises an antisense strand of at most about 50 nucleotides in length (e.g., at most 50, at most 40, at most 35, at most 30, at most 27, at most 25, at most 21, at most 19, at most 17, at most 15, or at most 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 the 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 strand that is 15 to 30 nucleotides in length. In some embodiments, the antisense strand of any of the oligonucleotides disclosed herein is 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.

[0330] 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) consecutive 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) consecutive 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) consecutive 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. 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) consecutive 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) consecutive 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) consecutive nucleotides of a sequence as shown in any one of SEQ ID NOs: 1588, 1595, 1599, 1586, and 1605.

[0331] Sense strand

[0332] In some embodiments, the oligonucleotide comprises a sense strand sequence as set forth 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) consecutive nucleotides of a sequence as set forth in any one of SEQ ID NOs: 1683-2066. In some embodiments, the sense strand comprises a sequence as 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) consecutive nucleotides of a sequence as set forth 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. In some embodiments, the sense strand is SEQ ID NO: 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) consecutive 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 SEQ ID NO: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. 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) consecutive nucleotides of the sequence shown in any one of. 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) consecutive 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 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) consecutive nucleotides of a sequence as set forth in any one of SEQ ID NOs: 1553, 1560, 1564, 1551, and 1570.

[0333] 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) consecutive 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) consecutive nucleotides of a sequence as set forth in any one of SEQ ID NOs: 1553, 1560, 1564, 1551, 1570, and 1681.

[0334] In some embodiments, the sense strand comprises at most about 50 nucleotides in length (e.g., at most 50, at most 40, at most 36, at most 30, at most 27, at most 25, at most 21, at most 19, at most 17, or at most 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 length of the sense strand ranges from 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 some embodiments, the length of the sense strand comprises 15 to 50 nucleotides. In some embodiments, the length of the sense strand comprises 18 to 36 nucleotides. 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.

[0335] In some embodiments, the sense strand comprises a stem-loop structure at its 3' end. In some embodiments, the stem-loop is formed by intrachain 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. In 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.

[0336] In some embodiments, the stem-loop provides oligonucleotide protection against degradation (e.g., enzymatic degradation), promotes or improves targeting and / or delivery to a target cell, tissue or organ (e.g., liver or brain), or both. For example, in some embodiments, the loop of the stem-loop provides a nucleotide comprising one or more modifications that promote, improve or increase targeting to a target mRNA (e.g., SNCA mRNA), inhibition of target gene expression (e.g., SNCA gene expression) and / or delivery to a target cell, tissue or organ (e.g., CNS), or a combination thereof. In some embodiments, the stem-loop itself or modifications to the stem-loop do not substantially affect the intrinsic gene expression inhibition activity of the oligonucleotide, but promote, improve or increase stability (e.g., provide protection against degradation) and / or delivery of the oligonucleotide to a target cell, tissue or organ (e.g., CNS). In certain embodiments, the oligonucleotide comprises a sense strand comprising (e.g., at its 3' end) a stem-loop as follows: S1-L-S2, wherein S1 is complementary to S2, and wherein L forms a single-stranded loop between S1 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 or triL). 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 length of loop (L) is 9 nucleotides (e.g., nona loop or nonaL). In some embodiments, the length of loop (L) is 10 nucleotides (e.g., deca loop or decaL).

[0337] In some embodiments, the oligonucleotide comprises a targeting sequence or a complementary region that is complementary to a contiguous nucleotide sequence of any one of SEQ ID NOs: 1683-2066, and the oligonucleotide comprises a sense strand that comprises (e.g., at its 3' end) a stem loop as follows: S1-L-S2, wherein S1 is complementary to S2, and wherein L forms an ss loop of up to about 10 nucleotides in length (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length) between S1 and S2. In some embodiments, the oligonucleotide comprises a targeting sequence or a complementary region that is complementary to a contiguous nucleotide sequence of any one of SEQ ID NOs: 1683-2066, and the oligonucleotide comprises a sense strand that comprises (e.g., at its 3' end) a stem loop as follows: S1-L-S2, wherein S1 is complementary to S2, and wherein L forms an ss loop of 4 nucleotides in length between S1 and S2. In some embodiments, the loop (L) of the stem-loop having the structure S1-L-S2 as described above is a tetraL as described in U.S. Patent No. 10,131,912 (e.g., within a nicked tetraL structure), which is incorporated herein by reference. In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region and a tetraL that is complementary to a continuous nucleotide sequence of any one of SEQ ID NOs: 1683-2066. 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).

[0338] In other embodiments, loop (L) is triL. In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region complementary to a contiguous nucleotide sequence of any one of SEQ ID NOs: 1683-2066 and triL. In some embodiments, triL comprises ribonucleotides, deoxyribonucleotides, modified nucleotides, delivery ligands, and combinations thereof.

[0339] Duplex length

[0340] In some embodiments, the length of the duplex formed between the sense strand and the antisense strand 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 some embodiments, the length of the duplex is in the range of about 12 to about 30 nucleotides (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 length of 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 some embodiments, the length of the duplex is 12 nucleotides. In some embodiments, the length of the duplex is 13 nucleotides. In some embodiments, the length of the duplex is 14 nucleotides. In some embodiments, the length of the duplex is 15 nucleotides. In some embodiments, the length of the duplex is 16 nucleotides. In some embodiments, the length of the duplex is 17 nucleotides. In some embodiments, the length of the duplex is 18 nucleotides. In some embodiments, the length of the duplex is 19 nucleotides. In some embodiments, the length of the duplex is 20 nucleotides. In some embodiments, the length of the duplex is 21 nucleotides. In some embodiments, the length of the duplex is 22 nucleotides. In some embodiments, the length of the duplex is 23 nucleotides. In some embodiments, the length of the duplex formed is 24 nucleotides. In some embodiments, the length of the duplex is 25 nucleotides. In some embodiments, the length of the duplex is 26 nucleotides. In some embodiments, the length of the duplex is 27 nucleotides. In some embodiments, the length of the duplex is 28 nucleotides. In some embodiments, the length of the duplex is 29 nucleotides. In some embodiments, the length of the duplex is 30 nucleotides. In some embodiments, the duplex does not span the entire length of the sense strand and / or antisense strand. In some embodiments, the duplex spans the entire length of either the sense strand or the antisense strand. In some embodiments, the duplex spans the entire length of both the sense strand and the antisense strand.

[0341] In some embodiments, the sense strand and the antisense strand comprise a nucleotide sequence selected from the group consisting of:

[0342] a) SEQ ID NO: 1537 and 1572, respectively;

[0343] b) SEQ ID NOs: 1538 and 1573, respectively;

[0344] c) SEQ ID NOs: 1539 and 1574, respectively;

[0345] d) SEQ ID NOs: 1540 and 1575, respectively;

[0346] e) SEQ ID NOs: 1541 and 1576, respectively;

[0347] f) SEQ ID NO: 1542 and 1577, respectively;

[0348] g) SEQ ID NOs: 1543 and 1578, respectively;

[0349] h) SEQ ID NOs: 1544 and 1579, respectively;

[0350] i) SEQ ID NOs: 1545 and 1580, respectively;

[0351] j) SEQ ID NOs: 1546 and 1581, respectively;

[0352] k) SEQ ID NOs: 1547 and 1582, respectively;

[0353] 1) are SEQ ID NOs: 1548 and 1583, respectively;

[0354] m) are SEQ ID NOs: 1549 and 1584, respectively;

[0355] n) are SEQ ID NOs: 1550 and 1585, respectively;

[0356] o) are SEQ ID NOs: 1551 and 1586, respectively;

[0357] p) are SEQ ID NOs: 1552 and 1587, respectively;

[0358] q) are SEQ ID NOs: 1553 and 1588, respectively;

[0359] r) are SEQ ID NOs: 1554 and 1589, respectively;

[0360] s) are SEQ ID NOs: 1555 and 1590, respectively;

[0361] t) are SEQ ID NOs: 1556 and 1591, respectively;

[0362] u) are SEQ ID NOs: 1557 and 1592, respectively;

[0363] v) SEQ ID NOs: 1558 and 1593, respectively;

[0364] w) are SEQ ID NOs: 1559 and 1594, respectively;

[0365] x) are SEQ ID NOs: 1560 and 1595, respectively;

[0366] y) are SEQ ID NOs: 1561 and 1596, respectively;

[0367] z) are SEQ ID NOs: 1562 and 1597, respectively;

[0368] aa) are SEQ ID NOs: 1563 and 1598, respectively;

[0369] bb) are SEQ ID NOs: 1564 and 1599, respectively;

[0370] cc) are SEQ ID NOs: 1565 and 1600, respectively;

[0371] dd) are SEQ ID NOs: 1566 and 1601, respectively;

[0372] ee) are SEQ ID NOs: 1567 and 1602, respectively;

[0373] ff) are SEQ ID NOs: 1568 and 1603, respectively;

[0374] gg) are SEQ ID NOs: 1569 and 1604, respectively;

[0375] hh) are SEQ ID NOs: 1570 and 1605, respectively;

[0376] ii) SEQ ID NO: 1571 and 1606, respectively; and

[0377] jj) are SEQ ID NOs: 1681 and 1586,

[0378] wherein the length of the duplex formed between the sense strand and the antisense strand ranges from about 12 to about 30 nucleotides (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).

[0379] In some embodiments, the sense strand and the antisense strand comprise a nucleotide sequence selected from the group consisting of:

[0380] a) SEQ ID NO: 1540 and 1575, respectively;

[0381] b) SEQ ID NOs: 1544 and 1579, respectively;

[0382] c) SEQ ID NOs: 1546 and 1581, respectively;

[0383] d) SEQ ID NOs: 1551 and 1586, respectively;

[0384] e) SEQ ID NOs: 1552 and 1587, respectively;

[0385] f) SEQ ID NOs: 1553 and 1588, respectively;

[0386] g) SEQ ID NOs: 1558 and 1594, respectively;

[0387] h) SEQ ID NOs: 1560 and 1595, respectively;

[0388] i) SEQ ID NOs: 1564 and 1599, respectively;

[0389] j) are SEQ ID NOs: 1565 and 1600, respectively;

[0390] k) are SEQ ID NOs: 1566 and 1601, respectively;

[0391] 1) are SEQ ID NOs: 1570 and 1605, respectively; and

[0392] m) are SEQ ID NOs: 1681 and 1586, respectively,

[0393] wherein the length of the duplex formed between the sense strand and the antisense strand ranges from about 12 to about 30 nucleotides (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).

[0394] In some embodiments, the sense strand and the antisense strand comprise a nucleotide sequence selected from the group consisting of:

[0395] a) SEQ ID NO: 1553 and 1588, respectively;

[0396] b) SEQ ID NOs: 1560 and 1595, respectively;

[0397] c) SEQ ID NOs: 1564 and 1599, respectively;

[0398] d) SEQ ID NOs: 1551 and 1586, respectively;

[0399] e) SEQ ID NOs: 1570 and 1605, respectively; and

[0400] f) are SEQ ID NOs: 1681 and 1586, respectively,

[0401] wherein the length of the duplex formed between the sense strand and the antisense strand ranges from about 12 to about 30 nucleotides (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).

[0402] Oligonucleotide ends

[0403] In some embodiments, an oligonucleotide (e.g., an RNAi oligonucleotide) comprises a sense strand and an antisense strand, wherein the end of any one or both strands comprises a flat end. In some embodiments, an oligonucleotide comprises a sense strand and an antisense strand, and the sense strand and the antisense strand are independent strands forming an asymmetric duplex region, and the duplex region has an overhang at the 3' end of the antisense strand. In some embodiments, an oligonucleotide comprises a sense strand and an antisense strand, wherein the end of any one or both strands comprises an overhang containing one or more nucleotides. In some embodiments, one or more nucleotides constituting the overhang are unpaired nucleotides. In some embodiments, an oligonucleotide herein comprises a sense strand and an antisense strand, wherein the 3' end of the sense strand and the 5' end of the antisense strand comprise a flat end. In some embodiments, an oligonucleotide comprises a sense strand and an antisense strand, wherein the 5' end of the sense strand and the 3' end of the antisense strand comprise a flat end.

[0404] In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand, wherein the 3' end 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.

[0405] 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. In 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.

[0406] In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand comprise a nucleotide sequence selected from the group consisting of:

[0407] a) SEQ ID NO: 1537 and 1572, respectively;

[0408] b) SEQ ID NOs: 1538 and 1573, respectively;

[0409] c) SEQ ID NOs: 1539 and 1574, respectively;

[0410] d) SEQ ID NOs: 1540 and 1575, respectively;

[0411] e) SEQ ID NOs: 1541 and 1576, respectively;

[0412] f) SEQ ID NO: 1542 and 1577, respectively;

[0413] g) SEQ ID NOs: 1543 and 1578, respectively;

[0414] h) SEQ ID NOs: 1544 and 1579, respectively;

[0415] i) SEQ ID NOs: 1545 and 1580, respectively;

[0416] j) SEQ ID NOs: 1546 and 1581, respectively;

[0417] k) SEQ ID NOs: 1547 and 1582, respectively;

[0418] 1) are SEQ ID NOs: 1548 and 1583, respectively;

[0419] m) are SEQ ID NOs: 1549 and 1584, respectively;

[0420] n) are SEQ ID NOs: 1550 and 1585, respectively;

[0421] o) are SEQ ID NOs: 1551 and 1586, respectively;

[0422] p) are SEQ ID NOs: 1552 and 1587, respectively;

[0423] q) are SEQ ID NOs: 1553 and 1588, respectively;

[0424] r) are SEQ ID NOs: 1554 and 1589, respectively;

[0425] s) are SEQ ID NOs: 1555 and 1590, respectively;

[0426] t) are SEQ ID NOs: 1556 and 1591, respectively;

[0427] u) are SEQ ID NOs: 1557 and 1592, respectively;

[0428] v) SEQ ID NOs: 1558 and 1593, respectively;

[0429] w) are SEQ ID NOs: 1559 and 1594, respectively;

[0430] x) are SEQ ID NOs: 1560 and 1595, respectively;

[0431] y) are SEQ ID NOs: 1561 and 1596, respectively;

[0432] z) are SEQ ID NOs: 1562 and 1597, respectively;

[0433] aa) are SEQ ID NOs: 1563 and 1598, respectively;

[0434] bb) are SEQ ID NOs: 1564 and 1599, respectively;

[0435] cc) are SEQ ID NOs: 1565 and 1600, respectively;

[0436] dd) are SEQ ID NOs: 1566 and 1601, respectively;

[0437] ee) are SEQ ID NOs: 1567 and 1602, respectively;

[0438] ff) are SEQ ID NOs: 1568 and 1603, respectively;

[0439] gg) are SEQ ID NOs: 1569 and 1604, respectively;

[0440] hh) are SEQ ID NOs: 1570 and 1605, respectively;

[0441] ii) SEQ ID NO: 1571 and 1606, respectively; and

[0442] jj) are SEQ ID NOs: 1681 and 1586,

[0443] wherein the antisense strand comprises a 3' overhang of 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.

[0444] In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand comprise a nucleotide sequence selected from the group consisting of:

[0445] a) SEQ ID NO: 1540 and 1575, respectively;

[0446] b) SEQ ID NOs: 1544 and 1579, respectively;

[0447] c) SEQ ID NOs: 1546 and 1581, respectively;

[0448] d) SEQ ID NOs: 1551 and 1586, respectively;

[0449] e) SEQ ID NOs: 1552 and 1587, respectively;

[0450] f) SEQ ID NOs: 1553 and 1588, respectively;

[0451] g) SEQ ID NOs: 1558 and 1594, respectively;

[0452] h) SEQ ID NOs: 1560 and 1595, respectively;

[0453] i) SEQ ID NOs: 1564 and 1599, respectively;

[0454] j) are SEQ ID NOs: 1565 and 1600, respectively;

[0455] k) are SEQ ID NOs: 1566 and 1601, respectively;

[0456] 1) are SEQ ID NOs: 1570 and 1605, respectively; and

[0457] m) are SEQ ID NOs: 1681 and 1586, respectively,

[0458] and wherein the antisense strand comprises a 3' overhang of 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.

[0459] In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand comprise a nucleotide sequence selected from the group consisting of:

[0460] a) SEQ ID NO: 1553 and 1588, respectively;

[0461] b) SEQ ID NOs: 1560 and 1595, respectively;

[0462] c) SEQ ID NOs: 1564 and 1599, respectively;

[0463] d) SEQ ID NOs: 1551 and 1586, respectively;

[0464] e) SEQ ID NOs: 1570 and 1605, respectively; and

[0465] f) are SEQ ID NOs: 1681 and 1586, respectively,

[0466] wherein the antisense strand comprises a 3' overhang of 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.

[0467] In some embodiments, an oligonucleotide comprises a sense strand and an antisense strand, wherein the sense strand comprises a 5' overhang comprising one or more nucleotides.

[0468] In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand comprise a nucleotide sequence selected from the group consisting of:

[0469] a) SEQ ID NO: 1540 and 1575, respectively;

[0470] b) SEQ ID NOs: 1544 and 1579, respectively;

[0471] c) SEQ ID NOs: 1546 and 1581, respectively;

[0472] d) SEQ ID NOs: 1551 and 1586, respectively;

[0473] e) SEQ ID NOs: 1552 and 1587, respectively;

[0474] f) SEQ ID NOs: 1553 and 1588, respectively;

[0475] g) SEQ ID NOs: 1558 and 1594, respectively;

[0476] h) SEQ ID NOs: 1560 and 1595, respectively;

[0477] i) SEQ ID NOs: 1564 and 1599, respectively;

[0478] j) are SEQ ID NOs: 1565 and 1600, respectively;

[0479] k) are SEQ ID NOs: 1566 and 1601, respectively;

[0480] 1) are SEQ ID NOs: 1570 and 1605, respectively; and

[0481] m) are SEQ ID NOs: 1681 and 1586, respectively,

[0482] wherein the antisense strand comprises a 5' overhang of 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.

[0483] In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand comprise a nucleotide sequence selected from the group consisting of:

[0484] a) SEQ ID NO: 1553 and 1588, respectively;

[0485] b) SEQ ID NOs: 1560 and 1595, respectively;

[0486] c) SEQ ID NOs: 1564 and 1599, respectively;

[0487] d) SEQ ID NOs: 1551 and 1586, respectively;

[0488] e) SEQ ID NOs: 1570 and 1605, respectively; and

[0489] f) are SEQ ID NOs: 1681 and 1586, respectively,

[0490] wherein the antisense strand comprises a 5' overhang of 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.

[0491] In some embodiments, one or more (e.g., 2, 3, 4, 5, or more) nucleotides that make up the 3' end or 5' end of the sense strand and / or antisense strand are modified. For example, in some embodiments, one or two terminal nucleotides at the 3' end of the antisense strand are modified. In some embodiments, the last nucleotide at the 3' end of the antisense strand is modified (e.g., comprises a 2' modification, such as 2'-O-methoxyethyl). In some embodiments, the last one or two terminal nucleotides at the 3' end of the antisense strand are complementary to the target. In some embodiments, the last one or two nucleotides at the 3' end of the antisense strand are not complementary to the target.

[0492] In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand, wherein the 3' end of the sense strand comprises a stem loop described herein and the 3' end of the antisense strand comprises a 3' overhang. In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand forming a nicked tetraL structure, wherein the 3' end of the sense strand comprises a stem loop, wherein the loop (L) is a tetraL described herein, and wherein the 3' end 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 constituting the 3' overhang both comprise a guanine (G) nucleobase. Typically, one or both of the nucleotides constituting the 3' overhang of the antisense strand are not complementary to the target mRNA.

[0493] Oligonucleotide modification

[0494] In some embodiments, the oligonucleotide comprises a modification. Oligonucleotides (e.g., RNAi oligonucleotides) can be modified in various ways to improve or control specificity, stability, delivery, bioavailability, resistance to nuclease degradation, immunogenicity, base pairing properties, RNA distribution and cellular uptake, and other features relevant to therapeutic research uses.

[0495] 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 bond. In some embodiments, the modification is a modified base. In some embodiments, the modification is a reversible modification. In some embodiments, the oligonucleotide may 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 bond, at least one modified base, and at least one reversible modification.

[0496] In some embodiments, the oligonucleotide comprises at least one modified sugar, a 5' terminal phosphate group, at least one modified internucleotide bond, and at least one modified base. In some embodiments, the sense strand and the antisense strand of the oligonucleotide comprise a nucleotide sequence selected from the group consisting of:

[0497] a) SEQ ID NO: 1537 and 1572, respectively;

[0498] b) SEQ ID NOs: 1538 and 1573, respectively;

[0499] c) SEQ ID NOs: 1539 and 1574, respectively;

[0500] d) SEQ ID NOs: 1540 and 1575, respectively;

[0501] e) SEQ ID NOs: 1541 and 1576, respectively;

[0502] f) SEQ ID NO: 1542 and 1577, respectively;

[0503] g) SEQ ID NOs: 1543 and 1578, respectively;

[0504] h) SEQ ID NOs: 1544 and 1579, respectively;

[0505] i) SEQ ID NOs: 1545 and 1580, respectively;

[0506] j) SEQ ID NOs: 1546 and 1581, respectively;

[0507] k) SEQ ID NOs: 1547 and 1582, respectively;

[0508] 1) are SEQ ID NOs: 1548 and 1583, respectively;

[0509] m) are SEQ ID NOs: 1549 and 1584, respectively;

[0510] n) are SEQ ID NOs: 1550 and 1585, respectively;

[0511] o) are SEQ ID NOs: 1551 and 1586, respectively;

[0512] p) are SEQ ID NOs: 1552 and 1587, respectively;

[0513] q) are SEQ ID NOs: 1553 and 1588, respectively;

[0514] r) are SEQ ID NOs: 1554 and 1589, respectively;

[0515] s) are SEQ ID NOs: 1555 and 1590, respectively;

[0516] t) are SEQ ID NOs: 1556 and 1591, respectively;

[0517] u) are SEQ ID NOs: 1557 and 1592, respectively;

[0518] v) SEQ ID NOs: 1558 and 1593, respectively;

[0519] w) are SEQ ID NOs: 1559 and 1594, respectively;

[0520] x) are SEQ ID NOs: 1560 and 1595, respectively;

[0521] y) are SEQ ID NOs: 1561 and 1596, respectively;

[0522] z) are SEQ ID NOs: 1562 and 1597, respectively;

[0523] aa) are SEQ ID NOs: 1563 and 1598, respectively;

[0524] bb) are SEQ ID NOs: 1564 and 1599, respectively;

[0525] cc) are SEQ ID NOs: 1565 and 1600, respectively;

[0526] dd) are SEQ ID NOs: 1566 and 1601, respectively;

[0527] ee) are SEQ ID NOs: 1567 and 1602, respectively;

[0528] ff) are SEQ ID NOs: 1568 and 1603, respectively;

[0529] gg) are SEQ ID NOs: 1569 and 1604, respectively;

[0530] hh) are SEQ ID NOs: 1570 and 1605, respectively;

[0531] ii) SEQ ID NO: 1571 and 1606, respectively; and

[0532] jj) are SEQ ID NOs: 1681 and 1586,

[0533] wherein the oligonucleotide comprises at least one modified sugar, a 5' terminal phosphate group, at least one modified internucleotide bond and at least one modified base.

[0534] In some embodiments, the sense strand and the antisense strand comprise a nucleotide sequence selected from the group consisting of:

[0535] a) SEQ ID NO: 1540 and 1575, respectively;

[0536] b) SEQ ID NOs: 1544 and 1579, respectively;

[0537] c) SEQ ID NOs: 1546 and 1581, respectively;

[0538] d) SEQ ID NOs: 1551 and 1586, respectively;

[0539] e) SEQ ID NOs: 1552 and 1587, respectively;

[0540] f) SEQ ID NOs: 1553 and 1588, respectively;

[0541] g) SEQ ID NOs: 1558 and 1594, respectively;

[0542] h) SEQ ID NOs: 1560 and 1595, respectively;

[0543] i) SEQ ID NOs: 1564 and 1599, respectively;

[0544] j) are SEQ ID NOs: 1565 and 1600, respectively;

[0545] k) are SEQ ID NOs: 1566 and 1601, respectively;

[0546] 1) are SEQ ID NOs: 1570 and 1605, respectively; and

[0547] m) are SEQ ID NOs: 1681 and 1586, respectively,

[0548] wherein the oligonucleotide comprises at least one modified sugar, a 5' terminal phosphate group, at least one modified internucleotide bond and at least one modified base.

[0549] In some embodiments, the sense strand and the antisense strand comprise a nucleotide sequence selected from the group consisting of:

[0550] a) SEQ ID NO: 1553 and 1588, respectively;

[0551] b) SEQ ID NOs: 1560 and 1595, respectively;

[0552] c) SEQ ID NOs: 1564 and 1599, respectively;

[0553] d) SEQ ID NOs: 1551 and 1586, respectively;

[0554] e) SEQ ID NOs: 1570 and 1605, respectively; and

[0555] f) are SEQ ID NOs: 1681 and 1586, respectively,

[0556] wherein the oligonucleotide comprises at least one modified sugar, a 5' terminal phosphate group, at least one modified internucleotide bond and at least one modified base.

[0557] The number of modifications on the oligonucleotide and the positions of those nucleotide modifications may affect the characteristics of the oligonucleotide. For example, the oligonucleotide can be delivered in vivo by being included in a lipid nanoparticle (LNP) or a similar carrier. However, when the oligonucleotide is not protected by LNP or a similar carrier, at least some nucleotides may be advantageously modified. Therefore, in some embodiments, all or substantially all 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 moieties of all nucleotides constituting the oligonucleotide are modified at the 2' position. The modification can be reversible or irreversible. In some embodiments, the oligonucleotide has the number and type of modified nucleotides that are sufficient to facilitate the desired characteristics (for example, to prevent enzymatic degradation, the ability and / or thermodynamic stability of the desired cells targeted after in vivo administration).

[0558] Sugar modification

[0559] In some embodiments, oligonucleotides include modified sugars. In some embodiments, modified sugars (also referred to herein as sugar analogs) include modified deoxyribose or ribose moieties, for example, wherein one or more modifications occur at 2', 3', 4' and / or 5' carbon positions of sugars. In some embodiments, modified sugars may also include non-natural alternative carbon structures, such as those present in the following structures: locked nucleic acids ("LNAs"; see, for example, Koshkin et al. (1998) Tetrahedon 54:3607-30), non-locked nucleic acids ("UNA"; see, for example, Snead et al. (2013) Mol. Ther-Nucl. Acids 2:e103) and bridged nucleic acids ("BNAs"; see, for example, Imanishi and Obika (2002) Chem Commun. (Camb) 21:1653-59).

[0560] In some embodiments, the nucleotide modification in the sugar includes a 2'-modification. In some embodiments, the 2'-modification can be 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, 2'-F, 2'-aminoethyl (EA), 2'-OMe, 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA) or 2'-deoxy-2'-fluoro-β-d-arabinose nucleic acid (2'-FANA). In some embodiments, the modification is 2'-F, 2'-OMe or 2'-MOE. In some embodiments, the modified sugar includes a modification of the sugar ring, which may include a modification of one or more carbons of the sugar ring. For example, the modification of the sugar of the nucleotide may include the 2'-oxygen of the sugar being connected to the 1'-carbon or 4'-carbon of the sugar, or the 2'-oxygen being connected to the 1'-carbon or 4'-carbon via an ethylene or methylene bridge. In some embodiments, the modified nucleotide has an acyclic sugar lacking a 2'-carbon to 3'-carbon bond. In some embodiments, the modified nucleotide has a thiol group (eg, at the 4'-position of the sugar).

[0561] 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).

[0562] In some embodiments, all nucleotides of the sense strand are modified. In some embodiments, all nucleotides of the antisense strand are modified. In some embodiments, all nucleotides (i.e., both the sense strand and the antisense strand) are modified. In some embodiments, the modified nucleotides comprise a 2'-modification (e.g., 2'-F or 2'-OMe, 2'-MOE, and 2'-deoxy-2'-fluoro-β-d-arabinose nucleic acid). In some embodiments, the modified nucleotides comprise a 2'-modification (e.g., 2'-F or 2'-OMe).

[0563] In some embodiments, the present disclosure provides oligonucleotides with different modification patterns.In some embodiments, the oligonucleotide comprises a sense strand sequence having a modification pattern as listed in the Examples and Sequence Listing and an antisense strand having a modification pattern as listed in the Examples and Sequence Listing.

[0564] In some embodiments, the oligonucleotide comprises an antisense strand having 2'-F modified nucleotides. In other embodiments, the oligonucleotide comprises an antisense strand having 2'-F and 2'-OMe modified nucleotides. In some embodiments, the oligonucleotide comprises a sense strand having 2'-F modified nucleotides. In other embodiments, the oligonucleotide comprises a sense strand having 2'-F and 2'-OMe modified nucleotides.

[0565] In some embodiments, the oligonucleotide comprises a sense strand, wherein about 10% to about 15%, or 10%, 11%, 12%, 13%, 14%, or 15% of the nucleotides of the sense strand comprise a 2'-F modification. In some embodiments, the oligonucleotide comprises a sense strand, wherein about 18% to about 23%, or 18%, 19%, 20%, 21%, 22%, or 23% of the nucleotides comprise a 2'-F modification. In some embodiments, the oligonucleotide comprises a sense strand, wherein about 38% to about 43%, or 38%, 39%, 40%, 41%, 42%, or 43% of the nucleotides comprise 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.

[0566] In some embodiments, the oligonucleotide comprises an antisense strand wherein about 25% to about 35%, or 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35% of the nucleotides comprise 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.

[0567] In some embodiments, one or more of positions 8, 9, 10, or 11 of the sense strand of 36 nucleotides is modified with a 2'-F group. In some embodiments, one or more of positions 8, 9, 10, or 11 of the sense strand comprising a stem-loop is modified with a 2'-F group. In some embodiments, the sugar moiety at each nucleotide at positions 1-7 and 12-20 of the sense strand of 36 nucleotides is modified with 2'-OMe. In some embodiments, the sugar moiety at each nucleotide at positions 1-7 and 12-20 of the sense strand comprising a stem-loop is modified with 2'-OMe. In some embodiments, the sugar moiety at each nucleotide at positions 1-7 and 12-36 in the sense strand is modified with 2'-OMe.

[0568] In some embodiments, one or more of positions 3, 5, 8, 10, 12, 13, 15, and 17 of the sense strand is modified with a 2'-F group.

[0569] In some embodiments, the antisense strand has 3 nucleotides modified with 2'-F at the 2'-position of the sugar moiety. In some embodiments, the sugar moieties at positions 2, 5, and 14 of the antisense strand and optionally up to 3 nucleotides at positions 1, 3, 7, and 10 are modified with 2'-F. In some embodiments, the sugar moieties at positions 2, 5, and 14 of the antisense strand and optionally up to 3 nucleotides at positions 3, 4, 7, and 10 are modified with 2'-F. In other embodiments, the sugar moieties at each of positions 2, 5, and 14 of the antisense strand are modified with 2'-F. In other embodiments, the sugar moieties at each of positions 1, 2, 5, and 14 of the antisense strand are modified with 2'-F. In other embodiments, the sugar moieties at each of positions 2, 4, 5, and 14 of the antisense strand are modified with 2'-F. In other embodiments, the sugar moieties at each of positions 1, 2, 3, 5, 7, and 14 of the antisense strand are modified with 2'-F. In other embodiments, the sugar moieties at each of positions 1, 2, 3, 5, 7, and 14 of the antisense strand are modified with 2'-F. In other embodiments, the sugar moiety at each of positions 2, 3, 4, 5, 7, and 14 of the antisense strand is modified with 2'-F. In another embodiment, the sugar moiety at each of positions 1, 2, 3, 5, 10, and 14 of the antisense strand is modified with 2'-F. In other embodiments, the sugar moiety at each of positions 2, 3, 4, 5, 10, and 14 of the antisense strand is modified with 2'-F. In another embodiment, the sugar moiety at each of positions 2, 3, 5, 7, 10, and 14 of the antisense strand is modified with 2'-F. In other embodiments, the sugar moiety at each of positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand that forms a duplex with the 36-nucleotide sense strand is modified with 2'-F. In some embodiments, the sugar moiety at each of positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand that forms a duplex with the sense strand comprising a stem-loop is modified with 2'-F.

[0570] In some embodiments, an oligonucleotide comprises an antisense strand having a sugar moiety modified with 2'-F at positions 2 and 14. In some embodiments, an oligonucleotide comprises an antisense strand having a sugar moiety modified with 2'-F at positions 2, 5, and 14. In some embodiments, an oligonucleotide comprises an antisense strand having a sugar moiety modified with 2'-F at positions 1, 2, 5, and 14. In some embodiments, an oligonucleotide comprises an antisense strand having a sugar moiety modified with 2'-F at positions 2, 4, 5, and 14. In some embodiments, an oligonucleotide comprises an antisense strand having a sugar moiety modified with 2'-F at positions 1, 2, 3, 5, 7, and 14. In some embodiments, an oligonucleotide comprises an antisense strand having a sugar moiety modified with 2'-F at positions 2, 3, 4, 5, 7, and 14. In some embodiments, the oligonucleotide comprises an antisense strand having a sugar moiety modified with 2'-F at positions 1, 2, 3, 5, 10, and 14. In some embodiments, the oligonucleotide comprises an antisense strand having a sugar moiety modified with 2'-F at positions 2, 3, 4, 5, 10, and 14. In some embodiments, the oligonucleotide comprises an antisense strand having a sugar moiety modified with 2'-F at positions 2, 3, 4, 5, 10, and 14. In some embodiments, the oligonucleotide comprises a 36-nucleotide sense strand and an antisense strand, wherein the antisense strand comprises a sugar moiety modified with 2'-F at positions 2, 3, 4, 5, 7, 10, and 14. In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand containing a stem-loop, wherein the antisense strand comprises a sugar moiety modified with 2'-F at positions 2, 3, 4, 5, 7, 10, and 14. In some embodiments, the oligonucleotide comprises an antisense strand having a sugar moiety modified with 2'-F at positions 2, 3, 4, 5, 7, 10, 14, 16, and 19.

[0571] In some embodiments, the oligonucleotide comprises an antisense strand having a sugar moiety with each nucleotide at positions 2, 5, and 14 modified with a 2'-F modification and each remaining nucleotide of the antisense strand modified with a sugar moiety selected from the group consisting of: 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, EA, 2'-OMe, 2'-MOE, 2'-O-NMA, and 2'-FANA.

[0572] In some embodiments, the oligonucleotide comprises an antisense strand having a sugar moiety with each nucleotide at positions 1, 2, 5, and 14 modified with a 2'-F and each remaining nucleotide of the antisense strand modified with a sugar moiety selected from the group consisting of: 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, EA, 2'-OMe, 2'-MOE, 2'-O-NMA, and 2'-FANA.

[0573] In some embodiments, the oligonucleotide comprises an antisense strand having a sugar moiety with each nucleotide at positions 2, 4, 5, and 14 modified with a 2'-F and each remaining nucleotide of the antisense strand modified with a sugar moiety selected from the group consisting of: 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, EA, 2'-OMe, 2'-MOE, 2'-O-NMA, and 2'-FANA.

[0574] In some embodiments, the oligonucleotide comprises an antisense strand having a sugar moiety with each nucleotide at positions 1, 2, 3, 5, 7, and 14 modified with a 2'-F and each remaining nucleotide of the antisense strand modified with a sugar moiety selected from the group consisting of: 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, EA, 2'-OMe, 2'-MOE, 2'-O-NMA, and 2'-FANA.

[0575] In some embodiments, the oligonucleotide comprises an antisense strand having a sugar moiety with each nucleotide at positions 2, 3, 4, 5, 7, and 14 modified with a 2'-F and each remaining nucleotide of the antisense strand modified with a sugar moiety selected from the group consisting of: 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, EA, 2'-OMe, 2'-MOE, 2'-O-NMA, and 2'-FANA.

[0576] In some embodiments, the oligonucleotide comprises an antisense strand having a sugar moiety with each nucleotide at positions 1, 2, 3, 5, 10, and 14 modified with a 2'-F and each remaining nucleotide of the antisense strand modified with a sugar moiety selected from the group consisting of: 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, EA, 2'-OMe, 2'-MOE, 2'-O-NMA, and 2'-FANA.

[0577] In some embodiments, the oligonucleotide comprises an antisense strand having a sugar moiety with each nucleotide at positions 2, 3, 4, 5, 10, and 14 modified with a 2'-F and each remaining nucleotide of the antisense strand modified with a sugar moiety selected from the group consisting of: 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, EA, 2'-OMe, 2'-MOE, 2'-O-NMA, and 2'-FANA.

[0578] In some embodiments, the oligonucleotide comprises an antisense strand having a sugar moiety with each nucleotide at positions 2, 3, 5, 7, 10, and 14 modified with a 2'-F and each remaining nucleotide of the antisense strand modified with a sugar moiety selected from the group consisting of: 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, EA, 2'-OMe, 2'-MOE, 2'-O-NMA, and 2'-FANA.

[0579] In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand of 36 nucleotides, wherein the antisense strand comprises a sugar moiety modified with 2'-F at positions 2, 3, 4, 5, 7, 10, and 14 and each of the remaining nucleotides of the antisense strand is modified with a sugar moiety modified with a modification selected from the group consisting of: 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, EA, 2'-OMe, 2'-MOE, 2'-O-NMA, and 2'-FANA. In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand comprising a stem-loop, wherein the antisense strand comprises a sugar moiety modified with 2'-F at positions 2, 3, 4, 5, 7, 10, and 14 and each of the remaining nucleotides of the antisense strand is modified with a sugar moiety modified with a modification selected from the group consisting of: 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, EA, 2'-OMe, 2'-MOE, 2'-O-NMA, and 2'-FANA.

[0580] In some embodiments, the oligonucleotide comprises an antisense strand having a sugar moiety with each nucleotide at positions 2, 3, 4, 5, 7, 10, 14, 16, and 19 modified with a 2'-F modification and each remaining nucleotide of the antisense strand modified with a sugar moiety selected from the group consisting of: 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, EA, 2'-OMe, 2'-MOE, 2'-O-NMA, and 2'-FANA.

[0581] In some embodiments, the oligonucleotide comprises an antisense strand having a sugar moiety modified with a 2'-F 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.

[0582] In some embodiments, the oligonucleotide comprises an antisense strand having a sugar moiety modified with 2'-OMe 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.

[0583] In some embodiments, the oligonucleotide comprises an antisense strand having a sugar moiety modified 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 with a modification selected from the group consisting of: 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, EA, 2'-OMe, 2'-MOE, 2'-O-NMA, and 2'-FANA.

[0584] In some embodiments, the oligonucleotide comprises a sense strand of 36 nucleotides having a sugar moiety modified with 2'-F at positions 8-11. In some embodiments, the oligonucleotide comprises a sense strand comprising a stem-loop and a sugar moiety modified with 2'-F at positions 8-11. In some embodiments, the oligonucleotide comprises a sense strand of 36 nucleotides having a sugar moiety modified with 2'-OMe at positions 1-7 and 12-17 or 12-20. In some embodiments, the oligonucleotide comprises a sense strand comprising a stem-loop and a sugar moiety modified with 2'-OMe at positions 1-7 and 12-17 or 12-20. In some embodiments, the oligonucleotide comprises a sense strand of 36 nucleotides having a sugar moiety modified with 2'-OMe at positions 1-7 and 12-17, 12-20, or 12-22. In some embodiments, the oligonucleotide comprises a sense strand comprising a stem-loop and a sugar moiety modified with 2'-OMe at positions 1-7 and 12-17, 12-20, or 12-22. In some embodiments, the oligonucleotide comprises a 36-nucleotide sense strand having a sugar moiety modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, EA, 2'-OMe, 2'-MOE, 2'-O-NMA, and 2'-FANA at each nucleotide at positions 1-7 and 12-17 or 12-20 of the sense strand. In some embodiments, the oligonucleotide comprises a sense strand comprising a stem-loop and having a sugar moiety modified with a modification selected from the group consisting of: 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, EA, 2'-OMe, 2'-MOE, 2'-O-NMA, and 2'-FANA at each nucleotide at positions 1-7 and 12-17, 12-20, or 12-22 of the sense strand. In some embodiments, the oligonucleotide comprises a 36-nucleotide sense strand having a sugar moiety modified with a modification selected from the group consisting of: 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, EA, 2'-OMe, 2'-MOE, 2'-O-NMA, and 2'-FANA at each nucleotide at positions 1-7 and 12-17, 12-20, or 12-22 of the sense strand. In some embodiments, the oligonucleotide comprises a sense strand comprising a stem-loop and a sugar moiety wherein each nucleotide at positions 1-7 and 12-17, 12-20, or 12-22 of the sense strand is modified with a modification selected from the group consisting of: 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, EA, 2'-OMe, 2'-MOE, 2'-O-NMA, and 2'-FANA.

[0585] In some embodiments, the oligonucleotide comprises a sense strand having a sugar moiety modified with 2'-F at positions 3, 5, 8, 10, 12, 13, 15, and 17. In some embodiments, the oligonucleotide comprises a sense strand having a sugar moiety modified with 2'-OMe at positions 1, 2, 4, 6, 7, 9, 11, 14, 16, and 18-20. In some embodiments, the oligonucleotide comprises a sense strand having a sugar moiety modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, EA, 2'-OMe, 2'-MOE, 2'-O-NMA, and 2'-FANA at each nucleotide at positions 1, 2, 4, 6, 7, 9, 11, 14, 16, and 18-20 of the sense strand. In some embodiments, the oligonucleotide comprises a sense strand having a sugar moiety modified with a modification selected from the group consisting of: 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, EA, 2'-OMe, 2'-MOE, 2'-O-NMA, and 2'-FANA at each nucleotide at positions 1-7 and 12-17, 12-20, or 12-22 of the sense strand.

[0586] In some embodiments, the oligonucleotide comprises a sense strand having a sugar moiety with a 2'-F modification 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.

[0587] In some embodiments, the oligonucleotide comprises a sense strand having a sugar moiety modified with 2'-OMe 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.

[0588] In some embodiments, the oligonucleotide comprises a sense strand having a sugar moiety modified 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 with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, EA, 2'-OMe, 2'-MOE, 2'-O-NMA, and 2'-FANA.

[0589] In some embodiments, the oligonucleotide comprises an antisense strand having a sugar moiety with each nucleotide at positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand being modified with a 2'-F modified sugar moiety and each remaining nucleotide of the antisense strand being modified with a modification selected from the group consisting of: 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, EA, 2'-OMe, 2'-MOE, 2'-O-NMA, and 2'-FANA; and a sense strand having a sugar moiety with each nucleotide at positions 8-11 of the sense strand being modified with a 2'-F modified sugar moiety and each remaining nucleotide of the sense strand being modified with a modification selected from the group consisting of: 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, EA, 2'-OMe, 2'-MOE, 2'-O-NMA, and 2'-FANA. In some embodiments, an oligonucleotide comprises an antisense strand having a sugar moiety with each nucleotide at positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand being modified with a 2'-F modified sugar moiety and each remaining nucleotide of the antisense strand being modified with a modification selected from the group consisting of: 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, EA, 2'-OMe, 2'-MOE, 2'-O-NMA, and 2'-FANA; and a sense strand having a stem-loop and a sugar moiety with each nucleotide at positions 8-11 of the sense strand being modified with a 2'-F modified sugar moiety and each remaining nucleotide of the sense strand being modified with a modification selected from the group consisting of: 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, 2'-EA, 2'-OMe, 2'-MOE, 2'-O-NMA, and 2'-FANA.

[0590] In some embodiments, the oligonucleotide comprises an antisense strand having a sugar moiety with each nucleotide at positions 2, 3, 4, 5, 7, 10, 14, 16, and 19 of the antisense strand modified with a 2'-F modified sugar moiety and each of the remaining nucleotides of the antisense strand modified with a modification selected from the group consisting of: 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, EA, 2'-OMe, 2'-MOE, 2'-O-NMA, and 2 '-FANA; and a sense strand having a sugar moiety with each nucleotide at positions 3, 5, 8, 10, 12, 13, 15, and 17 of the sense strand modified with a 2'-F modified sugar moiety and each remaining nucleotide of the sense strand modified with a sugar moiety selected from the group consisting of: 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, EA, 2'-OMe, 2'-MOE, 2'-O-NMA, and 2'-FANA.

[0591] In some embodiments, the sense strand and the antisense strand of the oligonucleotide comprise a nucleotide sequence selected from the group consisting of:

[0592] a) SEQ ID NO: 1537 and 1572, respectively;

[0593] b) SEQ ID NOs: 1538 and 1573, respectively;

[0594] c) SEQ ID NOs: 1539 and 1574, respectively;

[0595] d) SEQ ID NOs: 1540 and 1575, respectively;

[0596] e) SEQ ID NOs: 1541 and 1576, respectively;

[0597] f) SEQ ID NO: 1542 and 1577, respectively;

[0598] g) SEQ ID NOs: 1543 and 1578, respectively;

[0599] h) SEQ ID NOs: 1544 and 1579, respectively;

[0600] i) SEQ ID NOs: 1545 and 1580, respectively;

[0601] j) SEQ ID NOs: 1546 and 1581, respectively;

[0602] k) SEQ ID NOs: 1547 and 1582, respectively;

[0603] 1) are SEQ ID NOs: 1548 and 1583, respectively;

[0604] m) are SEQ ID NOs: 1549 and 1584, respectively;

[0605] n) are SEQ ID NOs: 1550 and 1585, respectively;

[0606] o) are SEQ ID NOs: 1551 and 1586, respectively;

[0607] p) are SEQ ID NOs: 1552 and 1587, respectively;

[0608] q) are SEQ ID NOs: 1553 and 1588, respectively;

[0609] r) are SEQ ID NOs: 1554 and 1589, respectively;

[0610] s) are SEQ ID NOs: 1555 and 1590, respectively;

[0611] t) are SEQ ID NOs: 1556 and 1591, respectively;

[0612] u) are SEQ ID NOs: 1557 and 1592, respectively;

[0613] v) SEQ ID NOs: 1558 and 1593, respectively;

[0614] w) are SEQ ID NOs: 1559 and 1594, respectively;

[0615] x) are SEQ ID NOs: 1560 and 1595, respectively;

[0616] y) are SEQ ID NOs: 1561 and 1596, respectively;

[0617] z) are SEQ ID NOs: 1562 and 1597, respectively;

[0618] aa) are SEQ ID NOs: 1563 and 1598, respectively;

[0619] bb) are SEQ ID NOs: 1564 and 1599, respectively;

[0620] cc) are SEQ ID NOs: 1565 and 1600, respectively;

[0621] dd) are SEQ ID NOs: 1566 and 1601, respectively;

[0622] ee) are SEQ ID NOs: 1567 and 1602, respectively;

[0623] ff) are SEQ ID NOs: 1568 and 1603, respectively;

[0624] gg) are SEQ ID NOs: 1569 and 1604, respectively;

[0625] hh) are SEQ ID NOs: 1570 and 1605, respectively;

[0626] ii) SEQ ID NO: 1571 and 1606, respectively; and

[0627] jj) are SEQ ID NOs: 1681 and 1586,

[0628] wherein one or more of positions 3, 5, 8, 10, 12, 13, 15, or 17 of the sense strand is modified with a 2'-F group.

[0629] In some embodiments, the sense strand and the antisense strand of the oligonucleotide comprise a nucleotide sequence selected from the group consisting of:

[0630] a) SEQ ID NO: 1540 and 1575, respectively;

[0631] b) SEQ ID NOs: 1544 and 1579, respectively;

[0632] c) SEQ ID NOs: 1546 and 1581, respectively;

[0633] d) SEQ ID NOs: 1551 and 1586, respectively;

[0634] e) SEQ ID NOs: 1552 and 1587, respectively;

[0635] f) SEQ ID NOs: 1553 and 1588, respectively;

[0636] g) SEQ ID NOs: 1558 and 1594, respectively;

[0637] h) SEQ ID NOs: 1560 and 1595, respectively;

[0638] i) SEQ ID NOs: 1564 and 1599, respectively;

[0639] j) are SEQ ID NOs: 1565 and 1600, respectively;

[0640] k) are SEQ ID NOs: 1566 and 1601, respectively;

[0641] 1) are SEQ ID NOs: 1570 and 1605, respectively; and

[0642] m) are SEQ ID NOs: 1681 and 1586, respectively,

[0643] wherein one or more of positions 3, 5, 8, 10, 12, 13, 15, or 17 of the sense strand is modified with a 2'-F group.

[0644] In some embodiments, the sense strand and the antisense strand of the oligonucleotide comprise a nucleotide sequence selected from the group consisting of:

[0645] a) SEQ ID NO: 1553 and 1588, respectively;

[0646] b) SEQ ID NOs: 1560 and 1595, respectively;

[0647] c) SEQ ID NOs: 1564 and 1599, respectively;

[0648] d) SEQ ID NOs: 1551 and 1586, respectively;

[0649] e) SEQ ID NOs: 1570 and 1605, respectively; and

[0650] f) are SEQ ID NOs: 1681 and 1586, respectively,

[0651] wherein one or more of positions 3, 5, 8, 10, 12, 13, 15, or 17 of the sense strand is modified with a 2'-F group.

[0652] 5' terminal phosphate

[0653] In some embodiments, the oligonucleotides described herein (e.g., RNAi oligonucleotides) include a sense strand and an antisense strand, wherein the antisense strand includes a 5' terminal phosphate. In some embodiments, the 5' terminal phosphate group enhances the interaction with Ago2. However, the oligonucleotides comprising a 5'-phosphate group may be susceptible to degradation via phosphatases or other enzymes, which may limit its in vivo bioavailability. In some embodiments, the oligonucleotides include 5'-phosphate analogs resistant to such degradation. In some embodiments, the phosphate analogs are oxygen methyl phosphonates, vinyl phosphonates or malonyl phosphonates or a combination thereof. In certain embodiments, the 5' end of the oligonucleotide chain is attached to a chemical moiety that simulates the electrostatic and steric properties of a natural 5'-phosphate group ("phosphate mimics").

[0654] In some embodiments, the oligonucleotide has a phosphate analog (referred to as a "4'-phosphate analog") at the 4'-carbon position of the sugar. See, for example, International Patent Application Publication No. WO 2018 / 045317. In some embodiments, the oligonucleotide comprises a 4'-phosphate analog at the 5' terminal nucleotide. In some embodiments, the phosphate analog is an oxymethylphosphonate, wherein the oxygen atom of the oxymethyl is bound to a sugar moiety (e.g., at its 4'-carbon) or an analog thereof. In other embodiments, the 4'-phosphate analog is a thiomethylphosphonate or an aminomethylphosphonate, wherein the sulfur atom of the thiomethyl or the nitrogen atom of the aminomethyl is bound to the 4'-carbon of a sugar moiety or its analog. In certain embodiments, the 4'-phosphate analog is an oxymethylphosphonate. In some embodiments, the oxymethylphosphonate is represented by the formula -O-CH2-PO(OH)2, -O-CH2-PO(OR)2, or -O-CH2-POOH(R), wherein R is independently selected from H, CH3, alkyl, CH2CH2CN, CH2OCOC(CH3)3, CHOCH2CH2Si(CH3)3, or a protecting group. In certain embodiments, the alkyl is CH2CH3. More typically, R is independently selected from H, CH3, or CH2CH3. In some embodiments, R is CH3. In some embodiments, the 4'-phosphate analog is 4'-oxymethylphosphonate. In some embodiments, the modified nucleotide having a 4'-phosphonate analog is uridine. In some embodiments, the modified nucleotide is 4'-O-monomethylphosphonate-2'-O-methyluridine.

[0655] In some embodiments, the sense strand and the antisense strand of the oligonucleotide comprise a nucleotide sequence selected from the group consisting of:

[0656] a) SEQ ID NO: 1537 and 1572, respectively;

[0657] b) SEQ ID NOs: 1538 and 1573, respectively;

[0658] c) SEQ ID NOs: 1539 and 1574, respectively;

[0659] d) SEQ ID NOs: 1540 and 1575, respectively;

[0660] e) SEQ ID NOs: 1541 and 1576, respectively;

[0661] f) SEQ ID NO: 1542 and 1577, respectively;

[0662] g) SEQ ID NOs: 1543 and 1578, respectively;

[0663] h) SEQ ID NOs: 1544 and 1579, respectively;

[0664] i) SEQ ID NOs: 1545 and 1580, respectively;

[0665] j) SEQ ID NOs: 1546 and 1581, respectively;

[0666] k) SEQ ID NOs: 1547 and 1582, respectively;

[0667] 1) are SEQ ID NOs: 1548 and 1583, respectively;

[0668] m) are SEQ ID NOs: 1549 and 1584, respectively;

[0669] n) are SEQ ID NOs: 1550 and 1585, respectively;

[0670] o) are SEQ ID NOs: 1551 and 1586, respectively;

[0671] p) are SEQ ID NOs: 1552 and 1587, respectively;

[0672] q) are SEQ ID NOs: 1553 and 1588, respectively;

[0673] r) are SEQ ID NOs: 1554 and 1589, respectively;

[0674] s) are SEQ ID NOs: 1555 and 1590, respectively;

[0675] t) are SEQ ID NOs: 1556 and 1591, respectively;

[0676] u) are SEQ ID NOs: 1557 and 1592, respectively;

[0677] v) SEQ ID NOs: 1558 and 1593, respectively;

[0678] w) are SEQ ID NOs: 1559 and 1594, respectively;

[0679] x) are SEQ ID NOs: 1560 and 1595, respectively;

[0680] y) are SEQ ID NOs: 1561 and 1596, respectively;

[0681] z) are SEQ ID NOs: 1562 and 1597, respectively;

[0682] aa) are SEQ ID NOs: 1563 and 1598, respectively;

[0683] bb) are SEQ ID NOs: 1564 and 1599, respectively;

[0684] cc) are SEQ ID NOs: 1565 and 1600, respectively;

[0685] dd) are SEQ ID NOs: 1566 and 1601, respectively;

[0686] ee) are SEQ ID NOs: 1567 and 1602, respectively;

[0687] ff) are SEQ ID NOs: 1568 and 1603, respectively;

[0688] gg) are SEQ ID NOs: 1569 and 1604, respectively;

[0689] hh) are SEQ ID NOs: 1570 and 1605, respectively;

[0690] ii) SEQ ID NO: 1571 and 1606, respectively; and

[0691] jj) are SEQ ID NOs: 1681 and 1586,

[0692] Wherein the oligonucleotide comprises a 5' terminal phosphate, optionally a 5' terminal phosphate analog.

[0693] In some embodiments, the sense strand and the antisense strand of the oligonucleotide comprise a nucleotide sequence selected from the group consisting of:

[0694] a) SEQ ID NO: 1540 and 1575, respectively;

[0695] b) SEQ ID NOs: 1544 and 1579, respectively;

[0696] c) SEQ ID NOs: 1546 and 1581, respectively;

[0697] d) SEQ ID NOs: 1551 and 1586, respectively;

[0698] e) SEQ ID NOs: 1552 and 1587, respectively;

[0699] f) SEQ ID NOs: 1553 and 1588, respectively;

[0700] g) SEQ ID NOs: 1558 and 1594, respectively;

[0701] h) SEQ ID NOs: 1560 and 1595, respectively;

[0702] i) SEQ ID NOs: 1564 and 1599, respectively;

[0703] j) are SEQ ID NOs: 1565 and 1600, respectively;

[0704] k) are SEQ ID NOs: 1566 and 1601, respectively;

[0705] 1) are SEQ ID NOs: 1570 and 1605, respectively; and

[0706] m) are SEQ ID NOs: 1681 and 1586, respectively,

[0707] Wherein the oligonucleotide comprises a 5' terminal phosphate, optionally a 5' terminal phosphate analog.

[0708] In some embodiments, the sense strand and the antisense strand of the oligonucleotide comprise a nucleotide sequence selected from the group consisting of:

[0709] a) SEQ ID NO: 1553 and 1588, respectively;

[0710] b) SEQ ID NOs: 1560 and 1595, respectively;

[0711] c) SEQ ID NOs: 1564 and 1599, respectively;

[0712] d) SEQ ID NOs: 1551 and 1586, respectively;

[0713] e) SEQ ID NOs: 1570 and 1605, respectively; and

[0714] f) are SEQ ID NOs: 1681 and 1586, respectively,

[0715] Wherein the oligonucleotide comprises a 5' terminal phosphate, optionally a 5' terminal phosphate analog.

[0716] In some embodiments, the oligonucleotide comprises an antisense strand comprising a 4'-phosphate analog at the 5' terminal nucleotide, wherein the 5' terminal nucleotide comprises the following structure:

[0717]

[0718] 4'-O-monomethylphosphonate-2'-O-methyluridine phosphorothioate [Mephosphonate-4O-mUs].

[0719] Modified internucleotide bonds

[0720] In some embodiments, the oligonucleotide (e.g., RNAi oligonucleotide) comprises a modified internucleotide bond. In some embodiments, the phosphate modification or substitution produces an oligonucleotide comprising at least about 1 (e.g., at least 1, at least 2, at least 3, at least 4, or at least 5) modified internucleotide bonds. In 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 internucleotide bonds. In some embodiments, the oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modified internucleotide bonds.

[0721] The modified internucleotide bond can be a phosphorodithioate bond, a phosphorothioate bond, a phosphotriester bond, a thiocarbonylalkylphosphonate bond, a thiocarbonylalkylphosphotriester bond, a phosphoramidite bond, a phosphonate bond, or a boranophosphate bond. In some embodiments, at least one modified internucleotide bond is a phosphorothioate bond.

[0722] In some embodiments, the oligonucleotide has a phosphorothioate bond 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 bond 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 bond 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.

[0723] In some embodiments, the sense strand and the antisense strand of the oligonucleotide comprise a nucleotide sequence selected from the group consisting of:

[0724] a) SEQ ID NO: 1537 and 1572, respectively;

[0725] b) SEQ ID NOs: 1538 and 1573, respectively;

[0726] c) SEQ ID NOs: 1539 and 1574, respectively;

[0727] d) SEQ ID NOs: 1540 and 1575, respectively;

[0728] e) SEQ ID NOs: 1541 and 1576, respectively;

[0729] f) SEQ ID NO: 1542 and 1577, respectively;

[0730] g) SEQ ID NOs: 1543 and 1578, respectively;

[0731] h) SEQ ID NOs: 1544 and 1579, respectively;

[0732] i) SEQ ID NOs: 1545 and 1580, respectively;

[0733] j) SEQ ID NOs: 1546 and 1581, respectively;

[0734] k) SEQ ID NOs: 1547 and 1582, respectively;

[0735] 1) are SEQ ID NOs: 1548 and 1583, respectively;

[0736] m) are SEQ ID NOs: 1549 and 1584, respectively;

[0737] n) are SEQ ID NOs: 1550 and 1585, respectively;

[0738] o) are SEQ ID NOs: 1551 and 1586, respectively;

[0739] p) are SEQ ID NOs: 1552 and 1587, respectively;

[0740] q) are SEQ ID NOs: 1553 and 1588, respectively;

[0741] r) are SEQ ID NOs: 1554 and 1589, respectively;

[0742] s) are SEQ ID NOs: 1555 and 1590, respectively;

[0743] t) are SEQ ID NOs: 1556 and 1591, respectively;

[0744] u) are SEQ ID NOs: 1557 and 1592, respectively;

[0745] v) SEQ ID NOs: 1558 and 1593, respectively;

[0746] w) are SEQ ID NOs: 1559 and 1594, respectively;

[0747] x) are SEQ ID NOs: 1560 and 1595, respectively;

[0748] y) are SEQ ID NOs: 1561 and 1596, respectively;

[0749] z) are SEQ ID NOs: 1562 and 1597, respectively;

[0750] aa) are SEQ ID NOs: 1563 and 1598, respectively;

[0751] bb) are SEQ ID NOs: 1564 and 1599, respectively;

[0752] cc) are SEQ ID NOs: 1565 and 1600, respectively;

[0753] dd) are SEQ ID NOs: 1566 and 1601, respectively;

[0754] ee) are SEQ ID NOs: 1567 and 1602, respectively;

[0755] ff) are SEQ ID NOs: 1568 and 1603, respectively;

[0756] gg) are SEQ ID NOs: 1569 and 1604, respectively;

[0757] hh) are SEQ ID NOs: 1570 and 1605, respectively;

[0758] ii) SEQ ID NO: 1571 and 1606, respectively; and

[0759] jj) are SEQ ID NOs: 1681 and 1586,

[0760] wherein the oligonucleotide comprises modified internucleotide bonds.

[0761] In some embodiments, the sense strand and the antisense strand of the oligonucleotide comprise a nucleotide sequence selected from the group consisting of:

[0762] a) SEQ ID NO: 1540 and 1575, respectively;

[0763] b) SEQ ID NOs: 1544 and 1579, respectively;

[0764] c) SEQ ID NOs: 1546 and 1581, respectively;

[0765] d) SEQ ID NOs: 1551 and 1586, respectively;

[0766] e) SEQ ID NOs: 1552 and 1587, respectively;

[0767] f) SEQ ID NOs: 1553 and 1588, respectively;

[0768] g) SEQ ID NOs: 1558 and 1594, respectively;

[0769] h) SEQ ID NOs: 1560 and 1595, respectively;

[0770] i) SEQ ID NOs: 1564 and 1599, respectively;

[0771] j) are SEQ ID NOs: 1565 and 1600, respectively;

[0772] k) are SEQ ID NOs: 1566 and 1601, respectively;

[0773] 1) are SEQ ID NOs: 1570 and 1605, respectively; and

[0774] m) are SEQ ID NOs: 1681 and 1586, respectively,

[0775] wherein the oligonucleotide comprises modified internucleotide bonds.

[0776] In some embodiments, the sense strand and the antisense strand of the oligonucleotide comprise a nucleotide sequence selected from the group consisting of:

[0777] a) SEQ ID NO: 1553 and 1588, respectively;

[0778] b) SEQ ID NOs: 1560 and 1595, respectively;

[0779] c) SEQ ID NOs: 1564 and 1599, respectively;

[0780] d) SEQ ID NOs: 1551 and 1586, respectively;

[0781] e) SEQ ID NOs: 1570 and 1605, respectively; and

[0782] f) are SEQ ID NOs: 1681 and 1586, respectively,

[0783] wherein the oligonucleotide comprises modified internucleotide bonds.

[0784] Base modification

[0785] In some embodiments, the oligonucleotides herein (e.g., RNAi oligonucleotides) have one or more modified nucleobases. In some embodiments, the modified nucleobases (also referred to herein as base analogs) are connected at the 1' position of the nucleotide sugar moiety. In certain embodiments, the modified nucleobases are nitrogenous bases. In certain embodiments, the modified nucleobases do not contain nitrogen atoms. See, e.g., U.S. Patent Application Publication No. 2008 / 0274462. In some embodiments, the modified nucleotides include universal bases. In some embodiments, the modified nucleotides do not contain nucleobases (abasic).

[0786] In some embodiments, the sense strand and the antisense strand of the oligonucleotide comprise a nucleotide sequence selected from the group consisting of:

[0787] a) SEQ ID NO: 1537 and 1572, respectively;

[0788] b) SEQ ID NOs: 1538 and 1573, respectively;

[0789] c) SEQ ID NOs: 1539 and 1574, respectively;

[0790] d) SEQ ID NOs: 1540 and 1575, respectively;

[0791] e) SEQ ID NOs: 1541 and 1576, respectively;

[0792] f) SEQ ID NO: 1542 and 1577, respectively;

[0793] g) SEQ ID NOs: 1543 and 1578, respectively;

[0794] h) SEQ ID NOs: 1544 and 1579, respectively;

[0795] i) SEQ ID NOs: 1545 and 1580, respectively;

[0796] j) SEQ ID NOs: 1546 and 1581, respectively;

[0797] k) SEQ ID NOs: 1547 and 1582, respectively;

[0798] 1) are SEQ ID NOs: 1548 and 1583, respectively;

[0799] m) are SEQ ID NOs: 1549 and 1584, respectively;

[0800] n) are SEQ ID NOs: 1550 and 1585, respectively;

[0801] o) are SEQ ID NOs: 1551 and 1586, respectively;

[0802] p) are SEQ ID NOs: 1552 and 1587, respectively;

[0803] q) are SEQ ID NOs: 1553 and 1588, respectively;

[0804] r) are SEQ ID NOs: 1554 and 1589, respectively;

[0805] s) are SEQ ID NOs: 1555 and 1590, respectively;

[0806] t) are SEQ ID NOs: 1556 and 1591, respectively;

[0807] u) are SEQ ID NOs: 1557 and 1592, respectively;

[0808] v) SEQ ID NOs: 1558 and 1593, respectively;

[0809] w) are SEQ ID NOs: 1559 and 1594, respectively;

[0810] x) are SEQ ID NOs: 1560 and 1595, respectively;

[0811] y) are SEQ ID NOs: 1561 and 1596, respectively;

[0812] z) are SEQ ID NOs: 1562 and 1597, respectively;

[0813] aa) are SEQ ID NOs: 1563 and 1598, respectively;

[0814] bb) are SEQ ID NOs: 1564 and 1599, respectively;

[0815] cc) are SEQ ID NOs: 1565 and 1600, respectively;

[0816] dd) are SEQ ID NOs: 1566 and 1601, respectively;

[0817] ee) are SEQ ID NOs: 1567 and 1602, respectively;

[0818] ff) are SEQ ID NOs: 1568 and 1603, respectively;

[0819] gg) are SEQ ID NOs: 1569 and 1604, respectively;

[0820] hh) are SEQ ID NOs: 1570 and 1605, respectively;

[0821] ii) SEQ ID NO: 1571 and 1606, respectively; and

[0822] jj) are SEQ ID NOs: 1681 and 1586,

[0823] The oligonucleotide comprises one or more modified nucleobases.

[0824] In some embodiments, the sense strand and the antisense strand of the oligonucleotide comprise a nucleotide sequence selected from the group consisting of:

[0825] a) SEQ ID NO: 1540 and 1575, respectively;

[0826] b) SEQ ID NOs: 1544 and 1579, respectively;

[0827] c) SEQ ID NOs: 1546 and 1581, respectively;

[0828] d) SEQ ID NOs: 1551 and 1586, respectively;

[0829] e) SEQ ID NOs: 1552 and 1587, respectively;

[0830] f) SEQ ID NOs: 1553 and 1588, respectively;

[0831] g) SEQ ID NOs: 1558 and 1594, respectively;

[0832] h) SEQ ID NOs: 1560 and 1595, respectively;

[0833] i) SEQ ID NOs: 1564 and 1599, respectively;

[0834] j) are SEQ ID NOs: 1565 and 1600, respectively;

[0835] k) are SEQ ID NOs: 1566 and 1601, respectively;

[0836] 1) are SEQ ID NOs: 1570 and 1605, respectively; and

[0837] m) are SEQ ID NOs: 1681 and 1586, respectively,

[0838] The oligonucleotide comprises one or more modified nucleobases.

[0839] In some embodiments, the sense strand and the antisense strand of the oligonucleotide comprise a nucleotide sequence selected from the group consisting of:

[0840] a) SEQ ID NO: 1553 and 1588, respectively;

[0841] b) SEQ ID NOs: 1560 and 1595, respectively;

[0842] c) SEQ ID NOs: 1564 and 1599, respectively;

[0843] d) SEQ ID NOs: 1551 and 1586, respectively;

[0844] e) SEQ ID NOs: 1570 and 1605, respectively; and

[0845] f) are SEQ ID NOs: 1681 and 1586, respectively,

[0846] The oligonucleotide comprises one or more modified nucleobases.

[0847] In some embodiments, the universal base is a heterocyclic moiety located at the 1' position of the nucleotide sugar moiety in the modified nucleotide, or at an equivalent position in a nucleotide sugar moiety substitution, which, when present in a duplex, can be positioned relative to more than one type of base without substantially altering the structure of the duplex. In some embodiments, the ss nucleic acid containing the universal base forms a duplex with the target nucleic acid at a lower Tm than a duplex formed with a complementary nucleic acid compared to a reference ss nucleic acid (e.g., an oligonucleotide) that is completely complementary to the target nucleic acid. In some embodiments, the ss nucleic acid containing the universal base forms a duplex with the target nucleic acid at a higher Tm than a duplex formed with a nucleic acid containing a mismatched base when compared to a reference ss nucleic acid in which the universal base has been replaced by a base to produce a single mismatch.

[0848] Non-limiting examples of universal binding nucleotides include, but are not limited to, inosine, 1-β-D-ribofuranosyl-5-nitroindole, and / or 1-β-D-ribofuranosyl-3-nitropyrrole (see U.S. 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 and Brown (1994) Nucleic Acids Res. 22:4039-43).

[0849] Targeting ligands

[0850] In some embodiments, it is desirable to target an oligonucleotide (e.g., an RNAi oligonucleotide) to one or more cells or one or more organs. This strategy can help avoid undesirable effects in other organs, or avoid excessive loss of oligonucleotides to cells, tissues, or organs that will not benefit from oligonucleotides. Therefore, in some embodiments, oligonucleotides are modified to promote targeting and / or delivery to specific tissues, cells, or organs (e.g., to promote delivery of oligonucleotides to CNS). In some embodiments, an oligonucleotide comprises at least one nucleotide (e.g., 1, 2, 3, 4, 5, 6, or more nucleotides) conjugated to one or more targeting ligands. In some embodiments, the sense strand and antisense strand of an oligonucleotide comprise a nucleotide sequence selected from the group consisting of:

[0851] a) SEQ ID NO: 1537 and 1572, respectively;

[0852] b) SEQ ID NOs: 1538 and 1573, respectively;

[0853] c) SEQ ID NOs: 1539 and 1574, respectively;

[0854] d) SEQ ID NOs: 1540 and 1575, respectively;

[0855] e) SEQ ID NOs: 1541 and 1576, respectively;

[0856] f) SEQ ID NO: 1542 and 1577, respectively;

[0857] g) SEQ ID NOs: 1543 and 1578, respectively;

[0858] h) SEQ ID NOs: 1544 and 1579, respectively;

[0859] i) SEQ ID NOs: 1545 and 1580, respectively;

[0860] j) SEQ ID NOs: 1546 and 1581, respectively;

[0861] k) SEQ ID NOs: 1547 and 1582, respectively;

[0862] 1) are SEQ ID NOs: 1548 and 1583, respectively;

[0863] m) are SEQ ID NOs: 1549 and 1584, respectively;

[0864] n) are SEQ ID NOs: 1550 and 1585, respectively;

[0865] o) are SEQ ID NOs: 1551 and 1586, respectively;

[0866] p) are SEQ ID NOs: 1552 and 1587, respectively;

[0867] q) are SEQ ID NOs: 1553 and 1588, respectively;

[0868] r) are SEQ ID NOs: 1554 and 1589, respectively;

[0869] s) are SEQ ID NOs: 1555 and 1590, respectively;

[0870] t) are SEQ ID NOs: 1556 and 1591, respectively;

[0871] u) are SEQ ID NOs: 1557 and 1592, respectively;

[0872] v) SEQ ID NOs: 1558 and 1593, respectively;

[0873] w) are SEQ ID NOs: 1559 and 1594, respectively;

[0874] x) are SEQ ID NOs: 1560 and 1595, respectively;

[0875] y) are SEQ ID NOs: 1561 and 1596, respectively;

[0876] z) are SEQ ID NOs: 1562 and 1597, respectively;

[0877] aa) are SEQ ID NOs: 1563 and 1598, respectively;

[0878] bb) are SEQ ID NOs: 1564 and 1599, respectively;

[0879] cc) are SEQ ID NOs: 1565 and 1600, respectively;

[0880] dd) are SEQ ID NOs: 1566 and 1601, respectively;

[0881] ee) are SEQ ID NOs: 1567 and 1602, respectively;

[0882] ff) are SEQ ID NOs: 1568 and 1603, respectively;

[0883] gg) are SEQ ID NOs: 1569 and 1604, respectively;

[0884] hh) are SEQ ID NOs: 1570 and 1605, respectively;

[0885] ii) SEQ ID NO: 1571 and 1606, respectively; and

[0886] jj) are SEQ ID NOs: 1681 and 1586,

[0887] wherein the oligonucleotide comprises a targeting ligand conjugated to at least one nucleotide.

[0888] 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 ligands. In some embodiments, the sense strand and the antisense strand of the oligonucleotide comprise a nucleotide sequence selected from the group consisting of:

[0889] a) SEQ ID NO: 1540 and 1575, respectively;

[0890] b) SEQ ID NOs: 1544 and 1579, respectively;

[0891] c) SEQ ID NOs: 1546 and 1581, respectively;

[0892] d) SEQ ID NOs: 1551 and 1586, respectively;

[0893] e) SEQ ID NOs: 1552 and 1587, respectively;

[0894] f) SEQ ID NOs: 1553 and 1588, respectively;

[0895] g) SEQ ID NOs: 1558 and 1594, respectively;

[0896] h) SEQ ID NOs: 1560 and 1595, respectively;

[0897] i) SEQ ID NOs: 1564 and 1599, respectively;

[0898] j) are SEQ ID NOs: 1565 and 1600, respectively;

[0899] k) are SEQ ID NOs: 1566 and 1601, respectively;

[0900] 1) are SEQ ID NOs: 1570 and 1605, respectively; and

[0901] m) are SEQ ID NOs: 1681 and 1586, respectively,

[0902] wherein the oligonucleotide comprises a targeting ligand conjugated to at least one nucleotide.

[0903] 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 ligands. In some embodiments, the sense strand and the antisense strand of the oligonucleotide comprise a nucleotide sequence selected from the group consisting of:

[0904] a) SEQ ID NO: 1553 and 1588, respectively;

[0905] b) SEQ ID NOs: 1560 and 1595, respectively;

[0906] c) SEQ ID NOs: 1564 and 1599, respectively;

[0907] d) SEQ ID NOs: 1551 and 1586, respectively;

[0908] e) SEQ ID NOs: 1570 and 1605, respectively; and

[0909] f) are SEQ ID NOs: 1681 and 1586, respectively,

[0910] wherein the oligonucleotide comprises a targeting ligand conjugated to at least one nucleotide.

[0911] In some embodiments, the targeting ligand comprises a carbohydrate, an amino sugar, cholesterol, a peptide, a polypeptide, or a portion of a protein or a protein (e.g., an antibody or antibody fragment). In some embodiments, the targeting ligand is an aptamer. For example, the targeting ligand can be an RGD peptide for targeting tumor vasculature or glioma cells, a CREKA peptide for targeting tumor vasculature or stoma, transferrin, lactoferrin, or an aptamer targeting transferrin receptor expressed on CNS vasculature, or an anti-EGFR antibody targeting 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.

[0912] In some embodiments, one or more (e.g., 1, 2, 3, 4, 5 or 6) nucleotides of an oligonucleotide are each conjugated to an independent targeting ligand. In some embodiments, 2 to 4 nucleotides of an oligonucleotide are each conjugated to an independent targeting ligand. In some embodiments, a targeting ligand is conjugated to 2 to 4 nucleotides at either end of a sense strand or an antisense strand (e.g., a targeting ligand is conjugated to 2 to 4 nucleotides at the 5' or 3' end of a sense strand or an antisense strand, overhanging or extending), so that the targeting ligand is similar to the bristles of a toothbrush and the oligonucleotide is similar to a toothbrush. For example, an oligonucleotide can include a stem loop at either end of the 5' or 3' end of the sense strand, and 1, 2, 3 or 4 nucleotides of the stem loop can be individually conjugated to a targeting ligand. In some embodiments, an oligonucleotide includes a stem loop at the 3' end of the sense strand, wherein the ring of the stem loop includes triL or tetraL, and wherein 3 or 4 nucleotides constituting triL or tetraL are individually conjugated to a targeting ligand, respectively. 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.

[0913] GalNAc conjugation

[0914] GalNAc is a high affinity ligand for ASGPR, which is expressed primarily on the sinusoidal surface of hepatocytes and plays a major role in binding, internalization, and subsequent clearance of circulating glycoproteins (asialoglycoproteins) containing terminal galactose or GalNAc residues. The conjugation (indirect or direct) of the GalNAc moiety to the oligonucleotides herein (e.g., RNAi oligonucleotides) can be used to target the oligonucleotides to the ASGPR expressed on the cell. In some embodiments, the oligonucleotides are conjugated to at least one or more GalNAc moieties, wherein the GalNAc moieties target the oligonucleotides to the ASGPR expressed on human liver cells (e.g., human hepatocytes). In some embodiments, the GalNAc moieties target the oligonucleotides to the liver.

[0915] In some embodiments, the oligonucleotide is directly or indirectly conjugated to a monovalent GalNAc. In some embodiments, the oligonucleotide is directly or indirectly conjugated to more than one monovalent GalNAc (i.e., conjugated to 2, 3 or 4 monovalent GalNAc moieties, and generally conjugated to 3 or 4 monovalent GalNAc moieties). In some embodiments, the oligonucleotide is conjugated to one or more divalent GalNAc, trivalent GalNAc or tetravalent GalNAc moieties. In some embodiments, the divalent, trivalent or tetravalent GalNAc moieties are conjugated to the oligonucleotide via a branched joint. In some embodiments, the monovalent GalNAc moiety is conjugated to the first nucleotide, and the divalent, trivalent or tetravalent GalNAc moiety is conjugated to the second nucleotide via a branched joint.

[0916] In some embodiments, one or more (e.g., 1, 2, 3, 4, 5, or 6) nucleotides of an oligonucleotide are each conjugated to a GalNAc moiety. In some embodiments, 2 to 4 nucleotides of a tetraL are each conjugated to an independent GalNAc. In some embodiments, 1 to 3 nucleotides of a triL are each conjugated to an independent GalNAc. In some embodiments, a targeting ligand is conjugated to 2 to 4 nucleotides at either end of a sense strand or an antisense strand (e.g., a ligand is conjugated to 2 to 4 nucleotides overhanging or extending at the 5' or 3' end of a sense strand or an antisense strand), such that the GalNAc moiety is similar to the bristles of a toothbrush and the oligonucleotide is similar to a toothbrush. In some embodiments, the GalNAc moiety is conjugated to the nucleotides of the sense strand. For example, 4 GalNAc moieties can be conjugated to the nucleotides in the tetraL of the sense strand, wherein each GalNAc moiety is conjugated to 1 nucleotide.

[0917] In some embodiments, the oligonucleotide comprises tetraL, wherein tetraL is any combination of adenine (A) and guanine (G) nucleotides. In some embodiments, 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):

[0918]

[0919] In some embodiments, tetraL has a monovalent GalNAc attached to any one or more adenine nucleotides of tetraL via any linker described herein, as depicted below (X = heteroatom):

[0920]

[0921] 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:

[0922]

[0923] 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: In some embodiments, the oligonucleotide comprises:

[0924]

[0925] An example of such a conjugation is shown below for a loop from 5' to 3' containing the nucleotide sequence GAAA (L = linker, X = heteroatom), wherein the stem attachment point is shown. Such a loop can be present, for example, at positions 27-30 of the sense strand of any of the sense strands listed in Tables 4 and 5. In the chemical formula, Used to describe the point of attachment to an oligonucleotide chain:

[0926]

[0927] The targeting ligand can be connected to the nucleotide using appropriate methods or chemistry (e.g., click chemistry). In some embodiments, the targeting ligand is conjugated to the nucleotide using a click linker. In some embodiments, an acetal-based linker is used to conjugate the targeting ligand to the nucleotide of any one of the oligonucleotides described herein. Acetal-based linkers are disclosed in, for example, International Patent Application Publication No. WO 2016 / 100401. In some embodiments, the linker is an unstable linker. However, in other embodiments, the linker is stable. The following shows an example of a loop comprising the nucleotide GAAA from 5' to 3', in which the GalNAc moiety is attached to 3 or 4 nucleotides of the loop using an acetal linker. Such a loop may be present at positions 27-30 of any one of the sense strands listed in, for example, Tables 4 and 5. In the chemical formula, is the point of attachment to the oligonucleotide chain:

[0928]

[0929]

[0930] As mentioned, the targeting ligand can be connected to the nucleotide using various appropriate methods or chemical synthesis techniques (e.g., click chemistry). In some embodiments, the targeting ligand is conjugated to the nucleotide using a click linker. In some embodiments, an acetal-based linker is used to conjugate the targeting ligand to the nucleotide of any one of the oligonucleotides described herein. Acetal-based linkers are disclosed in, for example, International Patent Application Publication No. WO 2016 / 100401. In some embodiments, the linker is an unstable linker. However, in other embodiments, the linker is a stable linker.

[0931] In some embodiments, a duplex extension (e.g., 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 GalNAc conjugated thereto.

[0932] In some embodiments, the sense strand and the antisense strand of the oligonucleotide comprise a nucleotide sequence selected from the group consisting of:

[0933] a) SEQ ID NO: 1537 and 1572, respectively;

[0934] b) SEQ ID NOs: 1538 and 1573, respectively;

[0935] c) SEQ ID NOs: 1539 and 1574, respectively;

[0936] d) SEQ ID NOs: 1540 and 1575, respectively;

[0937] e) SEQ ID NOs: 1541 and 1576, respectively;

[0938] f) SEQ ID NO: 1542 and 1577, respectively;

[0939] g) SEQ ID NOs: 1543 and 1578, respectively;

[0940] h) SEQ ID NOs: 1544 and 1579, respectively;

[0941] i) SEQ ID NOs: 1545 and 1580, respectively;

[0942] j) SEQ ID NOs: 1546 and 1581, respectively;

[0943] k) SEQ ID NOs: 1547 and 1582, respectively;

[0944] 1) are SEQ ID NOs: 1548 and 1583, respectively;

[0945] m) are SEQ ID NOs: 1549 and 1584, respectively;

[0946] n) are SEQ ID NOs: 1550 and 1585, respectively;

[0947] o) are SEQ ID NOs: 1551 and 1586, respectively;

[0948] p) are SEQ ID NOs: 1552 and 1587, respectively;

[0949] q) are SEQ ID NOs: 1553 and 1588, respectively;

[0950] r) are SEQ ID NOs: 1554 and 1589, respectively;

[0951] s) are SEQ ID NOs: 1555 and 1590, respectively;

[0952] t) are SEQ ID NOs: 1556 and 1591, respectively;

[0953] u) are SEQ ID NOs: 1557 and 1592, respectively;

[0954] v) SEQ ID NOs: 1558 and 1593, respectively;

[0955] w) are SEQ ID NOs: 1559 and 1594, respectively;

[0956] x) are SEQ ID NOs: 1560 and 1595, respectively;

[0957] y) are SEQ ID NOs: 1561 and 1596, respectively;

[0958] z) are SEQ ID NOs: 1562 and 1597, respectively;

[0959] aa) are SEQ ID NOs: 1563 and 1598, respectively;

[0960] bb) are SEQ ID NOs: 1564 and 1599, respectively;

[0961] cc) are SEQ ID NOs: 1565 and 1600, respectively;

[0962] dd) are SEQ ID NOs: 1566 and 1601, respectively;

[0963] ee) are SEQ ID NOs: 1567 and 1602, respectively;

[0964] ff) are SEQ ID NOs: 1568 and 1603, respectively;

[0965] gg) are SEQ ID NOs: 1569 and 1604, respectively;

[0966] hh) are SEQ ID NOs: 1570 and 1605, respectively;

[0967] ii) SEQ ID NO: 1571 and 1606, respectively; and

[0968] jj) are SEQ ID NOs: 1681 and 1586,

[0969] wherein the oligonucleotide comprises at least one GalNAc moiety conjugated to a nucleotide.

[0970] In some embodiments, the sense strand and the antisense strand of the oligonucleotide comprise a nucleotide sequence selected from the group consisting of:

[0971] a) SEQ ID NO: 1540 and 1575, respectively;

[0972] b) SEQ ID NOs: 1544 and 1579, respectively;

[0973] c) SEQ ID NOs: 1546 and 1581, respectively;

[0974] d) SEQ ID NOs: 1551 and 1586, respectively;

[0975] e) SEQ ID NOs: 1552 and 1587, respectively;

[0976] f) SEQ ID NOs: 1553 and 1588, respectively;

[0977] g) SEQ ID NOs: 1558 and 1594, respectively;

[0978] h) SEQ ID NOs: 1560 and 1595, respectively;

[0979] i) SEQ ID NOs: 1564 and 1599, respectively;

[0980] j) are SEQ ID NOs: 1565 and 1600, respectively;

[0981] k) are SEQ ID NOs: 1566 and 1601, respectively;

[0982] 1) are SEQ ID NOs: 1570 and 1605, respectively; and

[0983] m) are SEQ ID NOs: 1681 and 1586, respectively,

[0984] wherein the oligonucleotide comprises at least one GalNAc moiety conjugated to a nucleotide.

[0985] In some embodiments, the sense strand and the antisense strand of the oligonucleotide comprise a nucleotide sequence selected from the group consisting of:

[0986] a) SEQ ID NO: 1553 and 1588, respectively;

[0987] b) SEQ ID NOs: 1560 and 1595, respectively;

[0988] c) SEQ ID NOs: 1564 and 1599, respectively;

[0989] d) SEQ ID NOs: 1551 and 1586, respectively;

[0990] e) SEQ ID NOs: 1570 and 1605, respectively; and

[0991] f) are SEQ ID NOs: 1681 and 1586, respectively,

[0992] wherein the oligonucleotide comprises at least one GalNAc moiety conjugated to a nucleotide.

[0993] Lipid conjugation

[0994] In some embodiments, one or more lipid moieties are conjugated to the 5' terminal nucleotide of the 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 to a 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.

[0995] 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 a branched chain. In other embodiments, the hydrocarbon chain is a straight chain. In some embodiments, the lipid moiety is a C8-C30 hydrocarbon chain. In certain embodiments, the lipid moiety is C8:0, C10:0, C11:0, C12:0, C14:0, C16:0, C17:0, C18:0, C18:1, C18:2, C22:5, C22:0, C24:0, C26:0, C22:6, C24:1, diacyl C16:0 or diacyl C18:1. In some embodiments, the lipid moiety is a C16 hydrocarbon chain. In some embodiments, the C16 hydrocarbon chain is represented by:

[0996] In some embodiments, the sense strand is 20-22 nucleotides in length and the 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 the 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 the 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 the C16 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 the 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 the 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 the 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 the C16 hydrocarbon chain is conjugated to the 5' terminal nucleotide of the sense strand.

[0997] 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 portion 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 C16 hydrocarbon chain conjugated to the 5' terminal nucleotide of the sense strand.

[0998] In some embodiments, the oligonucleotide comprises (i) a sense strand of 20 nucleotides in length; (ii) an antisense strand of 22 nucleotides in length and 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 portion 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 and 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 and 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 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 and 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 C16 hydrocarbon chain conjugated to the 5' terminal nucleotide of the sense strand.

[0999] In some embodiments, the oligonucleotide comprises (i) an antisense strand of 19-30 nucleotides, the antisense strand comprising a complementary region to a SNCA mRNA target sequence selected from the group consisting of SEQ ID NO: 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 complementary region to a SNCA mRNA target sequence selected from the group consisting of 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, the antisense strand comprising a complementary region 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, the sense strand forming a duplex region with the antisense strand; and (iii) a lipid moiety conjugated to the 5' terminal nucleotide of the sense strand.

[1000] In some embodiments, the oligonucleotide comprises (i) an antisense strand of 19-30 nucleotides, the antisense strand comprising a complementary region to a SNCA mRNA target sequence selected from the group consisting of SEQ ID NO: 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 complementary region to a SNCA mRNA target sequence selected from the group consisting of SEQ ID NOs: 1798, 1817, 1718, 1846, 1852, 1865, 1804, 1721, 1847, 1855, 1864, and 1955; (ii) a sense strand of 19-25 nucleotides forming 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, the antisense strand comprising a complementary region to a SNCA mRNA target sequence selected from the group consisting of SEQ ID NOs: 1865, 1721, 1847, 1846, and 1955; (ii) a sense strand of 19-25 nucleotides, the sense strand forming a duplex region with the antisense strand; and (iii) a hydrocarbon chain conjugated to the 5' terminal nucleotide of the sense strand.

[1001] In some embodiments, the oligonucleotide comprises (i) an antisense strand of 19-30 nucleotides, the antisense strand comprising a complementary region to a SNCA mRNA target sequence selected from the group consisting of SEQ ID NO: 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 complementary region to a SNCA mRNA target sequence selected from the group consisting of SEQ ID NOs: 1798, 1817, 1718, 1846, 1852, 1865, 1804, 1721, 1847, 1855, 1864, and 1955; (ii) a sense strand of 19-25 nucleotides forming 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 complementary region to a SNCA mRNA target sequence selected from the group consisting of SEQ ID NOs: 1865, 1721, 1847, 1846, and 1955; (ii) a sense strand of 19-25 nucleotides forming a duplex region with the antisense strand; and (iii) a C14-C22 hydrocarbon chain conjugated to the 5' terminal nucleotide of the sense strand.

[1002] In some embodiments, the oligonucleotide comprises (i) an antisense strand of 19-30 nucleotides, the antisense strand comprising a complementary region to a SNCA mRNA target sequence selected from the group consisting of SEQ ID NO: 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 C16 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 complementary region to a SNCA mRNA target sequence selected from the group consisting of SEQ ID NOs: 1798, 1817, 1718, 1846, 1852, 1865, 1804, 1721, 1847, 1855, 1864, and 1955; (ii) a sense strand of 19-25 nucleotides forming a duplex region with the antisense strand; and (iii) a C16 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, the antisense strand comprising a complementary region 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, the sense strand forming a duplex region with the antisense strand; and (iii) a C16 hydrocarbon chain conjugated to the 5' terminal nucleotide of the sense strand.

[1003] In some embodiments, the oligonucleotide comprises a sense strand comprising a nucleotide sequence of SEQ ID NO: 1681 and an antisense strand comprising a 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 a nucleotide sequence of SEQ ID NO: 1681 and an antisense strand comprising a 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 a nucleotide sequence of SEQ ID NO: 1681 and an antisense strand comprising a 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 C16 hydrocarbon chain conjugated to the 5' terminal nucleotide of the sense strand.

[1004] Exemplary SNCA-targeting RNAi oligonucleotides

[1005] In some embodiments, the SNCA targeting RNAi oligonucleotide for reducing SNCA gene expression comprises a sense strand and an antisense strand, wherein all nucleotides constituting the sense strand and the antisense strand are modified, wherein the antisense strand comprises a complementary region to the SNCA mRNA target sequence of any one of SEQ ID NOs: 1683-2066, and wherein the length of the complementary region is at least 15 consecutive nucleotides. 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 bond. In some embodiments, the antisense strand and the sense strand comprise one or more 2'-F modified and 2'-OMe modified nucleotides and at least one phosphorothioate bond. In some embodiments, the antisense strand comprises 4 phosphorothioate bonds and the sense strand comprises 1 phosphorothioate bond. In some embodiments, the antisense strand comprises 5 phosphorothioate bonds and the sense strand comprises 1 phosphorothioate bond.

[1006] 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.

[1007] 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.

[1008] 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.

[1009] 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.

[1010] In some embodiments, the oligonucleotide consists of 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.

[1011] In some embodiments, the oligonucleotide consists of 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.

[1012] In some embodiments, the oligonucleotide is used to reduce SNCA gene expression and comprises:

[1013] a sense strand of 36 nucleotides, the sense strand comprising 2'-F modified nucleotides at positions 3, 5, 8, 10, 12, 13, 15, and 17; 2'-OMe modified nucleotides at positions 1, 2, 4, 6, 7, 9, 11, 14, 16, 18-27, and 31-36; GalNAc conjugated nucleotides at positions 28, 29, and 30; and a phosphorothioate bond between positions 1 and 2; and

[1014] An antisense strand of 22 nucleotides, the antisense strand comprising nucleotides modified with 2'-F at positions 2, 3, 4, 5, 7, 10, 14, 16 and 19; 2'-OMe at positions 1, 6, 8, 9, 11, 12, 13, 15, 17, 18 and 20-22; phosphorothioate bonds 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 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 a loop of the stem-loop, optionally wherein positions 27-30 comprise a tetraloop, wherein positions 21 and 22 of the antisense strand comprise an overhang, and wherein the sense strand and the antisense strand comprise a nucleotide sequence selected from the group consisting of:

[1015] a) SEQ ID NO: 1537 and 1572, respectively;

[1016] b) SEQ ID NOs: 1538 and 1573, respectively;

[1017] c) SEQ ID NOs: 1539 and 1574, respectively;

[1018] d) SEQ ID NOs: 1540 and 1575, respectively;

[1019] e) SEQ ID NOs: 1541 and 1576, respectively;

[1020] f) SEQ ID NO: 1542 and 1577, respectively;

[1021] g) SEQ ID NOs: 1543 and 1578, respectively;

[1022] h) SEQ ID NOs: 1544 and 1579, respectively;

[1023] i) SEQ ID NOs: 1545 and 1580, respectively;

[1024] j) SEQ ID NOs: 1546 and 1581, respectively;

[1025] k) SEQ ID NOs: 1547 and 1582, respectively;

[1026] 1) are SEQ ID NOs: 1548 and 1583, respectively;

[1027] m) are SEQ ID NOs: 1549 and 1584, respectively;

[1028] n) are SEQ ID NOs: 1550 and 1585, respectively;

[1029] o) are SEQ ID NOs: 1551 and 1586, respectively;

[1030] p) are SEQ ID NOs: 1552 and 1587, respectively;

[1031] q) are SEQ ID NOs: 1553 and 1588, respectively;

[1032] r) are SEQ ID NOs: 1554 and 1589, respectively;

[1033] s) are SEQ ID NOs: 1555 and 1590, respectively;

[1034] t) are SEQ ID NOs: 1556 and 1591, respectively;

[1035] u) are SEQ ID NOs: 1557 and 1592, respectively;

[1036] v) SEQ ID NOs: 1558 and 1593, respectively;

[1037] w) are SEQ ID NOs: 1559 and 1594, respectively;

[1038] x) are SEQ ID NOs: 1560 and 1595, respectively;

[1039] y) are SEQ ID NOs: 1561 and 1596, respectively;

[1040] z) are SEQ ID NOs: 1562 and 1597, respectively;

[1041] aa) are SEQ ID NOs: 1563 and 1598, respectively;

[1042] bb) are SEQ ID NOs: 1564 and 1599, respectively;

[1043] cc) are SEQ ID NOs: 1565 and 1600, respectively;

[1044] dd) are SEQ ID NOs: 1566 and 1601, respectively;

[1045] ee) are SEQ ID NOs: 1567 and 1602, respectively;

[1046] ff) are SEQ ID NOs: 1568 and 1603, respectively;

[1047] gg) are SEQ ID NOs: 1569 and 1604, respectively;

[1048] hh) are SEQ ID NOs: 1570 and 1605, respectively; and

[1049] ii) are SEQ ID NOs: 1571 and 1606, respectively.

[1050] In some embodiments, the oligonucleotide is used to reduce SNCA gene expression and comprises:

[1051] a sense strand of 36 nucleotides, the sense strand comprising 2'-F modified nucleotides at positions 3, 5, 8, 10, 12, 13, 15, and 17; 2'-OMe modified nucleotides at positions 1, 2, 4, 6, 7, 9, 11, 14, 16, 18-27, and 31-36; GalNAc conjugated nucleotides at positions 28, 29, and 30; and a phosphorothioate bond between positions 1 and 2; and

[1052] The invention relates to an antisense strand of 22 nucleotides, the antisense strand comprising nucleotides modified with 2'-F at positions 2, 3, 4, 5, 7, 10, 14, 16 and 19; 2'-OMe at positions 1, 6, 8, 9, 11, 12, 13, 15, 17, 18 and 20-22; phosphorothioate bonds 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' The 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 a loop of the stem-loop, optionally wherein positions 27-30 comprise tetraL, wherein positions 21 and 22 of the antisense strand comprise an overhang, and wherein the sense strand and the antisense strand comprise a nucleotide sequence selected from the group consisting of:

[1053] a) SEQ ID NO: 1540 and 1575, respectively;

[1054] b) SEQ ID NOs: 1544 and 1579, respectively;

[1055] c) SEQ ID NOs: 1546 and 1581, respectively;

[1056] d) SEQ ID NOs: 1551 and 1586, respectively;

[1057] e) SEQ ID NOs: 1552 and 1587, respectively;

[1058] f) SEQ ID NOs: 1553 and 1588, respectively;

[1059] g) SEQ ID NOs: 1558 and 1594, respectively;

[1060] h) SEQ ID NOs: 1560 and 1595, respectively;

[1061] i) SEQ ID NOs: 1564 and 1599, respectively;

[1062] j) are SEQ ID NOs: 1565 and 1600, respectively;

[1063] k) SEQ ID NOs: 1566 and 1601, respectively; and

[1064] 1) are SEQ ID NOs: 1570 and 1605, respectively.

[1065] In some embodiments, the oligonucleotide is used to reduce SNCA gene expression and comprises:

[1066] a sense strand of 36 nucleotides, the sense strand comprising 2'-F modified nucleotides at positions 3, 5, 8, 10, 12, 13, 15, and 17; 2'-OMe modified nucleotides at positions 1, 2, 4, 6, 7, 9, 11, 14, 16, 18-27, and 31-36; GalNAc conjugated nucleotides at positions 28, 29, and 30; and a phosphorothioate bond between positions 1 and 2; and

[1067] The invention relates to an antisense strand of 22 nucleotides, the antisense strand comprising nucleotides modified with 2'-F at positions 2, 3, 4, 5, 7, 10, 14, 16 and 19; 2'-OMe at positions 1, 6, 8, 9, 11, 12, 13, 15, 17, 18 and 20-22; phosphorothioate bonds 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' The 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 a loop of the stem-loop, optionally wherein positions 27-30 comprise tetraL, wherein positions 21 and 22 of the antisense strand comprise an overhang, and wherein the sense strand and the antisense strand comprise a nucleotide sequence selected from the group consisting of:

[1068] a) SEQ ID NO: 1553 and 1588, respectively;

[1069] b) SEQ ID NOs: 1560 and 1595, respectively;

[1070] c) SEQ ID NOs: 1564 and 1599, respectively;

[1071] d) SEQ ID NO: 1551 and 1586, respectively; and

[1072] e) are SEQ ID NOs: 1570 and 1605, respectively.

[1073] In some embodiments, the oligonucleotide is used to reduce SNCA gene expression and comprises:

[1074] a 20-nucleotide sense strand comprising 2'-F modified nucleotides at positions 3, 5, 8, 10, 12, 13, 15, and 17; 2'-OMe modified nucleotides at positions 2, 4, 6, 7, 9, 11, 14, 16, and 18-20; a C16 hydrocarbon chain conjugated to the nucleotide at position 1; and phosphorothioate bonds between positions 1 and 2, between positions 18 and 19, and between positions 19 and 20; and

[1075] The invention relates to an antisense strand of 22 nucleotides, the antisense strand comprising nucleotides modified with 2'-F at positions 2, 3, 4, 5, 7, 10, 14, 16 and 19; 2'-OMe at positions 1, 6, 8, 9, 11, 12, 13, 15, 17, 18 and 20-22; phosphorothioate bonds 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 overhangs, and wherein the sense strand and the antisense strand comprise the nucleotide sequences of SEQ ID NOs: 1681 and 1586, respectively.

[1076] In some embodiments, the oligonucleotide is used to reduce SNCA gene expression and comprises a sense strand comprising the nucleotide sequence set forth in SEQ ID NO: 1553 and an antisense strand comprising the nucleotide sequence set forth in SEQ ID NO: 1588. In some embodiments, the oligonucleotide is used to reduce SNCA gene expression and comprises a sense strand comprising the nucleotide sequence set forth in SEQ ID NO: 1560 and an antisense strand comprising the nucleotide sequence set forth in SEQ ID NO: 1595. In some embodiments, the oligonucleotide is used to reduce SNCA gene expression and comprises a sense strand comprising the nucleotide sequence set forth in SEQ ID NO: 1564 and an antisense strand comprising the nucleotide sequence set forth in SEQ ID NO: 1599. In some embodiments, the oligonucleotide is used to reduce SNCA gene expression and comprises a sense strand comprising the nucleotide sequence set forth in SEQ ID NO: 1551 and an antisense strand comprising the nucleotide sequence set forth in SEQ ID NO: 1586. In some embodiments, the oligonucleotide is used to reduce SNCA gene expression and comprises a sense strand comprising the nucleotide sequence shown in SEQ ID NO: 1570 and an antisense strand comprising the nucleotide sequence shown in SEQ ID NO: 1605. In some embodiments, the oligonucleotide is used to reduce SNCA gene expression and comprises a sense strand comprising the nucleotide sequence shown in SEQ ID NO: 1681 and an antisense strand comprising the nucleotide sequence shown in SEQ ID NO: 1586.

[1077] In some embodiments, the oligonucleotide is used to reduce 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 complementary region to a SNCA mRNA target sequence, wherein the complementary region is as set forth in SEQ ID NO: 1865; and (ii) a sense strand of 19-50 nucleotides in length, wherein the sense strand comprises a complementary region to the antisense strand, wherein the antisense strand and the sense strand are separate strands that form an asymmetric duplex region having an overhang of 1-4 nucleotides at the 3' end of the antisense strand.

[1078] In some embodiments, the oligonucleotide is used to reduce 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 complementary region to a SNCA mRNA target sequence, wherein the complementary region is as set forth in SEQ ID NO: 1721; and (ii) a sense strand of 19-50 nucleotides in length, wherein the sense strand comprises a complementary region to the antisense strand, wherein the antisense strand and the sense strand are separate strands that form an asymmetric duplex region having an overhang of 1-4 nucleotides at the 3' end of the antisense strand.

[1079] In some embodiments, the oligonucleotide is used to reduce SNCA expression and comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand comprises a nucleotide sequence comprising a complementary region to a SNCA mRNA target sequence, wherein the complementary region is as set forth in SEQ ID NO: 1847; and (ii) a sense strand of 19-50 nucleotides in length, wherein the sense strand comprises a complementary region to the antisense strand, wherein the antisense strand and the sense strand are separate strands that form an asymmetric duplex region having an overhang of 1-4 nucleotides at the 3' end of the antisense strand.

[1080] In some embodiments, the oligonucleotide is used to reduce 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 complementary region to a SNCA mRNA target sequence, wherein the complementary region is as set forth in SEQ ID NO: 1846; and (ii) a sense strand of 19-50 nucleotides in length, wherein the sense strand comprises a complementary region to the antisense strand, wherein the antisense strand and the sense strand are separate strands that form an asymmetric duplex region having an overhang of 1-4 nucleotides at the 3' end of the antisense strand.

[1081] In some embodiments, the oligonucleotide is used to reduce 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 complementary region to a SNCA mRNA target sequence, wherein the complementary region is as set forth in SEQ ID NO: 1955; and (ii) a sense strand of 19-50 nucleotides in length, wherein the sense strand comprises a complementary region to the antisense strand, wherein the antisense strand and the sense strand are separate strands that form an asymmetric duplex region having an overhang of 1-4 nucleotides at the 3' end of the antisense strand.

[1082] In some embodiments, the oligonucleotide is used to reduce 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 complementary region to a SNCA mRNA target sequence, wherein the complementary region is as set forth in SEQ ID NO: 1865; and (ii) a sense strand of 19-50 nucleotides in length, wherein the sense strand comprises the complementary region to the antisense strand and a stem-loop at the 3' end, wherein the stem-loop is as set forth in S1-L-S2, wherein S1 is complementary to S2, and wherein L forms a loop of 3 to 5 nucleotides in length between S1 and S2, wherein the antisense strand and the sense strand are independent strands forming an asymmetric duplex region having an overhang of 1-4 nucleotides at the 3' end of the antisense strand.

[1083] In some embodiments, the oligonucleotide is used to reduce 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 complementary region to a SNCA mRNA target sequence, wherein the complementary region is as set forth in SEQ ID NO: 1721; and (ii) a sense strand of 19-50 nucleotides in length, wherein the sense strand comprises the complementary region to the antisense strand and a stem-loop at the 3' end, wherein the stem-loop is as set forth in S1-L-S2, wherein S1 is complementary to S2, and wherein L forms a loop of 3 to 5 nucleotides in length between S1 and S2, wherein the antisense strand and the sense strand are independent strands forming an asymmetric duplex region having an overhang of 1-4 nucleotides at the 3' end of the antisense strand.

[1084] In some embodiments, the oligonucleotide is used to reduce 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 complementary region to a SNCA mRNA target sequence, wherein the complementary region is as set forth in SEQ ID NO: 1847; and (ii) a sense strand of 19-50 nucleotides in length, wherein the sense strand comprises the complementary region to the antisense strand and a stem-loop at the 3' end, wherein the stem-loop is as set forth in S1-L-S2, wherein S1 is complementary to S2, and wherein L forms a loop of 3 to 5 nucleotides in length between S1 and S2, wherein the antisense strand and the sense strand are independent strands forming an asymmetric duplex region having an overhang of 1-4 nucleotides at the 3' end of the antisense strand.

[1085] In some embodiments, the oligonucleotide is used to reduce 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 complementary region to a SNCA mRNA target sequence, wherein the complementary region is as set forth in SEQ ID NO: 1846; and (ii) a sense strand of 19-50 nucleotides in length, wherein the sense strand comprises the complementary region to the antisense strand and a stem-loop at the 3' end, wherein the stem-loop is as set forth in S1-L-S2, wherein S1 is complementary to S2, and wherein L forms a loop of 3 to 5 nucleotides in length between S1 and S2, wherein the antisense strand and the sense strand are independent strands forming an asymmetric duplex region having an overhang of 1-4 nucleotides at the 3' end of the antisense strand.

[1086] In some embodiments, the oligonucleotide is used to reduce 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 complementary region to a SNCA mRNA target sequence, wherein the complementary region is as set forth in SEQ ID NO: 1955; and (ii) a sense strand of 19-50 nucleotides in length, wherein the sense strand comprises the complementary region to the antisense strand and a stem-loop at the 3' end, wherein the stem-loop is as set forth in S1-L-S2, wherein S1 is complementary to S2, and wherein L forms a loop of 3 to 5 nucleotides in length between S1 and S2, wherein the antisense strand and the sense strand are independent strands forming an asymmetric duplex region having an overhang of 1-4 nucleotides at the 3' end of the antisense strand.

[1087] In some embodiments, an oligonucleotide is used to reduce 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 complementary region to a SNCA mRNA target sequence, wherein the complementary region is set forth in SEQ ID NO: 1846; and (ii) a sense strand of 19-25 nucleotides in length, wherein the sense strand comprises a complementary region to the antisense strand, wherein the oligonucleotide comprises a blunt end comprising the 3' end of the sense strand, and wherein the antisense strand and the sense strand are separate strands that form an asymmetric duplex region having an overhang of 1-4 nucleotides at the 3' end of the antisense strand.

[1088] In some embodiments, the oligonucleotide is used to reduce 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 complementary region to a SNCA mRNA target sequence, wherein the complementary region is set forth in SEQ ID NO: 1865; and (ii) a sense strand of 19-50 nucleotides in length, wherein the sense strand comprises a complementary region to the antisense strand, wherein the complementary region to the antisense strand is set forth in SEQ ID NO: 2249, and wherein the antisense strand and the sense strand are separate strands that form an asymmetric duplex region having an overhang of 1-4 nucleotides at the 3' end of the antisense strand.

[1089] In some embodiments, the oligonucleotide is used to reduce 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 complementary region to a SNCA mRNA target sequence, wherein the complementary region is set forth in SEQ ID NO: 1721; and (ii) a sense strand of 19-50 nucleotides in length, wherein the sense strand comprises a complementary region to the antisense strand, wherein the complementary region to the antisense strand is set forth in SEQ ID NO: 2105, and wherein the antisense strand and the sense strand are separate strands that form an asymmetric duplex region having an overhang of 1-4 nucleotides at the 3' end of the antisense strand.

[1090] In some embodiments, the oligonucleotide is used to reduce 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 complementary region to a SNCA mRNA target sequence, wherein the complementary region is set forth in SEQ ID NO: 1847; and (ii) a sense strand of 19-50 nucleotides in length, wherein the sense strand comprises a complementary region to the antisense strand, wherein the complementary region to the antisense strand is set forth in SEQ ID NO: 2231, and wherein the antisense strand and the sense strand are separate strands that form an asymmetric duplex region having an overhang of 1-4 nucleotides at the 3' end of the antisense strand.

[1091] In some embodiments, the oligonucleotide is used to reduce 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 complementary region to a SNCA mRNA target sequence, wherein the complementary region is set forth in SEQ ID NO: 1846; and (ii) a sense strand of 19-50 nucleotides in length, wherein the sense strand comprises a complementary region to the antisense strand, wherein the complementary region to the antisense strand is set forth in SEQ ID NO: 2230, and wherein the antisense strand and the sense strand are separate strands that form an asymmetric duplex region having an overhang of 1-4 nucleotides at the 3' end of the antisense strand.

[1092] In some embodiments, the oligonucleotide is used to reduce 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 complementary region to a SNCA mRNA target sequence, wherein the complementary region is set forth in SEQ ID NO: 1955; and (ii) a sense strand of 19-50 nucleotides in length, wherein the sense strand comprises a complementary region to the antisense strand, wherein the complementary region to the antisense strand is set forth in SEQ ID NO: 2339, and wherein the antisense strand and the sense strand are separate strands that form an asymmetric duplex region having an overhang of 1-4 nucleotides at the 3' end of the antisense strand.

[1093] In some embodiments, the oligonucleotide is used to reduce 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 complementary region to a SNCA mRNA target sequence, wherein the complementary region is as set forth in SEQ ID NO: 1865; and (ii) a sense strand of 19-50 nucleotides in length, the sense strand comprising the complementary region to the antisense strand and a stem-loop at the 3' end, wherein the complementary region to the antisense strand is as set forth in SEQ ID NO: 2249, wherein the stem-loop is as set forth in S1-L-S2, wherein S1 is complementary to S2, and wherein L forms a loop of 3 to 5 nucleotides in length between S1 and S2, wherein the antisense strand and the sense strand are independent strands forming an asymmetric duplex region having an overhang of 1-4 nucleotides at the 3' end of the antisense strand.

[1094] In some embodiments, the oligonucleotide is used to reduce 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 complementary region to a SNCA mRNA target sequence, wherein the complementary region is as set forth in SEQ ID NO: 1721; and (ii) a sense strand of 19-50 nucleotides in length, the sense strand comprising the complementary region to the antisense strand and a stem-loop at the 3' end, wherein the complementary region to the antisense strand is as set forth in SEQ ID NO: 2105, wherein the stem-loop is as set forth in S1-L-S2, wherein S1 is complementary to S2, and wherein L forms a loop of 3 to 5 nucleotides in length between S1 and S2, wherein the antisense strand and the sense strand are independent strands forming an asymmetric duplex region having an overhang of 1-4 nucleotides at the 3' end of the antisense strand.

[1095] In some embodiments, the oligonucleotide is used to reduce 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 complementary region to a SNCA mRNA target sequence, wherein the complementary region is as set forth in SEQ ID NO: 1847; and (ii) a sense strand of 19-50 nucleotides in length, the sense strand comprising the complementary region to the antisense strand and a stem-loop at the 3' end, wherein the complementary region to the antisense strand is as set forth in SEQ ID NO: 2231, wherein the stem-loop is as set forth in S1-L-S2, wherein S1 is complementary to S2, and wherein L forms a loop of 3 to 5 nucleotides in length between S1 and S2, wherein the antisense strand and the sense strand are independent strands forming an asymmetric duplex region having an overhang of 1-4 nucleotides at the 3' end of the antisense strand.

[1096] In some embodiments, the oligonucleotide is used to reduce 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 complementary region to a SNCA mRNA target sequence, wherein the complementary region is as set forth in SEQ ID NO: 1846; and (ii) a sense strand of 19-50 nucleotides in length, the sense strand comprising the complementary region to the antisense strand and a stem-loop at the 3' end, wherein the complementary region to the antisense strand is as set forth in SEQ ID NO: 2230, wherein the stem-loop is as set forth in S1-L-S2, wherein S1 is complementary to S2, and wherein L forms a loop of 3 to 5 nucleotides in length between S1 and S2, wherein the antisense strand and the sense strand are independent strands forming an asymmetric duplex region having an overhang of 1-4 nucleotides at the 3' end of the antisense strand.

[1097] In some embodiments, the oligonucleotide is used to reduce 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 complementary region to a SNCA mRNA target sequence, wherein the complementary region is as set forth in SEQ ID NO: 1955; and (ii) a sense strand of 19-50 nucleotides in length, the sense strand comprising the complementary region to the antisense strand and a stem-loop at the 3' end, wherein the complementary region to the antisense strand is as set forth in SEQ ID NO: 2339, wherein the stem-loop is as set forth in S1-L-S2, wherein S1 is complementary to S2, and wherein L forms a loop of 3 to 5 nucleotides in length between S1 and S2, wherein the antisense strand and the sense strand are independent strands forming an asymmetric duplex region having an overhang of 1-4 nucleotides at the 3' end of the antisense strand.

[1098] In some embodiments, an oligonucleotide is used to reduce 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 complementary region to a SNCA mRNA target sequence, wherein the complementary region is set forth in SEQ ID NO: 1846; and (ii) a sense strand of 19-25 nucleotides in length, wherein the sense strand comprises a complementary region to the antisense strand, wherein the complementary region 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 strand and the sense strand are separate strands that form an asymmetric duplex region having an overhang of 1-4 nucleotides at the 3' end of the antisense strand.

[1099] In some embodiments, the oligonucleotide is used to reduce SNCA gene expression and comprises a sense strand and an antisense strand according to:

[1100] Sense strand: 5'-mX-S-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'; hybridize to:

[1101] Antisense strand: 5'-[Me Phosphonate-4O-mX]-S-fX-S-fX-fX-fX-mX-fX-mX-mX-fX-mX-mX-mX-fX-mX-fX-mX-mX-fX-mX-S-mX-S-mX-3';

[1102] Where mX = 2'-OMe modified nucleotide, fX = 2'-F modified nucleotide, -S- = phosphorothioate bond, - = phosphodiester bond, [Mephosphonate-4O-mX] = 4'-O-monomethylphosphonate-2'-O-methyl modified nucleotide, and ademX-GalNAc = GalNAc attached to the nucleotide.

[1103] In some embodiments, the oligonucleotide is used to reduce SNCA gene expression and comprises a sense strand and an antisense strand according to:

[1104] Sense strand: 5'-[AdemX-L]-S-mX-fX-mX-fX-mX-fX-mX-fX-mX-fX-fX-fX-mX-fX-mX-fX-mX-S-mX-S-mX-3'; hybridizes to:

[1105] Antisense strand: 5'-[Me Phosphonate-4O-mX]-S-fX-S-fX-fX-fX-mX-fX-mX-mX-fX-mX-mX-mX-fX-mX-fX-mX-mX-fX-mX-S-mX-S-mX-3';

[1106] Where mX = 2'-OMe modified nucleotide, fX = 2'-F modified nucleotide, -S- = phosphorothioate bond, - = phosphodiester bond, [Mephosphonate-4O-mX] = 4'-O-monomethylphosphonate-2'-O-methyl modified nucleotide, and ademX-L = lipid moiety attached to the nucleotide.

[1107] In some embodiments, the oligonucleotide is used to reduce SNCA gene expression and comprises a sense strand and an antisense strand according to:

[1108] Sense strand: 5'-[AdemX-C16]-S-mX-fX-mX-fX-mX-fX-mX-fX-fX-mX-fX-fX-mX-fX-mX-fX-mX-S-mX-S-mX-3'; hybridizes to:

[1109] Antisense strand: 5′-[Me Phosphonate-4O-mX]-S-fX-S-fX-fX-fX-mX-fX-mX-mX-fX-mX-mX-mX-fX-mX-fX-mX-mX-fX-mX-S-mX-S-mX-3';

[1110] Where mX = 2'-OMe modified nucleotide, fX = 2'-F modified nucleotide, -S- = phosphorothioate bond, - = phosphodiester bond, [Mephosphonate-4O-mX] = 4'-O-monomethylphosphonate-2'-O-methyl modified nucleotide, and ademX-C16 = C16 hydrocarbon chain attached to the nucleotide.

[1111] In some embodiments, the oligonucleotide is used to reduce SNCA gene expression and comprises a sense strand and an antisense strand, the sense strand and the antisense strand comprising a nucleotide sequence selected from the group consisting of:

[1112] a) SEQ ID NO: 1607 and 1642, respectively;

[1113] b) SEQ ID NO: 1608 and 1643, respectively;

[1114] c) SEQ ID NOs: 1609 and 1644, respectively;

[1115] d) SEQ ID NO: 1610 and 1645, respectively;

[1116] e) SEQ ID NOs: 1611 and 1646, respectively;

[1117] f) SEQ ID NO: 1612 and 1647, respectively;

[1118] g) SEQ ID NO: 1613 and 1648, respectively;

[1119] h) SEQ ID NOs: 1614 and 1649, respectively;

[1120] i) SEQ ID NOs: 1615 and 1650, respectively;

[1121] j) SEQ ID NOs: 1616 and 1651, respectively;

[1122] k) are SEQ ID NOs: 1617 and 1652, respectively;

[1123] 1) are SEQ ID NOs: 1618 and 1653, respectively;

[1124] m) are SEQ ID NOs: 1619 and 1654, respectively;

[1125] n) are SEQ ID NOs: 1620 and 1655, respectively;

[1126] o) are SEQ ID NOs: 1621 and 1656, respectively;

[1127] p) are SEQ ID NOs: 1622 and 1657, respectively;

[1128] q) are SEQ ID NOs: 1623 and 1658, respectively;

[1129] r) are SEQ ID NOs: 1624 and 1659, respectively;

[1130] s) are SEQ ID NOs: 1625 and 1660, respectively;

[1131] t) are SEQ ID NOs: 1626 and 1661, respectively;

[1132] u) are SEQ ID NOs: 1627 and 1662, respectively;

[1133] v) SEQ ID NOs: 1628 and 1663, respectively;

[1134] w) are SEQ ID NOs: 1629 and 1664, respectively;

[1135] x) are SEQ ID NOs: 1630 and 1665, respectively;

[1136] y) are SEQ ID NOs: 1631 and 1666, respectively;

[1137] z) are SEQ ID NOs: 1632 and 1667, respectively;

[1138] aa) are SEQ ID NOs: 1633 and 1668, respectively;

[1139] bb) are SEQ ID NOs: 1634 and 1669, respectively;

[1140] cc) are SEQ ID NOs: 1635 and 1670, respectively;

[1141] dd) are SEQ ID NOs: 1636 and 1671, respectively;

[1142] ee) are SEQ ID NOs: 1637 and 1672, respectively;

[1143] ff) are SEQ ID NOs: 1638 and 1673, respectively;

[1144] gg) are SEQ ID NOs: 1639 and 1674, respectively;

[1145] hh) are SEQ ID NOs: 1640 and 1675, respectively;

[1146] ii) SEQ ID NO: 1641 and 1676, respectively; and

[1147] jj) are SEQ ID NOs: 1682 and 1656, respectively.

[1148] In some embodiments, the oligonucleotide is used to reduce SNCA gene expression and comprises a sense strand and an antisense strand, the sense strand and the antisense strand comprising a nucleotide sequence selected from the group consisting of:

[1149] a) SEQ ID NO: 1610 and 1645, respectively;

[1150] b) SEQ ID NOs: 1614 and 1649, respectively;

[1151] c) SEQ ID NOs: 1616 and 1651, respectively;

[1152] d) SEQ ID NO: 1621 and 1656, respectively;

[1153] e) SEQ ID NOs: 1622 and 1657, respectively;

[1154] f) SEQ ID NOs: 1623 and 1658, respectively;

[1155] g) SEQ ID NOs: 1629 and 1664, respectively;

[1156] h) SEQ ID NOs: 1630 and 1665, respectively;

[1157] i) SEQ ID NOs: 1634 and 1669, respectively;

[1158] j) SEQ ID NOs: 1635 and 1670, respectively;

[1159] k) are SEQ ID NOs: 1636 and 1671, respectively;

[1160] 1) are SEQ ID NOs: 1640 and 1675, respectively; and

[1161] m) are SEQ ID NOs: 1682 and 1656, respectively.

[1162] In some embodiments, the oligonucleotide is used to reduce SNCA gene expression and comprises a sense strand and an antisense strand, the sense strand and the antisense strand comprising a nucleotide sequence selected from the group consisting of:

[1163] a) SEQ ID NO: 1623 and 1658, respectively;

[1164] b) SEQ ID NOs: 1630 and 1665, respectively;

[1165] c) SEQ ID NOs: 1634 and 1669, respectively;

[1166] d) SEQ ID NO: 1621 and 1656, respectively;

[1167] e) SEQ ID NOs: 1640 and 1675, respectively; and

[1168] f) are SEQ ID NOs: 1682 and 1656, respectively.

[1169] In some embodiments, the oligonucleotide is used to reduce SNCA gene expression and comprises a sense strand comprising the nucleotide sequence set forth in SEQ ID NO: 1623 and an antisense strand comprising the nucleotide sequence set forth in SEQ ID NO: 1658. In some embodiments, the oligonucleotide is used to reduce SNCA gene expression and comprises a sense strand comprising the nucleotide sequence set forth in SEQ ID NO: 1630 and an antisense strand comprising the nucleotide sequence set forth in SEQ ID NO: 1665. In some embodiments, the oligonucleotide is used to reduce SNCA gene expression and comprises a sense strand comprising the nucleotide sequence set forth in SEQ ID NO: 1634 and an antisense strand comprising the nucleotide sequence set forth in SEQ ID NO: 1669. In some embodiments, the oligonucleotide is used to reduce SNCA gene expression and comprises a sense strand comprising the nucleotide sequence set forth in SEQ ID NO: 1621 and an antisense strand comprising the nucleotide sequence set forth in SEQ ID NO: 1656. In some embodiments, the oligonucleotide is used to reduce SNCA gene expression and comprises a sense strand comprising the nucleotide sequence shown in SEQ ID NO: 1640 and an antisense strand comprising the nucleotide sequence shown in SEQ ID NO: 1675. In some embodiments, the oligonucleotide is used to reduce SNCA gene expression and comprises a sense strand comprising the nucleotide sequence shown in SEQ ID NO: 1682 and an antisense strand comprising the nucleotide sequence shown in SEQ ID NO: 1656.

[1170] preparation

[1171] A variety of formulations have been developed to facilitate the use of oligonucleotides. For example, oligonucleotides (e.g., RNAi oligonucleotides) can be delivered to a subject or cell environment using formulations that minimize degradation, facilitate delivery and / or uptake, or provide another beneficial property to the oligonucleotide in the formulation. In some embodiments, the formulation is a composition comprising an oligonucleotide that reduces SNCA gene expression. Such a composition can be appropriately formulated so that when administered to the direct environment of a target cell or systemically to a subject, a sufficient portion of the oligonucleotide enters the cell to reduce SNCA gene expression. Any type of suitable oligonucleotide formulation can be used to deliver oligonucleotides for reducing SNCA gene expression. In some embodiments, the oligonucleotides are formulated in a buffered solution, such as a phosphate-buffered saline solution, liposomes, micellar structures, and capsids. In other embodiments, the oligonucleotides are formulated in a buffered solution, such as a phosphate-buffered saline solution.

[1172] The preparation of oligonucleotide and cationic lipid can be used to promote oligonucleotide transfection 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. In some embodiments, oligonucleotides are not formulated with components that promote transfection into cells.

[1173] Therefore, in some embodiments, the formulation comprises lipid nanoparticles. In some embodiments, the lipid nanoparticles comprise liposomes, lipids, lipid complexes, microspheres, microparticles, nanospheres or nanoparticles, or can be otherwise formulated for use in cells, tissues, organs or body administration to a subject in need thereof (see, e.g., Remington: THE SCIENCE AND PRACTICE OF PHARMACY, 22nd edition, Pharmaceutical Press, 2013).

[1174] In some embodiments, the formulation comprises an excipient that imparts improved stability, improved absorption, improved solubility, and / or therapeutic enhancement of the active ingredient to the composition. In some embodiments, the excipient is a buffer (e.g., sodium citrate, sodium phosphate, tris base, or sodium hydroxide) or a vehicle (e.g., a buffered solution, paraffin, dimethyl sulfoxide, or mineral oil). In some embodiments, the oligonucleotide is lyophilized to extend its shelf life and then made into a solution prior to use (e.g., administration to a subject). Thus, the excipient can be a lyoprotectant (e.g., mannitol, lactose, polyethylene glycol, or polyvinyl pyrrolidone) or a disintegration temperature regulator (e.g., dextran, Ficoll TM or gelatin).

[1175] 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.

[1176] In some embodiments, the formulation is formulated for administration into the CNS. In some embodiments, the formulation is formulated for administration into the cerebrospinal 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 intraventricular administration. In some embodiments, the formulation is formulated for use in the brainstem. In some embodiments, the formulation is formulated for administration within the cisterna magna.

[1177] 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 dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL TM (BASF; Parsippany, NJ) or phosphate buffered saline (PBS). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol and liquid polyethylene glycol, etc.), and suitable mixtures thereof. In many cases, it is preferred that isotonic agents, for example, sugars, polyols (such as mannitol, sorbitol), sodium chloride are included in the composition. Sterile injectable solutions can be prepared by incorporating the desired amount of the oligonucleotide into a selected solvent together with one or a combination of ingredients listed above, followed by sterilization by filtration as necessary.

[1178] In some embodiments, the formulation may contain at least about 0.1% oligonucleotide or more, but the percentage of active ingredient may be between about 1% to about 80% or more of the total composition weight or volume. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations will be anticipated by a skilled artisan preparing such pharmaceutical formulations, and therefore a variety of dosages and treatment regimens may be required.

[1179] How to use

[1180] Reduce SNCA expression

[1181] In some embodiments, the method of contacting or delivering to a cell or cell population comprises administering an effective amount of an oligonucleotide (e.g., an RNAi oligonucleotide) to reduce SNCA gene expression. In some embodiments, the reduction in SNCA gene expression is determined by measuring a reduction in the amount or level of SNCA mRNA, SNCA protein, SNCA activity, or a combination thereof in the cell. Such methods include those described herein and known to those of ordinary skill in the art.

[1182] In some embodiments, the method of reducing SNCA gene expression in the CNS comprises administering an effective amount of an oligonucleotide (e.g., an RNAi oligonucleotide) to reduce SNCA gene expression. In some embodiments, the CNS includes the brain and spinal cord. In some embodiments, SNCA gene expression is reduced in at least one brain region, including but 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 spinal cord region, including but 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 brain region and at least one spinal cord region. In some embodiments, SNCA gene expression is reduced in at least one of the following: 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 lumbar spinal cord, thoracic spinal cord, and cervical spinal cord. In some embodiments, SNCA gene expression is reduced in brain and / or spinal cord tissues associated with Parkinson's disease. In some embodiments, tissues associated with Parkinson's disease include but are not limited to putamen, midbrain tegmentum, substantia nigra, pons, and medulla. In some embodiments, SNCA gene expression is reduced in brain and / or spinal cord tissues associated with multiple system atrophy. In some embodiments, tissues associated with Parkinson's disease include, but are not limited to, the caudate nucleus, putamen, midbrain tegmentum, substantia nigra, pons, cerebellar cortex, cerebellar white matter, medulla, cervical spinal cord, thoracic spinal cord, and lumbar spinal cord.

[1183] In some embodiments, the reduction in SNCA gene expression persists from about 1 week to about 12 weeks after administration of the oligonucleotide or formulation comprising the oligonucleotide. In some embodiments, the reduction in SNCA gene expression persists for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks after administration of the oligonucleotide or formulation. In some embodiments, the reduction in SNCA gene expression persists from about 1 month to about 4 months after administration of the oligonucleotide or formulation. In some embodiments, the reduction in SNCA gene expression persists from about 1 month to about 6 months after administration of the oligonucleotide or formulation. In some embodiments, the reduction in SNCA gene expression persists for 1, 2, 3, or 4 months after administration of the oligonucleotide or formulation. In some embodiments, the reduction in SNCA gene expression persists for 1, 2, 3, 4, 5, or 6 months after administration of the oligonucleotide or formulation. In some embodiments, the reduction in SNCA gene expression persists for about 7 days to about 91 days after administration of the oligonucleotide or formulation. In some embodiments, the reduction in SNCA gene expression persists for 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, or 91 days after administration of the oligonucleotide or formulation.

[1184] In some embodiments, SNCA gene expression is reduced in at least one brain region and / or at least one spinal cord region for about 1 to about 12 weeks after administration of the oligonucleotide or formulation. In some embodiments, SNCA gene expression is reduced in at least one brain region and / or at least one spinal cord region for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 weeks after administration of the oligonucleotide or formulation. In some embodiments, SNCA gene expression is reduced in at least one brain region and / or at least one spinal cord region for about 1 month to about 4 months after administration of the oligonucleotide or formulation. In some embodiments, SNCA gene expression is reduced in at least one brain region and / or at least one spinal cord region for about 1 month to about 6 months after administration of the oligonucleotide or formulation. In some embodiments, SNCA gene expression is reduced in at least one brain region and / or at least one spinal cord region for 1, 2, 3 or 4 months after administration of the oligonucleotide or formulation. In some embodiments, SNCA gene expression is reduced in at least one brain region and / or at least one spinal cord region for 1, 2, 3, 4, 5 or 6 months after administration of the oligonucleotide or formulation. In some embodiments, SNCA gene expression is reduced in at least one brain region and / or at least one spinal cord region for about 7 days to about 91 days after administration of the oligonucleotide or formulation. In some embodiments, SNCA gene expression is reduced in at least one brain region and / or at least one spinal cord region for 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, or 91 days after administration of the oligonucleotide or formulation.

[1185] The methods herein can be used for any appropriate cell type. In some embodiments, the cell type is any cell (e.g., oligodendritic cell) expressing SNCA mRNA. 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 so that the cell generally maintains natural phenotypic properties. In some embodiments, the cell delivered by the oligonucleotide is ex vivo or in vitro (i.e., it can be delivered to the cell in culture or the organism in which the cell is located).

[1186] In some embodiments, the oligonucleotide is delivered to a cell or cell colony using a nucleic acid delivery method known in the art, including but not limited to injection of a solution or pharmaceutical composition (i.e., formulation) containing the oligonucleotide, bombardment of particles covered with the oligonucleotide, exposure of the cell or cell colony to a solution containing the oligonucleotide, or electroporation of the cell membrane in the presence of the oligonucleotide. Other methods known in the art for delivering the oligonucleotide to a cell may be used, such as lipid-mediated carrier transport, chemical-mediated transport, and cationic liposome transfection, such as calcium phosphate, etc.

[1187] In some embodiments, the 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 cell population associated with SNCA gene expression, or by an assay or technique that evaluates a molecule (e.g., SNCA mRNA or SNCA protein) that directly indicates SNCA gene expression in a cell or cell population. In some embodiments, the extent to which an oligonucleotide reduces SNCA gene expression is evaluated by comparing SNCA gene expression in a cell or cell population contacted with an oligonucleotide with a control cell or cell population (e.g., a cell or cell population not contacted with an oligonucleotide or contacted with a control oligonucleotide). In some embodiments, the control amount or level of SNCA gene expression in a control cell or cell population is predetermined, such that the control amount or level does not need to be measured in each instance of performing an assay or technique. The predetermined level or value can take a variety of forms. In some embodiments, the predetermined level or value can be a single cutoff value, such as a median or mean.

[1188] In some embodiments, contacting an oligonucleotide with a cell or cell population or delivering an oligonucleotide to a cell or cell population causes a decrease in SNCA gene expression. In some embodiments, the decrease in SNCA gene expression is relative to a control amount or level of SNCA gene expression in a cell or cell population that has not been contacted with an oligonucleotide or has been contacted with a control oligonucleotide. In some embodiments, the decrease in SNCA gene expression is about 1% or less, about 5% or less, about 10% or less, about 15% or less, about 20% or less, about 25% or less, about 30% or less, about 35% or less, about 40% or less, about 45% or less, about 50% or less, about 55% or less, about 60% or less, about 70% or less, about 80% or less, or about 90% or less relative to a control amount or level of SNCA gene expression. In some embodiments, the control amount or level of SNCA gene expression is the amount or level of SNCA mRNA and / or SNCA protein and / or SNCA activity / function in a cell or cell population that has not been contacted with an oligonucleotide. In some embodiments, the effect of delivering an oligonucleotide to a cell or cell population according to the methods herein is assessed after any limited period of time 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 cell population 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 an oligonucleotide to a cell or cell population. In some embodiments, SNCA gene expression is determined in a cell or cell population 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 the oligonucleotide with the cell or cell population or delivering the oligonucleotide to the cell or cell population.

[1189] In some embodiments, the oligonucleotide is delivered in the form of a transgenic engineered to express the oligonucleotide or a strand (e.g., its sense strand and antisense strand) of the oligonucleotide in the cell. In some embodiments, the oligonucleotide is delivered using a transgenic engineered to express any oligonucleotide. A viral vector (e.g., adenovirus, retrovirus, vaccinia virus, poxvirus, adeno-associated virus, or herpes simplex virus) or a non-viral vector (e.g., plasmid or synthetic mRNA) can be used to deliver the transgene. In some embodiments, the transgene can be injected directly into the subject.

[1190] Treatment

[1191] Also provided are oligonucleotides (e.g., RNAi oligonucleotides) for or suitable for treating a subject who would benefit from reducing SNCA gene expression (e.g., a person suffering from a disease, disorder, or condition associated with SNCA expression). In some aspects, the present disclosure provides oligonucleotides for or suitable for treating a subject suffering from a disease, disorder, or condition associated with SNCA expression. Also provided are oligonucleotides for or suitable for manufacturing a medicament or formulation / pharmaceutical composition for treating a disease, disorder, or condition associated with SNCA gene expression. In some embodiments, the oligonucleotide used or suitable for use targets SNCA mRNA and reduces SNCA gene expression (e.g., via an RNAi pathway). In some embodiments, the oligonucleotide used or suitable for use targets SNCA mRNA and reduces the amount or level of SNCA mRNA, SNCA protein, and / or SNCA activity / function.

[1192] Additionally, in some embodiments of the methods herein, a subject having a disease, disorder or condition associated with SNCA expression or susceptible to the disease, disorder or condition is selected for treatment with an oligonucleotide or formulation. In some embodiments, the methods include selecting a subject having a marker (e.g., biomarker) for a disease, disorder or condition associated with SNCA gene expression or susceptible to the disease, disorder or condition, such as, but not limited to, SNCA mRNA, SNCA protein, SNCA activity / function, or a combination thereof. Likewise, and as described in detail 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 administration of the oligonucleotide to the subject to assess the effectiveness of the treatment.

[1193] Provided herein are methods of treating a subject having, suspected of having, or at risk of having a disease, disorder, or condition associated with SNCA gene expression using an oligonucleotide or formulation. In some aspects, provided herein are methods of using an oligonucleotide or formulation to treat or mitigate the onset or progression of a disease, disorder, or condition associated with SNCA gene expression. In other aspects, provided herein are methods of using an oligonucleotide or formulation to achieve one or more therapeutic benefits in a subject having a disease, disorder, or condition associated with SNCA gene expression. In some embodiments, the subject is treated by administering a therapeutically effective amount of any one or more of the oligonucleotides provided herein. In some embodiments, treatment includes reducing SNCA gene expression. In some embodiments, the subject is treated therapeutically. In other embodiments, the subject is treated prophylactically.

[1194] In some embodiments of the methods herein, an 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 in the subject is reduced, thereby treating the subject. In some embodiments, the amount or level of SNCA mRNA in the subject is reduced. In some embodiments, the amount or level of SNCA protein in the subject is reduced. In other embodiments, the amount or level of SNCA activity / function in the subject is reduced.

[1195] In some embodiments, an oligonucleotide or a pharmaceutical composition / formulation comprising the oligonucleotide is administered to a subject having a disease, disorder, or condition associated with SNCA gene expression such that SNCA expression in the subject is reduced 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, an oligonucleotide or pharmaceutical composition is administered to a subject having a disease, disorder, or condition associated with SNCA gene expression such that SNCA expression in the subject is reduced 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 in a subject is reduced 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 not receiving the oligonucleotide or pharmaceutical composition or receiving a control oligonucleotide, pharmaceutical composition, or treatment (e.g., a reference or control subject). In some embodiments, SNCA expression in a subject is reduced 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 not receiving the oligonucleotide or pharmaceutical composition or receiving a control oligonucleotide, pharmaceutical composition, or treatment (e.g., a reference or control subject).

[1196] In some embodiments of the methods herein, an oligonucleotide or pharmaceutical composition is administered to a subject having a disease, disorder, or condition associated with SNCA gene expression such that the amount or level of SNCA mRNA in the subject is reduced 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, an oligonucleotide or pharmaceutical composition is administered to a subject having a disease, disorder, or condition associated with SNCA gene expression such that the amount or level of SNCA mRNA in the subject is reduced 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, as compared to the amount or level of SNCA mRNA prior to administration of the oligonucleotide or pharmaceutical composition. In some embodiments, the amount or level of SNCA mRNA in a subject is reduced 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 in a subject that did not receive the oligonucleotide or pharmaceutical composition or that received a control oligonucleotide, pharmaceutical composition, or treatment (e.g., a reference or control subject). In some embodiments, the amount or level of SNCA mRNA in a subject is reduced 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 in a subject that did not receive the oligonucleotide or pharmaceutical composition or received a control oligonucleotide, pharmaceutical composition, or treatment (e.g., a reference or control subject).

[1197] In some embodiments of the methods herein, an oligonucleotide or pharmaceutical composition is administered to a subject having a disease, disorder, or condition associated with expression of the SNCA gene such that the amount or level of SNCA protein in the subject is reduced 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, an oligonucleotide or pharmaceutical composition is administered to a subject having a disease, disorder, or condition associated with expression of the SNCA gene such that the amount or level of SNCA protein in the subject is reduced 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, as compared to the amount or level of SNCA protein prior to administration of the oligonucleotide or pharmaceutical composition. In some embodiments, the amount or level of SNCA protein in a subject is reduced 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 in a subject that did not receive the oligonucleotide or pharmaceutical composition or that received a control oligonucleotide, pharmaceutical composition, or treatment (e.g., a reference or control subject). In some embodiments, the amount or level of SNCA protein in a subject is reduced 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 in a subject that did not receive the oligonucleotide or pharmaceutical composition or received a control oligonucleotide, pharmaceutical composition, or treatment (e.g., a reference or control subject).

[1198] In some embodiments of the methods herein, an oligonucleotide or pharmaceutical composition is administered to a subject having a disease, disorder, or condition associated with SNCA gene expression such that the amount or level of SNCA activity / function in the subject is reduced 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, an oligonucleotide or pharmaceutical composition is administered to a subject having a disease, disorder, or condition associated with SNCA gene expression such that the amount or level of SNCA activity / function in the subject is reduced 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, as compared to the amount or level of SNCA activity / function prior to administration of the oligonucleotide or pharmaceutical composition. In some embodiments, the amount or level of SNCA activity / function in a subject is reduced 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 in a subject not receiving the oligonucleotide or pharmaceutical composition or receiving a control oligonucleotide, pharmaceutical composition, or treatment (e.g., a reference or control subject). In some embodiments, the amount or level of SNCA activity / function in a subject is reduced 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 in a subject not receiving the oligonucleotide or pharmaceutical composition or receiving a control oligonucleotide, pharmaceutical composition, or treatment (e.g., a reference or control subject).

[1199] Suitable methods for determining SNCA gene expression (such as SNCA expression), the amount or level of SNCA mRNA, the amount or level of SNCA protein, and / or the amount or level of SNCA activity / function in a subject or in a sample from a subject are known in the art. In addition, the Examples set forth herein illustrate exemplary methods for determining SNCA gene expression.

[1200] In some embodiments, SNCA gene expression (such as SNCA expression), amount or level of SNCA mRNA, amount or level of SNCA protein, amount or level of SNCA activity / function, or any combination thereof, is reduced in a cell (e.g., an oligodendrocyte), a cell population or a group of cells (e.g., an organoid), an organ (e.g., frontal cortex), blood or a portion 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 a subject. In some embodiments, SNCA expression, amount or level of SNCA mRNA, amount or level of SNCA protein, 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 types of cells), more than one group of cells, more than one organ (e.g., brain and one or more other organs), more than one blood portion (e.g., plasma and one or more other blood portions), more than one type of tissue (e.g., brain tissue and one or more other types of tissue), more than one type of sample (e.g., a brain biopsy sample and one or more other types of biopsy samples) obtained or isolated from a subject. In some embodiments, SNCA expression, amount or level of SNCA mRNA, amount or level of SNCA protein, 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, amount or level of SNCA mRNA, amount or level of SNCA protein, amount or level of SNCA activity / function, or any combination thereof, is reduced in brain and / or spinal cord tissues associated with Parkinson's disease. In some embodiments, tissues associated with Parkinson's disease include, but are not limited to, putamen, midbrain tegmentum, substantia nigra, pons, and medulla. In some embodiments, SNCA expression, the amount or level of SNCA mRNA, the amount or level of SNCA protein, the amount or level of SNCA activity / function, or any combination thereof, is reduced in brain and / or spinal cord tissue associated with multiple system atrophy. In some embodiments, tissues associated with multiple system atrophy include, but are not limited to, the caudate nucleus, putamen, midbrain tegmentum, substantia nigra, pons, cerebellar cortex, cerebellar white matter, medulla, cervical spinal cord, thoracic spinal cord, and lumbar spinal cord.

[1201] Examples of diseases, disorders or conditions associated with SNCA gene expression include multiple system atrophy, dementia with Lewy bodies, and Parkinson's disease.

[1202] Due to their high specificity, the oligonucleotides herein specifically target the SNCA mRNA of a target gene in a cell, tissue or organ (e.g., the brain). In preventing a disease, the target gene may be a gene that is required for initiation or maintenance of the disease or has been identified as being associated with a higher risk of contracting the disease. In treating a disease, the oligonucleotide may be contacted with a cell, tissue or organ (e.g., the brain) that exhibits or is responsible for mediating the disease. For example, an oligonucleotide substantially identical to all or part of a wild-type (i.e., native) or mutant gene associated with a disease, disorder or condition associated with SNCA gene expression may be contacted with or introduced into a cell or tissue type of interest (such as an oligodendritic cell or other brain cell).

[1203] In some embodiments, SNCA can be from any mammal, such as a human, and can be silenced according to the methods described herein.

[1204] The methods herein generally involve administering to a subject a therapeutically effective amount of an oligonucleotide, i.e., an amount capable of producing a desired therapeutic outcome. A therapeutically acceptable amount can be an amount that can treat a disease, condition, or illness in a therapeutic manner. The appropriate amount / dosage for any one subject will depend on certain factors, including the subject's size, body surface area, age, specific composition to be administered, active ingredients in the composition, time and route of administration, general health, and other drugs administered simultaneously.

[1205] In some embodiments, any one of the oligonucleotides or compositions herein is administered to a subject enterally (e.g., orally, by a gastric feeding tube, by a duodenal feeding tube, via gastrostomy, or rectally), parenterally (e.g., subcutaneous injection, intravenous injection or infusion, intraarterial injection or infusion, intraosseous infusion, intramuscular injection, intracerebral injection, intraventricular injection, intrathecal), topically (e.g., epidermal, inhaled, via eye drops, or transmucosal), or by direct injection into a target organ (e.g., the brain of a subject). Typically, the oligonucleotides or compositions are administered intravenously or subcutaneously. In some embodiments, the oligonucleotides or compositions are administered to the cerebrospinal fluid. In some embodiments, the oligonucleotides or compositions are administered intrathecally. In some embodiments, the oligonucleotides or compositions are administered intraventricularly. In some embodiments, the oligonucleotides or compositions are administered by injection into the cisterna magna.

[1206] As a non-limiting example, the oligonucleotides will be administered typically quarterly (once every three months), bimonthly (once every two months), monthly, or weekly. For example, the oligonucleotides may be administered weekly or at intervals of two or three weeks. Alternatively, the oligonucleotides may be administered daily. In some embodiments, one or more loading doses of the oligonucleotides are administered to the subject, followed by one or more maintenance doses of the oligonucleotides.

[1207] In some embodiments, the subject to be treated is a human or non-human primate or other mammalian subject. Other exemplary subjects include domestic 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.

[1208] Reagent test kit

[1209] In some embodiments, a kit comprising oligonucleotides described herein (e.g., RNAi oligonucleotides) and instructions for use thereof is provided. In some embodiments, the kit comprises oligonucleotides and package inserts containing instructions for use of the kit and / or any of its components. In some embodiments, the kit comprises oligonucleotides, one or more controls, and various buffers, reagents, enzymes, and other standard components well known in the art in a suitable container. In some embodiments, the container comprises at least one vial, well, test tube, flask, bottle, syringe, or other container tool, in which the oligonucleotides are placed and in some cases are appropriately aliquoted. In some embodiments providing additional components, the kit contains additional containers in which such components are placed. The kit may also include a tool for accommodating oligonucleotides and any other reagents in a confined space for commercial sale. Such containers may include injection-molded or blow-molded plastic containers, in which required vials are retained. The container and / or the kit may include a label with instructions for use and / or a warning.

[1210] In some embodiments, the kit comprises an oligonucleotide and a pharmaceutically acceptable carrier or pharmaceutical composition, and instructions for treating or delaying the progression of a disease, disorder, or condition associated with SNCA gene expression in a subject in need thereof.

[1211] In some embodiments, the kit comprises an oligonucleotide and a pharmaceutically acceptable carrier or pharmaceutical composition, and instructions for administering the oligonucleotide or pharmaceutical composition to the cerebrospinal fluid to reduce SNCA gene expression in at least one brain region and / or at least one spinal cord region of a subject in need thereof.

[1212] definition

[1213] As used herein, "approximately" or "about" when applied to one or more values ​​of interest refers to a value similar to the reference value recited. In certain embodiments, unless otherwise specified or otherwise apparent from the context (unless such a numerical value will exceed 100% of a possible value), "about" refers to a range of values ​​within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the reference value recited in either direction (greater than or less than).

[1214] As used herein, "administer," "administering," "administration," and the like refer to providing a substance (eg, an oligonucleotide) to a subject in a manner that is pharmacologically useful (eg, to treat a disease, disorder, or condition in the subject).

[1215] As used herein, "asialoglycoprotein receptor" or "ASGPR" refers to a bisected C-type lectin formed by a major 48 kDa subunit (ASGPR-1) and a minor 40 kDa subunit (ASGPR-2). ASGPR is primarily expressed on the sinusoidal surface of hepatocytes and plays a major role in the binding, internalization, and subsequent clearance of circulating glycoproteins containing terminal galactose or GalNAc residues (asialoglycoproteins).

[1216] As used herein, "attenuate", "attenuating", "attenuation" and the like refer to reducing or effectively preventing. As a non-limiting example, one or more treatments herein can reduce or effectively prevent the onset or progression of a disease, disorder or condition associated with SNCA gene expression in a subject. Such attenuation can be exemplified by, for example, a reduction in one or more aspects of a disease associated with SNCA gene expression (e.g., symptoms, tissue characteristics, and cellular, inflammatory or immune responses, etc.), no progression (worsening) of one or more aspects of a disease, disorder or condition is detected, or aspects of a disease that might otherwise be expected are not detected in a subject.

[1217] As used herein, "complementarity" refers to a structural relationship between two nucleotides (e.g., on two relative nucleic acids or on relative regions of single-stranded nucleic acids), which allows two nucleotides to form base pairs with each other. For example, the purine nucleotides of a nucleic acid complementary to the pyrimidine nucleotides of the relative nucleic acid can base pair together by forming hydrogen bonds with each other. In some embodiments, complementary nucleotides can base pair in a Watson-Crick manner or in any other manner that allows the formation of a stable duplex. In some embodiments, two nucleic acids can have a region of multiple nucleotides, and the regions are complementary to each other to form a complementary region, as described herein.

[1218] As used herein, "deoxyribonucleotide" refers to a nucleotide having a hydrogen replacing the hydroxyl group at the 2' position of its pentose when compared to a ribonucleotide. A modified deoxyribonucleotide is a deoxyribonucleotide having one or more modifications or substitutions of atoms other than the 2' position, including modifications or substitutions within or within the sugar, phosphate group, or base.

[1219] As used herein, "double-stranded oligonucleotide" or "ds oligonucleotide" refers to an oligonucleotide that is generally in the form of a duplex. In some embodiments, complementary base pairing of the duplex region of the ds oligonucleotide is formed between the antiparallel sequences of the nucleotides of the covalently separated nucleic acid chains. In some embodiments, complementary base pairing of the duplex region of the ds oligonucleotide is formed between the antiparallel sequences of the nucleotides of the covalently linked nucleic acid chains. In some embodiments, complementary base pairing of the duplex region of the ds oligonucleotide is formed by a single-stranded nucleic acid that is folded (e.g., via a hairpin) to provide a complementary antiparallel sequence of nucleotides that are base-paired together. In some embodiments, the ds oligonucleotide comprises two covalently separated nucleic acid chains that are completely duplexed with each other. However, in other embodiments, the ds oligonucleotide comprises two covalently separated nucleic acid chains that are partially duplexed (e.g., having an overhang at one or both ends). In some embodiments, the ds oligonucleotide comprises an antiparallel sequence of partially complementary nucleotides, and therefore may have one or more mismatches, which may include internal mismatches or terminal mismatches.

[1220] As used herein, "duplex" with respect to nucleic acids (eg, oligonucleotides) refers to the structure formed by complementary base pairing of two antiparallel sequences of nucleotides.

[1221] As used herein, "excipient" refers to a non-therapeutic agent that can be included in the composition, for example, to provide or aid in desired consistency or stabilization.

[1222] As used herein, a "labile linker" refers to a linker that can be cleaved (eg, by acidic pH). A "fairly stable linker" refers to a linker that is not cleavable.

[1223] As used herein, "loop" refers to an unpaired region of a nucleic acid (e.g., an oligonucleotide) that is flanked by two antiparallel regions of the nucleic acid that are sufficiently complementary to each other so that under appropriate hybridization conditions (e.g., in phosphate buffer, in cells), the two antiparallel regions flanking the unpaired region hybridize to form a duplex (referred to as the "stem").

[1224] As used herein, "modified internucleotide bonds" refers to internucleotide bonds having one or more chemical modifications when compared to a reference internucleotide bond comprising a phosphodiester bond. In some embodiments, the modified nucleotide is a non-naturally occurring bond. Typically, the modified internucleotide bond confers one or more desired properties to the nucleic acid in which the modified internucleotide bond is present. For example, the modified nucleotide can improve thermal stability, resistance to degradation, nuclease resistance, solubility, bioavailability, biological activity, reduced immunogenicity, etc.

[1225] As used herein, "modified nucleotides" refer to nucleotides having one or more chemical modifications compared to the corresponding reference nucleotides selected from the group consisting of adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, adenine deoxyribonucleotides, guanine deoxyribonucleotides, cytosine deoxyribonucleotides, and thymidine deoxyribonucleotides. In some embodiments, modified nucleotides are non-naturally occurring nucleotides. In some embodiments, modified nucleotides have one or more chemical modifications in their sugar, nucleobase, and / or phosphate groups. In some embodiments, modified nucleotides have one or more chemical moieties conjugated to the corresponding reference nucleotides. Typically, modified nucleotides confer one or more desired properties to the nucleic acid in which the modified nucleotides are present. For example, modified nucleotides can improve thermal stability, degradation resistance, nuclease resistance, solubility, bioavailability, biological activity, reduced immunogenicity, etc.

[1226] As used herein, a "nicked tetraloop structure" or "nicked tetraL structure" refers to the structure of an oligonucleotide (e.g., an RNAi oligonucleotide) characterized by independent sense (passenger) and antisense (guide) strands, wherein the sense strand has a complementary region to the antisense strand, and wherein at least one strand, typically the sense strand, has a tetraL configured to stabilize an adjacent stem region formed within at least one strand.

[1227] As used herein, "oligonucleotide" refers to a short nucleic acid (e.g., less than about 100 nucleotides in length). An oligonucleotide can be ss or ds. An oligonucleotide may or may not have a duplex region. As a set of non-limiting examples, an oligonucleotide can be, but is not limited to, a small interfering RNA (siRNA), a microRNA (miRNA), a short hairpin RNA (shRNA), a Dicer substrate interfering RNA (dsiRNA), an antisense oligonucleotide (ASO), a short siRNA, or a ss siRNA. In some embodiments, an oligonucleotide is a ds oligonucleotide and is an RNAi oligonucleotide.

[1228] As used herein, "overhang" refers to a terminal non-base paired nucleotide resulting from a strand or region extending beyond the end of a complementary strand with which the strand forms a duplex. In some embodiments, the overhang comprises one or more unpaired nucleotides extending from the duplex region at the 5' end or 3' end 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 oligonucleotide.

[1229] As used herein, "phosphate analogs" refer to chemical moieties that simulate the electrostatic and / or steric properties of a phosphate group. In some embodiments, a phosphate analog is located at the 5' terminal nucleotide of an oligonucleotide to replace a 5'-phosphate that is often easily removed enzymatically. In some embodiments, a 5'-phosphate analog contains a phosphatase-resistant bond. Examples of phosphate analogs include, but are not limited to, 5' phosphonates, such as 5' methylene phosphonates (5'-MP) and 5'-(E)-vinyl phosphonates (5'-VP). In some embodiments, an oligonucleotide has a phosphate analog at the 4'-carbon position of a sugar at the 5' terminal nucleotide (referred to as a "4'-phosphate analog"). An example of a 4'-phosphate analog is an oxymethylphosphonate, in which the oxygen atom of the oxymethyl group is bound to a sugar moiety (e.g., at its 4'-carbon) or an analog thereof. See, e.g., U.S. Provisional Patent Application Nos. 62 / 383,207 (filed September 2, 2016) and 62 / 393,401 (filed September 12, 2016). Other modifications have been developed for the 5' end of oligonucleotides (see, e.g., International Patent Application No. WO 2011 / 133871; U.S. Patent No. 8,927,513; and Prakash et al. (2015) Nucleic Acids Res. 43:2993-3011).

[1230] As used herein, "SNCA" refers to synuclein alpha. SNCA is found in large quantities in the brain and inhibits phospholipase D2. It functions in synaptic signaling and membrane trafficking. The mRNA encoding wild-type human SNCA is shown in SEQ ID NO: 1677. The mRNA encoding mouse SNCA is shown in SEQ ID NO: 1678. The mRNA encoding monkey SNCA is shown in SEQ ID NO: 1679.

[1231] As used herein, "reduced expression" of a gene (e.g., SNCA) refers to a decrease in the amount or level of an RNA transcript (e.g., SNCA mRNA) or protein encoded by the gene and / or a decrease in the amount or level of the activity / function of the gene and / or protein in a cell, cell population, sample, or subject when compared to an appropriate reference (e.g., a reference cell, cell population, sample, or subject). For example, the act of contacting a cell with an oligonucleotide (e.g., an oligonucleotide comprising an antisense strand having a nucleotide sequence complementary to a nucleotide sequence comprising SNCA mRNA, such as an RNAi oligonucleotide) can result in a decrease in the amount or level of SNCA mRNA, SNCA protein, and / or SNCA activity / function when compared to a cell not treated with the oligonucleotide (e.g., via an RNAi pathway). SNCA mRNA inactivation and / or degradation). Similarly and as used herein, "reducing expression" refers to the act of causing a decrease in the expression of a gene (e.g., SNCA).

[1232] As used herein, "reduced 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, cell population, sample or subject when compared to an appropriate reference (e.g., a reference cell, cell population, sample or subject).

[1233] 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 allow hybridization between the two sequences of nucleotides under appropriate hybridization conditions (e.g., in phosphate buffer, in a cell, etc.). In some embodiments, the oligonucleotide comprises a targeting sequence having a complementary region to an mRNA target sequence.

[1234] As used herein, "ribonucleotide" refers to a nucleotide having a ribose in the form of a pentose, containing a hydroxyl group at its 2' position. "Modified ribonucleotide" refers to a ribonucleotide having one or more modifications or substitutions of atoms other than the 2' position, including modifications or substitutions within or within the ribose, phosphate group, or base.

[1235] As used herein, "RNAi oligonucleotide" refers to (a) a ds oligonucleotide having a sense strand and an antisense strand, wherein the antisense strand or a portion of the antisense strand is used in the cleavage of a target mRNA (e.g., SNCA mRNA) by an Argonaute 2 (Ago2) endonuclease, or (b) an ss oligonucleotide having a single antisense strand, wherein that antisense strand (or a portion of that antisense strand) is used in the cleavage of a target mRNA (e.g., SNCA mRNA) by an Ago2 endonuclease.

[1236] As used herein, a "strand" refers to a single contiguous sequence of nucleotides linked together via internucleotide bonds (eg, phosphodiester bonds or phosphorothioate bonds). In some embodiments, a strand has two free ends (eg, a 5' end and a 3' end).

[1237] As used herein, "subject" means any mammal, including mice, rabbits, and humans. In one embodiment, the subject is a human or NHP. In addition, "individual" or "patient" can be used interchangeably with "subject".

[1238] As used herein, "synthetic" refers to nucleic acids or other molecules that are artificially synthesized (e.g., using a machine such as a solid-state nucleic acid synthesizer) or otherwise not derived from a natural source (e.g., a cell or organism) that normally produces the molecule.

[1239] As used herein, "targeting ligand" refers to a homologous molecule (e.g., a receptor) that selectively binds to a tissue or cell of interest, and can be conjugated to another substance to achieve the purpose of targeting another substance to a tissue or cell of interest (e.g., a carbohydrate, an amino sugar, cholesterol, or a polypeptide). For example, in some embodiments, a targeting ligand can be conjugated to an oligonucleotide to achieve the purpose of targeting an oligonucleotide to a specific tissue or cell of interest. In some embodiments, a targeting ligand selectively binds to a cell surface receptor. Therefore, in some embodiments, when a targeting ligand is conjugated to an oligonucleotide, it promotes the delivery of an oligonucleotide to a specific cell by selectively binding to a receptor expressed on the cell surface and endosomes of a cell comprising a complex of an oligonucleotide, a targeting ligand, and a receptor. In some embodiments, a targeting ligand is conjugated to an oligonucleotide via a linker that is cleaved after or during cell internalization so that the oligonucleotide is released from the targeting ligand in the cell.

[1240] As used herein, "tetraloop" or "tetraL" refers to a loop that increases the stability of adjacent duplexes formed by hybridization of flanking sequences of nucleotides. The increase in stability is detectable because the melting temperature (T m) is higher than the T of adjacent stem duplexes expected on average from a set of loops of comparable length and consisting of randomly selected nucleotide sequences. m For example, tetraL can confer a T 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 to a hairpin comprising a duplex of at least 2 base pairs (bp) in length in 10 mM NaHPO4. m. In some embodiments, tetraL can stabilize bp in adjacent stem duplexes by stacking interactions. In addition, interact...

Claims

1. An 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 complementary region to the SNCA mRNA target sequence of any one of SEQ ID NOs: 1683-2066, and wherein the length of the complementary region is at least 15 consecutive nucleotides.

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

3. The RNAi oligonucleotide of claim 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 the antisense strand and the sense strand form a duplex region having a length of at least 19 nucleotides, optionally at least 20 nucleotides in length.

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

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

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 complementary region to a SNCA mRNA target sequence, wherein the complementary region is selected from the group consisting of SEQ ID NOs: 2067-2450, and (ii) a sense strand of 19-50 nucleotides in length, the sense strand comprising a complementary region to the antisense strand, wherein the antisense strand and the sense strand are independent strands forming an asymmetric duplex region having an overhang of 1-4 nucleotides at the 3' end 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 as shown in S1-L-S2, wherein S1 is complementary to S2, and wherein L forms a loop of 3-5 nucleotides in length between S1 and S2.

10. The RNAi oligonucleotide of claim 9, wherein L is a tricyclic or tetracyclic ring.

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

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 to 12, wherein the S1 and S2 are 1 to 10 nucleotides in length and have the same length.

14. The RNAi oligonucleotide of claim 13, wherein the length of S1 and S2 is 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides or 10 nucleotides.

15. The RNAi oligonucleotide of claim 14, wherein S1 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 a purine nucleotide.

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 the group consisting of 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'-modification.

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-β-d-arabino nucleic acid.

30. The RNAi oligonucleotide of any one of claims 28-29, wherein the modification is a 2'-modification 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 from 5' to 3' at positions 1-36, wherein each of positions 3, 5, 8, 10, 12, 13, 15 and 17 comprises the 2'-fluoro modification.

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

38. The RNAi oligonucleotide of any one of claims 29-37, wherein the antisense strand comprises 22 nucleotides from 5' to 3' at positions 1-22, and wherein each of positions 2, 3, 4, 5, 7, 10, 14, 16 and 19 comprises 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 internucleotide bond.

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

42. The RNAi oligonucleotide of claim 41, wherein the antisense strand comprises the phosphorothioate bond at: (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 bond 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 bond 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 bond 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 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 oxymethylphosphonate, vinylphosphonate, or malonylphosphonate, optionally wherein the phosphate analog is a 4'-phosphate analog comprising 4'-oxymethylphosphonate.

48. The RNAi oligonucleotide of any 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, an amino sugar, a lipid, cholesterol, or a 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 are 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 the 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 an N-acetylgalactosamine (GalNAc) moiety.

54. The RNAi oligonucleotide of claim 53, wherein the GalNac moiety is a monovalent GalNAc moiety, a divalent 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 the 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 C8-C30 hydrocarbon chain.

60. The RNAi oligonucleotide of claim 58 or 59, wherein the hydrocarbon chain is a C16 hydrocarbon chain.

61. The RNAi oligonucleotide of claim 60, wherein the C16 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 1-62 of any one of claims 1-62, wherein the complementary region is fully complementary to the SNCA mRNA target sequence at nucleotide positions 2-8 of the antisense strand, and wherein nucleotide positions are numbered from 5' to 3'.

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

65. The RNAi oligonucleotide of any one of claims 1 to 64, wherein the sense strand comprises the 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 the 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 a nucleotide sequence selected from the group consisting of: a) SEQ ID NO: 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 NO: 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; 1) are SEQ ID NOs: 1548 and 1583, respectively; m) are SEQ ID NOs: 1549 and 1584, respectively; n) are SEQ ID NOs: 1550 and 1585, respectively; o) are SEQ ID NOs: 1551 and 1586, respectively; p) are SEQ ID NOs: 1552 and 1587, respectively; q) are SEQ ID NOs: 1553 and 1588, respectively; r) are SEQ ID NOs: 1554 and 1589, respectively; s) are SEQ ID NOs: 1555 and 1590, respectively; t) are SEQ ID NOs: 1556 and 1591, respectively; u) are SEQ ID NOs: 1557 and 1592, respectively; v) SEQ ID NOs: 1558 and 1593, respectively; w) are SEQ ID NOs: 1559 and 1594, respectively; x) are SEQ ID NOs: 1560 and 1595, respectively; y) are SEQ ID NOs: 1561 and 1596, respectively; z) are SEQ ID NOs: 1562 and 1597, respectively; aa) are SEQ ID NOs: 1563 and 1598, respectively; bb) are SEQ ID NOs: 1564 and 1599, respectively; cc) are SEQ ID NOs: 1565 and 1600, respectively; dd) are SEQ ID NOs: 1566 and 1601, respectively; ee) are SEQ ID NOs: 1567 and 1602, respectively; ff) are SEQ ID NOs: 1568 and 1603, respectively; gg) are SEQ ID NOs: 1569 and 1604, respectively; hh) are SEQ ID NOs: 1570 and 1605, respectively; ii) are SEQ ID NOs: 1571 and 1606, respectively; and jj) are 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 a nucleotide sequence selected from the group consisting of: a) SEQ ID NO: 1540 and 1575, respectively; b) SEQ ID NOs: 1544 and 1579, respectively; c) SEQ ID NOs: 1546 and 1581, respectively; d) SEQ ID NOs: 1551 and 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) are SEQ ID NOs: 1565 and 1600, respectively; k) are SEQ ID NOs: 1566 and 1601, respectively; 1) are SEQ ID NOs: 1570 and 1605, respectively; and m) are SEQ ID NOs: 1681 and 1586, respectively.

69. The RNAi oligonucleotide of any one of claims 1 to 66, wherein the sense strand and the antisense strand comprise a nucleotide sequence selected from the group consisting of: a) SEQ ID NO: 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) are SEQ ID NOs: 1681 and 1586, respectively.

70. The RNAi oligonucleotide of any one of claims 1 to 66, wherein the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 1553, and wherein the antisense strand comprises the nucleotide sequence shown 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 shown in SEQ ID NO: 1560, and wherein the antisense strand comprises the nucleotide sequence shown 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 shown in SEQ ID NO: 1564, and wherein the antisense strand comprises the nucleotide sequence shown 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 shown in SEQ ID NO: 1551, and wherein the antisense strand comprises the nucleotide sequence shown 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 shown in SEQ ID NO: 1570, and wherein the antisense strand comprises the nucleotide sequence shown 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 shown in SEQ ID NO: 1681, and wherein the antisense strand comprises the nucleotide sequence shown 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 the group consisting of 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 the group consisting of 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 the group consisting of 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 the 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 a nucleotide sequence selected from the group consisting of: a) SEQ ID NO: 1607 and 1642, respectively; b) SEQ ID NO: 1608 and 1643, respectively; c) SEQ ID NOs: 1609 and 1644, respectively; d) SEQ ID NO: 1610 and 1645, respectively; e) SEQ ID NOs: 1611 and 1646, respectively; f) SEQ ID NO: 1612 and 1647, respectively; g) SEQ ID NO: 1613 and 1648, respectively; h) are SEQ ID NOs: 1614 and 1649, respectively; i) are SEQ ID NOs: 1615 and 1650, respectively; j) are SEQ ID NOs: 1616 and 1651, respectively; k) are SEQ ID NOs: 1617 and 1652, respectively; 1) are SEQ ID NOs: 1618 and 1653, respectively; m) are SEQ ID NOs: 1619 and 1654, respectively; n) are SEQ ID NOs: 1620 and 1655, respectively; o) are SEQ ID NOs: 1621 and 1656, respectively; p) are SEQ ID NOs: 1622 and 1657, respectively; q) are SEQ ID NOs: 1623 and 1658, respectively; r) are SEQ ID NOs: 1624 and 1659, respectively; s) are SEQ ID NOs: 1625 and 1660, respectively; t) are SEQ ID NOs: 1626 and 1661, respectively; u) are SEQ ID NOs: 1627 and 1662, respectively; v) SEQ ID NOs: 1628 and 1663, respectively; w) are SEQ ID NOs: 1629 and 1664, respectively; x) are SEQ ID NOs: 1630 and 1665, respectively; y) are SEQ ID NOs: 1631 and 1666, respectively; z) are SEQ ID NOs: 1632 and 1667, respectively; aa) are SEQ ID NOs: 1633 and 1668, respectively; bb) are SEQ ID NOs: 1634 and 1669, respectively; cc) are SEQ ID NOs: 1635 and 1670, respectively; dd) are SEQ ID NOs: 1636 and 1671, respectively; ee) are SEQ ID NOs: 1637 and 1672, respectively; ff) are SEQ ID NOs: 1638 and 1673, respectively; gg) are SEQ ID NOs: 1639 and 1674, respectively; hh) are SEQ ID NOs: 1640 and 1675, respectively; ii) SEQ ID NO: 1641 and 1676, respectively; and jj) are 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 a nucleotide sequence selected from the group consisting of: a) SEQ ID NO: 1610 and 1645, respectively; b) SEQ ID NOs: 1614 and 1649, respectively; c) SEQ ID NOs: 1616 and 1651, respectively; d) SEQ ID NO: 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) are SEQ ID NOs: 1636 and 1671, respectively; 1) are SEQ ID NOs: 1640 and 1675, respectively; and m) are 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 a nucleotide sequence selected from the group consisting of: a) SEQ ID NO: 1623 and 1658, respectively; b) SEQ ID NOs: 1630 and 1665, respectively; c) SEQ ID NOs: 1634 and 1669, respectively; d) SEQ ID NO: 1621 and 1656, respectively; e) SEQ ID NOs: 1640 and 1675, respectively; and f) are 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 shown in SEQ ID NO: 1623, and wherein the antisense strand comprises the nucleotide sequence shown 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 shown in SEQ ID NO: 1630, and wherein the antisense strand comprises the nucleotide sequence shown 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 shown in SEQ ID NO: 1634, and wherein the antisense strand comprises the nucleotide sequence shown 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 shown in SEQ ID NO: 1621, and wherein the antisense strand comprises the nucleotide sequence shown 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 shown in SEQ ID NO: 1640, and wherein the antisense strand comprises the nucleotide sequence shown 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 shown in SEQ ID NO: 1682, and wherein the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 1656.

92. The RNAi oligonucleotide of claim 1, wherein the sense strand comprises the sequence 5'-[mCs][mA][fG][mC][fA][mG][mU][fG][mA][fU][mU][fG][fA][mA][fG][mU][fA][mU][mC][mA][mG][mC][mC][mG][ademA-GalNAc][ademA-GalNAc][ademA-GalNAc][mG][mC][mU][mG][mC]-3' (SEQ ID NO: 1623), wherein the antisense strand comprises the sequence 5'-[M ephosphonate-4O-mUs][fGs][fA][fU][fA][mC][fU][mU][mC][fA][mA][mU][mC][fA][mC][fU][mG][mC][fU][mGs][mGs][mG]-3' (SEQ ID NO: 1658), and wherein mC, mA, mG, mU = 2'-OMe ribonucleoside; fA, fC, fG, fU = 2'-F ribonucleoside; s = thiophosphate, 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][mA][fA][mU][fG][mU][mU][mA][mG][mC][mA][mG][mC][mC][mG][ademA-GalNAc][ademA-GalNAc][mG][mG][mC][mU][mG][mC]-3' (SEQ ID NO: 1630), wherein the antisense strand comprises the sequence 5'-[M e-phosphonate-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'-OMe ribonucleoside; fA, fC, fG, fU = 2'-F ribonucleoside; s = phosphorothioate, and wherein 94. The RNAi oligonucleotide of claim 1, wherein the sense strand comprises the sequence 5'-[mAs][mG][fU][mC][fA][mU][mG][fA][mC][fA][mU][fU][fU][mC][fU][mC][fA][mA][mA][mG][mC][mA][mG][mC][mC][mG][ademA-GalNAc][ademA-GalNAc][ademA-GalNAc][mG][mC][mU][mG][mC]-3' (SEQ ID NO: 1634), wherein the antisense strand comprises the sequence 5'-[M e-phosphonate-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'-OMe ribonucleoside; fA, fC, fG, fU = 2'-F ribonucleoside; s = phosphorothioate, and wherein 95. The RNAi oligonucleotide of claim 1, wherein the sense strand comprises the sequence 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][ademA-GalNAc][ademA-GalNAc][ademA-GalNAc][mG][mC][mU][mG][mC]-3' (SEQ ID NO: 1621), wherein the antisense strand comprises the sequence 5'-[Me phosphonate-4O-mUs][fUs][fU][fG][fA][mG][fA][mA][mA][fU][mG][m U][mC][fA][mU][fG][mA][mC][fU][mGs][mGs][mG]-3' (SEQ ID NO: 1656), and wherein mC, mA, mG, mU = 2'-OMe ribonucleoside; fA, fC, fG, fU = 2'-F ribonucleoside; s = phosphorothioate, and wherein 96. The RNAi oligonucleotide of claim 1, wherein the sense strand comprises the sequence 5'-[mAs][mG][fU][mU][fG][mU][mU][fA][mG][fU][mG][fA][fU][mU][fU][mG][fC][mU][mA][mA][mG][mC][mA][mG][mC][mC][mG][ademA-GalNAc][ademA-GalNAc][mG][mG][mC][mU][mG][mC]-3' (SEQ ID NO: 1640), wherein the antisense strand comprises the sequence 5'-[M e-phosphonate-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'-OMe ribonucleoside; fA, fC, fG, fU = 2'-F ribonucleoside; s = phosphorothioate, and wherein 97. The RNAi oligonucleotide of claim 1, wherein the sense strand comprises the sequence 5'-[ademCs-C16][mA][fG][mU][fC][mA][mU][fG][mA][fC][mA][f U][fU][mU][fC][mU][fC][mAs][mAs][mA]-3' (SEQ ID NO: 1682), wherein the antisense strand comprises the sequence 5'-[Me phosphonate-4O-mUs][fUs][fU][fG][fA][m G][fA][mA][mA][fU][mG][mU][mC][fA][mU][fG][mA][mC][fU][mGs][mGs][mG]-3' (SEQ ID NO: 1684). NO:1656), and wherein mC, mA, mG, mU = 2'-OMe ribonucleoside; fA, fC, fG, fU = 2'-F ribonucleoside; s = phosphorothioate, and 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 claims 1-97 or the 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 cell population, or a subject, the method comprising the steps of: i. contacting the cell or the cell population 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 the amount or level of SNCA mRNA, the amount or level of SNCA protein, SNCA activity / function, or a combination thereof.

103. The method of claim 101 or 102, wherein the subject suffers from 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's 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's disease.

106. The method of claim 105, wherein the tissue associated with Parkinson's disease is selected from the group consisting of 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 the tissue associated with multiple system atrophy is selected from the group consisting of: 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 decreased in one or more regions of the central nervous system (CNS) selected from the group consisting of 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 for the manufacture of a medicament for treating 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, which is used or suitable for treating 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, optionally a pharmaceutically acceptable carrier, and a package insert comprising instructions for administration to a subject suffering from a disease, disorder or condition associated with SNCA gene expression.

114. The use of claim 111, the RNAi oligonucleotide for use or the pharmaceutical composition suitable 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's disease.

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