SNCA siRNA and its uses

Abstract

This article provides small interfering RNA (siRNA) reagents targeting the SNCA gene and compositions containing such siRNA reagents.

Description

[0001] Cross-references to related applications This application claims the benefit of priority to U.S. Provisional Application No. 63 / 537,181, filed September 7, 2023, the entire contents of which are incorporated herein by reference and for all purposes. Background Technology

[0002] The accumulation of pathologically misfolded α-synuclein (α-synuclein) has been implicated in a range of neurodegenerative diseases known as “synucleinopathy.” These diseases include Parkinson’s disease (PD), Lewy body dementia (DLB), pure autonomic failure (PAF), and multiple system atrophy (MSA) (Goedert et al., 2017). In PD and DLB, α-synuclein aggregates are primarily present as neuronal cytoplasmic deposits (Lewy bodies) and neuritis deposits (Lewy neurites), varying across the brain regions involved (Coon et al., 2018). In MSA, α-synuclein aggregates are primarily detected as glial cytoplasmic inclusions in oligodendrocytes, and in PAF, the aggregates are primarily present as neuronal cytoplasmic deposits in the peripheral nervous system (ibid.).

[0003] In Parkinson's disease (PD), genetic evidence strongly supports the role of the SNCA locus encoding α-synuclein. Missense mutations and duplications of SNCA cause autosomal dominant familial forms of PD, suggesting that alterations in physical properties or increases in α-synuclein are sufficient to induce PD. The genetic dosage of SNCA influences disease severity and penetrance. SNCA triplication causes very rare familial PD cases with full penetrance, presenting as early-onset and rapidly progressive Parkinsonian syndrome with prominent nonmotor signs such as dementia, autonomic dysfunction, and psychotic features (Olgiati et al., 2015). Compared to triploidy or missense mutations, SNCA duplication is relatively more common in autosomal dominant PD, and patients with this condition do not exhibit a severe phenotype, and may resemble spontaneous PD (Konno et al., 2016). Causal SNCA mutations in DLB have been identified (Orme et al., 2018), but causal SNCA mutations in PAF (Coon, Singer et al., 2019) or MSA (Katzeff et al., 2019) have not been identified.

[0004] Beyond pathogenic mutations or doubling, genetic variants further highlight the crucial role of SNCA genes in PD. Genome-wide association studies have identified a group of non-coding variants located at the 3' end, promoter, and intron regions of SNCA (Maraganore et al., 2006; Linnertz et al., 2009; Satake et al., 2009; Simón-Sánchez et al., 2009; Lutz et al., 2015; Soldner et al., 2016; Pihlstrøm et al., 2018), and analysis of some of these non-coding variants indicates that upregulation of SNCA gene transcription increases the risk of PD. Compared to individuals carrying at least one copy of the PD-risk-associated Rep allele, the protective allele Rep1-259 bp located in the promoter region reduces SNCA mRNA in the temporal cortex and substantia nigra by 40% to 50% (Linnertz et al., 2009).

[0005] Studies in transgenic mouse models have also demonstrated the role of α-synuclein expression. In tetracycline-controlled transgenic mouse models, it has been reported that inhibiting α-synuclein expression for 3 months suppressed pathological progression, 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 septum (Lim et al., 2011). In a rat model of PD exposed to rotenone, adeno-associated virus-shRNA-mediated knockdown of endogenous α-synuclein by approximately 35% reduced the degeneration of dopaminergic neurons in the substantia nigra and attenuated progressive motor deficits (Zharikov et al., 2015). Furthermore, in a rodent model of PD preformed fibrils (PFF) injection, antisense oligonucleotides (ASO) mediated approximately 50% knockdown of endogenous α-synuclein, preventing and clearing existing α-synuclein pathology and preventing dopaminergic cell dysfunction (Cole et al., 2021).

[0006] Furthermore, mice with less than 50% reduction in SNCA mRNA have shown pathological relief and functional benefits (Zharikov et al., 2015; Cole et al., 2021). Such results suggest that partial knockdown of SNCA (close to or less than 50%) is desirable and likely to be tolerable for therapeutic applications.

[0007] Therefore, α-synuclein may be a key therapeutic target for PD and other related synuclein disorders. Consequently, there is a need for compounds and agents that inhibit SNCA gene expression to treat neurodegenerative diseases involving α-synuclein. Summary of the Invention

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

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

[0010] According to another aspect, the described invention provides a small interfering RNA (siRNA) reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a nucleotide sequence corresponding to any sense nucleotide sequence in Table 2, or the antisense strand comprises a nucleotide sequence corresponding to any antisense nucleotide sequence in the table.

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

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

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

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

[0015] According to some embodiments, the sense strand comprises a nucleotide sequence corresponding to a sense nucleotide sequence in one row of Table 4, and the antisense strand comprises a nucleotide sequence corresponding to an 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 sense nucleotide sequence in Table 4, or the antisense strand consists of a nucleotide sequence corresponding to any antisense nucleotide sequence in Table 4. According to some embodiments, the sense strand comprises a nucleotide having the sequence (InvAb)psmUpsmCmAmGmC(J2-CONC16A)mGmUfGfAfUmUmGmAmAmGmUmAmUmCpsmAps(InvAb) (SEQ ID NO:1535), and the antisense strand comprises a nucleotide having the sequence (VPmU)psfGpsmApsmUmAfCmUfUfCmAmAmUmCfAmCfUmGmCmUmGpsmA (SEQ ID NO:1536). According to some implementation schemes, 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 an antisense nucleotide sequence in the same row of Table 4.

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

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

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

[0019] According to some embodiments, the described invention provides a method for reducing α-synuclein levels in cells or cell populations, the method comprising contacting the cells or cell populations with the siRNA reagent described herein. According to some embodiments, the method of the invention described herein can be performed in vivo, and according to some embodiments, the method of the invention described herein can be performed in vitro or ex vivo.

[0020] According to some embodiments, the described invention provides a composition comprising an siRNA reagent and a carrier. Attached Figure Description

[0021] Figure 1 The chemical structures of the lipophilic moieties L1, L2, L3, L4 and L5, as well as the structure of cholesterol-triethylene glycol (CHOL4), are provided.

[0022] Figure 2 The knockdown efficiency of the selected cholesterol-siRNA conjugate in SNCA mRNA in human iPSC-derived cortical neurons treated for 7 days, as described in Example 3 (n=3), is shown (see also Table 8). Data are presented as residual SNCA mRNA (relative to control).

[0023] Figure 3 The knockdown efficiency of the selected cholesterol-siRNA conjugate in SNCA mRNA in human iPSC-derived cortical neurons treated for 14 days, as described in Example 3 (n=3), is shown (see also Table 9). Data are presented as residual SNCA mRNA (relative to control).

[0024] Figure 4 The knockdown efficiency of the selected cholesterol-siRNA conjugate in α-synuclein protein in human iPSC-derived cortical neurons treated for 14 days, as described in Example 3 (n=3), is shown (see also Table 10). Data are presented as residual α-synuclein protein (relative to control).

[0025] Figure 5 The knockdown efficiency of the selected lipid-siRNA conjugates in SNCA mRNA in human iPSC-derived cortical neurons treated for 7 days, as described in Example 3 (n=3), is shown (see also Table 12). Data are presented as residual SNCA mRNA (relative to control).

[0026] Figure 6The knockdown efficiency of the selected lipid-siRNA conjugate in α-synuclein protein in human iPSC-derived cortical neurons treated for 14 days, as described in Example 3 (n=3), is shown (see also Table 13). Data are presented as residual α-synuclein protein (relative to control). Detailed Implementation

[0027] Unless otherwise stated, the present invention may be practiced using conventional techniques of biotechnology, molecular biology, pharmaceutical science, formulation science, protein chemistry, cell biology, cell culture, genetics, recombinant DNA, immunology, clinical pharmacology, and clinical practice, which are within the scope of the art.

[0028] To facilitate a clearer understanding of the invention, certain terms are first defined. Additional definitions are set forth throughout this disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art related to this invention.

[0029] Any headings provided herein are not intended to limit the various aspects or embodiments of the invention, which can be obtained by referring to the specification as a whole. Therefore, the terms defined thereafter are defined more fully by referring to the entire specification.

[0030] All references cited in this disclosure are incorporated herein by reference in their entirety. Furthermore, any manufacturer's specifications or catalogues of any products referenced or mentioned herein are incorporated by reference. Documents incorporated herein by reference, or any teachings therein, may be used in the practice of this invention. Documents incorporated herein by reference are not acknowledged as prior art.

[0031] definition The wording or terminology used in this disclosure is for descriptive purposes and not for limitation, and the terminology or terminology in this specification should be interpreted by those skilled in the art based on the teachings and instructions.

[0032] As used in this specification and the appended claims, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” include plural references. The term “a” (or “an”) and the terms “one or more” and “at least one” are used interchangeably.

[0033] As used herein, the terms “comprising” and “including” are used interchangeably. The terms “comprising” and “including” should be construed as specifying the presence of the mentioned feature or component, but do not exclude the presence or addition of one or more features, components, or combinations thereof. Furthermore, the terms “comprising” and “including” are intended to include examples covered by the term “consisting of”. Therefore, the term “consisting of” can be used instead of the terms “comprising” and “including” to provide more specific embodiments.

[0034] As used herein, the term "or" should be interpreted as inclusive "or," meaning any one or any combination thereof. 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. Exceptions to this definition will only occur if the combination of elements, functions, steps, or actions is inherently mutually exclusive in some way.

[0035] Furthermore, "and / or" is considered to be a specific disclosure of each of two specified features or components with or without the other. Therefore, the term "and / or," as used in phrases such as "A and / or B," is intended to include A and B, A or B, A (alone) and B (alone). Similarly, the term "and / or," as used in phrases 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 represented in their International System of Units (SI) accepted form. A numerical range includes the number that defines the range, and any single value provided herein can be used as an endpoint of a range that includes other single values ​​provided herein. For example, a set of values ​​such as 1, 2, 3, 8, 9, and 10 is also a disclosure of numerical ranges from 1 to 10, from 1 to 8, from 3 to 9, etc. Similarly, a disclosed range is a disclosure of each individual value (i.e., intermediate value) covered by that range, including integers and fractions. For example, the specified range of 5 to 10 is also a disclosure of 5, 6, 7, 8, 9, and 10 individually, as well as 5.2, 7.5, 8.7, etc., individually.

[0037] Unless otherwise stated, the terms "at least" or "about" preceding a series of elements should be understood to refer to each element in the series. The term "about" preceding a numerical value includes ±10% of the stated value. For example, a concentration of about 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of about 1% (w / v) to 10% (w / v) includes 0.9% (w / v) to 11% (w / v).

[0038] The siRNA reagent of this invention inhibits the expression of the SNCA gene. As used herein, the term "SNCA gene" refers to the gene encoding a protein called α-synuclein.

[0039] α-synuclein, also referred to as "α-synuclein" in this paper, is a small protein (140 amino acids, 14.5 kDa) encoded by five exons of the SNCA locus on chromosome 4q21.3-q22. SNCA is expressed extensively throughout the brain (Jakes et al., 1994; Lavedan, 1998), with localization concentrated at presynaptic nerve endings (Iwai et al., 1995). Physiological α-synuclein exists in a dynamic equilibrium between unfolded monomers and α-helical tetramers (Lashuel et al., 2013), while pathological conditions drive α-synuclein aggregation, involving conformational changes to β-sheet-rich structures, thereby promoting the formation of oligomers, fibrils, and insoluble fibrils.

[0040] As used herein, the terms "small interfering RNA reagent" or "siRNA reagent" refer to RNA or RNA-like molecules (e.g., chemically modified RNA oligonucleotide molecules) containing both a sense strand and an antisense 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 antisense strand is 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, or 17 nucleotides in length. The sense and / or antisense strands of the siRNA reagent may include another portion (e.g., a lipophilic moiety). For example, the lipophilic moiety may be incorporated into the sense strand of the siRNA reagent. The sense and / or antisense strands of the siRNA reagent may be directly or indirectly linked or conjugated to another portion (e.g., a lipophilic moiety). For example, siRNA The sense strand of the reagent can be directly or indirectly linked or conjugated to another portion (e.g., a lipophilic moiety). In a specific embodiment, the siRNA reagent is a double-stranded RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide comprising a sense strand and an antisense strand forming a double-stranded region. The double-stranded region can be the entire length of the sense strand, the antisense strand, or both. Alternatively, the double-stranded region can be less than the entire length of the sense strand, the antisense strand, or both. The double-stranded region can be the result of the antisense strand being completely complementary, partially complementary, or substantially complementary to the sense strand. In a specific embodiment, the antisense strand of the siRNA reagent is partially complementary to the target RNA transcript (e.g., SNCA). In another specific embodiment, the antisense strand of the siRNA reagent is substantially complementary to the target RNA transcript (e.g., SNCA). In yet another specific embodiment, the antisense strand of the siRNA reagent is completely complementary to the target RNA transcript (e.g., SNCA).

[0041] The two strands that form a double-stranded region or double-stranded structure can be different parts of a larger RNA molecule, or they can be separate RNA molecules.

[0042] When the two substantially complementary strands of an siRNA reagent consist of separate RNA molecules, these molecules are not required, but can be covalently linked. In some embodiments, the two strands are covalently linked by a continuous nucleotide chain, different from the one forming the duplex structure, between the 3' end of one strand and the 5' end of the corresponding other strand; this linking structure is called a "connector." The RNA strands of the siRNA reagent can have the same or different numbers of nucleotides. In some embodiments, one or both strands of the siRNA reagent contain overhanging ends. In other embodiments, the siRNA reagent is blunt-ended.

[0043] In specific implementations, the siRNA reagents described herein mediate the degradation of messenger RNA (mRNA) or the inhibition of mRNA translation in a sequence-specific manner. In specific implementations, the siRNA reagents described herein inhibit SNCA gene expression via the RNA-induced silencing complex (RISC) pathway.

[0044] As used herein, when describing the relevance of a first nucleotide sequence (e.g., the sense strand or target sequence of an siRNA reagent) to a second nucleotide sequence (e.g., the antisense strand or single-stranded antisense oligonucleotide of an siRNA reagent), the term "complementary" means the ability of an oligonucleotide or polynucleotide containing the first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide containing the second nucleotide sequence (forming base-pair hydrogen bonds under mammalian physiological conditions (or similar in vitro conditions)) and, under certain conditions, to form a double-stranded or double-helix structure. Complementary sequences include Watson-Crick base pairs or non-Watson-Crick base pairs and include natural or modified nucleotides or nucleotide mimics that at least meet the hybridization requirements described above. Sequence identity or complementarity is independent of modification. For example, for the purpose of determining identity or complementarity, fA and mA are complementary to U (or T) and identical to A.

[0045] As used herein, the term "perfectly complementary" in the context of two nucleotide sequences means that all (100%) bases in a sequential sequence of the first nucleotide sequence will hybridize with the same number of bases in a sequential sequence of the second nucleotide sequence to form a double strand. When two nucleotide sequences are designed to form one or more single-stranded overhangs after hybridization, such overhangs should not be considered mismatches in determining complementarity. For example, for the purposes described herein, an siRNA reagent comprising one oligonucleotide of length 21 and another oligonucleotide of length 23 is considered "perfectly complementary," wherein the 23-nucleotide oligonucleotide comprises a sequence of 21 nucleotides perfectly complementary to the 21-nucleotide oligonucleotide. In a specific embodiment, two nucleotide sequences are "perfectly complementary" when all (100%) bases of the first nucleotide sequence hybridize with all (100%) bases of the second nucleotide sequence to form a double strand. In a 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 the sequential sequence of the first nucleotide sequence will hybridize with the same number of bases in the sequential sequence of the second nucleotide sequence to form a double strand. In a specific embodiment, the two nucleotide sequences 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 the sequential sequence of the first nucleotide sequence will hybridize with the same number of bases in the sequential sequence of the second nucleotide sequence to form a double strand. In some embodiments, the 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%, or at least 98% but less than 100% of the bases in the sequential sequence of the first oligonucleotide will hybridize with the same number of bases in the sequential sequence of the second oligonucleotide to form a double strand. In a 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, when referring to numerical values, the terms "about" and "approximately" cover the numerical value and variations within + / - 20%. For example, about 20% would cover values ​​between 16% and 24% and values ​​in between, including 20%. In one embodiment, when referring to numerical values, the terms "about" and "approximately" cover the numerical value and variations within + / - 15%. In another embodiment, when referring to numerical values, the terms "about" and "approximately" cover the numerical value and variations within + / - 10%. In yet another embodiment, when referring to numerical values, the terms "about" and "approximately" cover the numerical value and variations within + / - 5%.

[0049] As used herein, when referring to hybridization, the term "strict" means that under "strict conditions" or "strict hybridization conditions," the first nucleotide sequence will hybridize with the second nucleotide, with minimal hybridization to other sequences. In specific embodiments, the antisense sequence will hybridize with its target sequence under strict conditions, with minimal targeting to other sequences. Strict conditions are sequence-dependent (e.g., sequence length, complementarity) and vary under different environmental parameters (e.g., assay conditions, physiological environment). Examples of strict hybridization conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C for 12–16 hours, followed by washing. Those skilled in the art will understand that variations in hybridization strictness are inherent to the description.

[0050] As used herein, when referring to hybridization, the term "very stringent" means that under "very stringent conditions" or "very stringent hybridization conditions," hybridization of the first nucleotide sequence with the second nucleotide will be observed only. In specific embodiments, antisense sequences will be observed to hybridize with their target sequences only under very stringent conditions. Furthermore, very stringent conditions may not allow hybridization 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). Compared to stringent conditions under the same conditions, very stringent conditions may include higher temperatures, lower ionic strengths, and / or shorter reaction times. For example, very stringent conditions may include hybridization temperatures 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. Those skilled in the art will understand that variations in hybridization stringency are inherent to the description.

[0051] As used herein, a “target sequence” refers to a continuous portion of the nucleotide sequence of an RNA molecule formed during the transcription of a gene (e.g., the SNCA gene), including mRNA that is a primary transcription product (e.g., SNCA mRNA generated by alternative splicing). In one embodiment, the continuous portion of the nucleotide sequence is at least long enough to serve as a substrate for RNAi-guided cleavage at or near a portion of the nucleotide sequence of the mRNA molecule formed during the transcription of a gene (e.g., the SNCA gene). In a specific embodiment, the target sequence is approximately 15-30 nucleotides in length. For example, the length of the target sequence can be approximately 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 The target sequence is 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 long. In some embodiments, the target sequence is 19-25 nucleotides long. In some embodiments, the target sequence is 21-23 nucleotides long. Ranges and lengths between the above ranges and lengths are also considered part of this disclosure.

[0052] As used herein, the phrases “nucleotide sequence corresponding to any antisense nucleotide sequence in the antisense nucleotide sequence,” “nucleotide sequence corresponding to an antisense nucleotide sequence,” “nucleotide sequence corresponding to any sense nucleotide sequence in the sense nucleotide sequence,” “nucleotide sequence corresponding to a sense nucleotide sequence,” “nucleotide sequence corresponding to any nucleotide sequence in the nucleotide sequence,” or “nucleotide sequence corresponding to any SEQ ID NO” refer to an oligonucleotide comprising a nucleotide chain containing the described unmodified nucleotide, or one or more modified nucleotides, or one or more conjugated portions (e.g., portions described herein, such as lipids, or modified nucleotides conjugated to portions described herein). Those skilled in the art will recognize that the unmodified nucleotide can be replaced with other portions without substantially altering the base-pairing properties of the oligonucleotide comprising the nucleotide with such a replacement portion. For example, a nucleotide containing inosine as its base can pair with nucleotides containing adenine, cytosine, or uracil. Thus, a nucleotide containing uracil, guanine, or adenine can be replaced with a nucleotide containing, for example, inosine. In another example, adenine and cytosine can be replaced by guanine and uracil, respectively, to form a GU swing base pair with the target mRNA.

[0053] As used herein, the phrases “non-naturally occurring small interfering RNA (siRNA) reagent,” “non-naturally occurring small interfering RNA reagent,” or “non-naturally occurring siRNA reagent” refer to siRNA reagents not found in nature. Non-naturally occurring siRNA may contain one or more modified nucleotides.

[0054] As used herein, the term "protrusion" in the context of a 5' or 3' nucleotide protrusion refers to at least one unpaired nucleotide protruding from the double-stranded structure of the siRNA reagent. For example, a nucleotide protrusion exists when the 3' end of one strand of the siRNA reagent extends beyond the 5' end of the other strand, or vice versa. In some embodiments, the protrusion is present at the 3' end of the sense strand, the antisense strand, or both strands. In one embodiment, the 3' protrusion is present in the antisense strand. In another embodiment, the 3' protrusion is present in the sense strand. In some embodiments, the protrusion is present at the 5' end of the sense strand, the antisense strand, or both strands. In one embodiment, the 5' protrusion is present in the antisense strand. In another embodiment, the 5' protrusion is present in the sense strand. A protrusion may be the result of one strand being longer than the other, or of two strands of equal length being interleaved. In some embodiments, the protrusion forms a mismatch with the target sequence. In other embodiments, the protrusion is complementary to the targeted gene sequence. The nucleotides in the overhang region of the siRNA reagent can be modified or unmodified nucleotides (e.g., 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, deoxynucleotides, or combinations thereof).

[0055] In some embodiments, the 5' or 3' overhang of the sense or antisense strand of the siRNA reagent is phosphorylated. In some embodiments, the 5' or 3' overhang of both the sense and antisense strands of the siRNA reagent is phosphorylated. In some embodiments, the overhang region contains two nucleotides with a phosphate thioester between them, and these two nucleotides may be the same or different.

[0056] In some embodiments, the siRNA reagent contains only a single overhang, which can enhance the interfering activity of the siRNA reagent without affecting its overall stability. For example, the single-stranded overhang can be located at the 3' end of the sense strand of the siRNA reagent, or alternatively, at the 3' end of the antisense strand of the siRNA reagent. The siRNA reagent can 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 reagent has a nucleotide overhang at the 3' end and a blunt end at the 5' end.

[0057] In some embodiments, one strand of the siRNA reagent comprises at least one nucleotide, at least two nucleotides, or at least three nucleotides with a 5' end, a 3' end, or both a 5' end and a 3' end overhang. In some embodiments, one strand of the siRNA reagent comprises at least one nucleotide, at least two nucleotides, or at least three nucleotides but no more than five nucleotides with a 5' end, a 3' end, or both a 5' end and a 3' end overhang. In some embodiments, one strand of the siRNA reagent comprises one to two nucleotides, one to three nucleotides, one to four nucleotides, or one to five nucleotides with a 5' end, a 3' end, or both a 5' end and a 3' end overhang. In some embodiments, one strand of the siRNA reagent comprises two to three nucleotides, two to four nucleotides, or two to five nucleotides with a 5' end, a 3' end, or both a 5' end and a 3' end overhang. In some embodiments, one strand of the siRNA reagent comprises 3 to 4 nucleotides or 4 to 5 nucleotides with 5', 3', or both 5' and 3' overhangs. This strand can be an antisense strand or a sense strand. The nucleotide overhangs may comprise or consist of nucleotide analogs or nucleoside analogs.

[0058] In some embodiments, each strand of the siRNA reagent comprises at least one nucleotide, at least two nucleotides, or at least three nucleotides with a 5' end, a 3' end, or both a 5' end and a 3' end overhang. In some embodiments, each strand of the siRNA reagent comprises at least one nucleotide, at least two nucleotides, or at least three nucleotides but no more than five nucleotides with a 5' end, a 3' end, or both a 5' end and a 3' end overhang. In some embodiments, each strand of the siRNA reagent comprises one to two nucleotides, one to three nucleotides, one to four nucleotides, or one to five nucleotides with a 5' end, a 3' end, or both a 5' end and a 3' end overhang. In some embodiments, each strand of the siRNA reagent comprises two to three nucleotides, two to four nucleotides, or two to five nucleotides with a 5' end, a 3' end, or both a 5' end and a 3' end overhang. In some embodiments, each strand of the siRNA reagent contains 3 to 4 nucleotides or 4 to 5 nucleotides with 5', 3', or both 5' and 3' overhangs. The nucleotide overhangs may contain or consist of nucleotide analogs or nucleoside analogs.

[0059] As used herein, in the context of siRNA reagents, the term "blunt-ended" or "blunt-ended" means that there are no unpaired nucleotides or nucleotide analogs at a given end of the siRNA reagent; that is, there are no nucleotide overhangs. In some embodiments, one end of the siRNA reagent is blunt-ended. In other words, the 5' end of one strand and the 3' end of the other strand do not contain unpaired nucleotides or nucleotide analogs. In some embodiments, both ends of the siRNA reagent are blunt-ended. In other words, neither end of the siRNA reagent has a nucleotide overhang.

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

[0061] As used herein, in the context of siRNA reagents, the term "sense strand" or "passenger strand" refers to a strand of siRNA reagent that includes regions complementary to the regions of the antisense strand.

[0062] As used herein, in the context of siRNA reagent nucleobases, the term "modified" refers to nucleobases that are not naturally present in RNA molecules. Naturally present RNA sequences include purine bases adenine (A) and guanine (G), and pyrimidine bases cytosine (C) and uracil (U). See the section below on the chemical modifications of nucleotides, such as modified nucleobases.

[0063] As used herein, in the context of nucleotides in siRNA reagents, the term "modified" refers to a nucleotide that is not naturally present in RNA molecules. See the section below on the chemical modifications of nucleotides, for example, modified nucleotides.

[0064] As used herein, unless otherwise stated, the term "isomer" refers to different compounds having the same molecular formula. "Stereoisomers" are isomers that differ only in the spatial arrangement of their atoms. "Rotated isomers" are stereoisomers derived from rotationally blocked around a single bond. "Enantiomers" are a pair of stereoisomers that are non-overlapping mirror images of each other. A mixture of any proportion of a pair of enantiomers can be called a "racemic" mixture. "Diadiaomers" are stereoisomers that have at least two asymmetric atoms but are not mirror images of each other.

[0065] "Stereoisomers" may also include E and Z isomers or mixtures thereof, as well as cis and trans isomers or mixtures thereof. In some embodiments, the compounds described herein are isolated as E or Z isomers. In other embodiments, the compounds described herein are mixtures of E and Z isomers.

[0066] It should be noted that if there is a difference between the described structure and the name of the structure, the described structure will be given more weight.

[0067] The terms “inhibition,” “blocking,” “curbing,” and “reduction” are used interchangeably and refer to any statistically significant reduction in a given biological activity, including complete blockade of activity. For example, “inhibition” can refer to a reduction in biological activity of approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0068] nucleotide sequence This document provides siRNA reagents for (e.g., partially or completely) inhibiting the expression of SNCA genes (e.g., human SNCA genes). In specific embodiments, in assays known to those skilled in the art or described herein (e.g., in the Bioassays section below; or in the Examples section below), the siRNAs described herein inhibit the expression of SNCA genes (e.g., human SNCA genes) by at least about 25%, at least about 30%, at least about 40%, at least about 50%, or at least about 60%, relative to a negative control (e.g., PBS or siRNA against RNA molecules formed during transcription of genes other than SNCA). In another specific embodiment, in assays known to those skilled in the art or described herein (e.g., in the Bioassays section below; or in the Examples section below), the siRNAs described herein inhibit the expression of SNCA genes (e.g., human SNCA genes) 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 against RNA molecules formed during transcription of genes other than SNCA). In another specific embodiment, in assays known to those skilled in the art or described herein (e.g., in the Bioassays section below; or the Examples section below), the siRNA described herein inhibits the expression of the SNCA gene (e.g., the 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 against RNA molecules formed during transcription of genes other than SNCA). In another specific embodiment, in assays known to those skilled in the art or described herein (e.g., in the Bioassays section below; or the Examples section below), the siRNA described herein inhibits the expression of the SNCA gene (e.g., the 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 against RNA molecules formed during transcription of genes other than SNCA). In another specific embodiment, in assays known to those skilled in the art or described herein (e.g., in the Bioassays section below; or in the Examples section below), the siRNA described herein inhibits the expression of SNCA genes (e.g., human SNCA genes) by at least about 100% relative to a negative control (e.g., PBS or siRNA against RNA molecules formed during transcription of genes other than SNCA). In a specific embodiment, the inhibition of SNCA gene (e.g., human SNCA gene) expression is assessed using in vitro assays described in the Examples section below.In specific implementations, the siRNA reagents described herein exhibit in vitro or ex vivo knockdown efficiency, as described, for example, in the Examples section below. In specific implementations, the siRNA reagents described herein exhibit in vivo knockdown efficiency in mouse models, as described, for example, in the Examples section below. In specific implementations, the siRNA reagents described herein exhibit one, two, three, or more of the properties of the RNA molecules described in the Examples section below.

[0069] In specific implementations, the siRNA reagent described herein is used in the assays described herein (e.g., in the Examples section below) at an IC50 concentration of 6 nM to 5000 nM. 50 Inhibits the expression of SNCA genes (e.g., human SNCA genes) in neurons (e.g., human iPSC neurons). In specific embodiments, the siRNA reagent described herein is used in the assays described herein (e.g., in the Examples section below) at an IC50 concentration of 10 nM to 1000 nM. 50 Inhibits the expression of SNCA genes (e.g., human SNCA genes) in neurons (e.g., human iPSC neurons). In specific embodiments, the siRNA reagent described herein is used in the assays described herein (e.g., in the Examples section below) at an IC50 concentration of 10 nM to 50 nM. 50 Inhibits the expression of SNCA genes (e.g., human SNCA genes) in neurons (e.g., human iPSC neurons). In specific embodiments, the siRNA reagent described herein is used in the assays described herein (e.g., in the Examples section below) at an IC50 concentration of 150 nM to 1000 nM. 50 Inhibit the expression of SNCA genes (e.g., human SNCA genes) in neurons (e.g., human iPSC neurons).

[0070] In specific implementations, the siRNA reagents described herein are stable in vitro or ex vivo in one, two, or all of the following: mouse brain homogenate, human liver lysosomes, and rat liver tritosomes. The stability of such siRNA reagents can be assessed using methods known to those skilled in the art or as described herein (e.g., in the Bioassays section below; or in the Examples section below).

[0071] In specific embodiments, the siRNA reagent is a double-stranded RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide comprising a sense strand and an antisense strand that are annealed to form a double-stranded region or duplex. The antisense strand comprises a region substantially complementary to and typically perfectly complementary to the target sequence. The sense strand comprises a region 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 sequence of the siRNA reagent may also be contained as a self-complementary region of a single nucleic acid molecule, rather than located on a separate oligonucleotide. In specific embodiments, annealing of the sense and antisense strand sequences forms a stable duplex, as assessed by melting temperatures (Tm) of about 40°C, about 41°C, about 42°C, about 43°C, about 44°C, about 45°C, or higher. For melting temperature determination, see, for example, the bioassay section herein.

[0072] The sense and antisense strands of the siRNA reagent described herein may be of the same or different lengths. The length of each of the sense and antisense strands of the siRNA reagent described herein may be 10 to 30 nucleotides. In specific embodiments, the length of each of the sense and antisense strands of the siRNA reagent described herein does not exceed 30 nucleotides. In some embodiments, the length of each of the sense and antisense strands of the siRNA reagent described herein is independently 10 to 30 nucleotides. In some embodiments, the length of each of the sense and antisense strands of the siRNA reagent described herein is independently 10 to 25 nucleotides. In some embodiments, the length of each of the sense and antisense strands of the siRNA reagent described herein is independently 15 to 25 nucleotides. In some embodiments, the length of each of the sense and antisense strands of the siRNA reagent described herein is independently 16 to 25 nucleotides. In some embodiments, the length of each of the sense and antisense strands of the siRNA reagent described herein is independently 16 to 24 nucleotides. In some embodiments, the sense and antisense strands of the siRNA reagent described herein are each independently 16 to 23 nucleotides in length. In some embodiments, the sense and antisense strands of the siRNA reagent described herein are each independently 16 to 22 nucleotides in length. In some embodiments, the sense and antisense strands of the siRNA reagent described herein are each independently 17 to 22 nucleotides in length. In some embodiments, the sense and antisense strands of the siRNA reagent described herein are each independently 17 to 21 nucleotides in length. In some embodiments, the sense and antisense strands of the siRNA reagent described herein are each independently 17 to 20 nucleotides in length. In some embodiments, the sense and antisense strands of the siRNA reagent described herein are each independently 18 to 22 nucleotides in length. In some embodiments, the sense and antisense strands of the siRNA reagent described herein are each independently 18 to 21 nucleotides in length. In some embodiments, the sense and antisense strands of the siRNA reagent described herein are each independently 18 to 20 nucleotides in length. In some implementations, the sense and antisense strands of the siRNA reagent described herein are each independently about 19 nucleotides in length.

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

[0074] In some embodiments, the length of the double-stranded region of the siRNA reagent described herein is 10 to 30 base pairs. For example, the length of the double-stranded region of the siRNA reagent described herein can 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 some embodiments, the double-stranded region of the siRNA reagent described herein is 15 to 25 base pairs in length. In some embodiments, the double-stranded region of the siRNA reagent described herein is 16 to 25 base pairs in length. In some embodiments, the double-stranded region of the siRNA reagent described herein is 16 to 24 base pairs in length. In some embodiments, the double-stranded region of the siRNA reagent described herein is 16 to 23 base pairs in length. In some embodiments, the double-stranded region of the siRNA reagent described herein is 16 to 22 base pairs in length. In some embodiments, the double-stranded region of the siRNA reagent described herein is 17 to 23 base pairs in length. In some embodiments, the double-stranded region of the siRNA reagent described herein is 17 to 22 base pairs in length. In some embodiments, the double-stranded region of the siRNA reagent described herein is 17 to 21 base pairs in length. In some embodiments, the double-stranded region of the siRNA reagent described herein is 18 to 22 base pairs in length. In some embodiments, the double-stranded region of the siRNA reagent described herein is 18 to 21 base pairs in length. In some embodiments, the double-stranded region of the siRNA reagent described herein is 18 to 20 base pairs in length. In some embodiments, the double-stranded region of the siRNA reagent described herein is 19 to 22 base pairs in length. In some embodiments, the double-stranded region of the siRNA reagent described herein is 19 to 21 base pairs in length. In some embodiments, the double-stranded region of the siRNA reagent described herein is 19 or 20 base pairs in length.In some embodiments, the double-stranded region of the siRNA reagent described herein is approximately 19 base pairs in length. In some embodiments, the double-stranded region of the siRNA reagent described herein is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 base pairs in length. In specific embodiments, the double-stranded region of the siRNA reagent described herein is 17, 18, 19, 20, or 21 base pairs in length. Ranges and lengths intermediate to the above ranges and lengths are also considered part of this disclosure.

[0075] In some embodiments, the siRNA reagent described herein is blunt-ended. In other embodiments, the sense or antisense strand of the siRNA reagent described herein includes a bulge. The bulge may be located at the 5' end, 3' end, or both of the 5' and 3' ends of the sense or antisense strand of the siRNA reagent. In some embodiments, both the sense and antisense strands of the siRNA reagent described herein include bulges. The bulge of the sense strand may be located at the 5' end of the sense strand, and the bulge of the antisense strand may be located at the 3' end of the antisense strand. Alternatively, the bulge of the sense strand may be located at the 3' end of the sense strand, and the bulge of the antisense strand may be located at the 5' end of the antisense strand. In some embodiments, the bulge of the siRNA reagent strand includes at least one nucleotide. In some embodiments, the bulge of the siRNA reagent strand includes at least two nucleotides. In some embodiments, the bulge of the siRNA reagent strand includes at least three nucleotides. In some embodiments, the bulge of the siRNA reagent strand includes at least four nucleotides. In some embodiments, the overhang of the siRNA reagent strand contains at least 5 nucleotides. In some embodiments, the overhang of the siRNA reagent strand contains 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, or more. In some embodiments, the overhang of the siRNA reagent strand contains 1 to 5 nucleotides, 1 to 4 nucleotides, 1 to 3 nucleotides, or 1 to 2 nucleotides. In some embodiments, the overhang of the siRNA reagent strand contains 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 some embodiments, the sense strand of the siRNA reagent described herein is 17 nucleotides long, and the antisense strand of the siRNA reagent is 22 nucleotides long. In some embodiments, the sense strand of the siRNA reagent described herein is 17 nucleotides long, and the antisense strand of the siRNA reagent is 21 nucleotides long. In some embodiments, the sense strand of the siRNA reagent described herein is 18 nucleotides long, and the antisense strand of the siRNA reagent is 21 nucleotides long. In some embodiments, the sense strand of the siRNA reagent described herein is 19 nucleotides long, and the antisense strand of the siRNA reagent is 21 nucleotides long. In some embodiments, the sense strand of the siRNA reagent described herein is 20 nucleotides long, and the antisense strand of the siRNA reagent is 21 nucleotides long. In some embodiments, the sense strand of the siRNA reagent described herein is 21 nucleotides long, and the antisense strand of the siRNA reagent is 20 nucleotides long. In some embodiments, the sense strand of the siRNA reagent described herein is 22 nucleotides long, and the antisense strand of the siRNA reagent is 20 nucleotides long. In some embodiments, the sense strand of the siRNA reagent described herein is 22 nucleotides long, and the antisense strand of the siRNA reagent is 19 nucleotides long. In some embodiments, the sense strand of the siRNA reagent described herein is 22 nucleotides long, and the antisense strand of the siRNA reagent is 18 nucleotides long. In some embodiments, the sense strand of the siRNA reagent described herein is 22 nucleotides long, and the antisense strand of the siRNA reagent is 17 nucleotides long.

[0077] In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a nucleotide sequence corresponding to any of the sense strand nucleotide sequences in Table 1.

[0078] In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a nucleotide sequence corresponding to any of the antisense nucleotide sequences in Table 1.

[0079] In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a nucleotide sequence corresponding to a sense strand nucleotide sequence in a row of Table 1, and wherein the antisense strand comprises a nucleotide sequence corresponding to an antisense strand nucleotide sequence in the same row of Table 1. In a specific embodiment, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a nucleotide sequence corresponding to a sense strand nucleotide sequence of US14, US28, US58, US183, and US199 in Table 1, and wherein the antisense strand comprises a nucleotide sequence corresponding to an antisense strand nucleotide sequence of US14, US28, US58, US183, and US199 in Table 1, respectively.

[0080] In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a nucleotide sequence corresponding to any sense strand nucleotide sequence in 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, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a nucleotide sequence corresponding to any sense strand nucleotide sequence in the sense strand nucleotide sequences in Table 1, and the antisense strand comprises a nucleotide sequence substantially complementary to the sense strand. In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a nucleotide sequence corresponding to any sense strand nucleotide sequence in the sense strand nucleotide sequences in Table 1, and the antisense strand comprises a nucleotide sequence completely complementary to the sense strand.

[0081] In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a nucleotide sequence corresponding to any antisense nucleotide sequence in the antisense nucleotide sequences in Table 1, and the sense strand comprises a nucleotide sequence partially complementary to the antisense strand. In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a nucleotide sequence corresponding to any antisense nucleotide sequence in the antisense nucleotide sequences in Table 1, and the sense strand comprises a nucleotide sequence substantially complementary to the antisense strand. In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a nucleotide sequence corresponding to any antisense nucleotide sequence in the antisense nucleotide sequences in Table 1, and the sense strand comprises a nucleotide sequence completely complementary to the antisense strand.

[0082] In some embodiments, the siRNA reagent does not contain, for example, chemical modifications or conjugations known in the art and described herein. For information on siRNA reagents containing chemical modifications or conjugations, see, for example, the section on chemical modifications of nucleotides below and the section on examples below. In specific embodiments, the siRNA reagent contains, for example, chemical modifications or conjugations known in the art and described herein. In specific embodiments, the siRNA reagent contains one or more chemical modifications and / or conjugations described herein (e.g., in the section on chemical modifications of nucleotides below and the section on examples below). In some embodiments, the nucleobases of the nucleotide are modified. In some embodiments, the sugar of the nucleotide is modified. In some embodiments, the phosphate backbone of the nucleotide is modified.

[0083] siRNA reagents may contain only naturally occurring nucleotides, or they may contain one or more modified nucleotides. In some embodiments, the nucleobases of the nucleotides are modified. In some embodiments, the sugars of the nucleotides are modified. In some embodiments, the phosphate backbone of the nucleotides is modified.

[0084] In specific embodiments, the modified nucleotide comprises one or more of the modified nucleotides described herein (e.g., in the section on chemical modifications of nucleotides below and the section on examples below). In specific embodiments, the modified nucleotide is selected from the group consisting of 2'O-methyl modified nucleotides, deoxynucleotides, 2'-fluorinated modified nucleotides, 2'-O-methyl-uridine, 3'-O-methyl modified nucleotides, 3'-O-methyl modified nucleotides of phosphates having a 2'-5' link, reverse-base-free nucleotides, nucleotides containing S-ethylene glycol nucleic acids (GNA), unlocked nucleotides, vinylphosphonates, 5'-vinylphosphonate-2'-O-methyl-uridine, cis-cyclobutylphosphonate modified nucleotides, 5'-cis-cyclobutylphosphonate-2'-O-methyl modified nucleotides, (L)-α-threofuranyl modified nucleotides, and combinations thereof. In a specific implementation, the modified nucleotide is selected from the group consisting of 2'O-methyl modified nucleotides, deoxynucleotides, 2'-fluorine modified nucleotides, reverse abase-free nucleotides, nucleotides containing S-ethylene glycol nucleic acids (GNA), unlocking nucleotides, vinyl phosphonates, and combinations thereof.

[0085] In a specific implementation, the sense strand of the siRNA reagent described herein contains at least one modified nucleotide inter-bond.

[0086] In a specific implementation, the antisense strand of the siRNA reagent described herein contains at least one modified nucleoside interstrand.

[0087] In a specific embodiment, the antisense strand and sense strand of the siRNA reagent described herein each contain at least one modified nucleoside internucleotide bond. In a specific embodiment, the modified nucleoside internucleotide bond includes a phosphate thioester.

[0088] In specific implementations, the siRNA reagents described herein contain one or more of the following: phosphate thioester (PS) bonds, 2'-fluororibose (2'-F), 2'-methoxyribose (2'-OMe), vinylphosphonate, and reverse linkage (reverse no-base site, InvAb).

[0089] In specific embodiments, the siRNA reagents described herein exhibit stability, as assessed by assays described herein (e.g., in the Bioassays section and the Examples section below) or known to those skilled in the art. In some embodiments, the siRNA reagents described herein containing one or more modified nucleotides exhibit increased stability relative to the same siRNA reagent without those one or more modified nucleotides, as assessed using methods known to those skilled in the art or described herein (e.g., in the Bioassays section and the Examples section below).

[0090] In one embodiment, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a nucleotide sequence of any sense strand nucleotide sequence from Table 2. In another embodiment, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a nucleotide sequence of any antisense strand nucleotide sequence from Table 2.

[0091] In one embodiment, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a nucleotide sequence of an antisense strand nucleotide sequence in one row of Table 2, and wherein the antisense strand comprises a nucleotide sequence of a sense strand nucleotide sequence in the same row of Table 2.

[0092] In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a nucleotide sequence of any sense strand nucleotide sequence from 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, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a nucleotide sequence of any sense strand nucleotide sequence from the sense strand nucleotide sequences in Table 2, and the antisense strand comprises a nucleotide sequence substantially complementary to the sense strand. In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a nucleotide sequence of any sense strand nucleotide sequence from the sense strand nucleotide sequences in Table 2, and the antisense strand comprises a nucleotide sequence completely complementary to the sense strand.

[0093] In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a nucleotide sequence of any antisense nucleotide sequence from the antisense nucleotide sequences in Table 2, and the sense strand comprises a nucleotide sequence partially complementary to the antisense strand. In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a nucleotide sequence of any antisense nucleotide sequence from the antisense nucleotide sequences in Table 2, and the sense strand comprises a nucleotide sequence substantially complementary to the antisense strand. In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a nucleotide sequence of any antisense nucleotide sequence from the antisense nucleotide sequences in Table 2, and the sense strand comprises a nucleotide sequence completely complementary to the antisense strand.

[0094] In some embodiments, the siRNA reagents described herein are conjugated to one or more non-nucleotide groups, including but not limited to targeting groups, linking groups, delivery polymers, or delivery vectors. Additional information regarding siRNA reagents conjugated to one or more non-nucleotide groups can be found, for example, in the section on linking to nucleotide sequences below. In specific embodiments, the one or more non-nucleotide groups are the one or more non-nucleotide groups described herein (e.g., in the section on linking to nucleotide sequences below and the section on examples below).

[0095] In specific embodiments, the sense strand of the siRNA reagent described herein is conjugated directly or indirectly to one or more lipophilic moieties. For information on lipophilicity, see, for example, Kindt et al., (2007), pp. 531-554. In specific embodiments, the antisense strand of the siRNA reagent described herein is conjugated directly or indirectly to one or more lipophilic moieties. In specific embodiments, both the sense and antisense strands of the siRNA reagent described herein are each conjugated directly or indirectly to one or more lipophilic moieties. One or more lipophilic moieties may be conjugated to one or more terminal positions of the siRNA reagent. For example, the lipophilic moieties may be conjugated to the 5' and / or 3' ends of one or both strands of the siRNA reagent. Alternatively, or additionally, one or more lipophilic moieties may be conjugated to one or more internal positions of a double-stranded region of the siRNA reagent. In some embodiments, the lipophilic moieties are conjugated to one or more terminal positions and one or more internal positions of the siRNA reagent. In some embodiments, one or more lipophilic moieties are conjugated via a linker or carrier. In specific implementations, the adapter is the adapter described herein (e.g., in the section below on nucleotide sequence linkage).

[0096] In specific embodiments, the lipophilic portion comprises the lipophilic portion described herein (e.g., in the section below on linking nucleotide sequences, the section below on the synthesis of siRNA reagents, or the section below on examples). In specific embodiments, the lipophilic portion is cholesterol, such as cholesterol-triethylene glycol (CHOL4). In specific embodiments, the lipophilic portion is selected from one or more of the following: L1, L2, L3, L4, and L5 (see...). Figure 1 In a specific implementation, the lipophilic portion (such as that described in the Examples section) is conjugated to the sense strand of the siRNA reagent in the manner described in the Examples section.

[0097] In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a nucleotide sequence of any of the sense strand nucleotide sequences in Table 3. In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a nucleotide sequence of any of the antisense strand nucleotide sequences in Table 3.

[0098] In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a nucleotide sequence of a sense strand nucleotide sequence in one row of Table 3, and the antisense strand comprises a nucleotide sequence of an antisense strand nucleotide sequence in the same row of Table 3. In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a nucleotide sequence of any sense strand nucleotide sequence in Table 3, and wherein the sense strand is conjugated to cholesterol as indicated in Table 3, which is conjugated to the sense strand of the siRNA reagent. In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a nucleotide sequence of an antisense strand nucleotide sequence in one row of Table 3, and the sense strand comprises a nucleotide sequence of a sense strand nucleotide sequence in the same row of Table 3, and wherein the sense strand is conjugated to cholesterol as indicated in Table 3, which is conjugated to the sense strand of the siRNA reagent.

[0099] In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a nucleotide sequence of any sense strand nucleotide sequence from 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, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a nucleotide sequence of any sense strand nucleotide sequence from the sense strand nucleotide sequences in Table 3, and the antisense strand comprises a nucleotide sequence substantially complementary to the sense strand. In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a nucleotide sequence of any sense strand nucleotide sequence from the sense strand nucleotide sequences in Table 3, and the antisense strand comprises a nucleotide sequence completely complementary to the sense strand.

[0100] In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a nucleotide sequence of any sense strand nucleotide sequence from the sense strand nucleotide sequences in Table 3, wherein the sense strand is conjugated to cholesterol as indicated in Table 3, the cholesterol being conjugated to the sense strand of the siRNA reagent, and the antisense strand comprises a nucleotide sequence partially complementary to the sense strand. In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a nucleotide sequence of any sense strand nucleotide sequence from 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 some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a nucleotide sequence of any sense strand nucleotide sequence from 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 completely complementary to the sense strand.

[0101] In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a nucleotide sequence of any antisense nucleotide sequence from the antisense nucleotide sequences in Table 3, and the sense strand comprises a nucleotide sequence partially complementary to the antisense strand. In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a nucleotide sequence of any antisense nucleotide sequence from the antisense nucleotide sequences in Table 3, and the sense strand comprises a nucleotide sequence substantially complementary to the antisense strand. In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a nucleotide sequence of any antisense nucleotide sequence from the antisense nucleotide sequences in Table 3, and the sense strand comprises a nucleotide sequence completely complementary to the antisense strand.

[0102] In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a nucleotide sequence of any of the antisense nucleotide sequences in Table 3, and the sense strand comprises a nucleotide sequence complementary to the antisense portion, and wherein the sense strand of the siRNA reagent, as indicated in Table 3, is associated with cholesterol or, for example, cholesterol. Figure 1The description herein refers to conjugation with another lipophilic moiety. In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a nucleotide sequence of any of the antisense 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 reagent, as indicated in Table 3, is conjugated with cholesterol or, for example... Figure 1 The description herein refers to conjugation with another lipophilic moiety. In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a nucleotide sequence of any of the antisense nucleotide sequences in Table 3, and the sense strand comprises a nucleotide sequence that is completely complementary to the antisense strand, and wherein the sense strand of the siRNA reagent, as indicated in Table 3, is conjugated with cholesterol or, for example... Figure 1 The description in the text is related to the conjugation of another lipophilic part.

[0103] In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a nucleotide sequence of any sense strand nucleotide sequence from Table 21. In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a nucleotide sequence of any antisense strand nucleotide sequence from Table 21.

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

[0105] In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a nucleotide sequence of any sense strand nucleotide sequence from Table 21, and the antisense strand comprises a nucleotide sequence partially complementary to the sense strand. In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a nucleotide sequence of any sense strand nucleotide sequence from Table 21, and the antisense strand comprises a nucleotide sequence substantially complementary to the sense strand. In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a nucleotide sequence of any sense strand nucleotide sequence from Table 21, and the antisense strand comprises a nucleotide sequence completely complementary to the sense strand.

[0106] In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a nucleotide sequence of any sense strand nucleotide sequence from the sense strand nucleotide sequences in Table 21, wherein the sense strand is conjugated to cholesterol as indicated in Table 21, the cholesterol being conjugated to the sense strand of the siRNA reagent, and the antisense strand comprises a nucleotide sequence partially complementary to the sense strand. In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a nucleotide sequence of any sense strand nucleotide sequence from 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 some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a nucleotide sequence of any sense strand nucleotide sequence in Table 21, wherein the sense strand is conjugated to cholesterol as indicated in Table 21, and the antisense strand comprises a nucleotide sequence that is completely complementary to the sense strand.

[0107] In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a nucleotide sequence of any antisense nucleotide sequence from the antisense nucleotide sequences in Table 21, and the sense strand comprises a nucleotide sequence partially complementary to the antisense strand. In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a nucleotide sequence of any antisense nucleotide sequence from the antisense nucleotide sequences in Table 21, and the sense strand comprises a nucleotide sequence substantially complementary to the antisense strand. In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a nucleotide sequence of any antisense nucleotide sequence from the antisense nucleotide sequences in Table 21, and the sense strand comprises a nucleotide sequence completely complementary to the antisense strand.

[0108] In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a nucleotide sequence of any of the antisense nucleotide sequences in Table 21, and the sense strand comprises a nucleotide sequence complementary to the antisense portion, and wherein the sense strand of the siRNA reagent, as indicated in Table 21, is associated with cholesterol or, for example, cholesterol. Figure 1 The description herein refers to conjugation with another lipophilic moiety. In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a nucleotide sequence of any of the antisense 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 reagent, as indicated in Table 21, is conjugated with cholesterol or, for example... Figure 1 The description herein refers to conjugation with another lipophilic moiety. In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a nucleotide sequence of any of the antisense nucleotide sequences in Table 21, and the sense strand comprises a nucleotide sequence that is completely complementary to the antisense strand, and wherein the sense strand of the siRNA reagent, as indicated in Table 21, is conjugated with cholesterol or, for example, cholesterol. Figure 1 The description in the text is related to the conjugation of another lipophilic part.

[0109] In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a nucleotide sequence of any of the sense strand nucleotide sequences in Table 4. In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a nucleotide sequence of any of the antisense strand nucleotide sequences in Table 4.

[0110] In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a nucleotide sequence of a sense strand nucleotide sequence in one row of Table 4, and the antisense strand comprises a nucleotide sequence of an antisense strand nucleotide sequence in the same row of Table 4. In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a nucleotide sequence of any sense strand nucleotide sequence in Table 4, and wherein the sense strand is conjugated to a lipophilic moiety as indicated in Table 4, which is conjugated to the sense strand of the siRNA reagent. In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a nucleotide sequence of an antisense strand nucleotide sequence in one row of Table 4, and the sense strand comprises a nucleotide sequence of a sense strand nucleotide sequence in the same row of Table 4, and wherein the sense strand is conjugated to a lipophilic moiety as indicated in Table 4, which is conjugated to the sense strand of the siRNA reagent.

[0111] In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a nucleotide sequence of any sense strand nucleotide sequence from Table 4, and the antisense strand comprises a nucleotide sequence partially complementary to the sense strand. In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a nucleotide sequence of any sense strand nucleotide sequence from Table 4, and the antisense strand comprises a nucleotide sequence substantially complementary to the sense strand. In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a nucleotide sequence of any sense strand nucleotide sequence from Table 4, and the antisense strand comprises a nucleotide sequence completely complementary to the sense strand.

[0112] In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a nucleotide sequence of any sense strand nucleotide sequence from Table 4, wherein the sense strand is conjugated to a lipophilic moiety as indicated in Table 4, the lipophilic moiety being conjugated to the sense strand of the siRNA reagent, and the antisense strand comprises a nucleotide sequence complementary to the sense strand moiety. In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a nucleotide sequence of any sense strand nucleotide sequence from 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 some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a nucleotide sequence of any sense strand nucleotide sequence in Table 4, wherein the sense strand is conjugated to a lipophilic portion as indicated in Table 4, and the antisense strand comprises a nucleotide sequence that is completely complementary to the sense strand.

[0113] In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a nucleotide sequence of any antisense nucleotide sequence from the antisense nucleotide sequences in Table 4, and the sense strand comprises a nucleotide sequence partially complementary to the antisense strand. In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a nucleotide sequence of any antisense nucleotide sequence from the antisense nucleotide sequences in Table 4, and the sense strand comprises a nucleotide sequence substantially complementary to the antisense strand. In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a nucleotide sequence of any antisense nucleotide sequence from the antisense nucleotide sequences in Table 4, and the sense strand comprises a nucleotide sequence completely complementary to the antisense strand.

[0114] In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a nucleotide sequence of any of the antisense nucleotide sequences in Table 4, and the sense strand comprises a nucleotide sequence complementary to the antisense portion, and wherein the sense strand of the siRNA reagent, as indicated in Table 4, is associated with a lipophilic portion or, for example, a lipophilic portion. Figure 1The description herein refers to conjugation with another lipophilic moiety. In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a nucleotide sequence of any of the antisense 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 reagent, as indicated in Table 4, conjugates with a lipophilic moiety or, for example, a conjugation with another lipophilic moiety. Figure 1 The description herein refers to conjugation with another lipophilic moiety. In some embodiments, this document provides an siRNA reagent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a nucleotide sequence of any of the antisense nucleotide sequences in Table 4, and the sense strand comprises a nucleotide sequence that is completely complementary to the antisense strand, and wherein the sense strand of the siRNA reagent, as indicated in Table 4, conjugates with a lipophilic moiety or, for example, a conjugation with another lipophilic moiety. Figure 1 The description in the text is related to the conjugation of another lipophilic part.

[0115] In specific embodiments, the siRNA reagent described herein comprises a sense strand and an antisense strand of one of the names listed in Tables 1, 2, 3, or 4. In specific embodiments, the siRNA reagent described herein comprises a sense strand and an antisense strand corresponding to any of US14, US28, US58, US183, or US199 in Table 1. In specific embodiments, the siRNA reagent described herein comprises a sense strand and an antisense strand of any of S14, S28, S58, S183, or S199 in Table 2. In specific embodiments, the siRNA reagent described herein comprises a sense strand and an antisense strand of one of the names listed in Tables 3 or 4. In some embodiments, the sense strand of the siRNA reagent is conjugated to the lipophilic moiety described herein (e.g., Figure 1 In a specific implementation, the siRNA reagent is the reagent described in the Examples section below.

[0116] In some embodiments, the siRNA reagent described herein also comprises nonnucleotide groups, such as ligands (e.g., targeting ligands). Nonnucleotide groups (such as ligands (e.g., targeting ligands)) may be conjugated directly or indirectly to the antisense or sense strand of the siRNA reagent. Nonnucleotide groups (such as ligands (e.g., targeting ligands)) replace one or more nucleotides at internal positions within the double-stranded region of the siRNA reagent described herein. In some embodiments, nonnucleotide groups (such as ligands (e.g., targeting ligands)) are conjugated to the strand of the siRNA reagent via a linker or carrier (e.g., a delivery vector). In specific embodiments, the nonnucleotide groups (such as ligands (e.g., targeting ligands)) are the nonnucleotide groups described in the following section on nucleotide sequence linking.

[0117] Chemical modification of nucleotides In specific embodiments, the siRNA reagent described herein comprises one or more nucleotide modifications. Nucleotide modifications include: for example, terminal modifications, such as 5' end modifications (e.g., phosphorylation, conjugation, reverse linkage) or 3' end modifications (e.g., conjugation, DNA nucleotides, reverse linkage, etc.); base modifications, such as replacement with a stabilizing base, destabilizing a base, or a base paired with a base from an amplified partner library, base removal (base-free nucleotide), or conjugated bases; sugar modifications (e.g., at the 2' or 4' position) or sugar substitution; or backbone modifications, including modification or substitution of phosphodiester bonds. In some embodiments, the siRNA reagent comprises at least one modification selected from the group consisting of modified nucleotide internucleotides, modified nucleobases, modified sugars, and any combination thereof. Without limitation, such modifications may be present at any position in the siRNA reagent (e.g., in the sense strand, antisense strand, or both).

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

[0119] In some implementations, the siRNA reagent described herein comprises one or more ethylene glycol nucleic acids (GNAs). Typically, GNAs are acyclic nucleic acid analogs in which their repeating ethylene glycol units are linked by phosphodiester bonds, unlike the ribose-phosphodiester backbone of RNA.

[0120] In some embodiments, the siRNA reagent described herein comprises one or more end modifications, such as 5' end phosphorylation, conjugation, or reverse linkage. In some embodiments, the end modification comprises a 5'-phosphate, such as a 5'-terminal phosphate on the antisense strand of the siRNA reagent.

[0121] In some embodiments, the siRNA reagent comprises a sense strand and / or an antisense strand having an inverted abase-free nucleotide. In one embodiment, the sense strand contains an inverted abase-free nucleotide at its 3' end. In another embodiment, the sense strand contains an inverted abase-free nucleotide at its 5' end. In some embodiments, the sense strand contains inverted abase-free nucleotides at both the 5' and 3' ends.

[0122] In some embodiments, the siRNA reagent comprises a phosphate ester or phosphate ester mimic at the 5' end of the antisense strand. In one embodiment, the phosphate ester mimic is 5'-vinylphosphonate (VP).

[0123] In some embodiments, the siRNA reagent comprises one or more modified nucleotides. In specific embodiments, the modified nucleotides are selected from the group consisting of 2'O-methyl modified nucleotides, deoxynucleotides, 2'-fluorine modified nucleotides, 2'-O-methyl-uridine, 3'-O-methyl modified nucleotides, 3'-O-methyl modified nucleotides of phosphate esters having a 2'-5' link, inverse abase-free nucleotides, nucleotides containing S-ethylene glycol nucleic acid (GNA), unlocking nucleotides, 5'-vinylphosphonate-2'-O-methyl-uridine, cis-cyclobutylphosphonate modified nucleotides, 5'-cis-cyclobutylphosphonate-2'-O-methyl modified nucleotides, (L)-α-threofuranyl modified nucleotides, and combinations thereof. In specific embodiments, the modified nucleotides are selected from the group consisting of 2'O-methyl modified nucleotides, deoxynucleotides, 2'-fluorine modified nucleotides, inverse abase-free nucleotides, nucleotides containing GNA, unlocking nucleotides, vinylphosphonates, and combinations thereof.

[0124] In some embodiments, the siRNA reagent comprises one or more modified nucleoside internucleotides (i.e., modified RNA backbones). Modified nucleoside internucleotides include, for example, thiophosphates (e.g., monothiophosphates). Various salt, mixed salt, and free acid forms are also included. In some embodiments, the siRNA reagent described herein is in free acid form. In other embodiments, the siRNA reagent described herein is in salt form. In one embodiment, the siRNA reagent described herein is in sodium salt form. In some embodiments, when the siRNA reagent described herein is in salt form, the salt cation (e.g., sodium cation) is present in the reagent as a counterion of substantially all electronegative groups present in the electronegative groups (e.g., phosphodiester and / or thiophosphate groups) present in the reagent. In some embodiments, the counterion is a condensed counterion. In a specific embodiment, the counterion is a condensed sodium cation. In some embodiments, the condensed counterion is hydrated. In a specific embodiment, the condensed counterion is a hydrated sodium cation. Reagents in which virtually all phosphodiester and / or thiophosphate bonds have counterions include those with no more than 5, 4, 3, 2, or 1 phosphodiester and / or thiophosphate bonds without counterions. In other words, the electronegativity of the siRNA reagent is neutralized or substantially neutralized by condensation with counterions surrounding the siRNA. In some embodiments, when the siRNA reagent described herein is in sodium salt form, sodium ions are present around the reagent as counterions to virtually all phosphodiester and / or thiophosphate groups present in the reagent.

[0125] The phosphate group of an internucleotide phosphodiester bond can be modified by replacing one of the oxygen atoms with different substituents. One result of this modification is increased resistance of the oligonucleotide to nucleic acid degradation. Another result is improved stability of hybridized single-stranded RNA (ssRNA) in siRNA reagents. Examples of modified phosphate groups include thiophosphates (e.g., monothiophosphates).

[0126] In some embodiments, the siRNA reagent comprises an RNA mimic, wherein the sugar and nucleotide internucleotide bonds (i.e., the backbone) of the nucleotide units are replaced by substitution groups. In specific embodiments, the base units are maintained to hybridize with a suitable target sequence.

[0127] The siRNA reagents described herein may contain one or more asymmetric centers, and thus produce enantiomers, diastereomers, and other stereoisomers, which can be defined according to absolute stereochemistry as (R) or (S), such as for glycoterminal isomers, or as (D) or (L), such as for amino acids. The siRNA reagents provided herein include all such possible isomers, as well as their racemic and optically pure forms.

[0128] The part linked to the nucleotide sequence In some embodiments, the siRNA reagent described herein is conjugated to one or more nonnucleotide groups. The nonnucleotide groups can, for example, enhance the targeting, delivery, or attachment of the siRNA reagent. The nonnucleotide groups can be covalently linked to the 3' end, 5' end, and / or interior of the sense or antisense strand of the siRNA reagent. The nonnucleotide groups can be covalently linked to the 3' end, 5' end, both 3' and 5' ends, interior, both 3' and interior, 5' and interior, or both at the 3' end, 5' end, and interior of the sense and / or antisense strands of the siRNA reagent. In some embodiments, the siRNA reagent described herein contains a nonnucleotide group linked to the 3' end, 5' end, both 3' and 5' ends, interior, both 3' and interior, 5' and interior, or at the 3' end, 5' end, and interior of the sense strand. In some embodiments, the siRNA reagent described herein contains a nonnucleotide group linked to the 5' end of the sense strand. In some embodiments, the siRNA reagent described herein contains a nonnucleotide group linked to the 3' end of the sense strand. In some embodiments, the siRNA reagent described herein contains a non-nucleotide group linked to the inside of the sense strand. The non-nucleotide group can be linked to the siRNA reagent directly or indirectly via a linker / linker group.

[0129] In some embodiments, the siRNA reagent reacts with one or more lipophilic moieties, such as, for example, cholesterol moieties (e.g., Figure 1The CHOL4 shown is linked. In a specific embodiment, the lipophilic portion linked to the siRNA reagent contains... Figure 1 One or more of the following compounds, or their stereoisomers or pharmaceutically acceptable salts thereof, are described: L1 (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, the lipophilic moiety is selected from one or more of the following: L1, L2, L3, L4, and L5 (see [link to specific embodiment]). Figure 1 In specific embodiments, the lipophilic portion comprises CHOL4. In specific embodiments, the lipophilic portion (such as that described in the Examples section) is conjugated to the sense strand of the siRNA reagent in a manner described in the Examples section. In some embodiments, the lipid portion linked to the siRNA reagent comprises... Figure 1 One or more of the following compounds are described: L1, L2, L3, L4, L5 and / or Figure 1 CHOL4 in the middle. In some embodiments, the lipophilic portion is connected to the 3' end, 5' end and / or internally of the sense chain.

[0130] In one embodiment, the sense strand of the siRNA reagent is directly or indirectly conjugated to the lipophilic moiety at the 5' end. In one embodiment, the first nucleotide at the 5' end of the sense strand is linked to the lipophilic moiety. In one embodiment, the sense strand of the siRNA reagent is directly or indirectly conjugated internally to the lipophilic moiety.

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

[0132] In one embodiment, the sense strand is directly or indirectly conjugated to the lipophilic portion at the 5' end, and the sense strand is also directly or indirectly conjugated to the lipophilic portion at the 3' end, each as described herein and elsewhere. The lipophilic portions at the 5' and 3' ends may be the same or different.

[0133] In one embodiment, the sense strand is directly or indirectly conjugated to a lipophilic portion at the 5' end, 3' end, and / or internally. The lipophilic portions at the 5' end, 3' end, and / or internally may be the same or different.

[0134] In some embodiments, the linker group is conjugated to the siRNA reagent. The linker group facilitates the covalent attachment of the reagent to a targeting ligand, delivery polymer, or delivery vector. The linker group may be conjugated to the 3' end, 5' end, and / or internally of the sense strand of the siRNA reagent. In some embodiments, the linker group is conjugated to the sense strand of the siRNA reagent. In some embodiments, the linker group is conjugated to the 5' end, 3' end, and / or internally of the sense strand of the siRNA reagent. In some embodiments, the linker group is conjugated to the 5' end of the sense strand of the siRNA reagent. In some embodiments, the linker group is conjugated to the 3' end of the sense strand of the siRNA reagent. In some embodiments, the linker group is conjugated internally to the sense strand of the siRNA reagent.

[0135] Typically, a linker or connecting group is a connection between two atoms that links a chemical group (such as an siRNA reagent) 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. Unstable connections contain unstable bonds. Connections may optionally include spacers that increase the distance between the two connecting atoms. Spacers can further increase the flexibility and / or length of the connection.

[0136] Any of the siRNA reagent nucleotide sequences (whether modified or unmodified) listed in Tables 1, 2, 3, or 4 may contain a 3' end, a 5' end, and / or an internal targeting ligand and / or linker group. Any of the siRNA reagent duplexes (whether modified or unmodified) listed in Tables 1, 2, 3, or 4 may also contain a targeting ligand and / or linker group, and the targeting ligand or linker group may be attached to the 3' end, 5' end, and / or internal of the sense or antisense strand of the siRNA reagent duplex.

[0137] Synthesis of siRNA reagent siRNA reagents can be synthesized using standard methods known in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, SL et al. (eds.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference or described herein (e.g., in the Examples section below). For example, siRNA reagents can be prepared using a two-step procedure. First, the individual strands of the siRNA reagent are prepared separately. Then, the constituent strands are annealed. The individual strands of the siRNA reagent can be prepared using solution-phase or solid-phase organic synthesis, or both. Organic synthesis offers the advantage of readily preparing oligonucleotide chains containing non-natural or modified nucleotides. Similarly, single-stranded oligonucleotides 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, the oligonucleotide of the siRNA reagent described herein is synthesized such that it contains a reactive group, such as an amine group, at the 5' end, 3' end, and / or internally. Such a reactive group can be used to subsequently link a ligand (e.g., a targeting ligand) using methods typical in the art. In some embodiments, the oligonucleotide of the siRNA reagent is synthesized together with a linker group. This linker group can be used to conjugate a non-nucleotide group (e.g., a lipophilic moiety, ligand, or delivery group) to the siRNA reagent. In some embodiments, the oligonucleotide of the siRNA reagent is synthesized using an automated synthesizer with phosphorous amide (e.g., standard and non-standard phosphorous amides, which are commercially available). In a specific embodiment, the siRNA reagent is produced using the methods described in the Examples section below. In another specific embodiment, the methods described in the Examples section below are used to produce the siRNA reagent described herein, including siRNA reagents conjugated with non-nucleotide groups, such as those described in the section on linking nucleotide sequences above.

[0138] Delivery carrier In some implementations, a delivery vector can be used to deliver the siRNA reagents described herein to cells or tissues.

[0139] Composition In one aspect, this document provides compositions comprising the siRNA reagents described herein (e.g., in the nucleotide sequence section above or the examples section below). The compositions may also comprise a carrier, such as physiological saline, and / or one or more excipients, including but not limited to buffers, surfactants, stabilizers, and preservatives.

[0140] Uses of siRNA reagents The siRNA reagents and compositions of the present invention can be used to inhibit the expression of SNCA genes (e.g., human SNCA genes). As shown in the examples, the siRNA reagents inhibit the expression of SNCA genes. The siRNA reagents and compositions of the present invention can also be used to inhibit or reduce the level of α-synuclein. As shown in the examples, the siRNA reagents inhibit the level of α-synuclein. Certain assays and methods for measuring SNCA expression levels and for measuring α-synuclein levels are described herein. Other assays for measuring SNCA expression levels and for measuring α-synuclein levels are known in the art.

[0141] Inhibiting SNCA gene expression and / or suppressing α-synuclein levels can be used to study neurodegenerative diseases. α-synuclein levels are associated with diseases such as PD, DLB, PAF, and MSA. The siRNA of this invention can be used to investigate how the level of SNCA gene expression inhibition affects α-synuclein levels. The siRNA of this invention can be used in animal models to study how the level of SNCA gene expression inhibition affects α-synuclein levels. The siRNA of this invention can be used to investigate how the level of SNCA gene expression and / or α-synuclein levels affect one or more symptoms of neurodegenerative diseases such as PD, DLB, PAF, and MSA.

[0142] In one aspect, this document provides a method for inhibiting the expression of an SNCA gene (e.g., the human SNCA gene) in cells, comprising contacting the cells with an siRNA reagent as described herein or a composition comprising an siRNA reagent as described herein. In a specific embodiment, this document provides a method for inhibiting the expression of an SNCA gene (e.g., the human SNCA gene) in a cell population, comprising contacting the cell population with an siRNA reagent as described herein. The contact between the cells and the siRNA reagent as described herein can be direct or indirect. For example, the cells can be physically contacted with the siRNA reagent, or the cells can be placed in a situation that allows or leads to their subsequent contact with the siRNA reagent.

[0143] The expression of SNCA genes (e.g., human SNCA genes) can be measured directly or indirectly. For example, the level of SNCA RNA (e.g., pre-mRNA level, mRNA level, or both) can be measured, as can the level of proteins encoded by SNCA genes, the function of proteins encoded by SNCA genes, or combinations thereof. Alternatively or additionally, the expression of genes whose expression is indirectly or directly affected by the expression of SNCA genes, the function of proteins indirectly or directly affected by the expression of SNCA genes, or combinations thereof can be measured. For methods that can be used to directly or indirectly measure the expression of SNCA genes, see, for example, the Bioassays section below.

[0144] Bioassay Bioassays known to those skilled in the art or described herein (e.g., in the Examples section below) can be used to evaluate the ability of the siRNA reagents described herein to inhibit SNCA gene expression, the specificity of the siRNA reagents described herein, the stability of the siRNA reagents described herein, the off-target effects of the siRNA reagents described herein, the toxicity of the siRNA reagents described herein, the localization of the siRNA reagents to specific tissues, the pharmacokinetics of the siRNA reagents, and the immunogenicity of the siRNA reagents described herein.

[0145] In some embodiments, SNCA gene expression is measured at the RNA or protein level. The expression level of the SNCA gene can be assessed using any method known in the art for measuring RNA. In some embodiments, RNA can be isolated from a sample using RNA extraction methods (e.g., organic extraction, membrane-based spinning column, and paramagnetic particle techniques). In some embodiments, SNCA RNA levels can be measured using UV spectroscopy, in situ hybridization, fluorescence in situ hybridization (FISH), RNA blotting, microarrays, RT-PCR, qPCR, fluorescence dye-based quantification, and gel electrophoresis. SNCA protein expression levels can be measured using any method known in the art for characterizing proteins. In some embodiments, SNCA proteins can be purified using salt precipitation, dialysis, and chromatography (e.g., gel filtration, ion exchange, affinity purification). In some embodiments, crude samples containing SNCA proteins or purified SNCA proteins can be characterized using UV absorption and spectroscopy, electrophoresis, capillary electrophoresis, chromatography (e.g., gel filtration, high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), high-diffusion chromatography), and mass spectrometry. In some embodiments, crude samples containing SNCA protein or purified SNCA protein can be characterized by flow cytometry, immunodiffusion, immunoelectrophoresis, Western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, electrochemiluminescence assay, etc. In some embodiments, the expression of the gene can be measured by evaluating the function of the protein encoded by the SNCA gene. For example, the formation of α-synuclein oligomers, protofibrils, and insoluble protofibrils can be assessed. Gene expression in cells exposed to siRNA reagent in vitro or in vitro can be evaluated.

[0146] In some embodiments, assays known to those skilled in the art are used to assess the function of the α-synuclein protein encoded by the SNCA gene. Normal α-synuclein has multiple functions, the most well-established of which is microtubule stabilization.

[0147] In some embodiments, indirect measurements can be used to assess SNCA gene expression levels, including the expression of certain genes, functional assays of downstream proteins, and reporter gene assays. In some embodiments, SNCA gene expression can be assessed by detecting the expression of proteins regulated by the expression of the SNCA gene. In some embodiments, SNCA gene expression can be assessed by detecting the function of proteins regulated by the expression of the SNCA gene. In some embodiments, reporter genes can be introduced to facilitate the expression of indicated targets. For example, a reporter gene regulated by a protein downstream of a protein encoded by an SNCA gene can indicate the expression level of the SNCA gene. Reporter genes may include FLAG tags, green fluorescent protein (GFP), the chloramphenicol acetyltransferase gene (cat) from *E. coli* Tn9, the luciferase gene (luc) from *Photinus pyralis*, and β-glucuronidase (GUC). Easily detectable proteins encoded by the reporter gene indicate the SNCA gene expression level. Reporter gene assays may include fluorescence-based assays, such as fluorescence spectroscopy, fluorescence immunoassay, flow cytometry, fluorescence spectroscopy, and ELISA.

[0148] In some implementations, the specificity of the siRNA reagent can be analyzed by its effect on the repressed gene it targets or the affected off-target genes. This effect includes the inhibition of SNCA gene expression, wherein the siRNA reagent is used at a half-maximal inhibitory concentration (i.e., IC50). 50 This demonstrates its effectiveness. In terms of effectiveness, the use of IC... 50 The candidate siRNA reagent is selected based on the value. In some embodiments, the siRNA reagent described herein has an IC50 value of 6 nM to 5000 nM or 10 nM to 1000 nM in neurons (e.g., human iPSC neurons). 50In some embodiments, the specificity of the siRNA reagent is tested using a negative control, where scrambled or random sequences can be used. siRNA reagents that do not affect the negative target can be characterized as specific. In some embodiments, the specificity of the siRNA reagent is tested using a single target gene. Different siRNA reagents with comparable gene-suppressive efficacy for the same gene should induce similar changes in gene expression profiles or phenotypes. Any changes induced by one siRNA reagent but not others can be attributed to off-target effects or nonspecificity. In some embodiments, the specificity of the siRNA reagent is tested using titration. When the siRNA reagent is used at lower concentrations, nonspecific effects are mitigated. siRNA reagents exhibiting lower effective concentrations may have higher specificity. In some embodiments, the specificity of the siRNA reagent is tested by monitoring both RNA and protein levels. For example, a reduction in RNA genes observed without a corresponding decrease in protein levels indicates slow protein conversion. The siRNA reagent under investigation may have off-target effects. In some embodiments, transcriptomics assays can be used to assess off-target effects.

[0149] In some embodiments, agarose gel electrophoresis-based Förster resonance energy transfer (FRET) methods are used to assess the stability of siRNA reagents described herein in biological samples or fluids, such as, for example, serum or cerebrospinal fluid samples. For a description of such assays, see, for example, Tuttolomondo and Ditzel (2021). In some embodiments, this assay is also used to assess the interactions of the siRNA reagents described herein with serum proteins and enzymes. In some embodiments, agarose gel migration assays are used to assess the stability of the siRNA reagents described herein in biological samples or fluids, such as, for example, serum or cerebrospinal fluid samples.

[0150] In some implementations, the stability of the siRNA reagent can be studied under a variety of conditions, including temperature and RNase degradation in biological fluids. In some implementations, the stability of the siRNA reagent is monitored with increasing temperature (e.g., 1 °C / min) using equimolar concentrations of the two strands. 260 To measure the pyrolysis temperature of the siRNA reagent. m The melting curve (A) of the prehybridized duplex was measured. 260 The maximum value of the first derivative with respect to T). In terms of thermal stability, stable unmodified RNA can have a T value of 40°C to 60°C. mThe modified RNA may be stable or destabilized at approximately 10°C. In some embodiments, the stability of the siRNA reagent against RNase degradation is determined by incubation with different biological fluids. For example, serum stability can be determined by incubation with fetal bovine serum or human serum at 37°C for different time periods up to 48 hours. RNA in the sample can be detected by RNA blotting, in situ hybridization, qPCR, and any RNA detection method known in the art. The activity of the siRNA reagent can be tested. Stable siRNA reagents may exhibit slight degradation within 24 hours at 37°C. In some embodiments, the siRNA reagent exhibits sufficient stability for at least 2 hours. In other embodiments, the stability of the siRNA reagent exposed to, but not limited to, skin, saliva, topical ointments, or nanoparticles can be tested.

[0151] In a specific implementation scheme, as described in the Examples section below, the stability of the siRNA reagent described herein in mouse brain homogenate, human liver lysosomes, and rat liver trisomy was evaluated.

[0152] In some embodiments, the toxicity of the siRNA reagent to in vitro or ex vivo cell-based models was investigated. In some embodiments, toxicity was assessed by the extent of cell damage induced by the siRNA reagent. Damage could be necrosis (uncontrolled cell death), apoptosis (programmed cell death), autophagy, or cessation of active growth and division to reduce cell proliferation. In some embodiments, cytotoxicity assays were performed to measure the ability of the siRNA reagent to induce cell damage or cell death. For example, the release of lactate dehydrogenase (LDH) and glucose-6-phosphate dehydrogenase (G6PD) can be used as biomarkers of cell membrane damage. In another embodiment, cell viability under siRNA reagent treatment can be detected through a variety of mechanisms, such as membrane integrity, enzyme activity, or metabolic activity.

[0153] In some implementations, RNA in situ hybridization assays can be used to image siRNA reagents and assess the suppression of SNCA gene expression in tissue samples from subjects. RNAscope ® It can be used, for example, for RNA in situ hybridization assays.

[0154] In some embodiments, the siRNA reagents described herein are used to study, for example, PD, DLB, PAF, and MSA in animal models (e.g., mouse models). Animal models are known in the art. In specific embodiments, the animal model is the one described in the Examples section below.

[0155] Example Example 1: Bioinformatics and siRNA Synthesis SNCA siRNA bioinformatics computer hit ID A set of siRNAs targeting human SNCA was designed using all nine transcript variants of human synuclein α (SNCA), including GenBank accession numbers NM_000345.4, NM_001146054.2, NM_001146055.2, NM_001375287.1, NM_007308.2, NM_001375285.1, NM_001375288.1, NM_001375286.1, and NM_001375290.1, with Gene ID: 6622.

[0156] Identification of potential screening candidate siRNAs targeting human SNCA mRNA Bioinformatics methods assumed the classic siRNA structure. Positions 2-18 (5'-3') of the sense and antisense strands were used for specificity calculations. Positions 1-19 (5'-3') of the antisense strand were used for cross-reactivity and human single nucleotide polymorphism (SNP) analysis.

[0157] Evaluate the following parameters: Species cross-reactivity in humans, cynomolgus monkeys, rhesus monkeys, mice, and rats: analyses based on classic siRNA designs using 19 and 17 bases (ignoring positions 1 and 19) were used for cross-reactivity. Analysis included both perfect matches and single mismatch analyses.

[0158] Cross-reactivity was analyzed among humans, cynomolgus monkeys, rhesus monkeys, mice, and rats.

[0159] Predictive specificity in humans, rhesus monkeys, cynomolgus monkeys, mice, and rats: separate analysis of sense and antisense strands.

[0160] Potential match between siRNA seed regions and known miRNA seed regions.

[0161] Analysis of the Human SNP Database (NCBI-DB-SNP) was conducted to identify siRNAs targeting regions with known SNPs. Information included the location of the SNP within the target sequence and, where data was available, the minor allele frequency (MAF).

[0162] siRNA activity prediction based on classical siRNA design.

[0163] The following describes a bioinformatics procedure.

[0164] The possible siRNA was generated from the human SNCA mRNA sequence (NM_000345.4).

[0165] Predicting siRNA off-target hits in humans, rhesus monkeys, cynomolgus monkeys, mice, and rats.

[0166] Specificity scores are assigned to each siRNA strand.

[0167] The presence of miRNA seed regions in human, rhesus monkey, dog, pig, mouse, rat, and rabbit siRNA chains was analyzed.

[0168] Specificity categories were assigned to siRNAs (combined specificity score + miRNA seed analysis).

[0169] Calculate siRNA cross-reactivity: for transcript variants and different species, for 19mer and 17mer (nucleotides 2-18 of 19mer) and for 19mer and 17mer with a single mismatch.

[0170] Human SNPs were mapped to siRNA target sites in the SNCA transcript GenBank accession number NM_000345.4 and all nine transcript variants.

[0171] Predict siRNA activity and assign a score to each siRNA.

[0172] Based on the above procedure, 340 hit human SNCA sequences were selected from 4028 target sequences. Table 1 provides the unmodified sense and antisense nucleotide sequences of siRNAs US1 to US340 targeting human SNCA (gene ID: 6622). Table 2 provides the sense and antisense nucleotide sequences of siRNAs S1 to S340, which were generated by chemical modifications applied to siRNAs US1 to US340. These chemical modifications include 2'-fluorine (2'-F), 2'-O-methyl (2'-OMe), and phosphate thioesters.

[0173] Synthesis of SNCA siRNA duplexes 340 human SNCA siRNAs were synthesized and annealed as described below. In short, single-stranded sense and antisense oligonucleotides were synthesized at a scale of 0.2 μmol using standard solid-phase phosphoramide chemistry on a Dr. Oligo 192 synthesizer (Biolytic Lab Performance). Controlled-aperture glass (CPG, 500 Å) was used as the loading medium for the first base or UnyLinker. ™ Solid support was used. Detriphenylmethylation was performed using a 3% dichloroacetic acid solution in dichloromethane, followed by coupling of 2'-OMe and 2'-F modified nucleotides with the corresponding phosphorous amides. 5-Ethylthio-IH-tetrazole (ETT) was used as an activator (0.25 M acetonitrile solution), and the coupling time for all phosphorous amides was 6–8 minutes. Thiophosphate bonds were generated using a 0.2 M solution of hydrogenated xanthane in anhydrous pyridine. Oxidation was performed for 3 minutes using a 0.02 M tetrahydrofuran solution containing 10% water.

[0174] After synthesis, the solid support was transferred to 1.5 mL vials. Lysis and deprotection were performed using 500 µL AMA (concentrated ammonia / 40% methylamine aqueous solution, v / v = 1:1). After lysis and deprotection, the sample was filtered 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 purification system equipped with an autosampler and fraction collector. The fractions were analyzed by LC-MS to confirm quality. Appropriate fractions were combined and desalted using a CentriPure P10 column (emp Biotech). Samples for each sequence were analyzed by LC-MS to confirm identity, quantified by UV (260 nm), and purity determined by IP-RP chromatography. Distranded annealing was performed by mixing equimolar amounts of sense and antisense single strands. The final distranded sample solution was dried under vacuum using a GeneVac evaporator (SP Scientific). The final quality control (QC) specifications are ±0.05% of the calculated mass for single-strand identity (by LC / MS), >85% full-length oligonucleotides for single-strand purity (by HPLC), and >90% for double-strand purity (by non-denaturing HPLC).

[0175] siRNA-lipid conjugation A subset of siRNA compounds was synthesized using different novel lipid conjugates at various positions (5' / 3' end or interior). Lipids were incorporated into the sense chain via on-column and / or post-column synthesis. For on-column synthesis, the lipophilic moiety was modified at the corresponding phosphoramidite monomer or CPG, followed by a solid-phase phosphoramidite chemistry reaction as described above to synthesize a sense chain with lipid conjugates at the 5' / 3' end and / or interior. For post-column synthesis, lipids were introduced into the solution phase along with their corresponding N-hydroxysuccinimide esters and additional amine linkers to the nucleotides.

[0176] Conjugates of siRNA with CHOL4 are shown in Table 3 (and Table 21). Conjugates of siRNA with other lipophilic moieties are shown in Table 4.

[0177] Column synthesis For example, as shown in Scheme 1 below, compound 2 is introduced onto the column at the 3' end of the sense chain using a 3'-amino modifier (such as 2-dimethoxytriphenylmethyloxymethyl-6-fluorenylmethoxycarbonylamino-hexane-1-succinyl)-long-chain alkylamino-CPG 1). After deprotecting the Fmoc group with a solution of 20% piperidine in N,N-dimethylformamide (DMF), the CPG is thoroughly washed with DMF, methyl cyanide (MeCN or acetonitrile (ACN)), and diethyl ether and dried. A solution of 80 µmol of compound 2 in 2 mL of 1,4-dioxane is added to the above CPG, followed by the addition of 30 µl of diisopropylethylamine (DIPEA) and shaking for 12 hours to generate compound 3. The CPG is washed with methylene chloride (DCM), MeCN, and Et2O. A solution of CAP A and CAP B is added and the mixture is shaken for 30 minutes. Compound 3 was washed with DCM, MeCN, and Et2O, dried, and used for oligonucleotide synthesis. 3'-cholesterol-TEG CPG was used to synthesize the oligonucleotide conjugated to cholesterol at the 3' end.

[0178] Option 1 Compound 2 was introduced at the 5' end of the sense chain using a 5'-amino modifier (such as 6-(4-monomethoxytriphenylmethylamino)hexyl-(2-cyanoethyl)-(N,N'-diisopropyl)-phosphoramide). After synthesizing the sense chain on CPG according to standard procedure, in the penultimate step, the 5' end of the sense chain was coupled to 6-(4-monomethoxytriphenylmethylamino)hexyl-(2-cyanoethyl)-(N,N'-diisopropyl)-phosphoramide, followed by deblocking of the 4-monomethoxytriphenylmethyl group with 3% dichloroacetate (DCA) / DCM for 30 min. Subsequently, the CPG was washed with DCM and ACN and coupled with compound 2 for 12 h (Scheme 2), followed by chain cleavage and deprotection.

[0179] Option 2 During sense chain synthesis, lipids are introduced into internal positions along with the corresponding phosphoramidite monomer. For example, scheme 3 illustrates this using uridine-based phosphoramidite monomer 7.

[0180] Option 3 Following synthesis, lysis, and deprotection, lipid-conjugated oligonucleotides were purified by reversed-phase or ion-exchange chromatography. The buffers used for reversed-phase chromatography were 0.05 M sodium acetate (Buffer A) and acetonitrile (Buffer B) in 90% / 10% water / acetonitrile, while the buffers used for ion exchange were 20 mM sodium phosphate (Buffer A) in 90% / 10% water / acetonitrile and 20 mM sodium phosphate and 1.8 M sodium bromide (Buffer B). Fractions containing full-length oligonucleotides were combined, desalted, and analyzed by liquid chromatography-mass spectrometry (LC-MS).

[0181] Post-column synthesis After purification and desalting of the corresponding oligonucleotides, lipids were conjugated at the 5' / 3' end and internal positions of the sense chain in solution.

[0182] For example, incorporation at the internal position is carried out using a phosphoramidite monomer derived from an amine linker at the 2' position of the corresponding nucleotide. Scheme 4 illustrates this using uridine-based phosphoramidite monomer compound 9. After incorporating compound 9 at the internal position and completing synthesis, cleavage and deprotection, purification and desalting processes, the free oligonucleotide with a side-chain amine is coupled to the corresponding lipid-NHS ester in solution.

[0183] Option 4 In 60o At C, terminal / internal conjugation was performed using a 0.15 mM deprotected and desalted oligonucleotide solution in 0.1 M NaHCO3 solution (pH 8.4) and a corresponding 1.5 mM lipid-NHS ester solution in DMF. 0.6 mL of the lipid-NHS ester DMF solution was added to the oligonucleotide solution (0.25 mL), and the resulting mixture was 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, 30 °C). The reaction typically completed >90% in less than one hour. The reaction mixture was diluted with water and purified by reversed-phase (RP)-HPLC (C-8 Xbridge Waters column; A: 50 mM NaOAc, B: MeCN; 5%–100% B gradient, 60 °C). The separation yield of the lipid-conjugated oligonucleotides was 60%–70%. After purification, the product was desalted and double annealed, as described above.

[0184] Example 2: In vitro screening of SNCA siRNA The screening of SNCA siRNA reagents S1 to S340 listed in Table 2 was performed in transfected human neuroblastoma cells according to the protocol described below. Table 5 shows the remaining SNCA mRNA in human neuroblastoma cells after transfection with 2 pM siRNA. A set of 25 siRNAs was selected for potency evaluation in transfected human neuroblastoma cells; Table 6 summarizes the IC50 values ​​of the siRNAs shown. 50 The value and its 95% confidence interval.

[0185] method Cell culture and transfection Human neuroblastoma Kelly cells (DSMZ) were cultured in RPMI 1640 (Sigma R0883) supplemented with 10% fetal bovine serum (FBS) (Biowest S1810-500), 2 mM L-glutamine (Sigma G7513), and 50 μg / mL gentamicin (Gibco 15750) in a 37°C incubator containing 5% CO2. One day prior to transfection, cells were divided using 0.05% trypsin-EDTA (Gibco 25300) and seeded at a density of 30,000 cells per well in 96-well plates. The following day, cells were transfected with siRNA using the Lipofectamine RNAiMAX transfection reagent (ThermoFisher Scientific 13778150) according to the manufacturer's protocol. Cells were cultured at 37°C in 5% CO2 for 72 hours prior to RNA isolation.

[0186] Total RNA isolation : RNA extraction was performed using the RNeasy 96 kit (Qiagen) according to the manufacturer's protocol. Briefly, 125 µL of RLT buffer was added to each well of a 96-well plate, and cells were lysed by orbital oscillation at 200 rpm for 1 minute. The cell plate was then transferred to a -80°C freezer for storage until analysis. On the day of RNA extraction, the frozen cell plate was rapidly thawed in a 37°C oven, and immediately after thawing, an equal volume of 125 µL of 70% (v / v) ethanol was added to each well to mix with the RLT lysate. The mixture was then transferred to the wells of an RNeasy 96 plate placed on top of a QIAvac 96 vacuum block, and RNA was bound to the RNeasy 96 plate membrane by applying vacuum until liquid transfer was complete. The RNeasy 96 plate underwent a series of washes, including one wash with 800 µL RW1 buffer and two washes with 800 µL RPE buffer, using vacuum to remove liquid. After the final wash, the RNeasy 96 plate was centrifuged at 5600 × g for 3 minutes to remove residual lipids. RNA was eluted using 60 µL of RNase-free water at 5600 × g for 3 minutes at room temperature. RNA concentration was measured using a Nanodrop 8000 (ThermoFisher). The eluted RNA was stored at -80 °C until analysis.

[0187] Reverse transcription and real-time PCR : RNA was used as a template for reverse transcription, and reverse transcription was performed using a high-capacity cDNA reverse transcription kit (AppliedBiosystems) according to the manufacturer's instructions. In short, the final reaction of the 20 µL mixture was incubated at 25 °C for 10 min, followed by reverse transcription at 37 °C for 2 h, and enzyme inactivation at 85 °C for 5 min. For qPCR, the reverse-transcribed cDNA was diluted 10-fold and reacted with 2X PowerUp. ™ SYBR ™ Green Mater Mix (ThermoFisher A25743) and 500 nM qPCR primers were mixed to make a final reaction volume of 10 µL. The reaction was then performed using QuantStudio. ™ 12K Instruments (Applied Biosystems) ™ qPCR was performed using a standard thermal cycling protocol. Multiple validated primers were used to detect SNCA mRNA, and reference primers targeting the housekeeping genes ENOX2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used to normalize gene expression. Primer sequences are listed in Table 7. qPCR data were analyzed using qBase+ software (Biogazelle). IC50 was calculated using GraphPad Prism version 7.00 software by fitting a normalized response curve to log([drug]). 50 Value and IC 50 95% CI.

[0188] Example 3: In vitro knockdown efficiency of siRNA conjugates in neurons The in vitro knockdown efficiency of lipid-siRNA conjugates was evaluated in human iPSC-derived cortical neurons according to the following procedure.

[0189] Figures 2 to 4 Tables 8 through 10 show that the cholesterol-siRNA conjugates S356, S358, S362, S363, and S364 listed in Table 3 dose-dependently knocked down SNCA mRNA and α-synuclein protein in human iPSC neurons after 7 or 14 days of incubation.

[0190] The efficiency of the cholesterol-siRNA conjugates shown in Table 3 in knocking down SNCA mRNA in human iPSC neurons after 7 days of incubation was evaluated. Three concentrations of siRNA conjugates (200 nM, 40 nM, and 8 nM) were tested. The results are summarized in Table 11.

[0191] Furthermore, the effects of the lipid-siRNA conjugates S464, S465, S462, S467, S468, and S466 listed in Table 4 on the expression of SNCA mRNA and α-synuclein in human iPSC neurons after incubation for 7 or 14 days were investigated. Figure 5 and Figure 6 As shown in Tables 12 and 13, these lipid-siRNA conjugates dose-dependently knocked down SNCA mRNA and α-synuclein protein expression.

[0192] method Differentiation of human iPSCs into cortical neurons The human iPSC line (Sigma #iPSC0028) was obtained by reprogramming epithelial cells from a 24-year-old Caucasian female donor via OSKM retrovirus. The iPSCs were first differentiated into cortical neural stem cells (NSCs) using a modified dual SMAD inhibitor protocol (Shi et al., 2012). Briefly, the iPSCs were cultured at a density of 500,000 cells / cm². 2Inoculate onto wells coated with Matrigel (Corning 354230) in mTeSR medium (StemCell Technologies 5850) supplemented with 10 µM rho-associated protein kinase (ROCK) inhibitor (Sigma-Aldrich Y0503) and incubate at 37°C and 5% O2. Replace the medium with mTeSR on day -1. From day 0 to day 12, the cell culture medium was changed daily with cortical nerve induction medium, which consisted of 10 μM SB43142 (Tocris 1614) and 1 μM Morsomorphin (Tocris 3093), supplemented with nerve maintenance medium (1:1 DMEM:F12 Glutamax (ThermoFisher Scientific 10565108), Neurobasal (ThermoFisher Scientific 21103049), 2.5 μg / mL insulin (Sigma-Aldrich I9278-5ML), 50 μM 2-mercaptoethanol (ThermoFisher Scientific, 31350010), 0.5% non-essential amino acids (ThermoFisher Scientific 11140035), 0.5% glutamax supplement (ThermoFisher Scientific, 10565018), and 0.5 mM sodium pyruvate (ThermoFisher Scientific). The supplements included 1% penicillin-streptomycin (Sigma P4333), 0.5% N2 supplement (ThermoFisher Scientific 17502048), and 1% B27 supplement (ThermoFisher Scientific 17504044). On day 12, the neuroepithelial flaps were gently separated into large clumps of 300 to 500 cells using a needle and lifter, and collected in 15 mL of falcon by centrifugation at 160 g for 2 minutes. The cell pellet was gently resuspended in neural induction medium and passaged 1 / 2 or 1 / 3 of the culture medium into wells coated with 10 µg / mL laminin (Sigma-Aldrich L2020), with a total volume of 2 mL of neural induction medium per well in a 6-well plate. On days 13 and 15, the medium was replaced with neural maintenance medium supplemented with 20 ng / mL FGF2 (Stemcell Technologies 2634).On day 17, neural flower knots were isolated using a dispersant enzyme (ThermoFisher Scientific 17105041), passaged 1 / 3, and seeded into laminin-coated wells. An additional dispersant step (1 or 2) was performed for one week. Around days 25–30, neural stem cells were dissociated into single-cell suspensions using Accutase (ThermoFisher Scientific A1110501) and cryopreserved in freshly prepared neural cryopreservation medium supplemented with 10% (v / v) DMSO and 20 ng / mL FGF2. The cryopreserved vials of neural stem cells were stored in liquid nitrogen until use.

[0193] To generate iPSC neurons, frozen vials of neural stem cells (NSCs) were thawed and cultured at a density of 70,000 cells / cm². 2 Cells were seeded in laminin-coated wells containing nerve maintenance medium supplemented with 10 µM ROCK inhibitor and 20 ng / mL fibroblast growth factor-2 (FGF2). The medium was replaced daily for the next two days. On approximately day 4 post-thaw, cells were eluted with Accutase and seeded at 28,000 cells / well in 96-well plates pre-coated with poly-L-ornithine and laminin in nerve maintenance medium supplemented with 10 µM ROCK inhibitor. The day after re-platening, the culture medium was replaced with neural differentiation medium, which consisted of a maintenance medium supplemented with 20 ng / mL brain-derived neurotrophic factor (BDNF) (R&D Systems 212-BD-050 / CF), 20 ng / mL glial cell line-derived neurotrophic factor (GDNF) (R&D Systems 212-GD-050 / CF), 500 µM DB-cAMP (Sigma D0627), and 20 mM ascorbic acid (Sigma A4403). The cultures differentiated in the neural differentiation medium, with 50% of the medium replaced twice weekly. Two to three weeks after differentiation from neural stem cells (NSCs), neurons were treated with siRNA for 7 or 14 days. RNA analysis was performed by reverse transcription and real-time PCR, and protein analysis was performed by MSD immunoassay.

[0194] MSD Immunoassay Human iPSC neurons differentiated on a 96-well plate were supplemented with cOmplete ™ Protease inhibitor mixture (Roche) and PhosSTOP ™Lyse the cells in 100 µL of ice-cold RIPA buffer (Sigma) at Roche and gently oscillate at 4 °C for 30 min. Centrifuge the plate at 1,000 × g for 5 min, collect the cell lysates, dilute 5-fold, and then perform protein measurements using the MSD immunoassay according to standard procedures. In short, coat each well of the MSD plate with 30 µL of coating antibody diluted 2 µg / mL in phosphate-buffered saline (PBS) and incubate overnight at 4 °C. The next day, air dry the plates upside down on absorbent paper and then incubate at room temperature with 150 µL of 0.1% casein blocking buffer per well for 2 h while oscillating at 300 rpm. After blocking, incubate the plates with a solution of 0.1% casein buffer supplemented with 500 μL of RIPA buffer. ™ Cell lysates diluted in RIPA buffer with a protease inhibitor mixture (Roche) were incubated overnight at 4°C with slow orbital oscillation. The plates were washed 5x times using a Titertek Aquamax 4000 and air-dried upside down on absorbent paper. Then, 25 µL / well of secondary antibody or detection antibody diluted in 0.1% casein was added to the MSD plate and incubated at room temperature for 2 hours. After antibody incubation, the plates were washed 5x times and developed with 150 µL / well 2x MSD read buffer (Meso Scale Discovery) diluted in milliQ H2O. The plates were read immediately using an MSD instrument. For α-synuclein measurement, 4B12 (Biolegend 807801) was used for coating 96-well MSD plates, anti-syn MJFR1 (Abcam 138501) was used for primary detection, and SULFP-TAG-labeled anti-rabbit antibody (Meso Scale Discovery) was used for assay. For the 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 the primary detection antibody, and SULFP-TAG-labeled anti-mouse antibody (Meso Scale Discovery) was used as the secondary detection antibody.

[0195] Example 4: In vitro stability assessment of siRNA conjugates in various matrices The in vitro stability of siRNA conjugates in various matrices, including mouse brain homogenate, human liver lysosomes, and rat liver trimocytosomes, was evaluated using the methods described below.

[0196] Tables 14 to 17 summarize the LC-MS measurements of the antisense and sense strand stability of the siRNA conjugates in Tables 3 and 4 of mouse brain homogenates (Tables 14 and 15), human liver lysosomes (Table 16), and rat liver trimes (Table 17) after incubation at 37°C and 450 rpm for 24 hours. The reference compound MSC-2-V was added to the assays to benchmark the stringency of the assay conditions.

[0197] method Stability of siRNA conjugates in mouse brain homogenate Frozen brain tissue collected from C57BL / 6J wild-type mice was homogenized at a concentration of 200 mg / mL in ice-cold homogenization buffer containing 100 mM Tris-HCl, pH 6.0, supplemented with 1 mM MgCl2. The homogenization was performed by adding 1 mL of ice-cold homogenization buffer to a 2 mL tube containing lysed matrix D and one mouse brain hemisphere (MP Biomedicals). ™ In ), then in FastPrep ® The tube was homogenized for 30 seconds at a speed of 5 m / s in a -24 instrument. The sample was then placed on ice for 2 minutes and homogenized using FastPrep. ® The reaction was repeated twice using the -24 instrument. 100 µL of homogenate was added to each well of a 96-well PCR plate, along with 5 µL of 20 µM siRNA conjugate (final concentration in the reaction was 1 µM), leaving an equal number of blank wells containing brain homogenate without siRNA conjugate. The PCR plate containing the samples was placed in a Thermomixer and incubated at 37 °C for 24 hours with shaking at 450 rpm. After this reaction, the PCR plate was placed on ice until the samples cooled, and 5 µL of 20 µM siRNA conjugate was added to the blank wells containing brain homogenate (0-hour sample). The samples were diluted in 300 µL of 10 mM EDTA (4-fold dilution) containing 1 µg / mL internal standard oligonucleotide, and then the diluted samples were transferred to deep-phase 96-well plates. After thorough mixing, 50 µL of the sample was transferred to another deep-phase 96-well plate, and the solid-phase extraction procedure was started.

[0198] Stability of siRNA conjugates in human liver lysosomes and rat liver trisomy Human liver lysosomes (Xenotech H0610.L) and rat liver trisomy were thawed and diluted to 0.05 units / mL or 0.5 units / mL acid phosphatase levels, respectively, in 20 mM sodium citrate pH 5.0 buffer. 50 µL of the diluted human liver lysosomes and rat liver trisomy were added aliquoted into each well of a separate 96-format PCR plate, followed by 5 µL of 10 µM siRNA conjugate (final concentration in the reaction was 1 µM) to each well. An equal number of blank wells containing lysosomes / trisomy were left without siRNA conjugate. The PCR plates containing the samples were placed in a Thermomixer and incubated at 37 °C for 24 hours with shaking at 450 rpm. After this reaction, the PCR plates were placed on ice until the samples cooled, and 5 µL of 10 µM siRNA conjugate was added to the blank wells containing lysosomes / trisomy (0-hour samples). The sample was diluted in 300 µL of 10 mM EDTA (4-fold dilution) containing 1 µg / mL internal standard oligonucleotide, and the diluted sample was transferred to a deep-well 96-well plate. After thorough mixing, 50 µL of the sample was transferred to another deep-well 96-well plate, and then the solid-phase extraction procedure was started.

[0199] Sample preparation before solid phase extraction After stability testing, 50 µL of sample was digested by adding 10 µL of proteinase K solution (A4392,0010; PanReac Applichem) and 100 µL of Tris-EDTA buffer (10 mM Tris, 1 mM EDTA) at pH 8.0 (VWR; E112-500 ml), and incubated at 60 °C at 1100 rpm for 30 min. After incubation, the sample was diluted by adding 100 µL of lysis buffer (Phenomenex, CatAL0-8579) and 200 µL of equilibration buffer (500 mL of 3.45 g Na2HPO4 aqueous solution, adjusted to pH 5.5 with 1 N NaOH).

[0200] solid phase extraction Then, a Clarity OTX solid-phase extraction plate (Phenomenex, Cat. 8E-S103-EGA) was used. The plate was conditioned with 1 mL of methanol, followed by 1 mL of equilibration buffer (500 mL of 3.45 g Na₂HPO₄ aqueous solution, adjusted to pH 5.5 with 1 N NaOH). The sample was then loaded onto the column. The column was washed three times with 1 mL of wash buffer (equilibration buffer pH 5.5, 50% v / v acetonitrile), then once with 0.5 mL of water, and then once with 100 mM ammonium bicarbonate pH 10 supplemented with 0.56 g / L TCEP (tris(2-carboxyethyl)phosphine). The sample was eluted three times with 0.5 mL of elution buffer (200 mM ammonium bicarbonate pH 10, supplemented with TCEP (tris(2-carboxyethyl)phosphine), 50% v / v acetonitrile) and dried using a nitrogen stream (TurboVap, 65 psi N₂, 70 °C).

[0201] Analytical methods Following solid-phase extraction, the sample was reconstructed in a dissolution buffer (300 µL 100 mM ammonium bicarbonate adjusted to pH 10, 10% v / v acetonitrile) and analyzed using AB Sciex TripleTOF 6600 with liquid chromatography-mass spectrometry. Samples (20 µL) were injected and the sample was stored at 75°C using DNAPac. ™ Separation was performed using a 50 mm × 2.1 mm RP column with a 4 μm depth. Mobile phase A consisted of 0.2% dimethylbutylamine, 0.5% hexafluoroisopropanol, and 0.5% methanol, while mobile phase B was acetonitrile / isopropanol (95 / 5; v / v). Solvent gradients were as follows: 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, full scan, with a spray voltage of 4500 V and a source temperature of 350 °C. The flow rate was 0.25 mL / min. Data acquisition was performed using Analyst TF 1.8.1 software, and data processing was performed using Sciex OS 1.7.0 software.

[0202] Example 5: Evaluation of in vivo knockdown efficiency of siRNA conjugates in a mouse model The in vivo knockdown efficiency of certain lipid-siRNA conjugates in Tables 3 and 4 was evaluated in a mouse model by intraventricular (ICV) injection following the procedure described below.

[0203] Tables 18 and 19 summarize the RT-qPCR analysis of SNCA mRNA knockdown in seven brain regions (cortex, hippocampus, brainstem, cerebellum, striatum, midbrain, and cervical spinal cord) of WT mice 7 days after a single ICV injection of 15 nmol of the indicated siRNA conjugate.

[0204] Table 20 summarizes the 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 7 days after a single ICV injection of S468 at the indicated dose level.

[0205] method Intraventricular injection in mice Wild-type C57BL6 mice or hSNCA-BAC mice aged 2 to 3 months were randomly assigned to different treatment groups. Mice were anesthetized with isoflurane (induction: 4%–5%; maintenance: 1.8%–2.5%). They were then stereotactically injected into the bilateral ventricles using a power drill and microinjection robot (Neurostar, Germany, Sterodrive Software v 2019) at coordinates: AP: -0.62 mm, ML: + / -1.05 mm, and DV: 2.2 mm. Each injection volume was 5 µL, administered over 5 minutes, and the needle was withdrawn in three steps after injection (1. withdraw 1 mm within 60 seconds and hold for 5 minutes; 2. withdraw another 0.5 mm within 30 seconds and wait for 5 minutes; 3. withdraw from the brain very slowly) to prevent backflow of the compound along the needle tract. The amount of backflow was checked after each step. Several days after the selected injection, the animals were euthanized and dissected, including different brain regions such as the cortex, hippocampus, brainstem, cerebellum, striatum, midbrain, and cervical spinal cord, and then rapidly frozen and stored at -80°C until further analysis.

[0206] RNA extraction from tissues Tissues were collected in 2 mL tubes of Lysing Matrix D (MP Biomedicals 6913-500) containing 1.4 mm ceramic beads and stored at -80°C until analysis. The tissues were placed on ice under laminar flow and immediately 750 µL of Trizol (ThermoFisher 15596026 / 15596018) was added to each tube. FastPrep-24 was used. ™The tissue was homogenized and broken down using a 5G grinder at 5 m / s for 3 cycles of 30 seconds each. Between cycles, the sample was cooled on ice for 2 minutes. After homogenization, the tube was briefly swirld and 20% volume of chloroform was added (150 µL chloroform when using 750 µL Trizol). The tube was vortexed for 15 seconds and centrifuged at 14,000 × g for 15 minutes 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 thoroughly.

[0207] The following steps were performed using the RNeasy 96 kit (Qiagen) according to the manufacturer's protocol. The RNeasy 96 plate was placed on top of a Square-Well Block holder. Samples (Trizol / chloroform extraction) were applied to the wells of the RNeasy 96 plate, and the plate was sealed with an AirPore cap to prevent contamination. The plate was centrifuged at 5600×g for 3 minutes at room temperature and the solution was discarded. A series of washes, including one 800µL RW1 buffer wash and two 800µL RPE buffer washes, were applied to the RNeasy 96 plate. The wash buffer was then removed by centrifugation at 5600×g for 3 minutes at each step. After the final wash, the RNeasy 96 plate was centrifuged at 5600×g for 3 minutes to remove residual lipids. RNA was eluted using 60µL of RNase-free water by centrifugation at 5600×g for 3 minutes at room temperature. RNA concentration was measured using a Nanodrop 8000 (ThermoFisher). The eluted RNA was stored at -80°C until analysis.

[0208] RT-qPCR RNA was used as a template for reverse transcription, and reverse transcription was performed using a high-capacity cDNA reverse transcription kit (AppliedBiosystems) according to the manufacturer's instructions. In short, the final reaction of the 20 µL mixture was incubated at 25 °C for 10 min, followed by reverse transcription at 37 °C for 2 h, and enzyme inactivation at 85 °C for 5 min. For qPCR, the reverse-transcribed cDNA was diluted 10-fold and reacted with 2X PowerUp. ™ SYBR ™ Mix Green Mater Mix (ThermoFisher A25743) and 500 nM qPCR primers to a final reaction volume of 10 µL. Alternatively, dilute the reverse-transcribed cDNA 10-fold and mix with the PrimeTime qPCR probe assay (IDT Integrated DNA Technologies) according to the manufacturer's instructions. Use QuantStudio ™ 12K Instruments (Applied Biosystems)™ qPCR was performed using a 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 to normalize gene expression. Primer sequences and PrimeTime qPCR probe assays are listed in Table 5. qPCR data were analyzed using qBase+ software (Biogazelle).

[0209] Table 1. Unmodified sense and antisense nucleotide sequences (5' to 3') of unmodified SNCA siRNA .

[0210] Table 2. Modified sense and antisense nucleotide sequences (5' to 3') of modified SNCA siRNA .

[0211] (Modifications, where “X” represents a nucleotide: mX = 2'-OMe; fX = 2'-F; ps = phosphate thioester) Table 3. Sense and antisense nucleosides modified with cholesterol-conjugated SNCA siRNAs of various chemical variants. Acid sequence (5' to 3') .

[0212] (Modifications where “X” represents nucleotides: mX = 2'-OMe; fX = 2'-F; ps = phosphate thioester; VP = vinylphosphonate; dX = deoxynucleotide; UNA-X = unlocked nucleic acid; GNA-X = diol nucleic acid; invAb = reverse baseless; Chol4 = cholesterol) Table 4. Sense and antisense nuclei of SNCA siRNAs with chemical variants conjugated to lipids. nucleotide sequence (5' to 3') .

[0213] (Modifications where “X” represents a nucleotide: mX=2'-OMe; fX=2'-F; ps=phosphate thioester; VP=vinylphosphonate; invAb=reverse baseless; J2-CONC16X=L1; J2-C3NC16X=L2; J2-NCOOC16X=L3; J2-C2OC16X=L4; J2-C15AdaX=L5) Table 5. Single-dose screening of modified SNCA siRNA in transfected human neuroblastoma Kelly cells. Selected as described in Example 2. Data are expressed as a percentage of SNCA mRNA levels relative to the solvent group control. .

[0214] Table 6. Effects of selected modified SNCA siRNAs on transfected human neuroblastoma Kelly cells. IC 50 And 95% confidence intervals, as described in Example 2. Different groups of RT-qPCR primers SNCA_B08, SNCA_B03, and... SNCA_B05 estimates IC 50 value 。

[0215] Table 7. Sequences of qPCR primers and a catalog of PrimeTime qPCR assays (IDT), as described in Example 2. 。

[0216] Table 8. The selected cholesterol-siRNA conjugates, as described in Example 3 (n=3), showed efficacy in humans treated for 7 days. SNCA mRNA knockdown efficiency in iPSC-derived cortical neurons. Data are expressed as IC50. 50 And the 95% confidence interval, its use GraphPad Prism version 7.00 software calculates the drug's response by fitting the normalized response curve to log([drug]). 。

[0217] Table 9. The selected cholesterol-siRNA conjugates, as described in Example 3 (n=3), showed efficacy in humans treated for 14 days. SNCA mRNA knockdown efficiency in iPSC-derived cortical neurons. Data are expressed as IC50. 50 And the 95% confidence interval, its use GraphPad Prism version 7.00 software calculates the drug's response by fitting the normalized response curve to log([drug]). 。

[0218] Table 10. The selected cholesterol-siRNA conjugates, as described in Example 3 (n=3), after 14 days of treatment Knockdown efficiency of α-synuclein protein in human iPSC-derived cortical neurons. Data are expressed as IC50. 50 and 95% confidence zone In this process, GraphPad Prism version 7.00 software was used to calculate the response by fitting the normalized response curve with log([drug]). 。

[0219] Table 11. As described in Example 3 (n=3), the selected cholesterol-siRNA conjugates were used at 200 nM. The knockdown efficiency of SNCA mRNA in human iPSC-derived cortical neurons treated with 40 nM and 8 nM concentrations for 14 days. Data indicate Remaining SNCA mRNA relative to the control 。

[0220] Table 12. As described in Example 3 (n=3), the selected lipid-siRNA conjugates in humans treated for 7 days SNCA mRNA knockdown efficiency in iPSC-derived cortical neurons. Data are expressed as IC50. 50 And the 95% confidence interval, its use GraphPad Prism version 7.00 software calculates the drug's response by fitting the normalized response curve to log([drug]). 。

[0221] Table 13. As described in Example 3 (n=3), the selected lipid-siRNA conjugates in humans treated for 14 days Knockdown efficiency of α-synuclein protein in iPSC-derived cortical neurons. Data are expressed as IC50. 50 and 95% confidence zone In this process, GraphPad Prism version 7.00 software was used to calculate the response by fitting the normalized response curve with log([drug]). 。

[0222] Table 14. Cholesterol-siRNA conjugates selected from mouse brain homogenates as described in Example 4 (n=3) LC-MS measurements of the stability of the sense and antisense strands. Data are expressed as %stability. 。

[0223] Table 15. Lipid-siRNA conjugates selected from mouse brain homogenates as described in Example 4 (n=6) LC-MS measurements of the stability of the sense and antisense strands. Data are expressed as %stability. 。

[0224] Table 16. Lipid-siRNA conjugates selected in human liver lysosomes as described in Example 4 (n=6) LC-MS measurements of the stability of the sense and antisense strands. Data are expressed as %stability. 。

[0225] Table 17. Lipid-siRNA conjugates selected in rat liver trisomy, as described in Example 4 (n=6). LC-MS measurements of the stability of the sense and antisense strands. Data are expressed as %stability. .

[0226] Table 18. ICV injection at a dose of 15 nmol of conjugate, as described in Example 5 (n=6 / treatment group). Seven days after injection, the selected cholesterol-siRNA conjugates and lipid-siRNA conjugates showed various effects in wild-type mice. SNCA mRNA knockdown efficiency in tissues. Data are expressed as the percentage of remaining SNCA mRNA. .

[0227] Table 19. ICV injection at a dose of 15 nmol of conjugate, as described in Example 5 (n=6 / treatment group). Seven days after injection, the selected lipid-siRNA conjugate knocked down SNCA mRNA in various tissues of wild-type mice. Efficiency. Data are expressed as the percentage of remaining SNCA mRNA. .

[0228] Table 20. As described in Example 5 (n=5 / treatment group), with 60 nmol, 30 nmol, 15 nmol, Seven days after ICV injection at doses of 7.5 nmol, 3.8 nmol, and 1.9 nmol, the lipid-siRNA conjugate S468 in wild-type cells... The knockdown efficiency of SNCA mRNA in various tissues of mice. Data are expressed as the percentage of remaining MAPT mRNA. .

[0229] Table 21. Sense and antisense nucleosides of SNCA siRNAs with chemical variants conjugated to cholesterol. Acid sequence (5' to 3') .

[0230] (Modifications where “X” represents nucleotides: mX = 2'-OMe; fX = 2'-F; ps = phosphate thioester; VP = vinylphosphonate; dX = deoxynucleotide; UNA-X = unlocked nucleic acid; GNA-X = diol nucleic acid; invAb = reverse baseless; Chol4 = cholesterol) * * * * * This invention is not limited to the specific embodiments described herein. In fact, various modifications of the invention will become apparent to those skilled in the art from the foregoing description and drawings, in addition to those described. Such modifications are intended to fall within the scope of the appended claims.

[0231] All references cited in this paper are incorporated herein by reference in full for all purposes, to the extent that each individual publication, patent, or patent application is specifically and individually represented in full and incorporated herein by reference for all purposes.

Claims

1. A small interfering RNA (siRNA) reagent, said small interfering RNA (siRNA) reagent 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 sense nucleotide sequence in Table 1, or (a) The antisense strand comprises a nucleotide sequence corresponding to any of the antisense nucleotide sequences in Table 1.

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

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

4. A composition comprising a carrier and the siRNA reagent according to claim 1.

5. The siRNA reagent according to claim 2, wherein the sense strand consists of a nucleotide sequence corresponding to a sense nucleotide sequence in one 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 a carrier and the siRNA reagent according to claim 2.

7. A small interfering RNA (siRNA) reagent, said small interfering RNA (siRNA) reagent 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 sense nucleotide sequence in Table 2, or (b) The antisense strand comprises a nucleotide sequence corresponding to any of the antisense nucleotide sequences in Table 2.

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

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

10. A composition comprising a carrier and the siRNA reagent according to claim 7.

11. The siRNA reagent according to claim 8, wherein the sense strand consists of a nucleotide sequence corresponding to a sense nucleotide sequence in one 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 for inhibiting the expression of the SNCA gene in a cell or cell population, the method comprising contacting the cell or cell population with the siRNA reagent according to claim 11.

13. A method for reducing the level of α-synuclein in cells or cell populations, the method comprising contacting the cells or cell populations with the siRNA reagent according to claim 11.

14. A composition comprising a carrier and the siRNA reagent according to claim 8.

15. A small interfering RNA (siRNA) reagent, said small interfering RNA (siRNA) reagent 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 of the sense nucleotide sequences in Table 3, or (b) The antisense strand comprises a nucleotide sequence corresponding to any of the antisense nucleotide sequences in Table 3.

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

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

18. A composition comprising a carrier and the siRNA reagent according to claim 15.

19. The siRNA reagent of claim 16, wherein the sense strand consists of a nucleotide sequence corresponding to a sense nucleotide sequence in one 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 a carrier and the siRNA reagent according to claim 16.

21. A method for inhibiting the expression of an SNCA gene in a cell or cell population, the method comprising contacting the cell or cell population with the siRNA reagent according to claim 19.

22. A method for reducing the level of α-synuclein in cells or cell populations, the method comprising contacting the cells or cell populations with the siRNA reagent according to claim 19.

23. A small interfering RNA (siRNA) reagent, said small interfering RNA (siRNA) reagent 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 of the sense nucleotide sequences in Table 4, or (b) The antisense strand comprises a nucleotide sequence corresponding to any of the antisense nucleotide sequences in Table 4.

24. The siRNA reagent 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 an antisense nucleotide sequence in the same row of Table 4.

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

26. A composition comprising a carrier and the siRNA reagent according to claim 23.

27. The siRNA reagent of claim 24, wherein the sense strand comprises a nucleotide having the sequence (InvAb)psmUpsmCmAmGmC(J2-CONC16A)mGmUfGfAfUmUmGmAmAmGmUmAmUmCpsmAps(InvAb) (SEQ ID NO: 1535), and the antisense strand comprises a nucleotide having the sequence (VPmU)psfGpsmApsmUmAfCmUfUfCmAmAmUmCfAmCfUmGmCmUmGpsmA (SEQ ID NO: 1536).

28. A composition comprising a carrier and the siRNA reagent according to claim 27.

29. The siRNA reagent of claim 24, wherein the sense strand consists of a nucleotide sequence corresponding to a sense nucleotide sequence in one 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 a carrier and the siRNA reagent according to claim 24.

31. A method for inhibiting the expression of an SNCA gene in a cell or cell population, the method comprising contacting the cell or cell population with the siRNA reagent according to claim 27.

32. A method for reducing the level of α-synuclein in cells or cell populations, the method comprising contacting the cells or cell populations with the siRNA reagent according to claim 27.

33. A method for inhibiting the expression of an SNCA gene in a cell or cell population, the method comprising contacting the cell or cell population with the siRNA reagent according to claim 29.

34. A method for reducing the level of α-synuclein in cells or cell populations, the method comprising contacting the cells or cell populations with the siRNA reagent according to claim 29.

35. A small interfering RNA (siRNA) reagent, said small interfering RNA (siRNA) reagent 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 of the sense nucleotide sequences in Table 21, or (d) The antisense strand comprises a nucleotide sequence corresponding to any of the antisense nucleotide sequences in Table 21.

36. The siRNA reagent 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 an antisense nucleotide sequence in the same row of Table 21.

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

38. A composition comprising a carrier and the siRNA reagent according to claim 35.

39. The siRNA reagent of claim 36, wherein the sense strand consists of a nucleotide sequence corresponding to a sense nucleotide sequence in one 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 a carrier and the siRNA reagent according to claim 36.

41. A method for inhibiting the expression of the SNCA gene in a cell or cell population, the method comprising contacting the cell or cell population with the siRNA reagent according to claim 39.

42. A method for reducing the level of α-synuclein in cells or cell populations, the method comprising contacting the cells or cell populations with the siRNA reagent according to claim 39.

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