Compositions and Methods for Inhibiting the Expression of SNCA

Abstract

An oligonucleotide (RNAi oligonucleotide) that inhibits the expression of the SNCA gene and contains an oligonucleotide bound to a targeting ligand (e.g., a GalNAc moiety or a lipid moiety) is provided herein. Also provided are compositions containing the same and its use, particularly its use related to the treatment of diseases, disorders, and / or conditions associated with the expression of the SNCA gene.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63 / 364,639, filed May 13, 2022, the entire disclosure of which is incorporated herein by reference.

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

Background Art

[0003] A synapse is a functional compartment between cells where information is transmitted from one cell in the brain to another. SNCA is a protein mainly found in the brain and regulates synaptic vesicle transport and neurotransmitter release. In particular, SNCA acts as a molecular chaperone and helps fold synaptic fusion components (such as SNAREs). Mutations in SNCA (e.g., insertions or mismatches) that change the function or expression of SNCA, or general abnormal expression of SNCA, are known to cause several diseases that affect the central nervous system (e.g., Parkinson's disease and multiple system atrophy). To prevent such diseases, strategies targeting the SNCA gene are needed.

[0004] The mammalian CNS is a complex system of tissues that includes cells, fluids, and chemicals, which interact simultaneously to enable a diverse range of functions including movement, motor commands, cognition, speech, vision, and emotion. Unfortunately, various diseases and disorders of the CNS (e.g., neuropathy) are known, which affect or disrupt some or all of these functions. Typically, the treatment of CNS diseases and disorders is limited to small molecule drugs, antibodies, and / or adaptive or behavioral therapies. There continues to be a need to develop treatments for CNS diseases and disorders associated with inappropriate gene expression. SUMMARY OF THE INVENTION

[0005] To address this need, the present disclosure describes compositions and methods of treatment therefor for treating diseases, disorders, and / or conditions associated with the expression of the SNCA gene. The present disclosure is based, at least in part, on the discovery of RNAi oligonucleotides that effectively target and reduce the expression of the SNCA gene in CNS tissue. Specifically, target sequences within SNCA mRNA were identified and oligonucleotides were generated that bind to these target sequences and inhibit the expression of SNCA mRNA. As demonstrated herein, the oligonucleotides inhibited the expression of the SNCA gene in human and non-human primate (NHP) CNS tissue. Furthermore, the expression of SNCA mRNA was reduced in CNS tissue associated with Parkinson's disease and multiple system atrophy by oligonucleotides that target lipid-bound SNCA. Without being bound by theory, the oligonucleotides described herein are useful for treating diseases, disorders, or conditions associated with the expression of the SNCA gene.

[0006] Thus, in some aspects, the present disclosure provides RNAi oligonucleotides for reducing the expression of the SNCA gene, the oligonucleotides comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a double-stranded region, and the antisense strand comprises a region complementary to any one of the SNCA mRNA target sequences of SEQ ID NOs: 1683 to 2066, the complementary region being at least about 15 consecutive nucleotides in length.

[0007] In any of the foregoing aspects or related aspects, in some aspects, the sense strand is about 15 to about 50 (or about 15 to about 50 in length) nucleotides in length. In some aspects, the sense strand is about 18 to about 36 nucleotides in length. In some aspects, the antisense strand is about 15 to about 30 nucleotides in length. In some aspects, the antisense strand is 22 nucleotides in length, and the antisense strand and the sense strand form a double-stranded region of at least 19 nucleotides in length, optionally at least 20 nucleotides in length. In some aspects, the complementary region is at least 19 consecutive nucleotides in length. In some aspects, the complementary region is at least 20 consecutive nucleotides in length.

[0008] In other aspects, the present disclosure provides double-stranded (ds) RNAi oligonucleotides for reducing SNCA gene expression, the oligonucleotides comprising (i) an antisense strand about 19 to 30 nucleotides in length, the antisense strand comprising a nucleotide sequence comprising a region complementary to the SNCA mRNA target sequence, the complementary region being selected from SEQ ID NOs: 2067 to 2450, the antisense strand, and (ii) a sense strand about 19 to 50 nucleotides in length comprising a region complementary to the antisense strand, the antisense strand and the sense strand being separate strands that form an asymmetric double-stranded region having an overhang of 1 to 4 nucleotides at the 3' end of the antisense strand.

[0009] In some embodiments, the 3’ end of the sense strand comprises a stem-loop shown as S1-L-S2, where S1 is complementary to S2 and L forms a loop of 3 to 5 nucleotides in length between S1 and S2. In some embodiments, L is a triloop or a tetraloop. In some embodiments, L is a tetraloop. In some embodiments, the tetraloop comprises the sequence 5’-GAAA-3’. In some embodiments, S1 and S2 are nucleotides of 1 to 10 in length and have the same length. In some embodiments, S1 and S2 are 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, or 10 nucleotides in length. In some embodiments, S1 and S2 are 6 nucleotides in length. In some embodiments, the stem-loop comprises the sequence 5’-GCAGCCGAAAGGCUGC-3’ (SEQ ID NO: 1680).

[0010] In other embodiments, the oligonucleotide comprises blunt ends. In some embodiments, the blunt ends comprise the 3’ end of the sense strand. In some embodiments, the sense strand is 20 to 22 nucleotides in length. In some embodiments, the sense strand is 20 nucleotides in length.

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

[0012] In any of the foregoing embodiments or related embodiments, the oligonucleotide comprises at least one modified nucleotide. In some embodiments, the modified nucleotide comprises a 2'-modification. In some embodiments, the 2'-modification is a modification selected from 2'-aminoethyl, 2'-fluoro (2'-F), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl, and 2'-deoxy-2'-fluoro-β-d-arabinonucleic acid. In some embodiments, the modification is a 2'-modification selected from 2'-F and 2'-OMe. In some embodiments, 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 embodiments, about 38% to 43%, 38%, 39%, 40%, 41%, 42%, or 43% of the nucleotides of the sense strand comprise a 2'-F modification. In some embodiments, 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 embodiments, 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 embodiments, 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 embodiments, the sense strand comprises 36 nucleotides at positions 1 to 36 from 5' to 3', and each of positions 3, 5, 8, 10, 12, 13, 15, and 17 comprises a 2'-F modification. In some embodiments, the sense strand comprises 20 nucleotides at positions 1 to 20 from 5' to 3', and each of positions 3, 5, 8, 10, 12, 13, 15, and 17 comprises a 2'-F modification. In some embodiments, the antisense strand comprises 22 nucleotides at positions 1 to 22 from 5' to 3', and each of positions 2, 3, 4, 5, 7, 10, 14, 16, and 19 comprises a 2'-F modification. In some embodiments, the remaining nucleotides comprise a 2'-OMe modification.

[0013] In any of the foregoing or related embodiments, the oligonucleotide comprises at least one modified internucleotide linkage. In some embodiments, at least one modified internucleotide linkage is a phosphorothioate linkage. In some embodiments, the antisense strand comprises phosphorothioate linkages (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, where the positions are numbered 1 to 4 in the 5' to 3' direction. In some embodiments, the antisense strand is 22 nucleotides in length and comprises phosphorothioate linkages between positions 20 and 21 and between positions 21 and 22, where the positions are numbered 1 to 22 in the 5' to 3' direction. In some embodiments, the sense strand comprises a phosphorothioate linkage between positions 1 and 2, where the positions are numbered 1 to 2 in the 5' to 3' direction. In some embodiments, the sense strand is 20 nucleotides in length and comprises phosphorothioate linkages between positions 1 and 2, between positions 18 and 19, and between positions 19 and 20, where the positions are numbered 1 to 20 in the 5' to 3' direction.

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

[0015] In any of the foregoing or related aspects, at least one nucleotide of the oligonucleotide is bound 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 bound to one or more nucleotides of the stem-loop. In some aspects, one or more targeting ligands are bound to one or more nucleotides of the loop. In some aspects, the loop comprises four nucleotides numbered 1 to 4 from 5' to 3', wherein the nucleotides at positions 2, 3, and 4 each comprise one or more targeting ligands, and the targeting ligands are the same or different. In some aspects, each targeting ligand comprises an 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 four nucleotides of L of the stem-loop are each bound to a monovalent GalNac moiety.

[0016] In other aspects, one or more targeting ligands are lipid moieties. In some aspects, the lipid moiety is bound 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 C8-C 30 is a hydrocarbon chain. In some aspects, the hydrocarbon chain is C 16 is a hydrocarbon chain. In some aspects, C 16 The hydrocarbon chain is as follows

Chemical formula

[0017] In any of the foregoing embodiments or related embodiments, the complementary region is completely complementary to the SNCA mRNA target sequence at nucleotides 2 to 8 of the antisense strand, where the nucleotide positions are numbered 5' to 3'. In some embodiments, the complementary region is completely complementary to the SNCA mRNA target sequence at nucleotides 2 to 11 of the antisense strand, where the nucleotide positions are numbered 5' to 3'.

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

[0019] In some embodiments, the sense and antisense strands are: a) SEQ ID NOs: 1540 and 1575, respectively; b) SEQ ID NOs: 1544 and 1579, respectively; c) SEQ ID NOs: 1546 and 1581, respectively; d) SEQ ID NOs: 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 NO: 1560 and 1595, respectively; i) SEQ ID NO: 1564 and 1599, respectively; j) SEQ ID NO: 1565 and 1600, respectively; k) SEQ ID NO: 1566 and 1601, respectively; l) SEQ ID NO: 1570 and 1605, respectively; and m) It includes nucleotide sequences selected from the group consisting of SEQ ID NO: 1681 and 1586, respectively.

[0020] In some embodiments, the sense strand and the antisense strand are: a) SEQ ID NO: 1553 and 1588, respectively; b) SEQ ID NO: 1560 and 1595, respectively; c) SEQ ID NO: 1564 and 1599, respectively; d) SEQ ID NO: 1551 and 1586, respectively; e) SEQ ID NO: 1570 and 1605, respectively; f) It includes nucleotide sequences selected from the group consisting of SEQ ID NO: 1681 and 1586, respectively.

[0021] In some embodiments, the sense strand includes the nucleotide sequence shown in SEQ ID NO: 1553, and the antisense strand includes the nucleotide sequence shown in SEQ ID NO: 1588. In some embodiments, the sense strand includes the nucleotide sequence shown in SEQ ID NO: 1560, and the antisense strand includes the nucleotide sequence shown in SEQ ID NO: 1595. In some embodiments, the sense strand includes the nucleotide sequence shown in SEQ ID NO: 1564, and the antisense strand includes the nucleotide sequence shown in SEQ ID NO: 1599. In some embodiments, the sense strand includes the nucleotide sequence shown in SEQ ID NO: 1551, and the antisense strand includes the nucleotide sequence shown in SEQ ID NO: 1586. In some embodiments, the sense strand includes the nucleotide sequence shown in SEQ ID NO: 1570, and the antisense strand includes the nucleotide sequence shown in SEQ ID NO: 1605. In some embodiments, the sense strand includes the nucleotide sequence shown in SEQ ID NO: 1681, and the antisense strand includes the nucleotide sequence shown in SEQ ID NO: 1586.

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

[0023] In any of the foregoing embodiments or related embodiments, the sense strand comprises any one nucleotide sequence of SEQ ID NOs: 1607 to 1641 and 1682. In some embodiments, the antisense strand comprises any one nucleotide sequence of SEQ ID NOs: 1642 to 1676.

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

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

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

[0027] In some embodiments, 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 embodiments, 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 embodiments, 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 embodiments, 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 embodiments, 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 embodiments, 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.

[0028] In some embodiments, 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][mA][mG][mC][mC][mG][ademA-GalNAc][ademA-GalNAc][ademA-GalNAc][mG][mG][mC][mU][mG][mC]-3’ (SEQ ID NO: 1623) and all modifications thereof, where the antisense strand comprises the sequence 5’-[MePhosphonate-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 thereof, where mC, mA, mG, mU = 2’-OMe ribonucleoside; fA, fC, fG, fU = 2’-F ribonucleoside; s = phosphorothioate, and ademA-GalNAc = [Chemical Formula] It is.

[0029] In some embodiments, 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][ademA-GalNAc][mG][mG][mC][mU][mG][mC]-3’ (SEQ ID NO: 1630) and all modifications, wherein the antisense strand comprises the sequence 5’-[MePhosphonate-4O-mUs][fAs][fA][fC][fA][mU][fU][mU][mG][fU][mC][mA][mC][fU][mU][fG][mC][mU][fC][mUs][mGs][mG]-3’ (SEQ ID NO: 1665) and all modifications, wherein mC, mA, mG, mU = 2’-OMe ribonucleoside; fA, fC, fG, fU = 2’-F ribonucleoside; s = phosphorothioate, and ademA-GalNAc =

Chemical formula

[0030] In some embodiments, 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][mA][mG][mC][mA][mG][mC][mC][mG][ademA-GalNAc][ademA-GalNAc][ademA-GalNAc][mG][mG][mC][mU][mG][mC]-3’ (SEQ ID NO: 1634) and all modifications, wherein the antisense strand comprises the sequence 5’-[MePhosphonate-4O-mUs][fUs][fU][fU][fG][mA][fG][mA][mA][fA][mU][mG][mU][fC][mA][fU][mG][mA][fC][mUs][mGs][mG]-3’ (SEQ ID NO: 1669) and all modifications, wherein mC, mA, mG, mU = 2’-OMe ribonucleoside; fA, fC, fG, fU = 2’-F ribonucleoside; s = phosphorothioate, and ademA-GalNAc = [Chemical formula] is as follows.

[0031] In some embodiments, 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][mG][mC][mU][mG][mC]-3’ (SEQ ID NO: 1621) and all modifications, wherein the antisense strand comprises the sequence 5’-[MePhosphonate-4O-mUs][fUs][fU][fG][fA][mG][fA][mA][mA][fU][mG][mU][mC][fA][mU][fG][mA][mC][fU][mGs][mGs][mG]-3’ (SEQ ID NO: 1656) and all modifications, where mC, mA, mG, mU = 2’-OMe ribonucleoside; fA, fC, fG, fU = 2’-F ribonucleoside; s = phosphorothioate, and ademA-GalNAc = [Chemical formula] is.

[0032] In some embodiments, 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][ademA-GalNAc][mG][mG][mC][mU][mG][mC]-3’ (SEQ ID NO: 1640) and all modifications, At that time, the antisense strand contains the sequence 5’-[MePhosphonate-4O-mUs][fUs][fA][fG][fC][mA][fA][mA][mU][fC][mA][mC][mU][fA][mA][fC][mA][mA][fC][mUs][mGs][mG]-3’ (SEQ ID NO: 1675) and all modifications, where mC, mA, mG, mU = 2’-OMe ribonucleoside; fA, fC, fG, fU = 2’-F ribonucleoside; s = phosphorothioate, and ademA-GalNAc = [Chemical formula] is as follows. In some embodiments, the sense strand contains the sequence 5’-[ademCs-C 16 [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 the antisense strand contains the sequence 5’-[MePhosphonate-4O-mUs][fUs][fU][fG][fA][mG][fA][mA][mA][fU][mG][mU][mC][fA][mU][fG][mA][mC][fU][mGs][mGs][mG]-3’ (SEQ ID NO: 1656) and all modifications, where mC, mA, mG, mU = 2’-OMe ribonucleoside; fA, fC, fG, fU = 2’-F ribonucleoside; s = phosphorothioate, and [ademCs-C 16 = [Chemical formula] is as follows.

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

[0034] In another aspect, the present disclosure provides a method of treating a subject having a disease, disorder, or condition associated with the expression of the SNCA gene, 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.

[0035] In a further aspect, 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.

[0036] In yet a further aspect, the present disclosure provides a method of reducing the expression of the SNCA gene in a cell, a population of cells, or a subject, the method comprising: i. contacting the cell or population of cells with an RNAi oligonucleotide or pharmaceutical composition described herein; or ii. administering to the subject an RNAi oligonucleotide or pharmaceutical composition described herein.

[0037] In some aspects, reducing the expression of the SNCA gene comprises reducing the amount or level of SNCA mRNA, the amount or level of SNCA protein, or both. In some aspects, the subject has a disease, disorder, or condition associated with the expression of the SNCA gene. In some aspects, the disease, disorder, or condition associated with the expression of SNCA is multiple system atrophy, Lewy body dementia, or Parkinson's disease.

[0038] In any of the foregoing or related aspects, the expression of the SNCA gene is reduced in the tissue of one or more regions of the CNS by the RNAi oligonucleotide or pharmaceutical composition described herein, wherein the tissue is associated with Parkinson's disease. In some aspects, the tissue associated with Parkinson's disease is selected from the putamen, tegmentum, substantia nigra, pons, and medulla. In some aspects, the expression of the SNCA gene is reduced in the tissue of one or more regions of the CNS, wherein the tissue is associated with multiple system atrophy. In some aspects, the tissue associated with multiple system atrophy is selected from the caudate nucleus, putamen, tegmentum, substantia nigra, pons, cerebellar cortex, cerebellar white matter, medulla, cervical spinal cord, thoracic spinal cord, and lumbar spinal cord. In some aspects, the expression of the SNCA gene is reduced in one or more regions of the CNS selected from the cervical spinal cord, thoracic spinal cord, lumbar spinal cord, prefrontal 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, and dentate nucleus of the cerebellum. In some aspects, the expression of the SNCA gene is reduced in one or more regions of the CNS selected from the cervical spinal cord, thoracic spinal cord, lumbar spinal cord, prefrontal 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, dentate nucleus of the cerebellum, L1 dorsal nucleus, L2 dorsal nucleus, L3 dorsal nucleus, L4 dorsal nucleus, substantia nigra, cerebral cortex, cerebellar white matter, dentate nucleus of the cerebellum, 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.

[0039] In any of the foregoing or related aspects, the RNAi oligonucleotide or pharmaceutical composition is administered in combination with a second composition or therapeutic agent.

[0040] In other aspects, the present disclosure provides the use of the RNAi oligonucleotide or pharmaceutical composition described herein in the manufacture of a medicament for the treatment of a disease, disorder, or condition associated with the expression of the SNCA gene.

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

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

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

Brief Description of the Drawings

[0044]

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Modes for Carrying Out the Invention

[0045] According to some aspects, the present disclosure provides an oligonucleotide that reduces the expression of the SNCA gene 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 a method of treating a disease associated with SNCA expression by reducing the expression of the SNCA gene in a cell (e.g., a cell of the CNS).

[0046] Oligonucleotide inhibitors of SNCA expression The present disclosure provides, among other things, oligonucleotides (e.g., RNAi oligonucleotides) that inhibit the expression of the SNCA gene. In some embodiments, the oligonucleotide that inhibits the expression of the SNCA gene targets SNCA mRNA.

[0047] SNCA target sequence In some embodiments, the oligonucleotides herein (e.g., RNAi oligonucleotides) target target sequences that make up SNCA mRNA. In some embodiments, the oligonucleotides described herein target target sequences within the SNCA mRNA sequence.

[0048] In some embodiments, the oligonucleotides described herein correspond to target sequences within the SNCA mRNA sequence. In some embodiments, the oligonucleotide, or a portion, fragment, or strand thereof (e.g., the antisense strand or guide strand of an RNAi oligonucleotide) binds or anneals to a target sequence that makes up SNCA mRNA, thereby inhibiting the expression of the SNCA gene.

[0049] In some embodiments, the oligonucleotide targets the SNCA target sequence for the purpose of inhibiting the expression of the SNCA gene in vivo. In some embodiments, the amount or degree of inhibition of SNCA gene expression by an oligonucleotide targeting the SNCA target sequence correlates with the potency of the oligonucleotide. In some embodiments, the amount or degree of inhibition of SNCA gene expression by an oligonucleotide targeting the SNCA target sequence correlates with the amount or degree of therapeutic effect in a subject or patient having a disease, disorder, or condition associated with SNCA gene expression treated with the oligonucleotide.

[0050] Through examination of the nucleotide sequences of mRNAs encoding SNCA, including mRNAs of multiple different species (e.g., human, cynomolgus monkey, and mouse; see, e.g., Example 1), and through the results of in vitro and in vivo tests (see, e.g., Examples 2-5), it has been discovered that certain nucleotide sequences of SNCA mRNA are more suitable than others for oligonucleotide-based inhibition and are thus useful as target sequences for the oligonucleotides herein. In some embodiments, the sense strand of the oligonucleotides (e.g., RNAi oligonucleotides) described herein comprises the SNCA target sequence. In some embodiments, a part or region of the sense strand of the ds oligonucleotides described herein comprises the SNCA target sequence. In some embodiments, the SNCA target sequence comprises, or consists of, any one of the nucleotide sequences of SEQ ID NOs: 1683-2066. In some embodiments, the SNCA target sequence comprises, or consists of, any one of the nucleotide sequences 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, any one of the nucleotide sequences 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, any one of the nucleotide sequences 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.

[0051] Sequence targeting SNCA In some embodiments, the oligonucleotides herein target intracellular mRNA and have a region complementary to SNCA mRNA (e.g., within the target sequence of SNCA mRNA) for the purpose of inhibiting its expression. In some embodiments, the oligonucleotide comprises a sequence targeting SNCA (e.g., the antisense strand or guide strand of a ds oligonucleotide) having a complementary region that binds or anneals to the SNCA target sequence by complementary (Watson-Crick) base pairing. The complementary targeting sequence or targeting region is generally of a length and base content suitable to allow binding or annealing of the oligonucleotide (or its strand) to SNCA mRNA for the purpose of inhibiting SNCA mRNA expression. In some embodiments, 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 length. In some embodiments, the complementary targeting sequence or 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, at least 20 nucleotides in length. In some embodiments, the complementary targeting sequence or region is about 12-30 (e.g., 12-30, 12-22, 15-25, 17-21, 18-27, 19-27, or 15-30) nucleotides in length. In some embodiments, the complementary targeting sequence or 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 length. In some embodiments, the complementary targeting sequence or region is 18 nucleotides in length. In some embodiments, the complementary targeting sequence or region is 19 nucleotides in length. In some embodiments, the complementary targeting sequence or region is 20 nucleotides in length.In some embodiments, the complementary targeting sequence or region is 21 nucleotides in length. In some embodiments, the complementary targeting sequence or region is 22 nucleotides in length. In some embodiments, the complementary targeting sequence or region is 23 nucleotides in length. In some embodiments, the complementary targeting sequence or region is 24 nucleotides in length. In some embodiments, the oligonucleotide comprises a complementary targeting sequence or region that is complementary to any one of the sequences of SEQ ID NOs: 1683 to 2066, and the complementary targeting sequence or region is 18 nucleotides in length. In some embodiments, the oligonucleotide comprises a complementary targeting sequence or region that is complementary to any one of the sequences of SEQ ID NOs: 1683 to 2066, and the complementary targeting sequence or region is 19 nucleotides in length. In some embodiments, the oligonucleotide comprises a complementary targeting sequence or region that is complementary to any one of the sequences of SEQ ID NOs: 1 to 384, and the complementary targeting sequence or region is 20 nucleotides in length. In some embodiments, the oligonucleotide comprises a complementary targeting sequence or region that is complementary to any one of the sequences of SEQ ID NOs: 1 to 384, and the complementary targeting sequence or region is 21 nucleotides in length. In some embodiments, the oligonucleotide comprises a complementary targeting sequence or region that is complementary to any one of the sequences of SEQ ID NOs: 1 to 384, and the complementary targeting sequence or region is 22 nucleotides in length. In some embodiments, the oligonucleotide comprises a complementary targeting sequence or region that is complementary to any one of the sequences of SEQ ID NOs: 1 to 384, and the complementary targeting sequence or region is 23 nucleotides in length. In some embodiments, the oligonucleotide comprises a complementary targeting sequence or region that is complementary to any one of the sequences of SEQ ID NOs: 1 to 384, and the complementary targeting sequence or region is 24 nucleotides in length.

[0052] In some embodiments, the oligonucleotide comprises a complementary targeting sequence or targeting region (e.g., the antisense strand or guide strand of a ds oligonucleotide) that is fully complementary to the SNCA target sequence. In some embodiments, the complementary targeting sequence or targeting region is partially complementary to the SNCA target sequence. In some embodiments, the oligonucleotide comprises a complementary targeting sequence or targeting region that is fully complementary to any one of the sequences of SEQ ID NOs: 1683-2066. In some embodiments, the oligonucleotide comprises a complementary targeting sequence or targeting region that is partially complementary to any one of the sequences of SEQ ID NOs: 1683-2066. In some embodiments, the oligonucleotide comprises a complementary targeting sequence or targeting region that is fully complementary to any one of the sequences 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 complementary targeting sequence or targeting region that is partially complementary to any one of the sequences 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 complementary targeting sequence or targeting region that is fully complementary to any one of the sequences of SEQ ID NOs: 1798, 1817, 1718, 1846, 1852, 1865, 1804, 1721, 1847, 1855, 1864, and 1955.In some embodiments, the oligonucleotide comprises a complementary targeting sequence or targeting region that is partially complementary to any one of the sequences of SEQ ID NO: 1798, 1817, 1718, 1846, 1852, 1865, 1804, 1721, 1847, 1855, 1864, and 1955. In some embodiments, the oligonucleotide comprises a complementary targeting sequence or targeting region that is completely complementary to any one of the sequences of SEQ ID NO: 1865, 1721, 1847, 1846, and 1955. In some embodiments, the oligonucleotide comprises a complementary targeting sequence or targeting region that is partially complementary to any one of the sequences of SEQ ID NO: 1865, 1721, 1847, 1846, and 1955. In some embodiments, the oligonucleotide comprises a complementary targeting sequence or targeting region that is completely complementary to the sequence shown in SEQ ID NO: 1865. In some embodiments, the oligonucleotide comprises a complementary targeting sequence or targeting region that is completely complementary to the sequence shown in SEQ ID NO: 1721. In some embodiments, the oligonucleotide comprises a complementary targeting sequence or targeting region that is completely complementary to the sequence shown in SEQ ID NO: 1847. In some embodiments, the oligonucleotide comprises a complementary targeting sequence or targeting region that is completely complementary to the sequence shown in SEQ ID NO: 1846. In some embodiments, the oligonucleotide comprises a complementary targeting sequence or targeting region that is completely complementary to the sequence shown in SEQ ID NO: 1955. In some embodiments, the oligonucleotide comprises a complementary targeting sequence or targeting region that is partially complementary to any one of the sequences of SEQ ID NO: 1865. In some embodiments, the oligonucleotide comprises a complementary targeting sequence or targeting region that is partially complementary to the sequence of SEQ ID NO: 1721. In some embodiments, the oligonucleotide comprises a complementary targeting sequence or targeting region that is partially complementary to the sequence of SEQ ID NO: 1847. In some embodiments, the oligonucleotide comprises a complementary targeting sequence or targeting region that is partially complementary to the sequence of SEQ ID NO: 1846.In some embodiments, the oligonucleotide comprises a complementary targeting sequence or targeting region that is partially complementary to the sequence of SEQ ID NO: 1955.

[0053] In some embodiments, the oligonucleotides herein comprise a complementary targeting sequence or targeting region that is complementary to a contiguous sequence of nucleotides comprising SNCA mRNA, the contiguous sequence of nucleotides being from about 12 to about 30 nucleotides in length (e.g., 12 - 30, 12 - 28, 12 - 26, 12 - 24, 12 - 20, 12 - 18, 12 - 16, 14 - 22, 16 - 20, 18 - 20, or 18 - 19 nucleotides). In some embodiments, the oligonucleotide comprises a complementary targeting sequence or targeting region that is complementary to a contiguous sequence of nucleotides comprising SNCA mRNA, the contiguous sequence of nucleotides being 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In some embodiments, the oligonucleotide comprises a complementary targeting sequence or targeting region that is complementary to a contiguous sequence of nucleotides comprising SNCA mRNA, the contiguous sequence of nucleotides being 19 nucleotides in length. In some embodiments, the oligonucleotide comprises a complementary targeting sequence or targeting region that is complementary to a contiguous sequence of nucleotides comprising SNCA mRNA, the contiguous sequence of nucleotides being 20 nucleotides in length.

[0054] In some embodiments, the oligonucleotide comprises a complementary targeting sequence or targeting region that is complementary to a contiguous sequence of any one of nucleotides of SEQ ID NOs: 1683 to 2066, optionally wherein the contiguous sequence of nucleotides is 19 nucleotides in length. In some embodiments, the oligonucleotide comprises a complementary targeting sequence or targeting region that is complementary to a contiguous sequence of any one of nucleotides of SEQ ID NOs: 1781, 1782, 1796, 1798, 1802, 1808, 1814, 1817, 1713, 1718, 1726, 1830, 1839, 1742, 1846, 1852, 1865, 1784, 1804, 1721, 1822, 1840, 1735, 1847, 1855, 1864, 1901, 1902, 1938, 1947, 1955, 1964, 1973, and 1978, optionally wherein the contiguous sequence of nucleotides is 19 nucleotides in length. In some embodiments, the oligonucleotide comprises a complementary targeting sequence or targeting region that is complementary to a contiguous sequence of any one of nucleotides of SEQ ID NOs: 1798, 1817, 1718, 1846, 1852, 1865, 1804, 1721, 1847, 1855, 1864, and 1955, optionally wherein the contiguous sequence of nucleotides is 19 nucleotides in length. In some embodiments, the oligonucleotide comprises a complementary targeting sequence or targeting region that is complementary to a contiguous sequence of nucleotides of SEQ ID NOs: 1865, 1721, 1847, 1846, and 1955, optionally wherein the contiguous sequence of nucleotides is 19 nucleotides in length.

[0055] In some embodiments, the complementary targeting sequence or targeting region of the oligonucleotide (e.g., an RNAi oligonucleotide) is complementary to the contiguous nucleotides of the sequence shown in any one of SEQ ID NOs: 1683 - 2066 and spans the entire length of the antisense strand. In some embodiments, the complementary targeting sequence or targeting region of the oligonucleotide is complementary to the contiguous nucleotides of the sequence shown in any one of SEQ ID NOs: 1683 - 2066 and extends over a part of the entire length of the antisense strand. In some embodiments, the oligonucleotide comprises (e.g., on the antisense strand of a ds oligonucleotide) a complementary region that is at least partially (e.g., completely) complementary to a contiguous stretch of nucleotides spanning nucleotides 1 - 20 of the sequence shown in any one of SEQ ID NOs: 1683 - 2066. In some embodiments, the complementary targeting sequence or targeting region of the oligonucleotide is complementary to the contiguous nucleotides of the sequence 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 the oligonucleotide is complementary to the contiguous nucleotides of the sequence shown in any one of SEQ ID NOs: 1 - 384 and extends over a part of the entire length of the antisense strand. In some embodiments, the oligonucleotide comprises (e.g., on the antisense strand of a ds oligonucleotide) a complementary region that is at least partially (e.g., completely) complementary to a contiguous stretch of nucleotides spanning nucleotides 1 - 19 of the sequence shown in any one of SEQ ID NOs: 1 - 384.

[0056] In some embodiments, the oligonucleotide comprises a complementary targeting sequence or region having one or more base pair (bp) mismatches with the corresponding SNCA target sequence. In some embodiments, the complementary targeting sequence or 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 complementary targeting sequence or region to bind or anneal to SNCA mRNA under appropriate hybridization conditions, and / or the ability of the oligonucleotide to inhibit the expression of the SNCA gene is maintained. Alternatively, in some embodiments, the complementary targeting sequence or region comprises up to 1, up to 2, up to 3, up to 4, or up to 5 mismatches with the corresponding SNCA target sequence, provided that the ability of the complementary targeting sequence or region to bind or anneal to SNCA mRNA under appropriate hybridization conditions, and / or the ability of the oligonucleotide to inhibit the expression of the SNCA gene is maintained. In some embodiments, the oligonucleotide comprises a complementary targeting sequence or region having one mismatch with the corresponding target sequence. In some embodiments, the oligonucleotide comprises a complementary targeting sequence or region having two mismatches with the corresponding target sequence. In some embodiments, the oligonucleotide comprises a complementary targeting sequence or region having three mismatches with the corresponding target sequence. In some embodiments, the oligonucleotide comprises a complementary targeting sequence or region having four mismatches with the corresponding target sequence. In some embodiments, the oligonucleotide comprises a complementary targeting sequence or region having five mismatches with the corresponding target sequence. In some embodiments, the oligonucleotide comprises a complementary targeting sequence or region having more than one mismatch (e.g., 2, 3, 4, 5 or more mismatches) with the corresponding target sequence, wherein at least two (e.g., all) of the mismatches are arranged consecutively (e.g., 2, 3, 4, 5 or more consecutive mismatches), or the mismatches are scattered at any position throughout the complementary targeting sequence or region.In some embodiments, the oligonucleotide comprises a complementary targeting sequence or region having more than one mismatch (e.g., 2, 3, 4, 5 or more mismatches) with the corresponding target sequence, wherein at least two (e.g., all) of the mismatches are arranged consecutively (e.g., 2, 3, 4, 5 or more consecutive mismatches), or at least one non-mismatched base pair is located between the mismatches, or a combination thereof.

[0057] In some embodiments, the oligonucleotide comprises a complementary targeting sequence or targeting region that is complementary to a contiguous sequence of any one of nucleotides of SEQ ID NOs: 1683 to 2066, wherein the complementary targeting sequence or targeting 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. In some embodiments, the oligonucleotide comprises a complementary targeting sequence or targeting region that is complementary to a contiguous sequence of any one of nucleotides of SEQ ID NOs: 1683 to 2066, wherein the complementary targeting sequence or targeting region may have 1 or less, 2 or less, 3 or less, about 4 or less, about 5 or less mismatches with the corresponding SNCA target sequence. In some embodiments, the oligonucleotide comprises a complementary targeting sequence or targeting region that is complementary to a contiguous sequence of any one of nucleotides 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 complementary targeting sequence or targeting 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. In some embodiments, the oligonucleotide comprises a complementary targeting sequence or targeting region that is complementary to a contiguous sequence of any one of nucleotides 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 complementary targeting sequence or targeting region may have 1 or less, 2 or less, 3 or less, 4 or less, or 5 or less mismatches with the corresponding SNCA target sequence.In some embodiments, the oligonucleotide comprises a complementary targeting sequence or targeting region that is complementary to a contiguous sequence of any one of the nucleotides of SEQ ID NOs: 1798, 1817, 1718, 1846, 1852, 1865, 1804, 1721, 1847, 1855, 1864, and 1955, wherein the complementary targeting sequence or targeting 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. In some embodiments, the oligonucleotide comprises a complementary targeting sequence or targeting region that is complementary to a contiguous sequence of any one of the nucleotides of SEQ ID NOs: 1798, 1817, 1718, 1846, 1852, 1865, 1804, 1721, 1847, 1855, 1864, and 1955, wherein the complementary targeting sequence or targeting region may have 1 or less, 2 or less, 3 or less, 4 or less, or 5 or less mismatches with the corresponding SNCA target sequence. In some embodiments, the oligonucleotide comprises a complementary targeting sequence or targeting region that is complementary to a contiguous sequence of any one of the nucleotides of SEQ ID NOs: 1865, 1721, 1847, 1846, and 1955, wherein the complementary targeting sequence or targeting 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. In some embodiments, the oligonucleotide comprises a complementary targeting sequence or targeting region that is complementary to a contiguous sequence of any one of the nucleotides of SEQ ID NOs: 1865, 1721, 1847, 1846, and 1955, wherein the complementary targeting sequence or targeting region may have 1 or less, 2 or less, 3 or less, 4 or less, or 5 or less mismatches with the corresponding SNCA target sequence. In some embodiments, the oligonucleotide comprises a complementary targeting sequence or targeting region that is complementary to a contiguous sequence of the nucleotides of SEQ ID NO: 1865, wherein the complementary targeting sequence or targeting 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.In some embodiments, the oligonucleotide comprises a complementary targeting sequence or targeting region that is complementary to a contiguous sequence of nucleotides of SEQ ID NO: 1865, wherein the complementary targeting sequence or targeting region may have 1 or fewer, 2 or fewer, 3 or fewer, 4 or fewer, 5 or fewer mismatches with the corresponding SNCA target sequence. In some embodiments, the oligonucleotide comprises a complementary targeting sequence or targeting region that is complementary to a contiguous sequence of nucleotides of SEQ ID NO: 1721, wherein the complementary targeting sequence or targeting 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. In some embodiments, the oligonucleotide comprises a complementary targeting sequence or targeting region that is complementary to a contiguous sequence of nucleotides of SEQ ID NO: 1721, wherein the complementary targeting sequence or targeting region may have 1 or fewer, 2 or fewer, 3 or fewer, 4 or fewer, 5 or fewer mismatches with the corresponding SNCA target sequence. In some embodiments, the oligonucleotide comprises a complementary targeting sequence or targeting region that is complementary to a contiguous sequence of nucleotides of SEQ ID NO: 1847, wherein the complementary targeting sequence or targeting 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. In some embodiments, the oligonucleotide comprises a complementary targeting sequence or targeting region that is complementary to a contiguous sequence of nucleotides of SEQ ID NO: 1847, wherein the complementary targeting sequence or targeting region may have 1 or fewer, 2 or fewer, 3 or fewer, 4 or fewer, 5 or fewer mismatches with the corresponding SNCA target sequence. In some embodiments, the oligonucleotide comprises a complementary targeting sequence or targeting region that is complementary to a contiguous sequence of nucleotides of SEQ ID NO: 1846, wherein the complementary targeting sequence or targeting 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.In some embodiments, the oligonucleotide comprises a complementary targeting sequence or targeting region that is complementary to a contiguous sequence of nucleotides of SEQ ID NO: 1846, wherein the complementary targeting sequence or targeting region may have 1 or fewer, 2 or fewer, 3 or fewer, 4 or fewer, 5 or fewer mismatches with the corresponding SNCA target sequence. In some embodiments, the oligonucleotide comprises a complementary targeting sequence or targeting region that is complementary to a contiguous sequence of nucleotides of SEQ ID NO: 1955, wherein the complementary targeting sequence or targeting 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. In some embodiments, the oligonucleotide comprises a complementary targeting sequence or targeting region that is complementary to a contiguous sequence of nucleotides of SEQ ID NO: 1955, wherein the complementary targeting sequence or targeting region may have 1 or fewer, 2 or fewer, 3 or fewer, 4 or fewer, 5 or fewer mismatches with the corresponding SNCA target sequence.

[0058] Types of Oligonucleotides Various types and / or structures of oligonucleotides, including but not limited to RNAi oligonucleotides, antisense oligonucleotides, miRNAs, etc., are useful for targeting SNCA mRNA in the methods of the present specification. Any of the oligonucleotide types described herein or elsewhere are contemplated for use as a framework for incorporating into the present specification a sequence that targets SNCA mRNA for the purpose of inhibiting the expression of the SNCA gene.

[0059] In some embodiments, the oligonucleotides herein inhibit the expression of the SNCA gene (e.g., RNAi oligonucleotides) by participating in the RNA interference (RNAi) pathway upstream or downstream of Dicer involvement. For example, RNAi oligonucleotides have been developed where each strand has a size of about 19 to about 25 nucleotides and has at least one 3' overhang of about 1 to about 5 nucleotides (see, e.g., U.S. Patent No. 8,372,968). Longer oligonucleotides have also been developed that are processed by Dicer to yield active RNAi products (see, e.g., U.S. Patent No. 8,883,996). Further research has produced extended ds oligonucleotides in which at least one end of at least one strand extends beyond the double-stranded targeting region, including structures in which one strand contains a thermodynamically stable tetraloop structure (see, e.g., U.S. Patents Nos. 8,513,207 and 8,927,705, and International Patent Application Publication No. WO2010 / 033225). Such structures may include ds extensions as well as single-stranded extensions (ss) (on one or both sides of the molecule).

[0060] In some embodiments, the oligonucleotide is involved in the RNAi pathway downstream of the involvement of Dicer (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 a target mRNA (e.g., SNCA mRNA), and a complementary passenger sense strand that anneals to both strands to form a 19-bp double strand and forms a 2-nucleotide overhang at either or both 3' ends. Also contemplated are longer oligonucleotide designs that include an oligonucleotide having a 23-nucleotide antisense strand and a 21-nucleotide passenger strand, where the molecule has a blunt end on the right side (3' end of the sense strand / 5' end of the antisense strand) of the molecule and a 2-nucleotide 3' antisense strand overhang on the left side (5' end of the sense strand / 3' end of the antisense strand) of the molecule. Such molecules have a 21-bp double-stranded region. See, for example, U.S. Patent Nos. 9,012,138; 9,012,621, and 9,193,753.

[0061] In some embodiments, the oligonucleotides herein include a sense strand and an antisense strand, both having a length in the range of about 17 to about 36 (e.g., 17 - 26, 20 - 25, or 21 - 23) nucleotides. In some embodiments, the oligonucleotide includes an antisense strand having a length of 19 - 30 nucleotides and a sense strand having a length of 19 - 50 nucleotides, wherein the antisense strand and the sense strand are separate strands that form an asymmetric double-stranded region having an overhang of 1 - 4 nucleotides at the 3’ end of the antisense strand. In some embodiments, the oligonucleotide includes a sense strand and an antisense strand, both having a length in the range of about 19 to about 22 nucleotides. In some embodiments, the sense strand and the antisense strand are of equal length. In some embodiments, since the oligonucleotide includes a sense strand and an antisense strand, there is a 3’ overhang on either the sense strand or the antisense strand, or both the sense strand and the antisense strand. In some embodiments, for an oligonucleotide having a sense strand and an antisense strand, both having a length in the range of about 21 - 23 nucleotides, the 3’ overhang of the sense strand, the antisense strand, or both strands is 1 or 2 nucleotides in length. In some embodiments, the oligonucleotide has an antisense strand of 22 nucleotides in length and a sense strand of 20 nucleotides in length, with a blunt end on the right side of the molecule (3’ end of the sense strand / 5’ end of the antisense strand) and a 3’ antisense strand overhang of 2 nucleotides on the left side of the molecule (5’ end of the sense strand / 3’ end of the antisense strand). Such a molecule has a 20 bp double-stranded region.

[0062] Other oligonucleotide designs for use with the compositions and methods herein include 16-base siRNAs (see, e.g., “Nucleic Acids in Chemistry & Biology,”, Blackburn (ed.), Royal Society of Chemistry, 2006), shRNAs (e.g., those having a stem of 19 bp or shorter; see, e.g., Moore et al. (2010) Methods Mol. Biol. 629:141-58), blunt siRNAs (e.g., those of 19 bp in length; see, e.g., Kraynack & Baker (2006) RNA, 12:163-76), asymmetric siRNAs (aiRNAs; see, e.g., Sun et al. (2008) Nat. Biotechnol. 26:1379-82), asymmetric short double-stranded siRNAs (e.g., Chang et al. (2009) Mol. Ther. 17:725-32), fork siRNAs (see, e.g., Hohjoh (2004) FEBS Lett. 557:193-98), single-stranded siRNAs (see, e.g., Elsner (2012), Nat. Biotechnol. 30:1063), dumbbell-shaped circular siRNAs (see, e.g., Abe et al. (2007) J. Am. Chem. Soc. 129:15108-09), and small internally segmented interfering RNAs (siRNAs; see, e.g., Bramsen et al. (2007), Nucleic Acids Res. 35:5886-97). Further non-limiting examples of oligonucleotide designs that may be used in some embodiments to decrease or inhibit the expression of the SNCA gene are microRNAs (miRNAs), short hairpin RNAs (shRNAs), and short siRNAs (see, e.g., Hamilton et al. (2002) EMBO J. 21:4671-79; also see U.S. Patent Application Publication No. 2009 / 0099115).

[0063] However, in some embodiments, the oligonucleotides for reducing or inhibiting the expression of the SNCA gene herein may have a structure that is ss, such as ssRNAi molecules, but are not limited thereto. The activity of ssRNAi molecules has been demonstrated by recent efforts (see, e.g., Matsui et al. (2016) Mol. Ther. 24:946-955). However, in some embodiments, the oligonucleotide is an antisense oligonucleotide (ASO). An antisense oligonucleotide contains the complement of a targeted segment of a particular nucleic acid in the reverse direction when written or drawn in the 5' to 3' direction and is preferably modified to induce RNaseH-mediated cleavage of its target RNA in the cell (e.g., as a gapmer) or to inhibit translation of the target mRNA in the cell (e.g., as a mixmer), and has a nucleotide sequence of an ss oligonucleotide. The ASO for use herein may be modified in any suitable manner known in the art, including, for example, those shown in U.S. Patent No. 9,567,587 (including, for example, changes in the length of the nucleobases (pyrimidines, purines), the sugar moiety, and the heterocyclic moiety of the nucleobases). Further, ASOs have been used for decades to reduce the expression of specific target genes (see, e.g., Bennett et al. (2017) Annu. Rev. Pharmacol. 57:81-105).

[0064] In some embodiments, the antisense oligonucleotide (ASO) shares a region complementary to SNCA mRNA. In some embodiments, the ASO targets various regions of human SNCA identified as NM_000345.3. In some embodiments, the ASO is from about 15 to about 50 nucleotides in length. In some embodiments, the ASO is from 15 to 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 consecutive nucleotides in length. In some embodiments, the ASO is at least 19 consecutive nucleotides in length. In some embodiments, the ASO is at least 20 consecutive nucleotides in length. In some embodiments, the ASO differs from the target sequence by only 1, 2, or 3 nucleotides.

[0065] Double-stranded RNAi oligonucleotide In some aspects, the present disclosure provides a dsRNAi oligonucleotide that targets SNCA mRNA and inhibits the expression of the SNCA gene (e.g., via the RNAi pathway) and includes a sense strand (also referred to herein as the passenger strand) and an antisense strand (also referred to herein as the guide strand). In some embodiments, the sense strand and the antisense strand are separate strands and are not covalently linked. In some embodiments, the sense strand and the antisense strand are covalently linked. In some embodiments, the sense strand and the antisense strand form a double-stranded 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).

[0066] In some embodiments, the sense strand includes a first region (R1) and a second region (R2), where R2 includes a first sub-region (S1), a loop (L) such as a tetraloop (Tetral L) or a triloop (Trilo L), and a second sub-region (S2), L is located between S1 and S2, and S1 and S2 form a second double-strand (D2). D2 may have various lengths. In some embodiments, D2 is about 1 to about 6 bp in length. In other embodiments, D2 is 2 to 6, 3 to 6, 4 to 6, 5 to 6, 1 to 5, 2 to 5, 3 to 5, or 4 to 5 bp in length. In other embodiments, D2 is 1, 2, 3, 4, 5, or 6 bP in length. In some embodiments, D2 is 6 bp in length.

[0067] In some embodiments, the sense strand R1 and the antisense strand form a first double-strand (D1). In some embodiments, D1 is at least 15 nucleotides in length (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21). In some embodiments, D1 is about 12 to about 30 nucleotides in length (e.g., 12 to 30, 12 to 27, 15 to 22, 18 to 22, 18 to 25, 18 to 27, 18 to 30, or 21 to 30 nucleotides). In some embodiments, D1 is at least 12 nucleotides in length (e.g., at least 12, at least 15, at least 20, at least 25, or at least 30 nucleotides). In 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 that constitutes the sense strand and the antisense strand does not cover the entire length of the sense strand and / or the antisense strand. In some embodiments, D1 that constitutes the sense strand and the antisense strand covers the entire length of either or both of the sense strand and the antisense strand. In some embodiments, D1 that constitutes the sense strand and the antisense strand covers the entire length of both the sense strand and the antisense strand.

[0068] In some embodiments, the sense strand is 36 nucleotides in length and is numbered 1 to 36 from 5' to 3' in position. In some embodiments, the antisense strand is 22 nucleotides in length and is numbered 1 to 22 from 5' to 3' in position. In some embodiments, the position numbers described herein follow this numbering format.

[0069] In some embodiments, the oligonucleotide comprises a sense strand having any one of the sequences of SEQ ID NOs: 1 to 384, and an antisense strand comprising any one of the complementary sequences of SEQ ID NOs: 385 to 768. In some embodiments, the oligonucleotide comprises a sense strand having the sequence of SEQ ID NOs: 1683 to 2066, and an antisense strand comprising any one of the complementary sequences of SEQ ID NOs: 2067 to 2450.

[0070] In some embodiments, the oligonucleotide comprises a sense strand having any one of the sequences of SEQ ID NOs: 1537 to 1571, and an antisense strand comprising any one of the complementary sequences of SEQ ID NOs: 1572 to 1606. In some embodiments, the oligonucleotide comprises a sense strand having any one of the sequences of SEQ ID NOs: 1537 to 1571 and 1681, and an antisense strand comprising any one of the complementary sequences of SEQ ID NOs: 1572 to 1606.

[0071] In some embodiments, the oligonucleotide is: a) SEQ ID NOs: 1537 and 1572 respectively; b) SEQ ID NOs: 1538 and 1573 respectively; c) SEQ ID NOs: 1539 and 1574 respectively; d) SEQ ID NOs: 1540 and 1575 respectively; e) SEQ ID NOs: 1541 and 1576 respectively; f) SEQ ID NOs: 1542 and 1577 respectively; g) SEQ ID NOs: 1543 and 1578 respectively; h) SEQ ID NOs: 1544 and 1579 respectively; i) SEQ ID NOs: 1545 and 1580 respectively; j) SEQ ID NOs: 1546 and 1581 respectively; k) SEQ ID NOs: 1547 and 1582 respectively; l) SEQ ID NOs: 1548 and 1583 respectively; m) SEQ ID NOs: 1549 and 1584 respectively; n) SEQ ID NOs: 1550 and 1585 respectively; o) SEQ ID NOs: 1551 and 1586 respectively; p) SEQ ID NOs: 1552 and 1587, respectively; q) SEQ ID NOs: 1553 and 1588, respectively; r) SEQ ID NOs: 1554 and 1589, respectively; s) SEQ ID NOs: 1555 and 1590, respectively; t) SEQ ID NOs: 1556 and 1591, respectively; u) SEQ ID NOs: 1557 and 1592, respectively; v) SEQ ID NOs: 1558 and 1593, respectively; w) SEQ ID NOs: 1559 and 1594, respectively; x) SEQ ID NOs: 1560 and 1595, respectively; y) SEQ ID NOs: 1561 and 1596, respectively; z) SEQ ID NOs: 1562 and 1597, respectively; aa) SEQ ID NOs: 1563 and 1598, respectively; bb) SEQ ID NOs: 1564 and 1599, respectively; cc) SEQ ID NOs: 1565 and 1600, respectively; dd) SEQ ID NOs: 1566 and 1601, respectively; ee) SEQ ID NOs: 1567 and 1602, respectively; ff) SEQ ID NOs: 1568 and 1603, respectively; gg) SEQ ID NOs: 1569 and 1604, respectively; hh) SEQ ID NOs: 1570 and 1605, respectively; ii) SEQ ID NOs: 1571 and 1606, respectively; and jj) comprising a sense strand and an antisense strand comprising nucleotide sequences selected from SEQ ID NOs: 1681 and 1586, respectively.

[0072] In some embodiments, the oligonucleotide is: a) SEQ ID NOs: 1540 and 1575, respectively; b) SEQ ID NOs: 1544 and 1579, respectively; c) SEQ ID NOs: 1546 and 1581, respectively; d) SEQ ID NOs: 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) SEQ ID NOs: 1565 and 1600, respectively; k) SEQ ID NOs: 1566 and 1601, respectively; l) SEQ ID NOs: 1570 and 1605, respectively; and m) comprising a sense strand and an antisense strand comprising nucleotide sequences selected from SEQ ID NOs: 1681 and 1586, respectively.

[0073] In some embodiments, the oligonucleotide is: a) SEQ ID NOs: 1553 and 1588, respectively; b) SEQ ID NOs: 1560 and 1595, respectively; c) SEQ ID NOs: 1564 and 1599, respectively; d) SEQ ID NOs: 1551 and 1586, respectively; e) SEQ ID NOs: 1570 and 1605, respectively; and f) comprising a sense strand and an antisense strand comprising nucleotide sequences selected from SEQ ID NOs: 1681 and 1586, respectively.

[0074] 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.

[0075] In some embodiments, it should be understood that the sequences presented in the Sequence Listing may be referred to when describing the structure of oligonucleotides (e.g., RNAi oligonucleotides) or other nucleic acids. In such embodiments, the actual oligonucleotides or other nucleic acids retain complementary properties that are essentially the same as or similar to the specified sequence, while having one or more alternative nucleotides (e.g., the RNA counterpart of a DNA nucleotide or the DNA counterpart of an RNA nucleotide) and / or one or more modified nucleotides and / or one or more modified internucleotide linkages and / or one or more other modifications compared to the specified sequence.

[0076] In some embodiments, the RNAi oligonucleotides herein comprise a 25-nucleotide sense strand and a 27-nucleotide antisense strand that, when acted upon by the Dicer enzyme, yield an antisense strand that is incorporated into the mature RNA-induced silencing complex (RISC). In some embodiments, the 25-nucleotide sense strand comprises a sequence selected from SEQ ID NOs: 1-384. In some aspects, the 27-nucleotide antisense strand comprises a sequence selected from SEQ ID NOs: 385-768. In some embodiments, the sense strand is longer than 27 nucleotides (e.g., 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides). In some embodiments, the sense strand is longer than 25 nucleotides (e.g., 26, 27, 28, 29, or 30 nucleotides). In some embodiments, the sense strand comprises a nucleotide sequence selected from SEQ ID NOs: 1683-2066, and 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, and the nucleotide sequence is longer than 25 nucleotides (e.g., 26, 27, 28, 29, or 30 nucleotides).

[0077] In some embodiments, the oligonucleotide has one 5' end that is thermodynamically unstable compared to the other 5' end. In some embodiments, an asymmetric RNAi oligonucleotide is provided that includes a blunt 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 3' overhang on the antisense strand is from about 1 to about 8 nucleotides in length (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides in length). Typically, the oligonucleotide has a 2-nucleotide overhang at the 3' end of the antisense strand. However, other overhangs are possible. In some embodiments, the overhang is a 3' overhang that includes nucleotides between 1 and about, optionally between 1 and 5, 1 and 4, 1 and 3, 1 and 2, 2 and 6, 2 and 5, 2 and 4, 2 and 3, 3 and 6, 3 and 5, 3 and 4, 4 and 6, 4 and 5, 5 and 6 nucleotides, or 1, 2, 3, 4, 5, or 6 nucleotides. In other embodiments, the overhang is a 5' overhang that includes nucleotides between 1 and 6, optionally between 1 and 5, 1 and 4, 1 and 3, 1 and 2, 2 and 6, 2 and 5, 2 and 4, 2 and 3, 3 and 6, 3 and 5, 3 and 4, 4 and 6, 4 and 5, 5 and 6 nucleotides, or 1, 2, 3, 4, 5, or 6 nucleotides in length.

[0078] In some embodiments, two terminal nucleotides are modified at the 3'-end of the antisense strand. In some embodiments, the two terminal nucleotides at the 3'-end of the antisense strand are complementary to the target mRNA (e.g., the mRNA of SNCA). In some embodiments, the two terminal nucleotides at the 3'-end of the antisense strand are not complementary to the target mRNA. In some embodiments, the two terminal nucleotides at the 3'-end of the antisense strand of the oligonucleotide do not pair. In some embodiments, the two terminal nucleotides at the 3'-end of the antisense strand of the oligonucleotide contain unpaired GG. In some embodiments, the two terminal nucleotides at the 3'-end of the antisense strand of the oligonucleotide are not complementary to the target mRNA. In some embodiments, the two terminal nucleotides at the 3'-end of each of the oligonucleotides are GG. Usually, one or both of the two terminal GG nucleotides at the 3'-end of each of the ds oligonucleotides are not complementary to the target mRNA. In some embodiments, the two terminal nucleotides at the 3'-end of each of the oligonucleotides are GG. In some embodiments, one or both of the two terminal GG nucleotides at the 3'-end of each of the oligonucleotides are not complementary to the target mRNA. In some embodiments, the oligonucleotide comprises a complementary targeting sequence or targeting region that is complementary to a contiguous sequence of any one of nucleotides 2067 to 2045, wherein the two terminal nucleotides on the 3'-end of the antisense strand of the oligonucleotide contain unpaired GG. In some embodiments, the oligonucleotide comprises an antisense strand comprising a nucleotide sequence selected from SEQ ID NOs: 2067 to 2450, and the two terminal nucleotides on the 3'-end of the antisense strand of the oligonucleotide contain unpaired GG. In some embodiments, the oligonucleotide comprises a sense strand comprising a nucleotide sequence selected from SEQ ID NOs: 1683 to 2066 and an antisense strand comprising a nucleotide sequence selected from SEQ ID NOs: 2067 to 2450, and the two terminal nucleotides on the 3'-end of the antisense strand of the oligonucleotide contain unpaired GG.

[0079] In some embodiments, there are one or more (e.g., 1, 2, 3, 4, or 5) mismatches (plural possible) between the sense strand and the antisense strand that make up the oligonucleotides herein. If there are more than one mismatch between the sense strand and the antisense strand, they may be arranged continuously (e.g., 2, 3, or more in a row) or may be scattered 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 destabilizations of segments at the 3' end of the sense strand of the oligonucleotide improve or increase the potency of the oligonucleotide.

[0080] In some embodiments, the oligonucleotide is: a) SEQ ID NOs: 1537 and 1572, respectively; b) SEQ ID NOs: 1538 and 1573, respectively; c) SEQ ID NOs: 1539 and 1574, respectively; d) SEQ ID NOs: 1540 and 1575, respectively; e) SEQ ID NOs: 1541 and 1576, respectively; f) SEQ ID NOs: 1542 and 1577, respectively; g) SEQ ID NOs: 1543 and 1578, respectively; h) SEQ ID NOs: 1544 and 1579, respectively; i) SEQ ID NOs: 1545 and 1580, respectively; j) SEQ ID NOs: 1546 and 1581, respectively; k) SEQ ID NOs: 1547 and 1582, respectively; l) SEQ ID NOs: 1548 and 1583, respectively; m) SEQ ID NOs: 1549 and 1584, respectively; n) SEQ ID NOs: 1550 and 1585, respectively; o) SEQ ID NOs: 1551 and 1586, respectively; p) SEQ ID NOs: 1552 and 1587, respectively; q) SEQ ID NOs: 1553 and 1588, respectively; r) SEQ ID NOs: 1554 and 1589, respectively; s) SEQ ID NOs: 1555 and 1590, respectively; t) SEQ ID NOs: 1556 and 1591, respectively; u) SEQ ID NOs: 1557 and 1592, respectively; v) SEQ ID NOs: 1558 and 1593, respectively; w) SEQ ID NOs: 1559 and 1594, respectively; x) SEQ ID NOs: 1560 and 1595, respectively; y) SEQ ID NOs: 1561 and 1596, respectively; z) SEQ ID NOs: 1562 and 1597, respectively; aa) SEQ ID NOs: 1563 and 1598, respectively; bb) SEQ ID NOs: 1564 and 1599, respectively; cc) SEQ ID NOs: 1565 and 1600, respectively; dd) SEQ ID NOs: 1566 and 1601, respectively; ee) SEQ ID NOs: 1567 and 1602, respectively; ff) SEQ ID NOs: 1568 and 1603, respectively; gg) SEQ ID NOs: 1569 and 1604, respectively; hh) SEQ ID NOs: 1570 and 1605, respectively; ii) SEQ ID NOs: 1571 and 1606, respectively; and jj) a sense strand and an antisense strand comprising nucleotide sequences selected from SEQ ID NOs: 1681 and 1586, respectively, wherein there is one or more (e.g., 1, 2, 3, 4, 5) mismatches between the sense strand and the antisense strand.

[0081] In some embodiments, the oligonucleotide is: a) SEQ ID NOs: 1540 and 1575, respectively; b) SEQ ID NOs: 1544 and 1579, respectively; c) SEQ ID NOs: 1546 and 1581, respectively; d) SEQ ID NOs: 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) SEQ ID NOs: 1565 and 1600, respectively; k) SEQ ID NOs: 1566 and 1601, respectively; l) SEQ ID NOs: 1570 and 1605, respectively; and m) comprising a sense strand and an antisense strand comprising nucleotide sequences selected from SEQ ID NOs: 1681 and 1586, respectively, wherein there is one or more (e.g., 1, 2, 3, 4, 5) mismatches (plural possible) between the sense strand and the antisense strand.

[0082] In some embodiments, the oligonucleotide is: a) SEQ ID NOs: 1553 and 1588, respectively; b) SEQ ID NOs: 1560 and 1595, respectively; c) SEQ ID NOs: 1564 and 1599, respectively; d) SEQ ID NOs: 1551 and 1586, respectively; e) SEQ ID NOs: 1570 and 1605, respectively; f) comprising a sense strand and an antisense strand comprising nucleotide sequences selected from SEQ ID NOs: 1681 and 1586, respectively, wherein there is one or more (e.g., 1, 2, 3, 4, 5) mismatches (plural possible) between the sense strand and the antisense strand.

[0083] Antisense strand In some embodiments, the antisense strand of the oligonucleotide is referred to as the "guide strand," interacts with RISC, binds to an Argonaute protein such as Ago2, or interacts with 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."

[0084] In some embodiments, the oligonucleotide comprises an antisense strand of up to about 50 nucleotides in length (e.g., nucleotides of up to 50, up to 40, up to 35, up to 30, up to 27, up to 25, up to 21, up to 19, up to 17, up to 15, or up to 12 in length). In some embodiments, the oligonucleotide comprises an antisense strand of at least 12 nucleotides in length (e.g., nucleotides of 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 in length). In some embodiments, the antisense strand ranges from about 12 to about 40 nucleotides in length (e.g., 12 - 40, 12 - 36, 12 - 32, 12 - 28, 15 - 40, 15 - 36, 15 - 32, 15 - 30, 15 - 28, 17 - 22, 17 - 25, 19 - 27, 19 - 30, 20 - 40, 22 - 40, 25 - 40, or 32 - 40). In some embodiments, the oligonucleotide comprises an antisense of 15 - 30 nucleotides in length. In some embodiments, the antisense strand of any one 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.

[0085] In some embodiments, the antisense strand comprises, or consists of, a sequence shown in any one of SEQ ID NOs: 1683 to 2066. In some embodiments, the antisense strand comprises at least 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 shown in any one of SEQ ID NOs: 2067 to 2450. In some embodiments, the antisense strand comprises, or consists of, a sequence shown in any one of SEQ ID NOs: 385 to 768. In some embodiments, the antisense strand comprises at least 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 shown in any one of SEQ ID NOs: 385 to 768. In some embodiments, the antisense strand comprises, or consists of, a sequence shown in any one of SEQ ID NOs: 1572 to 1606. In some embodiments, the antisense strand comprises at least 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 shown in any one of SEQ ID NOs: 1572 to 1606. In some embodiments, the antisense strand comprises, or consists of, a sequence shown in any one of SEQ ID NOs: 2067 to 2450. In some embodiments, the antisense strand comprises at least 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 shown in any one of SEQ ID NOs: 2067 to 2450.In some embodiments, the antisense strand comprises, or consists of, the sequence shown 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 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 the sequence shown 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, the sequence shown in any one of SEQ ID NOs: 1588, 1595, 1599, 1586, and 1605. In some embodiments, the antisense strand comprises at least 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 the sequence shown in any one of SEQ ID NOs: 1588, 1595, 1599, 1586, and 1605.

[0086] Sense strand In some embodiments, the oligonucleotide comprises a sense strand sequence shown in any one of SEQ ID NOs: 1683 to 2066. In some embodiments, the sense strand comprises at least 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 the sequence shown in any one of SEQ ID NOs: 1683 to 2066. In some embodiments, the sense strand comprises the sequence shown in any one of SEQ ID NOs: 1 to 384. In some embodiments, the sense strand comprises at least 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 the sequence shown in any one of SEQ ID NOs: 1 to 384. In some embodiments, the sense strand comprises the sequence shown in any one of SEQ ID NOs: 1537 to 1571. In some embodiments, the sense strand comprises the sequence shown in any one of SEQ ID NOs: 1537 to 1571 and 1681. In some embodiments, the sense strand is SEQ ID NO: 1681. In some embodiments, the sense strand comprises at least 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 the sequence shown in any one of SEQ ID NOs: 1537 to 1571. In some embodiments, the sense strand comprises the sequence shown in any one of SEQ ID NOs: 1781, 1782, 1796, 1798, 1802, 1808, 1814, 1817, 1713, 1718, 1726, 1830, 1839, 1742, 1846, 1852, 1865, 1784, 1804, 1721, 1822, 1840, 1735, 1847, 1855, 1864, 1901, 1902, 1938, 1947, 1955, 1964, 1973, and 1978.In some embodiments, the sense strand comprises at least 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 the sequence shown 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 the sequence shown 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 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 the sequence shown 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 the sequence shown in any one of SEQ ID NOs: 1553, 1560, 1564, 1551, and 1570. In some embodiments, the oligonucleotide comprises at least 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 the sequence shown in any one of SEQ ID NOs: 1553, 1560, 1564, 1551, and 1570.

[0087] In some embodiments, the oligonucleotide comprises a sense strand sequence shown 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 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 the sequence shown 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 the sequence shown in any one of SEQ ID NOs: 1553, 1560, 1564, 1551, 1570, and 1681. In some embodiments, the sense strand comprises at least 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 the sequence shown in any one of SEQ ID NOs: 1553, 1560, 1564, 1551, 1570, and 1681.

[0088] In some embodiments, the sense strand comprises nucleotides up to about 50 in length (e.g., up to 50, up to 40, up to 36, up to 30, up to 27, up to 25, up to 21, up to 19, up to 17, or up to 12 nucleotides in length). In some embodiments, the sense strand comprises at least about 12 nucleotides in length (e.g., at least 12, at least 15, at least 19, at least 21, at least 25, at least 27, at least 30, at least 36, or at least 38 nucleotides in length). In some embodiments, the sense strand is in the range of about 12 to about 50 nucleotides in length (e.g., 12 - 50, 12 - 40, 12 - 36, 12 - 32, 12 - 28, 15 - 40, 15 - 36, 15 - 32, 15 - 28, 17 - 21, 17 - 25, 19 - 27, 19 - 30, 20 - 40, 22 - 40, 25 - 40, or 32 - 40). In some embodiments, the sense strand comprises nucleotides 15 - 50 in length. In some embodiments, the sense strand comprises nucleotides 18 - 36 in length. In some embodiments, the sense strand comprises 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In some embodiments, the sense strand is 36 nucleotides in length.

[0089] In some embodiments, the sense strand includes a stem-loop structure at its 3' end. In some embodiments, the stem-loop is formed by intramolecular base pairing. In some embodiments, the sense strand includes a stem-loop structure at its 5' end. In some embodiments, the stem of the stem-loop includes a double-stranded region 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 includes a double-stranded region of 2 nucleotides in length. In some embodiments, the stem of the stem-loop includes a double-stranded region of 3 nucleotides in length. In some embodiments, the stem of the stem-loop includes a double-stranded region of 4 nucleotides in length. In some embodiments, the stem of the stem-loop includes a double-stranded region of 5 nucleotides in length. In some embodiments, the stem of the stem-loop includes a double-stranded region of 6 nucleotides in length. In some embodiments, the stem of the stem-loop includes a double-stranded region of 7 nucleotides in length. In some embodiments, the stem of the stem-loop includes a double-stranded region of 8 nucleotides in length. In some embodiments, the stem of the stem-loop includes a double-stranded region of 9 nucleotides in length. In some embodiments, the stem of the stem-loop includes a double-stranded region of 10 nucleotides in length. In some embodiments, the stem of the stem-loop includes a double-stranded region of 11 nucleotides in length. In some embodiments, the stem of the stem-loop includes a double-stranded region of 12 nucleotides in length. In some embodiments, the stem of the stem-loop includes a double-stranded region of 13 nucleotides in length. In some embodiments, the stem of the stem-loop includes a double-stranded region of 14 nucleotides in length.

[0090] In some embodiments, the stem-loop provides protection of the oligonucleotide against degradation (e.g., enzymatic degradation), and facilitates and / or improves targeting and / or delivery to a target cell, tissue, or organ (e.g., the liver or the brain), or both. For example, in some embodiments, the loop of the stem-loop facilitates, improves, or increases targeting of 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., the CNS), or combinations thereof, by providing nucleotides comprising one or more modifications. In some embodiments, the stem-loop itself or modifications (s) to the stem-loop do not substantially affect the oligonucleotide's inherent gene expression inhibitory activity, but do facilitate, 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., the CNS). In certain embodiments, the oligonucleotide comprises a stem-loop shown as S1-L-S2 (e.g., at its 3' end), where S1 is complementary to S2 and L forms a single-stranded 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, and comprises a sense strand. In some embodiments, the loop (L) is 3 nucleotides in length (e.g., a triloop or triL). In some embodiments, the loop (L) is 4 nucleotides in length (e.g., a tetraloop or tetraL). In some embodiments, the loop (L) is 5 nucleotides in length (e.g., a pentaloop or pentaL). In some embodiments, the loop (L) is 6 nucleotides in length (e.g., a hexaloop or hexaL). In some embodiments, the loop (L) is 7 nucleotides in length (e.g., a heptaloop or heptaL). In some embodiments, the loop (L) is 8 nucleotides in length (e.g., an octaloop or octaL). In some embodiments, the loop (L) is 9 nucleotides in length (e.g., a nonaloop or nonaL).In some embodiments, the loop (L) is a nucleotide of length 10 (e.g., a decaloop or decaL).

[0091] In some embodiments, the oligonucleotide comprises a complementary targeting sequence or targeting region that is complementary to a contiguous sequence of any one of nucleotides of SEQ ID NOs: 1683 to 2066, and the oligonucleotide comprises a sense strand that comprises a stem-loop shown as S1-L-S2 (e.g., at its 3' end), wherein S1 is complementary to S2, and L forms a ss loop of nucleotides up to about 10 in length (e.g., nucleotides of length 3, 4, 5, 6, 7, 8, 9, or 10) between S1 and S2. In some embodiments, the oligonucleotide comprises a complementary targeting sequence or targeting region that is complementary to a contiguous sequence of any one of nucleotides of SEQ ID NOs: 1683 to 2066, and the oligonucleotide comprises a sense strand that comprises a stem-loop shown as S1-L-S2 (e.g., at its 3' end), wherein S1 is complementary to S2, and L forms a 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, which is incorporated herein by reference (e.g., within a tetraL structure having a nick). In some embodiments, the oligonucleotide comprises a complementary targeting sequence or targeting region that is complementary to a contiguous sequence of any one of nucleotides of SEQ ID NOs: 1683 to 2066 and a tetraL. In some embodiments, the tetraloop comprises the sequence 5'-GAAA-3'. In some embodiments, the stem-loop comprises the sequence 5'-GCAGCCGAAAGGCUGC-3' (SEQ ID NO: 1680).

[0092] In other embodiments, the loop (L) is a hairpin L. In some embodiments, the oligonucleotide comprises a complementary targeting sequence or targeting region that is complementary to a contiguous sequence of any one of nucleotides of SEQ ID NOs: 1683 to 2066, and a hairpin L. In some embodiments, the hairpin L comprises ribonucleotides, deoxyribonucleotides, modified nucleotides, delivery ligands, and combinations thereof.

[0093] Double-stranded length In some embodiments, the double-strand formed between the sense strand and the antisense strand is at least 12 (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21) nucleotides in length. In some embodiments, the double-strand is in the range of about 12 to about 30 nucleotides in length (e.g., 12 to 30, 12 to 27, 12 to 22, 15 to 25, 18 to 30, 18 to 22, 18 to 25, 18 to 27, 18 to 30, 19 to 30, or 21 to 30 nucleotides). In some embodiments, the double-strand 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, the double-strand is 12 nucleotides in length. In some embodiments, the double-strand is 13 nucleotides in length. In some embodiments, the double-strand is 14 nucleotides in length. In some embodiments, the double-strand is 15 nucleotides in length. In some embodiments, the double-strand is 16 nucleotides in length. In some embodiments, the double-strand is 17 nucleotides in length. In some embodiments, the double-strand is 18 nucleotides in length. In some embodiments, the double-strand is 19 nucleotides in length. In some embodiments, the double-strand is 20 nucleotides in length. In some embodiments, the double-strand is 21 nucleotides in length. In some embodiments, the double-strand is 22 nucleotides in length. In some embodiments, the double-strand is 23 nucleotides in length. In some embodiments, the double-strand is 24 nucleotides in length. In some embodiments, the double-strand is 25 nucleotides in length. In some embodiments, the double-strand is 26 nucleotides in length. In some embodiments, the double-strand is 27 nucleotides in length. In some embodiments, the double-strand is 28 nucleotides in length. In some embodiments, the double-strand is 29 nucleotides in length. In some embodiments, the double-strand is 30 nucleotides in length. In some embodiments, the double-strand does not extend over the entire length of the sense strand and / or the antisense strand.In some embodiments, the double strand extends over the entire length of either the sense strand or the antisense strand. In some embodiments, the double strand extends over the entire length of both the sense strand and the antisense strand.

[0094] In some embodiments, the sense strand and the antisense strand are: a) SEQ ID NO: 1537 and 1572, respectively; b) SEQ ID NO: 1538 and 1573, respectively; c) SEQ ID NO: 1539 and 1574, respectively; d) SEQ ID NO: 1540 and 1575, respectively; e) SEQ ID NO: 1541 and 1576, respectively; f) SEQ ID NO: 1542 and 1577, respectively; g) SEQ ID NO: 1543 and 1578, respectively; h) SEQ ID NO: 1544 and 1579, respectively; i) SEQ ID NO: 1545 and 1580, respectively; j) SEQ ID NO: 1546 and 1581, respectively; k) SEQ ID NO: 1547 and 1582, respectively; l) SEQ ID NO: 1548 and 1583, respectively; m) SEQ ID NO: 1549 and 1584, respectively; n) SEQ ID NO: 1550 and 1585, respectively; o) SEQ ID NO: 1551 and 1586, respectively; p) SEQ ID NO: 1552 and 1587, respectively; q) SEQ ID NO: 1553 and 1588, respectively; r) SEQ ID NO: 1554 and 1589, respectively; s) SEQ ID NO: 1555 and 1590, respectively; t) SEQ ID NO: 1556 and 1591, respectively; u) SEQ ID NO: 1557 and 1592, respectively; v) SEQ ID NO: 1558 and 1593, respectively; w) SEQ ID NO: 1559 and 1594, respectively; x) SEQ ID NO: 1560 and 1595, respectively; y) SEQ ID NO: 1561 and 1596, respectively; z) SEQ ID NOs: 1562 and 1597, respectively; aa) SEQ ID NOs: 1563 and 1598, respectively; bb) SEQ ID NOs: 1564 and 1599, respectively; cc) SEQ ID NOs: 1565 and 1600, respectively; dd) SEQ ID NOs: 1566 and 1601, respectively; ee) SEQ ID NOs: 1567 and 1602, respectively; ff) SEQ ID NOs: 1568 and 1603, respectively; gg) SEQ ID NOs: 1569 and 1604, respectively; hh) SEQ ID NOs: 1570 and 1605, respectively; ii) SEQ ID NOs: 1571 and 1606, respectively; and jj) a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1681 and 1586, wherein the double strand formed between the sense strand and the antisense strand is in the range of about 12 to about 30 nucleotides (e.g., 12 - 30, 12 - 27, 12 - 22, 15 - 25, 18 - 30, 18 - 22, 18 - 25, 18 - 27, 18 - 30, 19 - 30 or 21 - 30 nucleotides).

[0095] In some embodiments, the sense strand and the antisense strand are: a) SEQ ID NOs: 1540 and 1575, respectively; b) SEQ ID NOs: 1544 and 1579, respectively; c) SEQ ID NOs: 1546 and 1581, respectively; d) SEQ ID NOs: 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) SEQ ID NOs: 1565 and 1600, respectively; k) SEQ ID NOs: 1566 and 1601, respectively; l) SEQ ID NOs: 1570 and 1605, respectively; and m) a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1681 and 1586, respectively, wherein the double strand formed between the sense strand and the antisense strand is in the range of about 12 to about 30 nucleotides in length (e.g., 12 to 30, 12 to 27, 12 to 22, 15 to 25, 18 to 30, 18 to 22, 18 to 25, 18 to 27, 18 to 30, 19 to 30, or 21 to 30 nucleotides in length).

[0096] In some embodiments, the sense and antisense strands are: a) SEQ ID NOs: 1553 and 1588, respectively; b) SEQ ID NOs: 1560 and 1595, respectively; c) SEQ ID NOs: 1564 and 1599, respectively; d) SEQ ID NOs: 1551 and 1586, respectively; e) SEQ ID NOs: 1570 and 1605, respectively; and f) a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1681 and 1586, respectively, wherein the double strand formed between the sense strand and the antisense strand is in the range of about 12 to about 30 nucleotides in length (e.g., 12 to 30, 12 to 27, 12 to 22, 15 to 25, 18 to 30, 18 to 22, 18 to 25, 18 to 27, 18 to 30, 19 to 30, or 21 to 30 nucleotides in length).

[0097] The ends of the oligonucleotide In some embodiments, the oligonucleotide (e.g., an RNAi oligonucleotide) comprises a sense strand and an antisense strand, and the ends of either or both strands comprise blunt ends. In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand that are separate strands forming an asymmetric double-stranded region having an overhang at the 3’ end of the antisense strand. In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand, wherein the ends of either or both strands comprise an overhang comprising one or more nucleotides. In some embodiments, the one or more nucleotides comprising the overhang are unpaired nucleotides. In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, wherein the 3’ end of the sense strand and the 5’ end of the antisense strand comprise blunt ends. In some embodiments, the 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 blunt ends.

[0098] 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.

[0099] In some embodiments, the 3' overhang is from about 1 to about 20 nucleotides in length (e.g., from 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 from 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.

[0100] In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand are: a) SEQ ID NO: 1537 and 1572, respectively; b) SEQ ID NO: 1538 and 1573, respectively; c) SEQ ID NO: 1539 and 1574, respectively; d) SEQ ID NO: 1540 and 1575, respectively; e) SEQ ID NO: 1541 and 1576, respectively; f) SEQ ID NO: 1542 and 1577, respectively; g) SEQ ID NO: 1543 and 1578, respectively; h) SEQ ID NO: 1544 and 1579, respectively; i) SEQ ID NO: 1545 and 1580, respectively; j) SEQ ID NO: 1546 and 1581, respectively; k) SEQ ID NO: 1547 and 1582, respectively; l) SEQ ID NO: 1548 and 1583, respectively; m) SEQ ID NO: 1549 and 1584, respectively; n) SEQ ID NO: 1550 and 1585, respectively; o) SEQ ID NO: 1551 and 1586, respectively; p) SEQ ID NO: 1552 and 1587, respectively; q) SEQ ID NO: 1553 and 1588, respectively; r) SEQ ID NO: 1554 and 1589, respectively; s) SEQ ID NO: 1555 and 1590, respectively; t) SEQ ID NO: 1556 and 1591, respectively; u) SEQ ID NO: 1557 and 1592, respectively; v) SEQ ID NO: 1558 and 1593, respectively; w) SEQ ID NO: 1559 and 1594, respectively; x) SEQ ID NO: 1560 and 1595, respectively; y) SEQ ID NO: 1561 and 1596, respectively; z) SEQ ID NO: 1562 and 1597, respectively; aa) SEQ ID NO: 1563 and 1598, respectively; bb) SEQ ID NOs: 1564 and 1599, respectively; cc) SEQ ID NOs: 1565 and 1600, respectively; dd) SEQ ID NOs: 1566 and 1601, respectively; ee) SEQ ID NOs: 1567 and 1602, respectively; ff) SEQ ID NOs: 1568 and 1603, respectively; gg) SEQ ID NOs: 1569 and 1604, respectively; hh) SEQ ID NOs: 1570 and 1605, respectively; ii) SEQ ID NOs: 1571 and 1606, respectively; and jj) a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1681 and 1586, wherein the antisense strand comprises a 3' overhang of about 1 to about 20 nucleotides (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), and optionally, the 3' overhang is 2 nucleotides in length.

[0101] In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand are: a) SEQ ID NOs: 1540 and 1575, respectively; b) SEQ ID NOs: 1544 and 1579, respectively; c) SEQ ID NOs: 1546 and 1581, respectively; d) SEQ ID NOs: 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) SEQ ID NOs: 1565 and 1600, respectively; k) SEQ ID NOs: 1566 and 1601, respectively; l) SEQ ID NOs: 1570 and 1605, respectively; m) comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1681 and SEQ ID NO: 1586, wherein the antisense strand comprises a 3' overhang of about 1 to about 20 nucleotides (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), and optionally, the 3' overhang is 2 nucleotides in length.

[0102] In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand are: a) SEQ ID NO: 1553 and SEQ ID NO: 1588, respectively; b) SEQ ID NO: 1560 and SEQ ID NO: 1595, respectively; c) SEQ ID NO: 1564 and SEQ ID NO: 1599, respectively; d) SEQ ID NO: 1551 and SEQ ID NO: 1586, respectively; e) SEQ ID NO: 1570 and SEQ ID NO: 1605, respectively; and f) comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1681 and SEQ ID NO: 1586, wherein the antisense strand comprises a 3' overhang of about 1 to about 20 nucleotides (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), and optionally, the 3' overhang is 2 nucleotides in length.

[0103] In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand, wherein the sense strand comprises a 5' overhang comprising one or more nucleotides.

[0104] In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand are: a) SEQ ID NO: 1540 and SEQ ID NO: 1575, respectively; b) SEQ ID NO: 1544 and SEQ ID NO: 1579, respectively; c) SEQ ID NO: 1546 and SEQ ID NO: 1581, respectively; d) SEQ ID NO: 1551 and SEQ ID NO: 1586, respectively; e) SEQ ID NOs: 1552 and 1587, respectively; f) SEQ ID NOs: 1553 and 1588, respectively; g) SEQ ID NOs: 1558 and 1594, respectively; h) SEQ ID NOs: 1560 and 1595, respectively; i) SEQ ID NOs: 1564 and 1599, respectively; j) SEQ ID NOs: 1565 and 1600, respectively; k) SEQ ID NOs: 1566 and 1601, respectively; l) SEQ ID NOs: 1570 and 1605, respectively; m) comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1681 and 1586, wherein the antisense strand comprises a 5' overhang of about 1 to about 20 nucleotides (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), and optionally, the 5' overhang is 2 nucleotides in length.

[0105] In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand are: a) SEQ ID NOs: 1553 and 1588, respectively; b) SEQ ID NOs: 1560 and 1595, respectively; c) SEQ ID NOs: 1564 and 1599, respectively; d) SEQ ID NOs: 1551 and 1586, respectively; e) SEQ ID NOs: 1570 and 1605, respectively; and m) comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1681 and 1586, wherein the antisense strand comprises a 5' overhang of about 1 to about 20 nucleotides (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), and optionally, the 5' overhang is 2 nucleotides in length.

[0106] In some embodiments, one or more (e.g., 2, 3, 4, 5 or more) nucleotides including the 3' end or 5' end of the sense strand and / or the 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., 2'-modification, including, for example, 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.

[0107] In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand, the 3' end of the sense strand comprises the 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 that form a nicked tetra-L structure, wherein the 3' end of the sense strand comprises a stem-loop, the loop (L) is the tetra-L described herein, and the 3' end of the antisense strand comprises the 3' overhang described herein. In some embodiments, the 3' overhang is two nucleotides in length. In some embodiments, both of the two nucleotides constituting the 3' overhang contain the guanine (G) nucleobase. Usually, one or both of the nucleotides constituting the 3' overhang of the antisense strand are not complementary to the target mRNA.

[0108] Modification of oligonucleotides 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 properties relevant to therapeutic research applications.

[0109] 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 internucleotide linkage. In some embodiments, the modification is a modified base. In some embodiments, the modification is a reversible modification. In some embodiments, the oligonucleotide can comprise any one or any combination of the modifications described herein. For example, in some embodiments, the oligonucleotide comprises at least one modified sugar, a 5'-terminal phosphate group, at least one modified internucleotide linkage, at least one modified base, and at least one reversible modification.

[0110] In some embodiments, the oligonucleotide comprises at least one modified sugar, a 5'-terminal phosphate group, at least one modified internucleotide linkage, and at least one modified base. In some embodiments, the sense strand and the antisense strand of the oligonucleotide are: a) SEQ ID NO: 1537 and 1572, respectively; b) SEQ ID NO: 1538 and 1573, respectively; c) SEQ ID NO: 1539 and 1574, respectively; d) SEQ ID NO: 1540 and 1575, respectively; e) SEQ ID NO: 1541 and 1576, respectively; f) SEQ ID NO: 1542 and 1577, respectively; g) SEQ ID NO: 1543 and 1578, respectively; h) SEQ ID NO: 1544 and 1579, respectively; i) SEQ ID NO: 1545 and 1580, respectively; j) SEQ ID NO: 1546 and 1581, respectively; k) SEQ ID NO: 1547 and 1582, respectively; l) SEQ ID NO: 1548 and 1583, respectively; m) SEQ ID NO: 1549 and 1584, respectively; n) SEQ ID NO: 1550 and 1585, respectively; o) SEQ ID NO: 1551 and 1586, respectively; p) SEQ ID NOs: 1552 and 1587, respectively; q) SEQ ID NOs: 1553 and 1588, respectively; r) SEQ ID NOs: 1554 and 1589, respectively; s) SEQ ID NOs: 1555 and 1590, respectively; t) SEQ ID NOs: 1556 and 1591, respectively; u) SEQ ID NOs: 1557 and 1592, respectively; v) SEQ ID NOs: 1558 and 1593, respectively; w) SEQ ID NOs: 1559 and 1594, respectively; x) SEQ ID NOs: 1560 and 1595, respectively; y) SEQ ID NOs: 1561 and 1596, respectively; z) SEQ ID NOs: 1562 and 1597, respectively; aa) SEQ ID NOs: 1563 and 1598, respectively; bb) SEQ ID NOs: 1564 and 1599, respectively; cc) SEQ ID NOs: 1565 and 1600, respectively; dd) SEQ ID NOs: 1566 and 1601, respectively; ee) SEQ ID NOs: 1567 and 1602, respectively; ff) SEQ ID NOs: 1568 and 1603, respectively; gg) SEQ ID NOs: 1569 and 1604, respectively; hh) SEQ ID NOs: 1570 and 1605, respectively; ii) SEQ ID NOs: 1571 and 1606, respectively; and jj) a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1681 and 1586, wherein the oligonucleotide comprises at least one modified sugar, a 5' terminal phosphate group, at least one modified internucleotide linkage, and at least one modified base.

[0111] In some embodiments, the sense and antisense strands are: a) SEQ ID NOs: 1540 and 1575, respectively; b) SEQ ID NOs: 1544 and 1579, respectively; c) SEQ ID NOs: 1546 and 1581, respectively; d) SEQ ID NOs: 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) SEQ ID NOs: 1565 and 1600, respectively; k) SEQ ID NOs: 1566 and 1601, respectively; l) SEQ ID NOs: 1570 and 1605, respectively; and m) a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1681 and 1586, respectively, wherein the oligonucleotide comprises at least one modified sugar, a 5' terminal phosphate group, at least one modified internucleotide linkage, and at least one modified base.

[0112] In some embodiments, the sense and antisense strands are: a) SEQ ID NOs: 1553 and 1588, respectively; b) SEQ ID NOs: 1560 and 1595, respectively; c) SEQ ID NOs: 1564 and 1599, respectively; d) SEQ ID NOs: 1551 and 1586, respectively; e) SEQ ID NOs: 1570 and 1605, respectively; and f) a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1681 and 1586, respectively, wherein the oligonucleotide comprises at least one modified sugar, a 5' terminal phosphate group, at least one modified internucleotide linkage, and at least one modified base.

[0113] The number of modifications on an oligonucleotide and the positions of those nucleotide modifications may affect the properties of the oligonucleotide. For example, the oligonucleotide may be delivered in vivo by inclusion of the oligonucleotide in a lipid nanoparticle (LNP) or similar carrier. However, if the oligonucleotide is not protected by an LNP or similar carrier, it may be advantageous for at least some of the nucleotides to be modified. Thus, in some embodiments, all or substantially all of the nucleotides of the oligonucleotide are modified. In some embodiments, more than half of the nucleotides are modified. In some embodiments, less than half of the nucleotides are modified. In some embodiments, the sugar moiety of all of the nucleotides constituting the oligonucleotide is modified at the 2'-position. The modification may be reversible or irreversible. In some embodiments, the oligonucleotide has a sufficient number and type of modified nucleotides to produce the desired properties (e.g., protection from enzymatic degradation, the ability to target desired cells after in vivo administration, and / or thermodynamic stability).

[0114] Sugar modification In some embodiments, the oligonucleotide comprises a modified sugar. In some embodiments, the modified sugar (also referred to herein as a sugar analog) comprises a modified deoxyribose or ribose moiety, for example, such that one or more modifications occur at the 2'-, 3'-, 4'-, and / or 5'-position carbons of the sugar. In some embodiments, the modified sugar may also include non-natural alternative carbon structures such as those present in, for example, locked nucleic acids (“LNAs”; see, e.g., Koshkin et al. (1998), Tetrahedon, 54:3607-30), unlocked nucleic acids (“UNAs”; see, e.g., Snead et al. (2013), Mol. Ther-Nucl. Acids, 2:e103), and bridged nucleic acids (“BNAs”; see, e.g., Imanishi & Obika, (2002), Chem. Commun. (Camb), 21:1653-59).

[0115] In some embodiments, the modification of the nucleotide in the sugar comprises a 2'-modification. In some embodiments, the 2'-modification may be 2'-O-propynyl, 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-arabinonucleic acid (2'-FANA). In some embodiments, the modification is 2'-F, 2'-OMe, or 2'-MOE. In some embodiments, the modified sugar comprises 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 attached to the 1'-carbon or 4'-carbon of the sugar, or the 2'-oxygen attached to the 1'-carbon or 4'-carbon via an ethylene bridge or a methylene bridge. In some embodiments, the modified nucleotide has an acyclic sugar lacking a bond between the 2'-carbon and the 3'-carbon. In some embodiments, the modified nucleotide has a thiol group (e.g., at the 4'-position of the sugar).

[0116] In some embodiments, the oligonucleotide comprises at least about 1 (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) modified nucleotides. In some embodiments, the sense strand of the oligonucleotide comprises at least about 1 (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) modified nucleotides. In some embodiments, the antisense strand of the oligonucleotide comprises at least about 1 (e.g., at least 1, at least 5, at least 10, at least 15, at least 20, or more) modified nucleotides.

[0117] 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 include 2'-modifications (e.g., 2'-F or 2'-OMe, 2'-MOE, and 2'-deoxy-2'-fluoro-β-d-arabinonucleic acid). In some embodiments, the modified nucleotides include 2' modifications (e.g., 2'-F or 2'-OMe).

[0118] In some embodiments, the present disclosure provides oligonucleotides having different modification patterns. In some embodiments, the oligonucleotide includes a sense strand sequence having the modification pattern shown in the Examples and the Sequence Listing, and an antisense strand having the modification pattern shown in the Examples and the Sequence Listing.

[0119] In some embodiments, the oligonucleotide includes an antisense strand having nucleotides modified with 2'-F. In some embodiments, the oligonucleotide includes an antisense strand having nucleotides modified with 2'-F and 2'-OMe. In some embodiments, the oligonucleotide includes a sense strand having nucleotides modified with 2'-F. In some embodiments, the oligonucleotide includes a sense strand having nucleotides modified with 2'-F and 2'-OMe.

[0120] In some embodiments, the oligonucleotide comprises a sense strand in which about 10% to 15%, or 10%, 11%, 12%, 13%, 14%, or 15% of the nucleotides are 2'-F modified. In some embodiments, the oligonucleotide comprises a sense strand in which about 18% to about 23%, or 18%, 19%, 20%, 21%, 22%, or 23% of the nucleotides are 2'-F modified. In some embodiments, the oligonucleotide comprises a sense strand in which about 38% to about 43%, or 38%, 39%, 40%, 41%, 42%, or 43% of the nucleotides are 2'-F modified. In some embodiments, about 11% of the nucleotides of the sense strand are 2-F modified. In some embodiments, about 22% of the nucleotides of the sense strand are 2-F modified. In some embodiments, about 40% of the nucleotides of the sense strand are 2-F modified.

[0121] In some embodiments, the oligonucleotide comprises an antisense strand in which about 25% to about 35%, or 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35% of the nucleotides are 2'-F modified. In some embodiments, about 32% of the nucleotides of the antisense strand are 2'-F modified. 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 that are 2'-F modified. 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 that are 2'-F modified. In some embodiments, about 19% of the nucleotides are 2'-F modified. In some embodiments, about 29% of the nucleotides are 2'-F modified. In some embodiments, about 40% of the nucleotides are 2'-F modified.

[0122] In some embodiments, one or more of the 8th, 9th, 10th, or 11th positions of the 36-nucleotide sense strand are modified with a 2'-F group. In some embodiments, one or more of the 8th, 9th, 10th, or 11th positions of the sense strand containing a stem-loop are modified with a 2'-F group. In some embodiments, the respective sugar moieties of the nucleotides at positions 1 to 7 and 12 to 20 of the 36-nucleotide sense strand are modified with 2'-OMe. In some embodiments, the respective sugar moieties of the nucleotides at positions 1 to 7 and 12 to 20 of the sense strand containing a stem-loop are modified with 2'-OMe. In some embodiments, the respective sugar moieties of the nucleotides at positions 1 to 7 and 12 to 36 of the sense strand are modified with 2'-OMe.

[0123] In some embodiments, one or more of the 3rd, 5th, 8th, 10th, 12th, 13th, 15th, and 17th positions of the sense strand are modified with a 2'-F group.

[0124] In some embodiments, the antisense strand has three nucleotides modified with 2'-F at the 2'-position of the sugar moiety. In some embodiments, the sugar moieties at the 2nd, 5th, and 14th positions of the antisense strand, and optionally up to three of the nucleotides at the 1st, 3rd, 7th, and 10th positions are modified with 2'-F. In some embodiments, the sugar moieties at the 2nd, 5th, and 14th positions of the antisense strand, and optionally up to three of the nucleotides at the 3rd, 4th, 7th, and 10th positions are modified with 2'-F. In other embodiments, the sugar moiety at each of the 2nd, 5th, and 14th positions of the antisense strand is modified with 2'-F. In other embodiments, the sugar moieties at each of the 1st, 2nd, 5th, and 14th positions of the antisense strand are modified with 2'-F. In other embodiments, the sugar moieties at each of the 2nd, 4th, 5th, and 14th positions of the antisense strand are modified with 2'-F. In yet other embodiments, the sugar moieties at each of the 1st, 2nd, 3rd, 5th, 7th, and 14th positions of the antisense strand are modified with 2'-F. In other embodiments, the sugar moieties at each of the 2nd, 3rd, 4th, 5th, 7th, and 14th positions of the antisense strand are modified with 2'-F. In yet another embodiment, the sugar moieties at each of the 1st, 2nd, 3rd, 5th, 10th, and 14th positions of the antisense strand are modified with 2'-F. In other embodiments, the sugar moieties at each of the 2nd, 3rd, 4th, 5th, 10th, and 14th positions of the antisense strand are modified with 2'-F. In another embodiment, the sugar moieties at each of the 2nd, 3rd, 5th, 7th, 10th, and 14th positions of the antisense strand are modified with 2'-F. In other embodiments, the sugar moieties at each of the 2nd, 3rd, 4th, 5th, 7th, 10th, and 14th positions of the antisense strand that is double-stranded with a 36-nucleotide sense strand are modified with 2'-F. In some embodiments, the sugar moieties at each of the 2nd, 3rd, 4th, 5th, 7th, 10th, and 14th positions of the antisense strand that is double-stranded with a sense strand containing a stem-loop are modified with 2'-F.

[0125] In some embodiments, the oligonucleotide comprises an antisense strand having sugar moieties at positions 2 and 14 modified with 2'-F. In some embodiments, the oligonucleotide comprises an antisense strand having sugar moieties at positions 2, 5 and 14 modified with 2'-F. In some embodiments, the oligonucleotide comprises an antisense strand having sugar moieties at positions 1, 2, 5 and 14 modified with 2'-F. In some embodiments, the oligonucleotide comprises an antisense strand having sugar moieties at positions 2, 4, 5 and 14 modified with 2'-F. In some embodiments, the oligonucleotide comprises an antisense strand having sugar moieties at positions 1, 2, 3, 5, 7 and 14 modified with 2'-F. In some embodiments, the oligonucleotide comprises an antisense strand having sugar moieties at positions 2, 3, 4, 5, 7 and 14 modified with 2'-F. In some embodiments, the oligonucleotide comprises an antisense strand having sugar moieties at positions 1, 2, 3, 5, 10 and 14 modified with 2'-F. In some embodiments, the oligonucleotide comprises an antisense strand having sugar moieties at positions 2, 3, 4, 5, 10 and 14 modified with 2'-F. In some embodiments, the oligonucleotide comprises a 36-nucleotide sense strand and antisense strand, wherein the antisense strand comprises sugar moieties at positions 2, 3, 4, 5, 7, 10, and 14 modified with 2'-F. In some embodiments, the oligonucleotide comprises a sense strand and antisense strand comprising a stem-loop, wherein the antisense strand comprises sugar moieties at positions 2, 3, 4, 5, 7, 10, and 14 modified with 2'-F. In some embodiments, the oligonucleotide comprises an antisense strand having sugar moieties at positions 2, 3, 4, 5, 7, 10, 14, 16, and 19 modified with 2'-F.

[0126] In some embodiments, the oligonucleotide comprises an antisense strand having a sugar moiety of each nucleotide at positions 2, 5, and 14 modified with 2'-F, and a sugar moiety of each of the remaining nucleotides of the antisense strand modified with a modification selected from the group consisting of 2'-O-propynyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, EA, 2'-OMe, 2'-MOE, 2'-O-NMA, and 2'-FANA.

[0127] In some embodiments, the oligonucleotide comprises an antisense strand having a sugar moiety of each nucleotide at positions 1, 2, 5, and 14 of the antisense strand modified with 2'-F, and a sugar moiety of each of the remaining nucleotides of the antisense strand modified with a modification selected from the group consisting of 2'-O-propynyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, EA, 2'-OMe, 2'-MOE, 2'-O-NMA, and 2'-FANA.

[0128] In some embodiments, the oligonucleotide comprises an antisense strand having a sugar moiety of each nucleotide at positions 2, 4, 5, and 14 of the antisense strand modified with 2'-F, and a sugar moiety of each of the remaining nucleotides of the antisense strand modified with a modification selected from the group consisting of 2'-O-propynyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, EA, 2'-OMe, 2'-MOE, 2'-O-NMA, and 2'-FANA.

[0129] In some embodiments, the oligonucleotide comprises an antisense strand having a sugar moiety of each nucleotide at positions 1, 2, 3, 5, 7, and 14 of the antisense strand modified with 2'-F, and a sugar moiety of each of the remaining nucleotides of the antisense strand modified with a modification selected from the group consisting of 2'-O-propynyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, EA, 2'-OMe, 2'-MOE, 2'-O-NMA, and 2'-FANA.

[0130] In some embodiments, the oligonucleotide comprises an antisense strand having the sugar moiety of each nucleotide at positions 2, 3, 4, 5, 7, and 14 of the antisense strand modified with 2'-F, and the sugar moiety of each of the remaining nucleotides of the antisense strand modified with a modification selected from the group consisting of 2'-O-propynyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, EA, 2'-OMe, 2'-MOE, 2'-O-NMA, and 2'-FANA.

[0131] In some embodiments, the oligonucleotide comprises an antisense strand having the sugar moiety of each nucleotide at positions 1, 2, 3, 5, 10, and 14 of the antisense strand modified with 2'-F, and the sugar moiety of each of the remaining nucleotides of the antisense strand modified with a modification selected from the group consisting of 2'-O-propynyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, EA, 2'-OMe, 2'-MOE, 2'-O-NMA, and 2'-FANA.

[0132] In some embodiments, the oligonucleotide comprises an antisense strand having the sugar moiety of each nucleotide at positions 2, 3, 4, 5, 10, and 14 of the antisense strand modified with 2'-F, and the sugar moiety of each of the remaining nucleotides of the antisense strand modified with a modification selected from the group consisting of 2'-O-propynyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, EA, 2'-OMe, 2'-MOE, 2'-O-NMA, and 2'-FANA.

[0133] In some embodiments, the oligonucleotide comprises an antisense strand having the sugar moiety of each nucleotide at positions 2, 3, 5, 7, 10, and 14 of the antisense strand modified with 2'-F, and the sugar moiety of each of the remaining nucleotides of the antisense modified with a modification selected from the group consisting of 2'-O-propynyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, EA, 2'-OMe, 2'-MOE, 2'-O-NMA, and 2'-FANA.

[0134] In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand of 36 nucleotides, and the antisense strand has sugar moieties at positions 2, 3, 4, 5, 7, 10, and 14 modified with 2'-F, and sugar moieties of each of the remaining nucleotides of the antisense strand modified with a modification selected from the group consisting of 2'-O-propynyl, 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 that includes a stem-loop, and the antisense strand has sugar moieties at positions 2, 3, 4, 5, 7, 10, and 14 modified with 2'-F, and sugar moieties of each of the remaining nucleotides of the antisense modified with a modification selected from the group consisting of 2'-O-propynyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, EA, 2'-OMe, 2'-MOE, 2'-O-NMA, and 2'-FANA.

[0135] In some embodiments, the oligonucleotide comprises an antisense strand having sugar moieties of each of the nucleotides at positions 2, 3, 4, 5, 7, 10, 14, 16, and 19 modified with 2'-F, and sugar moieties of each of the remaining nucleotides of the antisense strand modified with a modification selected from the group consisting of 2'-O-propynyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, EA, 2'-OMe, 2'-MOE, 2'-O-NMA, and 2'-FANA.

[0136] In some embodiments, the oligonucleotide comprises an antisense strand having a sugar moiety at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 modified with 2'-F.

[0137] In some embodiments, the oligonucleotide comprises an antisense strand having a sugar moiety at the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, or 22nd position modified with 2'-OMe.

[0138] In some embodiments, the oligonucleotide comprises an antisense strand having a sugar moiety at the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, or 22nd position modified with a modification selected from the group consisting of 2'-O-propynyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, EA, 2'-OMe, 2'-MOE, 2'-O-NMA, and 2'-FANA.

[0139] In some embodiments, the oligonucleotide comprises a 36-nucleotide sense strand having a sugar moiety at positions 8 to 11 modified with 2'-F. In some embodiments, the oligonucleotide comprises a sense strand comprising a stem-loop and a sugar moiety at positions 8 to 11 modified with 2'-F. In some embodiments, the oligonucleotide comprises a 36-nucleotide sense strand having sugar moieties at positions 1 to 7 and 12 to 17 or 12 to 20 modified with 2'-OMe. In some embodiments, the oligonucleotide comprises a sense strand comprising a stem-loop and sugar moieties at positions 1 to 7 and 12 to 17 or 12 to 20 modified with 2'-OMe. In some embodiments, the oligonucleotide comprises a 36-nucleotide sense strand having sugar moieties at positions 1 to 7 and 12 to 17, 12 to 20 or 12 to 22 modified with 2'-OMe. In some embodiments, the oligonucleotide comprises a sense strand comprising a stem-loop and sugar moieties at positions 1 to 7 and 12 to 17, 12 to 20 or 12 to 22 modified with 2'-OMe. In some embodiments, the oligonucleotide comprises a 36-nucleotide sense strand having respective sugar moieties of nucleotides at positions 1 to 7 and 12 to 17 or 12 to 20 of the sense strand modified with a modification selected from the group consisting of 2'-O-propynyl, 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 comprising a stem-loop and having respective sugar moieties of nucleotides at positions 1 to 7 and 12 to 17 or 12 to 20 of the sense strand modified with a modification selected from the group consisting of 2'-O-propynyl, 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 36-nucleotide sense strand having the respective sugar moieties of the nucleotides at positions 1 to 7 and 12 to 17, 12 to 20, or 12 to 22 of the sense strand modified with a modification selected from the group consisting of 2'-O-propynyl, 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 comprising a stem-loop and the respective sugar moieties of the nucleotides at positions 1 to 7 and 12 to 17, 12 to 20, or 12 to 22 of the sense strand modified with a modification selected from the group consisting of 2'-O-propynyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, EA, 2'-OMe, 2'-MOE, 2'-O-NMA, and 2'-FANA.

[0140] In some embodiments, the oligonucleotide comprises a sense strand having sugar moieties at positions 3, 5, 8, 10, 12, 13, 15, and 17 modified with 2'-F. In some embodiments, the oligonucleotide comprises a sense strand having sugar moieties at positions 1, 2, 4, 6, 7, 9, 11, 14, 16, and 18 to 20 modified with 2'-OMe. In some embodiments, the oligonucleotide comprises a sense strand having the respective sugar moieties of the nucleotides at positions 1, 2, 4, 6, 7, 9, 11, 14, 16, and 18 to 20 of the sense strand modified with a modification selected from the group consisting of 2'-O-propynyl, 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 having the respective sugar moieties of the nucleotides at positions 1 to 7 and 12 to 17, 12 to 20, or 12 to 22 of the sense strand modified with a modification selected from the group consisting of 2'-O-propynyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, EA, 2'-OMe, 2'-MOE, 2'-O-NMA, and 2'-FANA.

[0141] In some embodiments, the oligonucleotide comprises a sense strand having a sugar moiety at the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st, 32nd, 33rd, 34th, 35th, or 36th position modified with 2'-F.

[0142] In some embodiments, the oligonucleotide comprises a sense strand having a sugar moiety at the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st, 32nd, 33rd, 34th, 35th, or 36th position modified with 2'-OMe.

[0143] In some embodiments, the oligonucleotide comprises a sense strand having a sugar moiety at the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st, 32nd, 33rd, 34th, 35th, or 36th position modified with a modification selected from the group consisting of 2'-O-propynyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, EA, 2'-OMe, 2'-MOE, 2'-O-NMA, and 2'-FANA.

[0144] In some embodiments, the oligonucleotide comprises an antisense strand having the sugar moiety of each of the nucleotides at positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand modified with 2'-F, and the sugar moiety of each of the remaining nucleotides of the antisense strand modified with a modification selected from the group consisting of 2'-O-propynyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, EA, 2'-OMe, 2'-MOE, 2'-O-NMA, and 2'-FANA; and a sense strand having the sugar moiety of each of the nucleotides at positions 8-11 of the sense strand modified with 2'-F, and the sugar moiety of each of the remaining nucleotides of the sense strand modified with a modification selected from the group consisting of 2'-O-propynyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, EA, 2'-OMe, 2'-MOE, 2'-O-NMA, and 2'-FANA. In some embodiments, the oligonucleotide comprises an antisense strand having the sugar moiety of each of the nucleotides at positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand modified with 2'-F, and the sugar moiety of each of the remaining nucleotides of the antisense strand modified with a modification selected from the group consisting of 2'-O-propynyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, EA, 2'-OMe, 2'-MOE, 2'-O-NMA, and 2'-FANA; and a stem-loop, and a sense strand having the sugar moiety of each of the nucleotides at positions 8-11 of the sense strand modified with 2'-F, and the sugar moiety of each of the remaining nucleotides of the sense strand modified with a modification selected from the group consisting of 2'-O-propynyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, EA, 2'-OMe, 2'-MOE, 2'-O-NMA, and 2'-FANA.

[0145] In some embodiments, the oligonucleotide comprises an antisense strand having a sugar moiety of each nucleotide at positions 2, 3, 4, 5, 7, 10, 14, 16, and 19 of the antisense strand modified with 2'-F, and a sugar moiety of each of the remaining nucleotides of the antisense strand modified with a modification selected from the group consisting of 2'-O-propynyl, 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 of each nucleotide at positions 3, 5, 8, 10, 12, 13, 15, and 17 of the sense strand modified with 2'-F, and a sugar moiety of each of the remaining nucleotides of the sense strand modified with a modification selected from the group consisting of 2'-O-propynyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, EA, 2'-OMe, 2'-MOE, 2'-O-NMA, and 2'-FANA.

[0146] In some embodiments, the sense and antisense strands of the oligonucleotide are: a) SEQ ID NO: 1537 and 1572, respectively; b) SEQ ID NO: 1538 and 1573, respectively; c) SEQ ID NO: 1539 and 1574, respectively; d) SEQ ID NO: 1540 and 1575, respectively; e) SEQ ID NO: 1541 and 1576, respectively; f) SEQ ID NO: 1542 and 1577, respectively; g) SEQ ID NO: 1543 and 1578, respectively; h) SEQ ID NO: 1544 and 1579, respectively; i) SEQ ID NO: 1545 and 1580, respectively; j) SEQ ID NO: 1546 and 1581, respectively; k) SEQ ID NO: 1547 and 1582, respectively; l) SEQ ID NO: 1548 and 1583, respectively; m) SEQ ID NO: 1549 and 1584, respectively; n) SEQ ID NO: 1550 and 1585, respectively; o) SEQ ID NOs: 1551 and 1586, respectively; p) SEQ ID NOs: 1552 and 1587, respectively; q) SEQ ID NOs: 1553 and 1588, respectively; r) SEQ ID NOs: 1554 and 1589, respectively; s) SEQ ID NOs: 1555 and 1590, respectively; t) SEQ ID NOs: 1556 and 1591, respectively; u) SEQ ID NOs: 1557 and 1592, respectively; v) SEQ ID NOs: 1558 and 1593, respectively; w) SEQ ID NOs: 1559 and 1594, respectively; x) SEQ ID NOs: 1560 and 1595, respectively; y) SEQ ID NOs: 1561 and 1596, respectively; z) SEQ ID NOs: 1562 and 1597, respectively; aa) SEQ ID NOs: 1563 and 1598, respectively; bb) SEQ ID NOs: 1564 and 1599, respectively; cc) SEQ ID NOs: 1565 and 1600, respectively; dd) SEQ ID NOs: 1566 and 1601, respectively; ee) SEQ ID NOs: 1567 and 1602, respectively; ff) SEQ ID NOs: 1568 and 1603, respectively; gg) SEQ ID NOs: 1569 and 1604, respectively; hh) SEQ ID NOs: 1570 and 1605, respectively; ii) SEQ ID NOs: 1571 and 1606, respectively; and jj) comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1681 and 1586, wherein one or more of positions 3, 5, 8, 10, 12, 13, 15, or 17 of the sense strand are modified with a 2'-F group.

[0147] In some embodiments, the sense and antisense strands of the oligonucleotide are: a) SEQ ID NOs: 1540 and 1575, respectively; b) SEQ ID NOs: 1544 and 1579, respectively; c) SEQ ID NOs: 1546 and 1581, respectively; d) SEQ ID NOs: 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) SEQ ID NOs: 1565 and 1600, respectively; k) SEQ ID NOs: 1566 and 1601, respectively; l) SEQ ID NOs: 1570 and 1605, respectively; and m) comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1681 and 1586, respectively, wherein one or more of positions 3, 5, 8, 10, 12, 13, 15, or 17 of the sense strand are modified with a 2'-F group.

[0148] In some embodiments, the sense and antisense strands of the oligonucleotide are: a) SEQ ID NOs: 1553 and 1588, respectively; b) SEQ ID NOs: 1560 and 1595, respectively; c) SEQ ID NOs: 1564 and 1599, respectively; d) SEQ ID NOs: 1551 and 1586, respectively; e) SEQ ID NOs: 1570 and 1605, respectively; and f) comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1681 and 1586, respectively, wherein one or more of positions 3, 5, 8, 10, 12, 13, 15, or 17 of the sense strand are modified with a 2'-F group.

[0149] 5'-terminal phosphate In some embodiments, the oligonucleotides described herein (e.g., RNAi oligonucleotides) include a sense strand and an antisense strand, and the antisense strand includes a 5' terminal phosphate. In some embodiments, the 5' terminal phosphate group enhances interaction with Ago2. However, oligonucleotides containing a 5' phosphate group may be susceptible to degradation by phosphatases or other enzymes, which may limit their bioavailability in vivo. In some embodiments, the oligonucleotide includes an analog of 5' phosphate that is resistant to such degradation. In some embodiments, the phosphate analog is phosphonate oxymethyl, phosphonate vinyl, or phosphonate malonyl, or a combination thereof. In certain embodiments, the 5' end of the oligonucleotide strand is linked to a chemical moiety (a "phosphate mimetic") that mimics the electrostatic and steric properties of the natural 5' phosphate group.

[0150] In some embodiments, the oligonucleotide has a phosphate analog at the 4'-carbon of the sugar (referred to as a 4'-phosphate analog). 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 phosphonic acid oxymethyl or an analog thereof in which the oxygen atom of the oxymethyl group is bonded to the sugar moiety (e.g., its 4'-carbon). In other embodiments, the 4'-phosphate analog is phosphonic acid thiomethyl or phosphonic acid aminomethyl or an analog thereof in which the sulfur atom of the thiomethyl group or the nitrogen atom of the aminomethyl group is bonded to the 4'-carbon of the sugar moiety. In certain embodiments, the 4'-phosphate analog is phosphonic acid oxymethyl. In some embodiments, phosphonic acid oxymethyl is represented by the formula -O-CH2-PO(OH)2, -O-CH2-PO(OR)2, or O-CH2-POOH(R), where R is independently selected from H, CH3, an alkyl group, CH2CH2CN, CH2OCOC(CH3)3, CH2OCH2CH2Si(CH3)3, or a protecting group. In certain embodiments, the alkyl group is CH2CH3. More typically, R is independently selected from H, CH3, or CH2CH3. In some embodiments, R is CH3. In some embodiments, the 4'-phosphate analog is 4'-oxymethyl phosphonic acid. In some embodiments, the modified nucleotide having a 4'-phosphonate analog is uridine. In some embodiments, the modified nucleotide is 4'-O-methylphosphonate-2'-O-methyluridine.

[0151] In some embodiments, the sense and antisense strands of the oligonucleotide are: a) SEQ ID NOs: 1537 and 1572, respectively; b) SEQ ID NOs: 1538 and 1573, respectively; c) SEQ ID NOs: 1539 and 1574, respectively; d) SEQ ID NOs: 1540 and 1575, respectively; e) SEQ ID NOs: 1541 and 1576, respectively; f) SEQ ID NOs: 1542 and 1577, respectively; g) SEQ ID NOs: 1543 and 1578, respectively; h) SEQ ID NOs: 1544 and 1579, respectively; i) SEQ ID NOs: 1545 and 1580, respectively; j) SEQ ID NOs: 1546 and 1581, respectively; k) SEQ ID NOs: 1547 and 1582, respectively; l) SEQ ID NOs: 1548 and 1583, respectively; m) SEQ ID NOs: 1549 and 1584, respectively; n) SEQ ID NOs: 1550 and 1585, respectively; o) SEQ ID NOs: 1551 and 1586, respectively; p) SEQ ID NOs: 1552 and 1587, respectively; q) SEQ ID NOs: 1553 and 1588, respectively; r) SEQ ID NOs: 1554 and 1589, respectively; s) SEQ ID NOs: 1555 and 1590, respectively; t) SEQ ID NOs: 1556 and 1591, respectively; u) SEQ ID NOs: 1557 and 1592, respectively; v) SEQ ID NOs: 1558 and 1593, respectively; w) SEQ ID NOs: 1559 and 1594, respectively; x) SEQ ID NOs: 1560 and 1595, respectively; y) SEQ ID NOs: 1561 and 1596, respectively; z) SEQ ID NOs: 1562 and 1597, respectively; aa) SEQ ID NOs: 1563 and 1598, respectively; bb) SEQ ID NOs: 1564 and 1599, respectively; cc) SEQ ID NOs: 1565 and 1600, respectively; dd) SEQ ID NOs: 1566 and 1601, respectively; ee) SEQ ID NOs: 1567 and 1602, respectively; ff) SEQ ID NOs: 1568 and 1603, respectively; gg) SEQ ID NOs: 1569 and 1604, respectively; hh) SEQ ID NOs: 1570 and 1605, respectively; ii) SEQ ID NOs: 1571 and 1606 respectively; and jj) a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1681 and 1586 respectively, wherein the oligonucleotide comprises a 5'-terminal phosphate group, optionally a 5'-terminal phosphate analog.

[0152] In some embodiments, the sense and antisense strands of the oligonucleotide are: a) SEQ ID NOs: 1540 and 1575 respectively; b) SEQ ID NOs: 1544 and 1579 respectively; c) SEQ ID NOs: 1546 and 1581 respectively; d) SEQ ID NOs: 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) SEQ ID NOs: 1565 and 1600 respectively; k) SEQ ID NOs: 1566 and 1601 respectively; l) SEQ ID NOs: 1570 and 1605 respectively; and m) a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1681 and 1586 respectively, wherein the oligonucleotide comprises a 5'-terminal phosphate group, optionally a 5'-terminal phosphate analog.

[0153] In some embodiments, the sense and antisense strands of the oligonucleotide are: a) SEQ ID NOs: 1553 and 1588 respectively; b) SEQ ID NOs: 1560 and 1595 respectively; c) SEQ ID NOs: 1564 and 1599 respectively; d) SEQ ID NOs: 1551 and 1586 respectively; e) SEQ ID NOs: 1570 and 1605 respectively; and f) comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1681 and 1586, wherein the oligonucleotide comprises a 5' terminal phosphate group and optionally a 5' terminal phosphate analog.

[0154] In some embodiments, the oligonucleotide comprises an antisense strand comprising a 4'-phosphate analog at the 5' terminal nucleotide, wherein the 5' terminal nucleotide has the following structure:

Chemical Formula

[0155] Modified internucleotide linkages In some embodiments, the oligonucleotide (e.g., an RNAi oligonucleotide) comprises modified internucleotide linkages. In some embodiments, the modification or substitution of phosphate results in an oligonucleotide comprising at least one (e.g., at least 1, at least 2, at least 3, at least 4, or at least 5) modified internucleotide linkages. In some embodiments, the oligonucleotide comprises about 1 to about 10 (e.g., 1 - 10, 2 - 8, 4 - 6, 3 - 10, 5 - 10, 1 - 5, 1 - 3, or 1 - 2) modified internucleotide linkages. In some embodiments, the oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modified internucleotide linkages.

[0156] The modified internucleotide linkages may be phosphorodithioate linkages, phosphorothioate linkages, phosphotriester linkages, thionoalkylphosphonate linkages, thionoalkylphosphotriester linkages, phosphoramidite linkages, phosphonic acid linkages, and / or boranophosphate linkages. In some embodiments, at least one modified internucleotide linkage is a phosphorothioate linkage.

[0157] In some embodiments, the oligonucleotide has phosphorothioate linkages 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 phosphorothioate linkages 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 phosphorothioate linkages between (i) positions 1 and 2 of the sense strand; and (ii) each of positions 1 and 2, 2 and 3, 3 and 4, 20 and 21, and 21 and 22 of the antisense strand.

[0158] In some embodiments, the sense and antisense strands of the oligonucleotide are: a) SEQ ID NO: 1537 and 1572, respectively; b) SEQ ID NO: 1538 and 1573, respectively; c) SEQ ID NO: 1539 and 1574, respectively; d) SEQ ID NO: 1540 and 1575, respectively; e) SEQ ID NO: 1541 and 1576, respectively; f) SEQ ID NO: 1542 and 1577, respectively; g) SEQ ID NO: 1543 and 1578, respectively; h) SEQ ID NO: 1544 and 1579, respectively; i) SEQ ID NO: 1545 and 1580, respectively; j) SEQ ID NO: 1546 and 1581, respectively; k) SEQ ID NO: 1547 and 1582, respectively; l) SEQ ID NO: 1548 and 1583, respectively; m) SEQ ID NO: 1549 and 1584, respectively; n) SEQ ID NO: 1550 and 1585, respectively; o) SEQ ID NO: 1551 and 1586, respectively; p) SEQ ID NO: 1552 and 1587, respectively; q) SEQ ID NOs: 1553 and 1588, respectively; r) SEQ ID NOs: 1554 and 1589, respectively; s) SEQ ID NOs: 1555 and 1590, respectively; t) SEQ ID NOs: 1556 and 1591, respectively; u) SEQ ID NOs: 1557 and 1592, respectively; v) SEQ ID NOs: 1558 and 1593, respectively; w) SEQ ID NOs: 1559 and 1594, respectively; x) SEQ ID NOs: 1560 and 1595, respectively; y) SEQ ID NOs: 1561 and 1596, respectively; z) SEQ ID NOs: 1562 and 1597, respectively; aa) SEQ ID NOs: 1563 and 1598, respectively; bb) SEQ ID NOs: 1564 and 1599, respectively; cc) SEQ ID NOs: 1565 and 1600, respectively; dd) SEQ ID NOs: 1566 and 1601, respectively; ee) SEQ ID NOs: 1567 and 1602, respectively; ff) SEQ ID NOs: 1568 and 1603, respectively; gg) SEQ ID NOs: 1569 and 1604, respectively; hh) SEQ ID NOs: 1570 and 1605, respectively; ii) SEQ ID NOs: 1571 and 1606, respectively; and jj) a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1681 and 1586, wherein the oligonucleotide comprises modified internucleotide linkages.

[0159] In some embodiments, the sense and antisense strands of the oligonucleotide are: a) SEQ ID NOs: 1540 and 1575, respectively; b) SEQ ID NOs: 1544 and 1579, respectively; c) SEQ ID NOs: 1546 and 1581, respectively; d) SEQ ID NOs: 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) SEQ ID NOs: 1565 and 1600, respectively; k) SEQ ID NOs: 1566 and 1601, respectively; l) SEQ ID NOs: 1570 and 1605, respectively; m) comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1681 and 1586, wherein the oligonucleotide comprises modified internucleotide linkages.

[0160] In some embodiments, the sense and antisense strands of the oligonucleotide are: a) SEQ ID NOs: 1553 and 1588, respectively; b) SEQ ID NOs: 1560 and 1595, respectively; c) SEQ ID NOs: 1564 and 1599, respectively; d) SEQ ID NOs: 1551 and 1586, respectively; e) SEQ ID NOs: 1570 and 1605, respectively; and f) comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1681 and 1586, wherein the oligonucleotide comprises modified internucleotide linkages.

[0161] Modification of Bases In some embodiments, the oligonucleotides herein (e.g., RNAi oligonucleotides) have one or more modified nucleic acid bases. In some embodiments, the modified nucleic acid bases (also referred to herein as base analogs) are linked at the 1'-position of the nucleotide sugar moiety. In certain embodiments, the modified nucleic acid base is a nitrogen-containing base. In certain embodiments, the modified nucleic acid base does not contain a nitrogen atom. See, e.g., U.S. Patent Application Publication No. 2008 / 0274462. In some embodiments, the modified nucleotide comprises a universal base. In some embodiments, the modified nucleotide is abasic (does not contain a nucleic acid base).

[0162] In some embodiments, the sense and antisense strands of the oligonucleotide are: a) SEQ ID NO: 1537 and 1572, respectively; b) SEQ ID NO: 1538 and 1573, respectively; c) SEQ ID NO: 1539 and 1574, respectively; d) SEQ ID NO: 1540 and 1575, respectively; e) SEQ ID NO: 1541 and 1576, respectively; f) SEQ ID NO: 1542 and 1577, respectively; g) SEQ ID NO: 1543 and 1578, respectively; h) SEQ ID NO: 1544 and 1579, respectively; i) SEQ ID NO: 1545 and 1580, respectively; j) SEQ ID NO: 1546 and 1581, respectively; k) SEQ ID NO: 1547 and 1582, respectively; l) SEQ ID NO: 1548 and 1583, respectively; m) SEQ ID NO: 1549 and 1584, respectively; n) SEQ ID NO: 1550 and 1585, respectively; o) SEQ ID NO: 1551 and 1586, respectively; p) SEQ ID NO: 1552 and 1587, respectively; q) SEQ ID NO: 1553 and 1588, respectively; r) SEQ ID NO: 1554 and 1589, respectively; s) SEQ ID NO: 1555 and 1590, respectively; t) SEQ ID NO: 1556 and 1591, respectively; u) SEQ ID NO: 1557 and 1592, respectively; v) SEQ ID NO: 1558 and 1593, respectively; w) SEQ ID NO: 1559 and 1594, respectively; x) SEQ ID NO: 1560 and 1595, respectively; y) SEQ ID NO: 1561 and 1596, respectively; z) SEQ ID NO: 1562 and 1597, respectively; aa) SEQ ID NO: 1563 and 1598, respectively; bb) SEQ ID NO: 1564 and 1599, respectively; cc) SEQ ID NO: 1565 and 1600, respectively; dd) SEQ ID NO: 1566 and 1601, respectively; ee) SEQ ID NO: 1567 and 1602, respectively; ff) SEQ ID NO: 1568 and 1603, respectively; gg) SEQ ID NO: 1569 and 1604, respectively; hh) SEQ ID NO: 1570 and 1605, respectively; ii) SEQ ID NO: 1571 and 1606, respectively; and jj) a nucleotide sequence selected from the group consisting of SEQ ID NO: 1681 and 1586, respectively, wherein the oligonucleotide comprises one or more modified nucleobases.

[0163] In some embodiments, the sense and antisense strands of the oligonucleotide are: a) SEQ ID NO: 1540 and 1575, respectively; b) SEQ ID NO: 1544 and 1579, respectively; c) SEQ ID NO: 1546 and 1581, respectively; d) SEQ ID NO: 1551 and 1586, respectively; e) SEQ ID NO: 1552 and 1587, respectively; f) SEQ ID NO: 1553 and 1588, respectively; g) SEQ ID NO: 1558 and 1594, respectively; h) SEQ ID NO: 1560 and 1595, respectively; i) SEQ ID NO: 1564 and 1599, respectively; j) SEQ ID NO: 1565 and 1600, respectively; k) SEQ ID NO: 1566 and 1601, respectively; l) SEQ ID NO: 1570 and 1605, respectively; and m) a nucleotide sequence selected from the group consisting of SEQ ID NO: 1681 and 1586, respectively, wherein the oligonucleotide comprises one or more modified nucleobases.

[0164] In some embodiments, the sense and antisense strands of the oligonucleotide are: a) SEQ ID NO: 1553 and 1588, respectively; b) SEQ ID NO: 1560 and 1595, respectively; c) SEQ ID NO: 1564 and 1599, respectively; d) SEQ ID NO: 1551 and 1586, respectively; e) SEQ ID NO: 1570 and 1605, respectively; and f) nucleotide sequences selected from the group consisting of SEQ ID NO: 1681 and 1586, respectively, wherein the oligonucleotide comprises one or more modified nucleobases.

[0165] In some embodiments, the universal base is a heterocyclic moiety located at the 1'-position of the nucleotide sugar moiety of a modified nucleotide, or at an equivalent position of a nucleotide sugar moiety substitution, which can be paired with multiple types of bases without significantly changing the double-stranded structure when present in a double strand. In some embodiments, compared to a reference ss nucleic acid (e.g., an oligonucleotide) that is completely complementary to the target nucleic acid, the ss nucleic acid containing the universal base has a lower T m for forming a double strand with a target nucleic acid having the same. In some embodiments, compared to a reference ss nucleic acid in which the universal base is replaced by a base resulting in one mismatch, the ss nucleic acid containing the universal base has a higher T m for forming a double strand with a target nucleic acid having the same.

[0166] 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, e.g., 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 & Brown, (1994), Nucleic Acids Res. 22:4039-43).

[0167] Targeting ligand In some embodiments, it is desirable to direct an oligonucleotide (e.g., an RNAi oligonucleotide) to one or more cells or one or more organs. Such strategies can help avoid unwanted effects in other organs or avoid unnecessary loss of the oligonucleotide to cells, tissues, or organs that do not benefit from the oligonucleotide. Thus, in some embodiments, the oligonucleotide is modified to facilitate targeting and / or delivery to a particular tissue, cell or organ (e.g., to facilitate delivery of the oligonucleotide to the CNS). In some embodiments, the oligonucleotide comprises at least one nucleotide (e.g., 1, 2, 3, 4, 5, 6, or more nucleotides) conjugated to one or more targeting ligands (s). In some embodiments, the sense and antisense strands of the oligonucleotide are: a) SEQ ID NOs: 1537 and 1572, respectively; b) SEQ ID NOs: 1538 and 1573, respectively; c) SEQ ID NOs: 1539 and 1574, respectively; d) SEQ ID NOs: 1540 and 1575, respectively; e) SEQ ID NOs: 1541 and 1576, respectively; f) SEQ ID NOs: 1542 and 1577, respectively; g) SEQ ID NOs: 1543 and 1578, respectively; h) SEQ ID NOs: 1544 and 1579, respectively; i) SEQ ID NOs: 1545 and 1580, respectively; j) SEQ ID NOs: 1546 and 1581, respectively; k) SEQ ID NOs: 1547 and 1582, respectively; l) SEQ ID NOs: 1548 and 1583, respectively; m) SEQ ID NOs: 1549 and 1584, respectively; n) SEQ ID NOs: 1550 and 1585, respectively; o) SEQ ID NOs: 1551 and 1586, respectively; p) SEQ ID NOs: 1552 and 1587, respectively; q) SEQ ID NOs: 1553 and 1588, respectively; r) SEQ ID NOs: 1554 and 1589, respectively; s) SEQ ID NOs: 1555 and 1590, respectively; t) SEQ ID NOs: 1556 and 1591, respectively; u) SEQ ID NOs: 1557 and 1592, respectively; v) SEQ ID NOs: 1558 and 1593, respectively; w) SEQ ID NOs: 1559 and 1594, respectively; x) SEQ ID NOs: 1560 and 1595, respectively; y) SEQ ID NOs: 1561 and 1596, respectively; z) SEQ ID NOs: 1562 and 1597, respectively; aa) SEQ ID NOs: 1563 and 1598, respectively; bb) SEQ ID NOs: 1564 and 1599, respectively; cc) SEQ ID NOs: 1565 and 1600, respectively; dd) SEQ ID NOs: 1566 and 1601, respectively; ee) SEQ ID NOs: 1567 and 1602, respectively; ff) SEQ ID NOs: 1568 and 1603, respectively; gg) SEQ ID NOs: 1569 and 1604, respectively; hh) SEQ ID NOs: 1570 and 1605, respectively; ii) SEQ ID NOs: 1571 and 1606, respectively; and jj) It contains a nucleotide sequence selected from the group consisting of SEQ ID NO: 1681 and 1586, wherein the oligonucleotide contains a targeting ligand bound to at least one nucleotide.

[0168] In some embodiments, the oligonucleotide contains at least one nucleotide (e.g., 1, 2, 3, 4, 5, 6, or more nucleotides) bound to one or more targeting ligand(s). In some embodiments, the sense and antisense strands of the oligonucleotide are: a) SEQ ID NO: 1540 and 1575, respectively; b) SEQ ID NO: 1544 and 1579, respectively; c) SEQ ID NO: 1546 and 1581, respectively; d) SEQ ID NO: 1551 and 1586, respectively; e) SEQ ID NO: 1552 and 1587, respectively; f) SEQ ID NO: 1553 and 1588, respectively; g) SEQ ID NO: 1558 and 1594, respectively; h) SEQ ID NO: 1560 and 1595, respectively; i) SEQ ID NO: 1564 and 1599, respectively; j) SEQ ID NO: 1565 and 1600, respectively; k) SEQ ID NO: 1566 and 1601, respectively; l) SEQ ID NO: 1570 and 1605, respectively; and m) It contains a nucleotide sequence selected from the group consisting of SEQ ID NO: 1681 and 1586, wherein the oligonucleotide contains a targeting ligand bound to at least one nucleotide.

[0169] In some embodiments, the oligonucleotide contains at least one nucleotide (e.g., 1, 2, 3, 4, 5, 6, or more nucleotides) bound to one or more targeting ligand(s). In some embodiments, the sense and antisense strands of the oligonucleotide are: 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) a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1681 and 1586, respectively, wherein the oligonucleotide comprises a targeting ligand attached to at least one nucleotide.

[0170] In some embodiments, the targeting ligand comprises a carbohydrate, an amino sugar, cholesterol, a peptide, a polypeptide, or a protein or a portion of a protein (e.g., an antibody or antibody fragment). In some embodiments, the targeting ligand is an aptamer. For example, the targeting ligand may be an RGD peptide used to target tumor vasculature or glioma cells, a CREKA peptide for targeting tumor vasculature or tumor stroma, transferrin, lactoferrin, or an aptamer for targeting the transferrin receptor expressed on the CNS vasculature, or an anti-EGFR antibody for 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.

[0171] In some embodiments, one or more (e.g., 1, 2, 3, 4, 5, or 6) nucleotides of the oligonucleotide are each conjugated to a separate targeting ligand. In some embodiments, 2 to 4 nucleotides of the oligonucleotide are each conjugated to a separate targeting ligand. In some embodiments, the targeting ligand is conjugated to 2 to 4 nucleotides at either the 5' or 3' end of either the sense or antisense strand (e.g., the targeting ligand is conjugated to an overhang or extension of 2 to 4 nucleotides at the 5' or 3' end of the sense or antisense strand), such that the targeting ligand resembles the bristles of a toothbrush and the oligonucleotide resembles the toothbrush. For example, the oligonucleotide may include a stem-loop at either the 5' or 3' end of the sense strand, and 1, 2, 3, or 4 nucleotides of the loop of the stem may be individually conjugated to the targeting ligand. In some embodiments, the oligonucleotide includes a stem-loop at the 3' end of the sense strand, wherein the loop of the stem-loop includes triL or tetraL, and the 3 or 4 nucleotides that make up triL or tetraL are each individually conjugated to the targeting ligand. In some embodiments, the oligonucleotide includes a blunt end at its 3' end and one or more targeting ligands conjugated to at least one nucleotide. In some embodiments, the oligonucleotide includes a blunt end at its 3' end and one or more targeting ligands conjugated to the 5' end nucleotide of the sense strand.

[0172] GalNAc conjugation GalNAc is a high-affinity ligand for the ASGPR that is primarily expressed on the sinusoidal surface of hepatocytes and plays a major role in binding, internalizing, and then eliminating circulating glycoproteins (asialoglycoproteins) containing terminal galactose or GalNAc residues. Binding of the GalNAc moiety (either indirectly or directly) to the oligonucleotides (e.g., RNAi oligonucleotides) herein can be used for the oligonucleotides to target ASGPR expressed on the cell surface. In some embodiments, the oligonucleotide is bound to at least one or more GalNAc moieties, wherein the GalNAc moiety targets the ASGPR expressed on human liver cells (e.g., human hepatocytes) for the oligonucleotide. In some embodiments, the GalNAc moiety targets the liver for the oligonucleotide.

[0173] In some embodiments, the oligonucleotide is directly or indirectly bound to monovalent GalNAc. In some embodiments, the oligonucleotide is directly or indirectly bound to more than one monovalent GalNAc (i.e., bound to 2, 3, or 4 monovalent GalNAc moieties, usually bound to 3 or 4 monovalent GalNAc moieties). In some embodiments, the oligonucleotide is bound to one or more divalent GalNAc, trivalent GalNAc, or tetravalent GalNAc moieties. In some embodiments, the divalent GalNAc, trivalent GalNAc, or tetravalent GalNAc moiety is bound to the oligonucleotide via a branched-chain linker. In some embodiments, the monovalent GalNAc moiety is bound to the first nucleotide, and the divalent, trivalent, or tetravalent GalNAc moiety is bound to the second nucleotide via a branched-chain linker.

[0174] In some embodiments, one or more (e.g., 1, 2, 3, 4, 5, or 6) nucleotides of the oligonucleotide are each attached to a GalNAc moiety. In some embodiments, 2 to 4 nucleotides of tetraL are each attached to a separate GalNAc. In some embodiments, 1 to 3 nucleotides of triL are each attached to a separate GalNAc. In some embodiments, the targeting ligand is attached to 2 to 4 nucleotides at either the 5' or 3' end of either the sense or antisense strand (e.g., the ligand is attached to an overhang or extension of 2 to 4 nucleotides at the 5' or 3' end of the sense or antisense strand), such that the GalNAc moiety resembles a toothbrush bristle and the oligonucleotide resembles a toothbrush. In some embodiments, the GalNAc moiety is attached to nucleotides of the sense strand. For example, four GalNAc moieties can be attached to the nucleotides of tetraL of the sense strand, with each GalNAc moiety attached to one nucleotide.

[0175] In some embodiments, the oligonucleotide comprises tetraL, where 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 of the linkers described herein, as illustrated below (X = heteroatom):

Chemical formula

[0176] In some embodiments, tetraL has a monovalent GalNAc attached to any one or more adenine nucleotides of tetraL via any of the linkers described herein, as illustrated below (X = heteroatom):

Chemical formula

[0177] In some embodiments, the oligonucleotides herein include a monovalent GalNAc conjugated to a guanine (G) nucleotide, such as [ademG-GalNAc] or 2'-aminodiethoxymethanol-guanidine-GalNAc as shown below:

Chemical formula

[0178] In some embodiments, the oligonucleotide includes a monovalent GalNAc conjugated to an adenine (A) nucleotide, such as [ademA-GalNAc] or 2'-aminodiethoxymethanol-adenine-GalNAc as illustrated below:

Chemical formula

[0179] An example of such a linkage is shown below for a loop containing the nucleotide sequence GAAA from 5' to 3' (L = linker, X = heteroatom). The stem attachment points are indicated. Such a loop may be present, for example, at positions 27 to 30 of any one of the sense strands listed in Table 4 or 5. In the chemical formula,

Chemical formula

Chemical formula

[0180] A targeting ligand can be linked to a nucleotide using a suitable method or chemical technique (e.g., click chemistry). In some embodiments, the targeting ligand is attached to the nucleotide using a click linker. In some embodiments, an acetal-based linker is used to attach the targeting ligand to any one of the nucleotides of the oligonucleotides described herein. Acetal-based linkers are disclosed, for example, in International Patent Application Publication No. WO2016 / 100401. In some embodiments, the linker is a labile linker. However, in other embodiments, the linker is stable. An example of a loop containing the nucleotides GAAA from 5' to 3' is shown below, in which the GalNAc moiety is attached to the 3rd or 4th nucleotide of the loop using an acetal linker. Such loops may be present, for example, at positions 27 to 30 of any one of the sense strands listed in Table 4 or 5. In chemical formula, [Chemical formula] is the point of attachment to the oligonucleotide chain: [Chemical formula] [Chemical formula]

[0181] As described above, a targeting ligand can be linked to a nucleotide using various suitable methods or chemical synthesis methods (e.g., click chemistry). In some embodiments, the targeting ligand is attached to the nucleotide using a click linker. In some embodiments, an acetal-based linker is used to attach the targeting ligand to any one of the nucleotides of the oligonucleotides described herein. Acetal-based linkers are disclosed, for example, in International Patent Application Publication No. WO2016 / 100401. In some embodiments, the linker is a labile linker. However, in other embodiments, the linker is stable.

[0182] In some embodiments, a double-stranded extension portion (e.g., up to 3, 4, 5, or 6 bp in length) is disposed between the targeting ligand (e.g., GalNAc moiety) and the oligonucleotide. In some embodiments, the oligonucleotide does not have GalNAc attached thereto.

[0183] In some embodiments, the sense and antisense strands of the oligonucleotide are: a) SEQ ID NO: 1537 and 1572, respectively; b) SEQ ID NO: 1538 and 1573, respectively; c) SEQ ID NO: 1539 and 1574, respectively; d) SEQ ID NO: 1540 and 1575, respectively; e) SEQ ID NO: 1541 and 1576, respectively; f) SEQ ID NO: 1542 and 1577, respectively; g) SEQ ID NO: 1543 and 1578, respectively; h) SEQ ID NO: 1544 and 1579, respectively; i) SEQ ID NO: 1545 and 1580, respectively; j) SEQ ID NO: 1546 and 1581, respectively; k) SEQ ID NO: 1547 and 1582, respectively; l) SEQ ID NO: 1548 and 1583, respectively; m) SEQ ID NO: 1549 and 1584, respectively; n) SEQ ID NO: 1550 and 1585, respectively; o) SEQ ID NO: 1551 and 1586, respectively; p) SEQ ID NO: 1552 and 1587, respectively; q) SEQ ID NO: 1553 and 1588, respectively; r) SEQ ID NO: 1554 and 1589, respectively; s) SEQ ID NO: 1555 and 1590, respectively; t) SEQ ID NO: 1556 and 1591, respectively; u) SEQ ID NO: 1557 and 1592, respectively; v) SEQ ID NO: 1558 and 1593, respectively; w) SEQ ID NOs: 1559 and 1594, respectively; x) SEQ ID NOs: 1560 and 1595, respectively; y) SEQ ID NOs: 1561 and 1596, respectively; z) SEQ ID NOs: 1562 and 1597, respectively; aa) SEQ ID NOs: 1563 and 1598, respectively; bb) SEQ ID NOs: 1564 and 1599, respectively; cc) SEQ ID NOs: 1565 and 1600, respectively; dd) SEQ ID NOs: 1566 and 1601, respectively; ee) SEQ ID NOs: 1567 and 1602, respectively; ff) SEQ ID NOs: 1568 and 1603, respectively; gg) SEQ ID NOs: 1569 and 1604, respectively; hh) SEQ ID NOs: 1570 and 1605, respectively; ii) SEQ ID NOs: 1571 and 1606, respectively; and jj) comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1681 and 1586, respectively, wherein the oligonucleotide comprises at least one GalNAc moiety attached to the nucleotide.

[0184] In some embodiments, the sense and antisense strands of the oligonucleotide are: a) SEQ ID NOs: 1540 and 1575, respectively; b) SEQ ID NOs: 1544 and 1579, respectively; c) SEQ ID NOs: 1546 and 1581, respectively; d) SEQ ID NOs: 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) SEQ ID NOs: 1565 and 1600, respectively; k) SEQ ID NOs: 1566 and 1601, respectively; l) SEQ ID NOs: 1570 and 1605, respectively; and m) a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1681 and 1586, respectively, wherein the oligonucleotide comprises at least one GalNAc moiety attached to a nucleotide.

[0185] In some embodiments, the sense and antisense strands of the oligonucleotide are: a) SEQ ID NOs: 1553 and 1588, respectively; b) SEQ ID NOs: 1560 and 1595, respectively; c) SEQ ID NOs: 1564 and 1599, respectively; d) SEQ ID NOs: 1551 and 1586, respectively; e) SEQ ID NOs: 1570 and 1605, respectively; and f) a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1681 and 1586, respectively, wherein the oligonucleotide comprises at least one GalNAc moiety attached to a nucleotide.

[0186] Lipid conjugation In some embodiments, one or more lipid moieties are attached to the 5'-terminal nucleotide of the sense strand. In some embodiments, one or more lipid moieties are attached to an adenine nucleotide. In some embodiments, one or more lipid moieties are attached to a guanine nucleotide. In some embodiments, one or more lipid moieties are attached to a cytosine nucleotide. In some embodiments, one or more lipid moieties are attached to a thymine (T) nucleotide. In some embodiments, one or more lipid moieties are attached to a uracil (U) nucleotide.

[0187] In some embodiments, the lipid moiety is a hydrocarbon chain. In some embodiments, the hydrocarbon chain is saturated. In other embodiments, the hydrocarbon chain is unsaturated. In some embodiments, the hydrocarbon chain is branched. In other embodiments, the hydrocarbon chain is linear. In some embodiments, the lipid moiety is C8-C 30It is a hydrocarbon chain. In certain embodiments, the lipid moiety is C8:0, C 10 :0, C 11 :0, C 12 :0, C 14 :0, C 16 :0, C 17 :0, C 18 :0, C 18 :1, C 18 :2, C 22 :5, C 22 :0, C 24 :0, C 26 :0, C 22 :6, C 24 :1, diacyl C 16 :0 or diacyl C 18 :1. In some embodiments, the lipid moiety is a C 16 hydrocarbon chain. In some embodiments, the C 16 hydrocarbon chain is [Chemical formula] represented as.

[0188] In some embodiments, the sense strand is 20 - 22 nucleotides in length and the lipid moiety is attached 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 attached to the 5'-terminal nucleotide of the sense strand. In some embodiments, the sense strand is 20 - 22 nucleotides in length and the C 14 - C 22 hydrocarbon chain is attached to the 5'-terminal nucleotide of the sense strand. In some embodiments, the sense strand is 20 - 22 nucleotides in length and the C 16 hydrocarbon chain is attached 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 attached 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 attached to the 5'-terminal nucleotide of the sense strand. In some embodiments, the sense strand is 20 nucleotides in length and the C 14 - C 22The hydrocarbon chain is attached to the 5'-terminal nucleotide of the sense strand. In some embodiments, the sense strand is 20 nucleotides in length and C 16 The hydrocarbon chain is attached to the 5'-terminal nucleotide of the sense strand.

[0189] In some embodiments, the oligonucleotide comprises (i) a sense strand that is 20 to 22 nucleotides in length; (ii) an antisense strand that comprises a 3'-overhang sequence that is 1 or more nucleotides in length; (iii) a blunt end that constitutes the 3'-end of the sense strand; and (iv) a lipid moiety attached to the 5'-terminal nucleotide of the sense strand. In some embodiments, the oligonucleotide comprises (i) a sense strand that is 20 to 22 nucleotides in length; (ii) an antisense strand that comprises a 3'-overhang sequence that is 1 or more nucleotides in length; (iii) a blunt end that constitutes the 3'-end of the sense strand; and (iv) a hydrocarbon chain attached to the 5'-terminal nucleotide of the sense strand. In some embodiments, the oligonucleotide comprises (i) a sense strand that is 20 to 22 nucleotides in length; (ii) an antisense strand that comprises a 3'-overhang sequence that is 1 or more nucleotides in length; (iii) a blunt end that constitutes the 3'-end of the sense strand; and (iv) a C 14 ~C 22 hydrocarbon chain attached to the 5'-terminal nucleotide of the sense strand. In some embodiments, the oligonucleotide comprises (i) a sense strand that is 20 to 22 nucleotides in length; (ii) an antisense strand that comprises a 3'-overhang sequence that is 1 or more nucleotides in length; (iii) a blunt end that constitutes the 3'-end of the sense strand; and (iv) a C 16 hydrocarbon chain attached thereto.

[0190] In some embodiments, the oligonucleotide comprises: (i) a sense strand that is 20 nucleotides in length; (ii) an antisense strand that is 22 nucleotides in length and includes a 3'-overhang sequence that is 2 nucleotides in length; (iii) a blunt end that constitutes the 3'-end of the sense strand; and (iv) a lipid moiety attached to the 5'-terminal nucleotide of the sense strand. In some embodiments, the oligonucleotide comprises: (i) a sense strand that is 20 nucleotides in length; (ii) an antisense strand that is 22 nucleotides in length and includes a 3'-overhang sequence that is 2 nucleotides in length; (iii) a blunt end that constitutes the 3'-end of the sense strand; and (iv) a hydrocarbon chain attached to the 5'-terminal nucleotide of the sense strand. In some embodiments, the oligonucleotide comprises: (i) a sense strand that is 20 nucleotides in length; (ii) an antisense strand that is 22 nucleotides in length and includes a 3'-overhang sequence that is 2 nucleotides in length; (iii) a blunt end that constitutes the 3'-end of the sense strand; and (iv) a C 14 ~C 22 hydrocarbon chain attached to the 5'-terminal nucleotide of the sense strand. In some embodiments, the oligonucleotide comprises: (i) a sense strand that is 20 nucleotides in length; (ii) an antisense strand that is 22 nucleotides in length and includes a 3'-overhang sequence that is 2 nucleotides in length; (iii) a blunt end that constitutes the 3'-end of the sense strand; and (iv) a C 16 hydrocarbon chain attached to the 5'-terminal nucleotide of the sense strand.

[0191] In some embodiments, the oligonucleotide comprises: (i) an antisense strand of 19 to 30 nucleotides comprising a region complementary to an SNCA mRNA target sequence selected from SEQ ID NOs: 1781, 1782, 1796, 1798, 1802, 1808, 1814, 1817, 1713, 1718, 1726, 1830, 1839, 1742, 1846, 1852, 1865, 1784, 1804, 1721, 1822, 1840, 1735, 1847, 1855, 1864, 1901, 1902, 1938, 1947, 1955, 1964, 1973, and 1978; (ii) a sense strand of 19 to 25 nucleotides that forms a double-stranded region with the antisense strand; and (iii) a lipid moiety attached to the 5'-terminal nucleotide of the sense strand. In some embodiments, the oligonucleotide comprises: (i) an antisense strand of 19 to 30 nucleotides comprising a region complementary to an SNCA mRNA target sequence selected from SEQ ID NOs: 1798, 1817, 1718, 1846, 1852, 1865, 1804, 1721, 1847, 1855, 1864, and 1955; (ii) a sense strand of 19 to 25 nucleotides that forms a double-stranded region with the antisense strand; and (iii) a lipid moiety attached to the 5'-terminal nucleotide of the sense strand. In some embodiments, the oligonucleotide comprises: (i) an antisense strand of 19 to 30 nucleotides comprising a region complementary to an SNCA mRNA target sequence selected from SEQ ID NOs: 1865, 1721, 1847, 1846, and 1955; (ii) a sense strand of 19 to 25 nucleotides that forms a double-stranded region with the antisense strand; and (iii) a lipid moiety attached to the 5'-terminal nucleotide of the sense strand.

[0192] In some embodiments, the oligonucleotide comprises: (i) an antisense strand of 19 to 30 nucleotides comprising a region complementary to an SNCA mRNA target sequence selected from SEQ ID NOs: 1781, 1782, 1796, 1798, 1802, 1808, 1814, 1817, 1713, 1718, 1726, 1830, 1839, 1742, 1846, 1852, 1865, 1784, 1804, 1721, 1822, 1840, 1735, 1847, 1855, 1864, 1901, 1902, 1938, 1947, 1955, 1964, 1973, and 1978; (ii) a sense strand of 19 to 25 nucleotides that forms a double-stranded region with the antisense strand; and (iii) a hydrocarbon chain attached to the 5'-terminal nucleotide of the sense strand. In some embodiments, the oligonucleotide comprises: (i) an antisense strand of 19 to 30 nucleotides comprising a region complementary to an SNCA mRNA target sequence selected from SEQ ID NOs: 1798, 1817, 1718, 1846, 1852, 1865, 1804, 1721, 1847, 1855, 1864, and 1955; (ii) a sense strand of 19 to 25 nucleotides that forms a double-stranded region with the antisense strand; and (iii) a hydrocarbon chain attached to the 5'-terminal nucleotide of the sense strand. In some embodiments, the oligonucleotide comprises: (i) an antisense strand of 19 to 30 nucleotides comprising a region complementary to an SNCA mRNA target sequence selected from SEQ ID NOs: 1865, 1721, 1847, 1846, and 1955; (ii) a sense strand of 19 to 25 nucleotides that forms a double-stranded region with the antisense strand; and (iii) a hydrocarbon chain attached to the 5'-terminal nucleotide of the sense strand.

[0193] In some embodiments, the oligonucleotide comprises: (i) an antisense strand of 19 to 30 nucleotides comprising a region complementary to an SNCA mRNA target sequence selected from SEQ ID NOs: 1781, 1782, 1796, 1798, 1802, 1808, 1814, 1817, 1713, 1718, 1726, 1830, 1839, 1742, 1846, 1852, 1865, 1784, 1804, 1721, 1822, 1840, 1735, 1847, 1855, 1864, 1901, 1902, 1938, 1947, 1955, 1964, 1973, and 1978; (ii) a sense strand of 19 to 25 nucleotides that forms a double-stranded region with the antisense strand; and (iii) a C 14 ~C 22 hydrocarbon chain attached to the 5'-terminal nucleotide of the sense strand. In some embodiments, the oligonucleotide comprises: (i) an antisense strand of 19 to 30 nucleotides comprising a region complementary to an SNCA mRNA target sequence selected from SEQ ID NOs: 1798, 1817, 1718, 1846, 1852, 1865, 1804, 1721, 1847, 1855, 1864, and 1955; (ii) a sense strand of 19 to 25 nucleotides that forms a double-stranded region with the antisense strand; and (iii) a C 14 ~C 22 hydrocarbon chain attached to the 5'-terminal nucleotide of the sense strand. In some embodiments, the oligonucleotide comprises: (i) an antisense strand of 19 to 30 nucleotides comprising a region complementary to an SNCA mRNA target sequence selected from SEQ ID NOs: 1865, 1721, 1847, 1846, and 1955; (ii) a sense strand of 19 to 25 nucleotides that forms a double-stranded region with the antisense strand; and (iii) a C 14 ~C 22 hydrocarbon chain attached to the 5'-terminal nucleotide of the sense strand.

[0194] In some embodiments, the oligonucleotide comprises: (i) an antisense strand of 19 to 30 nucleotides comprising a region complementary to an SNCA mRNA target sequence selected from SEQ ID NOs: 1781, 1782, 1796, 1798, 1802, 1808, 1814, 1817, 1713, 1718, 1726, 1830, 1839, 1742, 1846, 1852, 1865, 1784, 1804, 1721, 1822, 1840, 1735, 1847, 1855, 1864, 1901, 1902, 1938, 1947, 1955, 1964, 1973, and 1978; (ii) a sense strand of 19 to 25 nucleotides that forms a double-stranded region with the antisense strand; and (iii) a C 16 hydrocarbon chain attached to the 5'-terminal nucleotide of the sense strand. In some embodiments, the oligonucleotide comprises: (i) an antisense strand of 19 to 30 nucleotides comprising a region complementary to an SNCA mRNA target sequence selected from SEQ ID NOs: 1798, 1817, 1718, 1846, 1852, 1865, 1804, 1721, 1847, 1855, 1864, and 1955; (ii) a sense strand of 19 to 25 nucleotides that forms a double-stranded region with the antisense strand; and (iii) a C 16 hydrocarbon chain attached to the 5'-terminal nucleotide of the sense strand. In some embodiments, the oligonucleotide comprises: (i) an antisense strand of 19 to 30 nucleotides comprising a region complementary to an SNCA mRNA target sequence selected from SEQ ID NOs: 1865, 1721, 1847, 1846, and 1955; (ii) a sense strand of 19 to 25 nucleotides that forms a double-stranded region with the antisense strand; and (iii) a C 16 hydrocarbon chain attached to the 5'-terminal nucleotide of the sense strand.

[0195] In some embodiments, the oligonucleotide comprises a sense strand comprising the nucleotide sequence of SEQ ID NO: 1681 and an antisense strand comprising the nucleotide sequence of SEQ ID NO: 1586, wherein the sense strand comprises a lipid moiety attached to the 5'-terminal nucleotide of the sense strand. In some embodiments, the oligonucleotide comprises a sense strand comprising the nucleotide sequence of SEQ ID NO: 1681 and an antisense strand comprising the nucleotide sequence of SEQ ID NO: 1586, wherein the sense strand comprises a hydrocarbon chain attached 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 C 14 ~C 22 hydrocarbon chain attached 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 C 16 hydrocarbon chain attached to the 5'-terminal nucleotide of the sense strand.

[0196] Exemplary RNAi oligonucleotides targeting SNCA In some embodiments, the SNCA-targeting RNAi oligonucleotide for reducing the expression of the SNCA gene comprises a sense strand and an antisense strand, wherein all nucleotides constituting the sense strand and the antisense strand are modified, and the antisense strand comprises a region complementary to any one of the SNCA mRNA target sequences of SEQ ID NOs: 1683 to 2066, and the complementary region is at least 15 consecutive nucleotides in length. In some embodiments, the 5'-terminal nucleotide of the antisense strand comprises 4'-O-methylphosphonate-2'-O-methyluridine [MePhosphonate-40-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- and 2'-OMe modified nucleotides and at least one phosphorothioate bond. In some embodiments, the antisense strand comprises four phosphorothioate bonds and the sense strand comprises one phosphorothioate bond. In some embodiments, the antisense strand comprises five phosphorothioate bonds and the sense strand comprises one phosphorothioate bond.

[0197] In some embodiments, the oligonucleotide comprises a sense strand having any one of the sequences of SEQ ID NOs: 1683 to 2066 and an antisense strand comprising a complementary sequence selected from any one of SEQ ID NOs: 2067 to 2450.

[0198] In some embodiments, the oligonucleotide comprises a sense strand having any one of the sequences of SEQ ID NOs: 1 to 384 and an antisense strand comprising a complementary sequence selected from any one of SEQ ID NOs: 385 to 768.

[0199] In some embodiments, the oligonucleotide comprises a sense strand having any one of the sequences of SEQ ID NOs: 1537 to 1571 and an antisense strand comprising a complementary sequence selected from any one of SEQ ID NOs: 1572 to 1606.

[0200] In some embodiments, the oligonucleotide comprises a sense strand having any one of the sequences of SEQ ID NOs: 1537 to 1571 and 1681, and an antisense strand comprising a complementary sequence selected from any one of SEQ ID NOs: 1572 to 1606.

[0201] In some embodiments, the oligonucleotide comprises a sense strand having any one of the sequences of SEQ ID NOs: 1537 to 1571, and an antisense strand comprising a complementary sequence selected from any one of SEQ ID NOs: 1572 to 1606.

[0202] In some embodiments, the oligonucleotide comprises a sense strand having any one of the sequences of SEQ ID NOs: 1537 to 1571 and 1681, and an antisense strand comprising a complementary sequence selected from any one of SEQ ID NOs: 1572 to 1606.

[0203] In some embodiments, the oligonucleotide for reducing the expression of the SNCA gene comprises: 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-linked nucleotides at positions 28, 29 and 30; a 36-nucleotide sense strand comprising a phosphorothioate bond between positions 1 and 2; and 2'-F modified nucleotides at positions 2, 3, 4, 5, 7, 10, 14, 16 and 19; 2'-OMe modified nucleotides at positions 1, 6, 8, 9, 11, 12, 13, 15, 17, 18 and 20-22; phosphorothioate bonds between positions 1 and 2, 2 and 3, 20 and 21, and 21 and 22; a 5'-terminal nucleotide at position 1 comprising a 4'-phosphate analog; and an antisense strand of 22 nucleotides, optionally wherein the 5'-terminal nucleotide comprises 4-O-methylphosphonate-2'-O-methyluridine [MePhosphonate-4O-mU]; positions 1-20 of the antisense strand form a double-stranded region with positions 1-20 of the sense strand, positions 21-36 of the sense strand form a stem-loop, positions 27-30 form the loop of the stem-loop, optionally positions 27-30 comprise tetra L, positions 21 and 22 of the antisense strand comprise an overhang, and the sense strand and the antisense strand are: a) SEQ ID NO: 1537 and 1572, respectively; b) SEQ ID NO: 1538 and 1573, respectively; c) SEQ ID NO: 1539 and 1574, respectively; d) SEQ ID NO: 1540 and 1575, respectively; e) SEQ ID NO: 1541 and 1576, respectively; f) SEQ ID NO: 1542 and 1577, respectively; g) SEQ ID NO: 1543 and 1578, respectively; h) SEQ ID NO: 1544 and 1579, respectively; i) SEQ ID NO: 1545 and 1580, respectively; j) SEQ ID NO: 1546 and 1581, respectively; k) SEQ ID NOs: 1547 and 1582, respectively; l) SEQ ID NOs: 1548 and 1583, respectively; m) SEQ ID NOs: 1549 and 1584, respectively; n) SEQ ID NOs: 1550 and 1585, respectively; o) SEQ ID NOs: 1551 and 1586, respectively; p) SEQ ID NOs: 1552 and 1587, respectively; q) SEQ ID NOs: 1553 and 1588, respectively; r) SEQ ID NOs: 1554 and 1589, respectively; s) SEQ ID NOs: 1555 and 1590, respectively; t) SEQ ID NOs: 1556 and 1591, respectively; u) SEQ ID NOs: 1557 and 1592, respectively; v) SEQ ID NOs: 1558 and 1593, respectively; w) SEQ ID NOs: 1559 and 1594, respectively; x) SEQ ID NOs: 1560 and 1595, respectively; y) SEQ ID NOs: 1561 and 1596, respectively; z) SEQ ID NOs: 1562 and 1597, respectively; aa) SEQ ID NOs: 1563 and 1598, respectively; bb) SEQ ID NOs: 1564 and 1599, respectively; cc) SEQ ID NOs: 1565 and 1600, respectively; dd) SEQ ID NOs: 1566 and 1601, respectively; ee) SEQ ID NOs: 1567 and 1602, respectively; ff) SEQ ID NOs: 1568 and 1603, respectively; gg) SEQ ID NOs: 1569 and 1604, respectively; hh) SEQ ID NOs: 1570 and 1605, respectively; and ii) a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1571 and 1606, respectively.

[0204] In some embodiments, the oligonucleotide for reducing the expression of the SNCA gene comprises 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-linked nucleotides at positions 28, 29 and 30; a phosphorothioate bond between positions 1 and 2; and a 36-nucleotide sense strand; and 2'-F modified nucleotides at positions 2, 3, 4, 5, 7, 10, 14, 16 and 19; 2'-OMe modified nucleotides at positions 1, 6, 8, 9, 11, 12, 13, 15, 17, 18 and 20 - 22; phosphorothioate bonds between positions 1 and 2, 2 and 3, 20 and 21, and 21 and 22; a 5'-terminal nucleotide at position 1 containing a 4'-phosphate analog; and a 22-nucleotide antisense strand, optionally wherein the 5'-terminal nucleotide comprises 4-O-methylphosphonate-2'-O-methyluridine [MePhosphonate-4O-mU]; positions 1 - 20 of the antisense strand form a double-stranded region with positions 1 - 20 of the sense strand, positions 21 - 36 of the sense strand form a stem-loop, positions 27 - 30 form the loop of the stem-loop, optionally positions 27 - 30 contain tetraL, positions 21 and 22 of the antisense strand contain an overhang, and the sense and antisense strands are: a) SEQ ID NO: 1540 and 1575, respectively; b) SEQ ID NO: 1544 and 1579, respectively; c) SEQ ID NO: 1546 and 1581, respectively; d) SEQ ID NO: 1551 and 1586, respectively; e) SEQ ID NO: 1552 and 1587, respectively; f) SEQ ID NO: 1553 and 1588, respectively; g) SEQ ID NO: 1558 and 1594, respectively; h) SEQ ID NO: 1560 and 1595, respectively; i) SEQ ID NO: 1564 and 1599, respectively; j) SEQ ID NO: 1565 and 1600, respectively; k) SEQ ID NOs: 1566 and 1601, respectively; and l) It comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1570 and 1605, respectively.

[0205] In some embodiments, the oligonucleotide for reducing the expression of the SNCA gene comprises a 36-nucleotide sense strand containing 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-linked nucleotides at positions 28, 29 and 30; and a phosphorothioate bond between positions 1 and 2; and an antisense strand of 22 nucleotides containing 2'-F modified nucleotides at positions 2, 3, 4, 5, 7, 10, 14, 16 and 19; 2'-OMe modified nucleotides at positions 1, 6, 8, 9, 11, 12, 13, 15, 17, 18 and 20-22; phosphorothioate bonds between positions 1 and 2, 2 and 3, 20 and 21, and 21 and 22; and a 5'-terminal nucleotide at position 1 containing a 4'-phosphate analog, optionally wherein the 5'-terminal nucleotide comprises 4-O-methylphosphonate-2'-O-methyluridine [MePhosphonate-4O-mU]; positions 1-20 of the antisense strand form a double-stranded region with positions 1-20 of the sense strand, positions 21-36 of the sense strand form a stem-loop, positions 27-30 form the loop of the stem-loop, optionally positions 27-30 contain tetra L, positions 21 and 22 of the antisense strand contain an overhang, and the sense and antisense strands are: a) SEQ ID NOs: 1553 and 1588, respectively; b) SEQ ID NOs: 1560 and 1595, respectively; c) SEQ ID NOs: 1564 and 1599, respectively; d) SEQ ID NOs: 1551 and 1586, respectively; and e) It comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1570 and 1605, respectively.

[0206] In some embodiments, the oligonucleotide is for reducing the expression of the SNCA gene, 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 hydrocarbon chain linked to the nucleotide at position 1; and phosphorothioate linkages between positions 1 and 2, between positions 18 and 19, and between positions 19 and 20; and a 22-nucleotide antisense strand comprising 2'-F modified nucleotides at positions 2, 3, 4, 5, 7, 10, 14, 16 and 19; 2'-OMe modified nucleotides at positions 1, 6, 8, 9, 11, 12, 13, 15, 17, 18 and 20-22; phosphorothioate linkages between positions 1 and 2, between positions 2 and 3, between positions 20 and 21, and between 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-methylphosphonate-2'-O-methyluridine [MePhosphonate-4O-mU]; positions 1-20 of the antisense strand form a double-stranded region with positions 1-20 of the sense strand, positions 21 and 22 of the antisense strand comprise overhangs, and the sense strand and the antisense strand comprise the nucleotide sequences of SEQ ID NOs: 1681 and 1586, respectively. 16

[0207] ​In some embodiments, the oligonucleotide is for reducing the expression of the SNCA gene and comprises a sense strand comprising the nucleotide sequence shown in SEQ ID NO: 1553 and an antisense strand comprising the nucleotide sequence shown in SEQ ID NO: 1588. In some embodiments, the oligonucleotide is for reducing the expression of the SNCA gene and comprises a sense strand comprising the nucleotide sequence shown in SEQ ID NO: 1560 and an antisense strand comprising the nucleotide sequence shown in SEQ ID NO: 1595. In some embodiments, the oligonucleotide is for reducing the expression of the SNCA gene and comprises a sense strand comprising the nucleotide sequence shown in SEQ ID NO: 1564 and an antisense strand comprising the nucleotide sequence shown in SEQ ID NO: 1599. In some embodiments, the oligonucleotide is for reducing the expression of the SNCA gene and comprises a sense strand comprising the nucleotide sequence shown in SEQ ID NO: 1551 and an antisense strand comprising the nucleotide sequence shown in SEQ ID NO: 1586. In some embodiments, the oligonucleotide is for reducing the expression of the SNCA gene 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 for reducing the expression of the SNCA gene 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.

[0208] In some embodiments, the oligonucleotide is for reducing the expression of the SNCA gene and comprises: (i) an antisense strand comprising a nucleotide sequence comprising a region of complementarity to the SNCA mRNA target sequence, the region of complementarity being an antisense strand of 19 to 30 nucleotides in length shown in SEQ ID NO: 1865; and (ii) a sense strand of 19 to 50 nucleotides in length comprising a region of complementarity to the antisense strand, the antisense strand and the sense strand being separate strands that form an asymmetric double-stranded region having an overhang of 1 to 4 nucleotides at the 3' end of the antisense strand.

[0209] In some embodiments, the oligonucleotide is for reducing the expression of the SNCA gene and comprises: (i) an antisense strand comprising a nucleotide sequence having a region complementary to the SNCA mRNA target sequence, the complementary region being a 19- to 30-nucleotide antisense strand shown in SEQ ID NO: 1721; and (ii) a sense strand 19 to 50 nucleotides in length comprising a region complementary to the antisense strand, the antisense strand and the sense strand being separate strands that form an asymmetric double-stranded region having a 1- to 4-nucleotide overhang at the 3' end of the antisense strand.

[0210] In some embodiments, the oligonucleotide is for reducing the expression of SNCA and comprises: (i) an antisense strand comprising a nucleotide sequence having a region complementary to the SNCA mRNA target sequence, the complementary region being a 19- to 30-nucleotide antisense strand shown in SEQ ID NO: 1847; and (ii) a sense strand 19 to 50 nucleotides in length comprising a region complementary to the antisense strand, the antisense strand and the sense strand being separate strands that form an asymmetric double-stranded region having a 1- to 4-nucleotide overhang at the 3' end of the antisense strand.

[0211] In some embodiments, the oligonucleotide for reducing the expression of the SNCA gene comprises: (i) an antisense strand comprising a nucleotide sequence having a region complementary to the SNCA mRNA target sequence, the complementary region being shown in SEQ ID NO: 1846, a 19- to 30-nucleotide antisense strand; and (ii) a sense strand 19 to 50 nucleotides in length comprising a region complementary to the antisense strand, the antisense strand and the sense strand being separate strands that form an asymmetric double-stranded region having a 1- to 4-nucleotide overhang at the 3' end of the antisense strand.

[0212] In some embodiments, the oligonucleotide is for reducing the expression of the SNCA gene and comprises: (i) an antisense strand comprising a nucleotide sequence containing a region complementary to the SNCA mRNA target sequence, the complementary region being shown in SEQ ID NO: 1955, an antisense strand of 19 to 30 nucleotides in length; and (ii) a sense strand of 19 to 50 nucleotides in length containing a region complementary to the antisense strand, the antisense strand and the sense strand being separate strands that form an asymmetric double-stranded region having an overhang of 1 to 4 nucleotides at the 3'-end of the antisense strand.

[0213] In some embodiments, the oligonucleotide is for reducing the expression of the SNCA gene and comprises: (i) an antisense strand comprising a nucleotide sequence containing a region complementary to the SNCA mRNA target sequence, the complementary region being shown in SEQ ID NO: 1865, an antisense strand of 19 to 30 nucleotides in length; and (ii) a sense strand of 19 to 50 nucleotides in length containing a region complementary to the antisense strand and a stem-loop at the 3'-end, the stem-loop being shown as S1-L-S2, S1 being complementary to S2, L forming a loop of 3 to 5 nucleotides in length between S1 and S2, the antisense strand and the sense strand being separate strands that form an asymmetric double-stranded region having an overhang of 1 to 4 nucleotides at the 3'-end of the antisense strand.

[0214] In some embodiments, the oligonucleotide is for reducing the expression of the SNCA gene and comprises: (i) an antisense strand comprising a nucleotide sequence having a region complementary to the SNCA mRNA target sequence, the complementary region being shown in SEQ ID NO: 1721, an antisense strand of 19 to 30 nucleotides in length; and (ii) a sense strand of 19 to 50 nucleotides in length comprising a region complementary to the antisense strand and a stem-loop at the 3' end, the stem-loop being shown as S1-L-S2, S1 being complementary to S2, L forming a loop of 3 to 5 nucleotides in length between S1 and S2, and the antisense strand and the sense strand being separate strands that form an asymmetric double-stranded region having an overhang of 1 to 4 nucleotides at the 3' end of the antisense strand.

[0215] In some embodiments, the oligonucleotide is for reducing the expression of the SNCA gene and comprises: (i) an antisense strand comprising a nucleotide sequence having a region complementary to the SNCA mRNA target sequence, the complementary region being shown in SEQ ID NO: 1847, an antisense strand of 19 to 30 nucleotides in length; and (ii) a sense strand of 19 to 50 nucleotides in length comprising a region complementary to the antisense strand and a stem-loop at the 3' end, the stem-loop being shown as S1-L-S2, S1 being complementary to S2, L forming a loop of 3 to 5 nucleotides in length between S1 and S2, and the antisense strand and the sense strand being separate strands that form an asymmetric double-stranded region having an overhang of 1 to 4 nucleotides at the 3' end of the antisense strand.

[0216] In some embodiments, the oligonucleotide is for reducing the expression of the SNCA gene and comprises: (i) an antisense strand comprising a nucleotide sequence having a region complementary to the SNCA mRNA target sequence, the complementary region being shown in SEQ ID NO: 1846, the antisense strand being 19 to 30 nucleotides in length; and (ii) a sense strand 19 to 50 nucleotides in length comprising a region complementary to the antisense strand and a stem-loop at the 3' end, the stem-loop being shown as S1-L-S2, S1 being complementary to S2, L forming a loop 5 nucleotides in length between S1 and S2, and the antisense strand and the sense strand being separate strands that form an asymmetric double-stranded region having an overhang of 1 to 4 nucleotides at the 3' end of the antisense strand.

[0217] In some embodiments, the oligonucleotide is for reducing the expression of the SNCA gene and comprises: (i) an antisense strand comprising a nucleotide sequence having a region complementary to the SNCA mRNA target sequence, the complementary region being shown in SEQ ID NO: 1955, the antisense strand being 19 to 30 nucleotides in length; and (ii) a sense strand 19 to 50 nucleotides in length comprising a region complementary to the antisense strand and a stem-loop at the 3' end, the stem-loop being shown as S1-L-S2, S1 being complementary to S2, L forming a loop 3 to 5 nucleotides in length between S1 and S2, and the antisense strand and the sense strand being separate strands that form an asymmetric double-stranded region having an overhang of 1 to 4 nucleotides at the 3' end of the antisense strand.

[0218] In some embodiments, the oligonucleotide is for reducing the expression of the SNCA gene and comprises: (i) a nucleotide sequence in which the antisense strand contains a region complementary to the SNCA mRNA target sequence, the complementary region being a 19- to 30-nucleotide antisense strand shown in SEQ ID NO: 1846; and (ii) a sense strand of 19 to 25 nucleotides in length containing a region complementary to the antisense strand. The oligonucleotide contains a blunt end that constitutes the 3'-end of the sense strand, and the antisense strand and the sense strand are separate strands that form an asymmetric double-stranded region having an overhang of 1 to 4 nucleotides at the 3'-end of the antisense strand.

[0219] In some embodiments, the oligonucleotide is for reducing the expression of the SNCA gene and comprises: (i) a nucleotide sequence in which the antisense strand contains a region complementary to the SNCA mRNA target sequence, the complementary region being a 19- to 30-nucleotide antisense strand shown in SEQ ID NO: 1865; and (ii) a sense strand of 19 to 50 nucleotides in length containing a region complementary to the antisense strand, the region complementary to the antisense strand being shown in SEQ ID NO: 2249. The antisense strand and the sense strand are separate strands that form an asymmetric double-stranded region having an overhang of 1 to 4 nucleotides at the 3'-end of the antisense strand.

[0220] In some embodiments, the oligonucleotide is for reducing the expression of the SNCA gene and comprises: (i) a nucleotide sequence in which the antisense strand contains a region complementary to the SNCA mRNA target sequence, the complementary region being a 19- to 30-nucleotide antisense strand shown in SEQ ID NO: 1721; and (ii) a sense strand of 19 to 50 nucleotides in length containing a region complementary to the antisense strand, the region complementary to the antisense strand being shown in SEQ ID NO: 2105. The antisense strand and the sense strand are separate strands that form an asymmetric double-stranded region having an overhang of 1 to 4 nucleotides at the 3'-end of the antisense strand.

[0221] In some embodiments, the oligonucleotide is for reducing the expression of the SNCA gene and comprises: (i) an antisense strand comprising a nucleotide sequence containing a region complementary to the SNCA mRNA target sequence, the complementary region being shown in SEQ ID NO: 1847, an antisense strand of 19 to 30 nucleotides in length; and (ii) a sense strand comprising a region complementary to the antisense strand, the region complementary to the antisense strand being a sense strand of 19 to 50 nucleotides in length shown in SEQ ID NO: 2231, wherein the antisense strand and the sense strand are separate strands that form an asymmetric double-stranded region having an overhang of 1 to 4 nucleotides at the 3' end of the antisense strand.

[0222] In some embodiments, the oligonucleotide is for reducing the expression of the SNCA gene and comprises: (i) an antisense strand comprising a nucleotide sequence containing a region complementary to the SNCA mRNA target sequence, the complementary region being shown in SEQ ID NO: 1846, an antisense strand of 19 to 30 nucleotides in length; and (ii) a sense strand comprising a region complementary to the antisense strand, the region complementary to the antisense strand being a sense strand of 19 to 50 nucleotides in length shown in SEQ ID NO: 2230, wherein the antisense strand and the sense strand are separate strands that form an asymmetric double-stranded region having an overhang of 1 to 4 nucleotides at the 3' end of the antisense strand.

[0223] In some embodiments, the oligonucleotide is for reducing the expression of the SNCA gene and comprises: (i) an antisense strand comprising a nucleotide sequence containing a region complementary to the SNCA mRNA target sequence, the complementary region being shown in SEQ ID NO: 1955, an antisense strand of 19 to 30 nucleotides in length; and (ii) a sense strand comprising a region complementary to the antisense strand, the region complementary to the antisense strand being a sense strand of 19 to 50 nucleotides in length shown in SEQ ID NO: 2339, wherein the antisense strand and the sense strand are separate strands that form an asymmetric double-stranded region having an overhang of 1 to 4 nucleotides at the 3' end of the antisense strand.

[0224] In some embodiments, the oligonucleotide is for reducing the expression of the SNCA gene and comprises: (i) an antisense strand comprising a nucleotide sequence having a region complementary to the SNCA mRNA target sequence, the complementary region being shown in SEQ ID NO: 1865, an antisense strand of 19 to 30 nucleotides in length; and (ii) a sense strand of 19 to 50 nucleotides in length comprising a region complementary to the antisense strand and a stem-loop at the 3' end, the region complementary to the antisense strand being shown in SEQ ID NO: 2249, the stem-loop being shown as S1-L-S2, S1 being complementary to S2, L forming a loop of 3 to 5 nucleotides between S1 and S2, and the antisense strand and the sense strand being separate strands that form an asymmetric double-stranded region having an overhang of 1 to 4 nucleotides at the 3' end of the antisense strand.

[0225] In some embodiments, the oligonucleotide is for reducing the expression of the SNCA gene and comprises: (i) an antisense strand comprising a nucleotide sequence having a region complementary to the SNCA mRNA target sequence, the complementary region being shown in SEQ ID NO: 1721, an antisense strand of 19 to 30 nucleotides in length; and (ii) a sense strand of 19 to 50 nucleotides in length comprising a region complementary to the antisense strand and a stem-loop at the 3' end, the region complementary to the antisense strand being shown in SEQ ID NO: 2105, the stem-loop being shown as S1-L-S2, S1 being complementary to S2, L forming a loop of 3 to 5 nucleotides between S1 and S2, and the antisense strand and the sense strand being separate strands that form an asymmetric double-stranded region having an overhang of 1 to 4 nucleotides at the 3' end of the antisense strand.

[0226] In some embodiments, the oligonucleotide is for reducing the expression of the SNCA gene and comprises: (i) an antisense strand comprising a nucleotide sequence containing a region complementary to the SNCA mRNA target sequence, the complementary region being shown in SEQ ID NO: 1847, an antisense strand of 19 to 30 nucleotides in length; and (ii) a sense strand of 19 to 50 nucleotides in length containing a region complementary to the antisense strand and a stem-loop at the 3' end, the region complementary to the antisense strand being shown in SEQ ID NO: 2231, the stem-loop being shown as S1-L-S2, S1 being complementary to S2, L forming a loop of 3 to 5 nucleotides between S1 and S2, and the antisense strand and the sense strand being separate strands that form an asymmetric double-stranded region having an overhang of 1 to 4 nucleotides at the 3' end of the antisense strand.

[0227] In some embodiments, the oligonucleotide is for reducing the expression of the SNCA gene and comprises: (i) an antisense strand comprising a nucleotide sequence containing a region complementary to the SNCA mRNA target sequence, the complementary region being shown in SEQ ID NO: 1846, an antisense strand of 19 to 30 nucleotides in length; and (ii) a sense strand of 19 to 50 nucleotides in length containing a region complementary to the antisense strand and a stem-loop at the 3' end, the region complementary to the antisense strand being shown in SEQ ID NO: 2230, the stem-loop being shown as S1-L-S2, S1 being complementary to S2, L forming a loop of 3 to 5 nucleotides between S1 and S2, and the antisense strand and the sense strand being separate strands that form an asymmetric double-stranded region having an overhang of 1 to 4 nucleotides at the 3' end of the antisense strand.

[0228] In some embodiments, the oligonucleotide is for reducing the expression of the SNCA gene and comprises: (i) an antisense strand comprising a nucleotide sequence having a region complementary to the SNCA mRNA target sequence, the complementary region being a 19- to 30-nucleotide antisense strand shown in SEQ ID NO: 1955; and (ii) a sense strand comprising a region complementary to the antisense strand and a stem-loop at the 3′ end, the region complementary to the antisense strand being a 19- to 50-nucleotide sense strand shown in SEQ ID NO: 2339, the stem-loop being shown as S1-L-S2, where S1 is complementary to S2, L forms a loop 3 to 5 nucleotides in length between S1 and S2, and the antisense strand and the sense strand are separate strands that form an asymmetric double-stranded region having a 1- to 4-nucleotide overhang at the 3′ end of the antisense strand.

[0229] In some embodiments, the oligonucleotide is for reducing the expression of the SNCA gene and comprises: (i) an antisense strand comprising a nucleotide sequence having a region complementary to the SNCA mRNA target sequence, the complementary region being a 19- to 30-nucleotide antisense strand shown in SEQ ID NO: 1846; and (ii) a sense strand comprising a region complementary to the antisense strand, the region complementary to the antisense strand being a 19- to 25-nucleotide sense strand shown in SEQ ID NO: 2230, the oligonucleotide comprising a blunt end constituting the 3′ end of the sense strand, and the antisense strand and the sense strand being separate strands that form an asymmetric double-stranded region having a 1- to 4-nucleotide overhang at the 3′ end of the antisense strand.

[0230] In some embodiments, the oligonucleotide is for reducing the expression of the SNCA gene and comprises a sense strand and an antisense strand hybridized to the antisense strand: 5'-[MePhosphonate-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', where the 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', where mX = 2'-OMe modified nucleotide, fX = 2'-F modified nucleotide, -= phosphodiester bond, -S- = phosphorothioate bond, [MePhosphonate-4O-mX] = 4'-O-monomethylphosphonate-2'-O-methyl modified nucleotide, and ademX-GalNAc = GalNAc conjugated to a nucleotide.

[0231] In some embodiments, the oligonucleotide is for reducing the expression of the SNCA gene and comprises a sense strand and an antisense strand hybridized to the antisense strand: 5'-[MePhosphonate-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', where the sense strand: 5'-[AdemX-L]-S-mX-fX-mX-fX-mX-mX-fX-mX-fX-mX-fX-fX-mX-fX-mX-fX-mX-S-mX-S-mX-3', where mX = 2'-OMe modified nucleotide, fX = 2'-F modified nucleotide, -= phosphodiester bond, -S- = phosphorothioate bond, [MePhosphonate-4O-mX] = 4'-O-monomethylphosphonate-2'-O-methyl modified nucleotide, and ademX-L = lipid moiety conjugated to a nucleotide.

[0232] In some embodiments, the oligonucleotide is for reducing the expression of the SNCA gene, and hybridizes with an antisense strand: 5’-[MePhosphonate-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’ and a sense strand: 5’-[AdemX-C 16 -S-mX-fX-mX-fX-mX-mX-fX-mX-fX-mX-fX-fX-mX-fX-mX-fX-mX-S-mX-S-mX-3’, where mX = 2’-OMe modified nucleotide, fX = 2’-F modified nucleotide, -= phosphodiester bond, [MePhosphonate-4O-mX] = 4’-O-monomethylphosphonate-2’-O-methyl modified nucleotide, and ademX-C 16 = C attached to a nucleotide 16 is a hydrocarbon chain.

[0233] In some embodiments, the oligonucleotide is for reducing the expression of the SNCA gene, a) SEQ ID NOs: 1607 and 1642 respectively; b) SEQ ID NOs: 1608 and 1643 respectively; c) SEQ ID NOs: 1609 and 1644 respectively; d) SEQ ID NOs: 1610 and 1645 respectively; e) SEQ ID NOs: 1611 and 1646 respectively; f) SEQ ID NOs: 1612 and 1647 respectively; g) SEQ ID NOs: 1613 and 1648 respectively; h) SEQ ID NOs: 1614 and 1649 respectively; i) SEQ ID NOs: 1615 and 1650 respectively; j) SEQ ID NOs: 1616 and 1651 respectively; k) SEQ ID NOs: 1617 and 1652 respectively; l) SEQ ID NOs: 1618 and 1653 respectively; m) SEQ ID NOs: 1619 and 1654 respectively; n) SEQ ID NOs: 1620 and 1655, respectively; o) SEQ ID NOs: 1621 and 1656, respectively; p) SEQ ID NOs: 1622 and 1657, respectively; q) SEQ ID NOs: 1623 and 1658, respectively; r) SEQ ID NOs: 1624 and 1659, respectively; s) SEQ ID NOs: 1625 and 1660, respectively; t) SEQ ID NOs: 1626 and 1661, respectively; u) SEQ ID NOs: 1627 and 1662, respectively; v) SEQ ID NOs: 1628 and 1663, respectively; w) SEQ ID NOs: 1629 and 1664, respectively; x) SEQ ID NOs: 1630 and 1665, respectively; y) SEQ ID NOs: 1631 and 1666, respectively; z) SEQ ID NOs: 1632 and 1667, respectively; aa) SEQ ID NOs: 1633 and 1668, respectively; bb) SEQ ID NOs: 1634 and 1669, respectively; cc) SEQ ID NOs: 1635 and 1670, respectively; dd) SEQ ID NOs: 1636 and 1671, respectively; ee) SEQ ID NOs: 1637 and 1672, respectively; ff) SEQ ID NOs: 1638 and 1673, respectively; gg) SEQ ID NOs: 1639 and 1674, respectively; hh) SEQ ID NOs: 1640 and 1675, respectively; ii) SEQ ID NOs: 1641 and 1676, respectively; and jj) comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1682 and 1656, and comprising a sense strand and an antisense strand.

[0234] In some embodiments, the oligonucleotide is for reducing the expression of the SNCA gene, a) SEQ ID NOs: 1610 and 1645, respectively; b) SEQ ID NOs: 1614 and 1649, respectively; c) SEQ ID NOs: 1616 and 1651, respectively; d) SEQ ID NOs: 1621 and 1656, respectively; e) SEQ ID NOs: 1622 and 1657, respectively; f) SEQ ID NOs: 1623 and 1658, respectively; g) SEQ ID NOs: 1629 and 1664, respectively; h) SEQ ID NOs: 1630 and 1665, respectively; i) SEQ ID NOs: 1634 and 1669, respectively; j) SEQ ID NOs: 1635 and 1670, respectively; k) SEQ ID NOs: 1636 and 1671, respectively; l) SEQ ID NOs: 1640 and 1675, respectively; and m) comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1682 and 1656, respectively, and comprising a sense strand and an antisense strand.

[0235] In some embodiments, the oligonucleotide is for reducing the expression of the SNCA gene, a) SEQ ID NOs: 1623 and 1658, respectively; b) SEQ ID NOs: 1630 and 1665, respectively; c) SEQ ID NOs: 1634 and 1669, respectively; d) SEQ ID NOs: 1621 and 1656, respectively; e) SEQ ID NOs: 1640 and 1675, respectively, and, f) comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1682 and 1656, respectively, and comprising a sense strand and an antisense strand.

[0236] In some embodiments, the oligonucleotide is for reducing the expression of the SNCA gene 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 for reducing the expression of the SNCA gene 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 for reducing the expression of the SNCA gene 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 for reducing the expression of the SNCA gene 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 for reducing the expression of the SNCA gene and comprises a sense strand comprising the nucleotide sequence set forth in SEQ ID NO: 1640 and an antisense strand comprising the nucleotide sequence set forth in SEQ ID NO: 1675. In some embodiments, the oligonucleotide is for reducing the expression of the SNCA gene and comprises a sense strand comprising the nucleotide sequence set forth in SEQ ID NO: 1682 and an antisense strand comprising the nucleotide sequence set forth in SEQ ID NO: 1656.

[0237] Formulation Various formulations have been developed to facilitate the use of oligonucleotides. For example, an oligonucleotide (e.g., an RNAi oligonucleotide) can be delivered to a subject or a cellular environment using a formulation that minimizes degradation, promotes delivery and / or uptake, or confers another beneficial property to the oligonucleotide in the formulation. In some embodiments, the formulation is a composition comprising an oligonucleotide that reduces the expression of the SNCA gene. Such a composition can be suitably formulated such that when administered to the subject either locally adjacent to the target cells or systemically, a sufficient portion of the oligonucleotide enters the cells to reduce the expression of the SNCA gene. Any of a variety of suitable oligonucleotide formulations can be used to deliver an oligonucleotide for reducing the expression of the SNCA gene. In some embodiments, the oligonucleotide is formulated in a buffer such as phosphate buffered saline, liposomes, micellar structures, and capsids. In some embodiments, the oligonucleotide is formulated in a buffer solution such as phosphate buffered saline.

[0238] The use of a formulation of an oligonucleotide containing a cationic lipid can promote the transfection of the oligonucleotide 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 FuGene6 (Roche), all of which can be used according to the manufacturer's instructions. In some embodiments, the oligonucleotide is not formulated with a component that promotes transfection into cells.

[0239] Thus, in some embodiments, the formulation comprises lipid nanoparticles. In some embodiments, the lipid nanoparticles comprise liposomes, lipids, complex lipids, microspheres, microparticles, nanospheres, or nanoparticles, or may be formulated in other forms for administration to the cells, tissues, organs, or body of a subject in need thereof (see, e.g., Remington: The Science and Practice of Pharmacy, 22nd ed, Pharmaceutical Press, 2013).

[0240] In some embodiments, the formulation comprises excipients, which impart to the composition improved stability, improved absorbability, improved solubility, and / or a therapeutic enhancing effect of the active ingredient. In some embodiments, the excipient is a buffer (e.g., sodium citrate, sodium phosphate, tris base, or sodium hydroxide), or a vehicle (e.g., buffer solution, petrolatum, dimethyl sulfoxide, or mineral oil). In some embodiments, the oligonucleotide is lyophilized to extend its shelf life and then made into a solution before use (e.g., administration to a subject). Thus, the excipient may be a lyoprotectant (e.g., mannitol, lactose, polyethylene glycol, or polyvinylpyrrolidone), or a disintegration temperature regulator (e.g., dextran, Ficoll™, or gelatin).

[0241] In some embodiments, the formulation is a pharmaceutical composition adapted to its intended route of administration. Examples of routes of administration include, but are not limited to, parenteral (e.g., intravenous, intramuscular, intraperitoneal, intradermal, and subcutaneous), oral (e.g., inhalation), transdermal (e.g., topical), transmucosal, and rectal administration.

[0242] In some embodiments, the formulation is formulated for administration to the CNS. In some embodiments, the formulation is formulated for administration to 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 administration into the cerebral ventricle. In some embodiments, the formulation is formulated for the brainstem. In some embodiments, the formulation is formulated for intracisternal administration.

[0243] Pharmaceutical compositions suitable for injection use include sterile aqueous solutions (if water-soluble) or dispersions, and sterile powders for the immediate preparation of sterile injection solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. In many cases, it will be preferable to include in the composition isotonic agents such as sugars, polyhydric alcohols such as mannitol and sorbitol, and sodium chloride. A sterile injection solution can be prepared by incorporating the oligonucleotide in the required amount in the selected solvent, together with one or a combination of the ingredients listed above as appropriate, followed by sterile filtration.

[0244] In some embodiments, the proportion of the active ingredient(s) may be about 1% to about 80% or more by weight or volume of the total composition, but the formulation may contain at least about 0.1% or more of the oligonucleotide. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations are contemplated by those skilled in the art in the field of preparation of such pharmaceutical formulations, and thus various dosages and therapeutic dosing regimens may be desirable.

[0245] Method of Use Reduction of SNCA expression In some embodiments, the method of contacting or delivering to a cell or population of cells comprises administering an effective amount of an oligonucleotide (e.g., an RNAi oligonucleotide) to reduce the expression of the SNCA gene. In some embodiments, the reduction of SNCA gene expression is determined by measuring a decrease in the amount or level of SNCA mRNA, SNCA protein, SNCA activity, or combinations thereof in the cell. Methods include those described herein and known to those of skill in the art.

[0246] In some embodiments, a method of reducing SNCA gene expression in the CNS involves administering an effective amount of an oligonucleotide (e.g., an RNAi oligonucleotide) to reduce the expression of the SNCA gene. In some embodiments, the CNS includes the brain and spinal cord. In some embodiments, the expression of the SNCA gene is reduced in at least one region of the brain, 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, the expression of the SNCA gene is reduced in at least one region of the spinal cord, including, but not limited to, the cervical spinal cord, thoracic spinal cord, and lumbar spinal cord. In some embodiments, the expression of the SNCA gene is reduced in at least one region of the brain and at least one region of the spinal cord. In some aspects, the expression of the SNCA gene is reduced in at least one of the cervical spinal cord, thoracic spinal cord, lumbar spinal cord, frontal cortex, temporal cortex, cerebellum, midbrain, occipital cortex, parietal cortex, hippocampus, caudate nucleus, thalamus, brainstem, motor cortex, globus pallidus, tegmentum, substantia nigra, pons, cerebellar white matter, and dentate nucleus of the cerebellum. In some embodiments, the expression of the SNCA gene is reduced in at least one of the lumbar spinal cord, thoracic spinal cord, and cervical spinal cord. In some embodiments, the expression of the SNCA gene is reduced in brain and / or spinal cord tissue associated with Parkinson's disease. In some embodiments, tissues associated with Parkinson's disease include, but are not limited to, the putamen, tegmentum, substantia nigra, pons, and medulla. In some embodiments, the expression of the SNCA gene is reduced in brain and / or spinal cord tissue associated with multiple system atrophy. In some embodiments, tissues associated with Parkinson's disease include, but are not limited to, the caudate nucleus, putamen, tegmentum, substantia nigra, pons, cerebellar cortex, cerebellar white matter, medulla, cervical spinal cord, thoracic spinal cord, and lumbar spinal cord.

[0247] In some embodiments, the expression of the SNCA gene decreases over a period of about one week to twelve weeks after administration of the oligonucleotide or a formulation containing the same. In some embodiments, the expression of the SNCA gene decreases 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 expression of the SNCA gene decreases for about one month to about four months after administration of the oligonucleotide or formulation. In some embodiments, the expression of the SNCA gene decreases for about one month to about six months after administration of the oligonucleotide or formulation. In some embodiments, the expression of the SNCA gene decreases for 1, 2, 3, or 4 months after administration of the oligonucleotide or formulation. In some embodiments, the expression of the SNCA gene decreases for 1, 2, 3, 4, 5, or 6 months after administration of the oligonucleotide or formulation. In some embodiments, the expression of the SNCA gene decreases for about seven days to about ninety-one days after administration of the oligonucleotide or formulation. In some embodiments, the expression of the SNCA gene decreases for 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, or 91 days after administration of the oligonucleotide or formulation.

[0248] In some embodiments, the expression of the SNCA gene decreases in at least one region of the brain and / or at least one region of the spinal cord for about 1 to about 12 weeks after administration of the oligonucleotide or formulation. In some embodiments, the expression of the SNCA gene decreases in at least one region of the brain and / or at least one region of the spinal cord 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 expression of the SNCA gene decreases in at least one region of the brain and / or at least one region of the spinal cord for about 1 to about 4 months after administration of the oligonucleotide or formulation. In some embodiments, the expression of the SNCA gene decreases in at least one region of the brain and / or at least one region of the spinal cord for about 1 month to about 6 months after administration of the oligonucleotide or formulation. In some embodiments, the expression of the SNCA gene decreases in at least one region of the brain and / or at least one region of the spinal cord 1, 2, 3, or 4 months after administration of the oligonucleotide or formulation. In some embodiments, the expression of the SNCA gene decreases in at least one region of the brain and / or at least one region of the spinal cord 1, 2, 3, 4, 5, or 6 months after administration of the oligonucleotide or formulation. In some embodiments, the expression of the SNCA gene decreases in at least one region of the brain and / or at least one region of the spinal cord for about 7 days to about 91 days after administration of the oligonucleotide or formulation. In some embodiments, the expression of the SNCA gene decreases in at least one region of the brain and / or at least one region of the spinal cord 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, or 91 days after administration of the oligonucleotides described herein.

[0249] The methods of the present specification are useful in any suitable cell type. In some embodiments, the cell type is any cell that expresses SNCA mRNA (e.g., oligodendrocytes). In some embodiments, the cell type is primary cells obtained from a subject. In some embodiments, the primary cells have undergone a limited number of passages such that the cells maintain substantially their native phenotypic characteristics. In some embodiments, the cells to which the oligonucleotide is delivered are ex vivo or in vitro (i.e., cells in culture or can be delivered to an organism in which the cells are present).

[0250] In some embodiments, the oligonucleotide is delivered to a cell or cell population using nucleic acid delivery methods known in the art, including, but not limited to, injection of a solution or pharmaceutical composition (i.e., formulation) containing the oligonucleotide, particle bombardment with particles coated with the oligonucleotide, exposure of a cell or cell population 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 oligonucleotides to cells, such as lipid-mediated carrier transport, chemical-mediated transport, cationic liposome transfection such as calcium phosphate, may also be used.

[0251] In some embodiments, the reduction in the expression of the SNCA gene is determined by an assay or technique that evaluates one or more molecules, properties, or characteristics of a cell or cell population associated with the expression of the SNCA gene, or by an assay or technique that evaluates a molecule (e.g., SNCA mRNA or SNCA protein) that directly indicates the expression of the SNCA gene in a cell or cell population. In some embodiments, the degree to which an oligonucleotide reduces the expression of the SNCA gene is evaluated by comparing the expression of the SNCA gene in a cell or cell population contacted with the oligonucleotide to that in a control cell or cell population (e.g., a cell or cell population not contacted with the oligonucleotide or contacted with a control oligonucleotide). In some embodiments, since the control amount or level of the expression of the SNCA gene in the control cell or cell population is predetermined, it is not necessary to measure the control amount or level in every case where the assay or technique is performed. The predetermined level or value can be in various forms. In some embodiments, the predetermined level or value can be a single cutoff value such as a median or an average value.

[0252] In some embodiments, a decrease in the expression of the SNCA gene is effected by contacting or delivering an oligonucleotide to a cell or cell population. In some embodiments, the decrease in the expression of the SNCA gene is relative to a control amount or level of the expression of the SNCA gene in a cell or cell population that is not contacted with the oligonucleotide or is contacted with a control oligonucleotide. In some embodiments, the decrease in the expression of the SNCA gene 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 compared to the control amount or level of the expression of the SNCA gene. In some embodiments, the control amount or level of the expression of the SNCA gene is the amount or level of SNCA mRNA and / or SNCA protein and / or SNCA activity / function in a cell or cell population that is not in contact with the oligonucleotide. In some embodiments, the effect of the delivery of the oligonucleotide to a cell or cell population by the methods herein is evaluated after any finite period or time (e.g., minutes, hours, days, weeks, months). For example, in some embodiments, after contacting or delivering the oligonucleotide to 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, about 84 days, or more have elapsed, the expression of the SNCA gene is measured in the cell or cell population. In some embodiments, the expression of the SNCA gene is measured in the cell or cell population at least about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, or more after contacting or delivering the oligonucleotide to the cell or cell population.

[0253] In some embodiments, the oligonucleotide is delivered in the form of a transgene engineered to express the oligonucleotide or the strand(s) that make up the oligonucleotide (e.g., its sense and antisense strands) intracellularly. In some embodiments, the oligonucleotide is delivered using a transgene engineered to express any oligonucleotide. The transgene may be delivered using a viral vector (e.g., an adenovirus, retrovirus, vaccinia virus, poxvirus, adeno-associated virus, or herpes simplex virus), or a non-viral vector (e.g., a plasmid or synthetic mRNA). In some embodiments, the transgene can be directly injected into the subject.

[0254] Method of treatment Also provided are oligonucleotides (e.g., RNAi oligonucleotides) for use in, or adapted for use in, treating a subject (e.g., a human having a disease, disorder, or condition associated with SNCA gene expression) who would benefit from a reduction in SNCA gene expression. In some aspects, the disclosure provides oligonucleotides for use in, or adapted for use in, treating a subject having a disease, disorder, or condition associated with the expression of SNCA. Also provided are oligonucleotides for use in, or adapted for use in, the manufacture of a drug or formulation / pharmaceutical composition for treating a disease, disorder, or condition associated with SNCA gene expression. In some embodiments, the oligonucleotide for use in, or adapted for use in, reduces the expression of the SNCA gene by targeting SNCA mRNA (e.g., via the RNAi pathway). In some embodiments, the oligonucleotide for use in, or adapted for use in, reduces the amount or level of SNCA mRNA, SNCA protein, and / or SNCA activity / function by targeting SNCA mRNA.

[0255] Furthermore, in some embodiments of the methods herein, a subject having or susceptible to a disease, disorder, or condition associated with the expression of SNCA is selected for treatment with an oligonucleotide or formulation. In some embodiments, the method includes selecting an individual having a marker (e.g., a biomarker) for a disease, disorder, or condition associated with the expression of the SNCA gene, such as, but not limited to, SNCA mRNA, SNCA protein, SNCA activity / function, or combinations thereof, or an individual having a predisposition thereto. Similarly, as detailed below, some embodiments of the method measure or obtain a baseline value of a marker of SNCA gene expression and then compare such obtained value to one or more other baseline values or values obtained after administration of an oligonucleotide to the subject to evaluate the effectiveness of the treatment.

[0256] Methods are provided herein for treating a subject having, suspected of having, or at risk of developing a disease, disorder, or condition associated with SNCA gene expression with an oligonucleotide or formulation. In some aspects, methods are provided herein for treating or attenuating the onset or progression of a disease, disorder, or condition associated with SNCA gene expression using an oligonucleotide or formulation. In other aspects, methods are provided herein for achieving one or more therapeutic benefits in a subject having a disease, disorder, or condition associated with SNCA gene expression using an oligonucleotide or formulation. 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, the treatment includes reducing the expression of the SNCA gene. In some embodiments, the individual is treated therapeutically. In other embodiments, the individual is treated prophylactically.

[0257] In some embodiments of the methods herein, an oligonucleotide, or a pharmaceutical composition comprising an oligonucleotide, is administered to a subject having a disease, disorder or condition associated with the expression of SNCA such that the expression of SNCA is reduced in the subject, thereby treating the subject. In some embodiments, the amount or level of SNCA mRNA is reduced in the subject. In some embodiments, the amount or level of SNCA protein is reduced in the subject. In other embodiments, the amount or level of the activity / function of SNCA is reduced in the subject.

[0258] In some embodiments, an oligonucleotide or a pharmaceutical composition comprising an oligonucleotide is administered to a subject having a disease, disorder, or condition associated with the expression of the SNCA gene such that the expression of SNCA is reduced in the subject by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or more than 99% compared to the expression of SNCA before administration of the oligonucleotide or pharmaceutical composition. In some embodiments of the methods herein, an oligonucleotide or a pharmaceutical composition comprising an oligonucleotide is administered to a subject having a disease, disorder, or condition associated with the expression of the SNCA gene such that the expression of SNCA is reduced in the subject by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or more than 99% over a period of about 1 week to about 12 weeks, about 1 month to about 6 months, or about 7 days to about 91 days compared to the expression of SNCA before administration of the oligonucleotide or pharmaceutical composition. In some embodiments, the expression of SNCA is reduced in the subject by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or more than 99% compared to the expression of SNCA in a subject not administered the oligonucleotide or pharmaceutical composition or a subject receiving a control oligonucleotide, pharmaceutical composition, or treatment (e.g., a reference subject or control subject). In some embodiments, the expression of SNCA is reduced in the subject by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or more than 99% over a period of about 1 week to about 12 weeks, about 1 month to about 6 months, or about 7 days to about 91 days compared to the expression of SNCA in a subject not administered the oligonucleotide or pharmaceutical composition or a subject receiving a control oligonucleotide, pharmaceutical composition, or treatment (e.g., a reference subject or control subject).

[0259] In some embodiments of the methods herein, the amount or level of SNCA mRNA is 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 more than 99% lower in a subject having a disease, disorder, or condition associated with SNCA gene expression, compared to the amount or level of SNCA mRNA prior to administration of the oligonucleotide or pharmaceutical composition. In some embodiments of the methods, the amount or level of SNCA mRNA is 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 more than 99% lower in a subject having a disease, disorder, or condition associated with SNCA gene expression, compared to the amount or level of SNCA mRNA prior to administration of the oligonucleotide or pharmaceutical composition, over a period of about 1 week to about 12 weeks, about 1 month to about 6 months, or about 7 days to about 91 days in the subject. In some embodiments, the amount or level of SNCA mRNA is 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 more than 99% lower in a subject compared to the amount or level of SNCA mRNA in a subject not administered the oligonucleotide or pharmaceutical composition, or a subject receiving a control oligonucleotide, pharmaceutical composition, or treatment (reference or control subject). In some embodiments, the amount or level of SNCA mRNA is 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 more than 99% lower in a subject over a period of about 1 week to about 12 weeks, about 1 month to about 6 months, or about 7 days to about 91 days, compared to the amount or level of SNCA mRNA in a subject not administered the oligonucleotide or pharmaceutical composition, or a subject receiving a control oligonucleotide, pharmaceutical composition, or treatment (reference subject or control subject).

[0260] In some embodiments of the method, 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 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 more than 99% compared to the amount or level of SNCA protein before 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 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 more than 99% over a period of about 1 week to about 12 weeks, about 1 month to about 6 months, or about 7 days to about 91 days compared to the amount or level of SNCA protein before administration of the oligonucleotide or pharmaceutical composition. In some embodiments, 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 more than 99% compared to the amount or level of SNCA protein in a subject not administered the oligonucleotide or pharmaceutical composition or a subject receiving a control oligonucleotide, pharmaceutical composition, or treatment (reference subject or control subject). In some embodiments, 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 more than 99% over a period of about 1 week to about 12 weeks, about 1 month to about 6 months, or about 7 days to about 91 days compared to the amount or level of SNCA protein in a subject not administered the oligonucleotide or pharmaceutical composition or a subject receiving a control oligonucleotide, pharmaceutical composition, or treatment (e.g., reference subject or control subject).

[0261] 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 more than 99% 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 more than 99% over a period of about 1 week to about 12 weeks, about 1 month to about 6 months, or about 7 days to about 91 days 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 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 more than 99% in the subject compared to the amount or level of SNCA activity / function in a subject not administered the oligonucleotide or pharmaceutical composition or a subject receiving a control oligonucleotide, pharmaceutical composition, or treatment (e.g., a reference subject or control subject). In some embodiments, the amount or level of SNCA activity / function 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 more than 99% in the subject over a period of about 1 week to about 12 weeks, about 1 month to about 6 months, or about 7 days to about 91 days compared to the amount or level of SNCA activity / function in a subject not administered the oligonucleotide or pharmaceutical composition or a subject receiving a control oligonucleotide, pharmaceutical composition, or treatment (reference subject or control subject).

[0262] Suitable methods for determining the expression of the SNCA gene, such as the expression of SNCA, 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 or from a sample of a subject are known in the art. Further, the examples presented herein illustrate exemplary methods for determining the expression of the SNCA gene.

[0263] In some embodiments, the expression of SNCA, 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, such as the expression of the SNCA gene, is reduced in a cell (e.g., an oligodendrocyte), a cell population or group of cells (e.g., an organoid), an organ (e.g., the prefrontal cortex), blood or a fraction thereof (e.g., plasma), a tissue (e.g., brain tissue), a sample (e.g., a brain biopsy sample), or other biological material obtained or isolated from a subject. In some embodiments, the expression of SNCA, 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 multiple cell types (e.g., oligodendrocytes and one or more other types of cells), multiple cell populations, multiple organs (e.g., the brain and one or more other organs), multiple blood fractions (e.g., plasma and one or more other blood fractions), multiple types of tissue (e.g., brain tissue and one or more other types of tissue), multiple types of samples (e.g., a brain biopsy sample and one or more other types of biopsy samples obtained or isolated from a subject). In some embodiments, the expression of SNCA, 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 one or more of the cervical spinal cord, thoracic spinal cord, lumbar spinal cord, prefrontal 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, and dentate nucleus of the cerebellum. In some embodiments, the expression of SNCA, 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 tissues of the brain and / or spinal cord associated with Parkinson's disease. In some embodiments, tissues associated with Parkinson's disease include, but are not limited to, the putamen, tegmentum, substantia nigra, pons, and medulla.In some embodiments, the expression of SNCA, 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 decreased 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.

[0264] Examples of diseases, disorders or conditions associated with the expression of the SNCA gene include multiple system atrophy, Lewy body dementia, and Parkinson's disease.

[0265] Due to its high specificity, the oligonucleotides herein specifically target the SNCA mRNA of the target gene in cells, tissue(s), or organ(s) (e.g., the brain). In disease prevention, the target gene may be one that is required for the onset or maintenance of the disease, or one that has been identified as being associated with a high risk of developing the disease. In disease treatment, the oligonucleotide can be contacted with cells, tissue(s), or organ(s) (e.g., the brain) that exhibit the disease or are involved in mediating the disease. For example, an oligonucleotide that is substantially identical to all or part of the wild-type (i.e., native) or mutant gene associated with a disease, disorder or condition associated with the expression of the SNCA gene can be contacted with or introduced into a target cell type or tissue type, such as oligodendrocytes or other brain cells.

[0266] In some embodiments, SNCA may be derived from any mammal, such as a human, and may be silenced according to the methods described herein.

[0267] The methods of this specification typically involve administering to a subject a therapeutically effective amount of an oligonucleotide, i.e., an amount capable of producing a desired therapeutic result. A therapeutically tolerable amount may be an amount capable of treating a disease, disorder or condition. The appropriate amount / dosage for any one subject will depend on specific factors including the size of the subject, body surface area, age, the particular composition being administered, the active ingredient(s) in the composition, the time and route of administration, general health status, and other drugs being administered concurrently.

[0268] In some embodiments, a subject is administered any one of the oligonucleotides or compositions of this specification enterally (e.g., orally, by a gastrostomy tube, by a duodenal feeding tube, via a gastric fistula, or rectally), parenterally (e.g., by subcutaneous injection, intravenous injection or infusion, intra-arterial injection or infusion, intramedullary infusion, intramuscular injection, intracerebral injection, intraventricular injection, or intrathecal injection), topically (e.g., transdermally, by inhalation, via eye drops, or via mucosa), or by direct injection into a target organ (e.g., the brain of the subject). Usually, the oligonucleotide or composition is administered intravenously or subcutaneously. In some embodiments, the oligonucleotide or composition is administered into the cerebrospinal fluid. In some embodiments, the oligonucleotide or composition is administered intrathecally. In some embodiments, the oligonucleotide or composition is administered intraventricularly. In some embodiments, the oligonucleotide or composition is administered by intracisternal infusion.

[0269] As a non-limiting set of examples, oligonucleotides are typically administered quarterly (once every 3 months), bi-monthly (once every 2 months), monthly or weekly. For example, the oligonucleotide may be administered weekly, or at intervals of 2 or 3 weeks. Alternatively, the oligonucleotide may be administered daily. In some embodiments, a subject is administered one or more maintenance doses of the oligonucleotide following one or more loading doses of the oligonucleotide.

[0270] In some embodiments, the subject to be treated is a human or non-human primate or other mammalian subject. Other exemplary subjects include companion animals such as dogs and cats; livestock such as horses, cows, pigs, sheep, goats, and chickens; and animals such as mice, rats, guinea pigs, and hamsters.

[0271] Kit In some embodiments, a kit is provided that includes the oligonucleotides described herein (e.g., RNAi oligonucleotides) and instructions for use. In some embodiments, the kit includes an oligonucleotide and an accompanying document containing instructions for use of the kit and / or any of its components. In some embodiments, the kit includes the oligonucleotide, one or more controls, various buffers, reagents, enzymes, and other standard components well known in the art, within a suitable container. In some embodiments, the container includes at least one vial, well, test tube, flask, bottle, syringe, or other container means into which the oligonucleotide is placed and optionally dispensed appropriately. In some embodiments where additional components are provided, the kit contains an additional container into which the component is placed. The kit can also include means for hermetically containing the oligonucleotide and other reagents for commercial sale. Such containers may include injection-molded or blow-molded plastic containers that hold the desired vials. The container and / or the kit can include a label with instructions and / or warnings.

[0272] In some embodiments, the kit includes an oligonucleotide and a pharmaceutically acceptable carrier or a pharmaceutical composition, and instructions for treating or delaying the progression of a disease, disorder, or condition associated with the expression of the SNCA gene in a subject in need thereof.

[0273] In some embodiments, the kit comprises an oligonucleotide, a pharmaceutically acceptable carrier or a pharmaceutical composition, and instructions for administering the oligonucleotide or pharmaceutical composition into the cerebrospinal fluid to reduce the expression of the SNCA gene in at least one region of the brain and / or at least one region of the spinal cord in a subject in need thereof.

[0274] Definitions As used herein, "about" or "approximately" as applied to one or more values of interest refers to a value similar to the recited reference value. In certain embodiments, "about" means within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise apparent from the content (except where such number would exceed 100% of a possible value).

[0275] As used herein, "administer", "administering", "administration", etc. refer to providing a substance (e.g., an oligonucleotide) to a subject in a pharmacologically useful manner (e.g., to treat a disease, disorder or condition in the subject).

[0276] As used herein, "asialoglycoprotein receptor" or "ASGPR" refers to a dimeric C-type lectin formed by a 48 kDa major subunit (ASGPR-1) and a 40 kDa non-major subunit (ASGPR-2). ASGPR is mainly expressed on the sinusoidal surface of hepatocytes and plays a major role in the binding, internalization, and subsequent excretion of circulating glycoproteins (asialoglycoproteins) containing terminal galactose or GalNAc residues.

[0277] As used herein, "attenuate", "attenuating", "attenuation", etc. refer to weakening or effectively halting. By way of non-limiting example, one or more of the treatments herein may attenuate or effectively halt the onset or progression of a disease, disorder or condition associated with the expression of the SNCA gene in a subject. Such attenuation may be exemplified, for example, by a decrease in one or more aspects of a disease associated with SNCA gene expression (such as symptoms, tissue characteristics, and cellular inflammation or immune activity, etc.), the absence of a detectable progression (worsening) of one or more aspects of the disease, disorder or condition, or the non-detection in a subject of a detectable aspect of the disease that would otherwise be expected to be detected.

[0278] As used herein, "complementarity" refers to the structural relationship between two nucleotides that enables (for example, on two opposing nucleic acids or on opposing regions of a single nucleic acid strand) the two nucleotides to form base pairs with each other. For example, a purine nucleotide of one nucleic acid that is complementary to a pyrimidine nucleotide of an opposing nucleic acid may base pair together by forming hydrogen bonds with each other. In some embodiments, complementary nucleotides can base pair in a Watson-Crick manner or in any other manner that enables the formation of a stable double strand. In some embodiments, two nucleic acids may form a complementary region having regions of a plurality of nucleotides that are complementary to each other as described herein.

[0279] As used herein, "deoxyribonucleotide" refers to a nucleotide that has a hydrogen in place of a hydroxyl at the 2'-position of its pentose sugar as compared to a ribonucleotide. A modified deoxyribonucleotide is a deoxyribonucleotide having one or more modifications or substitutions at atoms other than the 2'-position and includes modifications or substitutions of the sugar, phosphate group or base.

[0280] As used herein, "double-stranded oligonucleotide" or "ds oligonucleotide" refers to an oligonucleotide that is substantially in a double-stranded form. In some embodiments, the complementary base pairing of the double-stranded region(s) of the ds oligonucleotide is formed between the antiparallel sequences of nucleotides of covalently separated nucleic acid strands. In some embodiments, the complementary base pairing of the double-stranded region(s) of the ds oligonucleotide is formed between the antiparallel sequences of nucleotides of covalently linked nucleic acid strands. In some embodiments, the complementary base pairing of the double-stranded region(s) of the ds oligonucleotide is formed from a single nucleic acid strand that is folded (e.g., via a hairpin) to provide the complementary antiparallel sequences of nucleotides that base pair together. In some embodiments, the ds oligonucleotide comprises two covalently separated nucleic acid strands that are fully double-stranded with each other. However, in other embodiments, the ds oligonucleotide comprises two covalently separated nucleic acid strands that are partially double-stranded (e.g., having overhangs at one or both ends). In some embodiments, the ds oligonucleotide may have one or more mismatches, including internal or terminal mismatches, since it comprises antiparallel sequences of nucleotides that are partially complementary.

[0281] As used herein, with respect to a nucleic acid (e.g., an oligonucleotide), "double-stranded" refers to a structure formed through complementary base pairing of two antiparallel sequences of nucleotides.

[0282] As used herein, "excipient" refers to a non-therapeutic agent that may be included in a composition, for example, to provide or contribute to a desired concentration or stabilizing effect.

[0283] As used herein, "labile linker" refers to a linker that can be cleaved (e.g., by an acidic pH). "Fairly stable linker" refers to a linker that cannot be cleaved.

[0284] As used herein, a "loop" refers to the unpaired region of a nucleic acid (e.g., an oligonucleotide) that is flanked by two anti-parallel regions of nucleic acids that are sufficiently complementary to each other such that under appropriate hybridization conditions (e.g., in a phosphate buffer solution, intracellularly), the two anti-parallel regions flanking the unpaired region hybridize to form a double strand (referred to as a "stem").

[0285] As used herein, a "modified internucleotide linkage" refers to an internucleotide linkage having one or more chemical modifications as compared to a reference internucleotide linkage that includes a phosphodiester linkage. In some embodiments, the modified nucleotide is a linkage that does not occur in nature. Typically, a modified internucleotide linkage imparts one or more desirable properties to a nucleic acid in which the modified internucleotide linkage is present. For example, the modified nucleotide may improve thermal stability, resistance to degradation, nuclease resistance, solubility, bioavailability, biological activity, reduction of immunogenicity, etc.

[0286] As used herein, a "modified nucleotide" refers to a nucleotide having one or more chemical modifications as compared to the corresponding reference nucleotide selected from adenosine ribonucleotide, guanosine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, adenosine deoxyribonucleotide, guanosine deoxyribonucleotide, cytosine deoxyribonucleotide, and thymidine deoxyribonucleotide. In some embodiments, the modified nucleotide is a nucleotide that does not occur in nature. In some embodiments, the modified nucleotide has one or more chemical modifications to its sugar, nucleobase, and / or phosphate group. In some embodiments, the modified nucleotide has one or more chemical moieties attached to the corresponding reference nucleotide. Typically, a modified nucleotide imparts one or more desirable properties to a nucleic acid in which the modified nucleotide is present. For example, the modified nucleotide may improve thermal stability, resistance to degradation, nuclease resistance, solubility, bioavailability, biological activity, reduction of immunogenicity, etc.

[0287] As used herein, the term "tetraloop structure with a nick" or "tetral L structure with a nick" refers to the structure of an oligonucleotide (RNAi oligonucleotide) characterized in that the sense (passenger) strand and the antisense (guide) strand are separate, the sense strand has a region complementary to the antisense strand, and at least one of the strands, generally the sense strand, has a tetral L configured to stabilize adjacent stem regions formed within at least one strand.

[0288] As used herein, the term "oligonucleotide" refers to a short nucleic acid (e.g., less than about 100 nucleotides in length). The oligonucleotide may be ss or ds. The oligonucleotide may or may not have a double-stranded region. By way of a non-limiting series of examples, the oligonucleotide may be, but is not limited to, small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), dicer substrate interfering RNA (dsiRNA), antisense oligonucleotide (ASO), short siRNA, or ss siRNA. In some embodiments, the oligonucleotide is a ds oligonucleotide and is an RNAi oligonucleotide.

[0289] As used herein, the term "overhang" refers to the terminal base pair non-forming nucleotide(s) resulting from one strand or region extending beyond the end of the complementary strand with which it forms a double strand. In some embodiments, the overhang comprises one or more non-pairing nucleotides extending from the double-stranded region at the 5' or 3' end of the ds oligonucleotide. In certain embodiments, the overhang is a 3' or 5' overhang on the antisense or sense strand of the ds oligonucleotide.

[0290] As used herein, "phosphate analog" refers to a chemical moiety that mimics the electrostatic and / or steric properties of a phosphate group. In some embodiments, the phosphate analog is placed at the 5'-terminal nucleotide of the oligonucleotide instead of the 5'-phosphate, which is often susceptible to enzymatic removal. In some embodiments, the 5'-phosphate analog contains phosphatase-resistant linkages. Examples of phosphate analogs include, but are not limited to, 5'-phosphonates such as 5'-methylenephosphonate (5'-MP) and 5'-(E)-vinylphosphonate (5'-VP). In some embodiments, the oligonucleotide has a phosphate analog (referred to as a "4'-phosphate analog") at the 4'-carbon position of the sugar of the 5'-terminal nucleotide. An example of a 4'-phosphate analog is oxyethylmethylphosphonate or an analog thereof in which the oxygen atom of the oxyethyl group is attached to the sugar moiety (e.g., its 4'-carbon). See, e.g., U.S. Provisional Patent Application Nos. 62 / 383,207 (filed Sep. 2, 2016) and 62 / 393,401 (filed Sep. 12, 2016). Other modifications to the 5'-terminus of oligonucleotides have been developed (see, e.g., International Patent Application No. WO2011 / 133871; U.S. Patent No. 8,927,513; and Prakash et al. (2015), Nucleic Acids Res. 43:2993-3011).

[0291] As used herein, "SNCA" refers to synuclein alpha. SNCA is abundant in the brain and inhibits phospholipase D2. It functions in resynaptic 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.

[0292] As used herein, "reduced expression" of a gene (e.g., SNCA) refers to a decrease in the amount or level of the RNA transcript (e.g., SNCA mRNA) or protein encoded by that gene, and / or a decrease in the amount or level of the activity / function of that gene, in a cell, cell population, sample, or subject, 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 such as an RNAi oligonucleotide comprising an antisense strand having a nucleotide sequence complementary to the nucleotide sequence that makes up SNCA mRNA) may result in a decrease in the amount or level of SNCA mRNA, SNCA protein, and / or SNCA activity / function (e.g., via inactivation and / or degradation of SNCA mRNA by the RNAi pathway) compared to a cell not treated with the oligonucleotide. Similarly, and as used herein, "decrease in expression" refers to an act that results in a decrease in the expression of a gene (e.g., SNCA).

[0293] As used herein, "reduction of SNCA gene expression" refers to a decrease in the amount or level of SNCA mRNA, SNCA protein, and / or SNCA activity / function in a cell, cell population, sample, or subject, compared to an appropriate reference (e.g., a reference cell, cell population, sample, or subject).

[0294] As used herein, "complementary region" refers to the nucleotide sequence of a nucleic acid (e.g., a ds oligonucleotide) that is sufficiently complementary to an antiparallel sequence of nucleotides to allow hybridization between two sequences of nucleotides under appropriate hybridization conditions (e.g., in a phosphate buffer, inside a cell, etc.). In some embodiments, the oligonucleotide comprises a targeting sequence having a region complementary to the mRNA target sequence.

[0295] As used herein, "ribonucleotide" refers to a nucleotide having ribose as its pentose sugar and containing a hydroxyl group at the 2'-position. "Modified ribonucleotide" refers to a ribonucleotide having one or more modifications or substitutions of atoms other than at the 2'-position, including modifications or substitutions with ribose, phosphate groups or bases, or including modifications or substitutions of itself.

[0296] As used herein, "RNAi oligonucleotide" refers to either (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 by Argonaute 2 (Ago2) endonuclease for cleavage of a target mRNA (e.g., SNCA mRNA), or (b) an ss oligonucleotide having a single-stranded antisense strand, wherein the antisense strand (or a portion of the antisense strand) is used by Ago2 endonuclease for cleavage of a target mRNA (e.g., SNCA mRNA).

[0297] As used herein, "strand" refers to a single continuous sequence of nucleotides linked to each other via nucleotide linkages (e.g., phosphodiester linkages, phosphorothioate linkages). In some embodiments, a strand has two free ends (e.g., a 5'-end and a 3'-end).

[0298] As used herein, "subject" means any mammal including mouse, rabbit, and human. In one embodiment, the subject is human or NHP. Further, "individual" or "patient" may be used interchangeably with "subject".

[0299] As used herein, "synthetic" refers to nucleic acids or other molecules that are artificially synthesized (e.g., using a machine, e.g., a solid-phase nucleic acid synthesizer) or are not derived from natural sources (e.g., cells or organisms) that normally produce the molecule.

[0300] As used herein, "targeting ligand" refers to a molecule (e.g., carbohydrate, amino sugar, cholesterol, or polypeptide) that selectively binds to a cognate molecule (e.g., receptor) of a target tissue or cell and is capable of binding to another substance for the purpose of directing the other substance to the target tissue or cell. For example, in some embodiments, a targeting ligand may be conjugated to an oligonucleotide for the purpose of targeting the oligonucleotide to a specific target tissue or cell of interest. In some embodiments, the targeting ligand selectively binds to a cell surface receptor. Thus, in some embodiments, when conjugated to an oligonucleotide, the targeting ligand promotes delivery of the oligonucleotide to a specific cell via selective binding to a receptor expressed on the surface of the cell and endosomal internalization of a complex comprising the oligonucleotide, the targeting ligand, and the receptor by the cell. In some embodiments, the targeting ligand is conjugated to the oligonucleotide via a linker that is cleaved following or during internalization of the cell such that the oligonucleotide is released from the targeting ligand within the cell.

[0301] As used herein, "tetraloop" or "tetraL" refers to a loop that enhances the stability of adjacent double-stranded regions formed by hybridization of adjacent sequences of nucleotides. The enhanced stability is detectable as an increase in the melting temperature (T m ) of the adjacent stem double-stranded regions that is higher than the average from a set of loops of equivalent length consisting of randomly selected nucleotide sequences. For example, tetraL has a T m of at least 50°C, at least 55°C, at least 56°C, at least 58°C, at least 60°C, at least 65°C, or at least 75°C in a hairpin containing at least a 2 base pair (bp) double-stranded region in 10 mM NaHPO4. mcan be imparted. In some embodiments, Tetra L may stabilize bp in adjacent stem double-strands by stacking interactions. In addition, interactions between nucleotides within Tetra L include, but are not limited to, non-Watson-Crick base pair formation, stacking interactions, hydrogen bonding, and contact interactions (Cheong et al. (1990), Nature, 346:680-82; Heus & Pardi (1991), Science, 253:191-94). In some embodiments, Tetra L comprises, or consists of, 3 to 6 nucleotides, and typically 4 to 5 nucleotides. In certain embodiments, Tetra L comprises, or consists of, 3, 4, 5, or 6 nucleotides, which may or may not be modified (e.g., may or may not be bound to a targeting moiety). In certain embodiments, Tetra L comprises, or consists of, 3, 4, 5, or 6 nucleotides, which may or may not be modified (e.g., may or may not be bound to a targeting ligand). In one embodiment, Tetra L consists of 4 nucleotides. Any nucleotide may be used in Tetra L, and the standard IUPAC-IUB symbols for such nucleotides may be used as described in Cornish-Bowden, (1985), Nucleic Acids Res. 13:3021-30. For example, the letter "N" may be used to mean that any base may be at that position, the letter "R" may be used to indicate that A (adenine) or G (guanine) may be at that position, and "B" may be used to indicate that C (cytosine), G (guanine), T (thymine) or U (uracil) may be at that position. Examples of tetraloops include the UNCG family of tetraloops (e.g., UUCG), the GNRA family of tetraloops (e.g., GAAA), and the CUUG tetraloop (Woese et al. (1990) Proc. Natl. Acad. Sci. USA, 87:8467-71; and Antao et al. (1991), Nucleic Acids Res. 19:5901-05).Examples of DNA tetraloops include the d(GNNA) family of tetraloops (e.g., d(GTTA)), the d(GNRA) family of tetraloops, the d(GNAB) family of tetraloops, the d(CNNG) family of tetraloops, and the d(TNCG) family of tetraloops (e.g., d(TTCG)). See, for example, Nakano et al. (2002), Biochem. 41:4281-92; Shinji et al. (2000), Nippon Kagakkai Koen Yokoshu, 78:731. In some embodiments, Tetra L is contained within a Tetra L structure with a nick.

[0302] As used herein, "treating" or "treatment" refers to the act of providing care to a subject in need of care by administering a therapeutic agent (e.g., an oligonucleotide of the present specification such as an RNAi oligonucleotide) to the subject, for example, for the purpose of improving the health and / or well-being of the subject with respect to an existing condition (e.g., a disease, disorder), or for the purpose of preventing or reducing the likelihood of the occurrence of the condition. In some embodiments, treatment includes reducing the frequency or severity of at least one sign, symptom, or contributing factor of a condition (e.g., a disease, disorder) experienced by the subject.

Examples

[0303] Example 1: Preparation of RNAi Oligonucleotides Oligonucleotide Synthesis and Purification The oligonucleotides (i.e., RNAi oligonucleotides) described in the foregoing examples are chemically synthesized using the methods described herein. Generally, RNAi oligonucleotides are synthesized using known phosphoramidite synthesis (e.g., Hughes & Ellington, (2017), Cold Spring Harb Perspect. Biol. 9(1):a023812; Beaucage & Caruthers (1981), Tetrahedron Lett. 22:1859-62) in addition to using solid-phase oligonucleotide synthesis methods as described for 19-23 base pair long siRNAs (e.g., see Scaringe et al. (1990), Nucleic Acids Res. 18:5433-5441 and Usman et al. (1987), J. Am. Chem. Soc. 109:7845-45; U.S. Patent Nos. 5,804,683; 5,831,071; 5,998,203; 6,008,400; 6,111,086; 6,117,657; 6,353,098; 6,362,323; 6,437,117 and 6,469,158). The dsRNAi oligonucleotides having a 19-base pair core sequence were constructed into constructs having a 25-base pair sense strand and a 27-base pair antisense strand so as to be processable by the RNAi apparatus. The 19-base pair core sequence is complementary to a region of the SNCA mRNA.

[0304] Individual RNA strands were synthesized and purified by HPLC according to standard methods (Integrated DNA Technologies, Coralville, IA). For example, RNA oligonucleotides were synthesized using solid-phase phosphoramidite chemistry, deprotected, and desalted on a NAP-5 column (Amersham Pharmacia Biotech; Piscataway, NJ) using standard techniques (Damha & Olgivie, (1993), Methods Mol. Biol. 20:81-114; Wincott et al. (1995), Nucleic Acids Res. 23:2677-84). Ion exchange high performance liquid chromatography (IE-HPLC) was used to purify the oligomers on an Amersham Source 15Q column (1.0 cm × 25 cm, Amersham Pharmacia Biotech) using a 15-minute stepwise linear gradient. The gradient varied from 90:10 buffer A:B to 52:48 buffer A:B, where buffer A was 100 mM Tris at pH 8.5 and buffer B was 100 mM Tris at pH 8.5, 1 M NaCl. Samples were monitored at 260 nm, and peaks corresponding to full-length oligonucleotide species were collected, pooled, desalted on a NAP-5 column, and lyophilized.

[0305] The purity of each oligomer was determined by capillary electrophoresis (CE) on a Beckman PACE 5000 (Beckman Coulter, Inc., Fullerton, CA). The CE capillary had an inner diameter of 100 μm and contained ssDNA 100R Gel (Beckman-Coulter). Typically, about 0.6 nmole of oligonucleotide was injected into the capillary, electrophoresed at an electric field of 444 V / cm, and detected by UV absorbance at 260 nm. Denaturing Tris-borate-7M-urea electrophoresis buffer was purchased from Beckman-Coulter. Oligoribonucleotides that were at least 90% pure were obtained as evaluated by CE for use in the experiments described below. The identity of the compounds was verified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry on a Voyager DE (trademark) Biospectometry Work Station (Applied Biosystems; Foster City, CA) according to the manufacturer's recommended protocol. The relative molecular weights of all oligomers were obtained, which were often within 0.2% of the predicted molecular weight.

[0306] Preparation of double-stranded The ssRNA oligomers were resuspended in a double-stranded buffer consisting of 100 mM potassium acetate, 30 mM HEPES, pH 7.5 (e.g., at a concentration of 100 μM). Complementary sense and antisense strands were mixed in equimolar amounts to obtain a final solution of, for example, 50 μM double-stranded. The sample was heated to 100 °C for 5 minutes in RNA buffer (IDT) and cooled to room temperature before use. The RNAi oligonucleotides were stored at -20 °C. The ssRNA oligomers were stored at -80 °C either lyophilized or in nuclease-free water.

[0307] Example 2: Generation of RNAi oligonucleotides targeting SNCA SNCA encodes SNCA, a neuronal protein that inhibits phospholipase D2. SNCA is involved in the regulation of synaptic vesicle transport and neurotransmitter release. Aberrant expression of SNCA can lead to various brain diseases, including but not limited to Parkinson's disease and multiple system atrophy. Oligonucleotides that can inhibit the expression of SNCA mRNA were identified and generated. Identification of SNCA mRNA target sequences ...

Claims

1. An RNAi oligonucleotide comprising an antisense strand and a sense strand forming a double-stranded region, The sense strand is 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-GalN Ac][ademA-GalN The sequence is Ac][ademA-GalNAc][mG][mG][mC][mU][mG][mC]-3' (sequence number 1621), The antisense strand is the sequence 5'-[MePhosphonate-4O-mUs][fUs][fU][fG][fA][mG][fA][mA][mA][fU][mG][mU][mC][fA][mU][fG][mA][mC][fU][mGs][mGs][mG]-3' (SEQ ID NO: 1656), mA is 2'-OMe adenosine, mC is 2'-OMe cytosine, mG is 2'-OMe guanosine, mU is 2'-OMe uridine, fA is 2'-F adenosine, fC is 2'-F cytosine, fG is 2'-F guanosine, fU is 2'-F-uridine, mCs are 2'-OMe cytosine having a 3'-phosphorothioate bond. mGs is 2'-OMe guanosine having a 3'-phosphorothioate bond, mUs is a 2'-OMe uridine having a 3'-phosphorothioate bond, fUs is a 2'-F uridine having a 3'-phosphorothioate bond, ademA-GalNAc is 2'-aminodiethoxymethanol-adenine-GalNAc, MePhosphonate-4O-mUs has the following structure: 【Chemistry 1】 This is an RNAi oligonucleotide.

2. A pharmaceutical composition comprising an RNAi oligonucleotide containing an antisense strand and a sense strand forming a double-stranded region, The sense strand is 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-GalN Ac][ademA-GalN The sequence is Ac][ademA-GalNAc][mG][mG][mC][mU][mG][mC]-3' (sequence number 1621), The antisense strand is the sequence 5'-[MePhosphonate-4O-mUs][fUs][fU][fG][fA][mG][fA][mA][mA][fU][mG][mU][mC][fA][mU][fG][mA][mC][fU][mGs][mGs][mG]-3' (SEQ ID NO: 1656), mA is 2'-OMe adenosine, mC is 2'-OMe cytosine, mG is 2'-OMe guanosine, mU is 2'-OMe uridine, fA is 2'-F adenosine, fC is 2'-F cytosine, fG is 2'-F guanosine, fU is 2'-F-uridine, mCs are 2'-OMe cytosine having a 3'-phosphorothioate bond. mGs is 2'-OMe guanosine having a 3'-phosphorothioate bond, mUs is a 2'-OMe uridine having a 3'-phosphorothioate bond, fUs is a 2'-F uridine having a 3'-phosphorothioate bond, ademA-GalNAc is 2'-aminodiethoxymethanol-adenine-GalNAc, MePhosphonate-4O-mUs has the following structure: 【Chemistry 2】 A pharmaceutical composition.

3. The pharmaceutical composition according to claim 2, further comprising a carrier suitable for intravenous administration.

4. The pharmaceutical composition according to claim 3, wherein the carrier comprises water.

5. The pharmaceutical composition according to claim 3, wherein the carrier comprises phosphate-buffered saline.

6. The pharmaceutical composition according to claim 2, further comprising a carrier suitable for intrathecal administration.

7. The pharmaceutical composition according to claim 6, wherein the carrier contains water.

8. The pharmaceutical composition according to claim 6, wherein the carrier comprises phosphate-buffered saline.

9. A pharmaceutical composition according to any one of claims 2 to 8 for reducing the expression of the alpha-synuclein (SNCA) gene in cells, cell populations, or subjects.

10. The pharmaceutical composition according to claim 9, wherein reducing the expression of the SNCA gene includes reducing the amount or level of SNCA mRNA, the amount or level of SNCA protein, SNCA activity / function, or a combination thereof.

11. The pharmaceutical composition according to claim 9, wherein the expression of the SNCA gene is reduced in tissue in one or more regions of the central nervous system (CNS), and the tissue is associated with Parkinson's disease.

12. The pharmaceutical composition according to claim 11, wherein the tissue associated with Parkinson's disease is selected from the putamen, midbrain tegmentum, substantia nigra, pons, and medulla.

13. The pharmaceutical composition according to claim 9, wherein the expression of the SNCA gene is reduced in tissue in one or more regions of the central nervous system (CNS), and the tissue is associated with multiple system atrophy.

14. The pharmaceutical composition according to claim 13, wherein the tissue associated with multiple system atrophy is selected from 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.

15. The pharmaceutical composition according to claim 9, wherein the expression of the SNCA gene is reduced in one or more regions of the central nervous system (CNS) selected from 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.

16. The pharmaceutical composition according to claim 9, wherein the subject has a disease, disorder, or condition related to the expression of the SNCA gene.

17. The pharmaceutical composition according to claim 16, wherein the disease, disorder, or condition related to SNCA activity is multiple system atrophy, Lewy body dementia, or Parkinson's disease.

18. The pharmaceutical composition according to claim 9, wherein the RNAi oligonucleotide or the pharmaceutical composition is administered in combination with a second composition or therapeutic agent.

19. A method for reducing the expression of an SNCA gene in cells or a population of cells in vitro, comprising contacting the cells or the population of cells with the RNAi oligonucleotide described in claim 1 or the pharmaceutical composition described in claim 2.

20. The method according to claim 19, wherein reducing the expression of the SNCA gene includes reducing the amount or level of SNCA mRNA, the amount or level of SNCA protein, SNCA activity / function, or a combination thereof.

21. A kit comprising an RNAi oligonucleotide according to claim 1 or a pharmaceutically acceptable salt thereof, an optional pharmaceutically acceptable carrier, and a package insert including instructions for use in administering to an individual having a disease, disorder, or condition related to alpha-synuclein (SNCA) activity.

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