SNCA siRNA AND USES THEREOF

Abstract

Provided herein are small interfering ribonucleic acid (siRNA) agents targeting an SNCA gene and compositions comprising such siRNA agents.

Description

TITLESNCA siRNA AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of United States Provisional Application No. 63 / 537,181, filed on September 7, 2023, which is incorporated by reference herein, in its entirety and for all purposes.BACKGROUND OF THE INVENTION

[0002] Accumulation of pathologically misfolded alpha-synuclein (a-synuclein) has been implicated in a range of neurodeg enerative diseases that are referred to as “synucleinopathies.” Such diseases include Parkinson’s disease (PD), dementia with Lewy bodies (DLB), pure autonomic failure (PAF), and multiple system atrophy (MSA) (Goedert et al., 2017). In PD and DLB, a-synuclein aggregates are predominantly present as neuronal cytoplasmic deposits (Lewy bodies) and neuritic deposits (Lewy neurites) with differences in the involved brain regions (Coon et al., 2018). In MSA a-synuclein aggregates are mainly detected as glial cytoplasmic inclusions in oligodendrocytes, and in PAF the aggregates primarily present as neuronal cytoplasmic deposits in the peripheral nerves system (Id.).

[0003] In PD, genetic evidence strongly supports the role of the SNCA locus that encodes a- synuclein protein. Missense mutations and multiplications of SNCA cause autosomal dominant familial forms of PD, indicating that alteration in the physical property or increase of a-synuclein protein is sufficient to cause PD. Gene dosage of SNCA affects the severity and penetrance of the disease. SNCA triplication causes very rare cases of familial PD with full penetrance, presenting early-onset and rapidly progressive Parkinsonism with prominent non-motor signs such as dementia, autonomic dysfunction, and psychiatric features (Olgiati etal., 2015). SNCA duplication is relatively more common in autosomal dominant PD as compared to triplication or missense mutations, and the phenotypes of patients are less severe than those with SNCA triplication and may resemble idiopathic PD (Konno etal., 2016). Causal SNCA mutations have also been identified for DLB (Orme et al., 2018), but not for PAF (Coon, Singer et al. 2019) or MSA (Katzeff etal., 2019).

[0004] In addition to pathogenic mutations or multiplications, genetic variants further highlight the pivotal role of the SNCA gene in PD. Genome-wide association studies have identified a panel of non-coding genetic variants located at the 3 ’-end, promoter, and intronic regions of SNCA (Maraganore et al., 2006, Linnertz et al., 2009, Satake et al., 2009; Simon- Sanchez et al., 2009; Lutz et al., 2015; Soldner et al., 2016; Pihlstrom et al., 2018), and analyses of some of these non-coding variants suggest upregulation of SNCA gene transcription increase the risk of PD. A protective allele Rep 1-259 bp located at the promoter region reduces 40%- 50% of SNCA mRNA in temporal-cortex and substantia-nigra, as compared to individuals carrying at least one copy of the PD-risk associated Rep alleles (Linnertz et al., 2009

[0005] Studies of transgenic mouse models also demonstrate the effects of a-synuclein expression. In a tetracycline-controllable transgenic mouse model, suppression of a-synuclein expression for 3 months was reported to inhibit progression of pathology, prevented reactive gliosis, reversed synaptic defects, improved memory function, and cleared existing pathology in the hippocampus but not in some other brain regions including the mammillary bodies, olfactory bulb, and septums (Lim et al., 2011). In a rotenone-exposed rat model of PD, adeno-associated virus-shRNA mediated knockdown of endogenous a-synuclein by -35%, reduced degeneration of nigral dopaminergic neurons, and attenuated progressive motor deficits (Zharikov et al., 2015). Further, in preformed fibril (PFF)-injected rodent models of PD, antisense- oligonucleotide (ASO)-mediated knockdown of endogenous a-synuclein by -50% resulted in prevention and removal of existing a-synuclein pathology, and prevented dopaminergic cell dysfunction (Cole et al., 2021).

[0006] Moreover, mice with less than 50% reduction of SNCA mRNA have exhibited a reduction of pathology and functional benefits (Zharikov et al., 2015; Cole et al., 2021). Such results suggest a partial knockdown (near or less than 50%) of SNCA is desirable and possibly tolerant for therapeutic application.

[0007] Thus, a-synuclein may be a key therapeutic target for PD and other related synucleinopathies. As such, there is a need for compounds and agents that inhibit the expression of the SNCA gene to treat neurodegenerative diseases in which a-synuclein is implicated.SUMMARY OF INVENTION

[0008] According to one aspect, the described invention provides a small interfering ribonucleic acid (siRNA) agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises a nucleotide sequence corresponding to any one of the sense nucleotide sequences in Table 1, or the antisense strand comprises a nucleotide sequence corresponding to any one of the antisense nucleotide sequences in Tablet.

[0009] According to some embodiments, the sense strand comprises a nucleotide sequence corresponding to a sense nucleotide sequence in a row of Table 1, and the antisense strand comprises a nucleotide sequence corresponding to the antisense nucleotide sequence in the same row of Table 1. According to some embodiments, the sense strand consists of a nucleotide sequence corresponding to any one of the sense nucleotide sequences in Table 1, or the antisense strand consists of a nucleotide sequence corresponding to any one of the antisense nucleotide sequences in Table 1. According to some embodiments, the sense strand consists of a nucleotide sequence corresponding to a sense nucleotide sequence in a row of Table 1, and the antisense strand consists of a nucleotide sequence corresponding to the antisense nucleotide sequence in the same row of Table 1.

[0010] According to another aspect, the described invention provides a small interfering ribonucleic acid (siRNA) agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises a nucleotide sequence corresponding to any one of the sense nucleotide sequences in Table 2, or the antisense strand comprises a nucleotide sequence corresponding to any one of the antisense nucleotide sequences in Table.

[0011] According to some embodiments, the sense strand comprises a nucleotide sequence corresponding to a sense nucleotide sequence in a row of Table 2, and the antisense strand comprises a nucleotide sequence corresponding to the antisense nucleotide sequence in the same row of Table 2. According to some embodiments, the sense strand consists of a nucleotide sequence corresponding to any one of the sense nucleotide sequences in Table 2, or the antisense strand consists of a nucleotide sequence corresponding to any one of the antisense nucleotide sequences in Table 2. According to some embodiments, the sense strand consists of a nucleotide sequence corresponding to a sense nucleotide sequence in a row of Table 2, and the antisense strand consists of a nucleotide sequence corresponding to the antisense nucleotide sequence in the same row of Table 2.

[0012] According to another aspect, the described invention provides a small interfering ribonucleic acid (siRNA) agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises a nucleotide sequence corresponding to any one of the sense nucleotide sequences in Table 3, or the antisense strand comprises a nucleotide sequence corresponding to any one of the antisense nucleotide sequences in Table 3.

[0013] According to some embodiments, the sense strand comprises a nucleotide sequence corresponding to a sense nucleotide sequence in a row of Table 3, and the antisense strand comprises a nucleotide sequence corresponding to the antisense nucleotide sequence in the same row of Table 3. According to some embodiments, the sense strand consists of a nucleotide sequence corresponding to any one of the sense nucleotide sequences in Table 3, or the antisense strand consists of a nucleotide sequence corresponding to any one of the antisense nucleotide sequences in Table 3. According to some embodiments, the sense strand consists of a nucleotide sequence corresponding to a sense nucleotide sequence in a row of Table 3, and the antisense strand consists of a nucleotide sequence corresponding to the antisense nucleotide sequence in the same row of Table 3.

[0014] According to another aspect, the described invention provides a small interfering ribonucleic acid (siRNA) agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises a nucleotide sequence corresponding to any one of the sense nucleotide sequences in Table 4, or the antisense strand comprises a nucleotide sequence corresponding to any one of the antisense nucleotide sequences in Table 4.

[0015] According to some embodiments, the sense strand comprises a nucleotide sequence corresponding to a sense nucleotide sequence in a row of Table 4, and the antisense strand comprises a nucleotide sequence corresponding to the antisense nucleotide sequence in the same row of Table 4. According to some embodiments, the sense strand consists of a nucleotide sequence corresponding to any one of the sense nucleotide sequences in Table 4, or the antisense strand consists of a nucleotide sequence corresponding to any one of the antisense nucleotide sequences in Table 4. According to some embodiments, the sense strand comprises nucleotides having the sequence (InvAb)psmUpsmCmAmGmC(J2- C0NC16A)mGmUfGfAfUmUmGmAmAmGmUmAmUmCpsmAps(InvAb) (SEQ ID NO: 1535), and the antisense strand comprises nucleotides having the sequence (VPmU)psfGpsmApsmUmAfCmUfUfCmAmAmUmCfAmCfUmGmCmUmGpsmA (SEQ IDNO: 1536). According to some embodiments, the sense strand consists of a nucleotide sequence corresponding to a sense nucleotide sequence in a row of Table 4, and the antisense strand consists of a nucleotide sequence corresponding to the antisense nucleotide sequence in the same row of Table 4.

[0016] According to another aspect, the described invention provides a small interfering ribonucleic acid (siRNA) agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises a nucleotide sequence corresponding to any one of the sense nucleotide sequences in Table 21, or the antisense strand comprises a nucleotide sequence corresponding to any one of the antisense nucleotide sequences in Table 21.

[0017] According to some embodiments, the sense strand comprises a nucleotide sequence corresponding to a sense nucleotide sequence in a row of Table 21, and the antisense strand comprises a nucleotide sequence corresponding to the antisense nucleotide sequence in the same row of Table 21. According to some embodiments, the sense strand consists of a nucleotide sequence corresponding to any one of the sense nucleotide sequences in Table 21, or the antisense strand consists of a nucleotide sequence corresponding to any one of the antisense nucleotide sequences in Table 21. According to some embodiments, the sense strand consists of a nucleotide sequence corresponding to a sense nucleotide sequence in a row of Table 21, and the antisense strand consists of a nucleotide sequence corresponding to the antisense nucleotide sequence in the same row of Table 21.

[0018] According to some embodiments, the described invention provides a method of inhibiting expression of SNCA gene in a cell or population of cells, the method comprising contacting the cell or population of cells with an siRNA agent described herein.

[0019] According to some embodiments, the described invention provides a method of reducing alpha-synuclein levels in a cell or population of cells, the method comprising contacting the cell or population of cells with an siRNA agent described herein. According to some embodiments methods of the invention described herein may be performed in vivo, According to some embodiments methods of the invention described herein may be performed in vitro or ex vivo.

[0020] According to some embodiments, the described invention provides a composition comprising an siRNA agent and a carrier.BRIEF DESCRIPTION OF THE FIGURES

[0021] FIG. 1 provides the chemical structures of lipophilic moieties LI, L2, L3, L4, and L5, as well as the structure of cholesterol-triethylene glycol (CHOL4).

[0022] FIG. 2 shows SNCA mRNA knockdown efficiency of select cholesterol-siRNA conjugates in human iPSC-derived cortical neurons treated for 7 days, as described in Example 3 (n=3) (see also Table 8). Data are presented as remaining SNCA mRNA (relative to control).

[0023] FIG. 3 shows SNCA mRNA knockdown efficiency of select cholesterol-siRNA conjugates in human iPSC-derived cortical neurons treated for 14 days, as described in Example 3 (n=3) (see also Table 9). Data are presented as remaining SNCA mRNA (relative to control).

[0024] FIG. 4 shows oc-synuclein protein knockdown efficiency of select cholesterol-siRNA conjugates in human iPSC-derived cortical neurons treated for 14 days, as described in Example 3 (n=3) (see also Table 10). Data are presented as remaining oc-synuclein protein (relative to control).

[0025] FIG. 5 shows SNCA mRNA knockdown efficiency of select lipid-siRNA conjugates in human iPSC-derived cortical neurons treated for 7 days, as described in Example 3 (n=3) (see also Table 12). Data are presented as remaining SNCA mRNA (relative to control).

[0026] FIG. 6 shows oc-synuclein protein knockdown efficiency of select lipid-siRNA conjugates in human iPSC-derived cortical neurons treated for 14 days, as described in Example 3 (n=3) (see also Table 13). Data are presented as remaining oc-synuclein protein (relative to control).DETAILED DESCRIPTION

[0027] The practice of the present invention can employ, unless otherwise indicated, conventional techniques of biotechnology, molecular biology, pharmaceutics, formulation science, protein chemistry, cell biology, cell culture, genetics, recombinant DNA, immunology, clinical pharmacology, and clinical practice, which are within the skill of the art.

[0028] In order that the present invention can be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention is related.

[0029] Any headings provided herein are not limitations of the various aspects or embodiments of the invention, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0030] All references cited in this disclosure are hereby incorporated by reference in their entireties. In addition, any manufacturers’ instructions or catalogues for any products cited or mentioned herein are incorporated by reference. Documents incorporated by reference into this text, or any teachings therein, can be used in the practice of the present invention. Documents incorporated by reference into this text are not admitted to be prior art.Definitions

[0031] The phraseology or terminology in this disclosure is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

[0032] As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents, unless the context clearly dictates otherwise. The terms “a” (or “an”) as well as the terms “one or more” and “at least one” can be used interchangeably.

[0033] As used herein, the terms “comprising” and “including” can be used interchangeably. The terms “comprising” and “including” are to be interpreted as specifying the presence of the stated features or components as referred to, but does not preclude the presence or addition of one or more features, or components, or groups thereof. Additionally, the terms “comprising” and “including” are intended to include examples encompassed by the term “consisting of’. Consequently, the term “consisting of’ can be used in place of the terms “comprising” and “including” to provide for more specific embodiments.

[0034] As used herein, the term “or” is to be interpreted as an inclusive “or” meaning any one or any combination. Therefore, “A, B, or C” means any of the following: A; B; C; A and B; A and C; B and C; A, B, and C. An exception to this definition will occur only when a combination of elements, functions, steps or acts are in some way inherently mutually exclusive.

[0035] Furthermore, “and / or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” is intended to include A and B, A or B, A (alone), and B (alone).Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to include A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone); and C (alone).

[0036] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range, and any individual value provided herein can serve as an endpoint for a range that includes other individual values provided herein. For example, a set of values such as 1, 2, 3, 8, 9, and 10 is also a disclosure of a range of numbers from 1-10, from 1-8, from 3-9, and so forth. Likewise, a disclosed range is a disclosure of each individual value (i.e., intermediate) encompassed by the range, including integers and fractions. For example, a stated range of 5-10 is also a disclosure of 5, 6, 7, 8, 9, and 10 individually, and of 5.2, 7.5, 8.7, and so forth.

[0037] Unless otherwise indicated, the terms “at least” or “about” preceding a series of elements is to be understood to refer to every element in the series. The term “about” preceding a numerical value includes ± 10% of the recited value. For example, a concentration of about 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Likewise, a concentration range of about 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v).

[0038] The siRNA agents of the invention inhibit the expression of the SNCA gene. As used herein, the term “SNCA” gene refers to the gene encoding a protein called alpha-synuclein.

[0039] Alpha-synuclein, also referred to herein as “oc-synuclein,” is a small protein (140 amino acids, 14.5 kDa) encoded by 5 exons of the SNCA locus in chromosome 4q21.3-q22. SNCA is abundantly expressed throughout the brain (Jakes et al., 1994; Lavedan, 1998) with localization concentrated to the presynaptic nerve terminals (Iwai et al., 1995). Physiological a- synuclein exists in a dynamic equilibrium between unfolded monomers and a-helically folded tetramers (Lashuel et al., 2013), whereas pathological conditions drive aggregation of a- synuclein involving a conformational change to a P-sheet-rich structure that promotes formation of oligomers, protofibrils and insoluble fibrils.

[0040] As used herein, the term “small interfering RNA agent” or “siRNA agent” means an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule that contains both a sense and an anti-sense strand. In some embodiments, the siRNA sense strand is 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18 or 17 nucleotides in length. In some embodiments, the siRNA anti-sense strand is 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18 or 17 nucleotides inlength. The sense strand and / or antisense strand of an siRNA agent may comprise another moiety (e.g., a lipophilic moiety). For example, a lipophilic moiety may be incorporated into the sense strand of an siRNA agent. The sense strand and / or antisense strand of an siRNA agent may be linked or conjugated, directly or indirectly, to another moiety (e.g., a lipophilic moiety). For example, the sense strand of the siRNA agent may be linked or conjugated, directly or indirectly, to another moiety (e.g., a lipophilic moiety). In a specific embodiment, an siRNA agent is a double-stranded RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide comprising a sense stand and an antisense strand forming a double-stranded region. The doublestranded region may be the entire length of the sense strand, antisense strand, or both. Alternatively, the double-stranded region may be less than the entire length of the sense strand, antisense strand, or both. The double-stranded region may be the result of the antisense strand being fully complementary, partially complementary, or substantially complementary to the sense strand. In a specific embodiment, the antisense strand of an siRNA agent is partially complementary to a target RNA transcript, e.g, SNCA. In another specific embodiment, the antisense strand of an siRNA agent is substantially complementary to a target RNA transcript, e.g., SNCA. In another specific embodiment, the antisense strand of an siRNA agent is fully complementary to a target RNA transcript, e.g, SNCA.

[0041] The two strands forming the double-stranded region or duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules.

[0042] Where the two substantially complementary strands of an siRNA agent are comprised by separate RNA molecules, those molecules need not be, but can be, covalently connected. In certain embodiments where the two strands are connected covalently by means other than a contiguous chain of nucleotides between the 3 ’-end of one strand and the 5 ’-end of the respective other strand forming the duplex structure, the connecting structure is referred to as a “linker”.The RNA strands of an siRNA agent may have the same or a different number of nucleotides. In some embodiments, one or both strands of an siRNA agent comprise an overhang. In other embodiments, the siRNA agent is blunt ended.

[0043] In a specific embodiment, an siRNA agent described herein mediates messenger RNA (mRNA) degradation or inhibition of translation of the mRNA in a sequence-specific manner. In specific embodiments, an siRNA agent described herein inhibits SNCA gene expression via an RNA-induced silencing complex (RISC) pathway

[0044] As used herein, term “complementary,” when used to describe a first nucleotide sequence (e.g., a sense strand of an siRNA agent, or targeted sequence) in relation to a second nucleotide sequence (e.g., antisense strand of an siRNA agent, or a single-stranded antisense oligonucleotide), means the ability of an oligonucleotide or polynucleotide including the first nucleotide sequence to hybridize (form base pair hydrogen bonds under mammalian physiological conditions (or similar conditions in vitro)) and form a duplex or double helical structure under certain conditions with an oligonucleotide or polynucleotide including the second nucleotide sequence. Complementary sequences include Watson-Crick base pairs or non- Watson-Crick base pairs and include natural or modified nucleotides or nucleotide mimics, at least to the extent that the above hybridization requirements are fulfilled. Sequence identity or complementarity is independent of modification. For example, fA and mA are complementary to U (or T) and identical to A for the purposes of determining identity or complementarity.

[0045] As used herein, the term “fully complementary” in the context of two nucleotide sequences means that all (100%) of the bases in a contiguous sequence of a first nucleotide sequence will hybridize to the same number of bases in a contiguous sequence of a second nucleotide sequence to form a duplex. Where two nucleotide sequences are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, an siRNA agent comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the 23 nucleotides oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the 21 nucleotides oligonucleotide, are considered “fully complementary” for the purposes described herein. In a specific embodiment, two nucleotide sequences are “fully complementary” when all (100%) of the bases of a first nucleotide sequence hybridize to all (100%) of the bases in a second nucleotide sequence to form a duplex. In one specific embodiment, the two nucleotide sequences hybridize under stringent conditions. In another specific embodiment, the two nucleotide sequences hybridize under very stringent conditions.

[0046] As used herein, the term “partially complementary” in the context of two nucleotide sequences means that at least 65% but less than 80% of the bases in a contiguous sequence of a first nucleotide sequence will hybridize to the same number of bases in a contiguous sequence of a second nucleotide sequence to form a duplex. In one specific embodiment, the two nucleotidesequences hybridize under stringent conditions. In another specific embodiment, the two nucleotide sequences hybridize under very stringent conditions.

[0047] As used herein, the term “substantially complementary” in the context of two nucleotide sequences means that at least 80% but less than 100% of the bases in a contiguous sequence of a first nucleotide sequence will hybridize to the same number of bases in a contiguous sequence of a second nucleotide sequence to form a duplex. In some embodiments, two nucleotide sequences are substantially complementary when at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, but less than 100% of the bases in a contiguous sequence of a first oligonucleotide will hybridize to the same number of bases in a contiguous sequence of a second oligonucleotide to form a duplex. In one specific embodiment, the two nucleotide sequences hybridize under stringent conditions. In another specific embodiment, the two nucleotide sequences hybridize under very stringent conditions.

[0048] As used herein, the terms “about” and “approximate” when referring to a numerical value encompass the recited numerical value and variations within + / - 20%. For example, about 20% would encompass 16% to 24% and values in between, including 20%. In one embodiment, the terms “about” and “approximate” when referring to a numerical value encompass the recited numerical value and variations within + / -15%. In another embodiment, the terms “about” and “approximate” when referring to a numerical value encompass the recited numerical value and variations within + / - 10%. In another embodiment, the terms “about” and “approximate” when referring to a numerical value encompass the recited numerical value and variations within + / - 5%.

[0049] As used herein, the term “stringent” when referring to hybridization means that under “stringent conditions”, or “stringent hybridization conditions”, a first nucleotide sequence will hybridize to a second nucleotide, with minimal hybridization to other sequences. In a specific embodiment, an antisense sequence will hybridize under stringent conditions to its target sequence, with minimal targeting to other sequences. Stringent conditions are sequence dependent (e.g., sequence length, complementarity), and vary under different environmental parameters (e.g., assay conditions, physiological environment). An example of stringent hybridization conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°Cor 70°C for 12-16 hours followed by washing. A skilled person will understand that variations in the stringency of hybridization are inherently described.

[0050] As used herein, the term “very stringent” when referring to hybridization means that under “very stringent conditions” or “very stringent hybridization conditions”, a first nucleotide sequence will only be observed to hybridize to a second nucleotide. In a specific embodiment, an antisense sequence will be observed to only hybridize to its target sequence under very stringent conditions. Also, very stringent conditions may not allow hybridization to occur between partially complementary sequences. Very stringent conditions are sequence dependent (e.g., sequence length, complementarity), and vary under different environmental parameters (e.g., assay conditions, physiological environment). Very stringent conditions may include a higher temperature, lower ionic strength, and / or shorter reaction time compared to stringent conditions under the same circumstance. For example, very stringent conditions may include a hybridization temperature of about 71° C, about 72° C, about 73° C, about 74° C, about 75° C, about 76° C, about 77° C, about 78° C, about 79° C, about 80° C, or higher. A skilled person will understand that variations in the stringency of hybridization are inherently described.

[0051] As used herein, “target sequence” refers to a contiguous portion of a nucleotide sequence of a RNA molecule formed during the transcription of a gene, e.g., an SNCA gene, including mRNA that is a product of RNA processing of a primary transcription product (e.g., SNCA mRNA resulting from alternate splicing). In one embodiment, the contiguous portion of the nucleotide sequence is at least long enough to serve as a substrate for RNAi-directed cleavage at or near that portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a gene, e.g, an SNCA gene. In specific embodiments, the target sequence is about 15-30 nucleotides in length. For example, the target sequence can be from about 15-30 nucleotides, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19- 29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21- 23, or 21-22 nucleotides in length. In certain embodiments, the target sequence is 19-25 nucleotides in length. In some embodiments, the target sequence is 21-23 nucleotides in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the disclosure.

[0052] As used herein, the phrase “nucleotide sequence corresponding to any one of the antisense strand nucleotide sequences”, “nucleotide sequence corresponding to the antisense strand nucleotide sequence”, “nucleotide sequence corresponding to any one of the sense strand nucleotide sequences”, “nucleotide sequence corresponding to the sense strand nucleotide sequence”, “nucleotide sequence corresponding to any one of the nucleotide sequences”, or “nucleotide sequence corresponding to any one of SEQ ID NOS” refers to an oligonucleotide comprising a chain of nucleotides comprising the recited unmodified nucleotides, or one or more modified nucleotides, or one or more conjugated moieties (e.g., a moiety described herein, such as, e.g., a lipid, or a modified nucleotide conjugated to a moiety described herein). A skilled person is aware that the recited unmodified nucleotide may be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, a nucleotide comprising inosine as its base can base pair with nucleotides containing adenine, cytosine, or uracil. Thus, a nucleotide containing uracil, guanine, or adenine can be replaced by a nucleotide containing, for example, inosine. In another example, adenine and cytosine may be replaced with guanine and uracil, respectively to form G-U Wobble base pairing with the target mRNA.

[0053] As used herein, the phrase “non-naturally occurring small interfering ribonucleic acid (siRNA) agent”, “non-naturally occurring small interfering ribonucleic acid agent”, or “non- naturally occurring siRNA agent” refers to an siRNA agent that is not found in nature. The non- naturally occurring siRNA may contain one or more modified nucleotides.

[0054] As used herein, the term “overhang” in the context of a 5’ or 3’ nucleotide overhang refers to at least one unpaired nucleotide that protrudes from the duplex structure of an siRNA agent. For example, when a 3'-end of one strand of an siRNA agent extends beyond the 5'-end of the other strand, or vice versa, there is a nucleotide overhang. In some embodiments, the overhang is present at the 3 ’-end of the sense strand, antisense strand, or both strands. In one embodiment, the 3 ’-overhang is present in the antisense strand. In another embodiment, the 3’- overhang is present in the sense strand. In some embodiments, the overhang is present at the 5’- end of the sense strand, antisense strand, or both strands. In one embodiment, the 5 ’-overhang is present in the antisense strand. In another embodiment, the 5 ’-overhang is present in the sense strand. The overhangs may be due to one strand being longer than the other, or the result of two strands of the same length being staggered. In some embodiments, the overhang forms amismatch with the target sequence. In other embodiments, the overhang is complementary to the gene sequences being targeted. The nucleotides in the overhang region of an siRNA agent may each independently be a modified or unmodified nucleotide (e.g., 2’-fluoro-modified nucleotide, 2’-O-methyl modified nucleotide, deoxynucleotide, or a combination thereof).

[0055] In some embodiments, the 5’- or 3’- overhangs of the sense strand or antisense strand of an siRNA agent are phosphorylated. In certain embodiments, the 5’- or 3’- overhangs of the sense strand and the antisense strand of an siRNA agent are phosphorylated. In some embodiments, the overhang region(s) contains two nucleotides having a phosphorothioate between the two nucleotides, and those two nucleotides can be the same or different.

[0056] In some embodiments, an siRNA agent contains only a single overhang, which can strengthen the interference activity of the siRNA agent, without affecting its overall stability. For example, the single-stranded overhang may be located at the 3'-terminal end of the sense strand of the siRNA agent, or, alternatively, at the 3 '-terminal end of the antisense strand of the siRNA agent. The siRNA agent may also have a blunt end, located at the 5 ’-end of the antisense strand (or the 3 ’-end of the sense strand) or vice versa. In some embodiments, the antisense strand of the siRNA agent has a nucleotide overhang at the 3 ’-end, and the 5 ’-end is blunt ended.

[0057] In some embodiments, one strand of an siRNA agent comprises a 5 ’-end, a 3 ’-end, or both a 5 ’-end and a 3 ’-end overhang of at least 1 nucleotide, at least 2 nucleotides, or at least 3 nucleotides. In certain embodiments, one strand of an siRNA agent comprises a 5 ’-end, a 3’- end, or both a 5 ’-end and a 3 ’-end overhang of at least 1 nucleotide, at least 2 nucleotides, or at least 3 nucleotides, but no more than 5 nucleotides. In some embodiments, one strand of an siRNA agent comprises a 5 ’-end, a 3 ’-end, or both a 5 ’-end and a 3 ’-end overhang of 1 nucleotide, 2 nucleotides, or 3 nucleotides. In certain embodiments, one strand of an siRNA agent comprises a 5 ’-end, a 3 ’-end, or both a 5 ’-end and a 3 ’-end overhang of 1 to 2 nucleotides, 1 to 3 nucleotides, 1 to 4 nucleotides, or 1 to 5 nucleotides. In some embodiments, one strand of an siRNA agent comprises a 5 ’-end, a 3 ’-end, or both a 5 ’-end and a 3 ’-end overhang of 2 to 3 nucleotides, 2 to 4 nucleotides, or 2 to 5 nucleotides. In certain embodiments, one strand of an siRNA agent comprises a 5 ’-end, a 3 ’-end, or both a 5 ’-end and a 3 ’-end overhang of 3 to 4 nucleotides, or 4 to 5 nucleotides. The strand may be an antisense strand or a sense strand. A nucleotide overhang may comprise or consist of a nucleotide analog or a nucleoside analog.

[0058] In some embodiments, each strand of an siRNA agent comprises a 5 ’-end, a 3 ’-end, or both a 5 ’-end and a 3 ’-end overhang of at least 1 nucleotide, at least 2 nucleotides, or at least 3 nucleotides. In certain embodiments, each strand of an siRNA agent comprises a 5 ’-end, a 3’- end, or both a 5 ’-end and a 3 ’-end overhang of at least 1 nucleotide, at least 2 nucleotides, or at least 3 nucleotides, but no more than 5 nucleotides. In some embodiments, each strand of an siRNA agent comprises a 5 ’-end, a 3 ’-end, or both a 5 ’-end and a 3 ’-end overhang of 1 nucleotide, 2 nucleotides, or 3 nucleotides. In certain embodiments, each strand of an siRNA agent comprises a 5 ’-end, a 3 ’-end, or both a 5 ’-end and a 3 ’-end overhang of 1 to 2 nucleotides, 1 to 3 nucleotides, 1 to 4 nucleotides, or 1 to 5 nucleotides. In some embodiments, each strand of an siRNA agent comprises a 5 ’-end, a 3 ’-end, or both a 5 ’-end and a 3 ’-end overhang of 2 to 3 nucleotides, 2 to 4 nucleotides, or 2 to 5 nucleotides. In certain embodiments, each strand of an siRNA agent comprises a 5 ’-end, a 3 ’-end, or both a 5 ’-end and a 3 ’-end overhang of 3 to 4 nucleotides, or 4 to 5 nucleotides. A nucleotide overhang may comprise or consist of a nucleotide analog or a nucleoside analog.

[0059] As used herein, the term “blunt” or “blunt ended” in the context of an siRNA agent means that there are no unpaired nucleotides or nucleotide analogs at a given terminal end of an siRNA agent, i.e., no nucleotide overhang. In some embodiments, one end of an siRNA agent is blunt ended. In other words, the 5 ’-end of one strand and the 3 ’-end of the other strand do not include an unpaired nucleotide or nucleotide analog. In some embodiments, both ends of an siRNA agent are blunt ended. In other words, there is no nucleotide overhang at either end of the siRNA agent.

[0060] As used herein, term “antisense strand” or “guide strand” in the context of an siRNA agent refers to the strand which includes a region that is complementary to a target sequence.

[0061] As used herein, the term “sense strand” or “passenger strand” in the context of an siRNA agent refers to the strand of an siRNA agent that includes a region that is complementary to a region of the antisense strand.

[0062] As used herein, the term “modified” in the context of a nucleobase of an siRNA agent, refers to a nucleobase not found in nature in an RNA molecule. Naturally occurring RNA sequences include purine bases adenine (A) and guanine (G), and pyrimidine bases cytosine (C) and uracil (U). See Chemical modifications to Nucleotides section, infra, for examples of modified nucleobases.

[0063] As used herein, the term “modified” in the context of a nucleotide of an siRNA agent, refers to a nucleotide not found in nature in an RNA molecule. See Chemical modifications to Nucleotides section, infra, for examples of modified nucleotides.

[0064] As used herein, and unless otherwise specified, the term “isomer” refers to different compounds that have the same molecular formula. “Stereoisomers” are isomers that differ only in the way the atoms are arranged in space. “Atropisomers” are stereoisomers from hindered rotation about single bonds. “Enantiomers” are a pair of stereoisomers that are non-superimposable mirror images of each other. A mixture of a pair of enantiomers in any proportion can be known as a “racemic” mixture. “Diastereoisomers” are stereoisomers that have at least two asymmetric atoms, but which are not mirror-images of each other.

[0065] Stereoisomers” can also include E and Z isomers, or a mixture thereof, and cis and trans isomers or a mixture thereof. In certain embodiments, a compound described herein is isolated as either the E or Z isomer. In other embodiments, a compound described herein is a mixture of the E and Z isomers.

[0066] It should be noted that if there is a discrepancy between a depicted structure and a name for that structure, the depicted structure is to be accorded more weight.

[0067] The terms “inhibit,” “block,” “suppress,” and “reduce” are used interchangeably and refer to any statistically significant decrease in a given biological activity, including full blocking of the activity. For example, “inhibition” can refer to a decrease of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% in biological activity.Nucleotide Sequences

[0068] Provided herein are siRNA agents which inhibit (e.g., partially or completely) the expression of an SNCA gene (e.g., a human SNCA gene). In a specific embodiment, an siRNA described herein inhibits expression of an SNCA gene (e.g, a human SNCA gene) by at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60% relative to a negative control (e.g, PBS or siRNA directed to an RNA molecule formed during the transcription of a gene other than SNCA) in an assay known to one of skill in the art, or described herein (e.g., in Biological Assays section, infra,' or EXAMPLES section, infra). In another specific embodiment, an siRNA described herein inhibits expression of an SNCA gene (e.g., a human SNCA gene) by at least about 70%, at least 75%, at least 80%, or at least 85%relative to a negative control (e.g., PBS or siRNA directed to an RNA molecule formed during the transcription of a gene other than SNCA) in an assay known to one of skill in the art, or described herein (e.g., in Biological Assays section, infra or EXAMPLES section, infra). In another specific embodiment, an siRNA described herein inhibits expression of an SNCA gene (e.g., a human SNCA gene) by at least about 90%, at least 95%, at least 96%, at least 97%, or at least 98% relative to a negative control (e.g., PBS or siRNA directed to an RNA molecule formed during the transcription of a gene other than SNCA) in an assay known to one of skill in the art, or described herein (e.g, in Biological Assays section, infra; or EXAMPLES section, infra). In another specific embodiment, an siRNA described herein inhibits expression of an SNCA gene (e.g., a human SNCA gene) by 25% to 50%, 50% to 75%, 75% to 85%, 85% to 95%, 90% to 95%, or 95% to 99% relative to a negative control (e.g., PBS or siRNA directed to an RNA molecule formed during the transcription of a gene other than SNCA) in an assay known to one of skill in the art, or described herein (e.g., in Biological Assays section, infra; or EXAMPLES section, infra). In another specific embodiment, an siRNA described herein inhibits expression of an SNCA gene (e.g., a human SNCA gene) by 100% relative to a negative control (e.g., PBS or siRNA directed to an RNA molecule formed during the transcription of a gene other than SNCA) in an assay known to one of skill in the art, or described herein (e.g., in Biological Assays section, infra; or EXAMPLES section, infra). In a specific embodiment, inhibition of an SNCA gene (e.g., a human SNCA gene) expression is assessed using an in vitro assay described in EXAMPLES section, infra. In a specific embodiment, an siRNA agent described herein exhibits knockdown efficiency in vitro or ex vivo, such as, e.g., described in EXAMPLES section, infra. In a specific embodiment, an siRNA agent described herein exhibits knockdown efficiency in vivo in a mouse model, such as, e.g, described in the Example, infra. In specific embodiments, an siRNA agent described herein exhibits one, two, three, or more of the properties of an RNA molecule described in EXAMPLES section, infra.

[0069] In a specific embodiment, an siRNA agent described herein inhibits the expression of an SNCA gene (e.g., human SNCA gene) in neurons (e.g., human iPSC-neurons) at an IC50 of 6 nM to 5000 nM in an assay described herein (e.g., EXAMPLES section, infra). In a specific embodiment, an siRNA agent described herein inhibits the expression of an SNCA gene (e.g., human SNCA gene) in neurons (e.g., human iPSC-neurons) at an IC50 of 10 nM to 1000 nM in an assay described herein (e.g., EXAMPLES section, infra). In a specific embodiment, ansiRNA agent described herein inhibits the expression of an SNCA gene (e.g., human SNCA gene) in neurons (e.g., human iPSC-neurons) at an IC50 of 10 nM to 50 nM in an assay described herein (e.g., EXAMPLES section, infra). In a specific embodiment, an siRNA agent described herein inhibits the expression of an SNCA gene (e.g, human SNCA gene) in neurons (e.g., human iPSC-neurons) at an IC50 of 150 nM to 1000 nM in an assay described herein (e.g., EXAMPLES section, infra).

[0070] In a specific embodiment, an siRNA agent described herein is stable in vitro or ex vivo in one, two, or all of the following: mouse brain homogenates, human liver lysosomes and rat liver tritosomes. The stability of such an siRNA agent may be assessed using a method known to one of skill in the art, or as described herein (e.g., in Biological Assays section, infra,' or EXAMPLES section, infra).

[0071] In a specific embodiment, an siRNA agent is a double-stranded RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide comprising a sense stand and an antisense strand, which anneal to form a double-stranded region or duplex. The antisense strand includes a region that is substantially complementary, and generally fully complementary, to the target sequence. The sense strand includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. As described herein and as known in the art, the complementary sequences of an siRNA agent can also be contained as self-complementary regions of a single nucleic acid molecule, as opposed to being on separate oligonucleotides. In a specific embodiment, the annealment of the sense strand and antisense strand sequences form a duplex that is stable as assessed by a melting temperature (Tm) of about 40° C, about 41° C, about 42° C, about 43° C, about 44° C, about 45° C, or higher. See, e.g., the Biological Assays section herein for melting temperature assays.

[0072] The sense strand and the antisense strand of an siRNA agent described herein may be the same length or different lengths. The sense strand and the antisense strand of an siRNA agent described herein can each be 10 to 30 nucleotides in length. In specific embodiments, the sense strand and the antisense strand of an siRNA agent described herein are each no more than 30 nucleotides in length. In some embodiments, the sense strand and the antisense strand of an siRNA agent described herein are each independently 10 to 30 nucleotides in length. In certain embodiments, the sense and the antisense strand of an siRNA agent described herein are eachindependently 10 to 25 nucleotides in length. In certain embodiments, the sense and the antisense strand of an siRNA agent described herein are each independently 15 to 25 nucleotides in length. In some embodiments, the sense and the antisense strand of an siRNA agent described herein are each independently 16 to 25 nucleotides in length. In certain embodiments, the sense and the antisense strand of an siRNA agent described herein are each independently 16 to 24 nucleotides in length. In some embodiments, the sense and the antisense strand of an siRNA agent described herein are each independently 16 to 23 nucleotides in length. In certain embodiments, the sense and the antisense strand of an siRNA agent described herein are each independently 16 to 22 nucleotides in length. In certain embodiments, the sense and the antisense strand of an siRNA agent described herein are each independently 17 to 22 nucleotides in length. In certain embodiments, the sense and the antisense strand of an siRNA agent described herein are each independently 17 to 21 nucleotides in length. In certain embodiments, the sense and the antisense strand of an siRNA agent described herein are each independently 17 to 20 nucleotides in length. In certain embodiments, the sense and the antisense strand of an siRNA agent described herein are each independently 18 to 22 nucleotides in length. In certain embodiments, the sense and the antisense strand of an siRNA agent described herein are each independently 18 to 21 nucleotides in length. In certain embodiments, the sense and the antisense strand of an siRNA agent described herein are each independently 18 to 20 nucleotides in length. In certain embodiments, the sense and the antisense strand of an siRNA agent described herein are each independently about 19 nucleotides in length.

[0073] The double stranded region formed by hybridization of the antisense strand and the sense strand of an siRNA agent described herein may be the entire length of both strands. For example, the antisense strand of an siRNA agent described herein may be 19 nucleotides in length, the sense strand of the siRNA agent may be 19 nucleotides in length, and the double strand region formed by hybridization of the strands is 19 nucleotides. In another example, the antisense strand of an siRNA agent described herein may be 21 nucleotides in length, the sense strand of the siRNA agent may be 21 nucleotides in length, and the double strand region formed by hybridization of the strands is 21 nucleotides. The double stranded region formed by hybridization of the antisense strand and the sense strand of an siRNA agent described herein may be less than entire length of one or both strands. For example, the antisense strand of an siRNA agent described herein may be 19 nucleotides in length, the sense strand of the siRNAagent may be 21 nucleotides in length, and the double strand region formed by hybridization of the strands is 19 nucleotides. In another example, the antisense strand of an siRNA agent described herein may be 21 nucleotides in length, the sense strand of the siRNA agent may be 19 nucleotides in length, and the double strand region formed by hybridization of the strands is 19 nucleotides. In another example, the antisense strand of an siRNA agent described herein may be 21 nucleotides in length, the sense strand of the siRNA agent may be 21 nucleotides in length, and the double strand region formed by hybridization of the strands is 19 nucleotides. In a specific embodiment, the double-strand region of an siRNA agent described herein is long enough to serve as a substrate for the Dicer enzyme.

[0074] In certain embodiments, the double strand region of an siRNA agent described herein is 10 to 30 base pairs in length. For example, the double strand region of an siRNA agent described herein may be 10-29, 10-28, 10-27, 10-26, 10-25, 10-24, 10-23, 10-22, 10-21, 10-20, 10-19, 10-18, 10-17, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15- 19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20- 28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, or 20-21, base pairs in length. In some embodiments, the double strand region of an siRNA agent described herein is 15 to 25 base pairs in length. In certain embodiments, the double strand region of an siRNA agent described herein is 16 to 25 base pairs in length. In some embodiments, the double strand region of an siRNA agent described herein is 16 to 24 base pairs in length. In some embodiments, the double strand region of an siRNA agent described herein is 16 to 23 base pairs in length. In some embodiments, the double strand region of an siRNA agent described herein is 16 to 22 base pairs in length. In some embodiments, the double strand region of an siRNA agent described herein is17 to 23 base pairs in length. In some embodiments, the double strand region of an siRNA agent described herein is 17 to 22 base pairs in length. In certain embodiments, the double strand region of an siRNA agent described herein is 17 to 21 base pairs in length. In some embodiments, the double strand region of an siRNA agent described herein is 18 to 22 base pairs in length. In some embodiments, the double strand region of an siRNA agent described herein is18 to 21 base pairs in length. In some embodiments, the double strand region of an siRNA agent described herein is 18 to 20 base pairs in length. In certain embodiments, the double strand region of an siRNA agent described herein is 19 to 22 base pairs in length. In someembodiments, the double strand region of an siRNA agent described herein is 19 to 21 base pairs in length. In some embodiments, the double strand region of an siRNA agent described herein is 19 or 20 base pairs in length. In some embodiments, the double strand region of an siRNA agent described herein is about 19 base pairs in length. In certain embodiments, the double strand region of an siRNA agent described herein is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 base pairs in length. In specific embodiments, the double strand region of an siRNA agent described herein is 17, 18, 19, 20 or 21 base pairs in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the disclosure.

[0075] In some embodiments, an siRNA agent described herein is blunt ended. In other embodiments, the sense strand or the antisense strand of an siRNA agent described herein comprises an overhang. The overhang may be at the 5’ end, the 3’ end, or both the 5’ and 3’ end of either the sense strand or antisense strand of the siRNA agent. In some embodiments, the sense strand and the antisense strand of an siRNA agent described herein comprise an overhang. The overhang of the sense strand may be at the 5’ end of the sense strand, and the overhang of the antisense strand may be at the 3’ end of the antisense strand. Alternatively, the overhang of the sense strand may be at the 3’ end of the sense strand, the overhang of the antisense strand may be at the 5’ end of the antisense strand. In certain embodiments, the overhang of a strand of an siRNA agent comprises at least 1 nucleotide. In some embodiments, the overhang of a strand of an siRNA agent comprises at least 2 nucleotides. In certain embodiments, the overhang of a strand of an siRNA agent comprises at least 3 nucleotides. In some embodiments, the overhang of a strand of an siRNA agent comprises at least 4 nucleotides. In certain embodiments, the overhang of a strand of an siRNA agent comprises at least 5 nucleotides. In some embodiments, the overhang of a strand of an siRNA agent comprises 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotide, 5 nucleotides, or more. In certain embodiments, the overhang of a strand of an siRNA agent comprises 1 to 5 nucleotides, 1 to 4 nucleotides, 1 to 3 nucleotides, or 1 to 2 nucleotides. In some embodiments, the overhang of a strand of an siRNA agent comprises 2 to 5 nucleotides, 2 to 4 nucleotides, 2 to 3 nucleotides, 3 to 4 nucleotides, 3 to 4 nucleotides, or 4 to 5 nucleotides.

[0076] In certain embodiments, the sense strand of an siRNA agent described herein is 17 nucleotides in length and the antisense strand of the siRNA agent is 22 nucleotides in length. In certain embodiments, the sense strand of an siRNA agent described herein is 17 nucleotides inlength and the antisense strand of the siRNA agent is 21 nucleotides in length. In some embodiments, the sense strand of an siRNA agent described herein is 18 nucleotides in length and the antisense strand of the siRNA agent is 21 nucleotides in length. In certain embodiments, the sense strand of an siRNA agent described herein is 19 nucleotides in length and the antisense strand of the siRNA agent is 21 nucleotides in length. In some embodiments, the sense strand of an siRNA agent described herein is 20 nucleotides in length and the antisense strand of the siRNA agent is 21 nucleotides in length. In certain embodiments, the sense strand of an siRNA agent described herein is 21 nucleotides in length and the antisense strand of the siRNA agent is 20 nucleotides in length. In some embodiments, the sense strand of an siRNA agent described herein is 22 nucleotides in length and the antisense strand of the siRNA agent is 20 nucleotides in length. In some embodiments, the sense strand of an siRNA agent described herein is 22 nucleotides in length and the antisense strand of the siRNA agent is 19 nucleotides in length. In some embodiments, the sense strand of an siRNA agent described herein is 22 nucleotides in length and the antisense strand of the siRNA agent is 18 nucleotides in length. In some embodiments, the sense strand of an siRNA agent described herein is 22 nucleotides in length and the antisense strand of the siRNA agent is 17 nucleotides in length.

[0077] In some embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises a nucleotide sequence corresponding to the nucleotide sequence of any one of the sense strand nucleotide sequences in Table 1.

[0078] In some embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises a nucleotide sequence corresponding to the nucleotide sequence of any one of the antisense strand nucleotide sequences in Table 1.

[0079] In some embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises a nucleotide sequence corresponding to the nucleotide sequence of the sense strand nucleotide sequence in a row in Table 1, and wherein the antisense strand comprises a nucleotide sequence corresponding to the nucleotide sequence of the antisense strand nucleotide sequence in the same row in Table 1. In specific embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the sense strandcomprises a nucleotide sequence corresponding to the nucleotide sequence of the sense strand nucleotide sequence of US14, US28, US58, US183, and US199 in Table 1, and the antisense strand comprises a nucleotide sequence corresponding to the nucleotide sequence of the antisense strand nucleotide sequence of US14, US28, US58, US183, and US199 in Table 1, respectively.

[0080] In certain embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises a nucleotide sequence corresponding to the nucleotide sequence of any one of the sense strand nucleotide sequences in Table 1, and the antisense strand comprises a nucleotide sequence partially complementary to the sense strand. In some embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises a nucleotide sequence corresponding to the nucleotide sequence of any one of the sense strand nucleotide sequences in Table 1, and the antisense strand comprises a nucleotide sequence substantially complementary to the sense strand. In certain embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises a nucleotide sequence corresponding to the nucleotide sequence of any one of the sense strand nucleotide sequences in Table 1, and the antisense strand comprises a nucleotide sequence fully complementary to the sense strand.

[0081] In certain embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises a nucleotide sequence corresponding to the nucleotide sequence of any one of the antisense strand nucleotide sequences in Table 1, and the sense strand comprises a nucleotide sequence partially complementary to the antisense strand. In some embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises a nucleotide sequence corresponding to the nucleotide sequence of any one of the antisense strand nucleotide sequences in Table 1, and the sense strand comprises a nucleotide sequence substantially complementary to the antisense strand. In certain embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises a nucleotide sequence corresponding to the nucleotide sequence of any one of the antisensestrand nucleotide sequences in Table 1, and the sense strand comprises a nucleotide sequence fully complementary to the antisense strand.

[0082] In some embodiments, an siRNA agent does not comprise, e.g., chemical modifications or conjugations known in the art and described herein. See, e.g., Chemical Modifications to Nucleotides section, infra, and EXAMPLES section, infra, for siRNA agents comprising chemical modifications or conjugations. In specific embodiments, an siRNA agent comprises, e.g, chemical modifications or conjugations known in the art and described herein. In a specific embodiment, an siRNA agent comprises one or more chemical modifications and / or conjugations described herein (e.g., in Chemical Modifications to Nucleotides section, infra, and EXAMPLES section, infra. In certain embodiments, the nucleobase of a nucleotide is modified. In some embodiments, the sugar of the nucleotide is modified. In certain embodiments, the phosphate backbone of the nucleotide is modified.

[0083] The siRNA agents may contain only naturally occurring nucleotides, or may contain one or more modified nucleotides. In certain embodiments, the nucleobase of a nucleotide is modified. In some embodiments, the sugar of the nucleotide is modified. In certain embodiments, the phosphate backbone of the nucleotide is modified.

[0084] In specific embodiments, the modified nucleotides comprise one or more modified nucleotides described herein (e.g., in Chemical Modifications to Nucleotides section, infra, and EXAMPLES section, infra'). In specific embodiments, the modified nucleotides are selected from the group consisting of a 2’O-methyl modified nucleotide, a deoxy-nucleotide, a 2’-fluoro modified nucleotide, a 2’-O-methyl-uridine, a 3’-O-methyl modified nucleotide, a 3’-O-methyl modified nucleotide with 2’ -5’ linked phosphate, an inverted abasic nucleotide, a nucleotide comprising S-glycol nucleic acid (GNA), an unlocked nucleotide, a vinyl phosphonate, a 5’- vinylphosphonate-2’-O-methyl-uridine, a cis-cyclobutyl phosphonate modified nucleotide, a 5’- cis-cyclobutyl phosphonate-2’-O-methyl modified nucleotide, a (L)-a-threofuranosyl modified nucleotide, and combinations thereof. In specific embodiments, the modified nucleotides are selected from the group consisting of a 2’O-methyl modified nucleotide, a deoxy-nucleotide, a 2’-fluoro modified nucleotide, an inverted abasic nucleotide, a nucleotide comprising S-glycol nucleic acid (GNA), an unlocked nucleotide, a vinyl phosphonate, and combinations thereof.

[0085] In specific embodiments, the sense strand of an siRNA agent described herein comprises at least one modified internucleoside linkage.

[0086] In specific embodiments, the antisense strand of an siRNA agent described herein comprises at least one modified internucleoside linkage.

[0087] In specific embodiments, the antisense strand and the sense strand of an siRNA agent described herein each comprise at least one modified internucleoside linkage In a specific embodiment, the modified internucleoside linkages include phosphorothioate.

[0088] In specific embodiments, siRNA agent described herein comprises one or more of the following: a phosphorothioate (PS) linkage, 2’ -fluororibose (2’-F), 2’-methoxyribose (2’-0Me), a vinyl phosphonate, and an inverted linkage (inverted abasic site, InvAb).

[0089] In specific embodiments, an siRNA agent described herein exhibits stability as assessed by an assay described herein (e.g., in Biological Assays section, infra, and EXAMPLES section, infra), or known to one skilled in the art. In certain embodiments, an siRNA agent described herein comprising one or more modified nucleotides exhibits increased stability relative to the same siRNA agent without those one or more modified nucleotides, as assessed using a method known to one skilled in the art, or described herein (e.g., in Biological Assays section, infra, and EXAMPLES section, infra).

[0090] In one embodiment, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises the nucleotide sequence of any one of the sense strand nucleotide sequences in Table 2. In one embodiment, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises the nucleotide sequence of any one of the antisense strand nucleotide sequences in Table 2.

[0091] In one embodiment, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises the nucleotide sequence of the antisense strand nucleotide sequence in a row in Table 2, and wherein the antisense strand comprises the nucleotide sequence of the sense strand nucleotide sequence in the same row in Table 2.

[0092] In certain embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises the nucleotide sequence of any one of the sense strand nucleotide sequences in Table 2, and the antisense strand comprises a nucleotide sequence partially complementary to the sense strand. In some embodiments, provided herein is an siRNA agent comprising a sense strand and anantisense strand forming a double stranded region, wherein the sense strand comprises the nucleotide sequence of any one of the sense strand nucleotide sequences in Table 2, and the antisense strand comprises a nucleotide sequence substantially complementary to the sense strand. In certain embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises the nucleotide sequence of any one of the sense strand nucleotide sequences in Table 2, and the antisense strand comprises a nucleotide sequence fully complementary to the sense strand.

[0093] In certain embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises the nucleotide sequence of any one of the antisense strand nucleotide sequences in Table 2, and the sense strand comprises a nucleotide sequence partially complementary to the antisense strand. In some embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises the nucleotide sequence of any one of the antisense strand nucleotide sequences in Table 2, and the sense strand comprises a nucleotide sequence substantially complementary to the antisense strand. In certain embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises the nucleotide sequence of any one of the antisense strand nucleotide sequences in Table 2, and the sense strand comprises a nucleotide sequence fully complementary to the antisense strand.

[0094] In some embodiments, an siRNA agent described herein is conjugated to one or more non-nucleotide groups including, but not limited to a targeting group, linking group, delivery polymer, or a delivery vehicle. See, e.g., Moieties Linked to Nucleotide Sequence section, infra, for additional information regarding siRNA agents conjugated to one or more non- nucleotide groups. In a specific embodiment, the one or more non-nucleotide groups is one or more of the ones described herein (e.g., in Moieties Linked to Nucleotide Sequence section, infra, and EXAMPLES section, infra, .

[0095] In specific embodiments, the sense strand of an siRNA agent described herein is conjugated directly or indirectly to one or more lipophilic moieties. See, e.g., Winiwarter el al. (2007), pages 531-554, regarding lipophilicity. In specific embodiments, the antisense strand of an siRNA agent described herein is conjugated directly or indirectly to one or more lipophilic moieties. In specific embodiments, the sense strand and antisense strand of an siRNA agentdescribed herein are each conjugated directly or indirectly to one or more lipophilic moieties.The one or more lipophilic moieties may be conjugated to one or more terminal positions of the siRNA agent. For example, the lipophilic moieties may be conjugated to the 5’ end and / or 3’ end of one or both strands of the siRNA agent. Alternatively, or in addition, the one or more lipophilic moieties may be conjugated to one or more internal positions of the double stranded region of the siRNA agent. In certain embodiments, the lipophilic moieties are conjugated to one or more terminal positions of the siRNA agent and one or more internal positions of the siRNA agent. In some embodiments, the one or more lipophilic moieties are conjugated via a linker or a carrier. In a specific embodiment, a linker is one described herein (e.g., in Moieties Linked to Nucleotide Sequence section, infra).

[0096] In specific embodiments, a lipophilic moiety comprises a lipophilic moiety described herein (e.g., in Moieties Linked to Nucleotide Sequence section, infra, Synthesis of siRNA Agents section, infra, or EXAMPLES section, infra). In specific embodiments, a lipophilic moiety is cholesterol, such as cholesterol-triethylene glycol (CHOL4). In specific embodiments, a lipophilic moiety is selected from one or more of the following: LI, L2, L3, L4, and L5 (see FIG. 1). In a specific embodiment, a lipophilic moiety, such as described in the EXAMPLES section, is conjugated to the sense strand of an siRNA agent in the manner described in the EXAMPLES section.

[0097] In some embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises the nucleotide sequence of any one of the sense strand nucleotide sequences in Table 3. In some embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises the nucleotide sequence of any one of the antisense strand nucleotide sequences in Table 3.

[0098] In some embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises the nucleotide sequence of the sense strand nucleotide sequence in a row in Table 3, and the antisense strand comprises the nucleotide sequence of the antisense strand nucleotide sequence in the same row in Table 3. In some embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises the nucleotide sequence of any one of the sense strand nucleotide sequences in Table3, and wherein the sense strand is conjugated to cholesterol as indicated in Table 3 conjugated to the sense strand of the siRNA agent. In some embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises the nucleotide sequence of the antisense strand nucleotide sequence in a row in Table 3, and the sense strand comprises the nucleotide sequence of the sense strand nucleotide sequence in the same row in Table 3, and wherein the sense strand is conjugated to cholesterol as indicated in Table 3 conjugated to the sense strand of the siRNA agent.

[0099] In certain embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises the nucleotide sequence of any one of the sense strand nucleotide sequences in Table 3, and the antisense strand comprises a nucleotide sequence partially complementary to the sense strand. In some embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises the nucleotide sequence of any one of the sense strand nucleotide sequences in Table 3, and the antisense strand comprises a nucleotide sequence substantially complementary to the sense strand. In certain embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises the nucleotide sequence of any one of the sense strand nucleotide sequences in Table 3, and the antisense strand comprises a nucleotide sequence fully complementary to the sense strand.

[0100] In certain embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises the nucleotide sequence of any one of the sense strand nucleotide sequences in Table 3, wherein the sense strand is conjugated to cholesterol as indicated in Table 3 conjugated to the sense strand of the siRNA agent, and the antisense strand comprises a nucleotide sequence partially complementary to the sense strand. In some embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises the nucleotide sequence of any one of the sense strand nucleotide sequences in Table 3, wherein the sense strand is conjugated to cholesterol as indicated in Table 3, and the antisense strand comprises a nucleotide sequence substantially complementary to the sense strand. In certain embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the sense strandcomprises the nucleotide sequence of any one of the sense strand nucleotide sequences in Table 3, wherein the sense strand is conjugated to cholesterol as indicated in Table 3, and the antisense strand comprises a nucleotide sequence fully complementary to the sense strand.

[0101] In certain embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises the nucleotide sequence of any one of the antisense strand nucleotide sequences in Table 3, and the sense strand comprises a nucleotide sequence partially complementary to the antisense strand. In some embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises the nucleotide sequence of any one of the antisense strand nucleotide sequences in Table 3, and the sense strand comprises a nucleotide sequence substantially complementary to the antisense strand. In certain embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises the nucleotide sequence of any one of the antisense strand nucleotide sequences in Table 3, and the sense strand comprises a nucleotide sequence fully complementary to the antisense strand.

[0102] In certain embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises the nucleotide sequence of any one of the antisense strand nucleotide sequences in Table 3, and the sense strand comprises a nucleotide sequence partially complementary to the antisense strand, and wherein the sense strand of the siRNA agent is conjugated to cholesterol as indicated in Table 3, or another lipophilic moiety, such as, e.g., described in FIG. 1. In some embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises the nucleotide sequence of any one of the antisense strand nucleotide sequences in Table 3, and the sense strand comprises a nucleotide sequence substantially complementary to the antisense strand, and wherein the sense strand of the siRNA agent is conjugated to cholesterol as indicated in Table 3, or another lipophilic moiety, such as, e.g., described in FIG. 1. In certain embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises the nucleotide sequence of any one of the antisense strand nucleotide sequences in Table 3, and the sense strand comprises a nucleotide sequence fully complementary to the antisense strand, and wherein the sense strand ofthe siRNA agent is conjugated to cholesterol as indicated in Table 3, or another lipophilic moiety, such as, e.g., described in FIG. 1.

[0103] In some embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises the nucleotide sequence of any one of the sense strand nucleotide sequences in Table 21. In some embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises the nucleotide sequence of any one of the antisense strand nucleotide sequences in Table 21.

[0104] In some embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises the nucleotide sequence of the sense strand nucleotide sequence in a row in Table 21, and the antisense strand comprises the nucleotide sequence of the antisense strand nucleotide sequence in the same row in Table 21. In some embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises the nucleotide sequence of any one of the sense strand nucleotide sequences in Table 21, and wherein the sense strand is conjugated to cholesterol as indicated in Table 21 conjugated to the sense strand of the siRNA agent. In some embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises the nucleotide sequence of the antisense strand nucleotide sequence in a row in Table 21, and the sense strand comprises the nucleotide sequence of the sense strand nucleotide sequence in the same row in Table 21, and wherein the sense strand is conjugated to cholesterol as indicated in Table 21 conjugated to the sense strand of the siRNA agent.

[0105] In certain embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises the nucleotide sequence of any one of the sense strand nucleotide sequences in Table 21, and the antisense strand comprises a nucleotide sequence partially complementary to the sense strand. In some embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises the nucleotide sequence of any one of the sense strand nucleotide sequences in Table 21, and the antisense strand comprises a nucleotide sequence substantially complementary to the sensestrand. In certain embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises the nucleotide sequence of any one of the sense strand nucleotide sequences in Table 21, and the antisense strand comprises a nucleotide sequence fully complementary to the sense strand.

[0106] In certain embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises the nucleotide sequence of any one of the sense strand nucleotide sequences in Table 21, wherein the sense strand is conjugated to cholesterol as indicated in Table 21 conjugated to the sense strand of the siRNA agent, and the antisense strand comprises a nucleotide sequence partially complementary to the sense strand. In some embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises the nucleotide sequence of any one of the sense strand nucleotide sequences in Table 21, wherein the sense strand is conjugated to cholesterol as indicated in Table 21, and the antisense strand comprises a nucleotide sequence substantially complementary to the sense strand. In certain embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises the nucleotide sequence of any one of the sense strand nucleotide sequences in Table 21, wherein the sense strand is conjugated to cholesterol as indicated in Table 21, and the antisense strand comprises a nucleotide sequence fully complementary to the sense strand.

[0107] In certain embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises the nucleotide sequence of any one of the antisense strand nucleotide sequences in Table 21, and the sense strand comprises a nucleotide sequence partially complementary to the antisense strand. In some embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises the nucleotide sequence of any one of the antisense strand nucleotide sequences in Table 21, and the sense strand comprises a nucleotide sequence substantially complementary to the antisense strand. In certain embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises the nucleotide sequence of any one of the antisense strand nucleotide sequences in Table 21, and the sense strand comprises a nucleotide sequence fully complementary to the antisense strand.

[0108] In certain embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises the nucleotide sequence of any one of the antisense strand nucleotide sequences in Table 21, and the sense strand comprises a nucleotide sequence partially complementary to the antisense strand, and wherein the sense strand of the siRNA agent is conjugated to cholesterol as indicated in Table 21, or another lipophilic moiety, such as, e.g., described in FIG. 1. In some embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises the nucleotide sequence of any one of the antisense strand nucleotide sequences in Table 21, and the sense strand comprises a nucleotide sequence substantially complementary to the antisense strand, and wherein the sense strand of the siRNA agent is conjugated to cholesterol as indicated in Table 21, or another lipophilic moiety, such as, e.g., described in FIG. 1. In certain embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises the nucleotide sequence of any one of the antisense strand nucleotide sequences in Table 21, and the sense strand comprises a nucleotide sequence fully complementary to the antisense strand, and wherein the sense strand of the siRNA agent is conjugated to cholesterol as indicated in Table 21, or another lipophilic moiety, such as, e.g., described in FIG. 1.

[0109] In some embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises the nucleotide sequence of any one of the sense strand nucleotide sequences in Table 4. In some embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises the nucleotide sequence of any one of the antisense strand nucleotide sequences in Table 4.

[0110] In some embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises the nucleotide sequence of the sense strand nucleotide sequence in a row in Table 4, and the antisense strand comprises the nucleotide sequence of the antisense strand nucleotide sequence in the same row in Table 4. In some embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises the nucleotide sequence of any one of the sense strand nucleotide sequences in Table4, and wherein the sense strand is conjugated to a lipophilic moiety as indicated in Table 4 conjugated to the sense strand of the siRNA agent. In some embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises the nucleotide sequence of the antisense strand nucleotide sequence in a row in Table 4, and the sense strand comprises the nucleotide sequence of the sense strand nucleotide sequence in the same row in Table 4, and wherein the sense strand is conjugated to a lipophilic moiety as indicated in Table 4 conjugated to the sense strand of the siRNA agent.

[0111] In certain embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises the nucleotide sequence of any one of the sense strand nucleotide sequences in Table 4, and the antisense strand comprises a nucleotide sequence partially complementary to the sense strand. In some embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises the nucleotide sequence of any one of the sense strand nucleotide sequences in Table 4, and the antisense strand comprises a nucleotide sequence substantially complementary to the sense strand. In certain embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises the nucleotide sequence of any one of the sense strand nucleotide sequences in Table 4, and the antisense strand comprises a nucleotide sequence fully complementary to the sense strand.

[0112] In certain embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises the nucleotide sequence of any one of the sense strand nucleotide sequences in Table 4, wherein the sense strand is conjugated to a lipophilic moiety as indicated in Table 4 conjugated to the sense strand of the siRNA agent, and the antisense strand comprises a nucleotide sequence partially complementary to the sense strand. In some embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises the nucleotide sequence of any one of the sense strand nucleotide sequences in Table 4, wherein the sense strand is conjugated to a lipophilic moiety as indicated in Table 4, and the antisense strand comprises a nucleotide sequence substantially complementary to the sense strand. In certain embodiments, provided herein is an siRNA agentcomprising a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises the nucleotide sequence of any one of the sense strand nucleotide sequences in Table 4, wherein the sense strand is conjugated to a lipophilic moiety as indicated in Table 4, and the antisense strand comprises a nucleotide sequence fully complementary to the sense strand.

[0113] In certain embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises the nucleotide sequence of any one of the antisense strand nucleotide sequences in Table 4, and the sense strand comprises a nucleotide sequence partially complementary to the antisense strand. In some embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises the nucleotide sequence of any one of the antisense strand nucleotide sequences in Table 4, and the sense strand comprises a nucleotide sequence substantially complementary to the antisense strand. In certain embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises the nucleotide sequence of any one of the antisense strand nucleotide sequences in Table 4, and the sense strand comprises a nucleotide sequence fully complementary to the antisense strand.

[0114] In certain embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises the nucleotide sequence of any one of the antisense strand nucleotide sequences in Table 4, and the sense strand comprises a nucleotide sequence partially complementary to the antisense strand, and wherein the sense strand of the siRNA agent is conjugated to a lipophilic moiety as indicated in Table 4, or another lipophilic moiety, such as, e.g., described in FIG. 1. In some embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises the nucleotide sequence of any one of the antisense strand nucleotide sequences in Table 4, and the sense strand comprises a nucleotide sequence substantially complementary to the antisense strand, and wherein the sense strand of the siRNA agent is conjugated to a lipophilic moiety as indicated in Table 4, or another lipophilic moiety, such as, e.g., described in FIG. 1. In certain embodiments, provided herein is an siRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises the nucleotidesequence of any one of the antisense strand nucleotide sequences in Table 4, and the sense strand comprises a nucleotide sequence fully complementary to the antisense strand, and wherein the sense strand of the siRNA agent is conjugated to a lipophilic moiety as indicated in Table 4, or another lipophilic moiety, such as, e.g., described in FIG. 1.

[0115] In specific embodiments, an siRNA agent described herein comprises the sense strand and the antisense strand of one named in Table 1, 2, 3, or 4. In specific embodiments, an siRNA agent described herein comprises a sense strand and an antisense strand corresponding to the sense strand and the antisense strand of any one of US14, US28, US58, US183, or US199 in Table 1. In specific embodiments, an siRNA agent described herein comprises the sense strand and the antisense strand of any one of S14, S28, S58, S183, or S199 in Table 2. In specific embodiments, an siRNA agent described herein comprises the sense strand and the antisense strand of one named in Tables 3 or 4. In some embodiments, the sense strand of the siRNA agent is conjugated to a lipophilic moiety described herein (e.g., FIG. 1). In specific embodiments, an siRNA agent is one described in the EXAMPLES section, infra.

[0116] In some embodiments, an siRNA agent described herein further comprises a nonnucleotide group, such as a ligand (e.g., a targeting ligand). The non-nucleotide group, such as a ligand (e.g., a targeting ligand) may be conjugated directly or directly to the antisense strand or sense strand of the siRNA agent. The non-nucleotide group, such as a ligand (e.g., a targeting ligand) replace one or more nucleotides in an internal position of the double stranded region of an siRNA agent described herein. In certain embodiments, the non-nucleotide group, such as a ligand (e.g., a targeting ligand) is conjugated to a strand of the siRNA agent via a linker or carrier (e.g., a delivery vehicle). In a specific embodiment, the non-nucleotide group, such as a ligand (e.g., a targeting ligand) is one described in the Moieties Linked to Nucleotide Sequence section, infra.Chemical Modifications to Nucleotides

[0117] In specific embodiments, an siRNA agent described herein comprises one or more nucleotide modifications. Nucleotide modifications include, for example, end modifications, e.g., 5’-end modifications (e.g., phosphorylation, conjugation, inverted linkages) or 3’-end modifications (e.g., conjugation, DNA nucleotides, inverted linkages, etc.); base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with anexpanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases; sugar modifications (e.g., at the 2’-position or deposition) or replacement of the sugar; or backbone modifications, including modification or replacement of the phosphodiester linkages. In some embodiments, an siRNA agent comprises at least one modification selected from the group consisting of modified internucleoside linkage, modified nucleobase, modified sugar, and any combinations thereof. Without limitations, such a modification can be present anywhere in the siRNA agent (e.g., in the sense strand, antisense strand, or both strands).

[0118] In some embodiments, an siRNA agent comprises one or more modified sugar modifications, such as one or more substituted sugar moieties. In some embodiments, an siRNA agent described herein includes one of the following at the 2’-position: H; F; or OCH3 (OMe).

[0119] In some embodiments, an siRNA agent described herein includes one or more glycol nucleic acids (GNA). Typically, GNA is an acyclic nucleic acid analogue, wherein its repeating glycol units are linked by phosphodi ester bonds, differing from RNA’s ribose sugarphosphodiester backbone composition.

[0120] In certain embodiments, an siRNA agent described herein includes one or more terminal modifications, for example, 5 ’-terminal phosphorylation, conjugation, or inverted linkages. In some embodiments, the terminal modifications include a 5 ’-phosphate, for example, a 5 ’-terminal phosphate on the antisense strand of an siRNA agent.

[0121] In some embodiments, an siRNA agent includes a sense strand and / or an antisense strand with an inverted abasic nucleotide. In one embodiment, the sense strand contains an inverted abasic nucleotide at the 3’ end. In another embodiment, the sense strand contains an inverted abasic nucleotide at the 5’ end. In some embodiments, the sense strand contains an inverted abasic nucleotide at the 5’ and 3’ end.

[0122] In some embodiments, an siRNA agent comprises a phosphate or phosphate mimic at the 5 ’-end of the antisense strand. In one embodiment, the phosphate mimic is a 5 ’-vinyl phosphonate (VP).

[0123] In some embodiments, an siRNA agent comprises one or more modified nucleotides. In specific embodiments, the modified nucleotides are selected from the group consisting of a 2’O-methyl modified nucleotide, a deoxy-nucleotide, a 2’ -fluoro modified nucleotide, a 2’-O- methyl-uridine, a 3’-O-methyl modified nucleotide, a 3’-O-methyl modified nucleotide with 2’- 5’ linked phosphate, an inverted abasic nucleotide, a nucleotide comprising S-glycol nucleic acid(GN A), an unlocked nucleotide, a 5’-vinylphosphonate-2’-O-methyl-uridine, a cis-cyclobutyl phosphonate modified nucleotide, a 5 ’-cis-cyclobutyl phosphonate-2’-O-methyl modified nucleotide, a (L)-a-threofuranosyl modified nucleotide, and combinations thereof. In specific embodiments, the modified nucleotides are selected from the group consisting of a 2’0-methyl modified nucleotide, a deoxy-nucleotide, a 2’-fluoro modified nucleotide, an inverted abasic nucleotide, a nucleotide comprising GNA, an unlocked nucleotide, a vinyl phosphonate, and combinations thereof.

[0124] In some embodiments, an siRNA agent comprises one or more modified internucleoside linkages (i.e., a modified RNA backbone). Modified internucleoside linkages include, for example, phosphorothioates (e.g., phosphoromonothioates). Various salts, mixed salts and free acid forms are also included. In some embodiments, an siRNA agent described herein is in a free acid form. In other embodiments, an siRNA agent described herein is in a salt form. In one embodiment, an siRNA agent described herein is in a sodium salt form. In certain embodiments, when an siRNA agent described herein is in the salt form, cations of the salt (e.g., sodium cations) are present at the agent as counterions for substantially all of the electronegative groups (e.g., phosphodiester and / or phosphor othioate groups) present in the agent. In some embodiments, the counterion is a condensed counterion. In specific embodiments, the counterion is a condensed sodium cation. In some embodiments, the condensed counterion is hydrated. In specific embodiments, the condensed counterion is a hydrated sodium cation. Agents in which substantially all of the phosphodiester and / or phosphorothioate linkages have a counterion include not more than 5, 4, 3, 2, or 1 phosphodiester and / or phosphorothioate linkages without a counterion. In other words, the electronegative potential of the siRNA agent is neutralized or substantially neutralized by counterion condensation around the siRNA. In some embodiments, when an siRNA agent described herein is in the sodium salt form, sodium ions are present around the agent as counterions for substantially all of the phosphodiester and / or phosphorothioate groups present in the agent.

[0125] The phosphate group of an internucleoside phosphodiester linkage can be modified by replacing one of the oxygens with a different substituent. One result of this modification can be increased resistance of the oligonucleotide to nucleolytic breakdown. Another result of this modification can be increased stability of hybridized single-stranded RNA (ssRNA) in thesiRNA agent. An example of a modified phosphate group includes phosphorothi oates (e.g., phosphoromonothi oates) .

[0126] In some embodiments, an siRNA agent comprises an RNA mimetic, in which both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with alternate groups. In specific embodiments, the base units are maintained for hybridization with an appropriate target sequence.

[0127] An siRNA agent described herein can contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations that may be defined, in terms of absolute stereochemistry, as (R) or (S), such as for sugar anomers, or as (D) or (L) such as for amino acids. Included in an siRNA agent provided herein are all such possible isomers, as well as their racemic and optically pure forms.Moieties Linked to Nucleotide Sequence

[0128] In some embodiments, an siRNA agent described herein is conjugated to one or more non-nucleotide groups. The non-nucleotide group can, e.g., enhance targeting, delivery or attachment of the siRNA agent. The non-nucleotide group can be covalently linked to the 3' end, 5' end, and / or internally to either the sense strand or the antisense strand of the siRNA agent. The non-nucleotide group can be covalently linked to the 3' end, 5' end, both the 3’ end and 5’ end, internally, both the 3’ end and internally, both 5’ end and internally, or at the 3’ end, the 5’ end, and internally of the sense strand and / or the antisense strand of the siRNA agent. In some embodiments, an siRNA agent described herein contains a non-nucleotide group linked to the 3' end, 5' end, both the 3’ end and 5’ end, internally, both the 3’ end and internally, both 5’ end and internally, or at the 3’ end, the 5’ end and internally of the sense strand. In certain embodiments, an siRNA agent described herein contains a non-nucleotide group linked to the 5' end of the sense strand. In certain embodiments, an siRNA agent described herein contains a non- nucleotide group linked to the 3’ end of the sense strand. In certain embodiments, an siRNA agent described herein contains a non-nucleotide group linked to the sense strand internally. A non-nucleotide group may be linked directly or indirectly to the siRNA agent via a linker / linking group.

[0129] In some embodiments, an siRNA agent is linked to one or more lipophilic moieties, such as, e.g., a cholesterol moiety (e.g., CHOL4 shown in FIG. 1). In specific embodiments, alipophilic moiety that is linked to an siRNA agent comprises one or more of the following compounds depicted FIG. 1, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof: LI (also referred to herein as “J2-CONC16”), L2 (also referred to herein as “J2- C3NC16”), L3 (also referred to herein as “J2-NCOOC16”), L4 (also referred to herein as “J2- C2OC16”), and / or L5 (also referred to herein as “J2-C15Ada”). In specific embodiments, a lipophilic moiety is selected from one or more of the following: LI, L2, L3, L4, and L5 (see FIG. 1). In specific embodiments, a lipophilic moiety comprises CH0L4. In a specific embodiment, a lipophilic moiety, such as described in the EXAMPLES section, is conjugated to the sense strand of an siRNA agent in the manner described in the EXAMPLES section. In some embodiments, a lipophilic moiety that is linked to an siRNA agent comprises one or more of the following compounds depicted FIG. 1: LI, L2, L3, L4, L5, and / or CH0L4 in FIG. 1. In some embodiments, the lipophilic moiety is linked to the 3’ end, 5’ end, and / or internally to the sense strand.

[0130] In one embodiment, the sense strand of an siRNA agent is conjugated, directly or indirectly, to a lipophilic moiety at the 5’ end. In one embodiment, the first nucleotide at the 5’ end of the sense strand is connected to a lipophilic moiety. In one embodiment, the sense strand of an siRNA agent is conjugated, directly or indirectly, to a lipophilic moiety internally.

[0131] In one embodiment, the sense strand is conjugated, directly or indirectly, to a lipophilic moiety at the 3’ end. In one embodiment, the first nucleotide at the 3’ end of the sense strand is connected to a lipophilic moiety. In one embodiment, the first nucleotide at the 3’ end of the sense strand is connected to a lipophilic moiety via a linker. In one embodiment, the linker is a nucleotide linker of the length of 1, 2, 3, 4, or 5 nucleotides. In one embodiment, the nucleotide linker is dTdT. In one embodiment, the linker is an InvAb.

[0132] In one embodiment, the sense strand is conjugated, directly or indirectly, to a lipophilic moiety at the 5’ end, and the sense strand is also conjugated, directly or indirectly, to a lipophilic moiety at the 3’ end, each of which is as described herein and elsewhere. The lipophilic moiety at the 5’ end and the 3’ end can be the same or different.

[0133] In one embodiment, the sense strand is conjugated, directly or indirectly, to a lipophilic moiety at the 5’ end, at the 3’ end, and / or internally. The lipophilic moieties at the 5’ end, the 3’ end, and / or internally can be the same or different.

[0134] In some embodiments, a linking group is conjugated to the siRNA agent. The linking group facilitates covalent linkage of the agent to a targeting ligand or delivery polymer or delivery vehicle. The linking group can be linked to the 3' end, the 5' end, and / or internally of the siRNA agent sense strand. In some embodiments, the linking group is linked to the siRNA agent sense strand. In some embodiments, the linking group is conjugated to the 5' end, 3' end, and / or internally to the sense strand of an siRNA agent. In some embodiments, a linking group is conjugated to the 5' end the sense strand of an siRNA agent. In some embodiments, a linking group is conjugated to the 3' end of the sense strand of an siRNA agent. In some embodiments, a linking group is conjugated internally to the sense strand of an siRNA agent.

[0135] Typically, a linker or linking group is a connection between two atoms that links one chemical group (such as an siRNA agent) or segment of interest to another chemical group (such as a targeting group or delivery polymer) or segment of interest via one or more covalent bonds. A labile linkage contains a labile bond. A linkage may optionally include a spacer that increases the distance between the two joined atoms. A spacer may further add flexibility and / or length to the linkage.

[0136] Any of the siRNA agent nucleotide sequences listed in Table 1, 2, 3, or 4, whether modified or unmodified, may contain a 3' end, 5' end, and / or internal targeting ligand and / or linking group. Any of the siRNA agent duplexes listed in Table 1, 2, 3, or 4, whether modified or unmodified, may further comprise a targeting ligand and / or linking group, and the targeting ligand or linking group may be attached to the 3' end, 5' end, and / or internally to either the sense strand or the antisense strand of the siRNA agent duplex.Synthesis of siRNA Agents

[0137] An siRNA agent can be synthesized by standard methods known in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S.L. et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which is hereby incorporated herein by reference, or described herein (e.g., in EXAMPLES section, infra). For example, an siRNA agent may be prepared using a two-step procedure. First, the individual strands of the siRNA agent are prepared separately. Then, the component strands are annealed. The individual strands of the siRNA agent can be prepared using solution-phase or solid-phase organic synthesis or both. Organic synthesis offers the advantage that the oligonucleotide strands comprising unnatural or modified nucleotides can be easily prepared. Similarly, single-strandedoligonucleotides can be prepared using solution-phase or solid-phase organic synthesis or both. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems® (Foster City, Calif). In some embodiments, an oligonucleotide(s) of an siRNA agent described herein is synthesized so that it contains a reactive group, such as an amine group, at the 5'-end, 3 ’-end and / or internally. Such a reactive group may be used to subsequently attach a ligand (e.g., targeting ligand) using methods typical in the art. In some embodiments, an oligonucleotide(s) of an siRNA agent is synthesized with a linking group. This linking group can be used to conjugate a non-nucleotide group (e.g., a lipophilic moiety, a ligand or delivery group) to the siRNA agent. In some embodiments, an oligonucleotide(s) of an siRNA agent is synthesized by an automated synthesizer using phosphoramidites (e.g., standard phosphoramidites and non-standard phosphoramidites, which are commercially available). In a specific embodiment, an siRNA agent is produced using a method described in EXAMPLES section, infra. In another specific embodiment, a method described in EXAMPLES section, infra, is used to produce an siRNA agent described herein, including one conjugated to a non- nucleotide group, such as described in Moieties Linked to Nucleotide Sequence section, supra.Delivery Vehicles

[0138] In some embodiments, a delivery vehicle may be used to deliver an siRNA agent described herein to a cell or tissue.Compositions

[0139] In one aspect, provided herein are compositions comprising an siRNA agent described herein (e.g, in Nucleotide Sequences section, supra, or EXAMPLES section, infra). The composition may further comprise a carrier such as physiological saline, and / or one or more excipients including, but not limited to a buffer, a surfactant, a stabilizing agent, and a preservative.Uses of siRNA Agents

[0140] The siRNA agents and compositions of the invention may be used to inhibit the expression of an SNCA gene, e.g., a human SNCA gene. As shown in the EXAMPLES, the siRNA agents inhibit expression of the SNCA gene. The siRNA agents and compositions of theinvention may also be used to inhibit or reduce the levels of oc-synuclein. As shown in the EXAMPLES, the siRNA agents inhibit oc-synuclein levels. Certain assays and methods for measuring SNCA expression levels and for measuring oc-synuclein levels are described here. Other assays for measuring SNCA expression levels and for measuring oc-synuclein levels are known in the art.

[0141] Inhibition of SNCA gene expression and / or inhibition of oc-synuclein levels is useful for studying neurodegenerative diseases, oc-synuclein levels have been associated with diseases such as PD, DLB, PAF, and MSA. The siRNAs of the invention may be used to study how levels of SNCA gene expression inhibition impact oc-synuclein levels. The siRNAs of the invention may be used in animal models to study how levels of SNCA gene expression inhibition impact oc-synuclein levels. The siRNAs of the invention may be used to study how levels of SNCA gene expression and / or oc-synuclein levels impact one or more symptoms of neurodegenerative diseases such as PD, DLB, PAF, and MSA.

[0142] In one aspect, provided herein is a method for inhibiting the expression of an SNCA gene (e.g., human SNCA gene) in a cell, comprising contacting a cell with an siRNA agent described herein, or a composition comprising an siRNA agent described herein. In a specific embodiment, provided herein is a method for inhibiting the expression of an SNCA gene (e.g., human SNCA gene) in a population of cells, comprising contacting a population of cells with an siRNA agent described herein. The contact between cell(s) and an siRNA agent described herein may be direct or indirect. For example, the cell(s) may be put into physical contact with the siRNA agent, or the cell(s) may be put into a situation that will permit or cause it to subsequently come into contact with the siRNA agent.

[0143] The expression of an SNCA gene (e.g., human SNCA gene) may be measured directly or indirectly. For example, the levels of SNCA RNA (e.g., pre-mRNA levels, mRNA levels, or both), the levels of a protein encoded by an SNCA gene, a function(s) of the protein encoded by an SNCA gene, or a combination thereof may be measured. Alternatively, or in addition, the expression of genes whose expression is indirectly or directly impacted by the expression of an SNCA gene, a function of a protein, which is indirectly or directly impacted by the expression of the SNCA gene, or a combination thereof may be measured. See, e.g, Biological Assays section, infra, for ways that may be used to measure the expression of an SNCA gene directly or indirectly.Biological Assays

[0144] Biological assays known to one of skill in the art or described herein (e.g., in EXAMPLES section, infra) may be used to assess the ability of an siRNA agent described herein to inhibit SNCA gene expression, the specificity of an siRNA agent described herein, the stability of an siRNA agent described herein, the off-target effects of an siRNA agent described herein, the toxicity of an siRNA agent described herein, the localization of an siRNA agent to specific tissues, the pharmacokinetics of an siRNA agent, and the immunogenicity of an siRNA agent described herein.

[0145] In some embodiments, SNCA gene expression is measured at the RNA or protein level. The expression level of SNCA gene can be assessed by any method known in the art for measuring RNA. In certain embodiments, the RNA may be isolated from samples by RNA extraction methods, for example, organic extraction, membrane-based spin column, and paramagnetic particle technology. In certain embodiments, SNCA RNA levels may be measured by UV spectroscopy, in situ hybridization, fluorescent in situ hybridization (FISH), northern blotting, microarray, RT-PCR, qPCR, fluorescent dye-based quantification, and gel electrophoresis. The SNCA protein expression levels can be measured by any method known in the art for characterizing proteins. In certain embodiments, SNCA protein can be purified by salt precipitation, dialysis, and chromatography (e.g., gel filtration, ion exchange, affinity purification). In certain embodiments, crude samples containing SNCA protein or purified SNCA protein can be characterized by UV absorbance and spectroscopy, electrophoresis, capillary electrophoresis, chromatography (e.g., gel filtration, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography), mass spectrometry. In certain embodiments, crude samples containing SNCA protein or purified SNCA protein can be characterized by flow cytometry, immunodiffusion, immunoelectrophoresis, western blot, immunoblot, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELIS As), immunofluor escent assays, electrochemiluminescence assays, and the like. In some embodiments, the function of the protein encoded by the SNCA gene may be assessed to measure expression of the gene. For example, formation of oligomers, protofibrils and insoluble fibrils of oc-synuclein may be assessed. Cells contacted in vitro or ex vivo with an siRNA agent may be assessed for expression of the gene.

[0146] In some embodiments, a function(s) of the oc-synuclein protein encoded by the SNCA gene is assessed using an assay known to one of skill in the art. Multiple functions have been proposed for normal oc-synuclein with the most established one being microtubule stabilization.

[0147] In some embodiments, the SNCA gene expression level may be assessed using indirect measurement including expression of certain genes, functional assays of downstream proteins, and reporter assays. In some embodiments, the expression of the SNCA gene may be assessed by detecting the expression of a protein regulated by the expression of the SNCA gene. In certain embodiments, the expression of the SNCA gene may be assessed by detecting the function of a protein regulated by the expression of the SNCA gene. In some embodiments, a reporter gene can be introduced to facilitate indication of target expression. For example, a reporter gene regulated by a protein downstream of the protein encoded by the SNCA gene can indicate the expression level of the SNCA gene. Reporter genes may include, FLAG-tag, green fluorescence protein (GFP), the chloramphenicol acetyltransferase gene (cat) from Tn9 of E. coli, the luciferase gene (luc) from firefly Photinus pyralis, and [3-glucuronidase (GUC). The easily detectable proteins encoded by the reporter gene indicate the SNCA gene expression level. The reporter assays may include fluorescence-based assays, for example, fluorescence spectroscopy, fluorescence immune assays, flow cytometry, fluorescence spectrometry, and ELISA.

[0148] In some embodiments, the specificity of an siRNA agent may be analyzed by the effect generated by the inhibited genes it targets or by the off-target genes that are affected. The effect includes inhibition of the SNCA gene expression, wherein the siRNA agent displays its potency by a half maximal inhibitory concentration, i.e., IC50. The candidate siRNA agents are selected using IC50 value in terms of effectiveness. In some embodiments, an siRNA agent described herein has an IC50 of 6 nM to 5000 nM, or 10 nM to 1000 nM, in neurons (e.g., human iPSC-neurons). In certain embodiments, the specificity of the siRNA agents is tested using a negative control, wherein a scrambled or random sequence may be used. The siRNA agent that does not affect the negative target may be qualified as specific. In certain embodiments, the specificity of the siRNA agents is tested using a single target gene. Different siRNA agents to the same gene with comparable gene inhibitory efficacy should induce similar changes in gene expression profiles or phenotypes. Any changes induced by one siRNA agent and not the other(s) may be attributed to off-target effects or non-specificity. In certain embodiments, thespecificity of the siRNA agents is tested using titration. Non-specific effect is mitigated when siRNA agents are used at lower concentrations. The siRNA agent that displays a lower effective concentration may have a higher specificity. In certain embodiments, the specificity of the siRNA agents is tested by monitoring both RNA and protein levels. For example, RNA gene reduction seen without a corresponding reduction in protein levels indicates that protein turnover is slow. The siRNA agent being investigated may have off-target effects. In some embodiments, a transcriptome assay may be used to assess off-target effects.

[0149] In some embodiments, the Forster resonance energy transfer (FRET) method based on agarose gel electrophoresis is to evaluate the stability of an siRNA agent described in a biological sample or fluid, such as, e.g., serum or cerebrospinal fluid sample. See, e.g, Tuttolomondo and Ditzel (2021) for a description of such an assay. In some embodiments, such an assay is also used to evaluate the interaction of siRNA agent described herein with serum proteins and enzymes. In certain embodiments, an agarose gel shift assay is used to evaluate the stability of an siRNA agent described herein in a biological sample or fluid, such as, e.g, serum or cerebrospinal fluid sample.

[0150] In some embodiments, the stability of an siRNA agent may be investigated under a variety of conditions, including temperature, and RNase degradation in biological fluids. In some embodiments, thermal melting temperature of an siRNA agent is measured with equimolar concentrations of both strands by monitoring A260 with increasing temperature (e.g, l°C / min). Tm is measured as the maximum of the first derivative of the melting curve (A260 vs T) of prehybridized duplexes. A stable unmodified RNA may have a Tmranging from 40°C to 60°C in terms of the thermal stability. A modified RNA may be stabilized or destabilized by ~10°C. In some embodiments, the stability of an siRNA agent toward RNase degradation is determined by incubating with different biological fluid. For example, the serum stability can be determined by incubating with fetal bovine serum or human serum at 37°C for various time periods up to 48 hours. The RNA in the samples can be examined by northern blot, in situ hybridization, qPCR, and any RNA detecting methods known in the art. The activity of siRNA agent can be tested. A stable siRNA agent may show minor degradation at 37°C for 24 hours. In some embodiments, an siRNA agent shows sufficiently stable for at least 2 hours. In other embodiments, the stability of an siRNA agent exposed to, but not limited to, skin, salvia, topical creams or nanoparticles may be tested.

[0151] In specific embodiments, stability of siRNA agents described herein in mouse brain homogenate, human liver lysosomes, and rat liver tritosomes are assessed as described in EXAMPLES section, infra.

[0152] In some embodiments, the toxicity of an siRNA agent to in vitro or ex vivo cell-based models is investigated. In certain embodiments, the toxicity is evaluated by the degree to which the siRNA agent can cause damage to a cell. The damage can be necrosis (uncontrolled cell death), apoptosis (programmed cell death), autophagy, or stop actively growing and dividing to decrease cell proliferation. In certain embodiment, cytotoxicity assays are performed to measure the ability of the siRNA agent to cause cell damage or cell death. For example, the release of lactate dehydrogenase (LDH) and glucose 6-phosphate dehydrogenase (G6PD) can be used as biomarkers for cellular plasma membrane damage. In another embodiment, cell viability under the treatment of the siRNA agent can be detected by various mechanisms, for example, membrane integrity, enzyme activity, or metabolic activity.

[0153] In some embodiments, RNA in situ hybridization assays may be used to image an siRNA agent and to assess the inhibition of SNCA gene expression in tissues samples from a subject. RNAscope® may be used, e.g., for RNA in situ hybridization assays.In some embodiments, an siRNA agent described herein is used to study, for example, PD, DLB, PAF, and MSA in an animal model (e.g., a mouse model). Animal models are known in the art. In a specific embodiment, the animal model is one described in EXAMPLES section, infra.EXAMPLESExample 1: Bioinformatics and siRNA SynthesisSNCA siRNA bioinformatics in silico hits ID

[0154] A set of siRNAs targeting human SNCA, Gene ID: 6622, were designed using all 9 transcript variants of Homo sapiens synuclein alpha (SNCA), including GenBank Accession Nos. NM_000345.4, NM_001146054.2, NM_001146055.2, NM_001375287.1, NM_007308.2, NM_001375285.1, NM_001375288.1, NM_001375286.1, NM_001375290.1.Identification of potential screening candidate siRNAs targeting human SNCA mRNA

[0155] The bioinformatical approach assumes a canonical siRNA structure. Positions 2-18 (5’-3 ’) of the sense and antisense strand were used for the specificity calculations. Positions 1-19 (5 ’-3’) of the antisense strand were used for cross-reactivity and human single nucleotide polymorphism (SNP) analysis.

[0156] The following parameters were assessed:■ Species cross-reactivity for human, cynomolgus monkey, rhesus monkey, mouse, and rat: Analysis based on a canonical siRNA design using 19 bases and 17 bases (without considering positions 1 and 19) for cross-reactivity. Analysis included full match as well as single mismatch analysis.■ Cross-reactivity for human, cynomolgus monkey, rhesus monkey, mouse and rat were analyzed.■ Predicted specificity in human, rhesus monkey, cynomolgus monkey, mouse, and rat: Analysis of sense and antisense strand separately.■ Potential match between the siRNA seed region and the seed regions of known miRNAs.■ Analysis of human SNP database (NCBI-DB-SNP) to identify siRNAs targeting regions with known SNPs. Information will include positions of SNPs within the target sequence as well as minor allele frequency (MAF) in cases where the data are available.■ siRNA activity prediction based on canonical siRNA design.

[0157] The bioinformatics procedure is described below.■ Possible siRNAs were created from human SNCA mRNA sequences (NM_000345.4).■ siRNA off-target hits were predicted for human, rhesus monkey, cynomolgus monkey, mouse, and rat.■ A specificity score was assigned to each siRNA strand.■ siRNA strands were analyzed for presence of human, rhesus monkey, dog, pig, mouse, rat, and rabbit miRNA seed regions.■ Specificity categories were assigned to siRNAs (combined specificity score + miRNA seed analysis).■ siRNA cross-reactivity was calculated: for transcript variants and different species, for 19mers and 17mers (nucleotides 2-18 of 19mer) and for 19mers and 17mers with a single mismatch.■ Human SNPs were mapped to siRNA target sites in SNCA transcript GenBank Accession No. NM_000345.4 and all 9 transcript variants.■ siRNA activity was predicted, and a score was assigned to each siRNA.

[0158] Based on the above procedure, 340 hits of SNCA human sequences were selected from 4028 target sequences. Table 1 provides the unmodified sense and antisense strand nucleotide sequences of siRNAs US1 to US340 targeting human SNCA, Gene ID: 6622. Table2 provides sense and antisense strand nucleotide sequences of siRNAs SI to S340, which result from chemical modifications applied to siRNAs US1 to US340, respectively. Such chemical modifications include 2’-fluoro (2’-F), 2’-O-methyl (2’-0Me), and phosphorothioate.Synthesis of SNCA siRNA duplex

[0159] The 340 human SNCA siRNAs were synthesized and annealed as described below. Briefly, single-stranded sense and antisense oligonucleotides were synthesized at 0.2 pmol scale on a Dr. Oligo 192 synthesizer (Biolytic Lab Performance) using the standard solid-phase phosphoramidite chemistry. Controlled pore glass (CPG, 500 A) was used as solid support loaded with first base or UnyLinker™. 3% dichloroacetic acid in dichloromethane was used for detritylation, and 2’-0Me and 2’-F modified nucleotides were coupled by using corresponding phosphoramidites. Coupling time for all phosphoramidites was 6-8 minutes using 5-Ethylthio- IH-tetrazole (ETT) as activator (0.25 M in acetonitrile). Phosphorothioate linkages were generated using a 0.2 M solution of xanthane hydride in anhydrous pyridine. Oxidation time was3 minutes using 0.02 M L in tetrahydrofuran with 10% water.

[0160] After synthesis, the solid support was transferred to a 1.5 mL vial. Cleavage and deprotection were performed using 500 pL AMA (concentrated ammonia / 40% aqueous methylamine, v / v = 1: 1). After the completion of cleavage and deprotection, the sample was filtrated to remove the solid support and washed once with water. The sample was purified using a 6 mL Source 15Q ion exchange column (Cytiva) on an AKTA Purifier System equipped with autosampler and fraction collector. Fractions were analyzed by LC-MS to confirm the quality. The appropriate fractions were pooled and desalted using CentriPure P10 Columns (emp Biotech). Samples from each sequence were analyzed by LC-MS to confirm the identity, UV (260 nm) for quantification and IP-RP chromatography to determine purity. Duplex annealing was performed by mixing equimolar of sense and antisense single strands. Final duplex sample solution was dried under vacuum using GeneVac evaporator (SP Scientific). The final quality control (QC) specifications are ± 0.05% of calculated mass (by LC / MS) for single strandidentity, >85% full length oligonucleotide (by HPLC) for single strand purity, and >90% by nondenaturing HPLC for duplex purity. siRNA-lipid conjugation

[0161] A subset of the siRNA compounds was synthesized with different novel lipid conjugates and at different positions (5’ / 3’-end, or internal). Lipids were incorporated to the sense strands via on-column and / or post-column synthesis. To perform on-column synthesis, lipophilic moiety was modified at the corresponding phosphoramidite monomer or CPG, followed by solid-phase phosphoramidite chemistry, as described above, to synthesize sense strand with 573’ terminal and / or the internal lipid conjugates. For post-column synthesis the lipids were introduced in solution phase with their corresponding N-hy dr oxy succinimide esters and the appended amine linker of the nucleotide.

[0162] Conjugates of siRNA with CHOL4 s are shown in Table 3 (and Table 21). Conjugates of siRNA conjugated with other lipophilic moieties are shown in Table 4.On-column synthesis

[0163] For example, as shown in Scheme 1 below, on-column introduction of compound 2 at the 3’ end of the sense strand was performed using 3 ’-amino modifier such as 2- dimethoxytrityloxymethyl-6-fluorenylmethoxy carbonylamino-hexane- 1 -succinoyl)-long chain alkylamino-CPG 1. Following the deprotection of the Fmoc group with 20% piperidine in N,N- dimethylformamide (DMF), the CPG was thoroughly washed with DMF, methyl cyanide (MeCN, or acetonitrile (ACN)), diethyl ether, and dried. A solution of 80 pmol of compound 2 in 2 ml of 1,4-dioxane was added to the above CPG followed by 30 pl of diisopropylethylamine (DIPEA) and shaken for 12 hours to produce compound 3. The CPG was washed with methylene chloride (DCM), MeCN, and EtiO. A solution of 1 ml of CAP A and CAP B was added and shaken for 30 minutes. Compound 3 was washed with DCM, MeCN, EtiO, and dried and used for the oligonucleotide synthesis. To synthesize oligonucleotide conjugated with cholesterol at 3’ end, 3'-Cholesteryl-TEG CPG was used.Scheme 1

[0164] Introduction of compound 2 at the 5’ end of the sense strand was carried out using 5’- amino modifier such as 6-(4-monomethoxytritylamino)hexyl-(2-cyanoethyl)-(N,N’-diisopropyl)- phosphoramidite. After the synthesis of the sense strand on the CPG following the standard procedure, in the penultimate step the 5’ end of the sense strand was coupled to 6-(4- monomethoxytritylamino)hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite followed by deblocking of 4-monomethoxytrityl group with 3% di chloroacetate (DCA) / DCM for 30 minutes. The CPG was subsequently washed with DCM, ACN and coupled with compound 2 for 12 hours (Scheme 2), followed by strand cleavage and deprotection.Scheme 2

[0165] The lipid at the internal position was introduced during the sense strand synthesis with corresponding phosphoramidite monomer. For example, Scheme 3 illustrates this with uridine based phosphoramidite monomer 7.Scheme 3

[0166] The synthesis, cleavage and deprotection, the lipid conjugated oligonucleotides were purified by reversed-phase or ion-exchange chromatography. The buffers for reverse phase were 0.05 M sodium acetate in 90 / 10% water / acetonitrile (Buffer A) and acetonitrile (Buffer B), and the buffers for ion-exchange are 20 mM sodium phosphate in 90 / 10% water / acetonitrile (bufferA) and 20 mM sodium phosphate in 90 / 10% water / acetonitrile, 1.8 M sodium bromide (bufferB). Fractions containing full-length oligonucleotides were pooled, desalted, and the compounds were analyzed by liquid chromatography - mass spectrometry (LC-MS).Post-column synthesis

[0167] Lipids were conjugated at the 573’ terminal position and at the internal position of the sense strand in the solution phase after the purification and desalting of the corresponding oligonucleotide.

[0168] For example, incorporation at the internal position was carried out with phosphoramidite monomer derivatized with an amine linker at the 2’ position of respective nucleotide. Scheme 4 illustrates this with uridine based phosphoramidite monomer compound 9. After incorporation of compound 9 at the internal position and complete synthesis, cleavage, and deprotection, purification and desalting process, the free oligonucleotide with the pendant amine was coupled with corresponding lipid-NHS esters in solution phase.Scheme 4

[0169] Conjugation at the terminal / internal positions was carried out with 0.15 mM solution of deprotected and desalted oligonucleotide in 0.1M solution of NaHCO (pH 8.4) with 1.5 mM solution of the corresponding lipid-NHS ester in DMF at 60 °C. To a solution (0.25 mL) of oligonucleotide was added DMF solution of lipid-NHS ester (0.6 mL) and the resulting mixture heated at 63 °C. Progress was monitored by RP-HPLC (C-18 column, A: 50 mM triethylammonium Acetate Solution (TEAA), B: MeCN; gradient 5-100% B at 30 °C). Reaction was > 90% completed usually in less than one hour. Reaction mixture was diluted with water and purified by reverse phase (RP)-HPLC (C-8 Xbridge Waters column; A: 50 mM NaOAc, B: MeCN; 5-100% B gradient, at 60 °C). Isolated yields of lipid conjugated oligonucleotides were 60-70%. After purification, the products were desalted and duplex annealed, as described above.Example 2: in vitro screening of SNCA siRNAs

[0170] The screenings of SNCA siRNA agents SI to S340 listed in Table 2 were performed in human neuroblastoma cells with transfection following the protocol described as below. Table 5 shows the remaining SNCA mRNA in human neuroblastoma cells after transfection of siRNAs at concentration of 2 pM. A set of 25 siRNAs were selected for potency assessment in human neuroblastoma cells with transfection; Table 6 summarizes the IC50 values and their 95% confidential intervals of indicated siRNAs.MethodsCell culture and transfections

[0171] Human neuroblastoma Kelly cells (DSMZ) were cultured in RPMI 1640 (Sigma R0883) supplemented with 10% fetal bovine serum (FBS) (Biowest SI 810-500), 2 mM L- glutamine (Sigma G7513), and 50 pg / mL gentamycin (Gibco 15750) at 37°C incubator with 5% CO2. One day before transfection, cells were split using 0.05% Trypsin-ethylenediamine tetraacetic acid (EDTA) (Gibco 25300) and plated at a density of 30,000 cells per well in 96-well plates. The next day, cells were transfected with siRNA using Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific 13778150) following manufacture’s protocol. Prior to RNA isolation, cells were cultured at 37°C incubator in 5% CO2 for 72 hours.Total RNA isolation:

[0172] RNA extraction was performed using the RNeasy 96 kit (Qiagen) following manufacture’s protocol. Briefly, 125 pL RLT buffer was added to each well of 96-well plate to lysate cells with orbital shaking at 200 rpm for 1 min, and cell plates were then transferred to - 80°C freezer for storage till analysis. On the day of RNA extraction, frozen cell plates were quickly thaw in 37 °C oven, and immediately after thawing an equal volume 125 pL of 70% (V / V) ethanol was added to each well to mix with RLT lysate. The mixture was subsequently transferred to wells of the RNeasy 96 plate placed on top of QIAvac 96 vacuum block, and RNA was bound to the RNeasy 96 plate membrane by applying vacuum till liquid transfer was complete. Serial wash steps including 1 time of 800 pL RW1 buffer, 2 times of 800 pL RPE buffer were applied to the RNeasy 96 plate using vacuum to remove the liquid. After the last wash, the RNeasy 96 plate was centrifuged at 5600 x g for 3 min to remove the residual lipid. RNA was eluted using 60 pL RNase-free water by centrifugation at 5600 x g for 3 min at RT. RNA concentration was measured by Nanodrop 8000 (ThermoFisher). The eluted RNA was stored at -80°C until analysis.Reverse transcription and Real time PCR:

[0173] RNA was used as template for reverse transcription using reversed transcribed using High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems) following manufacture’sprotocol. Briefly, a final reaction of 20 pL mixture was incubated at 25 °C for 10 min, followed by reverse transcription at 37 °C for 2 hours and enzyme inactivation at 85 °C for 5 min. For qPCR reaction, reverse transcribed cDNAs were diluted 10 times and mixed with 2X PowerUp™ SYBR™ Green Mater Mix (ThermoFisher A25743) and 500 nM qPCR primers to a final volume of 10 pL reaction. The qPCR runs were performed with a QuantStudio™ 12K instrument (Applied Biosystems™) using standard thermal cycling protocol. Multiple validated primers were used to detect SNCA mRNA, and reference primers targeting housekeeping genes EN0X2 and glyceraldehyde 3 -phosphate dehydrogenase (GAPDH) were used for normalization of gene expression. The primer sequences are listed in Table 7. qPCR data were analyzed using qBase+ software (Biogazelle). IC50 values and the 95% CI of IC50 were calculated from a log([drug]) versus normalized response curve fit using GraphPad Prism version 7.00 Software.Example 3: in vitro knockdown efficiency of siRNA conjugates in neurons

[0174] The in vitro knockdown efficiencies of lipid-siRNA conjugates were assessed in human iPSC-derived cortical neurons following the procedure described as below.

[0175] FIGS. 2-4 and Tables 8-10 show that cholesterol-siRNA conjugates S356, S358, S362, S363, and S364 listed in Table 3 dose-dependently knock down SNCA mRNA and a- synuclein protein in human iPSC-neurons after incubation for 7 days or 14 days.

[0176] The cholesterol-siRNA conjugates shown in Table 3 were assessed for their efficiency in knocking down SNCA mRNA in human iPSC-neurons after incubation for 7 days. Three concentrations of siRNA conjugates at 200 nM, 40 nM and 8 nM were tested. The results of the analysis are summarized in Table 11.

[0177] In addition, lipid-siRNA conjugates S464, S465, S462, S467, S468, and S466 as listed in Table 4 were studied for their effect on SNCA mRNA (and a-synuclein protein expression in human iPSC-neurons after incubation for 7 days or 14 days. As shown in FIGS. 5 and 6 and in Tables 12 and 13, these lipid-siRNA conjugates dose-dependently knocked down SNCA mRNA and a-Syn protein expression.MethodsDifferentiation of human iPSCs into cortical neurons

[0178] The human iPSC line (Sigma #iPSC0028) was derived with OSKM retroviral reprogramming of epithelial cells from a 24-years old Caucasian female donor. iPSCs were first differentiated into cortical neural stem cells (NSCs) following a dual SMAD inhibitor protocol (Shi etal., 2012) with some modifications. Briefly, iPSCs were plated at 500,000 cells / cm2on wells coated with Matrigel (Corning 354230) in mTeSR medium (StemCell Technologies 5850) supplemented with 10 pM rho-associated protein kinase (ROCK) inhibitor (Sigma- Aldrich Y0503) and cultured at 37 °C and with 5% O2. The media were replaced with mTeSR the next day (day -1). From day 0 until day 12, cell media were changed every day with cortical Neural Induction Medium comprised of 10 pM SB43142 (Tocris 1614) and 1 pM dorsomorphin (Tocris 3093) supplemented in Neural Maintenance Medium (1: 1 DMEM:F12 glutamax (ThermoFisher Scientific 10565108), neurobasal (ThermoFisher Scientific 21103049), 2.5 pg / mL insulin (Sigma-Aldrich I9278-5ML), 50 pM 2-mercaptoethanol (ThermoFisher Scientific, 31350010), 0.5% non-essential amino acids (ThermoFisher Scientific 11140035), 0.5% glutaMAX supplement (ThermoFisher Scientific, 10565018), 0.5 mM sodium pyruvate (ThermoFisher Scientific 11360070), 1% penicillin-streptomycin (Sigma P4333), 0.5% N2 supplement (ThermoFisher Scientific 17502048), 1% B27 supplement (ThermoFisher Scientific 17504044)). At day 12, the neuroepithelial sheet was gently detached into large aggregates of 300 to 500 cells using a needle and lifter and the clumps were collected in a 15 mL falcon by centrifugation at 160 g for 2 min. Cell pellets were gently resuspended in Neural Induction Medium for a 1 / 2 or 1 / 3 passage into wells coated with 10 pg / mL laminin (Sigma- Aldrich L2020) in a total volume of 2 mL of Neural Induction Medium per well of the 6- well plate. Media were changed at day 13 and day 15 into Neural Maintenance Medium supplemented with 20 ng / mL of FGF2 (Stemcell Technologies 2634). At day 17, the neural rosettes were detached with dispase (ThermoFisher Scientific 17105041) for a 1 / 3 passage and plated into laminin-coated wells. Extra dispase steps (1 or 2) were performed for another week. At around days 25-30, neural stem cells were dissociated with Accutase (ThermoFisher Scientific Al 110501) into single-cell suspension and cryopreserved in freshly prepared Neural Freezing Medium containing Neural Maintenance Medium supplemented with 10 % (V / V) DMSO and 20 ng / mL FGF2. The frozen vials of neural stem cells were stored in liquid nitrogen until use.

[0179] To generate iPSC-neurons, frozen vials of neural stem cells (NSCs) were thawed and plated on laminin-coated wells at 70, 000 cells / cm2in Neural Maintenance Mediumsupplemented with 10 pM ROCK inhibitor and 20 ng / mL fibroblast growth factor-2 (FGF2). In the following two days, media were replaced daily with neural maintenance medium. At around day 4 after thawing, cells were dissociated with Accutase and placed at 28.000 cells per well in 96- well plates pre-coated with poly-L-ornithine and laminin in Neural Maintenance Medium supplemented with 10 pM ROCK inhibitor. The next day after re-plating, culture media were replaced with Neural Differentiation Medium comprised of Neural Maintenance Medium supplemented with 20 ng / mL brain-derived neurotrophic factor (BDNF) (R&D Systems 212- BD-050 / CF), 20ng / mL glial cell line-derived neurotrophic factor (GDNF) (R&D Systems 212- GD-050 / CF), 500 pM DB-cAMP (Sigma D0627), and 20 mM ascorbic acid (Sigma A4403). Cultures were differentiated in Neural Differentiation Medium with 50% medium change twice per week. Two-to-three weeks after differentiation from neural stem cells (NSCs), neurons were treated with siRNA for 7 days or 14 days for RNA analysis by reverse transcription and real time PCR and protein analysis by MSD immunoassay.MSD immunoassay

[0180] Human iPSC-neurons differentiated on 96-well plate were lysed in 100 pL ice-cold RIPA buffer (Sigma) supplemented with cOmplete™ Protease inhibitor cocktail (Roche) and PhosSTOP™ (Roche) with slow orbital shaking for 30 min at 4 °C. The plates were centrifuged at 1,000 x g for 5 min, and the cell lysates were collected and diluted 5-fold for protein measurement using MSD immunoassays following a standardized procedure. Briefly, MSD plates were coated with 30 pL per well of coating antibody diluted in phosphate buffer solution (PBS) at 2 pg / mL overnight at 4 °C. The next day, plates were inverted on absorbent tissue to dry and afterwards incubated with 150 pL per well blocking buffer 0.1 % casein for 2 hours at RT with orbital shaking at 300 rpm. After blocking, the plates were incubated with cell lysate diluted in RIPA buffer supplemented with cOmplete™ Protease inhibitor cocktail (Roche) overnight with slow orbital shaking at 4 °C. Plates were washed 5x times using Titertek Aquamax 4000 and inverted on absorbent tissue to dry. Afterwards, 25 pL / per secondary or detection antibody diluted in 0.1% casein were added to MSD plates and incubated for 2 hours at room temperature. After antibody incubation, plates were washed 5x times and developed with 150 pL / well 2x MSD reading buffer (Meso Scale Discovery) diluted in milliQ H2O. Plates were read immediately using MSD instrument. For measurement of a-synuclein protein, 4B12(Biolegend 807801) was used for 96-well MSD plate coating, anti-syn MJFR1 (Abeam 138501) was primary detection and SULFP-TAG labeled anti-rabbit antibody (Meso Scale Discovery) was used for detection. For measurement of Histone H3 protein, recombinant rabbit monoclonal antibody 17H2L9 (ThermoFisher) was used for plate coating, mouse mAb 14221BF (Cell Signaling Technology) was used as primary detection and SULFP-TAG labeled anti-mouse antibody (Meso Scale Discovery) was used as secondary detection antibody.Example 4: in vitro stability assessment of siRNA conjugates in various matrices

[0181] The in vitro stability of siRNA conjugates was assessed in various matrices including mouse brain homogenate, human liver lysosomes, and rat liver tritosomes using the methods described below.

[0182] Tables 14-17 summarize the LC-MS measurement of antisense strand and sense strand stability of siRNA conjugates of Tables 3 and 4 in mouse brain homogenate (Tables 14 and 15), human liver lysosomes (Table 16), and rat liver tritosomes (Table 17) at 37 °C after 24 hours incubation with shaking at 450 rpm. A reference compound MSC-2-V was added in the assays for benchmarking the stringency of the assay conditions.MethodsStability of siRNA conjugates in mouse brain homogenate

[0183] Frozen brain tissues collected from C57BL / 6J wild type mice were homogenized at 200 mg / mL tissue concentration in ice-cold homogenization buffer containing 100 mM Tris- HC1, pH6.0 buffer supplemented with 1 mM MgCL. Tissue homogenization was performed by adding 1 mL ice-cold homogenization buffer to a 2 mL tube (MP Biomedicals™) containing lysing Matrix D and one mouse brain hemisphere, followed by processing the tube in the FastPrep®-24 Instrument for 30 sec at a speed of 5 m / s. The samples were placed on ice for 2 min and the processing was repeated by FastPrep®-24 Instrument for 2 rounds. 100 pL of the homogenate was added to each well of a 96-well PCR plate, and 5 pL of 20 pM siRNA conjugate (final concentration 1 pM in the reaction) was added to each well, leaving equal number of blank wells containing brain homogenates without adding siRNA conjugates. The PCR plate with samples were placed in a Thermomixer and incubated at 37 °C for 24 h with shaking at 450 rpm. After the reaction, the PCR plate was placed on ice until samples were cool,and 5 pL of 20 uM siRNA conjugates were added to the blank wells containing brain homogenates (0-hr samples). The samples were diluted in 300 pL 10 mM EDTA (4-fold dilution) containing 1 pg / mL internal standard oligo, and the diluted samples were then transferred to deep well 96 well plates. After well mixing, 50 pL samples were transferred into another deep well 96 well plates before starting the solid phase extraction procedure.Stability of siRNA conjugates in human liver lysosomes and rat liver tritosomes

[0184] Human Liver Lysosomes (Xenotech H0610.L) and rat liver tritosomes were thawed and diluted in 20 mM sodium citrate pH 5.0 buffer to 0.05 units / mL or 0.5 unites / mL acid phosphatase levels, respectively. 50 pL of diluted human liver lysosomes and rat liver tritosomes were aliquoted into each well of separate 96 format PCR plates, followed by addition of 5 pL of 10 pM siRNA conjugates (final concentration 1 pM in the reaction) into each well. An equal number of blank wells containing lysosomes / tritosomes were left without adding siRNA conjugates. The PCR plate with samples was placed in a Thermomixer and incubated at 37 °C for 24 h with shaking at 450 rpm. After the reaction, the PCR plate was placed on ice until samples were cool, and 5 pL of 10 pM siRNA conjugates were added to blank wells containing lysosomes / tritosomes (0-hr samples). The samples were diluted in 300 pL 10 mM EDTA (4-fold dilution) containing 1 pg / mL internal standard oligo, and the diluted samples were transferred to deep well 96 well plates. After well mixing, 50 pL samples were transferred into another deep well 96 well plates before starting the solid phase extraction procedure.Sample preparation before Solid Phase Extraction

[0185] After stability assay, 50 pL samples were digested by adding 10 pL proteinase K solution (A4392,0010; PanReac Applichem) and 100 pL Tris-EDTA buffer (10 mM Tris, ImM EDTA) pH 8.0 (VWR; El 12-500ml) incubated at 60 °C with vortex at 1100 rpm for 30 minutes. After incubation, samples were diluted by adding 100 pL lysis buffer (Phenomenex, Cat AL0- 8579) and 200 pL equilibration buffer (3.45 g NaiHPCL in 500 mL water, adjusted to pH 5.5 with 1 N NaOH)Solid Phase Extraction

[0186] Solid phase extraction was then performed using Clarity OTX solid phase extraction plates (Phenomenex, Cat. 8E-S103-EGA). The plates were conditioned by 1 mL methanol, followed by ImL equilibration buffer (3.45 g NaiHPCE in 500 mL water, adjusted to pH 5.5 with 1 N NaOH). The samples were then loaded onto the column. The column was washed with 1 mL wash buffer (equilibration buffer pH 5.5, 50% v / v acetonitrile) for 3 times, then washed once with 0.5 mL water, and then washed once in 100 mM ammonium bicarbonate pH 10 supplemented with 0.56 g / LTCEP (tris(2-carboxyethyl) phosphine). Samples were eluted 3 times each with 0.5 mL elution buffer (200 mM ammonium bicarbonate pH 10 supplemented with TCEP (tris(2-carboxy ethyl) phosphine), 50 % v / v acetonitrile), and dried using nitrogen flow (TurboVap, 65 psi N2 at 70 °C).Analytical Method

[0187] After solid phase extraction, samples were reconstituted in dissolving buffer (300 pL 100 mM ammonium bicarbonate adjust pH 10, 10 % v / v acetonitrile) and analyzed using liquid chromatography combined with mass spectrometry detection AB Sciex TripleTOL 6600.Samples were injected (20 pL) and separated using an DNAPac™ RP Column 50 mm x 2.1 mm, 4 pm maintained at 75 °C. Mobile phase A was 0.2% dimethyl butylamine, 0.5% hexafluoro isopropanol and 0.5% methanol and mobile phase B was acetonitrile / iso-propanol (95 / 5; v / v). Gradient of the solvent: time = 0 min, 98% A + 2% B; time = 1.5 min, 98% A + 2%B; time = 4.5 min, 5% A + 95% B; time = 7.5% A+ 95% B; time = 7.01 min, 98% A+2% B; time = 10 min, 98% A + 2% B. The ESI source was operated in negative ion mode, with full scan, using spray voltage = 4500 V, Source temperature = 350 °C. Plow speed = 0.25 mL per min. Analyst TF 1.8.1 software was used for data acquisition and Sciex OS 1.7.0 software was used for data processing.Example 5: in vivo knockdown efficiency assessment of siRNA conjugates in mouse models

[0188] The in vivo knockdown efficiencies of certain lipid-siRNA conjugates of Tables 3 and 4 were assessed in mouse models with intracerebroventricular (ICV) injection following the procedure described below.

[0189] Tables 18 and 19 summarize RT-qPCR analysis of SNCA mRNA knockdown in seven brain regions (cortex, hippocampus, brainstem, cerebellum, striatum, midbrain, andcervical spinal cord) of WT mice at 7 days after a single ICV injection of 15 nmol indicated siRNA conjugates.Table 20 summarizes RT-qPCR analysis of hSNCA mRNA knockdown in seven brain regions (cortex, hippocampus, brainstem, cerebellum, striatum, midbrain, and cervical spinal cord) of hSNCA-BAC mice at 7 days after a single ICV injection of S468 at indicated dose levels.MethodsMice and intracerebroventricular injection

[0190] Wild type C57BL6 mice or hSNCA-BAC mice at age of 2-to-3 -month were randomly assigned to different treatment groups. Mice were anesthetized with isoflurane (induction: 4-5 %; maintenance: 1.8-2.5 %). They were then stereotaxi cally injected using a motorized drill and microinjection robot (Neurostar, Germany, Sterodrive Sofeware v 2019) into the bilateral ventricles at coordinates: AP: -0.62 mm, ML: + / - 1.05 mm: and DV: 2.2 mm. Each injection was performed in 5 pL volume over 5 min and following injection the needle was withdrawn in three steps (1. 1 mm in 60 sec and kept there for 5 min; 2. another 0.5 mm in 30 sec, wait 5 min; 3. withdrawal out of the brain at very slow speed) to avoid compound reflux along the needle tract. After every step the amount of backflow was checked. At selected post-injection days, animals were sacrificed and different brain regions including cortex, hippocampus, brainstem, cerebellum, striatum, midbrain, and cervical spinal cord were dissected and snap frozen and stored at -80 °C until further analysis.RNA extraction from tissues

[0191] Tissues were collected in Lysing Matrix D (MP Biomedicals 6913-500) 2 mL tubes containing 1.4 mm ceramic spheres and stored in -80°C freezer until analysis. The tissues were placed on ice under laminar flow and immediately add 750 pL Trizol (ThermoFisher 15596026 / 15596018) per tube. The tissue was disrupted and homogenized using the FastPrep- 24™ 5G Grinder with 3 cycles of 30 sec at speed 5 m / sec. Between cycles, the samples were cooled on ice for 2 minutes. After homogenization, the tube was shortly spun and 20 % volume of chloroform (150 pL chloroform when 750 pL Trizol was used) was added. The tube was vortexed for 15 sec and centrifuged at 14,000 x g for 15 min at 4 °C. The upper aqueous phase was transferred to a deep 96-well plate and 1 volume of 70% ethanol was added and mixed well.

[0192] The next steps were performed using RNeasy 96 kit (Qiagen) following manufacture’s protocol. A RNeasy 96 plate was placed on top of the Square-Well Block holder, the samples (Trizol / Chloroform extraction) were applied into the wells of the RNeasy 96 plate, and the plate was sealed with AirPore cover to prevent contamination. The plate was centrifuged at 5600 x g for 3 min at RT and the solution was discarded. Serial wash steps were performed including 1 time of 800 pL RW1 buffer, 2 times of 800 pL RPE buffer that were applied to the RNeasy 96 plate. Wash buffers were then removed by centrifugation at 5600 x g for 3 min for each step. After the last wash, the RNeasy 96 plate was centrifuged at 5600 x g for 3 min to remove the residual lipid. RNA was eluted using 60 pL RNase-free water by centrifugation at 5600 x g for 3 min at RT. RNA concentration was measured by Nanodrop 8000 (ThermoFisher). The eluted RNA was stored at -80°C until analysis.RT-qPCR

[0193] RNA was used as template for reverse transcription using reversed transcribed using High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems) following manufacture’s protocol. Briefly, a final reaction of 20 pL mixture was incubated at 25 °C for 10 min, followed by reverse transcription at 37 °C for 2 hours and enzyme inactivation at 85 °C for 5 min. For qPCR reaction, reverse transcribed cDNAs were diluted 10 times and mixed with 2X PowerUp™ SYBR™ Green Mater Mix (ThermoFisher A25743) and 500 nM qPCR primers to a final volume of 10 pL reaction. Alternatively, reverse transcribed cDNAs were diluted 10 times and mixed with PrimeTime qPCR Probe Assays (IDT Integrated DNA Technologies) following manufacture’s protocol. The qPCR runs were performed with a QuantStudio™ 12K instrument (Applied Biosystems™) using standard thermal cycling protocol. Multiple primers were used to detect SNCA mRNA, and reference primers targeting mouse housekeeping genes AP3D1, PAK1IP1, RPLPO and ACTB were included for normalization of gene expression. The primer sequences and PrimeTime qPCR Probe Assays are listed in Table 5. qPCR data were analyzed using qBase+ software (Biogazelle).Table 1. Unmodified sense strand and antisense strand nucleotide sequences (5’ to 3’) of unmodified SNCA siRNA.Table 2. Modified sense strand and antisense strand nucleotide sequences (5’ to 3’) of modified SNCA siRNA.(modification, with “X” representing the nucleotide: mX = 2’-0Me; fX = 2’-F; ps = phosphorothioate)Table 3. Modified sense strand and antisense strand nucleotide sequences (5’ to 3’) of SNCA siRNA with chemical variants, conjugated to cholesterol.(modification, with “X” representing the nucleotide: mX = 2’-0Me; fX = 2’-F; ps = phosphorothioate; VP = vinyl phosphonate; dX = deoxynucleotide; UNA-X = unlocked nucleic acid; GNA-X = glycol nucleic acid; invAb = inverse abasic; Chol4 = Cholesterol)Table 4. Modified sense strand and antisense strand nucleotide sequences (5’ to 3’) of SNCA siRNA with chemical variants, conjugated to a lipid moiety.(modification, with “X” representing the nucleotide: mX = 2’-0Me; fX = 2’-F; ps = phosphorothioate; VP = vinyl phosphonate; invAb = inverse abasic; J2-CONC16X = LI; J2- C3NC16X = L2; J2-NCOOC16X = L3; J2-C2OC16X = L4; J2-C15AdaX = L5)Table 5. Single dose screen of modified SNCA siRNAs in human neuroblastoma Kelly cells with transfection, as described in Example 2. Data is expressed as percentage of SNCA mRNA level relative to vehicle control.Table 6. IC50 and 95% confidence intervals of select modified SNCA siRNAs in human neuroblastoma Kelly cells with transfection, as described in Example 2. The IC50 values are estimated using different sets of RT-qPCR primers SNCA_B08, SNCA_B03 and SNCA_B05.Table 7. Sequences of qPCR primers and catalogues of PrimeTime qPCR assays (IDT), as described in Example 2.Table 8. SNCA mRNA knockdown efficiency of select cholesterol-siRNA conjugates in human iPSC-derived cortical neurons treated for 7 days, as described in Example 3 (n=3). Data is presented as IC50 and 95% confidence intervals, which are calculated from a log([drug]) versus normalized response curve fit using GraphPad Prism version 7.00 Software.Table 9. SNCA mRNA knockdown efficiency of select cholesterol-siRNA conjugates in human iPSC-derived cortical neurons treated for 14 days, as described in Example 3 (n=3). Data is presented as IC50 and 95% confidence intervals, which are calculated from a log([drug]) versus normalized response curve fit using GraphPad Prism version 7.00 Software.Table 10. oc-synuclein protein knockdown efficiency of select cholesterol-siRNA conjugates in human iPSC-derived cortical neurons treated for 14 days, as described in Example 3 (n=3). Data is presented as IC50 and 95% confidence intervals, which are calculated from a log([drug]) versus normalized response curve fit using GraphPad Prism version 7.00 Software.Table 11. SNCA mRNA knockdown efficiency of select cholesterol-siRNA conjugates in human iPSC-derived cortical neurons treated for 14 days at concentrations of 200 nM, 40 nM, and 8 nM, as described in Example 3 (n=3). Data is presented as remaining SNCA mRNA relative to control.Table 12. SNCA mRNA knockdown efficiency of select lipid-siRNA conjugates in human iPSC-derived cortical neurons treated for 7 days, as described in Example 3 (n=3). Data is presented as IC50 and 95% confidence intervals, which are calculated from a log([drug]) versus normalized response curve fit using GraphPad Prism version 7.00 Software.Table 13. oc-synuclein protein knockdown efficiency of select lipid-siRNA conjugates in human iPSC-derived cortical neurons treated for 14 days, as described in Example 3 (n=3). Data is presented as IC50 and 95% confidence intervals, which are calculated from a log([drug]) versus normalized response curve fit using GraphPad Prism version 7.00 Software.Table 14. LC-MS measurement of sense strand and antisense strand stability of select cholesterol-siRNA conjugates in mouse brain homogenate, as described in Example 4 (n=3). Data is presented as % stability.Table 15. LC-MS measurement of sense strand and antisense strand stability of select lipid- siRNA conjugates in mouse brain homogenate, as described in Example 4 (n=6). Data is presented as % stability.Table 16. LC-MS measurement of sense strand and antisense strand stability of select lipid- siRNA conjugates in human liver lysosomes, as described in Example 4 (n=6). Data is presented as % stability.Table 17. LC-MS measurement of sense strand and antisense strand stability of select lipid- siRNA conjugates in rat liver tritosomes, as described in Example 4 (n=6). Data is presented as % stability.Table 18. SNCA mRNA knockdown efficiency of select cholesterol-siRNA conjugates and lipid-siRNA conjugates in various tissues in vivo in wildtype mice 7 days after ICV injection at a dose of 15 nmol of the conjugate, as described in Example 5 (n=6 per treatment group). Data is presented as remaining SNCA mRNA (%) levels.Table 19. SNCA mRNA knockdown efficiency of select lipid-siRNA conjugates in various tissues in vivo in wildtype mice 7 days after ICV injection at a dose of 15 nmol of the conjugate, as described in Example 5 (n=6 per treatment group). Data is presented as remaining SNCA mRNA (%) levels.Table 20. SNCA mRNA knockdown efficiency of lipid-siRNA conjugate S468 in various tissues in vivo in wildtype mice 7 days after ICV injection at doses of 60 nmol, 30 nmol, 15 nmol, 7.5 nmol, 3.8 nmol, and 1.9 nmol, as described in Example 5 (n=5 per treatment group). Data is presented as remaining hSNCA mRNA (%) levels.Table 21. Modified sense strand and antisense strand nucleotide sequences (5’ to 3’) of SNCA siRNA with chemical variants, conjugated to cholesterol.(modification, with “X” representing the nucleotide: mX = 2’-0Me; fX = 2’-F; ps = phosphorothioate; VP = vinyl phosphonate; dX = deoxynucleotide; UNA-X = unlocked nucleic acid; GNA-X = glycol nucleic acid; invAb = inverse abasic; Chol4 = Cholesterol)

[0194] The invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying Figures. Such modifications are intended to fall within the scope of the appended claims.

[0195] All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

Claims

CLAIMSWhat is claimed is:

1. A small interfering ribonucleic acid (siRNA) agent comprising a sense strand and an antisense strand forming a double stranded region, wherein(a) the sense strand comprises a nucleotide sequence corresponding to any one of the sense nucleotide sequences in Table 1, or(a) the antisense strand comprises a nucleotide sequence corresponding to any one of the antisense nucleotide sequences in Table 1.

2. The siRNA agent of claim 1, wherein the sense strand comprises a nucleotide sequence corresponding to a sense nucleotide sequence in a row of Table 1, and the antisense strand comprises a nucleotide sequence corresponding to the antisense nucleotide sequence in the same row of Table 1.

3. The siRNA agent of claim 1, wherein(a) the sense strand consists of a nucleotide sequence corresponding to any one of the sense nucleotide sequences in Table 1, or(b) the antisense strand consists of a nucleotide sequence corresponding to any one of the antisense nucleotide sequences in Table 1.

4. A composition comprising the siRNA agent of claim 1 , and a carrier.

5. The siRNA agent of claim 2, wherein the sense strand consists of a nucleotide sequence corresponding to a sense nucleotide sequence in a row of Table 1, and the antisense strand consists of a nucleotide sequence corresponding to the antisense nucleotide sequence in the same row of Table 1.

6. A composition comprising the siRNA agent of claim 2, and a carrier.

7. A small interfering ribonucleic acid (siRNA) agent comprising a sense strand and an antisense strand forming a double stranded region, wherein(a) the sense strand comprises a nucleotide sequence corresponding to any one of the sense nucleotide sequences in Table 2, or(b) the antisense strand comprises a nucleotide sequence corresponding to any one of the antisense nucleotide sequences in Table 2.

8. The siRNA agent of claim 7 wherein the sense strand comprises a nucleotide sequence corresponding to a sense nucleotide sequence in a row of Table 2, and the antisense strand comprises a nucleotide sequence corresponding to the antisense nucleotide sequence in the same row of Table 2.

9. The siRNA agent of claim 7, wherein(a) the sense strand consists of a nucleotide sequence corresponding to any one of the sense nucleotide sequences in Table 2, or(b) the antisense strand consists of a nucleotide sequence corresponding to any one of the antisense nucleotide sequences in Table 2.

10. A composition comprising the siRNA agent of claim 7, and a carrier.

11. The siRNA agent of claim 8, wherein the sense strand consists of a nucleotide sequence corresponding to a sense nucleotide sequence in a row of Table 2, and the antisense strand consists of a nucleotide sequence corresponding to the antisense nucleotide sequence in the same row of Table 2.

12. A method of inhibiting expression of SNCA gene in a cell or population of cells, the method comprising contacting the cell or population of cells with the siRNA agent of claim 11.

13. A method of reducing alpha-synuclein levels in a cell or population of cells, the method comprising contacting the cell or population of cells with the siRNA agent of claim 11.

14. A composition comprising the siRNA agent of claim 8, and a carrier.

15. A small interfering ribonucleic acid (siRNA) agent comprising a sense strand and an antisense strand forming a double stranded region, wherein(a) the sense strand comprises a nucleotide sequence corresponding to any one of the sense nucleotide sequences in Table 3, or(b) the antisense strand comprises a nucleotide sequence corresponding to any one of the antisense nucleotide sequences in Table 3.

16. The siRNA agent of claim 15, wherein the sense strand comprises a nucleotide sequence corresponding to a sense nucleotide sequence in a row of Table 3, and the antisense strand comprises a nucleotide sequence corresponding to the antisense nucleotide sequence in the same row of Table 3.

17. The siRNA agent of claim 15, wherein(a) the sense strand consists of a nucleotide sequence corresponding to any one of the sense nucleotide sequences in Table 3, or(b) the antisense strand consists of a nucleotide sequence corresponding to any one of the antisense nucleotide sequences in Table 3.

18. A composition comprising the siRNA agent of claim 15, and a carrier.

19. The siRNA agent of claim 16, wherein the sense strand consists of a nucleotide sequence corresponding to a sense nucleotide sequence in a row of Table 3, and the antisense strand consists of a nucleotide sequence corresponding to the antisense nucleotide sequence in the same row of Table 3.

20. A composition comprising the siRNA agent of claim 16, and a carrier.

21. A method of inhibiting expression of SNCA gene in a cell or population of cells, the method comprising contacting the cell or population of cells with the siRNA agent of claim 19.

22. A method of reducing alpha-synuclein levels in a cell or population of cells, the method comprising contacting the cell or population of cells with the siRNA agent of claim 19.

23. A small interfering ribonucleic acid (siRNA) agent comprising a sense strand and an antisense strand forming a double stranded region, wherein(a) the sense strand comprises a nucleotide sequence corresponding to any one of the sense nucleotide sequences in Table 4, or(b) the antisense strand comprises a nucleotide sequence corresponding to any one of the antisense nucleotide sequences in Table 4.

24. The siRNA agent of claim 23, wherein the sense strand comprises a nucleotide sequence corresponding to a sense nucleotide sequence in a row of Table 4, and the antisense strand comprises a nucleotide sequence corresponding to the antisense nucleotide sequence in the same row of Table 4.

25. The siRNA agent of claim 23, wherein(a) the sense strand consists of a nucleotide sequence corresponding to any one of the sense nucleotide sequences in Table 4, or(b) the antisense strand consists of a nucleotide sequence corresponding to any one of the antisense nucleotide sequences in Table 4.

26. A composition comprising the siRNA agent of claim 23, and a carrier.

27. The siRNA agent of claim 24, wherein the sense strand comprises nucleotides having the sequence (InvAb)psmUpsmCmAmGmC(J2- C0NC16A)mGmUfGfAfUmUmGmAmAmGmUmAmUmCpsmAps(InvAb) (SEQ ID NO: 1535), and the antisense strand comprises nucleotides having the sequence (VPmU)psfGpsmApsmUmAfCmUfUfCmAmAmUmCfAmCfUmGmCmUmGpsmA (SEQ ID NO: 1536).

28. A composition comprising the siRNA agent of claim 27, and a carrier.

29. The siRNA agent of claim 24, wherein the sense strand consists of a nucleotide sequence corresponding to a sense nucleotide sequence in a row of Table 4, and the antisense strand consists of a nucleotide sequence corresponding to the antisense nucleotide sequence in the same row of Table 4.

30. A composition comprising the siRNA agent of claim 24, and a carrier.

31. A method of inhibiting expression of SNCA gene in a cell or population of cells, the method comprising contacting the cell or population of cells with the siRNA agent of claim 27.

32. A method of reducing alpha-synuclein levels in a cell or population of cells, the method comprising contacting the cell or population of cells with the siRNA agent of claim 27.

33. A method of inhibiting expression of SNCA gene in a cell or population of cells, the method comprising contacting the cell or population of cells with the siRNA agent of claim 29.

34. A method of reducing alpha-synuclein levels in a cell or population of cells, the method comprising contacting the cell or population of cells with the siRNA agent of claim 29.

35. A small interfering ribonucleic acid (siRNA) agent comprising a sense strand and an antisense strand forming a double stranded region, wherein(c) the sense strand comprises a nucleotide sequence corresponding to any one of the sense nucleotide sequences in Table 21, or(d) the antisense strand comprises a nucleotide sequence corresponding to any one of the antisense nucleotide sequences in Table 21.

36. The siRNA agent of claim 35, wherein the sense strand comprises a nucleotide sequence corresponding to a sense nucleotide sequence in a row of Table 21, and the antisense strand comprises a nucleotide sequence corresponding to the antisense nucleotide sequence in the same row of Table 21.

37. The siRNA agent of claim 35, wherein(c) the sense strand consists of a nucleotide sequence corresponding to any one of the sense nucleotide sequences in Table 21, or(d) the antisense strand consists of a nucleotide sequence corresponding to any one of the antisense nucleotide sequences in Table 21.

38. A composition comprising the siRNA agent of claim 35, and a carrier.

39. The siRNA agent of claim 36, wherein the sense strand consists of a nucleotide sequence corresponding to a sense nucleotide sequence in a row of Table 21, and the antisense strand consists of a nucleotide sequence corresponding to the antisense nucleotide sequence in the same row of Table 21.

40. A composition comprising the siRNA agent of claim 36, and a carrier.

41. A method of inhibiting expression of SNCA gene in a cell or population of cells, the method comprising contacting the cell or population of cells with the siRNA agent of claim 39.

42. A method of reducing alpha-synuclein levels in a cell or population of cells, the method comprising contacting the cell or population of cells with the siRNA agent of claim 39.

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