siRNA COMPOSITIONS AND METHODS TARGETING ALPHA-SYNUCLEIN NUCLEIC ACIDS

1. SEQUENCE LISTING

[0001] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on September 27, 2023, is named “51436-

042WO2_Sequence_Listing_9_27_23” and is 820,156 bytes in size.

2. BACKGROUND

[0002] Expression of the alpha-synuclein (SNCA) gene produces the protein alpha-synuclein. While the alpha-synuclein protein is a soluble monomer normally localized at the presynaptic region of axons, the protein can form filamentous aggregates that are the major component of intracellular inclusions in neurodegenerative synucleinopathies. For example, mutations in the SNCA gene and SNCA gene multiplications have been linked to the formation of the filamentous aggregates that are a hallmark of familial Parkinson's disease. Additional neurodegenerative synucleinopathies associated with alpha-synuclein aggregates include sporadic Parkinson's disease, Alzheimer's disease, multiple system atrophy, and Lewy body dementia. Currently there are limited treatment options available for neurodegenerative synucleinopathies. Accordingly, there remains a need for therapeutics that can selectively diminish SNCA activity in a manner that provides effective treatment for neurodegenerative synucleinopathies.

3. SUMMARY

[0003] The instant application is directed to single- or double-stranded interfering RNA molecules (e.g., siRNA molecules) that target an SNCA gene. The interfering RNA molecules may contain specific patterns of nucleoside modifications and internucleoside linkage modifications. The disclosure also features pharmaceutical compositions including the same. The siRNA molecules may be branched siRNA molecules, such as di -branched, tri-branched, or tetra-branched siRNA molecules. The disclosed siRNA molecules may further feature a 5’ phosphorus stabilizing moiety and/or a hydrophobic moiety. Additionally, the disclosure provides methods for delivering the siRNA molecule of the disclosure to the central nervous system of a subject, such as a subject identified as having a synucleinopathy. In certain embodiments, an interfering RNA molecule of the present disclosure is a di-branched siRNA molecule. In certain embodiments, the di-branched siRNA molecules of the present disclosure comprise: a) a sense strand comprising the sequence (mU)#(mA)#(mC)(fC)(mA)(fC)(mU)(fU)(mA) (fU)(mU)(mU)(mC)(fU)#(mA)#(mA)-DIO (SEQ ID NO: 841) and an antisense strand comprising the sequence V(mU)#(fU)#(mA)(fG)(fA)(fA)(mA)(fU)(mA)(fA)(mG)(fU)(mG)(fG) (mU)(fA)#(mG)#(mU)#(mC)#(mA)#(mC) (SEQ ID NO: 842); b) a sense strand comprising the sequence (mC)#(mA)#(mA)(fG)(mU)(fG)(mC)(fU) (mC)(fA)(mG)(mU)(mU)(fC)#(mC)# (mA)-DIO (SEQ ID NO: 843) and an antisense strand comprising the sequence V(mU)#(fG)#(mG)(fA)(fA)(fC)(mU)(fG)(mA)(fG)(mC)(fA)(mC)(fU)( mU) (fG)#(mU)#(mA)#(mC)#(mA)#(mG) (SEQ ID NO: 844); c) a sense strand comprising the sequence (mA)#(mG)#(mU)(fG)(mG)(fU)(mG)(fC)(mA) (fU)(mG)(mG)(mU)(fG)#(mU)#(mA)-DIO (SEQ ID NO: 845) and an antisense strand comprising the sequence V(mU)#(fA)#(mC)(fA)(fC)(fC)(mA)(fU)(mG)(fC)(mA)(fC)(mC)(fA)( mC) (fU)#(mC)#(mC)#(mC)#(mU)#(mC) (SEQ ID NO: 846). d) a sense strand comprising the sequence (mC)#(mA)#(mA)(fU)(mG)(fA)(mG)(fG)(mC) (fU)(mU)(mA)(mU)(fG)#(mA)#(mA)-DIO (SEQ ID NO: 847) and an antisense strand comprising the sequence V(mU)#(fU)#(mU)(fG)(fG)(fA)(mA)(fC)(mU)(fG)(mA)(fG)(mC)(fA)( mC) (fU)#(mU)#(mG)#(mU)#(mA)#(mC) (SEQ ID NO: 848); e) a sense strand comprising the sequence (mU)#(mC)#(mA)(fG)(mU)(fU)(mC)(fC)(mA) (fA)(mU)(mG)(mU)(fG)#(mC)#(mA)-DIO (SEQ ID NO: 849) and an antisense strand comprising the sequence V(mU)#(fU)#(mU)(fG)(fG)(fA)(mA)(fC)(mU)(fG)(mA)(fG)(mC) (fA)(mC)(fU)#(mU)#(mG)#(mU)#(mA)#(mC) (SEQ ID NO: 850). f) a sense strand comprising the sequence (mC)#(mU)#(mA)(fC)(mG)(fA)(mU)(fG)(mU) (fU)(mA)(mA)(mA)(fA)#(mC)#(mA)-DIO (SEQ ID NO: 851) and an antisense strand comprising the sequence V(mU)#(fG)#(mC)(fA)(fC)(fA)(mU)(fU)(mG)(fG)(mA)(fA)(mC)(fU)( mG) (fA)#(mG)#(mC)#(mA)#(mC)#(mU) (SEQ ID NO: 852); or g) a sense strand comprising the sequence (mC)#(mU)#(mA)(fC)(mG)(fA)(mU)(fG)(mU) (fU)(mA)(mA)(mA)(fA)#(mC)#(mA)-DIO (SEQ ID NO: 853) and an antisense strand comprising the sequence V(mU)#(fG)#(mU)(fU)(fU)(fU)(mA)(fA)(mC)(fA)(mU)(fC)(mG)(fU) (mA)(fG)#(mA)#(mU)#(mU)#(mG)#(mA) (SEQ ID NO: 854); wherein m represents a 2’-0-Me ribonucleoside, f represents a 2’-F ribonucleoside, # represents a phosphorothioate internucleoside linkage, -DIO represents a divalent oligonucleotide (DIO) linker; and V represents a vinyl phosphonate.

[0004] In some embodiments, the siRNA molecule comprises: a) a sense strand comprising the sequence (mU)#(mA)#(mC)(fC)(mA)(fC)(mU)(fU)(mA) (fU)(mU)(mU)(mC)(fU)#(mA)#(mA)-DIO (SEQ ID NO: 841) and an antisense strand comprising the sequence V(mU)#(fU)#(mA)(fG)(fA)(fA)(mA)(fU)(mA)(fA)(mG)(fU)(mG)(fG) (mU)(fA)#(mG)#(mU)#(mC)#(mA)#(mC) (SEQ ID NO: 842); b) a sense strand comprising the sequence (mC)#(mA)#(mA)(fG)(mU)(fG)(mC)(fU) (mC)(fA)(mG)(mU)(mU)(fC)#(mC)# (mA)-DIO (SEQ ID NO: 843) and an antisense strand comprising the sequence V(mU)#(fG)#(mG)(fA)(fA)(fC)(mU)(fG)(mA)(fG)(mC)(fA)(mC)(fU)( mU) (fG)#(mU)#(mA)#(mC)#(mA)#(mG) (SEQ ID NO: 844); c) a sense strand comprising the sequence (mA)#(mG)#(mU)(fG)(mG)(fU)(mG)(fC)(mA) (fU)(mG)(mG)(mU)(fG)#(mU)#(mA)-DIO (SEQ ID NO: 845) and an antisense strand comprising the sequence V(mU)#(fA)#(mC)(fA)(fC)(fC)(mA)(fU)(mG)(fC)(mA)(fC)(mC)(fA)( mC) (fU)#(mC)#(mC)#(mC)#(mU)#(mC) (SEQ ID NO: 846). d) a sense strand comprising the sequence (mC)#(mA)#(mA)(fU)(mG)(fA)(mG)(fG)(mC) (fU)(mU)(mA)(mU)(fG)#(mA)#(mA)-DIO (SEQ ID NO: 847) and an antisense strand comprising the sequence

V(mU)#(fU)#(mC)(fA)(fU)(fA)(mA)(fG)(mC)(fC)(mU)(fC)(mA)(f U)(mU)(fG)#(mU )#(mC)#(mA)#(mG)#(mG) (SEQ ID NO: 855) e) a sense strand comprising the sequence (mA)#(mG)#(mU)(fG)(mC)(fU) (mC)(fA)(mG)(fU)(mU)(mC)(mC)(fA)#(mA)#(mA)-DIO (SEQ ID NO: 856) and an antisense strand comprising the sequence V(mU)#(fU)#(mU)(fG)(fG)(fA)(mA)(fC)(mU)(fG)(mA)(fG)(mC) (fA)(mC)(fU)#(mU)#(mG)#(mU)#(mA)#(mC) (SEQ ID NO: 850). f) a sense strand comprising the sequence (mU)#(mC)#(mA)(fG)(mU) (fU)(mC)(fC)(mA)(fA)(mU)(mG)(mU)(fG)#(mC)#(mA)-DIO (SEQ ID NO: 857) and an antisense strand comprising the sequence V(mU)#(fG)#(mC)(fA)(fC)(fA)(mU)(fU)(mG)(fG)(mA)(fA)(mC)(fU)( mG) (fA)#(mG)#(mC)#(mA)#(mC)#(mU) (SEQ ID NO: 852); or g) a sense strand comprising the sequence (mC)#(mU)#(mA)(fC)(mG)(fA)(mU)(fG)(mU) (fU)(mA)(mA)(mA)(fA)#(mC)#(mA)-DIO (SEQ ID NO: 853) and an antisense strand comprising the sequence V(mU)#(fG)#(mU)(fU)(fU)(fU)(mA)(fA)(mC)(fA)(mU)(fC)(mG)(fU) (mA)(fG)#(mA)#(mU)#(mU)#(mG)#(mA) (SEQ ID NO: 854); wherein m represents a 2’-O-Me ribonucleoside, f represents a 2’-F ribonucleoside, # represents a phosphorothioate internucleoside linkage, -DIO represents a divalent oligonucleotide (DIO) linker; and V represents a vinyl phosphonate.

[0005] In another aspect, the disclosure features a small interfering RNA (siRNA) molecule comprising an antisense strand and sense strand having complementarity to the antisense strand. The antisense strand may be, e.g., from 10 to 30 nucleotides in length and may have complementarity sufficient to hybridize to a region within an alpha-synuclein (SNCA) mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 421-840.

[0006] In some embodiments, the antisense strand has at least 70% complementarity to a region of 21 contiguous nucleobases within the SNCA mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 421-840. In some embodiments, the antisense strand has at least 75% complementarity to a region of 21 contiguous nucleobases within the SNCA mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 421-840. In some embodiments, the antisense strand has at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementarity to the region within the SNCA mRNA transcript having the nucleic acid sequence of any one of SEQ ID Nos: 421-840.

[0007] In some embodiments, the antisense strand comprises at least 10, at least 11, 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, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or 30 contiguous nucleotides that are fully complementary to a contiguous polynucleotide segment of equal length within the region of the SNCA mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 421-840.

[0008] In some embodiments, the antisense strand comprises from 10 to 30 contiguous nucleotides that are fully complementary to a contiguous polynucleotide segment of equal length within the region of the SNCA mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 421-840.

[0009] In some embodiments, the antisense strand comprises from 12 to 30 contiguous nucleotides that are fully complementary to a contiguous polynucleotide segment of equal length within the region of the SNCA mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 421-840.

[0010] In some embodiments, the antisense strand comprises from 15 to 30 contiguous nucleotides that are fully complementary to a contiguous polynucleotide segment of equal length within the region of the SNCA mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 421-840.

[0011] In some embodiments, the antisense strand comprises from 18 to 30 contiguous nucleotides that are fully complementary to a contiguous polynucleotide segment of equal length within the region of the SNCA mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 421-840.

[0012] In some embodiments, the antisense strand comprises from 21 to 30 contiguous nucleotides that are fully complementary to a contiguous polynucleotide segment of equal length within the region of the SNCA mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 421-840.

[0013] In some embodiments, the antisense strand comprises from 24 to 30 contiguous nucleotides that are fully complementary to a contiguous polynucleotide segment of equal length within the region of the SNCA mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 421-840.

[0014] In some embodiments, the antisense strand comprises 30 contiguous nucleotides that are fully complementary to a contiguous polynucleotide segment of equal length within the region of the SNCA mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 421-840.

[0015] In some embodiments, the antisense strand comprises 9 or fewer nucleotide mismatches relative to a region of 21 contiguous nucleobases of the SNCA mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 421-840. In some embodiments, the antisense strand comprises 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or only 1 mismatch relative to the region of the SNCA mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 421-840.

[0016] In some embodiments, the region of the SNCA mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 459, 535, 551, 599, 631, 663, 664, 666, 667, 671, 748, 750, 751, 752, 760, 762, and 777.

[0017] In some embodiments, the region of the SNCA mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 535, 599, 664, 666, 671, 750, 752, 760, and 762.

[0018] In some embodiments, the antisense strand has a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence of any one of SEQ ID NOs: 1-420.

[0019] In some embodiments, the antisense strand has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of any one of SEQ ID NOs: 1-420.

[0020] In some embodiments, the antisense strand has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NOs: 1-420. In some embodiments, the antisense strand has a nucleic acid sequence that is at least 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of any one of SEQ ID NOs: 1-420.

[0021] In some embodiments, the antisense strand has the nucleic acid sequence of any one of SEQ ID NOs: 1-420.

[0022] In some embodiments, the nucleic acid sequence is any one of SEQ ID NOs: 39, 115, 131, 179, 211, 243, 244, 246, 247, 251, 328, 330, 331, 332, 340, 342, and 357.

[0023] In some embodiments, the nucleic acid sequence is any one of SEQ ID NOs: 115, 179, 244, 246, 251, 330, 332, 340, and 342.

[0024] In some embodiments, the sense strand has a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence of any one of SEQ ID NOs: 421-840.

[0025] In some embodiments, the sense strand has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of any one of SEQ ID NOs: 421-840.

[0026] In some embodiments, the sense strand has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NOs: 421-840. In some embodiments, the sense strand has a nucleic acid sequence that is at least 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of any one of SEQ ID NOs: 421-840.

[0027] In some embodiments, the sense strand has the nucleic acid sequence of any one of SEQ ID NOs: 421-840.

[0028] In some embodiments, the nucleic acid sequence is any one of SEQ ID NOs: 459, 535, 551, 599, 631, 663, 664, 666, 667, 671, 748, 750, 751, 752, 760, 762, and 777.

[0029] In some embodiments, the nucleic acid sequence is any one of SEQ ID NOs: 535, 599, 664, 666, 671, 750, 752, 760, and 762.

[0030] In some embodiments, the antisense strand comprises a structure represented by Formula I, wherein Formula I is, in the 5’-to-3’ direction:

A-B-(AQj-C-P ² -D-Pk(C’-P C’

Formula I; wherein A is represented by the formula C-P^D-P ¹ ; each A’ is represented by the formula C-P ² -D-P ² ;

B is represented by the formula C-P ² -D-P ² -D-P ² -D-P ² ; each C is a 2’-O-methyl (2’-O-Me) ribonucleoside; each C’, independently, is a 2’-O-Me ribonucleoside or a 2’-fluoro (2’-F) ribonucleoside; each D is a 2’-F ribonucleoside; each P ¹ is a phosphorothioate internucleoside linkage; each P ² is a phosphodiester intemucleoside linkage; j is an integer from 1 to 7; and k is an integer from 1 to 7.

[0031] In some embodiments, the antisense strand comprises a structure represented by Formula Al, wherein Formula Al is, in the 5’-to-3’ direction:

A-S-B-S-A-O-B-O-B-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A -O-B-S-A-S-A-S- A-S-B-S-A

Formula Al; wherein A represents a 2’-O-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage. [0032] In some embodiments, the antisense strand comprises a structure represented by Formula II, wherein Formula II is, in the 5’-to-3’ direction:

Formula II; wherein A is represented by the formula C-P^D-P ¹ ; each A’ is represented by the formula C-P ² -D-P ² ;

B is represented by the formula C-P ² -D-P ² -D-P ² -D-P ² ; each C is a 2’-O-methyl (2’-O-Me) ribonucleoside; each C’, independently, is a 2’-O-Me ribonucleoside or a 2’-fluoro (2’-F) ribonucleoside; each D is a 2’-F ribonucleoside; each P ¹ is a phosphorothioate internucleoside linkage; each P ² is a phosphodiester intemucleoside linkage; j is an integer from 1 to 7; and k is an integer from 1 to 7.

[0033] In some embodiments, the antisense strand comprises a structure represented by Formula A2, wherein Formula A2 is, in the 5’-to-3’ direction:

A-S-B-S-A-O-B-O-B-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A -O-B-S-A-S-A-S- A-S-A-S-A

Formula A2; wherein A represents a 2’-O-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0034] In some embodiments, the sense strand comprises a structure represented by Formula III, wherein Formula III is, in the 5’-to-3’ direction:

E-(A’) _m -F

Formula III; wherein E is represented by the formula (C-P ¹ ^; F is represented by the formula (C-P ² )3-D-P ¹ -C-P ¹ -C, (C-P ² )3-D-P ² -C-P ² -C, (C-P ² )3-D-P ¹ -C- P ^x -D, or (C-P ² ) ₃ -D-P ² -C-P ² -D;

A’, C, D, P ¹ , and P ² are as defined in Formula II; and m is an integer from 1 to 7.

[0035] In some embodiments, the sense strand comprises a structure represented by Formula

51, wherein Formula SI is, in the 5’-to-3’ direction:

A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-A-O-A-O-B-S-A -S-A

Formula SI; wherein A represents a 2’-O-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0036] In some embodiments, the sense strand comprises a structure represented by Formula

52, wherein Formula S2 is, in the 5’-to-3’ direction:

A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-A-O-A-O-B-O-A -O-A Formula S2; wherein A represents a 2’-O-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0037] In some embodiments, the sense strand comprises a structure represented by Formula

53, wherein Formula S3 is, in the 5’-to-3’ direction:

A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-A-O-A-O-B-S-A -S-B Formula S3; wherein A represents a 2’-O-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0038] In some embodiments, the sense strand comprises a structure represented by Formula

54, wherein Formula S4 is, in the 5’-to-3’ direction: A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-A-O-A-O-B-O-A-O- B

Formula S4; wherein A represents a 2’-O-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0039] In some embodiments, the antisense strand comprises a structure represented by Formula IV, wherein Formula IV is, in the 5’-to-3’ direction:

Formula IV; wherein A is represented by the formula C-P^D-P ¹ ; each A’ is represented by the formula C-P ² -D-P ² ;

B is represented by the formula D-P^C-P^D-P ¹ ; each C is a 2’-O-Me ribonucleoside; each C’, independently, is a 2’-O-Me ribonucleoside or a 2’-F ribonucleoside; each D is a 2’-F ribonucleoside; each P ¹ is a phosphorothioate internucleoside linkage; each P ² is a phosphodiester intemucleoside linkage; j is an integer from 1 to 7; and k is an integer from 1 to 7.

[0040] In some embodiments, the antisense strand comprises a structure represented by Formula A3, wherein Formula A3 is, in the 5’-to-3’ direction:

A-S-B-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A -O-B-S-A-S-B-S- A-S-A-S-A

Formula A3; wherein A represents a 2’-O-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage. [0041] In some embodiments, the sense strand comprises a structure represented by Formula V, wherein Formula V is, in the 5’-to-3’ direction:

E-(A’) _m -C-P ² -F

Formula V; wherein E is represented by the formula (C-P ¹ )?;

F is represented by the formula D- or D-P ² -C-P ² -D;

A’, C, D, P ¹ and P ² are as defined in Formula IV; and m is an integer from 1 to 7.

[0042] In some embodiments, the sense strand comprises a structure represented by Formula

55, wherein Formula S5 is, in the 5’-to-3’ direction:

A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-S-A -S-A Formula S5; wherein A represents a 2’-O-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0043] In some embodiments, the sense strand comprises a structure represented by Formula

56, wherein Formula S6 is, in the 5’-to-3’ direction:

A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A -O-A Formula S6; wherein A represents a 2’-O-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0044] In some embodiments, the sense strand comprises a structure represented by Formula

57, wherein Formula S7 is, in the 5’-to-3’ direction:

A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-S-A -S-B

Formula S7; wherein A represents a 2’-0-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0045] In some embodiments, the sense strand comprises a structure represented by Formula S8, wherein Formula S8 is, in the 5’-to-3’ direction:

A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A -O-B Formula S8; wherein A represents a 2’-O-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0046] In some embodiments, the antisense strand comprises a structure represented by Formula VI, wherein Formula VI is, in the 5’-to-3’ direction:

A-Bj-E-Bk-E-F-Gi-D-Pkc’

Formula VI; wherein A is represented by the formula C-P^D-P ¹ ; each B is represented by the formula C-P ² ; each C is a 2’-O-Me ribonucleoside; each C’, independently, is a 2’-O-Me ribonucleoside or a 2’-F ribonucleoside; each D is a 2’-F ribonucleoside; each E is represented by the formula D-P ² -C-P ² ;

F is represented by the formula D-P^C-P ¹ ; each G is represented by the formula C-P ¹ ; each P ¹ is a phosphorothioate internucleoside linkage; each P ² is a phosphodiester intemucleoside linkage; j is an integer from 1 to 7; k is an integer from 1 to 7; and

1 is an integer from 1 to 7.

[0047] In some embodiments, the antisense strand comprises a structure represented by Formula A4, wherein Formula A4 is, in the 5’-to-3’ direction: A-S-B-S-A-O-A-O-A-O-B-O-A-O-A-O-A-O-A-O-A-O-A-O-A-O-B-O-A-O- B-S-A-S-A-S- A-S-B-S-A

Formula A4; wherein A represents a 2’-0-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0048] In some embodiments, the sense strand comprises a structure represented by Formula VII, wherein Formula VII is, in the 5’-to-3’ direction:

H-B _m -In-A’-Bo-H-C

Formula VII; wherein A’ is represented by the formula C-P ² -D-P ² ; each H is represented by the formula (C-P ¹ )?; each I is represented by the formula (D-P ² );

B, C, D, P ¹ and P ² are as defined in Formula VI; m is an integer from 1 to 7; n is an integer from 1 to 7; and o is an integer from 1 to 7.

[0049] In some embodiments, the sense strand comprises a structure represented by Formula S9, wherein Formula S9 is, in the 5’-to-3’ direction:

A-S-A-S-A-O-A-O-A-O-B-O-B-O-B-O-A-O-B-O-A-O-A-O-A-O-A-S-A -S-A

Formula S9; wherein A represents a 2’-O-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0050] In some embodiments, the antisense strand further comprises a 5’ phosphorus stabilizing moiety at the 5’ end of the antisense strand.

[0051] In some embodiments, the sense strand further comprises a 5’ phosphorus stabilizing moiety at the 5’ end of the sense strand. [0052] In some embodiments, each 5’ phosphorus stabilizing moiety is, independently, represented by any one of Formulas IX-XVI:

Formula IX Formula X Formula XI Formula XII

Formula XIII Formula XIV Formula XV Formula XVI wherein Nuc represents a nucleobase selected from the group consisting of adenine, uracil, guanine, thymine, and cytosine, and R represents an optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, phenyl, benzyl, hydroxy, or hydrogen.

[0053] In some embodiments, the nucleobase is an adenine, uracil, guanine, thymine, or cytosine.

[0054] In some embodiments, the 5’ phosphorus stabilizing moiety is (E)-vinylphosphonate represented by Formula XI.

[0055] In some embodiments, the siRNA molecule further comprises a hydrophobic moiety at the 5’ or the 3’ end of the siRNA molecule.

[0056] In some embodiments, the hydrophobic moiety is selected from a group consisting of cholesterol, vitamin D, or tocopherol.

[0057] In some embodiments, the length of the sense strand is between 12 and 30 nucleotides.

[0058] In some embodiments, the siRNA molecule is a branched siRNA molecule.

[0059] In some embodiments, the branched siRNA molecule is di-branched, tri-branched, or tetra-branched.

[0060] In some embodiments, the siRNA molecule is a di-branched siRNA molecule. In some embodiments, the di-branched siRNA molecule is represented by any one of Formulas XVII- XIX: RNA RNA RNA X-L-X X-L-X

RNA-L-RNA RNA' RNA RNA' RNA

Formula XVII; Formula XVIII; Formula XIX; wherein each RNA is, independently, an siRNA molecule, L is a linker, and each X, independently, represents a branch point moiety.

[0061] In some embodiments, the siRNA molecule is a tri-branched siRNA molecule, optionally wherein the tri-branched siRNA molecule is represented by any one of Formulas

XX-XXIII:

Formula XX; Formula XXI; Formula XXII; Formula XXIII; wherein each RNA is, independently, an siRNA molecule, L is a linker, and each X, independently, represents a branch point moiety.

[0062] In some embodiments, the siRNA molecule is a tetra-branched siRNA molecule, optionally wherein the tetra-branched siRNA molecule is represented by any one of Formulas XXIV-XXVIII:

Formula XXIV; Formula XXV; Formula XXVI; Formula XXVII; Formula XXVIII; wherein each RNA is, independently, an siRNA molecule, L is a linker, and each X, independently, represents a branch point moiety. [0063] In some embodiments, the linker is selected from a group consisting of one or more contiguous subunits of an ethylene glycol, alkyl, carbohydrate, block copolymer, peptide, RNA, and DNA.

[0064] In some embodiments, the one or more contiguous subunits is 2 to 20 contiguous subunits.

[0065] In another aspect, the disclosure features a pharmaceutical composition comprising the siRNA molecule of any of the foregoing aspects or embodiments of the disclosure, in combination with a pharmaceutically acceptable excipient, carrier, or diluent.

[0066] In another aspect, the disclosure features a method of delivering an siRNA molecule to a subject diagnosed as having a synucleinopathy, the method comprising administering to the subject a therapeutically effective amount of the siRNA molecule of any of the foregoing aspects or embodiments of the disclosure, optionally in combination with a pharmaceutically acceptable excipient, carrier, or diluent.

[0067] In another aspect, the disclosure features a method of treating a synucleinopathy in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the siRNA molecule of any of the foregoing aspects or embodiments of the disclosure, optionally in combination with a pharmaceutically acceptable excipient, carrier, or diluent.

[0068] In some embodiments, the synucleinopathy is Parkinson’s disease. In some embodiments, the synucleinopathy is Alzheimer's disease. In some embodiments, the synucleinopathy is Lewy body dementia. In some embodiments, the synucleinopathy is a multiple symptom atrophy.

[0069] In another aspect, the disclosure features a method of reducing SNCA expression in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the siRNA molecule of any of the foregoing aspects or embodiments of the disclosure, optionally in combination with a pharmaceutically acceptable excipient, carrier, or diluent.

[0070] In some embodiments, the siRNA molecule or the pharmaceutical composition is administered to the subject by way of intracerebroventricular, intrastriatal, intraparenchymal, or intrathecal injection. In some embodiments, the siRNA molecule or the pharmaceutical composition is administered to the subject by way of intravenous, intramuscular, or subcutaneous injection.

[0071] In some embodiments, the subject is a human. [0072] In a further aspect, the disclosure features a kit comprising (a) the siRNA molecule of any of the foregoing aspects or embodiments of the disclosure, optionally in combination with a pharmaceutically acceptable excipient, carrier, or diluent, and (b) a package insert. In some embodiments, the package insert instructs a user of the kit to perform the method of any of the above aspects or embodiments of the disclosure.

4. BRIEF DESCRIPTION OF THE FIGURES

[0073] FIG. 1 is a graph showing reduction in SNCA mRNA in hSNCA transgenic mice that were administered an siRNA molecule of the disclosure with a sense strand having SEQ ID No.: 664 and an antisense strand having SEQ ID No.: 244. mRNA was quantified in frontal cortex (fCTx), striatum (Cpu), hippocampus (Hp), temporal cortex (tCTx), midbrain, pons, medulla, and cerebellum (Cb), at a timepoint of 1 month, 2 months, 3 months, or 4 months following di-siRNA treatment at a dose level of 30 nmol.

[0074] FIG. 2 is a graph showing reduction in SNCA mRNA in hSNCA transgenic mice that were administered an siRNA molecule of the disclosure with a sense strand having SEQ ID No.: 664 and an antisense strand having SEQ ID No.: 244. mRNA was quantified in cervical spinal cord (SC-C), thoracic spinal cord (SC-T), and lumbar spinal cord (SC-L), at a timepoint of 1 month, 2 months, 3 months, or 4 months following di-siRNA treatment at a dose level of 30 nmol.

[0075] FIG. 3 is a graph showing reduction in human SNCA protein in hSNCA transgenic mice that were administered an siRNA molecule of the disclosure with a sense strand having SEQ ID No.: 664 and an antisense strand having SEQ ID No.: 244. Protein was quantified in frontal cortex (fCTx), striatum (Cpu), hippocampus (Hp), temporal cortex (tCTx), midbrain, and cerebellum (Cb), at a timepoint of 1 month, 2 months, 3 months, or 4 months following di-siRNA treatment at a dose level of 30 nmol.

[0076] FIG. 4 is a graph showing reduction in human SNCA protein in hSNCA transgenic mice that were administered an siRNA molecule of the disclosure with a sense strand having SEQ ID No.: 664 and an antisense strand having SEQ ID No.: 244. Protein was quantified in cervical spinal cord (SC-C), thoracic spinal cord (SC-T), and lumbar spinal cord (SC-L), at a timepoint of 1 month, 2 months, 3 months, or 4 months following di-siRNA treatment at a dose level of 30 nmol.

[0077] FIG. 5 is a graph showing reduction in mouse Snca mRNA in hSNCA transgenic mice that were administered an siRNA molecule of the disclosure with a sense strand having SEQ ID No.: 664 and an antisense strand having SEQ ID No.: 244. mRNA was quantified in frontal cortex, striatum, hippocampus, and temporal cortex, 3 months or 4 months following di-siRNA treatment at 2.5 nmol, 5 nmol, 15 nmol, or 20 nmol dose levels.

[0078] FIG. 6 is a graph showing reduction in human SNCA mRNA in hSNCA transgenic mice that were administered an siRNA molecule of the disclosure with a sense strand having SEQ ID No.: 664 and an antisense strand having SEQ ID No.: 244. mRNA was quantified in frontal cortex, striatum, hippocampus, and temporal cortex, 3 months or 4 months following di-siRNA treatment at 2.5 nmol, 5 nmol, 15 nmol, or 20 nmol dose levels.

[0079] FIG. 7 is a graph showing reduction in human SNCA protein in hSNCA transgenic mice that were administered an siRNA molecule of the disclosure with a sense strand having SEQ ID No.: 664 and an antisense strand having SEQ ID No.: 244. Protein was quantified in frontal cortex, striatum, hippocampus, and temporal cortex, 3 months or 4 months following di-siRNA treatment at 2.5 nmol, 5 nmol, 15 nmol, or 20 nmol dose levels.

5. DETAILED DESCRIPTION

[0080] The instant application is directed to single- or double-stranded interfering RNA molecules (e.g., siRNA) targeting an SNCA gene. The interfering RNA molecules may contain specific patterns of nucleoside modifications and internucleoside linkage modifications, as pharmaceutical compositions including the same. The siRNA molecules may be branched siRNA molecules, such as di-branched, tri-branched, or tetra-branched siRNA molecules. The disclosed siRNA molecules may further feature a 5’ phosphorus stabilizing moiety and/or a hydrophobic moiety. Additionally, the disclosure provides methods for delivering the siRNA molecule of the disclosure to the central nervous system of a subject, such as a subject identified as having a synucleinopathy.

5.1. Definitions

[0081] Unless otherwise defined herein, scientific, and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art. In the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The use of "or" means "and/or" unless stated otherwise. The use of the term "including," as well as other forms, such as "includes" and "included," is not limiting. [0082] As used herein, the term "nucleic acids" refers to RNA or DNA molecules consisting of a chain of ribonucleotides or deoxyribonucleotides, respectively. As used herein, the term "therapeutic nucleic acid" refers to a nucleic acid molecule (e.g., ribonucleic acid) that has partial or complete complementarity to, and interacts with, a disease-associated target mRNA and mediates silencing of expression of the mRNA.

[0083] As used herein, the term "carrier nucleic acid" refers to a nucleic acid molecule (e.g., ribonucleic acid) that has sequence complementarity with, and hybridizes with, a therapeutic nucleic acid. As used herein, the term "3' end" refers to the end of the nucleic acid that contains an unmodified hydroxyl group at the 3' carbon of the ribose ring.

[0084] As used herein, the term "nucleoside" refers to a molecule made up of a heterocyclic base and its sugar.

[0085] As used herein, the term "nucleotide" refers to a nucleoside having a phosphate group on its 3' or 5' sugar hydroxyl group.

[0086] As used herein, the term "siRNA" refers to small interfering RNA duplexes that induce the RNA interference (RNAi) pathway. siRNA molecules can vary in length (generally, between 18 and 30 base pairs) and contain varying degrees of complementarity to their target mRNA. The term "siRNA" includes duplexes of two separate strands, as well as single strands that optionally form hairpin structures including a duplex region.

[0087] As used herein, the term "antisense strand" refers to the strand of the siRNA duplex that contains some degree of complementarity to the target gene.

[0088] As used herein, the term "sense strand" refers to the strand of the siRNA duplex that contains complementarity to the antisense strand.

[0089] As used herein, the term "chemically modified nucleotide" refers to a non-standard nucleotide, including non-naturally occurring ribonucleotides or deoxyribonucleotides. Exemplary chemically modified nucleotides are modified at any position so as to alter certain chemical properties of the nucleotide yet retain the ability of the chemically modified nucleotide to perform its intended function.

[0090] As used herein, the term "metabolically stabilized" refers to RNA molecules that contain ribonucleotides that have been chemically modified from 2'-hydroxyl groups to 2’-O- methyl groups, 2’ fluoro groups, or other modifications known in the art to stabilized RNA to enzymatic and/or non-enzymatic degradation.

[0091] As used herein, the term "phosphorothioate" refers to a phosphate group of a nucleotide that is modified by substituting one or more of the oxygens of the phosphate group with sulfur. [0092] As used herein, the term "ethylene glycol chain" refers to a carbon chain with the formula ((CJfcOH^).

[0093] As used herein, “alkyl” refers to a saturated hydrocarbon group. Alkyl groups may be acyclic or cyclic and contain only C and H when unsubstituted. When an alkyl residue having a specific number of carbons is named, all geometric isomers having that number of carbons are intended to be encompassed and described; thus, for example, “butyl” is meant to include n-butyl, sec-butyl, and iso-butyl. Examples of alkyl include ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like. In some embodiments, alkyl may be substituted. Suitable substituents that may be introduced into an alkyl group include, for example, hydroxy, alkoxy, amino, alkylamino, and halo, among others.

[0094] As used herein, “alkenyl” refers to an acyclic or cyclic unsaturated hydrocarbon group having at least one site of olefinic unsaturation (i.e., having at least one moiety of the formula C=C). Alkenyl groups contain only C and H when unsubstituted. When an alkenyl residue having a specific number of carbons is named, all geometric isomers having that number of carbons are intended to be encompassed and described; thus, for example, “butenyl” is meant to include n-butenyl, sec-butenyl, and iso-butenyl. Examples of alkenyl include -CH=CH2, - CH2-CH=CH2, and -CH2-CH=CH-CH=CH2. In some embodiments, alkenyl may be substituted. Suitable substituents that may be introduced into an alkenyl group include, for example, hydroxy, alkoxy, amino, alkylamino, and halo, among others.

[0095] As used herein, “alkynyl” refers to an acyclic or cyclic unsaturated hydrocarbon group having at least one site of acetylenic unsaturation (i.e., having at least one moiety of the formula C=C). Alkynyl groups contain only C and H when unsubstituted. When an alkynyl residue having a specific number of carbons is named, all geometric isomers having that number of carbons are intended to be encompassed and described; thus, for example, “pentynyl” is meant to include n-pentynyl, sec-pentynyl, iso-pentynyl, and tert-pentynyl. Examples of alkynyl include -C=CH and -OC-CH3. In some embodiments, alkynyl may be substituted. Suitable substituents that may be introduced into an alkynyl group include, for example, hydroxy, alkoxy, amino, alkylamino, and halo, among others.

[0096] As used herein the term "phenyl" denotes a monocyclic arene in which one hydrogen atom from a carbon atom of the ring has been removed. A phenyl group can be unsubstituted or substituted with one or more suitable substituents, wherein the substituent replaces an H of the phenyl group. [0097] As used herein, the term “benzyl” refers to monovalent radical obtained when a hydrogen atom attached to the methyl group of toluene is removed. A benzyl generally has the formula of phenyl-CHz-.

[0098] A benzyl group can be unsubstituted or substituted with one or more suitable substituents. For example, the substituent may replace an H of the phenyl component and/or an H of the methylene (-CH2-) component.

[0099] As used herein, the term "amide" refers to an alkyl, alkenyl, alkynyl, or aromatic group that is attached to an amino-carbonyl functional group.

[0100] As used herein, the term "intemucleoside" and "intemucleotide" refer to the bonds between nucleosides and nucleotides, respectively.

[0101] As used herein, the term "triazol" refers to heterocyclic compounds with the formula (C2H3N3), having a five-membered ring of two carbons and three nitrogens, the positions of which can change resulting in multiple isomers.

[0102] As used herein, the term "terminal group" refers to the group at which a carbon chain or nucleic acid ends.

[0103] As used herein, the term "lipophilic amino acid" refers to an amino acid including a hydrophobic moiety (e.g., an alkyl chain or an aromatic ring).

[0104] As used herein, the term "target of delivery" refers to the organ or part of the body that is desired to deliver the branched oligonucleotide compositions to.

[0105] As used herein, the term “branched siRNA” refers to a compound containing two or more double-stranded siRNA molecules covalently bound to one another. Branched siRNA molecules may be “di-branched,” also referred to herein as “di-siRNA,” wherein the siRNA molecule includes 2 siRNA molecules covalently bound to one another, e.g., by way of a linker. Branched siRNA molecules may be “tri-branched,” also referred to herein as “tri-siRNA,” wherein the siRNA molecule includes 3 siRNA molecules covalently bound to one another, e.g., by way of a linker. Branched siRNA molecules may be “tetra-branched,” also referred to herein as “tetra-siRNA,” wherein the siRNA molecule includes 4 siRNA molecules covalently bound to one another, e.g., by way of a linker.

[0106] As used herein, the term “branch point moiety” refers to a chemical moiety of a branched siRNA structure of the disclosure that may be covalently linked to a 5’ end or a 3’ end of an antisense strand or a sense strand of an siRNA molecule and which may support the attachment of additional single- or double-stranded siRNA molecules. Non-limiting examples of branch point moieties suitable for use in conjunction with the disclosed methods and compositions include, e.g., phosphoroamidite, tosylated solketal, 1,3 -diaminopropanol, pentaerythritol, and any one of the branch point moieties described in US 10,478,503.

[0107] As used herein, the term “5' phosphorus stabilizing moiety” refers to a terminal phosphate group that includes phosphates as well as modified phosphates (e.g., phosphorothioates, phosphodiesters, phosphonates). The phosphate moiety can be located at either terminus, e.g., at the 5'-terminal nucleoside. In one aspect, the terminal phosphate is unmodified having the formula -O-P(=O)(OH)OH. In another aspect, the terminal phosphate is modified such that one or more of the O and OH groups are replaced with H, O, S, N(R’), or alkyl where R’ is H, an amino protecting group, or unsubstituted or substituted alkyl. In some embodiments, the 5' and or 3' terminal group can include from 1 to 3 phosphate moieties that are each, independently, unmodified (di- or tri-phosphates) or modified.

[0108] As used herein, the term “between X and Y” is inclusive of the values of X and Y. For example, “between X and Y” refers to the range of values between the value of X and the value of Y, as well as the value of X and the value of Y.

[0109] As used herein, an "amino acid" refers to a molecule containing amine and carboxyl functional groups and a side chain specific to the amino acid.

[0110] In some embodiments the amino acid is chosen from the group of proteinogenic amino acids. In some embodiments, the amino acid is an L-amino acid or a D-amino acid. In some embodiments, the amino acid is a synthetic amino acid (e.g., a beta-amino acid).

[OHl] It is understood that certain intemucleoside linkages provided herein, including, e.g., phosphodiester and phosphorothioate, include a formal charge of -1 at physiological pH, and that said formal charge will be balanced by a cationic moiety, e.g., an alkali metal such as sodium or potassium, an alkali earth metal such as calcium or magnesium, or an ammonium or guanidinium ion.

[0112] The phosphate group of the nucleotide may also be modified, e.g., by substituting one or more of the oxygens of the phosphate group with sulfur (e.g., phosphorothioates), or by making other substitutions which allow the nucleotide to perform its intended function such as described in, for example, Eckstein, Antisense Nucleic Acid Drug Dev. 10: 117-21, 2000; Rusckowski et al., Antisense Nucleic Acid Drug Dev. 10:333-45, 2000; Stein, Antisense Nucleic Acid Drug Dev. 11 :317-25, 2001; Vorobjev et al., Antisense Nucleic Acid Drug Dev . 11 :77-85, 2001; and US 5,684,143. Certain of the above-referenced modifications (e.g., phosphate group modifications) can decrease the rate of hydrolysis of, for example, polynucleotides including said modifications in vivo or in vitro. [0113] As used herein, the term “complementary” refers to two nucleotides that form canonical Watson-Crick base pairs. For the avoidance of doubt, Watson-Crick base pairs in the context of the present disclosure include adenine-thymine, adenine-uracil, and cytosine-guanine base pairs. A proper Watson-Crick base pair is referred to in this context as a “match,” while each unpaired nucleotide, and each incorrectly paired nucleotide, is referred to as a “mismatch.” Alignment for purposes of determining percent nucleic acid sequence complementarity can be achieved in various ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. [0114] “Percent (%) sequence complementarity” with respect to a reference polynucleotide sequence is defined as the percentage of nucleic acids in a candidate sequence that are complementary to the nucleic acids in the reference polynucleotide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence complementarity. A given nucleotide is considered to be “complementary” to a reference nucleotide as described herein if the two nucleotides form canonical Watson-Crick base pairs. For the avoidance of doubt, Watson-Crick base pairs in the context of the present disclosure include adenine-thymine, adenine-uracil, and cytosine-guanine base pairs. A proper Watson- Crick base pair is referred to in this context as a “match,” while each unpaired nucleotide, and each incorrectly paired nucleotide, is referred to as a “mismatch.” Alignment for purposes of determining percent nucleic acid sequence complementarity can be achieved in various ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal complementarity over the full length of the sequences being compared. As an illustration, the percent sequence complementarity of a given nucleic acid sequence, A, to a given nucleic acid sequence, B, (which can alternatively be phrased as a given nucleic acid sequence, A that has a certain percent complementarity to a given nucleic acid sequence, B) is calculated as follows:

100 multiplied by (the fraction X/Y) where X is the number of complementary base pairs in an alignment (e.g., as executed by computer software, such as BLAST) in that program’s alignment of A and B, and where Y is the total number of nucleic acids in B. It will be appreciated that where the length of nucleic acid sequence A is not equal to the length of nucleic acid sequence B, the percent sequence complementarity of A to B will not equal the percent sequence complementarity of B to A. As used herein, a query nucleic acid sequence is considered to be “completely complementary” to a reference nucleic acid sequence if the query nucleic acid sequence has 100% sequence complementarity to the reference nucleic acid sequence.

[0115] The term “gene silencing” refers to the suppression of gene expression, e.g., transgene, heterologous gene and/or endogenous gene expression, which may be mediated through processes that affect transcription and/or through processes that affect post-transcriptional mechanisms. In some embodiments, gene silencing occurs when an RNAi molecule initiates the inhibition or degradation of the mRNA transcribed from a gene of interest in a sequencespecific manner via RNA interference, thereby preventing translation of the gene's product.

[0116] The phrase “overactive disease driver gene,” as used herein, refers to a gene having increased activity and/or expression that contributes to or causes a disease state in a subject (e.g., a human). The disease state may be caused or exacerbated by the overactive disease driver gene directly or by way of an intermediate gene(s).

[0117] The term “negative regulator,” as used herein, refers to a gene that negatively regulates (e.g., reduces or inhibits) the expression and/or activity of another gene or set of genes.

[0118] The term “positive regulator,” as used herein, refers to a gene that positively regulates (e.g., increases or saturates) the expression and/or activity of another gene or set of genes.

[0119] The term “phosphate moiety” as used herein, refers to a terminal phosphate group that includes phosphates as well as modified phosphates. The phosphate moiety can be located at either terminus, e.g., at the 5'-terminal nucleoside. In one aspect, the terminal phosphate is unmodified having the formula — O — P(=O)(OH)OH. In another aspect, the terminal phosphate is modified such that one or more of the O and OH groups are replaced with H, O, S, N(R’) or alkyl where R’ is H, an amino protecting group or unsubstituted or substituted alkyl. In some embodiments, the 5' and or 3' terminal group can include from 1 to 3 phosphate moieties that are each, independently, unmodified (di or tri -phosphates) or modified.

[0120] In the context of this disclosure, the term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or other nucleic acids. The term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally- occurring (e.g., chemically modified) portions that function similarly. Such chemically modified oligonucleotides can exhibit desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases. [0121] The term “treatment” refers to clinical intervention designed to alter the natural course of the patient or cell being treated during the course of clinical pathology. Desirable effects of treatment include decreasing the rate of disease progression, ameliorating or palliating the disease state, and remission or improved prognosis. For example, a patient is successfully “treated” if one or more symptoms associated with a synucleinopathy described herein are mitigated or eliminated, including, but are not limited to, decreasing symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, and/or prolonging survival of patients. [0122] The term “delaying progression” of a disease refers to deferring, hindering, slowing, retarding, stabilizing, and/or postponing development of a cancer described herein. This delay can be of varying lengths of time, depending on the history of the synucleinopathy and/or patient being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the patient does not develop a synucleinopathy or relapse.

5.2. Anti-Alpha-Synuclein siRNAs

[0123] The instant application is directed to single- or double-stranded interfering RNA molecules (e.g., siRNA) targeting an SNCA gene. For example, but not by way of limitation, siRNA molecules targeting an SNCA gene can be designed to target an SNCA mRNA sequence.

[0124] In certain embodiments, the SNCA gene targeted by an siRNA of the instant disclosure expresses a mRNA comprising the sequence of NM_000345.3 (Homo sapiens synuclein, alpha (non A4 component of amyloid precursor) (SNCA), transcript variant 1, mRNA). The NM 000345.3 is: AGGAGAAGGAGAAGGAGGAGGACTAGGAGGAGGAGGACGG CGACGACCAGAAGGGGCCCAAGAGAGGGGGCGAGCGACCGAGCGCCGCGACGC GGAAGTGAGGTGCGTGCGGGCTGCAGCGCAGACCCCGGCCCGGCCCCTCCGAGA GCGTCCTGGGCGCTCCCTCACGCCTTGCCTTCAAGCCTTCTGCCTTTCCACCCTCG TGAGCGGAGAACTGGGAGTGGCCATTCGACGACAGTGTGGTGTAAAGGAATTCA TTAGCCATGGATGTATTCATGAAAGGACTTTCAAAGGCCAAGGAGGGAGTTGTG GCTGCTGCTGAGAAAACCAAACAGGGTGTGGCAGAAGCAGCAGGAAAGACAAA AGAGGGTGTTCTCTATGTAGGCTCCAAAACCAAGGAGGGAGTGGTGCATGGTGT GGCAACAGTGGCTGAGAAGACCAAAGAGCAAGTGACAAATGTTGGAGGAGCAG TGGTGACGGGTGTGACAGCAGTAGCCCAGAAGACAGTGGAGGGAGCAGGGAGC ATTGCAGCAGCCACTGGCTTTGTCAAAAAGGACCAGTTGGGCAAGAATGAAGAA

GGAGCCCCACAGGAAGGAATTCTGGAAGATATGCCTGTGGATCCTGACAATGAG

GCTTATGAAATGCCTTCTGAGGAAGGGTATCAAGACTACGAACCTGAAGCCTAA

GAAATATCTTTGCTCCCAGTTTCTTGAGATCTGCTGACAGATGTTCCATCCTGTAC

AAGTGCTCAGTTCCAATGTGCCCAGTCATGACATTTCTCAAAGTTTTTACAGTGT

ATCTCGAAGTCTTCCATCAGCAGTGATTGAAGTATCTGTACCTGCCCCCACTCAG

CATTTCGGTGCTTCCCTTTCACTGAAGTGAATACATGGTAGCAGGGTCTTTGTGTG

CTGTGGATTTTGTGGCTTCAATCTACGATGTTAAAACAAATTAAAAACACCTAAG

TGACTACCACTTATTTCTAAATCCTCACTATTTTTTTGTTGCTGTTGTTCAGAAGTT

GTTAGTGATTTGCTATCATATATTATAAGATTTTTAGGTGTCTTTTAATGATACTG

TCTAAGAATAATGACGTATTGTGAAATTTGTTAATATATATAATACTTAAAAATA

TGTGAGCATGAAACTATGCACCTATAAATACTAAATATGAAATTTTACCATTTTG

CGATGTGTTTTATTCACTTGTGTTTGTATATAAATGGTGAGAATTAAAATAAAAC

GTTATCTCATTGCAAAAATATTTTATTTTTATCCCATCTCACTTTAATAATAAAAA

TCATGCTTATAAGCAACATGAATTAAGAACTGACACAAAGGACAAAAATATAAA

GTTATTAATAGCCATTTGAAGAAGGAGGAATTTTAGAAGAGGTAGAGAAAATGG

AACATTAACCCTACACTCGGAATTCCCTGAAGCAACACTGCCAGAAGTGTGTTTT

GGTATGCACTGGTTCCTTAAGTGGCTGTGATTAATTATTGAAAGTGGGGTGTTGA

AGACCCCAACTACTATTGTAGAGTGGTCTATTTCTCCCTTCAATCCTGTCAATGTT

TGCTTTACGTATTTTGGGGAACTGTTGTTTGATGTGTATGTGTTTATAATTGTTAT

ACATTTTTAATTGAGCCTTTTATTAACATATATTGTTATTTTTGTCTCGAAATAATT

TTTTAGTTAAAATCTATTTTGTCTGATATTGGTGTGAATGCTGTACCTTTCTGACA

ATAAATAATATTCGACCATGAATAAAAAAAAAAAAAAAGTGGGTTCCCGGGAAC

TAAGCAGTGTAGAAGATGATTTTGACTACACCCTCCTTAGAGAGCCATAAGACA

CATTAGCACATATTAGCACATTCAAGGCTCTGAGAGAATGTGGTTAACTTTGTTT

AACTCAGCATTCCTCACTTTTTTTTTTTAATCATCAGAAATTCTCTCTCTCTCTCTC

TCTTTTTCTCTCGCTCTCTTTTTTTTTTTTTTTTTACAGGAAATGCCTTTAAACATC

GTTGGAACTACCAGAGTCACCTTAAAGGAGATCAATTCTCTAGACTGATAAAAA

TTTCATGGCCTCCTTTAAATGTTGCCAAATATATGAATTCTAGGATTTTTCCTTAG

GAAAGGTTTTTCTCTTTCAGGGAAGATCTATTAACTCCCCATGGGTGCTGAAAAT

AAACTTGATGGTGAAAAACTCTGTATAAATTAATTTAAAAATTATTTGGTTTCTCT

TTTTAATTATTCTGGGGCATAGTCATTTCTAAAAGTCACTAGTAGAAAGTATAAT

TTCAAGACAGAATATTCTAGACATGCTAGCAGTTTATATGTATTCATGAGTAATG

TGATATATATTGGGCGCTGGTGAGGAAGGAAGGAGGAATGAGTGACTATAAGGA TGGTTACCATAGAAACTTCCTTTTTTACCTAATTGAAGAGAGACTACTACAGAGT GCTAAGCTGCATGTGTCATCTTACACTAGAGAGAAATGGTAAGTTTCTTGTTTTA TTTAAGTTATGTTTAAGCAAGGAAAGGATTTGTTATTGAACAGTATATTTCAGGA AGGTTAGAAAGTGGCGGTTAGGATATATTTTAAATCTACCTAAAGCAGCATATTT TAAAAATTTAAAAGTATTGGTATTAAATTAAGAAATAGAGGACAGAACTAGACT GATAGCAGTGACCTAGAACAATTTGAGATTAGGAAAGTTGTGACCATGAATTTA AGGATTTATGTGGATACAAATTCTCCTTTAAAGTGTTTCTTCCCTTAATATTTATC TGACGGTAATTTTTGAGCAGTGAATTACTTTATATATCTTAATAGTTTATTTGGGA CCAAACACTTAAACAAAAAGTTCTTTAAGTCATATAAGCCTTTTCAGGAAGCTTG TCTCATATTCACTCCCGAGACATTCACCTGCCAAGTGGCCTGAGGATCAATCCAG TCCTAGGTTTATTTTGCAGACTTACATTCTCCCAAGTTATTCAGCCTCATATGACT CCACGGTCGGCTTTACCAAAACAGTTCAGAGTGCACTTTGGCACACAATTGGGA ACAGAACAATCTAATGTGTGGTTTGGTATTCCAAGTGGGGTCTTTTTCAGAATCT CTGCACTAGTGTGAGATGCAAACATGTTTCCTCATCTTTCTGGCTTATCCAGTATG TAGCTATTTGTGACATAATAAATATATACATATATGAAAATA (SEQ ID NO: 858). [0125] In certain embodiments, the SNCA gene targeted by an siRNA of the present disclosure expresses a mRNA comprising the sequence of NM_001146054.1 (Homo sapiens synuclein, alpha (non A4 component of amyloid precursor) (SNCA), transcript variant 2, mRNA). The NM_001146054.1 sequence is: GCCATTCGACGACAGGTTAGCGGGTTTGCCTCCCACT CCCCCAGCCTCGCGTCGCCGGCTCACAGCGGCCTCCTCTGGGGACAGTCCCCCCC GGGTGCCGCCTCCGCCCTTCCTGTGCGCTCCTTTTCCTTCTTCTTTCCTATTAAATA TTATTTGGGAATTGTTTAAATTTTTTTTTTAAAAAAAGAGAGAGGCGGGGAGGAG TCGGAGTTGTGGAGAAGCAGAGGGACTCAGTGTGGTGTAAAGGAATTCATTAGC CATGGATGTATTCATGAAAGGACTTTCAAAGGCCAAGGAGGGAGTTGTGGCTGC TGCTGAGAAAACCAAACAGGGTGTGGCAGAAGCAGCAGGAAAGACAAAAGAGG GTGTTCTCTATGTAGGCTCCAAAACCAAGGAGGGAGTGGTGCATGGTGTGGCAA CAGTGGCTGAGAAGACCAAAGAGCAAGTGACAAATGTTGGAGGAGCAGTGGTG ACGGGTGTGACAGCAGTAGCCCAGAAGACAGTGGAGGGAGCAGGGAGCATTGC

AGCAGCCACTGGCTTTGTCAAAAAGGACCAGTTGGGCAAGAATGAAGAAGGAGC CCCACAGGAAGGAATTCTGGAAGATATGCCTGTGGATCCTGACAATGAGGCTTA TGAAATGCCTTCTGAGGAAGGGTATCAAGACTACGAACCTGAAGCCTAAGAAAT ATCTTTGCTCCCAGTTTCTTGAGATCTGCTGACAGATGTTCCATCCTGTACAAGTG CTCAGTTCCAATGTGCCCAGTCATGACATTTCTCAAAGTTTTTACAGTGTATCTCG AAGTCTTCCATCAGCAGTGATTGAAGTATCTGTACCTGCCCCCACTCAGCATTTC GGTGCTTCCCTTTCACTGAAGTGAATACATGGTAGCAGGGTCTTTGTGTGCTGTG

GATTTTGTGGCTTCAATCTACGATGTTAAAACAAATTAAAAACACCTAAGTGACT

ACCACTTATTTCTAAATCCTCACTATTTTTTTGTTGCTGTTGTTCAGAAGTTGTTAG

TGATTTGCTATCATATATTATAAGATTTTTAGGTGTCTTTTAATGATACTGTCTAA

GAATAATGACGTATTGTGAAATTTGTTAATATATATAATACTTAAAAATATGTGA

GCATGAAACTATGCACCTATAAATACTAAATATGAAATTTTACCATTTTGCGATG

TGTTTTATTCACTTGTGTTTGTATATAAATGGTGAGAATTAAAATAAAACGTTATC

TCATTGCAAAAATATTTTATTTTTATCCCATCTCACTTTAATAATAAAAATCATGC

TTATAAGCAACATGAATTAAGAACTGACACAAAGGACAAAAATATAAAGTTATT

AATAGCCATTTGAAGAAGGAGGAATTTTAGAAGAGGTAGAGAAAATGGAACATT

AACCCTACACTCGGAATTCCCTGAAGCAACACTGCCAGAAGTGTGTTTTGGTATG

CACTGGTTCCTTAAGTGGCTGTGATTAATTATTGAAAGTGGGGTGTTGAAGACCC

CAACTACTATTGTAGAGTGGTCTATTTCTCCCTTCAATCCTGTCAATGTTTGCTTT

ACGTATTTTGGGGAACTGTTGTTTGATGTGTATGTGTTTATAATTGTTATACATTT

TTAATTGAGCCTTTTATTAACATATATTGTTATTTTTGTCTCGAAATAATTTTTTAG

TTAAAATCTATTTTGTCTGATATTGGTGTGAATGCTGTACCTTTCTGACAATAAAT

AATATTCGACCATGAATAAAAAAAAAAAAAAAGTGGGTTCCCGGGAACTAAGCA

GTGTAGAAGATGATTTTGACTACACCCTCCTTAGAGAGCCATAAGACACATTAGC

ACATATTAGCACATTCAAGGCTCTGAGAGAATGTGGTTAACTTTGTTTAACTCAG

CATTCCTCACTTTTTTTTTTTAATCATCAGAAATTCTCTCTCTCTCTCTCTCTTTTT C

TCTCGCTCTCTTTTTTTTTTTTTTTTTACAGGAAATGCCTTTAAACATCGTTGGAAC

TACCAGAGTCACCTTAAAGGAGATCAATTCTCTAGACTGATAAAAATTTCATGGC

CTCCTTTAAATGTTGCCAAATATATGAATTCTAGGATTTTTCCTTAGGAAAGGTTT

TTCTCTTTCAGGGAAGATCTATTAACTCCCCATGGGTGCTGAAAATAAACTTGAT

GGTGAAAAACTCTGTATAAATTAATTTAAAAATTATTTGGTTTCTCTTTTTAATTA

TTCTGGGGCATAGTCATTTCTAAAAGTCACTAGTAGAAAGTATAATTTCAAGACA

GAATATTCTAGACATGCTAGCAGTTTATATGTATTCATGAGTAATGTGATATATA

TTGGGCGCTGGTGAGGAAGGAAGGAGGAATGAGTGACTATAAGGATGGTTACCA

TAGAAACTTCCTTTTTTACCTAATTGAAGAGAGACTACTACAGAGTGCTAAGCTG

CATGTGTCATCTTACACTAGAGAGAAATGGTAAGTTTCTTGTTTTATTTAAGTTAT

GTTTAAGCAAGGAAAGGATTTGTTATTGAACAGTATATTTCAGGAAGGTTAGAA

AGTGGCGGTTAGGATATATTTTAAATCTACCTAAAGCAGCATATTTTAAAAATTT

AAAAGTATTGGTATTAAATTAAGAAATAGAGGACAGAACTAGACTGATAGCAGT

GACCTAGAACAATTTGAGATTAGGAAAGTTGTGACCATGAATTTAAGGATTTATG TGGATACAAATTCTCCTTTAAAGTGTTTCTTCCCTTAATATTTATCTGACGGTAAT TTTTGAGCAGTGAATTACTTTATATATCTTAATAGTTTATTTGGGACCAAACACTT AAACAAAAAGTTCTTTAAGTCATATAAGCCTTTTCAGGAAGCTTGTCTCATATTC ACTCCCGAGACATTCACCTGCCAAGTGGCCTGAGGATCAATCCAGTCCTAGGTTT ATTTTGCAGACTTACATTCTCCCAAGTTATTCAGCCTCATATGACTCCACGGTCGG CTTTACCAAAACAGTTCAGAGTGCACTTTGGCACACAATTGGGAACAGAACAAT CTAATGTGTGGTTTGGTATTCCAAGTGGGGTCTTTTTCAGAATCTCTGCACTAGTG TGAGATGCAAACATGTTTCCTCATCTTTCTGGCTTATCCAGTATGTAGCTATTTGT GACATAATAAATATATACATATATGAAAATA (SEQ ID NO: 859).

[0126] In certain embodiments, the SNCA gene targeted by an siRNA of the present disclosure expresses a mRNA comprising the sequence of NM_001146055.1 (Homo sapiens synuclein, alpha (non A4 component of amyloid precursor) (SNCA), transcript variant 3, mRNA). The NM_001146055.1 sequence is:

ATTCTGGTGTGATCCAGGAACAGCTGTCTTCCAGCTCTG AAAGAGTGTGGTGTAAAGGAATTCATTAGCCATGGATGTATTCATGAAAGGACT TTCAAAGGCCAAGGAGGGAGTTGTGGCTGCTGCTGAGAAAACCAAACAGGGTGT GGCAGAAGCAGCAGGAAAGACAAAAGAGGGTGTTCTCTATGTAGGCTCCAAAAC CAAGGAGGGAGTGGTGCATGGTGTGGCAACAGTGGCTGAGAAGACCAAAGAGC AAGTGACAAATGTTGGAGGAGCAGTGGTGACGGGTGTGACAGCAGTAGCCCAGA AGACAGTGGAGGGAGCAGGGAGCATTGCAGCAGCCACTGGCTTTGTCAAAAAGG ACCAGTTGGGCAAGAATGAAGAAGGAGCCCCACAGGAAGGAATTCTGGAAGAT ATGCCTGTGGATCCTGACAATGAGGCTTATGAAATGCCTTCTGAGGAAGGGTATC AAGACTACGAACCTGAAGCCTAAGAAATATCTTTGCTCCCAGTTTCTTGAGATCT GCTGACAGATGTTCCATCCTGTACAAGTGCTCAGTTCCAATGTGCCCAGTCATGA CATTTCTCAAAGTTTTTACAGTGTATCTCGAAGTCTTCCATCAGCAGTGATTGAAG TATCTGTACCTGCCCCCACTCAGCATTTCGGTGCTTCCCTTTCACTGAAGTGAATA CATGGTAGCAGGGTCTTTGTGTGCTGTGGATTTTGTGGCTTCAATCTACGATGTTA AAACAAATTAAAAACACCTAAGTGACTACCACTTATTTCTAAATCCTCACTATTT TTTTGTTGCTGTTGTTCAGAAGTTGTTAGTGATTTGCTATCATATATTATAAGATT TTTAGGTGTCTTTTAATGATACTGTCTAAGAATAATGACGTATTGTGAAATTTGTT AATATATATAATACTTAAAAATATGTGAGCATGAAACTATGCACCTATAAATACT AAATATGAAATTTTACCATTTTGCGATGTGTTTTATTCACTTGTGTTTGTATATAA ATGGTGAGAATTAAAATAAAACGTTATCTCATTGCAAAAATATTTTATTTTTATC CCATCTCACTTTAATAATAAAAATCATGCTTATAAGCAACATGAATTAAGAACTG ACACAAAGGACAAAAATATAAAGTTATTAATAGCCATTTGAAGAAGGAGGAATT

TTAGAAGAGGTAGAGAAAATGGAACATTAACCCTACACTCGGAATTCCCTGAAG

CAACACTGCCAGAAGTGTGTTTTGGTATGCACTGGTTCCTTAAGTGGCTGTGATT

AATTATTGAAAGTGGGGTGTTGAAGACCCCAACTACTATTGTAGAGTGGTCTATT

TCTCCCTTCAATCCTGTCAATGTTTGCTTTACGTATTTTGGGGAACTGTTGTTTGA

TGTGTATGTGTTTATAATTGTTATACATTTTTAATTGAGCCTTTTATTAACATATAT

TGTTATTTTTGTCTCGAAATAATTTTTTAGTTAAAATCTATTTTGTCTGATATTGGT

GTGAATGCTGTACCTTTCTGACAATAAATAATATTCGACCATGAATAAAAAAAAA

AAAAAAGTGGGTTCCCGGGAACTAAGCAGTGTAGAAGATGATTTTGACTACACC

CTCCTTAGAGAGCCATAAGACACATTAGCACATATTAGCACATTCAAGGCTCTGA

GAGAATGTGGTTAACTTTGTTTAACTCAGCATTCCTCACTTTTTTTTTTTAATCATC

AGAAATTCTCTCTCTCTCTCTCTCTTTTTCTCTCGCTCTCTTTTTTTTTTTTTTTTT A

CAGGAAATGCCTTTAAACATCGTTGGAACTACCAGAGTCACCTTAAAGGAGATC

AATTCTCTAGACTGATAAAAATTTCATGGCCTCCTTTAAATGTTGCCAAATATAT

GAATTCTAGGATTTTTCCTTAGGAAAGGTTTTTCTCTTTCAGGGAAGATCTATTAA

CTCCCCATGGGTGCTGAAAATAAACTTGATGGTGAAAAACTCTGTATAAATTAAT

TTAAAAATTATTTGGTTTCTCTTTTTAATTATTCTGGGGCATAGTCATTTCTAAAA

GTCACTAGTAGAAAGTATAATTTCAAGACAGAATATTCTAGACATGCTAGCAGTT

TATATGTATTCATGAGTAATGTGATATATATTGGGCGCTGGTGAGGAAGGAAGG

AGGAATGAGTGACTATAAGGATGGTTACCATAGAAACTTCCTTTTTTACCTAATT

GAAGAGAGACTACTACAGAGTGCTAAGCTGCATGTGTCATCTTACACTAGAGAG

AAATGGTAAGTTTCTTGTTTTATTTAAGTTATGTTTAAGCAAGGAAAGGATTTGTT

ATTGAACAGTATATTTCAGGAAGGTTAGAAAGTGGCGGTTAGGATATATTTTAAA

TCTACCTAAAGCAGCATATTTTAAAAATTTAAAAGTATTGGTATTAAATTAAGAA

ATAGAGGACAGAACTAGACTGATAGCAGTGACCTAGAACAATTTGAGATTAGGA

AAGTTGTGACCATGAATTTAAGGATTTATGTGGATACAAATTCTCCTTTAAAGTG

TTTCTTCCCTTAATATTTATCTGACGGTAATTTTTGAGCAGTGAATTACTTTATAT

ATCTTAATAGTTTATTTGGGACCAAACACTTAAACAAAAAGTTCTTTAAGTCATA

TAAGCCTTTTCAGGAAGCTTGTCTCATATTCACTCCCGAGACATTCACCTGCCAA

GTGGCCTGAGGATCAATCCAGTCCTAGGTTTATTTTGCAGACTTACATTCTCCCA

AGTTATTCAGCCTCATATGACTCCACGGTCGGCTTTACCAAAACAGTTCAGAGTG

CACTTTGGCACACAATTGGGAACAGAACAATCTAATGTGTGGTTTGGTATTCCAA

GTGGGGTCTTTTTCAGAATCTCTGCACTAGTGTGAGATGCAAACATGTTTCCTCAT CTTTCTGGCTTATCCAGTATGTAGCTATTTGTGACATAATAAATATATACATATAT GAAAATA (SEQ ID NO: 860)

[0127] In certain embodiments, the SNCA gene targeted by an siRNA of the present disclosure expresses a mRNA comprising the sequence of NM_007308.2 (Homo sapiens synuclein, alpha (non A4 component of amyloid precursor) (SNCA), transcript variant 4, mRNA). NM_007308.2 sequence is:

GCCATTCGACGACAGGTTAGCGGGTTTGCCTCCCACTCCCCCAGCCTC GCGTCGCCGGCTCACAGCGGCCTCCTCTGGGGACAGTCCCCCCCGGGTGCCGCCT CCGCCCTTCCTGTGCGCTCCTTTTCCTTCTTCTTTCCTATTAAATATTATTTGGGAA TTGTTTAAATTTTTTTTTTAAAAAAAGAGAGAGGCGGGGAGGAGTCGGAGTTGTG GAGAAGCAGAGGGACTCAGTGTGGTGTAAAGGAATTCATTAGCCATGGATGTAT TCATGAAAGGACTTTCAAAGGCCAAGGAGGGAGTTGTGGCTGCTGCTGAGAAAA CCAAACAGGGTGTGGCAGAAGCAGCAGGAAAGACAAAAGAGGGTGTTCTCTAT GTAGGCTCCAAAACCAAGGAGGGAGTGGTGCATGGTGTGGCAACAGTGGCTGAG AAGACCAAAGAGCAAGTGACAAATGTTGGAGGAGCAGTGGTGACGGGTGTGAC AGCAGTAGCCCAGAAGACAGTGGAGGGAGCAGGGAGCATTGCAGCAGCCACTG GCTTTGTCAAAAAGGACCAGTTGGGCAAGGAAGGGTATCAAGACTACGAACCTG AAGCCTAAGAAATATCTTTGCTCCCAGTTTCTTGAGATCTGCTGACAGATGTTCC ATCCTGTACAAGTGCTCAGTTCCAATGTGCCCAGTCATGACATTTCTCAAAGTTTT TACAGTGTATCTCGAAGTCTTCCATCAGCAGTGATTGAAGTATCTGTACCTGCCC CCACTCAGCATTTCGGTGCTTCCCTTTCACTGAAGTGAATACATGGTAGCAGGGT CTTTGTGTGCTGTGGATTTTGTGGCTTCAATCTACGATGTTAAAACAAATTAAAA ACACCTAAGTGACTACCACTTATTTCTAAATCCTCACTATTTTTTTGTTGCTGTTG TTCAGAAGTTGTTAGTGATTTGCTATCATATATTATAAGATTTTTAGGTGTCTTTT AATGATACTGTCTAAGAATAATGACGTATTGTGAAATTTGTTAATATATATAATA CTTAAAAATATGTGAGCATGAAACTATGCACCTATAAATACTAAATATGAAATTT TACCATTTTGCGATGTGTTTTATTCACTTGTGTTTGTATATAAATGGTGAGAATTA AAATAAAACGTTATCTCATTGCAAAAATATTTTATTTTTATCCCATCTCACTTTAA TAATAAAAATCATGCTTATAAGCAACATGAATTAAGAACTGACACAAAGGACAA AAATATAAAGTTATTAATAGCCATTTGAAGAAGGAGGAATTTTAGAAGAGGTAG AGAAAATGGAACATTAACCCTACACTCGGAATTCCCTGAAGCAACACTGCCAGA AGTGTGTTTTGGTATGCACTGGTTCCTTAAGTGGCTGTGATTAATTATTGAAAGTG GGGTGTTGAAGACCCCAACTACTATTGTAGAGTGGTCTATTTCTCCCTTCAATCCT GTCAATGTTTGCTTTACGTATTTTGGGGAACTGTTGTTTGATGTGTATGTGTTTAT AATTGTTATACATTTTTAATTGAGCCTTTTATTAACATATATTGTTATTTTTGTCTC GAAATAATTTTTTAGTTAAAATCTATTTTGTCTGATATTGGTGTGAATGCTGTACC TTTCTGACAATAAATAATATTCGACCATGAATAAAAAAAAAAAAAAAGTGGGTT CCCGGGAACTAAGCAGTGTAGAAGATGATTTTGACTACACCCTCCTTAGAGAGC CATAAGACACATTAGCACATATTAGCACATTCAAGGCTCTGAGAGAATGTGGTT

AACTTTGTTTAACTCAGCATTCCTCACTTTTTTTTTTTAATCATCAGAAATTCTCTC TCTCTCTCTCTCTTTTTCTCTCGCTCTCTTTTTTTTTTTTTTTTTACAGGAAATGCCT TTAAACATCGTTGGAACTACCAGAGTCACCTTAAAGGAGATCAATTCTCTAGACT GATAAAAATTTCATGGCCTCCTTTAAATGTTGCCAAATATATGAATTCTAGGATT

TTTCCTTAGGAAAGGTTTTTCTCTTTCAGGGAAGATCTATTAACTCCCCATGGGTG CTGAAAATAAACTTGATGGTGAAAAACTCTGTATAAATTAATTTAAAAATTATTT GGTTTCTCTTTTTAATTATTCTGGGGCATAGTCATTTCTAAAAGTCACTAGTAGAA AGTATAATTTCAAGACAGAATATTCTAGACATGCTAGCAGTTTATATGTATTCAT

GAGTAATGTGATATATATTGGGCGCTGGTGAGGAAGGAAGGAGGAATGAGTGAC TATAAGGATGGTTACCATAGAAACTTCCTTTTTTACCTAATTGAAGAGAGACTAC TACAGAGTGCTAAGCTGCATGTGTCATCTTACACTAGAGAGAAATGGTAAGTTTC TTGTTTTATTTAAGTTATGTTTAAGCAAGGAAAGGATTTGTTATTGAACAGTATAT

TTCAGGAAGGTTAGAAAGTGGCGGTTAGGATATATTTTAAATCTACCTAAAGCAG CATATTTTAAAAATTTAAAAGTATTGGTATTAAATTAAGAAATAGAGGACAGAA CTAGACTGATAGCAGTGACCTAGAACAATTTGAGATTAGGAAAGTTGTGACCAT GAATTTAAGGATTTATGTGGATACAAATTCTCCTTTAAAGTGTTTCTTCCCTTAAT

ATTTATCTGACGGTAATTTTTGAGCAGTGAATTACTTTATATATCTTAATAGTTTA TTTGGGACCAAACACTTAAACAAAAAGTTCTTTAAGTCATATAAGCCTTTTCAGG AAGCTTGTCTCATATTCACTCCCGAGACATTCACCTGCCAAGTGGCCTGAGGATC AATCCAGTCCTAGGTTTATTTTGCAGACTTACATTCTCCCAAGTTATTCAGCCTCA

TATGACTCCACGGTCGGCTTTACCAAAACAGTTCAGAGTGCACTTTGGCACACAA TTGGGAACAGAACAATCTAATGTGTGGTTTGGTATTCCAAGTGGGGTCTTTTTCA GAATCTCTGCACTAGTGTGAGATGCAAACATGTTTCCTCATCTTTCTGGCTTATCC AGTATGTAGCTATTTGTGACATAATAAATATATACATATATGAAAATA (SEQ ID

NO: 861).

[0128] In certain embodiments, the present disclosure is directed to an siRNA molecule comprising an antisense strand and sense strand having complementarity to the antisense strand, wherein the antisense strand is from 10 to 30 nucleotides in length and has complementarity sufficient to hybridize to a region within an SNCA mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 421-840. In certain embodiments, the antisense strand has at least 70% complementarity to a region of 21 contiguous nucleobases within the SNCA mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 421-840. In certain embodiments, the antisense strand has at least 75% complementarity to a region of 21 contiguous nucleobases within the SNCA mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 421-840. For example, but not by way of limitation, the antisense strand can have at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementarity to the region within the SNCA mRNA transcript having the nucleic acid sequence of any one of SEQ ID Nos: 421-840.

[0129] In certain embodiments, the antisense strand comprises at least 10, at least 11, 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, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or 30 contiguous nucleotides that are fully complementary to a contiguous polynucleotide segment of equal length within the region of the SNCA RNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 421-840. In certain embodiments, the antisense strand comprises from 10 to 30 contiguous nucleotides that are fully complementary to a contiguous polynucleotide segment of equal length within the region of the SNCA RNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 421-840. In certain embodiments, the antisense strand comprises from 12 to 30 contiguous nucleotides that are fully complementary to a contiguous polynucleotide segment of equal length within the region of the SNCA RNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 421-840. In certain embodiments, the antisense strand comprises from 15 to 30 contiguous nucleotides that are fully complementary to a contiguous polynucleotide segment of equal length within the region of the SNCA RNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 421-840. In certain embodiments, the antisense strand comprises from 18 to 30 contiguous nucleotides that are fully complementary to a contiguous polynucleotide segment of equal length within the region of the SNCA RNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 421-840. In certain embodiments, the antisense strand comprises from 21 to 30 contiguous nucleotides that are fully complementary to a contiguous polynucleotide segment of equal length within the region of the SNCA RNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 421-840. In certain embodiments, the antisense strand comprises from 24 to 30 contiguous nucleotides that are fully complementary to a contiguous polynucleotide segment of equal length within the region of the SNCA RNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 421-840. In certain embodiments, the antisense strand comprises 30 contiguous nucleotides that are fully complementary to a contiguous polynucleotide segment of equal length within the region of the SNCA RNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 421- 840.

[0130] In certain embodiments, the antisense strand comprises 9 or fewer nucleotide mismatches relative to a region of 21 contiguous nucleobases of the SNCA RNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 421-840. For example, but not by way of limitation, the antisense strand can comprise 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or only 1 mismatch relative to the region of the SNCA RNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 421-840.

[0131] In certain embodiments, the siRNA molecule of the present disclosure targets a region of an SNCA RNA transcript that has the nucleic acid sequence of any one of SEQ ID NOs: 459, 535, 551, 599, 631, 663, 664, 666, 667, 671, 748, 750, 751, 752, 760, 762, and 777.

[0132] In certain embodiments, the present disclosure is directed to an siRNA molecule comprising a sense strand comprising an unmodified sense strand of Table 1A. In certain embodiments, the present disclosure is directed to an siRNA molecule comprising an antisense strand comprising an unmodified antisense strand of Table 1 A. In certain embodiments the sense strand is UACCACUUAUUUCUAA (SEQ ID NO: 760) and the antisense strand is UUAGAAAUAAGUGGUAGUCAC (SEQ ID NO: 340). In certain embodiments the sense strand is CAAGUGCUCAGUUCCA (SEQ ID NO: 664) and the antisense strand is UGGAACUGAGCACUUGUACAG (SEQ ID NO: 244). In certain embodiments the sense strand is AGUGGUGCAUGGUGUA (SEQ ID NO: 535) and the antisense strand is UACACCAUGCACCACUCCCUC (SEQ ID NO: 115). In certain embodiments the sense strand is CAAUGAGGCUUAUGAA (SEQ ID NO: 599) and the antisense strand is UUCAUAAGCCUCAUUGUCAGG (SEQ ID NO: 179). In certain embodiments the sense strand is AGUGCUCAGUUCCAAA (SEQ ID NO: 666) and the antisense strand is UUUGGAACUGAGCACUUGUAC (SEQ ID NO: 246). In certain embodiments the sense strand is UCAGUUCCAAUGUGCA (SEQ ID NO: 671) and the antisense strand is UGCACAUUGGAACUGAGCACU (SEQ ID NO: 251). In certain embodiments the sense strand is CUACGAUGUUAAAACA (SEQ ID NO: 748) and the antisense strand is UGUUUUAACAUCGUAGAUUGA (SEQ ID NO: 328).

[0133] In certain embodiments, the present disclosure is directed to an siRNA molecule comprising a sense strand comprising a modified sense strand of Table IB. In certain embodiments, the present disclosure is directed to an siRNA molecule comprising an antisense strand comprising a modified antisense strand of Table IB. In certain embodiments the sense strand is

(mU)#(mA)#(mC)(fC)(mA)(fC)(mU)(fU)(mA)(fU)(mU)(mU)(mC)(fU )#(mA)#(mA)-DIO (SEQ ID NO: 841) and the antisense strand is

V(mU)#(fU)#(mA)(fG)(fA)(fA)(mA)(fU)(mA)(fA) (mG)(fU)(mG)(fG)(mU)(fA)#(mG)#(mU)#(mC)#(mA)#(mC) (SEQ ID NO: 842) (where m = 2'0me; f = 2'F; # = phosphorothioate; -DIO = a divalent oligonucleotide (DIO) linker; V = vinyl phosphonate In certain embodiments the sense strand is (mC)#(mA)#(mA)(fG)(mU)(fG)(mC)(fU) (mC)(fA)(mG)(mU)(mU)(fC)#(mC)#(mA)-DIO (SEQ ID NO: 843) and the antisense strand is

V(mU)#(fG)#(mG)(fA)(fA)(fC)(mU)(fG)(mA)(fG)(mC)(fA)(mC)(f U)(mU)(fG)#(mU)#(mA) #(mC)#(mA)#(mG) (SEQ ID NO: 844). In certain embodiments the sense strand is (mA)#(mG)#(mU) (fG)(mG)(fU)(mG)(fC)(mA)(fU)(mG)(mG)(mU)(fG)#(mU)#(mA)-DIO (SEQ ID NO: 845) and the antisense strand is

V(mU)#(fA)#(mC)(fA)(fC)(fC)(mA)(fU)(mG)(fC)(mA)(fC)(mC)(f A)(mC)(fU)#(mC)# (mC)#(mC)#(mU)#(mC) (SEQ ID NO: 846). In certain embodiments the sense strand is (mC)#(mA)#(mA)(fU)(mG)(fA)(mG)(fG)(mC)(fU)(mU)(mA)(mU)(fG)#( mA)#(mA)-DIO (SEQ ID NO: 847) and the antisense strand is

V(mU)#(fU)#(mU)(fG)(fG)(fA)(mA)(fC)(mU)(fG)(mA)(fG)(mC)(f A) (mC)(fU)#(mU)#(mG)#(mU)#(mA)#(mC) (SEQ ID NO: 848). In certain embodiments the sense strand is

(mU)#(mC)#(mA)(fG)(mU)(fU)(mC)(fC)(mA)(fA)(mU)(mG)(mU)(fG )#(mC)#(mA)-DIO (SEQ ID NO: 849) and the antisense strand is

V(mU)#(fU)#(mU)(fG)(fG)(fA)(mA)(fC)(mU)(fG)(mA)(fG)(mC)(f A) (mC)(fU)#(mU)#(mG)#(mU)#(mA)#(mC) (SEQ ID NO: 850). In certain embodiments the sense strand is

(mC)#(mU)#(mA)(fC)(mG)(fA)(mU)(fG)(mU)(fU)(mA)(mA)(mA)(fA )#(mC)#(mA)-DIO (SEQ ID NO: 851) and the antisense strand is

V(mU)#(fG)#(mC)(fA)(fC)(fA)(mU)(fU)(mG)(fG)(mA)(fA)(mC)(f U) (mG)(fA)#(mG)#(mC)#(mA)#(mC)#(mU) (SEQ ID NO: 852). In certain embodiments the sense strand is

(mC)#(mU)#(mA)(fC)(mG)(fA)(mU)(fG)(mU)(fU)(mA)(mA)(mA)(fA )#(mC)#(mA)-DIO (SEQ ID NO: 853) and the antisense strand is V(mU)#(fG)#(mU)(fU)(fU)(fU)(mA)(fA)(mC)(fA)(mU)(fC)(mG)(fU) (mA)(fG)#(mA)#(mU)#(mU)#(mG)#(mA) (SEQ ID NO: 854). In certain embodiments, the sense strand is (mC)#(mA)#(mA)(fU)(mG)(fA)(mG)(fG)(mC)

(fU)(mU)(mA)(mU)(fG)#(mA)#(mA)-DIO (SEQ ID NO: 847) and the antisense strand is V(mU)#(fU)#(mC)(fA)(fU)(fA)(mA)(fG)(mC)(fC)(mU)(fC)(mA)(fU)( mU)(fG)#(mU)#(mC)

#(mA)#(mG)#(mG) (SEQ ID NO: 855). In certain embodiments, the sense strand is (mA)#(mG)#(mU)(fG)(mC)(fU) (mC)(fA)(mG)(fU)(mU)(mC)(mC)(fA)#(mA)#(mA)-DIO (SEQ ID NO: 856) and the antisense strand is

V(mU)#(fU)#(mU)(fG)(fG)(fA)(mA)(fC)(mU)(fG)(mA)(fG)(mC)(f A)(mC)(fU)#(mU)#(mG) #(mU)#(mA)#(mC) (SEQ ID NO: 850). In certain embodiments, the sense strand is

(mU)#(mC)#(mA)(fG)(mU) (fU)(mC)(fC)(mA)(fA)(mU)(mG)(mU)(fG)#(mC)#(mA)-DIO (SEQ ID NO: 857) and the antisense strand is

V(mU)#(fG)#(mC)(fA)(fC)(fA)(mU)(fU)(mG)(fG)(mA)(fA)(mC)(f U)(mG) (fA)#(mG)#(mC)#(mA)#(mC)#(mU) (SEQ ID NO: 852).

Table 1A.

Table IB.

Note: m = 2'0me; f = 2'F; # = phosphorothioate; -DIO = a divalent oligonucleotide (DIO) linker; V = vinyl phosphonate.

5.3. siRNA Structure

[0134] The siRNA molecules of the disclosure may be in the form of a single-stranded (ss) or double-stranded (ds) oligonucleotide structure. In some embodiments, the siRNA molecules may be di-branched, tri-branched, or tetra-branched molecules. Furthermore, the siRNA molecules of the disclosure may contain one or more phosphodiester internucleoside linkages and/or an analog thereof, such as a phosphorothioate internucleoside linkage. The siRNA molecules of the disclosure may further contain chemically modified nucleosides having 2’ sugar modifications.

[0135] The simplest siRNAs consist of a ribonucleic acid, including a ss- or ds- structure, formed by a first strand (i.e., antisense strand), and in the case of a ds-siRNA, a second strand (i.e., sense strand). The first strand includes a stretch of contiguous nucleotides that is at least partially complementary to a target nucleic acid. The second strand also includes a stretch of contiguous nucleotides where the second stretch is at least partially identical to a target nucleic acid. The first strand and said second strand may be hybridized to each other to form a doublestranded structure. The hybridization typically occurs by Watson Crick base pairing.

[0136] Depending on the sequence of the first and second strand, the hybridization or base pairing is not necessarily complete or perfect, which means that the first and second strand are not 100% base-paired due to mismatches. One or more mismatches may also be present within the duplex without necessarily impacting the siRNA RNAi activity.

[0137] The first strand contains a stretch of contiguous nucleotides which is essentially complementary to a target nucleic acid. Typically, the target nucleic acid sequence is, in accordance with the mode of action of interfering ribonucleic acids, a ss-RNA, e.g., an mRNA. Such hybridization occurs most likely through Watson Crick base pairing but is not necessarily limited thereto. The extent to which the first strand has a complementary stretch of contiguous nucleotides to a target nucleic acid sequence may be between 80% and 100%, e.g., 80%, 85%, 90%, 95%, or 100% complementary.

[0138] The siRNA molecules described herein may employ modifications to the nucleobase, phosphate backbone, ribose core, 5'- and 3 '-ends, and branching, wherein multiple strands of siRNA may be covalently linked.

[0139] The siRNA molecules described herein may contain one or more phosphodiester internucleoside linkages and/or an analog thereof, such as a phosphorothioate internucleoside linkage, in which oxyanion moieties are electrostatically neutralized by ionic bonding to a divalent metal cation, such as Ba ²⁺ , Be ²⁺ , Ca ²⁺ , Cu ²⁺ , Mg ²⁺ , Mn ²⁺ , Ni ²⁺ , or Zn ²⁺ . In certain embodiments, the one or more divalent cations includes Ca ²⁺ and Mg ²⁺ , optionally wherein the ratio of Ca ²⁺ to Mg ²⁺ is from 1 : 100 to 100:1 (e.g., 1 :75, 1 :50, 1 :25, 1 : 10, 1:5, 1 : 1, 5: 1, 10: 1, 25: 1. 50:1, 75: 1, or 100: 1). In certain embodiments, the Ca ²⁺ and Mg ²⁺ are present in a 1 : 1 ratio. In certain embodiments, the one or more divalent cations displace water from a cationic binding site of the siRNA molecule. In certain embodiments, the degree of saturation of the cationic binding sites by the one or more divalent cations is from about 10% to about 100% (e.g., from about 20% to about 100%, from about 30% to about 100%, from about 40% to about 100%, from about 50% to about 100%, from about 60% to about 100%, from about 70% to about 100%, from about 80% to about 100%, or from about 90% to about 100%). In certain embodiments, the cationic binding site is located within an intemucleoside linkage, such as a phosphodiester linkage and/or a phosphorothioate linkage. For example, the cationic binding site may be an oxyanion moiety within a phosphodiester linage or phosphorothioate linkage. In certain embodiments, the one or more divalent cations are characterized as having an ionic radius ranging from about 30 picometers to about 150 picometers (e.g., from about 30 picometers to about 140 picometers, from about 40 picometers to about 130 picometers, from about 50 picometers to about 120 picometers, from about 60 picometers to about 110 picometers, from about 60 picometers to about 100 picometers, or from about 60 picometers to about 90 picometers).

5.3.1. Lengths of siRNA Molecules

[0140] It is within the scope of the disclosure that any length, known and previously unknown in the art, may be employed for the current invention. As described herein, potential lengths for an antisense strand of the siRNA molecules of the present disclosure is between 10 and 30 nucleotides (e.g., 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides), 15 and 25 nucleotides (e.g., 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, or 25 nucleotides), or 18 and 23 nucleotides (e.g., 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, or 23 nucleotides). In some embodiments, the antisense strand is 20 nucleotides. In some embodiments, the antisense strand is 21 nucleotides. In some embodiments, the antisense strand is 22 nucleotides. In some embodiments, the antisense strand is 23 nucleotides. In some embodiments, the antisense strand is 24 nucleotides. In some embodiments, the antisense strand is 25 nucleotides. In some embodiments, the antisense strand is 26 nucleotides. In some embodiments, the antisense strand is 27 nucleotides. In some embodiments, the antisense strand is 28 nucleotides. In some embodiments, the antisense strand is 29 nucleotides. In some embodiments, the antisense strand is 30 nucleotides.

[0141] In some embodiments, the sense strand of the siRNA molecules of the present disclosure is between 12 and 30 nucleotides (e.g., 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides), or 14 and 23 nucleotides (e.g., 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, or 23 nucleotides). In some embodiments, the sense strand is 15 nucleotides. In some embodiments, the sense strand is 16 nucleotides. In some embodiments, the sense strand is 17 nucleotides. In some embodiments, the sense strand is 18 nucleotides. In some embodiments, the sense strand is 19 nucleotides. In some embodiments, the sense strand is 20 nucleotides. In some embodiments, the sense strand is 21 nucleotides. In some embodiments, the sense strand is 22 nucleotides. In some embodiments, the sense strand is 23 nucleotides. In some embodiments, the sense strand is 24 nucleotides. In some embodiments, the sense strand is 25 nucleotides. In some embodiments, the sense strand is 26 nucleotides. In some embodiments, the sense strand is 27 nucleotides. In some embodiments, the sense strand is 28 nucleotides. In some embodiments, the sense strand is 29 nucleotides. In some embodiments, the sense strand is 30 nucleotides.

5.3.2. 2’ Sugar Modifications

[0142] The present disclosure may include ss- and ds- siRNA molecule compositions including at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or more) nucleosides having 2’ sugar modifications. Possible 2'-modifications include all possible orientations of OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Cl to CIO alkyl or C2 to CIO alkenyl and alkynyl. In some embodiments, the modification includes a 2’-O-methyl (2’-O-Me) modification. Other potential sugar substituent groups include: Cl to CIO lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF ₃ , OCF ₃ , SOCH3, SO2CH3, ONO2, NO ₂ , N ₃ , NH ₂ , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. In some embodiments, the modification includes 2 '-methoxy ethoxy (2'-O- CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-M0E). In some embodiments, the modification includes 2 '-dimethylaminooxy ethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2'-DMA0E, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O- dimethylamino-ethoxy-ethyl or 2'-DMAE0E), i.e., 2'-O-CH2OCH2N(CH3)2. Other potential sugar substituent groups include, e.g., aminopropoxy (-OCH2CH2CH2NH2), allyl (-CH2- CH=CH2), -O-allyl (-O-CH2-CH=CH2) and fluoro (F). 2'-sugar substituent groups may be in the arabino (up) position or ribo (down) position. In some embodiments, the 2'-arabino modification is 2'-F. Similar modifications may also be made at other positions on the siRNA molecule, particularly the 3' position of the sugar on the 3 ' terminal nucleoside or in 2'-5 ' linked oligonucleotides and the 5' position of 5' terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.

5.3.3. Nucleobase Modifications

[0143] The siRNA molecules of the disclosure may also include nucleosides or other surrogate or mimetic monomeric subunits that include a nucleobase (often referred to in the art simply as "base" or "heterocyclic base moiety"). The nucleobase is another moiety that has been extensively modified or substituted and such modified and or substituted nucleobases are amenable to the present disclosure. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Chemically modified nucleobases also referred herein as heterocyclic base moieties include other synthetic and natural nucleobases such as 5- methylcytosine (5-me-C), 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2- aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5- halouracil and cytosine, 5-propynyl (-C=C-CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4- thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8- azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3 -deazaguanine and 3- deazaadenine. Nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2- aminopyridine and 2-pyridone. Further nucleobases include those disclosed in US 3,687,808, those disclosed in Kroschwitz, J.I., ed. The Concise Encyclopedia of Polymer Science and Engineering, New York, John Wiley & Sons, 1990, pp. 858-859; those disclosed by Englisch et \.,Angewandte Chemie, International Edition 30:613, 1991; and those disclosed by Sanghvi, Y.S., Chapter 16, Antisense Research and Applications, CRC Press, Gait, M.J. ed., 1993, pp. 289-302. The siRNA molecules of the present disclosure may also include polycyclic heterocyclic compounds in place of one or more heterocyclic base moieties. A number of tricyclic heterocyclic compounds have been previously reported. These compounds are routinely used in antisense applications to increase the binding properties of the modified strand to a target strand.

[0144] Representative cytosine analogs that make three hydrogen bonds with a guanosine in a second strand include l,3-diazaphenoxazine-2-one (Kurchavov et al., Nucleosides and Nucleotides, 16: 1837-46, 1997), l,3-diazaphenothiazine-2-one (Lin et al. Am. Chem. Soc., 117:3873-4, 1995), and 6,7,8,9-tetrafluoro-l,3-diazaphenoxazine-2-one (Wang et al., Tetrahedron Lett., 39:8385-8, 1998). Incorporated into oligonucleotides, these base modifications were shown to hybridize with complementary guanine and the latter was also shown to hybridize with adenine and to enhance helical thermal stability by extended stacking interactions (also see US 10/155,920 and US 10/013,295, both of which are herein incorporated by reference in their entirety). Further helix-stabilizing properties have been observed when a cytosine analog/substitute has an aminoethoxy moiety attached to the rigid 1,3- diazaphenoxazine-2-one scaffold (Lin et al., Am. Chem. Soc., 120:8531-2, 1998).

5.3.4. Internucleoside Linkage Modifications

[0145] Another variable in the design of the present disclosure is the internucleoside linkage making up the phosphate backbone of the siRNA molecule. Although the natural RNA phosphate backbone may be employed here, derivatives thereof may be used which enhance desirable characteristics of the siRNA molecule. In certain embodiments, the siRNA molecule will be modified to exhibit reduced hydrolysis relative to the unmodified siRNA. One example of a modification that decreases the rate of hydrolysis is phosphorothioates. Any portion or the whole of the backbone may contain phosphate substitutions (e.g., phosphorothioates). For instance, the internucleoside linkages may be between 0 and 100% phosphorothioate, e.g., between 0 and 100%, 10 and 100%, 20 and 100%, 30 and 100%, 40 and 100%, 50 and 100%, 60 and 100%, 70 and 100%, 80 and 100%, 90 and 100%, 0 and 90%, 0 and 80%, 0 and 70%, 0 and 60%, 0 and 50%, 0 and 40%, 0 and 30%, 0 and 20%, 0 and 10%, 10 and 90%, 20 and 80%, 30 and 70%, 40 and 60%, 10 and 40%, 20 and 50%, 30 and 60%, 40 and 70%, 50 and 80%, or 60 and 90% phosphorothioate linkages. Similarly, the internucleoside linkages may be between 0 and 100% phosphodiester linkages, e.g., between 0 and 100%, 10 and 100%, 20 and 100%, 30 and 100%, 40 and 100%, 50 and 100%, 60 and 100% 70 and 100%, 80 and 100%, 90 and 100%, 0 and 90%, 0 and 80%, 0 and 70%, 0 and 60%, 0 and 50%, 0 and 40%, 0 and 30%, 0 and 20%, 0 and 10%, 10 and 90%, 20 and 80%, 30 and 70%, 40 and 60%, 10 and 40%, 20 and 50%, 30 and 60%, 40 and 70%, 50 and 80%, or 60 and 90% phosphodiester linkages.

[0146] Specific examples of some potential siRNA molecules useful in this invention include oligonucleotides containing modified e.g., non-naturally occurring intemucleoside linkages. As defined in this specification, oligonucleotides having modified internucleoside linkages include internucleoside linkages that retain a phosphorus atom and intemucleoside linkages that do not have a phosphorus atom. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their intemucleoside backbone can also be considered to be oligonucleosides. An exemplary phosphorus containing modified intemucleoside linkage is the phosphorothioate intemucleoside linkage. In some embodiments, the modified oligonucleotide backbones containing a phosphorus atom therein include, for example, phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkyl -phosphonates, thionoalkylphosphotriesters, selenophosphates, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage. Exemplary U.S. patents describing the preparation of phosphorus-containing linkages include but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050;

6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199;

6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294;

6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat. RE39464, the entire contents of each of which are hereby incorporated herein by reference.

[0147] In some embodiments, the modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioform acetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts. Non-limiting examples of U. S. patents that teach the preparation of non-phosphorus backbones include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, the entire contents of each of which are hereby incorporated herein by reference.

5.4. Patterns of Modifications of siRNA Molecules

[0148] The following section provides a set of exemplary scaffolds into which the siRNA molecules of the disclosure may be incorporated.

[0149] In some embodiments of the disclosure, the siRNA may contain an antisense strand including a region represented by Formula I, wherein Formula I is, in the 5’-to-3’ direction

A-B-(A C-P ² -D-Pk(C’-P C’ Formula I; wherein A is represented by the formula C-P^D-P ¹ ; each A’ is represented by the formula C- P ² -D-P ² ; B is represented by the formula C-P ² -D-P ² -D-P ² -D-P ² ; each C is a 2’-O-methyl (2’- O-Me) ribonucleoside; each C’, independently, is a 2’-0-Me ribonucleoside or a 2’-fluoro (2’- F) ribonucleoside; each D is a 2’-F ribonucleoside; each P ¹ is a phosphorothioate internucleoside linkage; each P ² is a phosphodiester internucleoside linkage; j is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7); and k is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7). In some embodiments, j is 4. In some embodiments, k is 4. In some embodiments, j is 4 and k is 4. The antisense is complementary (e.g., fully or partially complementary) to a target nucleic acid sequence.

[0150] In some embodiments, the antisense strand includes a structure represented by Formula Al, wherein Formula Al is, in the 5’-to-3’ direction:

A-S-B-S-A-O-B-O-B-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A -O-B-S-A-S-A-S- A-S-B-S-A

Formula Al; wherein A represents a 2’-O-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0151] In some embodiments of the disclosure, the siRNA may contain an antisense strand including a region represented by Formula II, wherein Formula II is, in the 5’-to-3’ direction: Formula II; wherein A is represented by the formula C-P^D-P ¹ ; each A’ is represented by the formula C- P ² -D-P ² ; B is represented by the formula C-P ² -D-P ² -D-P ² -D-P ² ; each C is a 2’-O-methyl (2’- O-Me) ribonucleoside; each C’, independently, is a 2’-O-Me ribonucleoside or a 2’-fluoro (2’- F) ribonucleoside; each D is a 2’-F ribonucleoside; each P ¹ is a phosphorothioate internucleoside linkage; each P ² is a phosphodiester internucleoside linkage; j is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7); and k is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7). In some embodiments, j is 4. In some embodiments, k is 4. In some embodiments, j is 4 and k is 4. The antisense is complementary (e.g., fully or partially complementary) to a target nucleic acid sequence.

[0152] In some embodiments of the disclosure, the antisense strand includes a structure represented by Formula A2, wherein Formula A2 is, in the 5’-to-3’ direction: A-S-B-S-A-O-B-O-B-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O- B-S-A-S-A-S- A-S-A-S-A

Formula A2;

[0153] wherein A represents a 2’-O-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0154] In some embodiments of the disclosure, the sense strand includes a structure represented by Formula III, wherein Formula III is, in the 5’-to-3’ direction:

E-(A’) _m -F

Formula III; wherein E is represented by the formula (C-P ¹ ^; F is represented by the formula (C-P^-D-P ¹ - P ² are as defined in Formula I; and m is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7). In some embodiments, m is 4. The sense strand is complementary (e.g., fully or partially complementary) to the antisense strand.

[0155] In some embodiments of the disclosure, the sense strand includes a structure represented by Formula SI, wherein Formula SI is, in the 5’-to-3’ direction:

A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-A-O-A-O-B-S-A -S-A

Formula SI; wherein A represents a 2’-O-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0156] In some embodiments of the disclosure, the sense strand includes a structure represented by Formula S2, wherein Formula S2 is, in the 5’-to-3’ direction:

A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-A-O-A-O-B-O-A -O-A

Formula S2; wherein A represents a 2’-0-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester intemucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0157] In some embodiments of the disclosure, the sense strand includes a structure represented by Formula S3, wherein Formula S3 is, in the 5’-to-3’ direction:

A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-A-O-A-O-B-S-A -S-B

Formula S3; wherein A represents a 2’-O-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0158] In some embodiments of the disclosure, the sense strand includes a structure represented by Formula S4, wherein Formula S4 is, in the 5’-to-3’ direction:

A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-A-O-A-O-B-O-A -O-B

Formula S4; wherein A represents a 2’-O-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0159] In some embodiments of the disclosure, the siRNA may contain an antisense strand including a region represented by Formula IV, wherein Formula IV is, in the 5’-to-3’ direction:

Formula IV; wherein A is represented by the formula C-P^D-P ¹ ; each A’ is represented by the formula C- P ² -D-P ² ; B is represented by the formula D-P^C-P^D-P ¹ ; each C is a 2’-O-Me ribonucleoside; each C’, independently, is a 2’-O-Me ribonucleoside or a 2’-F ribonucleoside; each D is a 2’- F ribonucleoside; each P ¹ is a phosphorothioate intemucleoside linkage; each P ² is a phosphodiester intemucleoside linkage; j is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7); and k is an integer from 1 to7 (e.g., 1, 2, 3, 4, 5, 6, or 7). In some embodiments, j is 6. In some embodiments, k is 4. In some embodiments, j is 6 and k is 4. The antisense strand is complementary (e.g., fully or partially complementary) to a target nucleic acid.

[0160] In some embodiments of the disclosure, the antisense strand includes a structure represented by Formula A3, wherein Formula A3 is, in the 5’-to-3’ direction:

A-S-B-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A -O-B-S-A-S-B-S-

A-S-A-S-A

Formula A3; wherein A represents a 2’-O-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0161] In some embodiments of the disclosure, the siRNA of the disclosure may have a sense strand represented by Formula V, wherein Formula V is, in the 5’-to-3’ direction:

E-(A’) _m -C-P ² -F

Formula V;

[0162] wherein E is represented by the formula (C-P ¹ ^; F is represented by the formula D-P ¹ - c-pkc, D-P ² -C-P ² -C, D-PkC-PkD, or D-P ² -C-P ² -D; A’, C, D, P ¹ , and P ² are as defined in Formula IV; and m is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7). In some embodiments, m is 5. The sense strand is complementary (e.g., fully or partially complementary) to the antisense strand.

[0163] In some embodiments of the disclosure, the sense strand includes a structure represented by Formula S5, wherein Formula S5 is, in the 5’-to-3’ direction:

A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-S-A -S-A

Formula S5; wherein A represents a 2’-O-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0164] In some embodiments of the disclosure, the sense strand includes a structure represented by Formula S6, wherein Formula S6 is, in the 5’-to-3’ direction: A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O- A

Formula S6; wherein A represents a 2’-0-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0165] In some embodiments of the disclosure, the sense strand includes a structure represented by Formula S7, wherein Formula S7 is, in the 5’-to-3’ direction:

A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-S-A -S-B

Formula S7; wherein A represents a 2’-O-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0166] In some embodiments of the disclosure, the sense strand includes a structure represented by Formula S8, wherein Formula S8 is, in the 5’-to-3’ direction:

A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A -O-B

Formula S8; wherein A represents a 2’-O-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0167] In some embodiments of the disclosure, the siRNA may contain an antisense strand including a region represented by Formula VI, wherein Formula VI is, in the 5’-to-3’ direction:

2- B -E-Bk-E-F-Gi-D-P'-C”

Formula VI; wherein A is represented by the formula C-P^D-P ¹ ; each B is represented by the formula C- P ² ; each C is a 2’-O-Me ribonucleoside; each C’, independently, is a 2’-O-Me ribonucleoside or a 2’-F ribonucleoside; each D is a 2’-F ribonucleoside; each E is represented by the formula D-P ² -C-P ² ; F is represented by the formula D-P^C-P ¹ ; each G is represented by the formula C-P ¹ ; each P ¹ is a phosphorothioate internucleoside linkage; each P ² is a phosphodiester internucleoside linkage; j is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7); k is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7); and 1 is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7). In some embodiments, j is 3. In some embodiments, k is 6. In some embodiments, 1 is 2. In some embodiments, j is 3, k is 6, and 1 is 2. The antisense strand is complementary (e.g., fully or partially complementary) to a target nucleic acid.

[0168] In some embodiments of the disclosure, the antisense strand includes a structure represented by Formula A4, wherein Formula A4 is, in the 5’-to-3’ direction:

A-S-B-S-A-O-A-O-A-O-B-O-A-O-A-O-A-O-A-O-A-O-A-O-A-O-B-O-A -O-B-S-A-S-A-S- A-S-B-S-A

Formula A4; wherein A represents a 2’-O-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0169] In some embodiments of the disclosure, the siRNA may contain a sense strand including a region represented by Formula VII, wherein Formula VII is, in the 5’-to-3’ direction:

H-B _m -In-A’-Bo-H-C Formula VII; wherein A’ is represented by the formula C-P ² -D-P ² ; each H is represented by the formula (C- P ^x )2; each I is represented by the formula (D-P ² ); B, C, D, P ¹ , and P ² are as defined in Formula VI; m is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7); n is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7); and o is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7). In some embodiments, m is 3. In some embodiments, n is 3. In some embodiments, o is 3. In some embodiments, m is 3, n is 3, and o is 3. The sense strand is complementary (e.g., fully or partially complementary) to the antisense strand.

[0170] In some embodiments of the disclosure, the sense strand includes a structure represented by Formula S9, wherein Formula S9 is, in the 5’-to-3’ direction: A-S-A-S-A-O-A-O-A-O-B-O-B-O-B-O-A-O-B-O-A-O-A-O-A-O-A-S-A-S- A Formula S9; wherein A represents a 2’-0-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0171] In some embodiments of the disclosure, the siRNA may contain an antisense strand including a region that is represented by Formula VIII:

Z-((A-P-) _n (B-P-) _m ) _q ;

Formula VIII wherein Z is a 5’ phosphorus stabilizing moiety; each A is a 2’-O-methyl (2'-O-Me) ribonucleoside; each B is a 2'-fluoro-ribonucleoside; each P is, independently, an internucleoside linkage selected from a phosphodi ester linkage and a phosphorothioate linkage; n is an integer from 1 to 5 (e.g., 1, 2, 3, 4, or 5); m is an integer from 1 to 5 (e.g., 1, 2, 3, 4, or 5); and q is an integer between 1 and 30 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30).

5.5. Methods of siRNA Synthesis

[0172] The siRNA molecules of the disclosure can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc.

[0173] The siRNA agent can be prepared using solution-phase or solid-phase organic synthesis or both. Organic synthesis offers the advantage that the oligonucleotide including unnatural or chemically modified nucleotides can be easily prepared. siRNA molecules of the disclosure can be prepared using solution-phase or solid-phase organic synthesis or both.

[0174] Further, it is contemplated that for any siRNA agent disclosed herein, further optimization could be achieved by systematically either adding or removing linked nucleosides to generate longer or shorter sequences. Further still, such optimized sequences can be adjusted by, e.g., the introduction of chemically modified nucleosides, and/or modified internucleoside linkages as described herein or as known in the art, including alternative nucleosides, alternative sugar moieties, and/or alternative internucleoside linkages as known in the art and/or discussed herein to further optimize the molecule (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, and/or targeting to a particular location or cell type).

5.6. 5' Phosphorus Stabilizing Moieties

[0175] To further protect the siRNA molecules of this disclosure from degradation, a 5'- phosphorus stabilizing moiety may be employed. A 5 '-phosphorus stabilizing moiety replaces the 5 '-phosphate to prevent hydrolysis of the phosphate. Hydrolysis of the 5 '-phosphate prevents binding to RISC, a necessary step in gene silencing. Any replacement for phosphate that does not impede binding to RISC is contemplated in this disclosure. In some embodiments, the replacement for the 5 '-phosphate is also stable to in vivo hydrolysis. Each strand of a siRNA molecule may independently and optionally employ any suitable 5 '-phosphorus stabilizing moiety.

Formula IX Formula X Formula XI Formula XII

Formula XIII Formula XIV Formula XV Formula XVI

[0176] Some exemplary endcaps are demonstrated in Formulas IX-XVI. Nuc in Formulas IX- XVI represents a nucleobase or nucleobase derivative or replacement as described herein. X in formula IX-XVI represents a 2 ’-modification as described herein. Some embodiments employ hydroxy as in Formula IX, phosphate as in Formula X, vinylphosphonates as in Formula XI and XIV, 5’-methyl-substitued phosphates as in Formula XII, XIII, and XVI, methylenephosphonates as in Formula XV, or vinyl 5'-vinylphsophonate as a 5 '-phosphorus stabilizing moiety as demonstrated in Formula XI. [0177] The present disclosure further provides siRNA molecules having one or more hydrophobic moieties attached thereto. The hydrophobic moiety may be covalently attached to the 5’ end or the 3’ end of the siRNA molecules of the disclosure. Non-limiting examples of hydrophobic moieties suitable for use with the siRNA molecules of the disclosure may include cholesterol, vitamin D, tocopherol, phosphatidylcholine (PC), docosahexaenoic acid, docosanoic acid, PC-docosanoic acid, eicosapentaenoic acid, lithocholic acid or any combination of the aforementioned hydrophobic moieties with PC.

5.7. siRNA Branching

[0178] The siRNA molecules of the disclosure may be branched. For example, the siRNA molecules of the disclosure may have one of several branching patterns, as described herein.

[0179] According to the present disclosure, the siRNA molecules disclosed herein may be branched siRNA molecules. The siRNA molecule may not be branched, or may be dibranched, tri-branched, or tetra-branched, connected through a linker. Each main branch may be further branched to allow for 2, 3, 4, 5, 6, 7, or 8 separate RNA single- or double-strands. The branch points on the linker may stem from the same atom, or separate atoms along the linker. Some exemplary embodiments are listed in Table 2.

Table 2. Branched siRNA structures

[0180] In some embodiments, the siRNA molecule is a branched siRNA molecule. In some embodiments, the branched siRNA molecule is di-branched, tri-branched, or tetra-branched. In some embodiments, the di-branched siRNA molecule is represented by any one of Formulas XVII-XIX, wherein each RNA, independently, is an siRNA molecule, L is a linker, and each X, independently, represents a branch point moiety (e.g., phosphoroamidite, tosylated solketal, 1,3-diaminopropanol, pentaerythritol, or any one of the branch point moieties described in US 10,478,503).

[0181] In some embodiments, the tri-branched siRNA molecule represented by any one of Formulas XX-XXIII, wherein each RNA, independently, is an siRNA molecule, L is a linker, and each X, independently, represents a branch point moiety.

[0182] In some embodiments, the tetra-branched siRNA molecule represented by any one of Formulas XXIV-XXVIII, wherein each RNA, independently, is an siRNA molecule, L is a linker, and each X, independently, represents a branch point moiety.

[0183] Multiple strands of siRNA described herein may be covalently attached by way of a linker. The effect of this branching improves, inter alia, cell permeability allowing better access into cells (e.g., neurons or glial cells) in the CNS. Any linking moiety may be employed which is not incompatible with the siRNAs of the present invention. Linkers include ethylene glycol chains of 2 to 10 subunits (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 subunits), alkyl chains, carbohydrate chains, block copolymers, peptides, RNA, DNA, and others. In some embodiments, any carbon or oxygen atom of the linker is optionally replaced with a nitrogen atom, bears a hydroxyl substituent, or bears an oxo substituent. In some embodiments, the linker is a poly-ethylene glycol (PEG) linker. The PEG linkers suitable for use with the disclosed compositions and methods include linear or non-linear PEG linkers. Examples of non-linear PEG linkers include branched PEGs, linear forked PEGs, or branched forked PEGs. [0184] PEG linkers of various weights may be used with the disclosed compositions and methods. For example, the PEG linker may have a weight that is between 5 and 500 Daltons. In some embodiments, a PEG linker having a weight that is between 500 and 1,000 Dalton may be used. In some embodiments, a PEG linker having a weight that is between 1,000 and 10,000 Dalton may be used. In some embodiments, a PEG linker having a weight that is between 200 and 20,000 Dalton may be used. In some embodiments, the linker is covalently attached to a sense strand of the siRNA. In some embodiments, the linker is covalently attached to an antisense strand of the siRNA. In some embodiments, the PEG linker is a triethylene glycol (TrEG) linker. In some embodiments, the PEG linker is a tetraethylene glycol (TEG) linker. [0185] In some embodiments, the linker is an alkyl chain linker. In some embodiments, the linker is a peptide linker. In some embodiments, the linker is an RNA linker. In some embodiments, the linker is a DNA linker.

[0186] Linkers may covalently link 2, 3, 4, or 5 unique siRNA strands. The linker may covalently bind to any part of the siRNA oligomer. In some embodiments, the linker attaches to the 3' end of nucleosides of each siRNA strand. In some embodiments, the linker attaches to the 5' end of nucleosides of each siRNA strand. In some embodiments, the linker attaches to a nucleoside of an siRNA strand (e.g., sense or antisense strand) by way of a covalent bondforming moiety. In some embodiments, the covalent-bond-forming moiety is selected from the group consisting of an alkyl, ester, amide, carbonate, carbamate, triazole, urea, formacetal, phosphonate, phosphate, and phosphate derivative (e.g., phosphorothioate, phosphoramidate, etc.). In certain embodiments the linker is a divalent oligonucleotide (DIO) linker.

[0187] In some embodiments, the linker has a structure of Formula LI :

(Formula LI)

[0188] In some embodiments, the linker has a structure of Formula L2:

(Formula L2)

[0189] In some embodiments, the linker has a structure of Formula L3:

(Formula L3)

[0190] In some embodiments, the linker has a structure of Formula L4:

(Formula L4)

[0191] In some embodiments, the linker has a structure of Formula L5:

(Formula L5)

[0192] In some embodiments, the linker has a structure of Formula L6:

(Formula L6)

[0193] In some embodiments, the linker has a structure of Formula L7, as is shown below:

(Formula L7)

[0194] In some embodiments, the linker has a structure of Formula L8:

(Formula L8)

[0195] In some embodiments, the linker has a structure of Formula L9:

(Formula L9)

[0196] In some embodiments, the selection of a linker for use with one or more of the branched siRNA molecules disclosed herein may be based on the hydrophobicity of the linker, such that, e.g., desirable hydrophobicity is achieved for the one or more branched siRNA molecules of the disclosure. For example, a linker containing an alkyl chain may be used to increase the hydrophobicity of the branched siRNA molecule as compared to a branched siRNA molecule having a less hydrophobic linker or a hydrophilic linker.

[0197] The siRNA agents disclosed herein may be synthesized and/or modified by methods well established in the art, such as those described in Beaucage, S. L. et al. (edrs.), Current Protocols in Nucleic Acid Chemistry, John Wiley & Sons, Inc., New York, N.Y., 2000, which is hereby incorporated herein by reference.

5.8. Methods of Treatment

[0198] The SNCA -targeting siRNA molecules of the disclosure may be delivered to a subject, thereby treating a synucleinopathy. In certain embodiments, the synucleinopathy is Parkinson’s disease, Alzheimer's disease, Lewy body dementia, multiple symptom atrophy, or any other medical risk(s) associated with the SNCA gene. For example, the siRNA molecules may be delivered to a subject, thereby treating a synucleinopathy and/or mitigating synucleinopathy associated phenotypes. Exemplary synucleinopathies include Parkinson’s disease, Alzheimer's disease, Lewy body dementia, multiple symptom atrophy, or any other medical risk(s) associated with the SNCA gene. Furthermore, the siRNA molecules of the disclosure may also be delivered to a subject having a variant of the SNCA gene for which siRNA-mediated gene silencing of the SNCA variant gene reduces the expression level of SNCA transcript, thereby treating a synucleinopathy, e.g., Parkinson’s disease, Alzheimer's disease, Lewy body dementia, multiple symptom atrophy, or any other medical risk(s) associated with the SNCA gene. [0199] The disclosure provides methods of treating a subject by way of SNCA gene silencing with one or more of the siRNA molecules described herein. The gene silencing may be performed in a subject to silence wild type SNCA transcripts, mutant SNCA transcripts, splice isoforms of SNCA transcripts, and/or overexpressed SNCA transcripts thereof, relative to a healthy subject. The method may include delivering to the CNS or affected tissues of the subject (e.g., a human) the siRNA molecules of the disclosure or a pharmaceutical composition containing the same by any appropriate route of administration (e.g., intracerebroventricular, intrathecal injection, intrastriatal injection, intra-cistema magna injection by catheterization, intraparenchymal injection, intravenous injection, subcutaneous injection, or intramuscular injection). The active compound can be administered in any suitable dose. The actual dosage amount of a composition of the present disclosure administered to a patient can be determined by physical and physiological factors such as body weight, severity of condition, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. Depending upon the dosage and the route of administration, the number of administrations of an exemplary dosage and/or an effective amount may vary according to the response of the subject. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject. Administration may occur any suitable number of times per day, and for as long as necessary. Subjects may be adult or pediatric humans, with or without comorbid diseases.

5.8.1. Selection of Subjects

[0200] Subjects that may be treated with the siRNA molecules disclosed herein are subjects in need of treatment of, for example, a synucleinopathy. In certain embodiments, the synucleinopathy is Parkinson’s disease, Alzheimer's disease, Lewy body dementia, multiple symptom atrophy, or any other medical risk(s) associated with the SNCA gene. Subjects that may be treated with the siRNA molecules disclosed herein may include, for example, humans, monkeys, rats, mice, pigs, and other mammals containing at least one orthologous copy of the SNCA gene. Subjects may be adult or pediatric humans, with or without comorbid diseases.

5.8.2. Pharmaceutical Compositions

[0201] The siRNA molecules in the present disclosure may be formulated into a pharmaceutical composition for administration to a subject in a biologically compatible form suitable for administration in vivo. Accordingly, the present disclosure provides a pharmaceutical composition containing a siRNA molecule of the disclosure in admixture with a suitable diluent, carrier, or excipient. The siRNA molecules may be administered, for example, directly into the CNS or affected tissues of the subject (e.g., by way of intracerebroventricular, intrastriatally, intrathecal injection, intra-ci sterna magna injection by catheterization, intraparenchymal injection, intravenous injection, subcutaneous injection, or intramuscular injection).

[0202] Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington, J.P. The Science and Practice of Pharmacy, Easton, PA. Mack Publishers, 2012, 22 ^nd ed. and in The United States Pharmacopeial Convention, The National Formulary, United States Pharmacopeial, 2015, USP 38 NF 33).

[0203] Under ordinary conditions of storage and use, a pharmaceutical composition may contain a preservative, e.g., to prevent the growth of microorganisms. Pharmaceutical compositions may include sterile aqueous solutions, dispersions, or powders, e.g., for the extemporaneous preparation of sterile solutions or dispersions. In all cases the form may be sterilized using techniques known in the art and may be fluidized to the extent that may be easily administered to a subject in need of treatment.

[0204] A pharmaceutical composition may be administered to a subject, e.g., a human subject, alone or in combination with pharmaceutically acceptable carriers, as noted herein, the proportion of which may be determined by the solubility and/or chemical nature of the compound, chosen route of administration, and standard pharmaceutical practice.

5.8.3. Dosing Regimens

[0205] A physician having ordinary skill in the art can readily determine an effective amount of the siRNA molecule for administration to a mammalian subject (e.g., a human) in need thereof. For example, a physician could start prescribing doses of one the siRNA molecules of the disclosure at levels lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. Alternatively, a physician may begin a treatment regimen by administering one of the siRNA molecules of the disclosure at a high dose and subsequently administer progressively lower doses until reaching a minimal dosage at which a therapeutic effect is achieved (e.g., a reduction in expression of a target gene sequence). In general, a suitable daily dose of one of the siRNA molecules of the disclosure will be an amount of the siRNA molecule which is the lowest dose effective to produce a therapeutic effect. The ss- or ds-siRNA molecules of the disclosure may be administered by injection, e.g., intrathecally, intracerebroventricularly, by intra-cistema magna injection by catheterization, intraparenchy-mally, intravenously, subcutaneously, or intramuscularly. A daily dose of a therapeutic composition of the siRNA molecules of the disclosure may be administered as a single dose or as two, three, four, five, six or more doses administered separately at appropriate intervals throughout the day, week, month, or year, optionally, in unit dosage forms. While it is possible for the siRNA molecules of the disclosure to be administered alone, it may also be administered as a pharmaceutical formulation in combination with excipients, carriers, and optionally, additional therapeutic agents.

5.8.4. Routes of Administration

[0206] The method of the disclosure contemplates any route of administration tolerated by the therapeutic composition. Some embodiments of the method include injection intrathecally, intracerebroventricularly, intrastriatally, intraparenchymally, or by intra-cistema magna injection by catheterization.

[0207] Intrathecal injection is the direct injection into the spinal column or subarachnoid space. By injecting directly into the CSF of the spinal column the siRNA molecules of the disclosure have direct access to cells (e.g., neurons and glial cells) in the spinal column and a route to access the cells in the brain by bypassing the blood brain barrier.

[0208] Intracerebroventricular (ICV) injection is a method to directly inject into the CSF of the cerebral ventricles. Similar to intrathecal injection, ICV is a method of injection which bypasses the blood brain barrier. Using ICV allows the advantage of access to the cells of the brain and spinal column without the danger of the therapeutic being degraded in the blood.

[0209] Intrastriatal injection is the direct injection into the striatum, or corpus striatum. The striatum is an area in the subcortical basal ganglia in the brain. Injecting into the striatum bypasses the blood brain barrier and the pharmacokinetic challenges of injection into the blood stream and allows for direct access to the cells of the brain.

[0210] Intraparenchymal administration is the direct injection into the parenchyma (e.g., the brain parenchyma). Injection into the brain parenchyma allows for injection directly into brain regions affected by a disease or disorder while bypassing the blood brain barrier.

[0211] Intra-cisterna magna injection by catheterization is the direct injection into the cisterna magna. The cisterna magna is the area of the brain located between the cerebellum and the dorsal surface of the medulla oblongata. Injecting into the cisterna magna results in more direct delivery to the cells of the cerebellum, brainstem, and spinal cord.

[0212] In some embodiments of the methods described herein, the therapeutic composition may be delivered to the subject by way of systemic administration, e.g., intravenously, intramuscularly, or subcutaneously.

[0213] Intravenous (IV) injection is a method to directly inject into the bloodstream of a subject. The IV administration may be in the form of a bolus dose or by way of continuous infusion, or any other method tolerated by the therapeutic composition.

[0214] Intramuscular (IM) injection is injection into a muscle of a subject, such as the deltoid muscle or gluteal muscle. IM may allow for rapid absorption of the therapeutic composition. [0215] Subcutaneous injection is injection into subcutaneous tissue. Absorption of compositions delivered subcutaneously may be slower than IV or IM injection, which may be beneficial for compositions requiring continuous absorption.

6. ENUMERATED EMBODIMENTS OF THE DISCLOSURE

[0216] The compositions, methods, and kits described herein include the following nonlimiting, exemplary, enumerated embodiments of the disclosure. In some embodiments, the disclosure provides:

Embodiment 1. A small interfering RNA (siRNA) molecule comprising an antisense strand and sense strand having complementarity to the antisense strand, wherein the antisense strand is from 10 to 30 nucleotides in length and has complementarity sufficient to hybridize to a region within an alpha-synuclein (SNCA) mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 421-840.

Embodiment 2. The siRNA molecule of embodiment 1, wherein the antisense strand has at least 70% complementarity to a region of 21 contiguous nucleobases within the SNCA mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 421-840.

Embodiment 3. The siRNA molecule of embodiment 2, wherein the antisense strand has at least 75% complementarity to a region of 21 contiguous nucleobases within the SNCA mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 421-840, optionally wherein the antisense strand has at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementarity to the region within the 421-840 mRNA transcript having the nucleic acid sequence of any one of SEQ ID Nos: 421-840. Embodiment 4. The siRNA molecule of any one of embodiments 1-3, wherein the antisense strand comprises at least 10, at least 11, 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, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or 30 contiguous nucleotides that are fully complementary to a contiguous polynucleotide segment of equal length within the region of the SNCA mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 421-840.

Embodiment 5. The siRNA molecule of embodiment 4, wherein the antisense strand comprises from 10 to 30 contiguous nucleotides that are fully complementary to a contiguous polynucleotide segment of equal length within the region of the SNCA mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 421-840.

Embodiment 6. The siRNA molecule of embodiment 5, wherein the antisense strand comprises from 12 to 30 contiguous nucleotides that are fully complementary to a contiguous polynucleotide segment of equal length within the region of the SNCA mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 421-840.

Embodiment 7. The siRNA molecule of embodiment 6, wherein the antisense strand comprises from 15 to 30 contiguous nucleotides that are fully complementary to a contiguous polynucleotide segment of equal length within the region of the SNCA mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 421-840.

Embodiment 8. The siRNA molecule of embodiment 7, wherein the antisense strand comprises from 18 to 30 contiguous nucleotides that are fully complementary to a contiguous polynucleotide segment of equal length within the region of the SNCA mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 421-840.

Embodiment 9. The siRNA molecule of embodiment 8, wherein the antisense strand comprises from 21 to 30 contiguous nucleotides that are fully complementary to a contiguous polynucleotide segment of equal length within the region of the SNCA mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 421-840.

Embodiment 10. The siRNA molecule of any one of embodiments 1-9, wherein the antisense strand comprises from 24 to 30 contiguous nucleotides that are fully complementary to a contiguous polynucleotide segment of equal length within the region of the SNCA mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 421-840.

Embodiment 11. The siRNA molecule of embodiment 10, wherein the antisense strand comprises 30 contiguous nucleotides that are fully complementary to a contiguous polynucleotide segment of equal length within the region of the SNCA mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 421-840.

Embodiment 12. The siRNA molecule of any one of embodiments 1-11, wherein the antisense strand comprises 9 or fewer nucleotide mismatches relative to a region of 21 contiguous nucleobases of the SNCA mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 421-840, optionally wherein the antisense strand comprises 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or only 1 mismatch relative to the region of the SNCA mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 421-840.

Embodiment 13. The siRNA molecule of any one of embodiments 1-12, wherein the region of the SNCA mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 459, 535, 551, 599, 631, 663, 664, 666, 667, 671, 748, 750, 751, 752, 760, 762, and 777.

Embodiment 14. The siRNA molecule of embodiment 13, wherein the region of the SNCA mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 535, 599, 664, 666, 671, 750, 752, 760, and 762.

Embodiment 15. The siRNA molecule of any one of embodiments 1-14, wherein the antisense strand has a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence of any one of SEQ ID NOs: 1-420.

Embodiment 16. The siRNA molecule of embodiment 15, wherein the antisense strand has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of any one of SEQ ID NOs: 1-420.

Embodiment 17. The siRNA molecule of embodiment 16, wherein the antisense strand has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NOs: 1-420, optionally wherein the antisense strand has a nucleic acid sequence that is at least 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of any one of SEQ ID NOs: 1-420.

Embodiment 18. The siRNA molecule of embodiment 17, wherein the antisense strand has the nucleic acid sequence of any one of SEQ ID NOs: 1-420.

Embodiment 19. The siRNA molecule of any one of embodiments 15-18, wherein the nucleic acid sequence is any one of SEQ ID NOs: 39, 115, 131, 179, 211, 243, 244, 246, 247, 251, 328, 330, 331, 332, 340, 342, and 357.

Embodiment 20. The siRNA molecule of embodiment 19, wherein the nucleic acid sequence is any one of SEQ ID NOs: 115, 179, 244, 246, 251, 330, 332, 340, and 342. Embodiment 21. The siRNA molecule of any one of embodiments 1-20, wherein the sense strand has a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence of any one of SEQ ID NOs: 421-840.

Embodiment 22. The siRNA molecule of embodiment 21, wherein the sense strand has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of any one of SEQ ID NOs: 421-840.

Embodiment 23. The siRNA molecule of embodiment 22, wherein the sense strand has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NOs: 421-840, optionally wherein the sense strand has a nucleic acid sequence that is at least 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of any one of SEQ ID NOs: 421-840.

Embodiment 24. The siRNA molecule of embodiment 23, wherein the sense strand has the nucleic acid sequence of any one of SEQ ID NOs: 421-840.

Embodiment 25. The siRNA molecule of any one of embodiments 21-24, wherein the nucleic acid sequence is any one of SEQ ID NOs: 459, 535, 551, 599, 631, 663, 664, 666, 667, 671, 748, 750, 751, 752, 760, 762, and 777.

Embodiment 26. The siRNA molecule of embodiment 25, wherein the nucleic acid sequence is any one of SEQ ID NOs: 535, 599, 664, 666, 671, 750, 752, 760, and 762. Embodiment 27. The siRNA molecule of any one of embodiments 1-26, wherein the antisense strand comprises a structure represented by Formula I, wherein Formula I is, in the 5’-to-3’ direction:

A-B-(AQj-C-P ² -D-Pk(C’-P C’ Formula I; wherein A is represented by the formula C-P^D-P ¹ ; each A’ is represented by the formula C-P ² -D-P ² ;

B is represented by the formula C-P ² -D-P ² -D-P ² -D-P ² ; each C is a 2’-O-methyl (2’-O-Me) ribonucleoside; each C’, independently, is a 2’-O-Me ribonucleoside or a 2’-fluoro (2’-F) ribonucleoside; each D is a 2’-F ribonucleoside; each P ¹ is a phosphorothioate internucleoside linkage; each P ² is a phosphodiester internucleoside linkage; j is an integer from 1 to 7; and k is an integer from 1 to 7.

Embodiment 28. The siRNA molecule of embodiment 27, wherein the antisense strand comprises a structure represented by Formula Al, wherein Formula Al is, in the 5’-to-3’ direction:

A-S-B-S-A-O-B-O-B-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A -O-B-S-A-S-A-S- A-S-B-S-A

Formula Al; wherein A represents a 2’-0-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

Embodiment 29. The siRNA molecule of any one of embodiments 1-26, wherein the antisense strand comprises a structure represented by Formula II, wherein Formula II is, in the 5’-to-3’ direction:

A-B-(A C-P ² -D-Pk(C-P C’

Formula II; wherein A is represented by the formula C-P^D-P ¹ ; each A’ is represented by the formula C-P ² -D-P ² ;

B is represented by the formula C-P ² -D-P ² -D-P ² -D-P ² ; each C is a 2’-O-methyl (2’-0-Me) ribonucleoside; each C’, independently, is a 2’-0-Me ribonucleoside or a 2’-fluoro (2’-F) ribonucleoside; each D is a 2’-F ribonucleoside; each P ¹ is a phosphorothioate internucleoside linkage; each P ² is a phosphodiester internucleoside linkage; j is an integer from 1 to 7; and k is an integer from 1 to 7. Embodiment 30. The siRNA molecule of embodiment 29, wherein the antisense strand comprises a structure represented by Formula A2, wherein Formula A2 is, in the 5’-to-3’ direction: A-S-B-S-A-O-B-O-B-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O- B-S-A-S-A-S-

A-S-A-S-A

Formula A2; wherein A represents a 2’-O-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

Embodiment 31. The siRNA molecule of any one of embodiments 1-30, wherein the sense strand comprises a structure represented by Formula III, wherein Formula III is, in the 5’-to- 3’ direction:

E-(A’) _m -F Formula III; wherein E is represented by the formula (C-P ¹ )?;

F is represented by the formula (C-P ² )3-D-P ¹ -C-P ¹ -C, (C-P ² )3-D-P ² -C-P ² -C, (C-P ² )3-D-P ¹ -C- pkD, or (C-P ² ) ₃ -D-P ² -C-P ² -D;

A’, C, D, P ¹ , and P ² are as defined in Formula II; and m is an integer from 1 to 7.

Embodiment 32. The siRNA molecule of embodiment 31, wherein the sense strand comprises a structure represented by Formula SI, wherein Formula SI is, in the 5’-to-3’ direction:

A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-A-O-A-O-B-S-A -S-A Formula SI; wherein A represents a 2’-O-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

Embodiment 33. The siRNA molecule of embodiment 31, wherein the sense strand comprises a structure represented by Formula S2, wherein Formula S2 is, in the 5’-to-3’ direction:

A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-A-O-A-O-B-O-A -O-A Formula S2; wherein A represents a 2’-O-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

Embodiment 34. The siRNA molecule of embodiment 31, wherein the sense strand comprises a structure represented by Formula S3, wherein Formula S3 is, in the 5’-to-3’ direction:

A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-A-O-A-O-B-S-A -S-B Formula S3; wherein A represents a 2’-O-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

Embodiment 35. The siRNA molecule of embodiment 31, wherein the sense strand comprises a structure represented by Formula S4, wherein Formula S4 is, in the 5’-to-3’ direction:

A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-A-O-A-O-B-O-A -O-B Formula S4; wherein A represents a 2’-O-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

Embodiment 36. The siRNA molecule of any one of embodiments 1-26 and 31-35, wherein the antisense strand comprises a structure represented by Formula IV, wherein Formula IV is, in the 5’-to-3’ direction:

Formula IV; wherein A is represented by the formula C-P^D-P ¹ ; each A’ is represented by the formula C-P ² -D-P ² ; B is represented by the formula D-P^C-P^D-P ¹ ; each C is a 2’-0-Me ribonucleoside; each C’, independently, is a 2’-0-Me ribonucleoside or a 2’-F ribonucleoside; each D is a 2’-F ribonucleoside; each P ¹ is a phosphorothioate internucleoside linkage; each P ² is a phosphodiester internucleoside linkage; j is an integer from 1 to 7; and k is an integer from 1 to 7. Embodiment 37. The siRNA molecule of embodiment 36, wherein the antisense strand comprises a structure represented by Formula A3, wherein Formula A3 is, in the 5’-to-3’ direction:

A-S-B-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A -O-B-S-A-S-B-S- A-S-A-S-A

Formula A3; wherein A represents a 2’-0-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

Embodiment 38. The siRNA molecule of any one of embodiments 1-30, 36, and 37, wherein the sense strand comprises a structure represented by Formula V, wherein Formula V is, in the 5’-to-3’ direction:

E-(A’) _m -C-P ² -F

Formula V; wherein E is represented by the formula (C-P ¹ )?;

F is represented by the formula D- or D-P ² -C-P ² -D;

A’, C, D, P ¹ and P ² are as defined in Formula IV; and m is an integer from 1 to 7.

Embodiment 39. The siRNA molecule of embodiment 38, wherein the sense strand comprises a structure represented by Formula S5, wherein Formula S5 is, in the 5’-to-3’ direction: A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-S-A-S- A

Formula S5; wherein A represents a 2’-O-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

Embodiment 40. The siRNA molecule of embodiment 38, wherein the sense strand comprises a structure represented by Formula S6, wherein Formula S6 is, in the 5’-to-3’ direction:

A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A -O-A Formula S6; wherein A represents a 2’-O-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

Embodiment 41. The siRNA molecule of embodiment 38, wherein the sense strand comprises a structure represented by Formula S7, wherein Formula S7 is, in the 5’-to-3’ direction:

A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-S-A -S-B Formula S7; wherein A represents a 2’-O-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

Embodiment 42. The siRNA molecule of embodiment 38, wherein the sense strand comprises a structure represented by Formula S8, wherein Formula S8 is, in the 5’-to-3’ direction:

A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A -O-B

Formula S8; wherein A represents a 2’-0-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

Embodiment 43. The siRNA molecule of any one of embodiments 1-26, 31-35 and 38-42, wherein the antisense strand comprises a structure represented by Formula VI, wherein Formula VI is, in the 5’-to-3’ direction:

A-Bj-E-Bk-E-F-Gi-D-PkC’

Formula VI; wherein A is represented by the formula C-P^D-P ¹ ; each B is represented by the formula C-P ² ; each C is a 2’-0-Me ribonucleoside; each C’, independently, is a 2’-0-Me ribonucleoside or a 2’-F ribonucleoside; each D is a 2’-F ribonucleoside; each E is represented by the formula D-P ² -C-P ² ;

F is represented by the formula D-P^C-P ¹ ; each G is represented by the formula C-P ¹ ; each P ¹ is a phosphorothioate internucleoside linkage; each P ² is a phosphodiester internucleoside linkage; j is an integer from 1 to 7; k is an integer from 1 to 7; and

1 is an integer from 1 to 7.

Embodiment 44. The siRNA molecule of embodiment 43, wherein the antisense strand comprises a structure represented by Formula A4, wherein Formula A4 is, in the 5’-to-3’ direction:

A-S-B-S-A-O-A-O-A-O-B-O-A-O-A-O-A-O-A-O-A-O-A-O-A-O-B-O-A -O-B-S-A-S-A-S- A-S-B-S-A

Formula A4; wherein A represents a 2’-0-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage. Embodiment 45. The siRNA molecule of any one of embodiments 1-30, 36, 37, 43, and 44, wherein the sense strand comprises a structure represented by Formula VII, wherein Formula VII is, in the 5’-to-3’ direction:

H-B _m -In-A’-Bo-H-C

Formula VII; wherein A’ is represented by the formula C-P ² -D-P ² ; each H is represented by the formula (C-P ¹ )?; each I is represented by the formula (D-P ² );

B, C, D, P ¹ and P ² are as defined in Formula VI; m is an integer from 1 to 7; n is an integer from 1 to 7; and o is an integer from 1 to 7. Embodiment 46. The siRNA molecule of embodiment 45, wherein the sense strand comprises a structure represented by Formula S9, wherein Formula S9 is, in the 5’-to-3’ direction:

A-S-A-S-A-O-A-O-A-O-B-O-B-O-B-O-A-O-B-O-A-O-A-O-A-O-A-S-A -S-A Formula S9; wherein A represents a 2’-O-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

Embodiment 47. The siRNA molecule of any one of embodiments 1-46, wherein the antisense strand further comprises a 5’ phosphorus stabilizing moiety at the 5’ end of the antisense strand.

Embodiment 48. The siRNA molecule of any one of embodiments 1-47, wherein the sense strand further comprises a 5’ phosphorus stabilizing moiety at the 5’ end of the sense strand. Embodiment 49. The siRNA molecule of embodiment 47 or 48, wherein each 5’ phosphorus stabilizing moiety is, independently, represented by any one of Formulas IX- XVI:

Formula IX Formula X Formula XI Formula XII

Formula XIII Formula XIV Formula XV Formula XVI wherein Nuc represents a nucleobase selected from the group consisting of adenine, uracil, guanine, thymine, and cytosine, and R represents an optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, phenyl, benzyl, hydroxy, or hydrogen. Embodiment 50. The siRNA molecule of embodiment 49, wherein the nucleobase is an adenine, uracil, guanine, thymine, or cytosine.

Embodiment 51. The siRNA molecule of any one of embodiments 47-50, wherein the 5’ phosphorus stabilizing moiety is (E)-vinylphosphonate represented by Formula XI.

Embodiment 52. The siRNA molecule of any one of embodiments 1-51, wherein the siRNA molecule further comprises a hydrophobic moiety at the 5’ or the 3’ end of the siRNA molecule.

Embodiment 53. The siRNA molecule of embodiment 52, wherein the hydrophobic moiety is selected from a group consisting of cholesterol, vitamin D, or tocopherol.

Embodiment 54. The siRNA molecule of any one of embodiments 1-53, wherein the length of the sense strand is between 12 and 30 nucleotides.

Embodiment 55. The siRNA molecule of any one of embodiments 1-54, wherein the siRNA molecule is a branched siRNA molecule.

Embodiment 56. The siRNA molecule of embodiment 55, wherein the branched siRNA molecule is di -branched, tri-branched, or tetra-branched.

Embodiment 57. The siRNA molecule of embodiment 56, wherein the siRNA molecule is a di-branched siRNA molecule, optionally wherein the di-branched siRNA molecule is represented by any one of Formulas XVII-XIX: RNA RNA RNA

X-L-X X-L-X

RNA-L-RNA RNA' RNA RNA' RNA

Formula XVII; Formula XVIII; Formula XIX; wherein each RNA is, independently, an siRNA molecule, L is a linker, and each X, independently, represents a branch point moiety.

Embodiment 58. The siRNA molecule of embodiment 56, wherein the siRNA molecule is a tri-branched siRNA molecule, optionally wherein the tri-branched siRNA molecule is represented by any one of Formulas XX-XXIII:

Formula XX; Formula XXI; Formula XXII; Formula XXIII; wherein each RNA is, independently, an siRNA molecule, L is a linker, and each X, independently, represents a branch point moiety.

Embodiment 59. The siRNA molecule of embodiment 56, wherein the siRNA molecule is a tetra-branched siRNA molecule, optionally wherein the tetra-branched siRNA molecule is represented by any one of Formulas XXIV-XXVIII:

Formula XXIV; Formula XXV; Formula XXVI; Formula XXVII; Formula

XXVIII; wherein each RNA is, independently, an siRNA molecule, L is a linker, and each X, independently, represents a branch point moiety. Embodiment 60. The siRNA molecule of any one of embodiments 57-59, wherein the linker is selected from a group consisting of one or more contiguous subunits of an ethylene glycol, alkyl, carbohydrate, block copolymer, peptide, RNA, and DNA.

Embodiment 61. The siRNA molecule of embodiment 60, wherein the one or more contiguous subunits is 2 to 20 contiguous subunits.

Embodiment 62. An siRNA molecule comprising:

(I) a) a sense strand comprising the sequence (mU)#(mA)#(mC)(fC)(mA)(fC)(mU)(fU)(mA) (fU)(mU)(mU)(mC)(fU)#(mA)#(mA)-DIO (SEQ ID NO: 841) and an antisense strand comprising the sequence V(mU)#(fU)#(mA)(fG)(fA)(fA)(mA)(fU)(mA)(fA)(mG)(fU)(mG)(fG) (mU)(fA)#(mG)#(mU)#(mC)#(mA)#(mC) (SEQ ID NO: 842); b) a sense strand comprising the sequence (mC)#(mA)#(mA)(fG)(mU)(fG)(mC)(fU) (mC)(fA)(mG)(mU)(mU)(fC)#(mC)# (mA)-DIO (SEQ ID NO: 843) and an antisense strand comprising the sequence V(mU)#(fG)#(mG)(fA)(fA)(fC)(mU)(fG)(mA)(fG)(mC)(fA)(mC)(fU)( mU) (fG)#(mU)#(mA)#(mC)#(mA)#(mG) (SEQ ID NO: 844); c) a sense strand comprising the sequence (mA)#(mG)#(mU)(fG)(mG)(fU)(mG)(fC)(mA) (fU)(mG)(mG)(mU)(fG)#(mU)#(mA)-DIO (SEQ ID NO: 845) and an antisense strand comprising the sequence V(mU)#(fA)#(mC)(fA)(fC)(fC)(mA)(fU)(mG)(fC)(mA)(fC)(mC)(fA)( mC) (fU)#(mC)#(mC)#(mC)#(mU)#(mC) (SEQ ID NO: 846). d) a sense strand comprising the sequence (mC)#(mA)#(mA)(fU)(mG)(fA)(mG)(fG)(mC) (fU)(mU)(mA)(mU)(fG)#(mA)#(mA)-DIO (SEQ ID NO: 847) and an antisense strand comprising the sequence V(mU)#(fU)#(mU)(fG)(fG)(fA)(mA)(fC)(mU)(fG)(mA)(fG)(mC)(fA)( mC) (fU)#(mU)#(mG)#(mU)#(mA)#(mC) (SEQ ID NO: 848); e) a sense strand comprising the sequence (mU)#(mC)#(mA)(fG)(mU)(fU)(mC)(fC)(mA) (fA)(mU)(mG)(mU)(fG)#(mC)#(mA)-DIO (SEQ ID NO: 849) and an antisense strand comprising the sequence V(mU)#(fU)#(mU)(fG)(fG)(fA)(mA)(fC)(mU)(fG)(mA)(fG)(mC) (fA)(mC)(fU)#(mU)#(mG)#(mU)#(mA)#(mC) (SEQ ID NO: 850). f) a sense strand comprising the sequence (mC)#(mU)#(mA)(fC)(mG)(fA)(mU)(fG)(mU) (fU)(mA)(mA)(mA)(fA)#(mC)#(mA)-DIO (SEQ ID NO: 851) and an antisense strand comprising the sequence V(mU)#(fG)#(mC)(fA)(fC)(fA)(mU)(fU)(mG)(fG)(mA)(fA)(mC)(fU)( mG) (fA)#(mG)#(mC)#(mA)#(mC)#(mU) (SEQ ID NO: 852); or g) a sense strand comprising the sequence (mC)#(mU)#(mA)(fC)(mG)(fA)(mU)(fG)(mU) (fU)(mA)(mA)(mA)(fA)#(mC)#(mA)-DIO (SEQ ID NO: 853) and an antisense strand comprising the sequence V(mU)#(fG)#(mU)(fU)(fU)(fU)(mA)(fA)(mC)(fA)(mU)(fC)(mG)(fU) (mA)(fG)#(mA)#(mU)#(mU)#(mG)#(mA) (SEQ ID NO: 854); wherein m represents a 2’-O-Me ribonucleoside, f represents a 2’-F ribonucleoside, # represents a phosphorothioate internucleoside linkage, -DIO represents a divalent oligonucleotide (DIO) linker; and V represents a vinyl phosphonate; or

(II) a) a sense strand comprising the sequence (mU)#(mA)#(mC)(fC)(mA)(fC)(mU)(fU)(mA) (fU)(mU)(mU)(mC)(fU)#(mA)#(mA)-DIO (SEQ ID NO: 841) and an antisense strand comprising the sequence V(mU)#(fU)#(mA)(fG)(fA)(fA)(mA)(fU)(mA)(fA)(mG)(fU)(mG)(fG) (mU)(fA)#(mG)#(mU)#(mC)#(mA)#(mC) (SEQ ID NO: 842); b) a sense strand comprising the sequence (mC)#(mA)#(mA)(fG)(mU)(fG)(mC)(fU) (mC)(fA)(mG)(mU)(mU)(fC)#(mC)# (mA)-DIO (SEQ ID NO: 843) and an antisense strand comprising the sequence V(mU)#(fG)#(mG)(fA)(fA)(fC)(mU)(fG)(mA)(fG)(mC)(fA)(mC)(fU)( mU) (fG)#(mU)#(mA)#(mC)#(mA)#(mG) (SEQ ID NO: 844); c) a sense strand comprising the sequence (mA)#(mG)#(mU)(fG)(mG)(fU)(mG)(fC)(mA) (fU)(mG)(mG)(mU)(fG)#(mU)#(mA)-DIO (SEQ ID NO: 845) and an antisense strand comprising the sequence V(mU)#(fA)#(mC)(fA)(fC)(fC)(mA)(fU)(mG)(fC)(mA)(fC)(mC)(fA)( mC) (fU)#(mC)#(mC)#(mC)#(mU)#(mC) (SEQ ID NO: 846). d) a sense strand comprising the sequence (mC)#(mA)#(mA)(fU)(mG)(fA)(mG)(fG)(mC) (fU)(mU)(mA)(mU)(fG)#(mA)#(mA)-DIO (SEQ ID NO: 847) and an antisense strand comprising the sequence V(mU)#(fU)#(mC)(fA)(fU)(fA)(mA)(fG)(mC)(fC)(mU)(fC)(mA)(fU)( mU)(fG)#(mU )#(mC)#(mA)#(mG)#(mG) (SEQ ID NO: 855) e) a sense strand comprising the sequence (mA)#(mG)#(mU)(fG)(mC)(fU) (mC)(fA)(mG)(fU)(mU)(mC)(mC)(fA)#(mA)#(mA)-DIO (SEQ ID NO: 856) and an antisense strand comprising the sequence V(mU)#(fU)#(mU)(fG)(fG)(fA)(mA)(fC)(mU)(fG)(mA)(fG)(mC) (fA)(mC)(fU)#(mU)#(mG)#(mU)#(mA)#(mC) (SEQ ID NO: 850). f) a sense strand comprising the sequence (mU)#(mC)#(mA)(fG)(mU) (fU)(mC)(fC)(mA)(fA)(mU)(mG)(mU)(fG)#(mC)#(mA)-DIO (SEQ ID NO: 857) and an antisense strand comprising the sequence V(mU)#(fG)#(mC)(fA)(fC)(fA)(mU)(fU)(mG)(fG)(mA)(fA)(mC)(fU)( mG) (fA)#(mG)#(mC)#(mA)#(mC)#(mU) (SEQ ID NO: 852); or g) a sense strand comprising the sequence (mC)#(mU)#(mA)(fC)(mG)(fA)(mU)(fG)(mU) (fU)(mA)(mA)(mA)(fA)#(mC)#(mA)-DIO (SEQ ID NO: 853) and an antisense strand comprising the sequence V(mU)#(fG)#(mU)(fU)(fU)(fU)(mA)(fA)(mC)(fA)(mU)(fC)(mG)(fU) (mA)(fG)#(mA)#(mU)#(mU)#(mG)#(mA) (SEQ ID NO: 854); wherein m represents a 2’-O-Me ribonucleoside, f represents a 2’-F ribonucleoside, # represents a phosphorothioate internucleoside linkage, -DIO represents a divalent oligonucleotide (DIO) linker; and V represents a vinyl phosphonate

Embodiment 63. A pharmaceutical composition comprising the siRNA molecule of any one of embodiments 1-62 and a pharmaceutically acceptable excipient, carrier, or diluent.

Embodiment 64. A method of delivering an siRNA molecule to a subject diagnosed as having an , the method comprising administering a therapeutically effective amount of the siRNA molecule of any one of embodiments 1-62 or the pharmaceutical composition of embodiment 63 to the subject. Embodiment 65. A method of treating a synucleinopathy in a subject in need thereof, the method comprising administering a therapeutically effective amount of the siRNA molecule of any one of embodiments 1-62 or the pharmaceutical composition of embodiment 63 to the subject.

Embodiment 66. The method of embodiment 65, wherein the synucleinopathy is Parkinson’s disease.

Embodiment 67. The method of embodiment 65, wherein the synucleinopathy is Alzheimer's disease.

Embodiment 68. The method of embodiment 65, wherein the synucleinopathy is Lewy body dementia.

Embodiment 69. The method of embodiment 65, wherein the synucleinopathy is a multiple symptom atrophy.

Embodiment 70. A method of reducing SNCA expression in a subject in need thereof, the method comprising administering a therapeutically effective amount of the siRNA molecule of any one of embodiments 1-62 or the pharmaceutical composition of embodiment 63 to the subject.

Embodiment 71. The method of any one of embodiments 64-70, wherein the siRNA molecule or the pharmaceutical composition is administered to the subject by way of intracerebroventricular, intrastriatal, intraparenchymal, or intrathecal injection.

Embodiment 72. The method of any one of embodiments 64-71, wherein the siRNA molecule or the pharmaceutical composition is administered to the subject by way of intravenous, intramuscular, or subcutaneous injection.

Embodiment 73. The method of any one of embodiments 64-72, wherein the subject is a human.

Embodiment 74. A kit comprising the siRNA molecule of any one of embodiments 1-62, or the pharmaceutical composition of embodiment 63, and a package insert, wherein the package insert instructs a user of the kit to perform the method of any one of embodiments 64-73. 7. EXAMPLES

[0217] The following examples are put forth so as to provide those of ordinary skill in the art with a description of how the compositions and methods described herein may be used, made, and evaluated, and are intended to be purely exemplary of the disclosure and are not intended to limit the scope of what the inventors regard as their disclosure

Example 1. In Vitro Screening

[0218] In vitro screening was performed using human MeWo cells in a 96-well format. Briefly, cholesterol-conjugated, mono-siRNA was passively administered to cells in culture for 72 hours at 2 uM or 0.5 uM concentrations (single point format) or at serial 1 :2.5 dilutions from 3 uM to 0.00196608 uM (IC50 format). Cells were lysed using the Cells-to-Ct kit (Thermo) and quantitative reverse transcriptase polymerase change reaction (QRT-PCR) was performed to assess target (SNCA) gene expression relative to housekeeping (ATP5B) gene expression using TaqMan primer/probes and Fast Advanced master mix (Thermo). Data are presented as mean % SNCA target expression (N=2-3 biological replicates, 2 technical replicates each) relative to untreated control cells, where 100% represents no knockdown and 0% represents complete target knockdown.

Table 2. In Vitro Screening

Example 2. In Vivo Screening

[0219] In vivo screening was performed using 12 week old hSNCA transgenic mice. Briefly, PBS or divalent siRNA was administered via bilateral stereotactic ICV injection at a dose of 1 nmol or 5 nmol in a total volume of 5 uL. One week after compound administration, animals were euthanized, perfused, and the brain was sliced into 1mm thick cortical slices. Biopsy punches (l-2mm diameter) were taken of the relevant brain regions (Striatum = Cpu, hippocampus = HP, temporal cortex = tCTx) and were snap frozen. Tissue punches were homogenized in Trizol (Invitrogen) using a Qiagen TissueLyser and total RNA was isolated the RNeasy kit (Qiagen). Quantitative reverse transcriptase polymerase change reaction (QRT- PCR) was performed to assess target (human SNCA) gene expression relative to housekeeping (mouse ATP5B) gene expression using TaqMan primer/probes and Fast Advanced master mix (Thermo). Data are presented as % SNCA target knockdown (N=8 animals, 4 male, 4 female, 2 technical replicates each) relative to PBS treated control animals, where 100% represents complete target knockdown and 0% represents no target knockdown.

Table 3. In Vivo Screening

% SNCA Knock-Down

Example 3. In vivo inhibition of SNCA gene expression using a di-siRNA sequence in mouse

Objective

[0220] This Example describes the results of a series of experiments undertaken to investigate the ability of an siRNA molecule complementary to a specific region within a human SNCA transcript to effectuate reduced expression of the SNCA gene.

In vivo administration of di-siRNA

[0221] An siRNA molecule of the disclosure was synthesized as a di -branched siRNA molecule having the structure of Formula XVII. The sense strand had the sequence of SEQ ID No. : 664 and had a pattern of chemical modifications having the broad structure of Formula III and the specific structure of Formula SI. The antisense strand had the sequence of SEQ ID No. : 244 and had a pattern of chemical modifications having the broad structure of Formula II and the specific structure of Formula A2. The antisense strand further included a 5’ vinyl phosphonate moiety of Formula XI.

[0222] In vivo durability was assessed in mouse using similar methods as described in Example 2. Briefly, PBS or the di -branched siRNA molecule described above was administered via bilateral stereotactic intracerebroventricular (ICV) injection at a dose of 30 nmol in a total volume of 10 pL.

[0223] One month, two months, three months, or four months after compound administration, animals were euthanized, perfused, and the brain was sliced into 1 mm thick cortical slices. Biopsy punches (1-2 mm diameter) were taken of the relevant brain regions (frontal cortex = fCTx, striatum = Cpu, hippocampus = HP, temporal cortex = tCTx, midbrain = Mb, pons = Pons, medulla = Med, cerebellum = CB, cervical spinal cord = SC-C, thoracic spinal cord = SC-T, and lumbar spinal cord = SC-L) and snap frozen.

RNA quantification analysis

[0224] mRNA quantification was performed as described in Example 2. Comparative analysis was performed for relative SNCA mRNA quantitation against samples taken from time-matched PBS control mice. Results are shown in Figure 1 and Figure 2.

Protein expression analysis

[0225] Human a-synuclein protein knockdown was quantified by colorimetric sandwich ELISA (Invitrogen KHB0061). Biological samples were lysed using tissue lysis buffer (CST Cell Lysis Buffer #9803) and bead-beater homogenization on a Qiagen Tissuelyser II. The ELISA was performed using the tissue lysates in accordance with the manufacturer’s instructions. The ELISA utilizes a target-specific antibody-coated immunoassay plate to bind to the analyte which in turn binds to the capture antibody. An HRP conjugated secondary detection antibody is used in conjunction with a substrate to generate the measurable signal which is read on a colorimetric plate reader. Data are presented as % SNCA target knockdown (N=8 animals, 4 male, 4 female, 2 technical replicates each), with each siRNA treatment group normalized to time-matched PBS control animals. Results are shown in Figure 3 and Figure 4.

Statistical assessment

[0226] Statistical analysis was Mann-Whitney test, two-tailed. **** = p<0.0001, *** = p<0.001, ** = p<0.01, * = p<0.05. BQL = values below the limit of quantification but above the limit of detection in the ELISA; values were included in the graph.

Conclusion

[0227] The results in this example demonstrate that an siRNA molecule of the disclosure, with a sense strand having SEQ ID No.: 664 and an antisense strand having SEQ ID No.: 244, successfully reduced SNCA expression in vivo.

Example 4: Dose response of SNCA knockdown in vivo

Objective

[0228] This Example describes the results of a series of experiments undertaken to investigate the in vivo dose response of an siRNA molecule complementary to a specific region within an SNCA transcript to effectuate reduced expression of the SNCA gene.

Materials and Methods

[0229] In vivo dose response was assessed in mouse using similar methods as described in Example 3. Briefly, PBS or the di -branched siRNA molecule described in Example 3 was administered via bilateral stereotactic ICV injection at a dose of 2.5 nmol, 5 nmol, 10 nmol, 15 nmol, or 20 nmol in a total volume of 10 pL. Three months after compound administration, animals were euthanized and tissue harvest, mRNA quantification (human and mouse Snca), and protein quantitation (human SNCA) were performed as described in Example 2 and Example 3. Results

[0230] Statistical analysis was two-way ANOVA with Dunnett’s multiple comparisons test. **** ₌ p<o 0001, *** = p<0.001, ** = p<0.01, * = p<0.05. Results are shown in Figures 5, 6, and 7.

Conclusion

[0231] The results reported in this example demonstrate that an siRNA molecule of the disclosure successfully silences SNCA in a dose-dependent manner in various regions of the brain.