RNAi AGENTS FOR INHIBITING EXPRESSION OF SYNUCLEIN ALPHA (SNCA), COMPOSITIONS THEREOF, AND METHODS OF USE

Abstract

Described are RNAi agents, compositions that include RNAi agents, and methods for inhibition of a synuclein alpha (SNCA) gene. The SNCA RNAi agents and RNAi agent conjugates disclosed herein inhibit the expression of an SNCA gene. The SNCA RNAi agents are conjugated to an antigen binding protein that may enable subcutaneous delivery of the RNAi agents by facilitating crossing of the blood brain barrier (BBB). Pharmaceutical compositions that include one or more SNCA RNAi agents, optionally with one or more additional therapeutics, are also described. Delivery of the described SNCA RNAi agents to central nervous system (CNS) tissue, in vivo, provides for inhibition of SNCA gene expression and a reduction in SNCA activity, which can provide a therapeutic benefit to subjects, including human subjects, for the treatment of various diseases including Parkinson's Disease.

Description

RNAi Agents for Inhibiting Expression of Synuclein Alpha (SNCA), Compositions Thereof, and Methods of UseCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to United States Provisional Patent Application Serial No. 63 / 695,051, filed on September 16, 2024, and United States Provisional Patent Application Serial No. 63 / 786,589, filed on April 10, 2025, the contents of each of which are incorporated herein by reference in their entirety.FIELD OF THE INVENTION

[0002] The present disclosure relates to RNA interference (RNAi) agents, e.g., double stranded RNAi agents such as chemically modified small interfering RNAs (siRNAs), for inhibition of synuclein alpha (“SNCA”) gene expression, compositions that include SNCA RNAi agents, and methods of use thereof.SEQUENCE LISTING

[0003] This application contains a Sequence Listing (in compliance with Standard ST26), which has been submitted in xml format and is hereby incorporated by reference in its entirety. The xml sequence listing file is named 30748-WO_SeqListing.xml, created August 28, 2025, and is 4,865,199 bytes in size.BACKGROUND

[0004] The present disclosure relates to RNA interference (RNAi) agents, e.g., double stranded RNAi agents such as chemically modified small interfering RNAs (siRNAs), for inhibition of synuclein alpha (“SNCA”) gene expression, compositions that include SNCA RNAi agents, and methods of use thereof. Synuclein alpha may also be referred to as alpha-synuclein, a-syn, a-syn.

[0005] Synuclein alpha is a small (14 kDa) acidic protein expressed in neurons of the central and peripheral nervous systems, as well as in blood cells and other tissues. Calabresi, P., Mechelli, A., Natale, G. et al. Alpha-synuclein in Parkinson’s disease and othersynucleinopathies: from overt neurodegeneration back to early synaptic dysfunction. Cell Death Dis 14, 176 (2023). SNCA encodes for the protein alpha-synuclein. Synuclein alpha has been identified as playing a critical role in the pathogenesis of synucleinopathies, such as Parkinson’s disease (PD). Abnormal forms of synuclein alpha result in selective and progressive neuronal death through mitochondrial impairment, lysosomal dysfunction, and alteration of calcium homeostasis. SNCA is abundant in the brain, and smaller amounts are also present in the heart, muscles, and other tissues. MedlinePlus, National Library of Medicine 2021. SNCA has been shown to maintain an adequate supply of synaptic vesicles in presynaptic terminals, and may also help regulate the release of dopamine, as well as play a role in movement of microtubules to help cells maintain their shape.

[0006] Under normal conditions, synuclein alpha is natively unfolded monomer, or is bound to membranes with alpha-helical structure secondary structure. There are at least 30 variants of the SNCA gene that can alter the SNCA protein. These mutations can result in misfolding or overproduction of the alpha-synuclein protein. SNCA has been associated with neurodegenerative diseases such as Parkinson’s disease, Lewy body dementia, multiple system atrophy (MSA), and others. Parkinson’s Foundation, Genetics Behind Parkinson’s 2024.SUMMARY

[0007] There exists a need for novel RNA interference (RNAi) agents (termed RNAi agents, RNAi triggers, or triggers), e.g, double stranded RNAi agents such as siRNAs, that are able to selectively and efficiently inhibit the expression of SNCA gene, including for use as a therapeutic or medicament. Further, there exists a need for compositions of novel SNCA- specific RNAi agents for the treatment of diseases or disorders associated with mutant or overexpressed SNCA gene and / or disorders that can be mediated at least in part by a reduction in SNCA gene expression.

[0008] The nucleotide sequences and chemical modifications of the SNCA RNAi agents disclosed herein, as well as their combination with certain specific antigen binding proteins suitable for selectively and efficiently delivering the SNCA RNAi agents to relevant CNS cells in vivo, differ from those previously disclosed or known in the art. The SNCA RNAi agents disclosed herein provide for highly potent and efficient inhibition of the expression of a SNCA gene.

[0009] In general, the present disclosure features SNCA gene-specific RNAi agents, compositions that include SNCA RNAi agents, and methods for inhibiting expression of a SNCA gene in vitro and / or in vivo using the SNCA RNAi agents and compositions that include SNCA RNAi agents described herein. The SNCA RNAi agents described herein are able to selectively and efficiently decrease expression of a SNCA gene, and thereby reduce the expression of the SNCA protein.

[0010] The described SNCA RNAi agents can be used in methods for therapeutic treatment (including preventative or prophylactic treatment) of symptoms and diseases including, but not limited to, various central nervous system diseases and neurodegenerative diseases (including Parkinson’s Disease and MSA).

[0011] In one aspect, the disclosure features RNAi agents for inhibiting expression of a SNCA gene, wherein the RNAi agent includes a sense strand (also referred to as a passenger strand) and an antisense strand (also referred to as a guide strand). The sense strand and the antisense strand can be partially, substantially, or fully complementary to each other. The length of the RNAi agent sense strands described herein each can be 15 to 49 nucleotides in length. The length of the RNAi agent antisense strands described herein each can be 18 to 49 nucleotides in length. In some embodiments, the sense and antisense strands are independently 18 to 26 nucleotides in length. The sense and antisense strands can be either the same length or different lengths. In some embodiments, the sense and antisense strands are independently 21 to 26 nucleotides in length. In some embodiments, the sense and antisense strands are independently 21 to 24 nucleotides in length. In some embodiments, both the sense strand and the antisense strand are 21 nucleotides in length. In some embodiments, the antisense strands are independently 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, the sense strands are independently 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 nucleotides in length. The RNAi agents described herein, upon delivery to a cell expressing SNCA such as endothelial cells, neurons, microglia, and astrocytes, inhibit the expression of one or more SNCA gene variants in vivo and / or in vitro.

[0012] The SNCA RNAi agents disclosed herein target a human SNCA gene (see, e.g., SEQ ID NO: 1). In some embodiments, the SNCA RNAi agents disclosed herein target a portion of a SNCA gene having the sequence of any of the sequences disclosed in Table 1.

[0013] In another aspect, the disclosure features compositions, including pharmaceutical compositions, that include one or more of the disclosed SNCA RNAi agents that are able toselectively and efficiently decrease expression of an SNCA gene. The compositions that include one or more SNCA RNAi agents described herein can be administered to a subject, such as a human or animal subject, for the treatment (including prophylactic treatment or inhibition) of symptoms and diseases associated with SNCA protein or enzyme levels.

[0014] Examples of SNCA RNAi agent sense strands and antisense strands that can be used in a SNCA RNAi agent are provided in Tables 3, 4, 5, and 6. Examples of SNCA RNAi agent duplexes are provided in Tables 7, 8, and 9. Examples of 19-nucleotide core stretch sequences that may consist of or may be included in the sense strands and antisense strands of certain SNCA RNAi agents disclosed herein, are provided in Table 2.

[0015] In another aspect, the disclosure features methods for delivering SNCA RNAi agents to neurons, astrocytes, microglia and endothelial cells in a subject, such as a mammal, in vivo. Also described herein are compositions for use in such methods. In some embodiments, disclosed herein are methods for delivering SNCA RNAi agents to central nervous system cells (neurons, astrocytes, microglia and endothelial cells) to a subject in vivo. In some embodiments, the subject is a human subject.

[0016] The methods disclosed herein include the administration of one or more SNCA RNAi agents to a subject, e.g., a human or animal subject, by any suitable means known in the art. The pharmaceutical compositions disclosed herein that include one or more SNCA RNAi agents can be administered in a number of ways depending upon whether local or systemic treatment is desired. Administration can be, but is not limited to, for example, intravenous, intraarterial, subcutaneous, intraperitoneal, subdermal (e.g., via an implanted device), and intraparenchymal administration. In some embodiments, the pharmaceutical compositions described herein are administered by intrathecal injection, intracerebroventricular injection, or subcutaneous injection.

[0017] In some embodiments, it is desired that the SNCA RNAi agents described herein inhibit the expression of an SNCA gene in central nervous system cells.

[0018] The one or more SNCA RNAi agents can be delivered to target cells or tissues using any oligonucleotide delivery technology known in the art. In some embodiments, a SNCA RNAi agent is delivered to cells or tissues by covalently linking the RNAi agent to a targeting group or an antigen binding protein .

[0019] An antigen binding protein can be linked to the 3' or 5' end of a sense strand or an antisense strand of a SNCA RNAi agent. In some embodiments, an antigen binding protein is linked to the 3' or 5' end of the sense strand. In some embodiments, an antigen binding proteinis linked to the 5' end of the sense strand. In some embodiments, an antigen binding protein is linked internally to a nucleotide on the sense strand and / or the antisense strand of the RNAi agent. In some embodiments, an antigen binding protein is linked to the RNAi agent via a linker.

[0020] In another aspect, the disclosure features compositions that include one or more SNCA RNAi agents that have the duplex structures disclosed in Tables 7, 8, and 9.

[0021] The use of SNCA RNAi agents provides methods for therapeutic (including prophylactic) treatment of diseases or disorders for which a reduction in SNCA protein levels can provide a therapeutic benefit. The SNCA RNAi agents disclosed herein can be used to treat various neurodegenerative diseases, including synucleinopathies such as Parkinson’s Disease. Such methods of treatment include administration of a SNCA RNAi agent to a human being or animal having elevated or mutant SNCA protein or mutant SNCA activity beyond desirable levels.DETAILED DESCRIPTIONDefinitions

[0022] As used herein, the terms “oligonucleotide” and “polynucleotide” mean a polymer of linked nucleosides each of which can be independently modified or unmodified.

[0023] As used herein, an “RNAi agent” (also referred to as an “RNAi trigger”) means a chemical composition of matter that contains an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule that is capable of degrading or inhibiting (e.g., degrades or inhibits under appropriate conditions) translation of messenger RNA (mRNA) transcripts of a target mRNA in a sequence specific manner. As used herein, RNAi agents may operate through the RNA interference mechanism (i.e., inducing RNA interference through interaction with the RNA interference pathway machinery (RNA-induced silencing complex or RISC) of mammalian cells), or by any alternative mechanism(s) or pathway(s). While it is believed that RNAi agents, as that term is used herein, operate primarily through the RNA interference mechanism, the disclosed RNAi agents are not bound by or limited to any particular pathway or mechanism of action. RNAi agents disclosed herein are comprised of a sense strand and an antisense strand, and include, but are not limited to: small (or short) interfering RNAs (siRNAs), double stranded RNAs (dsRNA), micro RNAs (miRNAs), short hairpin RNAs (shRNA), and dicer substrates. The antisense strand of the RNAi agents described herein is atleast partially complementary to the mRNA being targeted (i.e. SNCA mRNA). RNAi agents can include one or more modified nucleotides and / or one or more non-phosphodiester linkages.

[0024] As used herein, the terms “silence,” “reduce,” “inhibit,” “down-regulate,” or “knockdown” when referring to expression of a given gene, mean that the expression of the gene, as measured by the level of RNA transcribed from the gene or the level of polypeptide, protein, or protein subunit translated from the mRNA in a cell, group of cells, tissue, organ, or subject in which the gene is transcribed, is reduced when the cell, group of cells, tissue, organ, or subject is treated with the RNAi agents described herein as compared to a second cell, group of cells, tissue, organ, or subject that has not or have not been so treated.

[0025] As used herein, the terms “sequence” and “nucleotide sequence” mean a succession or order of nucleobases or nucleotides, described with a succession of letters using standard nomenclature.

[0026] As used herein, a “base,” “nucleotide base,” or “nucleobase,” is a heterocyclic pyrimidine or purine compound that is a component of a nucleotide, and includes the primary purine bases adenine and guanine, and the primary pyrimidine bases cytosine, thymine, and uracil. A nucleobase may further be modified to include, without limitation, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases. (See, e.g., Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley - VCH, 2008). The synthesis of such modified nucleobases (including phosphoramidite compounds that include modified nucleobases) is known in the art.

[0027] As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleobase or nucleotide sequence (e.g., RNAi agent sense strand or targeted mRNA) in relation to a second nucleobase or nucleotide sequence (e.g., RNAi agent antisense strand or a single-stranded antisense oligonucleotide), means the ability of an oligonucleotide or polynucleotide including the first nucleotide sequence to hybridize (form base pair hydrogen bonds under mammalian physiological conditions (or otherwise suitable in vivo or in vitro conditions)) and form a duplex or double helical structure under certain standard conditions with an oligonucleotide that includes the second nucleotide sequence. The person of ordinary skill in the art would be able to select the set of conditions most appropriate for a hybridization test. Complementary sequences include Watson-Crick base pairs or non-Watson-Crick base pairs and include natural or modified nucleotides or nucleotide mimics, at least to the extent that the above hybridization requirements are fulfilled. Sequence identity or complementarityis independent of modification. For example, a and Af, as defined herein, are complementary to U (or T) and identical to A for the purposes of determining identity or complementarity.

[0028] As used herein, “perfectly complementary” or “fully complementary” means that in a hybridized pair of nucleobase or nucleotide sequence molecules, all (100%) of the bases in a contiguous sequence of a first oligonucleotide will hybridize with the same number of bases in a contiguous sequence of a second oligonucleotide. The contiguous sequence may comprise all or a part of a first or second nucleotide sequence.

[0029] As used herein, “partially complementary” means that in a hybridized pair of nucleobase or nucleotide sequence molecules, at least 70%, but not all, of the bases in a contiguous sequence of a first oligonucleotide will hybridize with the same number of bases in a contiguous sequence of a second oligonucleotide. The contiguous sequence may comprise all or a part of a first or second nucleotide sequence.

[0030] As used herein, “substantially complementary” means that in a hybridized pair of nucleobase or nucleotide sequence molecules, at least 85%, but not all, of the bases in a contiguous sequence of a first oligonucleotide will hybridize with the same number of bases in a contiguous sequence of a second oligonucleotide. The contiguous sequence may comprise all or a part of a first or second nucleotide sequence.

[0031] As used herein, the terms “complementary,” “fully complementary,” “partially complementary,” and “substantially complementary” are used with respect to the nucleobase or nucleotide matching between the sense strand and the antisense strand of an RNAi agent, or between the antisense strand of an RNAi agent and a sequence of an SNCA mRNA.

[0032] As used herein, the term “substantially identical” or “substantial identity,” as applied to a nucleic acid sequence means the nucleotide sequence (or a portion of a nucleotide sequence) has at least about 85% sequence identity or more, e.g., at least 90%, at least 95%, or at least 99% identity, compared to a reference sequence. Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window. The percentage is calculated by determining the number of positions at which the same type of nucleic acid base occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. The inventions disclosed herein encompass nucleotide sequences substantially identical to those disclosed herein.

[0033] As used herein, the terms “treat,” “treatment,” and the like, mean the methods or steps taken to provide relief from or alleviation of the number, severity, and / or frequency of one or more symptoms of a disease in a subject. As used herein, “treat” and “treatment” may include the prevention, management, prophylactic treatment, and / or inhibition or reduction of the number, severity, and / or frequency of one or more symptoms of a disease in a subject.

[0034] As used herein, the phrase “introducing into a cell,” when referring to an RNAi agent, means functionally delivering the RNAi agent into a cell. The phrase “functional delivery,” means delivering the RNAi agent to the cell in a manner that enables the RNAi agent to have the expected biological activity, e.g., sequence-specific inhibition of gene expression.

[0035] Unless stated otherwise, use of the symbolas used herein means that any group or groups may be linked thereto that is in accordance with the scope of the inventions described herein.

[0036] As used herein, the term “isomers” refers to compounds that have identical molecular formulae, but that differ in the nature or the sequence of bonding of their atoms or in the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space are termed “stereoisomers.” Stereoisomers that are not mirror images of one another are termed “diastereoisomers,” and stereoisomers that are non-superimposable mirror images are termed “enantiomers,” or sometimes optical isomers. A carbon atom bonded to four nonidentical substituents is termed a “chiral center.”

[0037] As used herein, unless specifically identified in a structure as having a particular conformation, for each structure in which asymmetric centers are present and thus give rise to enantiomers, diastereomers, or other stereoisomeric configurations, each structure disclosed herein is intended to represent all such possible isomers, including their optically pure and racemic forms. For example, the structures disclosed herein are intended to cover mixtures of diastereomers as well as single stereoisomers.

[0038] As used in a claim herein, the phrase “consisting of’ excludes any element, step, or ingredient not specified in the claim. When used in a claim herein, the phrase “consisting essentially of’ limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention.

[0039] The person of ordinary skill in the art would readily understand and appreciate that the compounds and compositions disclosed herein may have certain atoms (e.g., N, O, or S atoms) in a protonated or deprotonated state, depending upon the environment in which the compoundor composition is placed. Accordingly, as used herein, the structures disclosed herein envisage that certain functional groups, such as, for example, OH, SH, or NH, may be protonated or deprotonated. The disclosure herein is intended to cover the disclosed compounds and compositions regardless of their state of protonation based on the environment (such as pH), as would be readily understood by the person of ordinary skill in the art. Correspondingly, compounds described herein with labile protons or basic atoms should also be understood to represent salt forms of the corresponding compound. Compounds described herein may be in a free acid, free base, or salt form. Pharmaceutically acceptable salts of the compounds described herein should be understood to be within the scope of the invention.

[0040] As used herein, the term “linked” or “conjugated” when referring to the connection between two compounds or molecules means that two compounds or molecules are joined by a covalent bond. Unless stated, the terms “linked” and “conjugated” as used herein may refer to the connection between a first compound and a second compound either with or without any intervening atoms or groups of atoms.

[0041] As used herein, the term “including” is used to herein mean, and is used interchangeably with, the phrase “including but not limited to.” The term “or” is used herein to mean, and is used interchangeably with, the term “and / or,” unless the context clearly indicates otherwise.

[0042] 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. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0043] Other objects, features, aspects, and advantages of the invention will be apparent from the following detailed description, and from the claims.RNAi Agents

[0044] Described herein are RNAi agents for inhibiting expression of the SNCA (or SNCA) gene (referred to herein as SNCA RNAi agents or SNCA RNAi triggers). Each SNCA RNAi agent disclosed herein comprises a sense strand and an antisense strand. The sense strand can be 15 to 49 nucleotides in length. The antisense strand can be 18 to 30 nucleotides in length.The sense and antisense strands can be either the same length or they can be different lengths. In some embodiments, the sense and antisense strands are each independently 18 to 27 nucleotides in length. In some embodiments, both the sense and antisense strands are each 21- 26 nucleotides in length. In some embodiments, the sense and antisense strands are each 21-24 nucleotides in length. In some embodiments, the sense and antisense strands are each independently 19-21 nucleotides in length. In some embodiments, the sense strand is about 19 nucleotides in length while the antisense strand is about 21 nucleotides in length. In some embodiments, the sense strand is about 21 nucleotides in length while the antisense strand is about 23 nucleotides in length. In some embodiments, a sense strand is 23 nucleotides in length and an antisense strand is 21 nucleotides in length. In some embodiments, both the sense and antisense strands are each 21 nucleotides in length. In some embodiments, the RNAi agent sense strands are each independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 nucleotides in length. In some embodiments, the RNAi agent antisense strands are each independently 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, a double-stranded RNAi agent has a duplex length of about 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides.

[0045] Examples of nucleotide sequences used in forming SNCA RNAi agents are provided in Tables 2, 3, 4, 5, 6, and 10. Examples of RNAi agent duplexes, that include the sense strand and antisense strand sequences in Tables 2, 3, 4, 5, 6, are shown in Tables 7, 8, 9, and 10.

[0046] In some embodiments, the region of perfect, substantial, or partial complementarity between the sense strand and the antisense strand is 16-26 (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26) nucleotides in length and occurs at or near the 5' end of the antisense strand (e.g., this region may be separated from the 5' end of the antisense strand by 0, 1, 2, 3, or 4 nucleotides that are not perfectly, substantially, or partially complementary).

[0047] A sense strand of the SNCA RNAi agents described herein includes at least 15 consecutive nucleotides that have at least 85% identity to a core stretch sequence (also referred to herein as a “core stretch” or “core sequence”) of the same number of nucleotides in an SNCA mRNA. In some embodiments, a sense strand core stretch sequence is 100% (perfectly) complementary or at least about 85% (substantially) complementary to a core stretch sequence in the antisense strand, and thus the sense strand core stretch sequence is typically perfectly identical or at least about 85% identical to a nucleotide sequence of the same length (sometimes referred to, e.g., as a target sequence) present in the SNCA mRNA target. In someembodiments, this sense strand core stretch is 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides in length. In some embodiments, this sense strand core stretch is 17 nucleotides in length. In some embodiments, this sense strand core stretch is 19 nucleotides in length.

[0048] An antisense strand of a SNCA RNAi agent described herein includes at least 16 consecutive nucleotides that have at least 85% complementarity to a core stretch of the same number of nucleotides in an SNCA mRNA and to a core stretch of the same number of nucleotides in the corresponding sense strand. In some embodiments, an antisense strand core stretch is 100% (perfectly) complementary or at least about 85% (substantially) complementary to a nucleotide sequence (e.g, target sequence) of the same length present in the SNCA mRNA target. In some embodiments, this antisense strand core stretch is 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides in length. In some embodiments, this antisense strand core stretch is 19 nucleotides in length. In some embodiments, this antisense strand core stretch is 17 nucleotides in length. A sense strand core stretch sequence can be the same length as a corresponding antisense core sequence or it can be a different length.

[0049] The SNCA RNAi agent sense and antisense strands anneal to form a duplex. A sense strand and an antisense strand of a SNCA RNAi agent can be partially, substantially, or fully complementary to each other. Within the complementary duplex region, the sense strand core stretch sequence is at least 85% complementary or 100% complementary to the antisense core stretch sequence. In some embodiments, the sense strand core stretch sequence contains a sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23 nucleotides that is at least 85% or 100% complementary to a corresponding 16, 17, 18, 19, 20, 21, 22, or 23 nucleotide sequence of the antisense strand core stretch sequence (i.e., the sense and antisense core stretch sequences of a SNCA RNAi agent have a region of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23 nucleotides that is at least 85% base paired or 100% base paired.)

[0050] In some embodiments, the antisense strand of a SNCA RNAi agent disclosed herein differs by 0, 1, 2, or 3 nucleotides from any of the antisense strand sequences in Table 2, Table 3, or Table 9. In some embodiments, the sense strand of a SNCA RNAi agent disclosed herein differs by 0, 1, 2, or 3 nucleotides from any of the sense strand sequences in Table 2, Table 4, Table 5, Table 6, or Table 9.

[0051] In some embodiments, the sense strand and / or the antisense strand can optionally and independently contain an additional 1, 2, 3, 4, 5, or 6 nucleotides (extension) at the 3' end, the 5' end, or both the 3' and 5' ends of the core stretch sequences. The antisense strand additionalnucleotides, if present, may or may not be complementary to the corresponding sequence in the SNCA mRNA. The sense strand additional nucleotides, if present, may or may not be identical to the corresponding sequence in the SNCA mRNA. The antisense strand additional nucleotides, if present, may or may not be complementary to the corresponding sense strand’s additional nucleotides, if present.

[0052] As used herein, an extension comprises 1, 2, 3, 4, 5, or 6 nucleotides at the 5' and / or 3' end of the sense strand core stretch sequence and / or antisense strand core stretch sequence. The extension nucleotides on a sense strand may or may not be complementary to nucleotides, either core stretch sequence nucleotides or extension nucleotides, in the corresponding antisense strand. Conversely, the extension nucleotides on an antisense strand may or may not be complementary to nucleotides, either core stretch nucleotides or extension nucleotides, in the corresponding sense strand. In some embodiments, both the sense strand and the antisense strand of an RNAi agent contain 3' and 5' extensions. In some embodiments, one or more of the 3' extension nucleotides of one strand base pairs with one or more 5' extension nucleotides of the other strand. In other embodiments, one or more of 3' extension nucleotides of one strand do not base pair with one or more 5' extension nucleotides of the other strand. In some embodiments, a SNCA RNAi agent has an antisense strand having a 3' extension and a sense strand having a 5' extension. In some embodiments, the extension nucleotide(s) are unpaired and form an overhang. As used herein, an “overhang” refers to a stretch of one or more unpaired nucleotides located at a terminal end of either the sense strand or the antisense strand that does not form part of the hybridized or duplexed portion of an RNAi agent disclosed herein.

[0053] In some embodiments, a SNCA RNAi agent comprises an antisense strand having a 3' extension of 1, 2, 3, 4, 5, or 6 nucleotides in length. In other embodiments, a SNCA RNAi agent comprises an antisense strand having a 3' extension of 1, 2, or 3 nucleotides in length. In some embodiments, one or more of the antisense strand extension nucleotides comprise nucleotides that are complementary to the corresponding SNCA mRNA sequence. In some embodiments, one or more of the antisense strand extension nucleotides comprise nucleotides that are not complementary to the corresponding SNCA mRNA sequence.

[0054] In some embodiments, a SNCA RNAi agent comprises a sense strand having a 3' extension of 1, 2, 3, 4, or 5 nucleotides in length. In some embodiments, one or more of the sense strand extension nucleotides comprises adenosine, uracil, or thymidine nucleotides, AT dinucleotide, or nucleotides that correspond to or are the identical to nucleotides in the SNCA mRNA sequence. In some embodiments, the 3' sense strand extension includes or consists ofone of the following sequences, but is not limited to: T, UT, TT, UU, UUT, TTT, or TTTT (each listed 5' to 3').

[0055] A sense strand can have a 3' extension and / or a 5' extension. In some embodiments, a SNCA RNAi agent comprises a sense strand having a 5' extension of 1, 2, 3, 4, 5, or 6 nucleotides in length. In some embodiments, one or more of the sense strand extension nucleotides comprise nucleotides that correspond to or are identical to nucleotides in the SNCA mRNA sequence.

[0056] Examples of sequences used in forming SNCA RNAi agents are provided in Tables 2, 3, 4, 5, 6, and 9. In some embodiments, a SNCA RNAi agent antisense strand includes a sequence of any of the sequences in Tables 2, 3, or 9. In certain embodiments, a SNCA RNAi agent antisense strand comprises or consists of any one of the modified sequences in Table 3. In some embodiments, a SNCA RNAi agent antisense strand includes the sequence of nucleotides (from 5' end3' end) 1-17, 2-15, 2-17, 1-18, 2-18, 1-19, 2-19, 1-20, 2-20, 1-21, or 2-21, of any of the sequences in Tables 2 or 3. In some embodiments, a SNC A RNAi agent sense strand includes the sequence of any of the sequences in Tables 2, 4, 5, or 6. In some embodiments, a SNCA RNAi agent sense strand includes the sequence of nucleotides (from 5' end 3' end) 1-18, 1-19, 1-20, 1-21, 2-19, 2-20, 2-21, 3-20, 3-21, or 4-21 of any of the sequences in Tables 2, 4, 5, or 6. In certain embodiments, a SNCA RNAi agent sense strand comprises or consists of a modified sequence of any one of the modified sequences in Table 4, 5, 6, or 9.

[0057] In some embodiments, the sense and antisense strands of the RNAi agents described herein contain the same number of nucleotides. In some embodiments, the sense and antisense strands of the RNAi agents described herein contain different numbers of nucleotides. In some embodiments, the sense strand 5' end and the antisense strand 3' end of an RNAi agent form a blunt end. In some embodiments, the sense strand 3' end and the antisense strand 5' end of an RNAi agent form a blunt end. In some embodiments, both ends of an RNAi agent form blunt ends. In some embodiments, neither end of an RNAi agent is blunt-ended. As used herein a “blunt end” refers to an end of a double stranded RNAi agent in which the terminal nucleotides of the two annealed strands are complementary (form a complementary base-pair).

[0058] In some embodiments, the sense strand 5' end and the antisense strand 3' end of an RNAi agent form a frayed end. In some embodiments, the sense strand 3' end and the antisense strand 5' end of an RNAi agent form a frayed end. In some embodiments, both ends of an RNAi agent form a frayed end. In some embodiments, neither end of an RNAi agent is a frayed end.As used herein a frayed end refers to an end of a double stranded RNAi agent in which the terminal nucleotides of the two annealed strands form a pair (i.e., do not form an overhang) but are not complementary (i.e. form a non-complementary pair). In some embodiments, one or more unpaired nucleotides at the end of one strand of a double stranded RNAi agent form an overhang. The unpaired nucleotides may be on the sense strand or the antisense strand, creating either 3' or 5' overhangs. In some embodiments, the RNAi agent contains: a blunt end and a frayed end, a blunt end and 5' overhang end, a blunt end and a 3' overhang end, a frayed end and a 5' overhang end, a frayed end and a 3' overhang end, two 5' overhang ends, two 3' overhang ends, a 5' overhang end and a 3' overhang end, two frayed ends, or two blunt ends. Typically, when present, overhangs are located at the 3 ' terminal ends of the sense strand, the antisense strand, or both the sense strand and the antisense strand.

[0059] The SNCA RNAi agents disclosed herein may also be comprised of one or more modified nucleotides. In some embodiments, substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand of the SNCA RNAi agent are modified nucleotides. The SNCA RNAi agents disclosed herein may further be comprised of one or more modified intemucleoside linkages, e.g., one or more phosphorothioate linkages. In some embodiments, a SNCA RNAi agent contains one or more modified nucleotides and one or more modified intemucleoside linkages. In some embodiments, a 2'-modified nucleotide is combined with modified intemucleoside linkage.

[0060] In some embodiments, a SNCA RNAi agent is prepared or provided as a salt, mixed salt, or a free-acid. In some embodiments, a SNCA RNAi agent is prepared as a pharmaceutically acceptable salt. In some embodiments, a SNCA RNAi agent is prepared as a pharmaceutically acceptable sodium salt. Such forms that are well known in the art are within the scope of the inventions disclosed herein.Modified Nucleotides

[0061] Modified nucleotides, when used in various oligonucleotide constructs, can preserve activity of the compound in cells while at the same time increasing the serum stability of these compounds, and can also minimize the possibility of activating interferon activity in humans upon administration of the oligonucleotide construct.

[0062] In some embodiments, a SNCA RNAi agent contains one or more modified nucleotides. As used herein, a “modified nucleotide” is a nucleotide other than a ribonucleotide (2'-hydroxyl nucleotide). In some embodiments, at least 50% (e.g., at least 60%, at least 70%, at least 80%,at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) of the nucleotides are modified nucleotides. As used herein, modified nucleotides can include, but are not limited to, deoxyribonucleotides, nucleotide mimics, abasic nucleotides, 2'-modified nucleotides, inverted nucleotides, modified nucleobase-comprising nucleotides, bridged nucleotides, peptide nucleic acids (PNAs), 2', 3 '-seco nucleotide mimics (unlocked nucleobase analogues), locked nucleotides, 3'-O-methoxy (2' intemucleoside linked) nucleotides, 2'-F-Arabino nucleotides, 5 '-Me, 2'-fhioro nucleotide, morpholino nucleotides, vinyl phosphonate deoxyribonucleotides, vinyl phosphonate containing nucleotides, and cyclopropyl phosphonate containing nucleotides. 2'-modified nucleotides (z.e., a nucleotide with a group other than a hydroxyl group at the 2' position of the five-membered sugar ring) include, but are not limited to, 2'-O-methyl nucleotides (also referred to herein or in the art as 2'-methoxy nucleotides), 2'- fluoro nucleotides (also referred to herein or in the art as 2 '-deoxy-2 '-fluoro nucleotides), 2'- deoxy nucleotides, 2 '-methoxy ethyl (2'-O-2-methoxylethyl) nucleotides (also referred herein or in the art as 2'-M0E nucleotides), 2'-amino nucleotides, and 2'-alkyl nucleotides. It is not necessary for all positions in a given compound to be uniformly modified. Conversely, more than one modification can be incorporated in a single SNCA RNAi agent or even in a single nucleotide thereof. The SNCA RNAi agent sense strands and antisense strands can be synthesized and / or modified by methods known in the art. Modification at one nucleotide is independent of modification at another nucleotide.

[0063] Modified nucleobases include synthetic and natural nucleobases, such as 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, (e.g., 2-aminopropyladenine, 5-propynyluracil, or 5-propynylcytosine), 5-methylcytosine (5-me-C), 5 -hydroxymethyl cytosine, inosine, xanthine, hypoxanthine, 2-aminoadenine, 6-alkyl (e.g., 6- methyl, 6-ethyl, 6-isopropyl, or 6-n-butyl) derivatives of adenine and guanine, 2-alkyl (e.g., 2- methyl, 2-ethyl, 2-isopropyl, or 2-n-butyl) and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2 -thiothymine, 2-thiocytosine, 5-halouracil, cytosine, 5-propynyl uracil, 5-propynyl cytosine, 6-azo uracil, 6-azo cytosine, 6-azo thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-sulfhydryl, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo (e.g., 5-bromo), 5 -trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3 -deazaguanine, and 3 -deazaadenine.

[0064] In some embodiments, the 5’ and / or 3' end of the antisense strand can include abasic residues (Ab), which can also be referred to as an “abasic site” or “abasic nucleotide.” Anabasic residue (Ab) is a nucleotide or nucleoside that lacks a nucleobase at the 1' position of the sugar moiety. (See, e.g., U.S. Patent No. 5,998,203). In some embodiments, an abasic residue can be placed internally in a nucleotide sequence. In some embodiments, Ab or Ab Ab can be added to the 3' end of the antisense strand. In some embodiments, the 5' end of the sense strand can include one or more additional abasic residues (e.g., (Ab) or (AbAb)). In some embodiments, UUAb, UAb, or Ab are added to the 3' end of the sense strand. In some embodiments, an abasic (deoxyribose) residue can be replaced with a ribitol (abasic ribose) residue.

[0065] In some embodiments, all or substantially all of the nucleotides of an RNAi agent are modified nucleotides. As used herein, an RNAi agent wherein substantially all of the nucleotides present are modified nucleotides is an RNAi agent having four or fewer (i.e., 0, 1, 2, 3, or 4) nucleotides in both the sense strand and the antisense strand being ribonucleotides (i.e., unmodified). As used herein, a sense strand wherein substantially all of the nucleotides present are modified nucleotides is a sense strand having two or fewer (i.e., 0, 1, or 2) nucleotides in the sense strand being unmodified ribonucleotides. As used herein, an antisense strand wherein substantially all of the nucleotides present are modified nucleotides is an antisense strand having two or fewer (i.e., 0, 1, or 2) nucleotides in the antisense strand being unmodified ribonucleotides. In some embodiments, one or more nucleotides of an RNAi agent is an unmodified ribonucleotide. Chemical structures for certain modified nucleotides are set forth in Table 10 herein.Modified Internudeoside Linkages

[0066] In some embodiments, one or more nucleotides of a SNCA RNAi agent are linked by non-standard linkages or backbones (i.e., modified internucleoside linkages or modified backbones). Modified internudeoside linkages or backbones include, but are not limited to, phosphorothioate groups (represented herein as a lower case “s”), chiral phosphorothioates, thiophosphates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, alkyl phosphonates (e.g., methyl phosphonates or 3 '-alkylene phosphonates), chiral phosphonates, phosphinates, phosphoramidates (e.g., 3 '-amino phosphoramidate, aminoalkylphosphoramidates, or thionophosphoramidates), thionoalkyl-phosphonates, thionoalkylphosphotriesters, morpholino linkages, boranophosphates having normal 3 '-5' linkages, 2'-5' linked analogs of boranophosphates, or boranophosphates having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3 '-5' to 5 '-3' or 2'-5' to 5 '-2'.In some embodiments, a modified internucleoside linkage or backbone lacks a phosphorus atom. Modified intemucleoside linkages lacking a phosphorus atom include, but are not limited to, short chain alkyl or cycloalkyl inter-sugar linkages, mixed heteroatom and alkyl or cycloalkyl inter-sugar linkages, or one or more short chain heteroatomic or heterocyclic inter- sugar linkages. In some embodiments, modified intemucleoside backbones include, but are not limited to, siloxane backbones, sulfide backbones, sulfoxide backbones, sulfone backbones, formacetyl and thioformacetyl backbones, methylene formacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and other backbones having mixed N, O, S, and CH2 components.

[0067] In some embodiments, a sense strand of a SNCA RNAi agent can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages, an antisense strand of a SNCA RNAi agent can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages, or both the sense strand and the antisense strand independently can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages. In some embodiments, a sense strand of a SNCA RNAi agent can contain 1, 2, 3, or 4 phosphorothioate linkages, an antisense strand of a SNCA RNAi agent can contain 1, 2, 3, or 4 phosphorothioate linkages, or both the sense strand and the antisense strand independently can contain 1, 2, 3, or 4 phosphorothioate linkages.

[0068] In some embodiments, a SNCA RNAi agent sense strand contains at least two phosphorothioate intemucleoside linkages. In some embodiments, the phosphorothioate intemucleoside linkages are between the nucleotides at positions 1-3 from the 3' end of the sense strand. In some embodiments, one phosphorothioate intemucleoside linkage is at the 5’ end of the sense strand nucleotide sequence, and another phosphorothioate linkage is at the 3’ end of the sense strand nucleotide sequence. In some embodiments, two phosphorothioate intemucleoside linkage are located at the 5’ end of the sense strand, and another phosphorothioate linkage is at the 3’ end of the sense strand. In some embodiments, the sense strand does not include any phosphorothioate intemucleoside linkages between the nucleotides, but contains one, two, or three phosphorothioate linkages between the terminal nucleotides on both the 5’ and 3’ ends and the optionally present inverted abasic residue terminal caps. In some embodiments, a targeting ligand is linked to the sense strand via a phosphorothioate linkage.

[0069] In some embodiments, a SNCA RNAi agent antisense strand contains four phosphorothioate intemucleoside linkages. In some embodiments, the four phosphorothioateinternucleoside linkages are between the nucleotides at positions 1-3 from the 5' end of the antisense strand and between the nucleotides at positions 19-21, 20-22, 21-23, 22-24, 23-25, or 24-26 from the 5' end. In some embodiments, three phosphorothioate internucleoside linkages are located between positions 1-4 from the 5’ end of the antisense strand, and a fourth phosphorothioate internucleoside linkage is located between positions 20-21 from the 5’ end of the antisense strand. In some embodiments, a SNCA RNAi agent contains at least three or four phosphorothioate internucleoside linkages in the antisense strand.Capping Residues or Moieties

[0070] In some embodiments, the sense strand may include one or more capping residues or moieties, sometimes referred to in the art as a “cap,” a “terminal cap,” or a “capping residue.” As used herein, a “capping residue” is a non-nucleotide compound or other moiety that can be incorporated at one or more termini of a nucleotide sequence of an RNAi agent disclosed herein. A capping residue can provide the RNAi agent, in some instances, with certain beneficial properties, such as, for example, protection against exonuclease degradation. In some embodiments, inverted abasic residues (invAb) (also referred to in the art as “inverted abasic sites”) are added as capping residues (see Table 10). (See, e.g., F. Czauderna, Nucleic Acids Res., 2003, 31(11), 2705-16). Capping residues are generally known in the art, and include, for example, inverted abasic residues as well as carbon chains such as a terminal C3H7 (propyl), CeHi3 (hexyl), or C12H25 (dodecyl) groups. In some embodiments, a capping residue is present at either the 5' terminal end, the 3' terminal end, or both the 5' and 3' terminal ends of the sense strand. In some embodiments, the 5’ end and / or the 3' end of the sense strand may include more than one inverted abasic deoxyribose moiety as a capping residue.

[0071] In some embodiments, one or more inverted abasic residues (invAb) are added to the 3' end of the sense strand. In some embodiments, one or more inverted abasic residues (invAb) are added to the 5' end of the sense strand. In some embodiments, one or more inverted abasic residues or inverted abasic sites are inserted between a targeting ligand and the nucleotide sequence of the sense strand of the RNAi agent. In some embodiments, the inclusion of one or more inverted abasic residues or inverted abasic sites at or near the terminal end or terminal ends of the sense strand of an RNAi agent allows for enhanced activity or other desired properties of an RNAi agent.

[0072] In some embodiments, one or more inverted abasic residues (invAb) are added to the 5' end of the sense strand. In some embodiments, one or more inverted abasic residues can beinserted between a targeting ligand and the nucleotide sequence of the sense strand of the RNAi agent. The inverted abasic residues may be linked via phosphate, phosphorothioate (e.g., shown herein as (invAb)s)), or other internucleoside linkages. In some embodiments, the inclusion of one or more inverted abasic residues at or near the terminal end or terminal ends of the sense strand of an RNAi agent may allow for enhanced activity or other desired properties of an RNAi agent. In some embodiments, an inverted abasic (deoxyribose) residue can be replaced with an inverted ribitol (abasic ribose) residue. In some embodiments, the 3' end of the antisense strand core stretch sequence, or the 3' end of the antisense strand sequence, may include an inverted abasic residue. The chemical structures for inverted abasic deoxyribose residues are shown in Table 10 below.SNCA RNAi Agents

[0073] T he SNCA RNAi agents disclosed herein are designed to target specific positions on a SNCA gene (e.g., SEQ ID NO: 1 (NM_000345.4)). As defined herein, an antisense strand sequence is designed to target a SNCA gene at a given position on the gene when the 5' terminal nucleobase of the antisense strand is aligned with a position that is 21 nucleotides downstream (towards the 3' end) from the position on the gene when base pairing to the gene. For example, as illustrated in Tables 1 and 2 herein, an antisense strand sequence designed to target a SNCA gene at position 216 requires that when base pairing to the gene, the 5' terminal nucleobase of the antisense strand is aligned with position 236 of a SNCA gene.

[0074] As provided herein, a SNCA RNAi agent does not require that the nucleobase at position 1 (5' - 3') of the antisense strand be complementary to the gene, provided that there is at least 85% complementarity (e.g., at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% complementarity) of the antisense strand and the gene across a core stretch sequence of at least 16 consecutive nucleotides. For example, for a SNCA RNAi agent disclosed herein that is designed to target position 216 of a SNCA gene, the 5' terminal nucleobase of the antisense strand of the of the SNCA RNAi agent must be aligned with position 236 of the gene; however, the 5' terminal nucleobase of the antisense strand may be, but is not required to be, complementary to position 236 of a SNCA gene, provided that there is at least 85% complementarity (e.g., at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% complementarity) of the antisense strand and the gene transcript across a core stretch sequence of at least 16 consecutive nucleotides. As shown by, among other things, the various examples disclosed herein, the specific site of binding of the gene by the antisensestrand of the SNCA RNAi agent (e.g., whether the SNCA RNAi agent is designed to target a SNCA gene at position 216, at position 220, at position 225, or at some other position) is an important factor to the level of inhibition achieved by the SNCA RNAi agent. (See, e.g., Kamola et al., The siRNA Non-seed Region and Its Target Sequences are Auxiliary Determinants of Off-Target Effects, PLOS Computational Biology, 11(12), Figure 1 (2015)).

[0075] In some embodiments, the SNCA RNAi agents disclosed herein target a SNCA gene at or near the positions of the SNCA sequence shown in Table 1. In some embodiments, the antisense strand of a SNCA RNAi agent disclosed herein includes a core stretch sequence that is fully, substantially, or at least partially complementary to a target SNCA 19-mer sequence disclosed in Table 1.Table 1. SNCA 19-mer mRNA Target Sequences (taken from Homo sapiens synuclein alpha(SNCA) transcript, mRNA, 3177 bases, GenBank NM_000345.4 (SEQ ID NO:1))

[0076] Homo sapiens synuclein alpha (SNCA), transcript variant 1, mRNA, GenBank NM_000345.4 (SEQ ID NO: 1), gene transcript (3177 bases):1 ggcgacgacc agaaggggcc caagagaggg ggcgagcgac cgagcgccgc gacgcggaag61 tgaggtgcgt gcgggctgca gcgcagaccc cggcccggcc cctccgagag cgtcctgggc121 gctccctcac gccttgcctt caagccttct gcctttccac cctcgtgagc ggagaactgg181 gagtggccat tcgacgacag tgtggtgtaa aggaattcat tagccatgga tgtattcatg241 aaaggacttt caaaggccaa ggagggagtt gtggctgctg ctgagaaaac caaacagggt301 gtggcagaag cagcaggaaa gacaaaagag ggtgttctct atgtaggctc caaaaccaag361 gagggagtgg tgcatggtgt ggcaacagtg gctgagaaga ccaaagagca agtgacaaat421 gttggaggag cagtggtgac gggtgtgaca gcagtagccc agaagacagt ggagggagca481 gggagcattg cagcagccac tggctttgtc aaaaaggacc agttgggcaa gaatgaagaa541 ggagccccac aggaaggaat tctggaagat atgcctgtgg atcctgacaa tgaggcttat601 gaaatgcctt ctgaggaagg gtatcaagac tacgaacctg aagcctaaga aatatctttg661 ctcccagttt cttgagatct gctgacagat gttccatcct gtacaagtgc tcagttccaa721 tgtgcccagt catgacattt ctcaaagttt ttacagtgta tctcgaagtc ttccatcagc781 agtgattgaa gtatctgtac ctgcccccac tcagcatttc ggtgcttccc tttcactgaa841 gtgaatacat ggtagcaggg tctttgtgtg ctgtggattt tgtggcttca atctacgatg901 ttaaaacaaa ttaaaaacac ctaagtgact accacttatt tctaaatcct cactattttt961 ttgttgctgt tgttcagaag ttgttagtga tttgctatca tatattataa gatttttagg 1021 tgtcttttaa tgatactgtc taagaataat gacgtattgt gaaatttgtt aatatatata 1081 atacttaaaa atatgtgagc atgaaactat gcacctataa atactaaata tgaaatttta 1141 ccattttgcg atgtgtttta ttcacttgtg tttgtatata aatggtgaga attaaaataa 1201 aacgttatct cattgcaaaa atattttatt tttatcccat ctcactttaa taataaaaat 12 1 catgcttata agcaacatga attaagaact gacacaaagg acaaaaatat aaagttatta 1321 atagccattt gaagaaggag gaattttaga agaggtagag aaaatggaac attaacccta 1381 cactcggaat tccctgaagc aacactgcca gaagtgtgtt ttggtatgca ctggttcctt 1441 aagtggctgt gattaattat tgaaagtggg gtgttgaaga ccccaactac tattgtagag 1501 tggtctattt ctcccttcaa tcctgtcaat gtttgcttta cgtattttgg ggaactgttg 1561 tttgatgtgt atgtgtttat aattgttata catttttaat tgagcctttt attaacatat 1621 attgttattt ttgtctcgaa ataatttttt agttaaaatc tattttgtct gatattggtg 1681 tgaatgctgt acctttctga caataaataa tattcgacca tgaataaaaa aaaaaaaaaa 1741 gtgggttccc gggaactaag cagtgtagaa gatgattttg actacaccct ccttagagag 1801 ccataagaca cattagcaca tattagcaca ttcaaggctc tgagagaatg tggttaactt 1861 tgtttaactc agcattcctc actttttttt tttaatcatc agaaattctc tctctctctc 1921 tctctttttc tctcgctctc tttttttttt tttttttaca ggaaatgcct ttaaacatcg 1981 ttggaactac cagagtcacc ttaaaggaga tcaattctct agactgataa aaatttcatg 2041 gcctccttta aatgttgcca aatatatgaa ttctaggatt tttccttagg aaaggttttt 2101 ctctttcagg gaagatctat taactcccca tgggtgctga aaataaactt gatggtgaaa 2161 aactctgtat aaattaattt aaaaattatt tggtttctct ttttaattat tctggggcat 2221 agtcatttct aaaagtcact agtagaaagt ataatttcaa gacagaatat tctagacatg 2281 ctagcagttt atatgtattc atgagtaatg tgatatatat tgggcgctgg tgaggaagga 2341 aggaggaatg agtgactata aggatggtta ccatagaaac ttcctttttt acctaattga 2401 agagagacta ctacagagtg ctaagctgca tgtgtcatct tacactagag agaaatggta 2461 agtttcttgt tttatttaag ttatgtttaa gcaaggaaag gatttgttat tgaacagtat 2521 atttcaggaa ggttagaaag tggcggttag gatatatttt aaatctacct aaagcagcat 2581 attttaaaaa tttaaaagta ttggtattaa attaagaaat agaggacaga actagactga 2641 tagcagtgac ctagaacaat ttgagattag gaaagttgtg accatgaatt taaggattta 2701 tgtggataca aattctcctt taaagtgttt cttcccttaa tatttatctg acggtaattt 2761 ttgagcagtg aattacttta tatatcttaa tagtttattt gggaccaaac acttaaacaa 2821 aaagttcttt aagtcatata agccttttca ggaagcttgt ctcatattca ctcccgagac 2881 attcacctgc caagtggcct gaggatcaat ccagtcctag gtttattttg cagacttaca2941 ttctcccaag ttattcagcc tcatatgact ccacggtcgg ctttaccaaa acagttcaga3001 gtgcactttg gcacacaatt gggaacagaa caatctaatg tgtggtttgg tattccaagt 3061 ggggtctttt tcagaatctc tgcactagtg tgagatgcaa acatgtttcc tcatctttct 3121 ggcttatcca gtatgtagct atttgtgaca taataaatat atacatatat gaaaata

[0077] In some embodiments, a SNCA RNAi agent includes an antisense strand wherein position 19 of the antisense strand (5'- 3') is capable of forming a base pair with position 1 of a 19-mer target sequence disclosed in Table 1. In some embodiments, a SNCA agent includes an antisense strand wherein position 1 of the antisense strand (5 '->3') is capable of forming a base pair with position 19 of a 19-mer target sequence disclosed in Table 1.

[0078] In some embodiments, a SNCA agent includes an antisense strand wherein position 2 of the antisense strand (5' - 3') is capable of forming a base pair with position 18 of a 19-mer target sequence disclosed in Table 1. In some embodiments, a SNCA agent includes an antisense strand wherein positions 2 through 18 of the antisense strand (5' - 3') are capable of forming base pairs with each of the respective complementary bases located at positions 18 through 2 of the 19-mer target sequence disclosed in Table 1.

[0079] For the RNAi agents disclosed herein, the nucleotide at position 1 of the antisense strand (from 5' end3' end) can be perfectly complementary to a SNCA gene, or can be non- complementary to a SNCA gene. In some embodiments, the nucleotide at position 1 of the antisense strand (from 5' end3' end) is a U, A, or dT. In some embodiments, the nucleotide at position 1 of the antisense strand (from 5' end -> 3' end) forms an A:U or U:A base pair with the sense strand.

[0080] In some embodiments, a SNCA RNAi agent antisense strand comprises the sequence of nucleotides (from 5' end3' end) 2-18 or 2-19 of any of the antisense strand sequences in Table 2 or Table 3. In some embodiments, a SNCA RNAi sense strand comprises the sequence of nucleotides (from 5' end - 3' end) 1-17, 1-18, or 2-18 of any of the sense strand sequences in Table 2, Table 4, Table 5, or Table 6.

[0081] In some embodiments, a SNCA RNAi agent is comprised of (i) an antisense strand comprising the sequence of nucleotides (from 5' end -> 3' end) 2-18 or 2-19 of any of the antisense strand sequences in Table 2 or Table 3, and (ii) a sense strand comprising the sequence of nucleotides (from 5' end3' end) 1-17 or 1-18 of any of the sense strand sequences in Table 2, Table 4, Table 5, or Table 6.

[0082] In some embodiments, the SNCA RNAi agents include core 19-mer nucleotide sequences shown in the following Table 2.

[0083] Table 2. SNCA RNAi Agent Antisense Strand and Sense Strand Core Stretch Base Sequences (N=any nucleobase; I = inosine(hypoxanthine nucleobase)

[0084] The SNCA RNAi agent sense strands and antisense strands that comprise or consist of the nucleotide sequences in Table 2 can be modified nucleotides or unmodified nucleotides. In some embodiments, the SNCA RNAi agents having the sense and antisense strand sequences that comprise or consist of any of the nucleotide sequences in Table 2 are all or substantially all modified nucleotides.

[0085] In some embodiments, the antisense strand of a SNCA RNAi agent disclosed herein differs by 0, 1, 2, or 3 nucleotides from any of the antisense strand sequences in Table 2. In some embodiments, the sense strand of a SNCA RNAi agent disclosed herein differs by 0, 1,2, or 3 nucleotides from any of the sense strand sequences in Table 2.

[0086] As used herein, each N listed in a sequence disclosed in Table 2 may be independently selected from any and all nucleobases (including those found on both modified and unmodified nucleotides). In some embodiments, an N nucleotide listed in a sequence disclosed in Table 2 has a nucleobase that is complementary to the N nucleotide at the corresponding position on the other strand. In some embodiments, an N nucleotide listed in a sequence disclosed in Table 2 has a nucleobase that is not complementary to the N nucleotide at the corresponding position on the other strand. In some embodiments, an N nucleotide listed in a sequence disclosed in Table 2 has a nucleobase that is the same as the N nucleotide at the corresponding position on the other strand. In some embodiments, an N nucleotide listed in a sequence disclosed in Table 2 has a nucleobase that is different from the N nucleotide at the corresponding position on the other strand .

[0087] Certain modified SNCA RNAi agent sense and antisense strands are provided in Table3, Table 4, Table 5, Table 6, and Table 9. Certain modified SNCA RNAi agent antisense strands, as well as their underlying unmodified nucleobase sequences, are provided in Table 3. Certain modified SNCA RNAi agent sense strands, as well as their underlying unmodified nucleobase sequences, are provided in Tables 4, 5, and 6. In forming SNCA RNAi agents, each of the nucleotides in each of the underlying base sequences listed in Tables 3, 4, 5, and 6, as well as in Table 2, above, can be a modified nucleotide.

[0088] The SNCA RNAi agents described herein are formed by annealing an antisense strand with a sense strand. A sense strand containing a sequence listed in Table 2, Table 4, Table 5, or Table 6, can be hybridized to any antisense strand containing a sequence listed in Table 2 or Table 3, provided the two sequences have a region of at least 85% complementarity over a contiguous 16, 17, 18, 19, 20, or 21 nucleotide sequence.

[0089] In some embodiments, a SNCA RNAi agent antisense strand comprises a nucleotide sequence of any of the sequences in Table 2 or Table 3.

[0090] In some embodiments, a SNCA RNAi agent comprises or consists of a duplex having the nucleobase sequences of the sense strand and the antisense strand of any of the sequences in Table 2, Table 3, Table 4, Table 5, Table 6, or Table 9.

[0091] Examples of antisense strands containing modified nucleotides are provided in Table 3. Examples of sense strands containing modified nucleotides are provided in Tables 4, 5 and 6.

[0092] As used in Tables 3, 4, 5, 6, and 10, the following notations are used to indicate modified nucleotides, targeting groups, and linking groups:A = adenosine-3 '-phosphateC = cytidine-3 '-phosphateG = guanosine-3 '-phosphateU = uridine-3 '-phosphateI = inosine-3 '-phosphate a = 2'-O-methyladenosine-3 '-phosphate as = 2'-O-methyladenosine-3'-phosphorothioate c = 2'-O-methylcytidine-3 '-phosphate cs = 2'-O-methylcytidine-3'-phosphorothioate g = 2 '-O-methylguanosine-3 '-phosphate gs = 2'-O-methylguanosine-3'-phosphorothioate i = 2 '-O-methylinosine-3 '-phosphate is = 2'-O-methylinosine-3'-phosphorothioate t = 2'-O-methyl-5-methyluridine-3 '-phosphate ts = 2'-O-methyl-5-methyluridine-3'-phosphorothioate u = 2'-O-methyluridine-3 '-phosphate us = 2'-O-methyluridine-3'-phosphorothioateAf = 2 '-fluoroadenosine-3 '-phosphateAfs = 2'-fluoroadenosine-3'-phosporothioateCf = 2'-fluorocytidine-3 '-phosphateCfs = 2'-fluorocytidine-3'-phosphorothioateGf = 2 '-fluoroguanosine-3 '-phosphateGfs = 2'-fluoroguanosine-3'-phosphorothioateTf = 2'-fluoro-5'-methyluridine-3 '-phosphateTfs = 2'-fluoro-5'-methyluridine-3'-phosphorothioateUf = 2'-fluorouridine-3 '-phosphateUfs = 2'-fluorouridine-3'-phosphorothioate dT = 2'-deoxythymidine-3 '-phosphate dU = 2'-deoxyuridine-3 '-phosphate dUs = 2'-deoxyuridine-3'-phosphorothioate dA = 2 '-deoxy adenosine-3 '-phosphatedAs = 2'-deoxyadenosine-3'-phosphorothioate dG = 2 '-deoxy guanosine-3 '-phosphate dGs = 2'-deoxyguanosine-3'-phosphorothioate dC = 2'-deoxycytidine-3 '-phosphate dCs = 2'-deoxycytidine-3'-phosphorothioateAUNA = 2',3'-seco-adenosine-3 '-phosphateAUNAS = 2',3'-seco-adenosine-3'-phosphorothioateCUNA = 2',3'-seco-cytidine-3 '-phosphateCUNAS = 2',3'-seco-cytidine-3'-phosphorothioateGUNA = 2',3'-seco-guanosine-3 '-phosphateGUNAS = 2',3'-seco-guanosine-3'-phosphorothioateUUNA = 2',3'-seco-uridine-3 '-phosphateUUNAS = 2',3'-seco-uridine-3'-phosphorothioateAGNA = 2',3'-dihydroxypropyl-adenosine a_2N = see Table 10 a_2Ns = see Table 10 aC16 = see Table 10 uC16 = see Table 10 cC16 = see Table 10 gC16 = see Table 10(invAb) = inverted abasic deoxyribonucleotide-5 '- phosphate, see Table 10(invAb)s = inverted abasic deoxyribonucleotide-5 '- phosphorothioate, see Table 10 s = phosphorothioate linkage ss = phosphorodithioate linkage p = terminal phosphate (as synthesized) vpu = vinyl phosphonate 2'-O-methyluridine-3 '-phosphate cPrpa = 5 ’-cyclopropyl phosphonate-2'-O-methyladenosine-3 '-phosphate(see Table 10) cPrpas = 5 ’ -cyclopropyl phosphonate-2'-O-methyladenosine-3'- phosphorothioate (see Table 10) cPrpu = 5 ’-cyclopropyl phosphonate-2'-O-methyluridine-3 '-phosphate (seeTable 10) cPrpus = 5 ’ -cyclopropyl phosphonate-2'-O-methyluridine-3 '- phosphorothioate (see Table 10)(NH2-C6) = see Table 10(NH-C6) = see Table 10(NH-C6)s = see Table 10LP293 = see Table 10LP310 = see Table 10LP429 = see Table 10LP462 = see Table 10LP183 = see Table 10L-1026 = see Table 10 uC16 = see Table 10[CP-1113] = Fabs were capped according to the procedure in Example IF;(see also Table 10 for structure)Fab0061 = see Antigen Binding Proteins, infraFab0070 = see Antigen Binding Proteins, infraFab0016 = see Antigen Binding Proteins, infra

[0093] As the person of ordinary skill in the art would readily understand, unless otherwise indicated by the sequence (such as, for example, by a phosphorothioate linkage “s”), when present in an oligonucleotide, the nucleotide monomers are mutually linked by 5 ’-3’- phosphodiester bonds. As the person of ordinary skill in the art would clearly understand, the inclusion of a phosphorothioate linkage as shown in the modified nucleotide sequences disclosed herein replaces the phosphodiester linkage typically present in oligonucleotides. Further, the person of ordinary skill in the art would readily understand that the terminal nucleotide at the 3’ end of a given oligonucleotide sequence would typically have a hydroxyl (-OH) group at the respective 3’ position of the given monomer instead of a phosphate moiety ex vivo. Additionally, for the embodiments disclosed herein, when viewing the respective strand 5’ 3’, the inverted abasic residues are inserted such that the 3’ position of the deoxyribose is linked at the 3’ end of the preceding monomer on the respective strand (see, e.g., Table 10). Moreover, as the person of ordinary skill would readily understand and appreciate, while the phosphorothioate chemical structures depicted herein typically show the anion on the sulfur atom, the inventions disclosed herein encompass all phosphorothioate tautomers (e.g., where the sulfur atom has a double-bond and the anion is on an oxygen atom). Unless expressly indicated otherwise herein, such understandings of the person of ordinary skill in the art are used when describing the SNCA RNAi agents and compositions of SNCA RNAi agents disclosed herein.

[0094] Certain examples of antigen binding proteins and linking groups used with the SNCA RNAi agents disclosed herein are included in the chemical structures provided below in Table 10. Each sense strand and / or antisense strand can have any antigen binding protein or linking group listed herein, as well as other targeting groups, antigen binding proteins, linking groups, conjugated to the 5' and / or 3' end of the sequence.Table 3. SNCA RNAi Agent Antisense Strand Sequencesa_2N = 2-aminoadenosine nucleotide, (A2N) = 2-aminoadenine nucleotideI = hypoxanthine (inosine) nucleotideTable 4. SNCA Agent Sense Strand Sequences (Shown Without Linkers, Conjugates, or Capping Moieties)a_2N = 2-aminoadenosine nucleotide, (A2N) = 2-aminoadenine nucleotideI = hypoxanthine (inosine) nucleotideTable 5. SNCA Agent Sense Strand Sequences (Shown without antigen binding protein conjugate and with terminal caps (see Table 10 for structure information.))a_2N = 2-aminoadenosine nucleotide, (A2N) = 2-aminoadenine nucleotideI = hypoxanthine (inosine) nucleotideTable 6. SNCA Agent Sense Strand Sequences (shown with lipid binding moiety or antigen binding moiety). The structure of the lipid binding moieties and antigen binding moieties are shown in Tables A and B.

[0095] The SNCA RNAi agents disclosed herein are formed by annealing an antisense strand with a sense strand. A sense strand containing a sequence listed in Table 2, Table 4, Table 5, or Table 6 can be hybridized to any antisense strand containing a sequence listed in Table 2 or Table 3, provided the two sequences have a region of at least 85% complementarity over a contiguous 16, 17, 18, 19, 20, or 21 nucleotide sequence.

[0096] As shown in Table 5 above, certain of the example SNCA RNAi agent nucleotide sequences are shown to further include reactive linking groups at one or both of the 5’ terminal end and the 3’ terminal end of the sense strand. For example, many of the SNCA RNAi agent sense strand sequences shown in Table 5 above have a (NH2-C6) linking group at the 5’ end of the nucleotide sequence. Other linking groups, such as a (6-SS-6) linking group or a (C6-SS-C6) linking group, may be present as well or alternatively in certain embodiments. Such reactive linking groups are positioned to facilitate the linking of targeting ligands, targeting groups, and / or antigen binding proteins to the SNCA RNAi agents disclosed herein. Linking or conjugation reactions are well known in the art and provide for formation of covalent linkages between two molecules or reactants. Suitable conjugation reactions for use in the scope of the inventions herein include, but are not limited to, amide coupling reaction, Michael addition reaction, hydrazone formation reaction, inverse-demand Diels-Alder cycloaddition reaction, oxime ligation, and Copper (I)- catalyzed or strain-promoted azide-alkyne cycloaddition reaction cycloaddition reaction.

[0097] In some embodiments, targeting ligands, can be synthesized as activated esters, such as tetrafluorophenyl (TFP) esters, which can be displaced by a reactive amino group (e.g., NFL-Ce) to attach the targeting ligand to the SNCA RNAi agents disclosed herein. In some embodiments, targeting ligands are synthesized as azides, which can be conjugated to a propargyl or DBCO group, for example, via Copper (I)- catalyzed or strain-promoted azide-alkyne cycloaddition reaction.

[0098] Additionally, certain of the nucleotide sequences can be synthesized with a dT nucleotide at the 3’ terminal end of the sense strand, followed by (3’5’) a linker (e.g., C6-SS-C6). The linker can, in some embodiments, facilitate the linkage to additional components, such as, for example, an antigen binding protein or one or more targeting ligands. As described herein, the disulfide bond of C6-SS-C6 is first reduced, removing the dT from the molecule, which can then facilitate the conjugation of the desired component. The terminal dT nucleotide therefore is not a part of the fully conjugated construct.

[0099] Additionally, certain of the nucleotide sequences can be synthesized with a dT nucleotide at the 3’ terminal end of the sense strand, followed by (3’5’) a linker (e.g., C6-SS-C6). The linker can, in some embodiments, facilitate the linkage to additional components, such as, for example, a lipid or one or more targeting ligands. As described herein, the disulfide bond of C6- SS-C6 is first reduced, removing the dT from the molecule, which can then facilitate the conjugation of the desired component. The terminal dT nucleotide therefore is not a part of the fully conjugated construct.

[0100] In some embodiments, the antisense strand of a SNCA RNAi agent disclosed herein differs by 0, 1, 2, or 3 nucleotides from any of the antisense strand sequences in Table 3 or Table 9. In some embodiments, the sense strand of a SNCA RNAi agent disclosed herein differs by 0, 1, 2, or 3 nucleotides from any of the sense strand sequences in Table 4, Table 5, Table 6, or Table 9.

[0101] In some embodiments, a SNCA RNAi agent antisense strand comprises a nucleotide sequence of any of the sequences in Table 2 or Table 3. In some embodiments, a SNCA RNAi agent antisense strand comprises the sequence of nucleotides (from 5’ end3’ end) 1-17, 2-17, 1-18, 2-18, 1-19, 2-19, 1-20, 2-20, 1-21, 2-21, 1-22, 2-22, 1-23, 2-23, 1-24, or 2-24 of any of the sequences in Table 2, Table 3, or Table 9. In certain embodiments, a SNCA RNAi agent antisense strand comprises or consists of a modified sequence of any one of the modified sequences in Table 3 or Table 9.

[0102] In some embodiments, a SNCA RNAi agent sense strand comprises the nucleotide sequence of any of the sequences in Table 2 or Table 4. In some embodiments, a SNCA RNAi agent sense strand comprises the sequence of nucleotides (from 5’ end3’ end) 1-17, 2-17, 3- 17, 4-17, 1-18, 2-18, 3-18, 4-18, 1-19, 2-19, 3-19, 4-19, 1-20, 2-20, 3-20, 4-20, 1-21, 2-21, 3-21, 4-21, 1-22, 2-22, 3-22, 4-22, 1-23, 2-23, 3-23, 4-23, 1-24, 2-24, 3-24, or 4-24, of any of the sequences in Table 2, Table 4, Table 5, Table 6, or Table 9. In certain embodiments, a SNCA RNAi agent sense strand comprises or consists of a modified sequence of any one of the modified sequences in Table 6 or Table 9.

[0103] For the RNAi agents disclosed herein, the nucleotide at position 1 of the antisense strand (from 5' end 3' end) can be perfectly complementary to a SNCA gene, or can be non- complementary to a SNCA gene. In some embodiments, the nucleotide at position 1 of the antisense strand (from 5' end3' end) is a U, A, or dT (or a modified version of U, A or dT). In some embodiments, the nucleotide at position 1 of the antisense strand (from 5’ end3’ end) forms an A:U or U:A base pair with the sense strand.

[0104] In some embodiments, a SNCA RNAi agent antisense strand comprises the sequence of nucleotides (from 5' end3' end) 2-18 or 2-19 of any of the antisense strand sequences in Table 2, Table 3, or Table 9. In some embodiments, a SNCA RNAi sense strand comprises the sequence of nucleotides (from 5' end3' end) 1-17 or 1-18 of any of the sense strand sequences in Table 2, Table 4, Table 5, Table 6, or Table 9.

[0105] In some embodiments, a SNCA RNAi agent includes (i) an antisense strand comprising the sequence of nucleotides (from 5' end3' end) 2-18 or 2-19 of any of the antisense strand sequences in Table 2, Table 3, or Table 9, and (ii) a sense strand comprising the sequence of nucleotides (from 5' end3' end) 1-17 or 1-18 of any of the sense strand sequences in Table 2, Table 4, Table 5, Table 6, or Table 9.

[0106] A sense strand containing a sequence listed in Table 2 or Table 4 can be hybridized to any antisense strand containing a sequence listed in Table 2 or Table 3 provided the two sequences have a region of at least 85% complementarity over a contiguous 16, 17, 18, 19, 20, or 21 nucleotide sequence. In some embodiments, the SNCA RNAi agent has a sense strand consisting of the modified sequence of any of the modified sequences in Table 4, Table 5, Table 6, or Table 9, and an antisense strand consisting of the modified sequence of any of the modified sequences in Table 3 or Table 9. Certain representative sequence pairings are exemplified by the Duplex ID Nos. shown in Tables 7, 8, and 9.

[0107] In some embodiments, a SNCA RNAi agent comprises, consists of, or consists essentially of a duplex represented by any one of the Duplex ID Nos. presented herein. In some embodiments, a SNCA RNAi agent consists of any of the Duplex ID Nos. presented herein. In some embodiments, a SNCA RNAi agent comprises the sense strand and antisense strand nucleotide sequences of any of the Duplex ID Nos. presented herein. In some embodiments, a SNCA RNAi agent comprises the sense strand and antisense strand nucleotide sequences of any of the Duplex ID Nos. presented herein and a targeting group, linking group, antigen binding protein and / or other non-nucleotide group wherein the targeting group, linking group, antigen binding protein and / or other non-nucleotide group is covalently linked (i.e., conjugated) to the sense strand or the antisense strand. In some embodiments, a SNCA RNAi agent includes the sense strand and antisense strand modified nucleotide sequences of any of the Duplex ID Nos. presented herein. In some embodiments, a SNCA RNAi agent comprises the sense strand and antisense strand modified nucleotide sequences of any of the Duplex ID Nos. presented herein and a targeting group, linking group, and / or other non-nucleotide group, wherein the targetinggroup, linking group, antigen binding protein and / or other non-nucleotide group is covalently linked to the sense strand or the antisense strand.

[0108] In some embodiments, a SNCA RNAi agent comprises an antisense strand and a sense strand having the nucleotide sequences of any of the antisense strand / sense strand duplexes of Tables 2, 7, 8, or 9, and comprises an antigen binding protein. In some embodiments, a SNCA RNAi agent comprises an antisense strand and a sense strand having the nucleotide sequences of any of the antisense strand / sense strand duplexes of Tables 2, 7, 8, or 9, and comprises one or more antigen binding proteins.

[0109] In some embodiments, a SNCA RNAi agent comprises an antisense strand and a sense strand having the nucleotide sequences of any of the antisense strand / sense strand duplexes of Tables 2, 7, 8, or 9, and comprises an antigen binding protein. In some embodiments, a SNCA RNAi agent comprises an antisense strand and a sense strand having the nucleotide sequences of any of the antisense strand / sense strand duplexes of Tables 2, 7, 8, or 9, and comprises one or more antigen binding protein.

[0110] In some embodiments, a SNCA RNAi agent comprises an antisense strand and a sense strand having the nucleotide sequences of any of the antisense strand / sense strand duplexes of Tables 2, 7, 8, or 9, and comprises a PK / PD modulator. In some embodiments, a SNCA RNAi agent comprises an antisense strand and a sense strand having the nucleotide sequences of any of the antisense strand / sense strand duplexes of Tables 2, 7, 8, or 9, and comprises one or more lipid moieties.[OHl] In some embodiments, a SNCA RNAi agent comprises an antisense strand and a sense strand having the nucleotide sequences of any of the antisense strand / sense strand duplexes of Tables 2, 7, 8, or 9, and comprises a lipid moiety. In some embodiments, a SNCA RNAi agent comprises an antisense strand and a sense strand having the nucleotide sequences of any of the antisense strand / sense strand duplexes of Tables 2, 7, 8, or 9, and comprises one or more lipid moieties.

[0112] In some embodiments, a SNCA RNAi agent comprises an antisense strand and a sense strand having the modified nucleotide sequences of any of the antisense strand / sense strand duplexes of Tables 7, 8, and 9.

[0113] In some embodiments, a SNCA RNAi agent comprises an antisense strand and a sense strand having the modified nucleotide sequences of any of the antisense strand / sense strand duplexes of Tables 7, 8, and 9, and comprises an antigen binding protein.

[0114] In some embodiments, a SNCA RNAi agent comprises an antisense strand and a sense strand having the modified nucleotide sequences of any of the antisense strand / sense strand duplexes of Tables 7, 8, and 9, and comprises a lipid moiety.

[0115] In some embodiments, a SNCA RNAi agent comprises, consists of, or consists essentially of any of the duplexes of Tables 7, 8, and 9.

[0116] Table 7. SNCA RNAi Agent Duplexes with Corresponding Sense and Antisense StrandID Numbers and Sequence ID numbers for the modified and unmodified nucleotide sequences.

[0117] Table 8. Conjugate Duplex ID Numbers Referencing Position Targeted On SNCA Gene

[0118] Table 9. Conjugate ID Numbers With Chemically Modified Antisense and Sense Strands (including Linkers and Conjugates)

[0119] In some embodiments, a SNCA RNAi agent is prepared or provided as a salt, mixed salt, or a free-acid. In some embodiments, a SNCA RNAi agent is prepared or provided as a pharmaceutically acceptable salt. In some embodiments, a SNCA RNAi agent is prepared or provided as a pharmaceutically acceptable sodium or potassium salt The RNAi agents described herein, upon delivery to a cell expressing an SNCA gene, inhibit or knockdown expression of one or more SNCA genes in vivo and / or in vitro.Targeting Groups, Linking Groups, Lipid PK / PD modulators and Delivery Vehicles

[0120] In some embodiments, a SNCA RNAi agent contains or is conjugated to one or more non-nucleotide groups including, but not limited to, a targeting group, a linking group, a delivery polymer, a pharmacokinetic / pharmacodynamic (PK / PD) modulator, or a delivery vehicle. The non-nucleotide group can enhance targeting, delivery, or attachment of the RNAi agent. The non-nucleotide group can be covalently linked to the 3' and / or 5' end of either the sense strand and / or the antisense strand. In some embodiments, a SNCA RNAi agent contains a non-nucleotide group linked to the 3' and / or 5' end of the sense strand. In some embodiments, a non-nucleotide group is linked to the 5' end of a SNCA RNAi agent sense strand. A non- nucleotide group can be linked directly or indirectly to the RNAi agent via a linker / linking group. In some embodiments, a non-nucleotide group is linked to the RNAi agent via a labile, cleavable, or reversible bond or linker.

[0121] In some embodiments, a non-nucleotide group enhances the pharmacokinetic or biodistribution properties of an RNAi agent or conjugate to which it is attached to improve cell- or tissue-specific distribution and cell-specific uptake of the conjugate. In some embodiments, a non-nucleotide group enhances endocytosis of the RNAi agent.

[0122] Targeting groups or targeting moieties enhance the pharmacokinetic or biodistribution properties of a conjugate or RNAi agent to which they are attached to improve cell-specific (including, in some cases, organ specific) distribution and cell-specific (or organ specific) uptake of the conjugate or RNAi agent. A targeting group can be monovalent, divalent, trivalent, tetravalent, or have higher valency for the target to which it is directed. Representative targeting groups include, without limitation, compounds with affinity to cell surface molecule, cell receptor ligands, hapten, antibodies, monoclonal antibodies, antibody fragments, and antibody mimics with affinity to cell surface molecules. In some embodiments, a targeting group is linked to an RNAi agent using a linker, such as a PEG linker or one, two,or three abasic and / or ribitol (abasic ribose) residues, which in some instances can serve as linkers.

[0123] A targeting group, with or without a linker, can be attached to the 5' or 3' end of any of the sense and / or antisense strands disclosed in Tables 2, 3, 4, 5, 6, and 9. A linker, with or without a targeting group, can be attached to the 5 ' or 3 ' end of any of the sense and / or antisense strands disclosed in Tables 2, 3, 4, 5, 6, and 9.

[0124] The SNCA RNAi agents described herein can be synthesized having a reactive group, such as an amino group (also referred to herein as an amine), at the 5 '-terminus and / or the 3'- terminus. The reactive group can be used subsequently to attach a targeting moiety using methods typical in the art.

[0125] For example, in some embodiments, the SNCA RNAi agents disclosed herein are synthesized having an NH2-C6 group at the 5 '-terminus of the sense strand of the RNAi agent. The terminal amino group subsequently can be reacted to form a conjugate with, for example, a group that includes a lipid moiety or an antigen binding protein. In some embodiments, the SNCA RNAi agents disclosed herein are synthesized having one or more alkyne groups at the 5 '-terminus of the sense strand of the RNAi agent.

[0126] In some embodiments, targeting groups are linked to the SNCA RNAi agents without the use of an additional linker. In some embodiments, the targeting group is designed having a linker readily present to facilitate the linkage to a SNCA RNAi agent. In some embodiments, when two or more RNAi agents are included in a composition, the two or more RNAi agents can be linked to their respective targeting groups using the same linkers. In some embodiments, when two or more RNAi agents are included in a composition, the two or more RNAi agents are linked to their respective targeting groups using different linkers.

[0127] In some embodiments, a linking group is conjugated to the RNAi agent. The linking group facilitates covalent linkage of the agent to a targeting group, pharmacokinetic modulator, delivery polymer, or delivery vehicle. The linking group can be linked to the 3' and / or the 5' end of the RNAi agent sense strand or antisense strand. In some embodiments, the linking group is linked to the RNAi agent sense strand. In some embodiments, the linking group is conjugated to the 5' or 3' end of an RNAi agent sense strand. In some embodiments, a linking group is conjugated to the 5' end of an RNAi agent sense strand. Examples of linking groups, include but are not limited to: C6-SS-C6, 6-SS-6, reactive groups such a primary amines (e.g., NH2-C6) and alkynes, alkyl groups, abasic residues / nucleotides, amino acids, tri-alkynefunctionalized groups, ribitol, and / or PEG groups. Examples of certain linking groups are provided in Table 10.

[0128] A linker or linking group is a connection between two atoms that links one chemical group (such as an RNAi agent) or segment of interest to another chemical group (such as a targeting group, pharmacokinetic modulator, or delivery polymer) or segment of interest via one or more covalent bonds. A labile linkage contains a labile bond. A linkage can optionally include a spacer that increases the distance between the two joined atoms. A spacer may further add flexibility and / or length to the linkage. Spacers include, but are not to be limited to, alkyl groups, alkenyl groups, alkynyl groups, aryl groups, aralkyl groups, aralkenyl groups, and aralkynyl groups; each of which can contain one or more heteroatoms, heterocycles, amino acids, nucleotides, and saccharides. Spacer groups are well known in the art and the preceding list is not meant to limit the scope of the description. In some embodiments, a SNCA RNAi agent is conjugated to a polyethylene glycol (PEG) moiety, or to a hydrophobic group having 12 or more carbon atoms, such as a cholesterol or palmitoyl group.

[0129] In some embodiments, a SNCA RNAi agent is linked to one or more antigen binding proteins. Antigen binding proteins may enhance the bioavailability of the RNAi agent, the delivery of the RNAi agent to a cell of interest, or the facilitation of shuttling the RNAi agent across the blood brain barrier. In some embodiments, the antigen binding protein may be conjugated to a linker at the 3' or 5' end of a sense strand or an antisense strand of an RNAi agent described herein. In some embodiments, an antigen binding protein may be linked at both the 3' or 5' end of either the sense strand or the antisense strand of an RNAi agent described herein.

[0130] In some embodiments, an antigen binding protein may be conjugated to a SNCA RNAi agent by reacting a SNCA RNAi agent comprising an amine-comprising linker, for example, (NH2-C6) (see Table 10). In some embodiments, the amine-comprising linker may be located on the 5' end of the sense strand or the antisense strand of a SNCA RNAi agent. In some embodiments, the amine-comprising linker may be located on the 3' end of the sense strand or the antisense strand of an RNAi agent.

[0131] Any of the SNCA RNAi agent nucleotide sequences listed in Tables 2, 3, 4, 5, 6, and 9, whether modified or unmodified, can contain 3' and / or 5' targeting group(s), linking group(s), and / or antigen binding fragments. Any of the SNCA RNAi agent duplexes listed in Tables 7, 8, and 9, whether modified or unmodified, can further comprise a targeting group or linking group, but not limited to, those depicted in Table 10, and the targeting group or linkinggroup can be attached to the 3' or 5' terminus of either the sense strand or the antisense strand of the SNCA RNAi agent duplex.

[0132] In some embodiments, a SNCA RNAi agent is linked to one or more lipid PK / PD moi eties (referred to herein as “lipid moi eties” or “PK / PD modulators”.) Lipid PK / PD moi eties may enhance the pharmacodynamic or pharmacokinetic properties of the RNAi agent. In some embodiments, the lipid moiety may be conjugated to a linker at the 3' or 5' end of a sense strand or an antisense strand of an RNAi agent described herein. In some embodiments, a lipid moiety may be linked at both the 3' or 5' end of either the sense strand or the antisense strand of an RNAi agent described herein. Examples of PK / PD modulators may be found, for example, in PCT Publication No. WO2023 / 245061, which is incorporated by reference in its entirety herein.

[0133] In some embodiments, a lipid moiety may be conjugated to a SNCA RNAi agent by reacting a SNCA RNAi agent comprising an amine-comprising linker, for example, (NH2-C6) (see Table 10). In some embodiments, the amine-comprising linker may be located on the 5' end of the sense strand or the antisense strand of a SNCA RNAi agent. In some embodiments, the amine-comprising linker may be located on the 3' end of the sense strand or the antisense strand of an RNAi agent.

[0134] Any of the SNCA RNAi agent nucleotide sequences listed in Tables 2, 3, 4, 5, 6, and 9, whether modified or unmodified, can contain 3' and / or 5' targeting group(s), linking group(s), and / or antigen binding fragments. Any of the SNCA RNAi agent duplexes listed in Tables 7, 8, and 9, whether modified or unmodified, can further comprise a targeting group or linking group, but not limited to, those depicted in Table 10, and the targeting group or linking group can be attached to the 3' or 5' terminus of either the sense strand or the antisense strand of the SNCA RNAi agent duplex.

[0135] In some embodiments, an RNAi agent comprising an amine-comprising linker, such as (NH2-C6) or (NH2-C6)s, may be reacted with a lipid comprising an activated ester moiety. Example lipids with activated ester moi eties include LP-183-p and LP293-p as shown in Table 10 below.

[0136] In some embodiments, a SNCA RNAi agent may be conjugated to a lipid moiety using phosphoramidite synthesis. Synthesizing oligonucleotides using phosphoramidites is well- known in the art. In some embodiments, a lipid moiety may be conjugated to the 5' end of the sense strand or the antisense strand of a SNCA RNAi agent using a phosphoramidite. In some embodiments, a lipid moiety may be conjugated to the 3 ' end of the sense strand or the antisensestrand of a SNCA RNAi agent using a phosphoramidite. In some embodiments, a phosphoramidite such as LP-183 phosphoramidite, shown in Table 10 below, may be used to conjugate a lipid moiety to a SNCA RNAi agent.

[0137] In some embodiments, SNCA RNAi agents may comprise a lipid moiety on an internal nucleotide (i.e., not on the 3' or 5' terminal nucleotides.) In some embodiments, an internal nucleotide may be linked to the 2' position of ribose.

[0138] Any of the SNCA RNAi agent nucleotide sequences listed in Tables 2, 3, 4, 5, 6, and 9, whether modified or unmodified, can contain 3' and / or 5' targeting group(s), linking group(s), and / or lipid PK / PD moieties. Any of the SNCA RNAi agent sequences listed in Tables 3, 4, 5, 6, and 9, or are otherwise described herein, which contain a 3' or 5' targeting group, linking group, and / or lipid PK / PD moiety can alternatively contain no 3' or 5' targeting group, linking group, or lipid PK / PD moiety, or can contain a different 3' or 5' targeting group, linking group, or lipid PK / PD moiety including, but not limited to, those depicted in Table 10. Any of the SNCA RNAi agent duplexes listed in Tables 7, 8, and 9, whether modified or unmodified, can further comprise a targeting group, linking group, or PK / PD moiety including, but not limited to, those depicted in Table 10, and the targeting group, linking group or PK / PD moiety can be attached to the 3' or 5' terminus of either the sense strand or the antisense strand of the SNCA RNAi agent duplex.

[0139] Examples of certain modified nucleotides, capping moieties, PK / PD modulators and linking groups are provided in Table 10.Table 10. Structures Representing Various Modified Nucleotides, Capping Moieties, LipidPK / PD Moieties, and Linking Groups (wherein indicates the point of connection)

[0140] Alternatively, other linking groups known in the art may be used. In many instances, linking groups can be commercially acquired or alternatively, are incorporated into commercially available nucleotide phosphoramidites. (See, e.g., International Patent Application Publication No. WO 2019 / 161213, which is incorporated herein by reference in its entirety).

[0141] In some embodiments, a SNCA RNAi agent is delivered without being conjugated to a targeting ligand or pharmacokinetic / pharmacodynamic (PK / PD) modulator (referred to as being “naked” or a “naked RNAi agent”).

[0142] In some embodiments, a SNCA RNAi agent is delivered without being conjugated to an antigen binding protein or other targeting group (referred to as being “naked” or a “naked RNAi agent”).

[0143] In some embodiments, a SNCA RNAi agent is conjugated to a targeting group, a linking group, a PK modulator, and / or another non-nucleotide group to facilitate delivery of the SNCA RNAi agent to the cell or tissue of choice, for example, to a CNS cell in vivo. In some embodiments, a SNCA RNAi agent is conjugated to an antigen binding protein. In some embodiments, a SNCA RNAi agent is conjugated to a lipid moiety.

[0144] In some embodiments, a delivery vehicle may be used to deliver an RNAi agent to a cell or tissue. A delivery vehicle is a compound that improves delivery of the RNAi agent to a cell or tissue. A delivery vehicle can include, or consist of, but is not limited to: a polymer,such as an amphipathic polymer, a membrane active polymer, a peptide, a melittin peptide, a melittin-like peptide (MLP), a lipid, a reversibly modified polymer or peptide, or a reversibly modified membrane active polyamine.

[0145] In some embodiments, the RNAi agents can be combined with lipids, nanoparticles, polymers, liposomes, micelles, DPCs or other delivery systems available in the art for nucleic acid delivery. The RNAi agents can also be chemically conjugated to targeting groups, lipids (including, but not limited to cholesteryl and cholesteryl derivatives), encapsulating in nanoparticles, liposomes, micelles, conjugating to polymers or DPCs (see, for example WO 2000 / 053722, WO 2008 / 022309, WO 2011 / 104169, and WO 2012 / 083185, WO 2013 / 032829, WO 2013 / 158141, each of which is incorporated herein by reference), by iontophoresis, or by incorporation into other delivery vehicles or systems available in the art such as hydrogels, cyclodextrins, biodegradable nanocapsules, bioadhesive microspheres, or proteinaceous vectors. In some embodiments the RNAi agents can be conjugated to antibodies having affinity for CNS cells. In some embodiments, the RNAi agents can be linked to targeting ligands that have affinity for CNS cells or receptors present on CNS cells.Antigen Binding Proteins

[0146] In one aspect, SNCA RNAi agents are conjugates to antigen binding proteins. In some embodiments, the antigen binding protein may be selected from the group consisting of an antibody, an antibody fragment (e.g., an antigen binding fragment, or Fab), scFv, or other functional component or derivative of an antibody encompassing a Fab and / or complementary- determining regions (CDRs) disclosed herein.

[0147] In some embodiments, the antigen binding protein may act as a shuttle to facilitate the crossing of the blood brain barrier (BBB) of the RNAi agent, such that the RNAi agent may be administered subcutaneously and reach CNS tissue. In some embodiments, the antigen binding protein is an anti-Transferrin 1 (TfRl) antibody or Fab.

[0148] In some embodiments, the antigen binding protein is a Fab. In some embodiments, the Fab comprises (i) 6 complementary determining regions (CDRs), (ii) 3 CDRs on the variable light chain (VL), or (iii) 3 CDRs on the variable heavy chain (VH).

[0149] In some embodiments, the Fab comprises a light chain and a heavy chain. In some embodiments, the light chain comprises a variable light chain (VL) and a light constant chain 1 (CL). In some embodiments, the VL comprises three CDRs. In some embodiments, the VL comprises a VL CDR1, a VL CDR2, and a VL CDR3. In some embodiments, the heavy chaincomprises a variable heavy chain (VH) and a heavy constant chain 1 (CH). In some embodiments, the VH comprises three CDRs. In some embodiments, the VH comprises a VH CDR1, a VH CDR2, and a VH CDR3.

[0150] In some embodiments, the light constant chain 1 (CL) comprises or consists of the sequence:RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 2).

[0151] In some embodiments, the light chain comprises or consists of the sequence: DIQLTQSPSSLSASVGDRVTITCRASDKLYSNLAWYQQKPGKAPKLLIYDATLLASG VPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHFWGTPLTFGQGTKVEIKRTVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 3).

[0152] In some embodiments, the heavy constant chain 1 (CH) comprises or consists of the sequence:ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH (SEQ ID NO: 4).

[0153] In some embodiments the heavy chain comprises or consists of the sequence: EVQLVESGGGLVQPGGSLRLSCATSGFTFTSYWMHWVRQAPGKGLEWVAEINPTN GRTNYIEKFKSRITLSVDKSKSTVYLQMNSLRAEDTAVYYCARGTRAYHYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH (SEQ ID NO: 5).

[0154] In some embodiments, the antigen binding protein may have a VL CDR1 sequence selected from the group consisting of: RASDGLYSNLA (SEQ ID NO: 6), RASDNLYRNLA (SEQ ID NO: 7), and RASDKLYSNLA (SEQ ID NO: 8).

[0155] In some embodiments, the antigen binding protein may have a VL CDR2 sequence selected from the group consisting of: DATLLAS (SEQ ID NO: 9), DARNLAS (SEQ ID NO: 10), DAFNLAS (SEQ ID NO: 11), DATRLAS (SEQ ID NO: 12), DATKLAS (SEQ ID NO: 13), and DAKNLAS (SEQ ID NO: 14).

[0156] In some embodiments, the antigen binding protein may have a VL CDR 3 sequence of QHFWGTPLT (SEQ ID NO: 15).

[0157] In some embodiments, the antigen binding protein may have a VH CDR1 sequence selected from the group consisting of: GYTFNSYWMH (SEQ ID NO: 16), GYTFKSYWMH (SEQ ID NO: 17), GFTFTSYWMH (SEQ ID NO: 18), GYTFTSYWVH (SEQ ID NO: 19), and GYTFTSYWMH (SEQ ID NO: 20).

[0158] In some embodiments, the antigen binding protein may have a VH CDR2 sequence selected from the group consisting of: EINPTNGRVNYIEKFKS (SEQ ID NO: 21), EINPTNGRFNYIEKFKS (SEQ ID NO: 22), EINPTNGRTNYIEKFKS (SEQ ID NO: 23), and EINPTNGRSNYIEKFKS (SEQ ID NO: 24).

[0159] In some embodiments, the antigen binding protein may have a VH CDR3 sequence of: GTRAYHY (SEQ ID NO: 25).

[0160] In some embodiments, the antigen binding protein may have a VL CDR1 sequence of RASDKLYSNLA (SEQ ID NO: 8), a VL CDR2 sequence of DATLLAS (SEQ ID NO: 9), and a VL CDR 3 sequence of QHFWGTPLT (SEQ ID NO: 15).

[0161] In some embodiments, the antigen binding protein may have a VH CDR1 sequence of GFTFTSYWMH (SEQ ID NO: 18), a VH CDR2 sequence of EINPTNGRTNYIEKFKS (SEQ ID NO: 23), and a VH CDR 3 sequence of GTRAYHY (SEQ ID NO: 25).

[0162] In some embodiments, the antigen binding protein may have a VL CDR1 sequence of RASDKLYSNLA (SEQ ID NO: 8), a VL CDR2 sequence of DATLLAS (SEQ ID NO: 9), a VL CDR 3 sequence of QHFWGTPLT (SEQ ID NO: 15), a VH CDR1 sequence of GFTFTSYWMH (SEQ ID NO: 18), a VH CDR2 sequence of EINPTNGRTNYIEKFKS (SEQ ID NO: 23), and a VH CDR 3 sequence of GTRAYHY (SEQ ID NO: 25).

[0163] In some embodiments, the VL comprises a sequence of any one of the sequences listed in Table A. Each of the Fabs described in Table A may have a light chain constant region that comprises or consists of the sequence: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 2).

[0164] In some embodiments, the antigen binding protein may have a VL sequence of: DIQLTQSPSSLSASVGDRVTITCRASDKLYSNLAWYQQKPGKAPKLLIYDATLLASG VPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHFWGTPLTFGQGTKVEIK (SEQ ID NO: 32).

[0165] In some embodiments, the antigen binding protein may have a VH sequence of: EVQLVESGGGLVQPGGSLRLSCATSGFTFTSYWMHWVRQAPGKGLEWVAEINPTNGRTNYIEKFKSRITLSVDKSKSTVYLQMNSLRAEDTAVYYCARGTRAYHYWGQGTL VTVSS (SEQ ID NO: 40).

[0166] In some embodiments, the antigen binding protein may have a VL sequence of: DIQLTQSPSSLSASVGDRVTITCRASDKLYSNLAWYQQKPGKAPKLLIYDATLLASG VPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHFWGTPLTFGQGTKVEIK (SEQ ID NO: 32) and a VH sequence of:EVQLVESGGGLVQPGGSLRLSCATSGFTFTSYWMHWVRQAPGKGLEWVAEINPTN GRTNYIEKFKSRITLSVDKSKSTVYLQMNSLRAEDTAVYYCARGTRAYHYWGQGTL VTVSS (SEQ ID NO: 40).Table A. VL chains with CDR mutation combinations in bold.

[0167] In some embodiments, the VL comprises a sequence of any one of the sequences listed in Table B. Each of the Fabs described in Table B may have a heavy chain constant region that comprises or consists of the sequence:ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH (SEQ ID NO: 4).Table B. VH chains with CDR mutation combinations in bold.

[0168] Tables C-H show the CDR1, CDR2, and CDR3 variants from VL and VH with the combined beneficial mutations.Table C. VL CDR1 variantTable D. VL CDR2 variantsTable E. VL CDR3 variantTable F. VH CDR1 variantsTable G. VH CDR2 variantsTable H. VH CDR3 variants

[0169] In some embodiments, RNAi agents may be conjugated to an antigen binding protein specific to a non-human mammal to carry out studies. RNAi agents may be conjugated to a mouse-specific anti-transferrin antibody such as Fab0016. Fab0016 as used herein refers to an antibody fragment having a light chain sequence ofDIQMTQSPASLSASLEEIVTITCQASQDIGNWLAWYQQKPGKSPQLLIYGATSLADGV PSRFSGSRSGTQFSLKISRVQVEDIGIYYCLQAYNTPWTFGGGTKLELKRADAAPTVS IFPPSTEQLATGGASVVCLMNNFYPRDISVKWKIDGTERRDGVLDSVTDQDSKDSTY SMSSTLSLTKADYESHNLYTCEWHKTSSSPVVKSFNRNEC (SEQ ID NO: 44), and a heavy chain sequence of:EVQLVESGGGLVQPGNSLTLSCVASGFTFSNYGMFIWIRQAPKKGLEWIAMIYYDSS KMNYADTVKGRFTISRDNSKNTLYLEMNSLRSEDTAMYYCAVPTSHYVVDVWGQGVSVTVSSAETTAPSVYPLAPGTALKSNSMVTLGCLVKGYFPEPVTVTWNSGALSSG VHTFPAVLQSGLYTLTSSVTVPSSTWSSQAVTCNVAHPASSTKVDKKIVPREC (SEQ ID NO: 45).

[0170] In some embodiments, the Fab binds TfRl. In some embodiments, the Fab binds TfRl with an affinity of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 nM. In some embodiments, the Fab binds TfRl with an affinity of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 nM. . In some embodiments, the Fab binds TfRl with an affinity of at least about 1 nM. In some embodiments, the Fab binds TfRl with a KD value of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 nM. In someembodiments, the Fab binds TfRl with a KD value of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 nM. In some embodiments, the Fab binds TfRl with a KD value of at least about 1 nM.

[0171] In some embodiments, the Fab is conjugated to an RNAi agent disclosed herein. In some embodiments, the RNAi agent is conjugated to the Fab using a covalent or non-covalent bond, ionic bond, hydrogen bond, hydrophobic interaction, peptide, polymer, or a nucleic acid binding protein. In some embodiments, the RNAi agent is conjugated to the Fab using a covalent bond. In some embodiments, the RNAi agent is conjugated to the Fab via a lysine residue or a cysteine residue. In some embodiments, the RNAi agent is conjugated to the Fab via a lysine residue. In some embodiments, the RNAi agent is conjugated to the Fab via a cysteine residue. In some embodiments, the RNAi agent is conjugated to the Fab in a sitespecific manner. In some embodiments, the RNAi agent is conjugated to the Fab in a non-sitespecific manner.

[0172] In some embodiments, the RNAi agent is conjugated to the Fab at the 5’ terminus or the 3’ terminus of the RNAi agent. In some embodiments, the RNAi agent is conjugated to the Fab at the 5’ terminus of the RNAi agent. In some embodiments, the RNAi agent is conjugated to the Fab at the 3’ terminus of the RNAi agent. In some embodiments, the RNAi agent is conjugated to the Fab at the 5’ terminus or the 3’ terminus of the sense strand of the RNAi agent. In some embodiments, the RNAi agent is conjugated to the Fab at the 5’ terminus of the sense strand of the RNAi agent. In some embodiments, the RNAi agent is conjugated to the Fab at the 3’ terminus of the sense strand of the RNAi agent.Pharmaceutical Compositions and Formulations

[0173] The SNCA RNAi agents disclosed herein can be prepared as pharmaceutical compositions or formulations (also referred to herein as “medicaments”). In some embodiments, pharmaceutical compositions include at least one SNCA RNAi agent. These pharmaceutical compositions are particularly useful in the inhibition of the expression of SNCA mRNA in a target cell, a group of cells, a tissue, or an organism. The pharmaceutical compositions can be used to treat a subject having a disease, disorder, or condition that would benefit from reduction in the level of the target mRNA, or inhibition in expression of the target gene. The pharmaceutical compositions can be used to treat a subject at risk of developing a disease or disorder that would benefit from reduction of the level of the target mRNA or an inhibition in expression the target gene. In one embodiment, the method includes administeringa SNCA RNAi agent linked to a PK / PD modulator as described herein, to a subject to be treated. In one embodiment, the method includes administering a SNCA RNAi agent linked to an antigen binding protein as described herein, to a subject to be treated. In some embodiments, one or more pharmaceutically acceptable excipients (including vehicles, earners, diluents, and / or delivery polymers) are added to the pharmaceutical compositions that include a SNCA RNAi agent, thereby forming a pharmaceutical formulation or medicament suitable for in vivo delivery to a subject, including a human.

[0174] The pharmaceutical compositions that include a SNCA RNAi agent and methods disclosed herein decrease the level of the target mRNA in a cell, group of cells, tissue, organ, or subject, including by administering to the subject a therapeutically effective amount of a herein described SNCA RNAi agent, thereby inhibiting the expression of SNCA mRNA in the subject. In some embodiments, the subject has been previously identified or diagnosed as having a disease or disorder that can be mediated at least in part by a reduction in SNCA gene expression. In some embodiments, the subject has been previously diagnosed with having one or more neurodegenerative diseases, such as one or more synucleinopathies. In some embodiments the neurodegenerative disease is Parkinson’s Disease.

[0175] In some embodiments the subject has been previously diagnosed with having neurodegenerative disease.

[0176] Embodiments of the present disclosure include pharmaceutical compositions for delivering a SNCA RNAi agent to a CNS cell in vivo. Such pharmaceutical compositions can include, for example, a SNCA RNAi agent conjugated to an antigen binding protein. In other embodiments, a pharmaceutical composition can include a SNCA RNAi agent conjugated to a lipid moiety

[0177] In some embodiments, the described pharmaceutical compositions including a SNCA RNAi agent are used for treating or managing clinical presentations in a subject that would benefit from the inhibition of expression of SNCA. In some embodiments, a therapeutically or prophylactically effective amount of one or more of pharmaceutical compositions is administered to a subject in need of such treatment. In some embodiments, administration of any of the disclosed SNCA RNAi agents can be used to decrease the number, severity, and / or frequency of symptoms of a disease in a subject.

[0178] In some embodiments, the described SNCA RNAi agents are optionally combined with one or more additional (i.e., second, third, etc.) therapeutics. A second therapeutic can be another SNCA RNAi agent (e.g., a SNCA RNAi agent that targets a different sequence withina SNCA gene). In some embodiments, a second therapeutic can be an RNAi agent that targets the SNCA gene. An additional therapeutic can also be a small molecule drug, antibody, antibody fragment, and / or aptamer. The SNCA RNAi agents, with or without the one or more additional therapeutics, can be combined with one or more excipients to form pharmaceutical compositions.

[0179] The described pharmaceutical compositions that include a SNCA RNAi agent can be used to treat at least one symptom in a subject having a disease or disorder that would benefit from reduction or inhibition in expression of SNCA mRNA. In some embodiments, the subject is administered a therapeutically effective amount of one or more pharmaceutical compositions that include a SNCA RNAi agent thereby treating the symptom. In other embodiments, the subject is administered a prophylactically effective amount of one or more SNCA RNAi agents, thereby preventing or inhibiting the at least one symptom.

[0180] In some embodiments, one or more of the described SNCA RNAi agents are administered to a mammal in a pharmaceutically acceptable carrier or diluent. In some embodiments, the mammal is a human.

[0181] The route of administration is the path by which a SNCA RNAi agent is brought into contact with the body. In general, methods of administering drugs, oligonucleotides, and nucleic acids including the CNS, for treatment of a mammal are well known in the art and can be applied to administration of the compositions described herein. The SNCA RNAi agents disclosed herein can be administered via any suitable route in a preparation appropriately tailored to the particular route. Thus, in some embodiments, the herein described pharmaceutical compositions are administered via inhalation, intranasal administration, intratracheal administration, or oropharyngeal aspiration administration. In some embodiments, the pharmaceutical compositions can be administered by injection, for example, intravenously, intramuscularly, intracutaneously, intracerebroventricularly, intraarticularly, intraocularly, intraperitoneally, topically, or subcutaneously.

[0182] The pharmaceutical compositions including a SNCA RNAi agent described herein can be delivered to a cell, group of cells, tissue, or subject using oligonucleotide delivery technologies known in the art. In general, any suitable method recognized in the art for delivering a nucleic acid molecule (in vitro or in vivo) can be adapted for use with the compositions described herein. For example, delivery can be by local administration, (e.g., direct injection, implantation, or topical administering), systemic administration, or subcutaneous, intravenous, intraperitoneal, or parenteral routes, including intracranial (e.g.,intraventricular, intraparenchymal and intrathecal), intracerebroventricular, intramuscular, transdermal, airway (aerosol), nasal, oral, rectal, subcutaneous, or topical (including buccal and sublingual) administration. In some embodiments, the compositions are administered via inhalation, intranasal administration, oropharyngeal aspiration administration, or intratracheal administration. For example, in some embodiments, it is desired that the SNCA RNAi agents described herein inhibit the expression of an SNCA gene in the CNS.

[0183] In some embodiments, the pharmaceutical compositions described herein comprise one or more pharmaceutically acceptable excipients. The pharmaceutical compositions described herein are formulated for administration to a subject.

[0184] As used herein, a pharmaceutical composition or medicament includes a pharmacologically effective amount of at least one of the described therapeutic compounds and one or more pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients (excipients) are substances other than the Active Pharmaceutical Ingredient (API, therapeutic product, e.g., SNCA RNAi agent) that are intentionally included in the drug delivery system. Excipients do not exert or are not intended to exert a therapeutic effect at the intended dosage. Excipients can act to a) aid in processing of the drug delivery system during manufacture, b) protect, support or enhance stability, bioavailability or patient acceptability of the API, c) assist in product identification, and / or d) enhance any other attribute of the overall safety, effectiveness, of delivery of the API during storage or use. A pharmaceutically acceptable excipient may or may not be an inert substance.

[0185] Excipients include, but are not limited to: absorption enhancers, anti-adherents, antifoaming agents, anti-oxidants, binders, buffering agents, carriers, coating agents, colors, delivery enhancers, delivery polymers, detergents, dextran, dextrose, diluents, disintegrants, emulsifiers, extenders, fillers, flavors, glidants, humectants, lubricants, oils, polymers, preservatives, saline, salts, solvents, sugars, surfactants, suspending agents, sustained release matrices, sweeteners, thickening agents, tonicity agents, vehicles, water-repelling agents, and wetting agents.

[0186] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor® ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). It should be stable under the conditions of manufacture and storage and should be preserved against the contaminatingaction of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0187] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation include vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0188] Formulations suitable for intra-articular administration can be in the form of a sterile aqueous preparation of the drug that can be in microcrystalline form, for example, in the form of an aqueous microcrystalline suspension. Liposomal formulations or biodegradable polymer systems can also be used to present the drug for both intra-articular and ophthalmic administration.

[0189] The active compounds can be prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0190] The SNCA RNAi agents can be formulated in compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form refers to physically discreteunits suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the disclosure are dictated by and directly dependent on the unique characteristics of the active compound and the therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

[0191] A pharmaceutical composition can contain other additional components commonly found in pharmaceutical compositions. Such additional components include, but are not limited to: anti -pruri tics, astringents, local anesthetics, or anti-inflammatory agents (e.g., antihistamine, diphenhydramine, etc.). It is also envisioned that cells, tissues, or isolated organs that express or comprise the herein defined RNAi agents may be used as “pharmaceutical compositions.” As used herein, “pharmacologically effective amount,” “therapeutically effective amount,” or simply “effective amount” refers to that amount of an RNAi agent to produce a pharmacological, therapeutic, or preventive result.

[0192] In some embodiments, SNCA RNAi agent pharmaceutical compositions may contain salts such as sodium chloride, calcium chloride, magnesium chloride, potassium chloride, sodium phosphate dibasic, sodium phosphate monobasic, or combinations thereof.

[0193] In some embodiments, the methods disclosed herein further comprise the step of administering a second therapeutic or treatment in addition to administering an RNAi agent disclosed herein. In some embodiments, the second therapeutic is another SNCA RNAi agent (e.g., a SNCA RNAi agent that targets a different sequence within the SNCA target). In other embodiments, the second therapeutic can be a small molecule drug, an antibody, an antibody fragment, and / or an aptamer.

[0194] In some embodiments, described herein are compositions that include a combination or cocktail of at least two SNCA RNAi agents having different sequences. In some embodiments, the two or more SNCA RNAi agents are each separately and independently linked to antigen binding proteins. In some embodiments, the two or more SNCA RNAi agents are each separately and independently linked to lipids.

[0195] Described herein are compositions for delivery of SNCA RNAi agents to central nervous system (CNS) cells. Furthermore, compositions for delivery of SNCA RNAi agents to cells, including neurons, astrocytes, microglia and endothelial cells, in vivo, are generally described herein.

[0196] Generally, an effective amount of a SNCA RNAi agent disclosed herein will be in the range of from about 0.0001 to about 20 mg / kg of body weight dose, e.g., from about 0.001 to about 5 mg / kg of body weight dose. In some embodiments, an effective amount of a SNCA RNAi agent will be in the range of from about 0.01 mg / kg to about 3.0 mg / kg of body weight per dose. In some embodiments, an effective amount of a SNCA RNAi agent will be in the range of from about 0.03 mg / kg to about 2.0 mg / kg of body weight per dose. In some embodiments, an effective amount of a SNCA RNAi agent will be in the range of from about 0.01 to about 1.0 mg / kg. In some embodiments, an effective amount of a SNCA RNAi agent will be in the range of from about 0.50 to about 1.0 mg / kg. In some embodiments, a fixed dose of SNCA RNAi agent is administered to the subject. In some embodiments the dose administered to the human subject is between about 1.0 mg and about 750 mg. In some embodiments, the dose of SNCA RNAi agent administered to the human subject is between about 10 mg and about 450 mg. In some embodiments, the dose of SNCA RNAi agent administered to the human subject is between about 25 mg and about 450 mg. In some embodiments, the dose of SNCA RNAi agent administered to the human subject is about 50 mg, about 75 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, or about 450 mg. The amount administered will also likely depend on such variables as the overall health status of the patient, the relative biological efficacy of the compound delivered, the formulation of the drug, the presence and types of excipients in the formulation, and the route of administration. Also, it is to be understood that the initial dosage administered can be increased beyond the above upper level to rapidly achieve the desired blood-level or tissue level, or the initial dosage can be smaller than the optimum. In some embodiments, a dose is administered daily. In some embodiments, a dose is administered weekly. In further embodiments, a dose is administered bi-weekly, tri-weekly, once monthly, or once quarterly (i.e., once every three months).

[0197] For treatment of disease or for formation of a medicament or composition for treatment of a disease, the pharmaceutical compositions described herein including a SNCA RNAi agent can be combined with an excipient or with a second therapeutic agent or treatment including, but not limited to: a second or other RNAi agent, a small molecule drug, an antibody, an antibody fragment, peptide, and / or an aptamer.

[0198] The described SNCA RNAi agents, when added to pharmaceutically acceptable excipients or adjuvants, can be packaged into kits, containers, packs, or dispensers.Methods of Treatment and Inhibition of SNCA Gene Expression

[0199] The SNCA RNAi agents disclosed herein can be used to treat a subject (e.g., a human or other mammal) having a disease or disorder that would benefit from administration of the RNAi agent. In some embodiments, the RNAi agents disclosed herein can be used to treat a subject (e.g., a human) that would benefit from a reduction and / or inhibition in expression of SNCA mRNA and / or a reduction in SNCA protein and / or enzyme levels.

[0200] In some embodiments, the RNAi agents disclosed herein can be used to treat a subject (e.g., a human) having a disease or disorder for which the subject would benefit from reduction in SNCA protein, including but not limited to, synucleinopathies such as Parkinson’s Disease. Treatment of a subject can include therapeutic and / or prophylactic treatment. The subject is administered a therapeutically effective amount of any one or more SNCA RNAi agents described herein. The subject can be a human, patient, or human patient. The subject may be an adult, adolescent, child, or infant. Administration of a pharmaceutical composition described herein can be to a human being or animal.

[0201] Mutant SNCA activity is known to promote neurodegen erative disorders. In some embodiments, the described SNCA RNAi agents are used to treat at least one symptom mediated at least in part by a reduction in mutant SNCA enzyme levels, in a subject. The subject is administered a therapeutically effective amount of any one or more of the described SNCA RNAi agents. In some embodiments, the subject is administered a prophylactically effective amount of any one or more of the described RNAi agents, thereby treating the subject by preventing or inhibiting the at least one symptom.

[0202] In certain embodiments, the present disclosure provides methods for treatment of diseases, disorders, conditions, or pathological states mediated at least in part by SNCA gene expression, in a patient in need thereof, wherein the methods include administering to the patient any of the SNCA RNAi agents described herein.

[0203] In some embodiments, the SNCA RNAi agents are used to treat or manage a clinical presentation or pathological state in a subject, wherein the clinical presentation or pathological state is mediated at least in part by a reduction in SNCA gene expression. The subject is administered a therapeutically effective amount of one or more of the SNCA RNAi agents or SNCA RNAi agent-containing compositions described herein. In some embodiments, the method comprises administering a composition comprising a SNCA RNAi agent described herein to a subject to be treated.

[0204] In a further aspect, the disclosure features methods of treatment (including prophylactic or preventative treatment) of diseases or symptoms that may be addressed by a reduction in SNCA protein and / or enzyme levels, the methods comprising administering to a subject in need thereof a SNCA RNAi agent that includes an antisense strand comprising the sequence of any of the sequences in Table 2, Table 3, or Table 9. Also described herein are compositions for use in such methods.

[0205] The described SNCA RNAi agents and / or compositions that include SNCA RNAi agents can be used in methods for therapeutic treatment of disease or conditions caused by enhanced or elevated SNCA protein and / or enzyme activity levels. Such methods include administration of a SNCA RNAi agent as described herein to a subject, e.g., a human or animal subject.

[0206] In another aspect, the disclosure provides methods for the treatment (including prophylactic treatment) of a pathological state (such as a condition or disease) mediated at least in part by SNCA gene expression, wherein the methods include administering to a subject a therapeutically effective amount of an RNAi agent that includes an antisense strand comprising the sequence of any of the sequences in Table 2, Table 3, or Table 9.

[0207] In some embodiments, methods for inhibiting expression of an SNCA gene are disclosed herein, wherein the methods include administering to a cell an RNAi agent that includes an antisense strand comprising the sequence of any of the sequences in Table 2, Table 3, or Table 9.

[0208] In some embodiments, methods for the treatment (including prophylactic treatment) of a pathological state mediated at least in part by SNCA gene expression are disclosed herein, wherein the methods include administering to a subject a therapeutically effective amount of an RNAi agent that includes a sense strand comprising the sequence of any of the sequences in Table 2, Table 4, Table 5, Table 6, or Table 9.

[0209] In some embodiments, methods for inhibiting expression of an SNCA gene are disclosed herein, wherein the methods comprise administering to a cell an RNAi agent that includes a sense strand comprising the sequence of any of the sequences in Table 2, Table 4, Table 5, Table 6, or Table 9.

[0210] In some embodiments, methods for the treatment (including prophylactic treatment) of a pathological state mediated at least in part by SNCA gene expression are disclosed herein, wherein the methods include administering to a subject a therapeutically effective amount of an RNAi agent that includes a sense strand comprising the sequence of any of the sequencesin Table 4, Table 5, Table 6, or Table 9, and an antisense strand comprising the sequence of any of the sequences in Table 3 or Table 9.

[0211] In some embodiments, methods for inhibiting expression of a SNCA gene are disclosed herein, wherein the methods include administering to a cell an RNAi agent that includes a sense strand comprising the sequence of any of the sequences in Table 4, Table 5, Table 6, or Table 9, and an antisense strand comprising the sequence of any of the sequences in Table 3 or Table 9.

[0212] In some embodiments, methods of inhibiting expression of a SNCA gene are disclosed herein, wherein the methods include administering to a subject a SNCA RNAi agent that includes a sense strand consisting of the nucleobase sequence of any of the sequences in Table 4, Table 5, Table 6, or Table 9, and the antisense strand consisting of the nucleobase sequence of any of the sequences in Table 3 or Table 9. In other embodiments, disclosed herein are methods of inhibiting expression of a SNCA gene, wdierein the methods include administering to a subject a SNCA RNAi agent that includes a sense strand consisting of the modified sequence of any of the modified sequences in Table 4, Table 5, Table 6, or Table 9, and the antisense strand consisting of the modified sequence of any of the modified sequences in Table 3 or Table 9.

[0213] In some embodiments, methods for inhibiting expression of an SNCA gene in a cell are disclosed herein, wherein the methods include administering one or more SNCA RNAi agents comprising a duplex structure of one of the duplexes set forth in Tables 7, 8, and 9.

[0214] In some embodiments, the gene expression level and / or mRNA level of an SNCA gene in certain CNS cells of subject to whom a described SNCA RNAi agent is administered is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99%, relative to the subject prior to being administered the SNCA RNAi agent or to a subject not receiving the SNCA RNAi agent. In some embodiments, the SNCA protein and / or enzyme levels in certain CNS cells of a subject to whom a described SNCA RNAi agent is administered is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99%, relative to the subject prior to being administered the SNCA RNAi agent or to a subject not receiving the SNCA RNAi agent. The gene expression level, protein level, and / or mRNA level in the subject may be reduced in a cell, group of cells, and / or tissue of the subject. In some embodiments, the SNCA mRNA levels in certain CNS cells subject to whom a describedSNCA RNAi agent has been administered is reduced by at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% relative to the subject prior to being administered the SNCA RNAi agent or to a subject not receiving the SNCA RNAi agent.

[0215] A reduction in gene expression, mRNA, and protein levels can be assessed by any methods known in the art. Reduction or decrease in SNCA protein and or enzyme levels are collectively referred to herein as a decrease in, reduction of, or inhibition of SNCA expression. The Examples set forth herein illustrate known methods for assessing inhibition of SNCA gene expression, including but not limited to determining SNCA enzyme levels.Cells, Tissues, Organs, and Non-Human Organisms

[0216] Cells, tissues, organs, and non-human organisms that include at least one of the SNCA RNAi agents described herein are contemplated. The cell, tissue, organ, or non-human organism is made by delivering the RNAi agent to the cell, tissue, organ, or non-human organism.Additional Illustrative Embodiments

[0217] Provided here are certain additional illustrative embodiments of the disclosed technology. These embodiments are illustrative only and do not limit the scope of the present disclosure or of the claims attached hereto.

[0218] Embodiment 1. An RNAi agent for inhibiting expression of a synuclein alpha (SNCA) gene, comprising: an antisense strand comprising at least 17 contiguous nucleotides differing by 0 or 1 nucleotides from any one of the sequences provided in Table 2 or Table 3; and a sense strand comprising a nucleotide sequence that is at least partially complementary to the antisense strand.

[0219] Embodiment 2. The RNAi agent of embodiment 1, wherein the antisense strand comprises nucleotides 2-18 of any one of the sequences provided in Table 2 or Table 3.

[0220] Embodiment 3. The RNAi agent of embodiment 1 or embodiment 2, wherein the sense strand comprises a nucleotide sequence of at least 17 contiguous nucleotides differing by 0 or 1 nucleotides from any one of the sequences provided in Table 2 or Table 4, and wherein the sense strand has a region of at least 85% complementarity over the 17 contiguous nucleotides to the antisense strand.

[0221] Embodiment 4. The RNAi agent of any one of embodiments 1-3, wherein at least one nucleotide of the SNCA RNAi agent is a modified nucleotide or includes a modified internucleoside linkage.

[0222] Embodiment 5. The RNAi agent of any one of embodiments 1-4, wherein all or substantially all of the nucleotides are modified nucleotides.

[0223] Embodiment 6. The RNAi agent of any one of embodiments 4-5, wherein the modified nucleotide is selected from the group consisting of: 2'-O-methyl nucleotide, 2'-fluoro nucleotide, 2'-deoxy nucleotide, 2', 3 '-seco nucleotide mimic, locked nucleotide, 2'-F-arabino nucleotide, 2 '-methoxy ethyl nucleotide, abasic nucleotide, ribitol, inverted nucleotide, inverted 2'-O-methyl nucleotide, inverted 2'-deoxy nucleotide, 2'-amino-modified nucleotide, 2'-alkyl- modified nucleotide, morpholino nucleotide, vinyl phosphonate-containing nucleotide, cyclopropyl phosphonate-containing nucleotide, and 3'-O-methyl nucleotide.

[0224] Embodiment 7. The RNAi agent of embodiment 5, wherein all or substantially all of the nucleotides are modified with 2'-O-methyl nucleotides, 2'-fluoro nucleotides, or combinations thereof.

[0225] Embodiment 8. The RNAi agent of any one of embodiments 1-7, wherein the antisense strand comprises the nucleotide sequence of any one of the modified sequences provided in Table 3.

[0226] Embodiment 9. The RNAi agent of any one of embodiments 1-8, wherein the sense strand comprises the nucleotide sequence of any one of the modified sequences provided in Table 4.

[0227] Embodiment 10. The RNAi agent of embodiment 1, wherein the antisense strand comprises the nucleotide sequence of any one of the modified sequences provided in Table 3 and the sense strand comprises the nucleotide sequence of any one of the modified sequences provided in Table 4.

[0228] Embodiment 11. The RNAi agent of any one of embodiments 1-10, wherein the sense strand is between 18 and 30 nucleotides in length, and the antisense strand is between 18 and 30 nucleotides in length.

[0229] Embodiment 12. The RNAi agent of embodiment 11, wherein the sense strand and the antisense strand are each between 18 and 27 nucleotides in length.

[0230] Embodiment 13. The RNAi agent of embodiment 12, wherein the sense strand and the antisense strand are each between 18 and 24 nucleotides in length.

[0231] Embodiment 14. The RNAi agent of embodiment 13, wherein the sense strand and the antisense strand are each 21 nucleotides in length.

[0232] Embodiment 15. The RNAi agent of embodiment 14, wherein the RNAi agent has two blunt ends.

[0233] Embodiment 16. The RNAi agent of any one of embodiments 1-15, wherein the sense strand comprises one or two terminal caps.

[0234] Embodiment 17. The RNAi agent of any one of embodiments 1-16, wherein the sense strand comprises one or two inverted abasic residues.

[0235] Embodiment 18. The RNAi agent of embodiment 1, wherein the RNAi agent is comprised of a sense strand and an antisense strand that form a duplex having the structure of any one of the duplexes in Table 7, Table 8, or Table 9.

[0236] Embodiment 19. The RNAi agent of embodiment 18, wherein all or substantially all of the nucleotides are modified nucleotides.

[0237] Embodiment 20. The RNAi agent of embodiment 1, comprising an antisense strand that consists of, consists essentially of, or comprises a nucleotide sequence that differs by 0 or 1 nucleotides from any one of the following sequences (5’ - 3'):UGAUACUUCAAUCACUGCU (SEQ ID NO: 217);UUAGAGAACACCCUCUUUU (SEQ ID NO: 110);UUUGAGAAAUGUCAUGACU (SEQ ID NO: 196);UGAUACUUCAAUCACUGCUGU (SEQ ID NO: 891);UUAGAGAACACCCUCUUUUGU (SEQ ID NO: 880); or UUUGAGAAAUGUCAUGACUGG (SEQ ID NO: 890).

[0238] Embodiment 21. The RNAi agent of embodiment 20, comprising an sense strand that consists of, consists essentially of, or comprises a nucleotide sequence that differs by 0 or 1 nucleotides from any one of the following sequences (5’ - 3 '):AGCAGUGAUUGAAGUAUCA (SEQ ID NO: 414) AAAAGAGGGUGUUCUCUAA (SEQ ID NO: 307) AGUCAUGACAUUUCUCAAA (SEQ ID NO: 393) AC AGCAGUGAUUGAAGUAUCA (SEQ ID NO: 958) AC AAAAGAGGGUGUUCUCUAA (SEQ ID NO: 974) CCAGUCAUGACAUUUCUCAAA (SEQ ID NO: 957).

[0239] Embodiment 22. The RNAi agent of embodiment 20 or 21, wherein all or substantially all of the nucleotides are modified nucleotides.

[0240] Embodiment 23. The RNAi agent of embodiment 1, comprising an antisense strand that comprises, consists of, or consists essentially of a modified nucleotide sequence that differs by 0 or 1 nucleotides from one of the following nucleotide sequences (5' 3'): cPrpusGfsauacUfucaaUfcAfcUfgcusgsu (SEQ ID NO: 572); cPrpusUfsagagAfacacCfcUfcUfuuusgsu (SEQ ID NO: 535); or cPrpusUfsugagAfaaugUfcAfuGfacugsg (SEQ ID NO: 495); wherein a represents 2'-O-methyl adenosine, c represents 2'-O-methyl cytidine, g represents 2'-O-methyl guanosine, u represents 2'-O-methyl uridine; Af represents 2'- fluoro adenosine, Cf represents 2'-fluoro cytidine, Gf represents 2'-fluoro guanosine, Uf represents 2'-fluoro uridine; cPrpu represents 5 ’-cyclopropyl phosphonate-2’-O-m ethyl uridine; s represents a phosphorothioate linkage; and wherein all or substantially all of the nucleotides on the sense strand are modified nucleotides.

[0241] Embodiment 24. The RNAi agent of embodiment 1, wherein the sense strand comprises, consists of, or consists essentially of a modified nucleotide sequence that differs by 0 or 1 nucleotides from one of the following nucleotide sequences (5' - 3 '): acagcaguGfAfUfugaaguauca (SEQ ID NO: 691); a_2NcaaaagaGfgGfUfguucucuaa (SEQ ID NO: 698); or ccagucauGfaCfAfuuucucaaa (SEQ ID NO: 631); wherein a represents 2'-O-methyl adenosine, c represents 2'-O-methyl cytidine, g represents 2'-O-methyl guanosine, u represents 2'-O-methyl uridine; Af represents 2'- fluoro adenosine, Cf represents 2'-fluoro cytidine, Gf represents 2'-fluoro guanosine, Uf represents 2'-fluoro uridine, and a_2N represents 2-aminoadenosine.

[0242] Embodiment 25. The RNAi agent of any one of embodiments 20-24, wherein the sense strand further includes inverted abasic residues at the 3’ terminal end of the nucleotide sequence, at the 5’ terminal end of the nucleotide sequence, or both.

[0243] Embodiment 26. The RNAi agent of any one of the preceding embodiments, wherein the RNAi agent is conjugated to a targeting ligand and has the duplex structure of any one of the duplex structures in Table 9.

[0244] Embodiment 27. The RNAi agent of any one of embodiments 1-25, wherein the RNAi agent is conjugated to an antigen binding protein.

[0245] Embodiment 28. The RNAi agent of embodiment 27, wherein the antigen binding protein is conjugated to the sense strand.

[0246] Embodiment 29. The RNAi agent of embodiment 27 or 28, wherein the antigen binding protein is an antibody fragment (Fab) that specifically binds to one or more epitopes on a transferrin receptor (TfRl).

[0247] Embodiment 30. The RNAi agent of embodiment 29, wherein the Fab comprises (i) 6 complementary determining regions (CDRs), (ii) 3 CDRs on the variable light chain (VL), and / or (iii) 3 CDRs on the variable heavy chain (VH).

[0248] Embodiment 31. The RNAi agent of embodiment 30, wherein the variable light chain has a VL CDR1 sequence selected from the group consisting of: RASDGLYSNLA (SEQ ID NO: 6), RASDNLYRNLA (SEQ ID NO: 7), and RASDKLYSNLA (SEQ ID NO: 8); a VL CDR2 sequence selected from the group consisting of: DATLLAS (SEQ ID NO: 9), DARNLAS (SEQ ID NO: 10), DAFNLAS (SEQ ID NO: 11), DATRLAS (SEQ ID NO: 12), DATKLAS (SEQ ID NO: 13), and DAKNLAS (SEQ ID NO: 14); and / or a VL CDR 3 sequence of QHFWGTPLT (SEQ ID NO: 15).

[0249] Embodiment 32. The RNAi agent of embodiment 30 or 31, wherein the variable light chain is selected from any one of the VL chains shown in Table A.

[0250] Embodiment 33. The RNAi agent of any one of embodiments 23-25, wherein the variable light chain comprises the sequence: DIQLTQSPSSLSASVGDRVTITCRASDKLYSNLAWYQQKPGKAPKLLIYDATLLASG VPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHFWGTPLTFGQGTKVEIK (SEQ ID NO: 32).

[0251] Embodiment 34. The RNAi agent of any one of embodiments 30-33, wherein the variable heavy chain has a VH CDR1 sequence selected from the group consisting of: GYTFNSYWMH (SEQ ID NO: 16), GYTFKSYWMH (SEQ ID NO: 17), GFTFTSYWMH (SEQ ID NO: 18), GYTFTSYWVH (SEQ ID NO: 19), and GYTFTSYWMH (SEQ ID NO: 20), a VH CDR2 sequence selected from the group consisting of: EINPTNGRVNYIEKFKS (SEQ ID NO: 21), EINPTNGRFNYIEKFKS (SEQ ID NO: 22), EINPTNGRTNYIEKFKS (SEQ ID NO: 23), and EINPTNGRSNYIEKFKS (SEQ ID NO: 24); and / or a VH CDR3 sequence of: GTRAYHY (SEQ ID NO: 25).

[0252] Embodiment 35. The RNAi agent of any one of embodiments 30-34, wherein the variable heavy chain is selected from any one of the VH chains shown in Table B.

[0253] Embodiment 36. The RNAi agent of any one of embodiments 30-35, wherein the variable heavy chain comprises the sequence:EVQLVESGGGLVQPGGSLRLSCATSGFTFTSYWMHWVRQAPGKGLEWVAEINPTN GRTNYIEKFKSRITLSVDKSKSTVYLQMNSLRAEDTAVYYCARGTRAYHYWGQGTL VTVSS (SEQ ID NO: 40).

[0254] Embodiment 37. The RNAi agent of any one of embodiments 29-36, wherein the Fab further comprises a light constant chain 1 (CL).

[0255] Embodiment 38. The RNAi agent of embodiment 37, wherein the light constant chain 1 (CL) sequence is:RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 2).

[0256] Embodiment 39. The RNAi agent of any one of embodiments 29-38, wherein the Fab further comprises a heavy constant chain 1 (CH).

[0257] Embodiment 40. The RNAi agent of embodiment 39, wherein the heavy constant chain 1 (CH) sequence is:ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH (SEQ ID NO: 4).

[0258] Embodiment 41. The RNAi agent of any one of embodiments 29-40, wherein the antibody fragment (Fab) binds TfRl with an affinity of at least 1 nM KD.

[0259] Embodiment 42. The RNAi agent of any one of embodiments 1-26, wherein the RNAi agent is conjugated to a lipid.

[0260] Embodiment 43. The RNAi agent of embodiment 42, wherein the lipid is selected from any one of the lipid moi eties shown in Table 10.

[0261] Embodiment 44. The RNAi agent of embodiment 42 or 43, wherein the lipid moiety is conjugated to the sense strand.

[0262] Embodiment 45. The RNAi agent of embodiment 44, wherein the lipid moiety is conjugated to the 5’ terminal end of the sense strand.

[0263] Embodiment 46. A conjugate comprising the RNAi agent of any one of embodiments 1-26 conjugated to an antibody fragment (Fab) that specifically binds to one or more epitopes on a transferrin receptor (TfRl).

[0264] Embodiment 47. The conjugate of embodiment 46, wherein the Fab comprises (i) 6 complementary determining regions (CDRs), (ii) 3 CDRs on the variable light chain (VL), or (iii) 3 CDRs on the variable heavy chain (VH).

[0265] Embodiment 48. The conjugate of embodiment 47, wherein the variable light chain has a VL CDR1 sequence selected from the group consisting of: RASDGLYSNLA (SEQ ID NO: 6), RASDNLYRNLA (SEQ ID NO: 7), and RASDKLYSNLA (SEQ ID NO: 8); a VL CDR2 sequence selected from the group consisting of: DATLLAS (SEQ ID NO: 9), DARNLAS (SEQ ID NO: 10), DAFNLAS (SEQ ID NO: 11), DATRLAS (SEQ ID NO: 12), DATKLAS (SEQ ID NO: 13), and DAKNLAS (SEQ ID NO: 14); and / or a VL CDR 3 sequence of QHFWGTPLT (SEQ ID NO: 15).

[0266] Embodiment 49. The conjugate of embodiment 47 or 48, wherein the variable light chain is selected from any one of the VL chains shown in Table A.

[0267] Embodiment 50. The conjugate of any one of embodiments 47-49, wherein the variable light chain comprises the sequence:DIQLTQSPSSLSASVGDRVTITCRASDKLYSNLAWYQQKPGKAPKLLIYDATLLASG VPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHFWGTPLTFGQGTKVEIK (SEQ ID NO: 32).

[0268] Embodiment 51. The conjugate of any one of embodiments 47-50, wherein the variable heavy chain has a VH CDR1 sequence selected from the group consisting of: GYTFNSYWMH (SEQ ID NO: 16), GYTFKSYWMH (SEQ ID NO: 17), GFTFTSYWMH (SEQ ID NO: 18), GYTFTSYWVH (SEQ ID NO: 19), and GYTFTSYWMH (SEQ ID NO: 20), a VH CDR2 sequence selected from the group consisting of: EINPTNGRVNYIEKFKS (SEQ ID NO: 21), EINPTNGRFNYIEKFKS (SEQ ID NO: 22), EINPTNGRTNYIEKFKS (SEQ ID NO: 23), and EINPTNGRSNYIEKFKS (SEQ ID NO: 24); and / or a VH CDR3 sequence of: GTRAYHY (SEQ ID NO: 25).

[0269] Embodiment 52. The conjugate of any one of embodiments 47-51, wherein the variable heavy chain is selected from any one of the VH chains shown in Table B.

[0270] Embodiment 53. The conjugate of any one of embodiments 47-52, wherein the variable heavy chain comprises the sequence: EVQLVESGGGLVQPGGSLRLSCATSGFTFTSYWMHWVRQAPGKGLEWVAEINPTN GRTNYIEKFKSRITLSVDKSKSTVYLQMNSLRAEDTAVYYCARGTRAYHYWGQGTL VTVSS (SEQ ID NO: 40).

[0271] Embodiment 54. The conjugate of any one of embodiments 46-53, wherein the Fab further comprises a light constant chain 1 (CL).

[0272] Embodiment 55. The conjugate of embodiment 54, wherein the light constant chain 1 (CL) sequence is:RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:2).

[0273] Embodiment 56. The conjugate of any one of embodiments 46-55, wherein the Fab further comprises a heavy constant chain 1 (CH).

[0274] Embodiment 57. The conjugate of embodiment 56, wherein the heavy constant chain 1 (CH) sequence is: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH (SEQ ID NO:4).

[0275] Embodiment 58. The conjugate of any one of embodiments 46-57, wherein the antibody fragment (Fab) binds TfRl with an affinity of at least 1 nM KD.

[0276] Embodiment 59. The conjugate of any one of embodiments 46-58, wherein the RNAi agent is conjugated to the Fab using a covalent or non-covalent bond, ionic bond, hydrogen bond, hydrophobic interaction, peptide, polymer, or a nucleic acid binding protein.

[0277] Embodiment 60. The conjugate of any one of embodiments 46-59, wherein the RNAi agent is conjugated to the Fab through a linker comprising a structure selected from thewherein G represents a point of attachment to the Fab, and <R represents a point of attachment to the RNAi agent portion of the conjugate.

[0278] Embodiment 61. A composition comprising the RNAi agent of any one of embodiments 1-45, or the conjugate of any one of embodiments 46-60, wherein the composition further comprises a pharmaceutically acceptable excipient.

[0279] Embodiment 62. The composition of embodiment 61, further comprising a second RNAi agent capable of inhibiting the expression of SNCA gene expression.

[0280] Embodiment 63. The composition of any one of embodiments 61-62, further comprising one or more additional therapeutics.

[0281] Embodiment 64. The composition of any of embodiments 61-63, wherein the RNAi agent is a sodium salt.

[0282] Embodiment 65. The composition of any of embodiments 61-64, wherein the pharmaceutically acceptable excipient is water for injection.

[0283] Embodiment 66. The composition of any of embodiments 61-64, wherein the pharmaceutically acceptable excipient is a buffered saline solution.

[0284] Embodiment 67. A method for inhibiting expression of a SNCA gene in a cell, the method comprising introducing into a cell an effective amount of an RNAi agent of any one of embodiments 1-45, the conjugate of any one of embodiments 46-60, or the composition of any one of embodiments 61-66.

[0285] Embodiment 68. The method of embodiment 67, wherein the cell is within a subject.

[0286] Embodiment 69. The method of embodiment 68, wherein the subject is a human subject.

[0287] Embodiment 70. The method of any one of embodiments 67-69, wherein following the administration of the RNAi agent the SNCA gene expression is inhibited by at least about 30%.

[0288] Embodiment 71. A method of treating one or more symptoms or diseases associated with enhanced or elevated mutant SNCA activity levels, the method comprising administering to a human subject in need thereof a therapeutically effective amount of the RNAi agent of any one of embodiments 1-45, the conjugate of any one of embodiments 46-60, or the composition of any one of embodiments 61-66.

[0289] Embodiment 72. The method of embodiment 71, wherein the disease is a neurodegenerative disease.

[0290] Embodiment 73. The method of embodiment 72, wherein the neurodegenerative disease is a synucleinopathy.

[0291] Embodiment 74. The method of any one of embodiments 71-73, wherein the disease is Parkinson’s Disease.

[0292] Embodiment 75. The method of any one of embodiments 67-74, wherein the RNAi agent is administered at a dose of about 0.01 mg / kg to about 5.0 mg / kg of body weight of the subject.

[0293] Embodiment 76. The method of any one of embodiments 67-74, wherein the RNAi agent is administered at a dose of about 0.03 mg / kg to about 2.0 mg / kg of body weight of the subject.

[0294] Embodiment 77. The method of any of embodiments 67-76, wherein the RNAi agent is administered in two or more doses.

[0295] Embodiment 78. Use of the RNAi agent of any one of embodiments 1-45, or the conjugate of any one of embodiments 46-60, for the treatment of a disease, disorder, or symptom that is mediated at least in part by mutant SNCA activity and / or SNCA gene expression.

[0296] Embodiment 79. Use of the composition according to any one of embodiments 61-66, for the treatment of a disease, disorder, or symptom that is mediated at least in part by SNCA activity and / or SNCA gene expression.

[0297] Embodiment 80. Use of the composition according to any one of embodiments 61-66, for the manufacture of a medicament for treatment of a disease, disorder, or symptom that is mediated at least in part by SNCA activity and / or SNCA gene expression.

[0298] Embodiment 81. The use of any one of embodiments 78-80, wherein the disease is a neurodegenerative disease.

[0299] Embodiment 82. A method of making an RNAi agent of any one of embodiments 1- 45, comprising annealing a sense strand and an antisense strand to form a double-stranded ribonucleic acid molecule.EXAMPLESExample 1. Synthesis of SNCA RNAi Agents.

[0300] SNCA RNAi agent duplexes disclosed herein were synthesized in accordance with the following:

[0301] A. Synthesis. The sense and antisense strands of the SNCA RNAi agents were synthesized according to phosphoramidite technology on solid phase used in oligonucleotide synthesis. Depending on the scale, a MerMade96E® (Bioautomation), a MerMadel2® (Bioautomation), or an OP Pilot 100 (GE Healthcare) was used. Syntheses were performed ona solid support made of controlled pore glass (CPG, 500 A or 600A, obtained from Prime Synthesis, Aston, PA, USA). All RNA and 2'-modified RNA phosphoramidites were purchased from Thermo Fisher Scientific (Milwaukee, WI, USA). Specifically, the 2'-O- methyl phosphoramidites that were used included the following: (5'-O-dimethoxytrityl-N6- (benzoyl)-2'-O-methyl-adenosine-3 -O-(2-cyanoethyl-N,N-diisopropylamino) phosphorami dite, 5 O-dimethoxy -tri tyl-N4-(acetyl)-2'-O-methyl-cytidine-3'-O-(2-cy anoethyl -N,N-diisopropyl- amino) phosphorami dite, (5'-O-dimethoxytrityl-N2-(isobutyryl)-2'-O-methyl-guanosine-3'-O- (2-cyanoethyl-N,N-diisopropylamino) phosphorami dite, and 5'-O-dimethoxytrityl-2'-O- methyl-uridine-3'-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite. The 2'-deoxy-2'- fluoro-phosphoramidites carried the same protecting groups as the 2'-O-methyl RNA amidites. 5 '-dimethoxytri tyl-2'-O-methyl-inosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidites were purchased from Glen Research (Virginia). The inverted abasic (3'-O- dimethoxytrityl-2'-deoxyribose-5'-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidites were purchased from ChemGenes (Wilmington, MA, USA). The following UNA phosphoramidites were used: 5 '-(4, 4'-Dimethoxytrityl)-N6-(benzoyl)-2', 3 '-seco-adenosine, 2'- benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-(4,4'-Dimethoxytrityl)-N- acetyl -2 ',3 '-seco-cytosine, 2'-benzoyl-3 '-[(2-cyanoethyl)-(N,N-diiso-propyl)]- phosphoramidite, 5 '-(4, 4'-Dimethoxytrityl)-N-isobutyryl-2', 3 '-seco-guanosine, 2'-benzoyl-3'- [(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, and 5'-(4,4'-Dimethoxy-trityl)-2',3'- seco-uridine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N- diiso-propyl)]-phosphoramidite. TFA aminolink phosphoramidites were also commercially purchased (ThermoFisher). Linker L6 was purchased as propargyl -PEG5-NHS from BroadPharm (catalog # BP-20907) and coupled to the NH2-C6 group from an aminolink phosphoramidite to form -L6-C6-, using standard coupling conditions. The linker Alk-cyHex was similarly commercially purchased from Lumiprobe (alkyne phosphoramidite, 5 ’-terminal) as a propargyl -containing compound phosphoramidite compound to form the linker -Alk-cyHex-. In each case, phosphorothioate linkages were introduced as specified using the conditions set forth herein. The cyclopropyl phosphonate phosphoramidites were synthesized in accordance with International Patent Application Publication No. WO 2017 / 214112 (see also Altenhofer et. al., Chem. Communications (Royal Soc. Chem.), 57(55):6808-6811 (July 2021)).

[0302] Tri-alkyne-containing phosphoramidites were dissolved in anhydrous dichloromethane or anhydrous acetonitrile (50 mM), while all other amidites were dissolved in anhydrous acetonitrile (50 mM) and molecular sieves (3 A) were added. 5-Benzylthio-lH-tetrazole (BTT,250 mM in acetonitrile) or 5-Ethylthio-lH-tetrazole (ETT, 250 mM in acetonitrile) was used as activator solution. Coupling times were 10 minutes (RNA), 90 seconds (2' O-Me), and 60 seconds (2' F). In order to introduce phosphorothioate linkages, a 100 mM solution of 3-phenyl l,2,4-dithiazoline-5-one (POS, obtained from PolyOrg, Inc., Leominster, MA, USA) in anhydrous acetonitrile was employed.

[0303] Alternatively, tri-alkyne moieties were introduced post-synthetically (see section E, below). For this route, the sense strand was functionalized with a 5' and / or 3' terminal nucleotide containing a primary amine. TFA aminolink phosphoramidite was dissolved in anhydrous acetonitrile (50 mM) and molecular sieves (3 A) were added. 5-Benzylthio-lH- tetrazole (BTT, 250 mM in acetonitrile) or 5-Ethylthio-lH-tetrazole (ETT, 250 mM in acetonitrile) was used as activator solution. Coupling times were 10 minutes (RNA), 90 seconds (2' O-Me), and 60 seconds (2' F). In order to introduce phosphorothioate linkages, a 100 mM solution of 3-phenyl l,2,4-dithiazoline-5-one (POS, obtained from PolyOrg, Inc., Leominster, MA, USA) in anhydrous acetonitrile was employed.

[0304] B. Cleavage and deprotection of support bound oligomer. After finalization of the solid phase synthesis, the dried solid support was treated with a 1 : 1 volume solution of 40 wt. % methylamine in water and 28% to 31% ammonium hydroxide solution (Aldrich) for 1.5 hours at 30°C The solution was evaporated and the solid residue was reconstituted in water (see below).

[0305] C. Purification. Crude oligomers were purified by anionic exchange HPLC using a TSKgel SuperQ-5PW 13 pm column and Shimadzu LC-8 system. Buffer A was 20 mM Tris, 5 mM EDTA, pH 9.0 and contained 20% Acetonitrile and buffer B was the same as buffer A with the addition of 1.5 M sodium chloride. UV traces at 260 nm were recorded. Appropriate fractions were pooled then run on size exclusion HPLC using a GE Healthcare XK 16 / 40 column packed with Sephadex G-25 fine with a running buffer of lOOmM ammonium bicarbonate, pH 6.7 and 20% Acetonitrile or filtered water. Alternatively, pooled fractions were desalted and exchanged into an appropriate buffer or solvent system via tangential flow filtration.

[0306] D. Annealing. Complementary strands were mixed by combining equimolar RNA solutions (sense and antisense) in 1 * PBS (Phosphate-Buffered Saline, 1 *, Corning, Cellgro) to form the RNAi agents. Some RNAi agents were lyophilized and stored at -15 to -25°C. Duplex concentration was determined by measuring the solution absorbance on a UV-Vis spectrometer in 1 * PBS. The solution absorbance at 260 nm was then multiplied by aconversion factor (0.050 mg / (mL-cm)) and the dilution factor to determine the duplex concentration.

[0307] E. Synthesis of antibody-siRNA linkers

[0308] The synthesis of various Fab linkers used throughout the present application are provided below.

[0309] Synthesis of 2,3,5,6-tetrafluorophenyl 16-((3,5-bis(5-(methylsulfonyl)-l,3,4- oxadiazol-2-yl)phenyl)amino)-16-oxo-4,7,10,13-tetraoxahexadecanoate (i.e., L-1026-p)

[0310] Compound 6 (2.35 g, 7.31 mmol; prepared according to Sarbisheh et al. Bioconjugate Chemistry 2020 31 (12), 2789-2806), EDC-HC1 (2.38 g, 12.43 mmol), and K-Oxyma (2.50 g, 13.9 mmol) were combined as solids and slurried in DMF (190 mL) under N2 at ambient temperature. Compound 7 (1.1.92 g, 5.48 mmol) was added as a solution in DMF (10 mL). After 5 m, tri ethylamine (4.5 mL, 32.2 mmol) was added dropwise at ambient temperature. The reaction mixture was heated at 50°C for 2 days. The reaction mixture was concentrated under reduced pressure to a red oil which was slurried in DCM (250 mL) and washed with sat. aq. sodium bicarbonate (200 mL). The layers were separated, and the aqueous layer was further extracted with DCM (100 mL). The combined organic phase was washed with water (200 mL) and brine (200 mL). The organic phase was dried over sodium sulfate, filtered, and concentrated. The residue was purified by normal phase SiCh chromatography with a gradient of ethyl acetate in DCM (0-100%). Yield of compound 8: 1.77 g (49%), partially contaminated with compound 6. Calculated mw for compound 8: 653.77 g / mol, found m / z (ESI, positive mode): 654.83.

[0311] Compound 8 (1.77 g, 2.71 mmol) was dissolved in TFA:DCM [1 : 1] (18 mL) and stirred at ambient temperature for 1 hour. The reaction mixture was concentrated under reduced pressure then co-evaporated with toluene (3 x 30 mL). The residue was purified by normal phase SiCh chromatography with a gradient of DCM containing 0.1% formic acid and methanol (0-7%). Yield of compound 9: 1.30 g (80%). Calculated mw for compound 9: 597.66g / mol, found m / z (ESI, positive mode): 598.79.

[0312] Compound 9 (1.30 g, 2.18 mmol) was dissolved in DCM (50 mL) and cooled to 0°C. A 100 mg / mL solution of m-CPBA solution was prepared by dissolving 10.38 g m-CPBA (77 wt%) in 80 mL DCM and drying with sodium sulfate until clear. To the solution of compound 9 was added 58 mL m-CPBA (5.85 g, 26.1 mmol) dropwise at 0°C. The reaction mixture was warmed to ambient temperature and allowed to proceed overnight. The reaction mixture was concentrated, slurried in DCM 0.1% formic acid (50 mL), and filtered. The filtrate was purified by normal phase SiCh chromatography with a gradient of DCM containing 0.1% formic acid and methanol (0-10%). Yield of compound 10: 1.03 g (72%). Calculated mw for compound 10: 661.65 g / mol, found m / z (ESI, positive mode): 662.65.

[0313] To a solution of compound 10 (1.03 g, 1.56 mmol) in DCM:ACN [4: 1] (15 mL) at 0°C was added EDC (0.448 g, 2.34 mmol) followed by a solution of TFP (0.310 g, 1.87 mmol) in DCM: ACN [4: 1] (5 mL). After 5 m, the reaction mixture was warmed to ambient temperature. After 1.5 h, the reaction mixture was concentrated to dryness. The crude was purified by preparative reverse phase HPLC (Phenomenex Gemini C18 50 mm x 250 mm, 10 um) using a gradient of water / acetonitrile containing 0.1% TFA. Product-containing fractions were concentrated under reduced pressure. Yield of L-1026-p: 1.10 g (87%). Calculated mw for compound L-1026-p: 809.71 g / mol, found m / z (ESI, positive mode): 810.62. *H NMR (400 MHz,[D6]DMSO, 25°C): 5 = 2.64 (t, 2H), 3.00 (t, 2H), 3.49 (m, 12H), 3.74 (m, 10H), 7.92 (m, 1H), 8.34 (t, 1H), 8.68 (d, 2H), 10.67 (s, 1H).

[0314] Synthesis of L20-p

[0315] To a suspension of compound 1 (5.00 g, 22.50 mmol) and CS2CO3 (25.66 g, 78.75 mmol) in anhydrous DMF (80 mL) was added methyl iodide (4.20 mL, 67.50 mmol) at room temperature. The reaction mixture was stirred at room temperature for 48 hours. The reaction mixture was quenched with water (200 mL) and the mixture was extracted with EtOAc (3 x 100 mL). The organic phase was combined and washed with water and brine. The organic layer was dried over anhydrous Na2SO4 and concentrated. Compound 2 was obtained as a light yellow solid, 5.41 g, 96%. Compound 2 was used directly without further purification. LC- MS: [M+H] calculated 251.05, found 251.18.

[0316] To a solution of compound 2 (5.41 g, 21.62 mmol) in THF / H2O (50 mL / 50 mL) was added LiOH (2.59 g, 108.08 mmol) at room temperature. The reaction mixture was stirred at room temperature for 1 hour. After removing THF under vacuum, the pH was adjusted to ~2 by [C] HC1. Then EtOAc (3 x 60 mL) was used to extract. The organic layers were combined, washed with brine, then dried over anhydrous Na2SO4, and concentrated. Compound 3 was obtained as an off-white solid, 5 g, 98%. Compound 3 was used directly without further purification. LC-MS: calculated [M+H] 237.03, found 237.26.

[0317] To a solution of compound 3 (5.81 g, 24.60 mmol) in THF / DMF (80 mL / 20 mL) was added EDC (7.07 g, 36.90 mmol), DMAP (0.30 g, 2.46 mmol) and compound 4 (6.13 g, 36.90 mmol) at room temperature. The reaction mixture was stirred at room temperature overnight. After removing solvent under vacuum, the residue was loaded on a 120 g column and compound 5 was eluted with 0-50% EtOAc in hexanes. Compound 5 was obtained as a white solid, 9.36 g, 99%. LC-MS: calculated [M+H] 385.03, found 385.46.

[0318] To a solution of compound 5 (2.29 g, 5.96 mmol) in DCM (110 mL) was added 70% m-CPBA (5.14 g, 27.79 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 6 hours. Another 1.8 g m-CPBA was added at room temperature. The reaction mixture was stirred at room temperature overnight. After filtration, the solvent was removed under vacuum. The residue was recrystallized from DCM / EtOAc (50 mL / 50 mL) twice. Compound L20-p was obtained as white needle crystals, 1.93 g, 78%. LC-MS: calculated [M+H] 417, found 417.

[0319] F Conjugation of RNAi agents to Fabs.

[0320] RNAi agents described herein comprising a free amine were conjugated to L20-pusing standard amide reaction chemistry following cleavage from the solid phase. To a solution of Fab in PBS (0.2 pmol, 1.0-10.0 mg / mL in PBS) was added a freshly prepared solution of (tris(2-carboxyethyl)phosphine) hydrochloride(TCEP-HC1) in PBS (5-20 eq, 70 mM). The reaction was held overnight at room temperature and covered from light. The next day, TCEP was removed by loading the reaction mixture on a PD-10 desalting column equilibrated with PBS and eluted with PBS. The concentration of Fab in the eluate was determined using the theoretical absorptivity factor at 280 nm. A solution of L20-modified sense strand in sodium phosphate buffer was prepared, and the concentration was determined using the theoretical absorptivity factor at 260 nm. To the desalted Fab solution was added L20-modified sense strand (1-1.3 eq, 0.5-2.5 mM), and the reaction was mixed endover-end. Analysis by SEC Method 1 and AIEX Method 1 show a mixture of starting Fab, DARI, and DAR2. After 1 hour, a solution of CP-1113-p (see Table 10 for structure) in DMSO and added to the reaction mixture (3 eq, 36 mM). After 1 hour, a solution of L-cysteine in PBS was added to the reaction mixture (6-10 eq, 165 mM). Finally, the conjugate was annealed by addition of antisense strand (1.2-1.5 eq, 0.5-2.5 mM). The conjugate was purified by an AKTA Pure FPLC system equipped with 20 mM tris pH 8 (Buffer A), 20 mM tris 1500 mM NaCl (Buffer B), and a 5 x 200 mm column packed with Tosoh SuperQ 5PW (20 micron). The crude reaction mixture was pump loaded onto the column and eluted with a gradient of 10-40% Buffer B. DARI and DAR2 fractions were differentiated by SEC Method 1, AEX Method 1, and Nanodrop 260 / 280 readings. DARI fractions were pooled and buffer exchanged to PBS using a PD-10 desalting column. The purified conjugate was analyzed by SEC Method 1 and eluted as a monomeric peak with a retention time of 13.2 minutes.SEC Method 1AIEX Method 1

[0321] RNAi agents described herein comprising a free amine were conjugated to L1026-p:following cleavage from the solid phase according to the following procedure:

[0322] To a solution of Fab0070 (28 mg, 0.59 pmol, 5.55 mg / mL in PBS) was added a freshly prepared solution of TCEP-HC1 in PBS (5 eq, 70 mM, 42 pL). The reduction was mixed endover-end at ambient temperature for 15 minutes then held at 5°C overnight without agitation. The next day, TCEP was removed by loading the reaction mixture on two PD-10 desalting columns (Cytiva) equilibrated with 20 mM tris 50 mM NaCl pH 7.6 (alternatively, 20 mM tris pH 8 or PBS buffer can be used) and eluted with the same buffer. The concentration of the Fab in the eluate was determined using the theoretical absorptivity factor at 280 nm. A solution of L-1026-modified sense strand (CS915332) in 10 mM sodium phosphate buffer pH 6.0-6.5 was prepared, and the concentration was determined using the theoretical absorptivity factor at 260 nm. To the desalted Fab solution was added L-1026-modified CS915332 (1.15 eq, 2.75 mM, 240 pL), and the reaction was mixed end-over-end at ambient temperature. Analysis by SEC Method 1 and AIEX Method 1 show a mixture of starting Fab0070, DARI product, and DAR2product. After 30 m, a solution of L-cysteine in 20 mM tris 50 mM NaCl pH 7.6 (alternatively, some L-1026 conjugates have been prepared in 20 mM tris pH 8 or PBS buffer solutions) was added to the reaction mixture (10 eq, 165 mM, 36 pL). After 30 m, the conjugate was annealed by addition of antisense strand (CA003820) (1.3 eq, 1.45 mM in water, 529 pL). The conjugate was purified by an AKTA Pure FPLC system equipped with 20 mM tris pH 8 (Buffer A), 20 mM tris 1500 mM NaCl (Buffer B), and a 5 x 200 mm column packed with Tosoh SuperQ 5PW (20 micron). The crude reaction mixture was loaded onto the column and eluted with a gradient of 10-40% Buffer B. DARI and DAR2 fractions were differentiated by SEC Method 1, AIEX Method 1, and UV-Vis 260 / 280 measurements. DARI fractions were pooled and buffer exchanged to PBS using two PD-10 columns. The purified conjugate was analyzed by SEC Method 1 and eluted as a monomeric peak with a retention time of 7.2 minutes.SEC Method 1AIEX Method 1Example 2. Synthesis of Lipids

[0323] Synthesis of LP-183 phosphoramidite

[0324] To a solution of compound 2 (2.00 g) in DCM was added TEA (2.27 mL) followed by compound 1 (4.931 g) dropwise at room temperature. Then the mixture was stirred at room temperature for 2h. The mixture was then filtered. The white solid was dried overnight. Product is as white solid, yield, 4.267g, 74%. LC-MS: calculated [M+H] 356.35, found 356.63.

[0325] To a mixture of compound 1 (2.54 g) in 120 mL DCM was added compound 3 (0.61 g) followed by compound 2 (5.37 g) dropwise at room temperature. Then the mixture was stirred at room temperature overnight. 5 mL TEA was added followed by Celite. After removing solvent in vacuo, the residue was loaded on a 40g column by dry method. Hexanes (2% TEA) to 50% EtOAc (2% TEA) in Hexanes (2% TEA) as gradient was used to purify the product. Product is a white waxy solid, yield 3.462 g, 87%. LC-MS: calculated [M+H] 556.46, found 556.64.

[0326] Synthesis of LP293-p U

[0327] To a solution of compound 1 (73 mg), NEt3 (0.112 mL), and COMU (126 mg) in DMF was added compound 2 (48.9 mg) under ambient conditions. The reaction was stirred until full conversion was observed by LC-MS. Conversion was not able to be clearly observed by LC-MS, and instead, reaction was allowed to stir for 30 min. until bright yellow color (before the addition of compound 2) transitioned to a honey orange color and all material was observed to be mainly dissolved. The reaction mixture was then washed with water, extracted with DCM, dried over Na2SO4, filtered, and concentrated under vacuum. The residue was purified by CombiFlash® via DCM liquid-load onto a 12-g column with a gradient hexanes to 100% EtOAc in which product eluted at 30% B. The product was concentrated under vacuum to provide a white solid residue and confirmed by 1H NMR in CDCh.

[0328] Conjugation of lipid PK / PD modulator precursors

[0329] Either prior to or after annealing, one or more lipid PK / PD modulator precursors can be linked to the RNAi agents disclosed herein. The following describes the general conjugation process used to link lipid PK / PD modulator precursors to the constructs set forth in the Examples depicted herein.

[0330] A. Conjugation of Activated Ester PK / PD modulators

[0331] The following procedure was used to conjugate PK / PD modulators having an activated ester moiety such as TFP (tetrafluorophenoxy) or PNP (para-nitrophenol) to an RNAi agent with an amine-functionalized sense strand, such as C6-NH2, NH2-C6, or (NH2-C6). An annealed RNAi Agent dried by lyophilization was dissolved in DMSO and 10% water (v / v%) at 25 mg / mL. Then 50-100 equivalents of TEA and 3 equivalents of activated ester PK / PD modulator were added to the solution. The solution was allowed to react for 1-2 hours, while monitored by RP- HPLC-MS (mobile phase A 100 mM HFIP, 14 mM TEA; mobile phase B: acetonitrile on an Waters™ XBridge Cl 8 column, Waters Corp.)

[0332] The product was then precipitated by adding 12 mL acetonitrile and 0.4 mL PBS and centrifuging the solid to a pellet. The pellet was then re-dissolved in 0.4 mL of 1XPBS and 12 mL of acetonitrile. The resulting pellet was dried on high vacuum for one hour.

[0333] B. Conjugation of phosphoramidite PK / PD modulators

[0334] PK / PD modulators having a phosphoramidite moiety may be attached on resin using typical oligonucleotide manufacturing conditions.

[0335] C. Hydrolysis of PK / PD modulators

[0336] Certain PK / PD modulators are hydrolyzed in the cleavage and deprotection conditions described in Example 1, above.Example 3. In Vivo Administration of SNCA RNAi Agents in SNCA Transgenic Mice.

[0337] SNCA RNAi agents were evaluated in vivo in mice. On Day 1, four (n=4) FBV-129S6- SncatalNbmTg(SNCA)lNbm / J mice (2 male, 2 female) for each group were dosed, via intracerebroventricular (ICV) injection, with SNCA RNAi agents formulated in artificial cerebrospinal fluid (aCSF) (at 100 pg total SNCA RNAi agent) or with aCSF. The SNCA RNAi agents were formulated at 10 mg / mL at 10 pL total injection volume. The dosing was in accordance with the following Table 11.

[0338] FBV-129S6-SncatmlNbmTg(SNCA)lNbm / J mice can also be referred to as Snca'; PAC- Tg(SNCAWT), or PAC-Tg(SNCAWT); Snca- / -. These PAC-Tg(SNCAWT); Snca- / - mice harbor a Snca knock-out allele and a transgene encoding the human SNCA.

[0339] Table 11. Dosing for mice of Example 3.

[0340] Each of the SNCA RNAi agents included modified nucleotides and were conjugated at the 5’ terminal end of the sense strand to a targeting ligand that included a lipid moiety or antigen binding moiety having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5, 6, 7, 8, 9, and 10 for specific modifications and structure information related to the SNCA RNAi agents, including the lipid and antigen binding moieties).

[0341] On Day 8, the mice were euthanized. From the mice, right half of the brain and thoracic spinal cord were harvested and collected for analysis. hSNCA expression was analyzed via qPCR in the cortex, thoracic spinal cord, cerebellum, and midbrain, with mPPIA as endogenous gene, normalized to Group 1 mice dosed with aCSF. The hSNCA expression data is shown in the following Table 12.

[0342] Table 12. SNCA expression in mice brain tissues of Example 3.

[0343] SNCA RNAi agents achieved inhibition of SNC A transcripts in SNCA transgenic mice out to at least Day 8. In the cortex, Groups 2-20 showed reduction in SNCA transcripts. In the thoracic spinal cord, Groups 2-20 showed reduction in SNCA transcripts. In the cerebellum, Groups 5 and 9-14 showed reduction in SNCA transcripts; Groups 2-4, 6-8, and 15-20 showed negligible to no reduction in SNCA transcripts. In the midbrain, Groups 2-6, 10-15, and 17-20 showed reduction in SNCA transcripts; Groups 7-9 and 16 showed negligible to no reduction in SNCA transcripts. Most notably, a single dose 100 pg AC003880 achieved -95% SNCA transcript inhibition (0.051) in the cortex.Example 4. In Vivo Administration of SNCA RNAi Agents in SNCA Transgenic Mice.

[0344] SNCA RNAi agents were evaluated in vivo in mice. On Days 1, 2, 3, and 4, four (n=4) FBV-129S6-SncatmlNbmTg(SNCA)lNbm / J mice (2 male, 2 female) for each group were dosed, via subcutaneous (SC) injection, with SNCA RNAi agents formulated in PBS (at 1.0 mg / kg or 3.0 mg / kg) or with aCSF . The SNCA RNAi agent doses were adjusted for individual body weight. The SNCA RNAi agents were formulated at 250 pL / 25 g at 10 pL total injection volume. The dosing was in accordance with the following Table 13.

[0345] FBV-129S6-SncatmlNbmTg(SNCA)lNbm / J mice can also be referred to as Snca'; PAC- Tg(SNCAWT), or PAC-Tg(SNCAWT); Snca- / -. These PAC-Tg(SNCAWT); Snca- / - mice harbor a Snca knock-out allele and a transgene encoding the human SNCA.

[0346] Table 13. Dosing for mice of Example 4.

[0347] * Groups 14 and 15 were dosed with SNCA RNAi agent AC007343 that is of a different lot from Groups 5 and 11, and therefore Groups 14 and 15 are denoted as “AC007343-2.”

[0348] Each of the SNCA RNAi agents included modified nucleotides and were conjugated at the 5’ terminal end of the sense strand to a targeting ligand that included a lipid moiety or antigen binding moiety having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5, 6, 7, 8, 9, and 10 for specific modifications and structure information related to the SNCA RNAi agents, including the lipid and antigen binding moieties).

[0349] On Day 15, the mice were euthanized. From the mice, right half of the brain and thoracic spinal cord were harvested and collected for analysis. hSNCA expression was analyzed via qPCR in the cortex, thoracic spinal cord, cerebellum, and midbrain, with mPPIA as endogenous gene, normalized to Group 1 mice dosed with aCSF. The hSNCA expression data is shown in the following Table 14.

[0350] Table 14. SNCA expression in mice brain tissues of Example 4.

[0351] SNCA RNAi agents achieved inhibition of SNC A transcripts in SNCA transgenic mice out to at least Day 15. In the cortex, Groups 2-15 showed reduction in SNCA transcripts. In the thoracic spinal cord, Groups 2-15 showed reduction in SNCA transcripts. In the cerebellum, Groups 3-15 showed reduction in SNCA transcripts. In the midbrain, Groups 2- 15 showed reduction in SNCA transcripts. Most notably, four (4) doses of 3.0 mg / kgAC007340 achieved -81% SNCA transcript inhibition (0.190) in the cortex. In the cortex, a dose-response was observed for all test groups. In the thoracic spinal cord, a dose-response was observed for AC007341, AC007342, AC007343, AC007344, AC007345, and AC007343- 2. In the cerebellum, a dose-response was observed for AC007340, AC007341, AC007342, and AC007343-2. In the midbrain, a dose-response was observed for AC007340, AC007341, AC007342, AC007343, AC007344, and AC007345.Example 5. In Vivo Administration of SNCA RNAi Agents in Mice.

[0352] SNCA RNAi agents were evaluated in vivo in mice. On Day 1, five (n=5) female C57B1 / 6 mice for each group were dosed, via intracerebroventricular (ICV) injection, with SNCA RNAi agents formulated in artificial cerebrospinal fluid (aCSF) (at 45 pg total SNCA RNAi agent) or with aCSF. The SNCA RNAi agents were formulated at 4.5 mg / mL at 10 pL total injection volume. The dosing was in accordance with the following Table 15.

[0353] Table 15. Dosing for mice of Example 5.

[0354] The SNCA RNAi agents evaluated in this Example target both human and mouse SNCA.

[0355] Each of the SNCA RNAi agents included modified nucleotides and were conjugated at the 5’ terminal end of the sense strand to a targeting ligand that included a lipid moiety or antigen binding moiety having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5, 6, 7, 8, 9, and 10 for specific modifications and structureinformation related to the SNCA RNAi agents, including the lipid and antigen binding moieties).

[0356] On Day 8, the mice were euthanized. From the mice, right half of the brain and thoracic spinal cord were harvested and collected for analysis. mSNCA expression was analyzed via qPCR in the cortex, thoracic spinal cord, cerebellum, and midbrain, with mPPIA as endogenous gene, normalized to Group 1 mice dosed with aCSF. The mSNCA expression data is shown in the following Table 16.

[0357] Table 16. SNCA expression in mice brain tissues of Example 5.

[0358] SNCA RNAi agents achieved inhibition of SNCA transcripts in mice out to at least Day 8. In the cortex, thoracic spinal cord, and midbrain, Groups 2-10 showed reduction in SNCA transcripts. In the cerebellum, Groups 2-7, 9, and 10 showed reduction in SNCA transcripts; Group 8 showed negligible to no reduction in SNCA transcripts. Most notably, a single dose 45 pg AC008875 achieved -72% SNCA transcript inhibition (0.278) in the thoracic spinal cord.Example 6. In Vivo Administration of SNCA RNAi Agents in SNCA Transgenic Mice.

[0359] SNCA RNAi agents were evaluated in vivo in mice. On Day 1, four (n=4) FBV-129S6- SncatalNbmTg(SNCA)lNbm / J mice (2 male, 2 female) for each group were dosed, via intracerebroventricular (ICV) injection, with SNCA RNAi agents formulated in artificial cerebrospinal fluid (aCSF) (at 30 pg total SNCA RNAi agent) or with aCSF. The SNCA RNAi agents were formulated at 3 mg / mL at 10 pL total injection volume. The dosing was in accordance with the following Table 17.

[0360] FBV-129S6-SncatmlNbmTg(SNCA)lNbm / J mice can also be referred to as Snca'; PAC- Tg(SNCAWT), or PAC-Tg(SNCAWT); Snca- / -. These PAC-Tg(SNCAWT); Snca- / - mice harbor a Snca knock-out allele and a transgene encoding the human SNCA.

[0361] Table 17. Dosing for mice of Example 6.

[0362] Each of the SNCA RNAi agents included modified nucleotides and were conjugated at the 5’ terminal end of the sense strand to a targeting ligand that included a lipid moiety or antigen binding moiety having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5, 6, 7, 8, 9, and 10 for specific modifications and structure information related to the SNCA RNAi agents, including the lipid and antigen binding moieties).

[0363] On Day 8, the mice were euthanized. From the mice, right half of the brain and thoracic spinal cord were harvested and collected for analysis. hSNCA expression was analyzed via qPCR in the cortex, thoracic spinal cord, cerebellum, and midbrain, with mPPIA as endogenous gene, normalized to Group 1 mice dosed with aCSF. The hSNCA expression data is shown in the following Table 18.

[0364] Table 18. SNCA expression in mice brain tissues of Example 6.

[0365] SNCA RNAi agents achieved inhibition of SNC A transcripts in SNCA transgenic mice out to at least Day 8. In the cortex and thoracic spinal cord, Groups 2-10 showed reduction in SNCA transcripts. In the cerebellum, Groups 2-6, 8, and 9 showed reduction in SNCA transcripts; Groups 7 and 10 showed negligible to no reduction in SNCA transcripts. In the midbrain, Groups 2-6 and 8-10 showed reduction in SNCA transcripts; Group 7 showed negligible to no reduction in SNCA transcripts. Most notably, a single dose 30 pg AC009465 achieved -81% SNCA transcript inhibition (0.194) in the cortex.Example 7. In Vivo Administration of SNCA RNAi Agents in SNCA Transgenic Mice.

[0366] SNCA RNAi agents were evaluated in vivo in mice. On Day 1, four (n=4) FBV-129S6- SncatalNbmTg(SNCA)lNbm / J mice (2 male, 2 female) for each group were dosed, via intracerebroventricular (ICV) injection, with SNCA RNAi agents formulated in artificial cerebrospinal fluid (aCSF) (at 30 pg total SNCA RNAi agent) or with aCSF. The SNCA RNAi agents were formulated at 3 mg / mL at 10 pL total injection volume. The dosing was in accordance with the following Table 19.

[0367] FBV-129S6-SncatmlNbmTg(SNCA)lNbm / J mice can also be referred to as Snca'; PAC- Tg(SNCAWT), or PAC-Tg(SNCAWT); Snca- / -. These PAC-Tg(SNCAWT); Snca- / - mice harbor a Snca knock-out allele and a transgene encoding the human SNCA.

[0368] Table 19. Dosing for mice of Example 7.

[0369] Each of the SNCA RNAi agents included modified nucleotides and were conjugated at the 5’ terminal end of the sense strand to a targeting ligand that included a lipid moiety or antigen binding moiety having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5, 6, 7, 8, 9, and 10 for specific modifications and structure information related to the SNCA RNAi agents, including the lipid and antigen binding moieties).

[0370] On Day 8, the mice were euthanized. From the mice, right half of the brain and thoracic spinal cord were harvested and collected for analysis. hSNCA expression was analyzed via qPCR in the cortex, thoracic spinal cord, cerebellum, and midbrain, with mPPIA as endogenous gene, normalized to Group 1 mice dosed with aCSF. The hSNCA expression data is shown in the following Table 20.

[0371] Table 20. SNCA expression in mice brain tissues of Example 7.

[0372] SNCA RNAi agents achieved inhibition of SNC A transcripts in SNCA transgenic mice out to at least Day 8. In the cortex, thoracic spinal cord, and midbrain, Groups 2-9 showed reduction in SNCA transcripts. Most notably, a single 30 pg dose of AC009317 achieved -77% SNCA transcript inhibition (0.230) in the cortex.Example 8. In Vivo Administration of SNCA RNAi Agents in SNCA Transgenic Mice.

[0373] SNCA RNAi agents were evaluated in vivo in mice. On Day 1, four (n=4) FBV-129S6- SncatalNbmTg(SNCA)lNbm / J mice (2 male, 2 female) for each group were dosed, via intracerebroventricular (ICV) injection, with SNCA RNAi agents formulated in artificial cerebrospinal fluid (aCSF) (at 30 pg or 60 pg total SNCA RNAi agent) or with aCSF. The SNCA RNAi agents were formulated at 3 mg / mL or 6 mg / mL at 10 pL total injection volume. The dosing was in accordance with the following Table 21.

[0374] FBV-129S6-SncatmlNbmTg(SNCA)lNbm / J mice can also be referred to as Snca'; PAC- Tg(SNCAWT), or PAC-Tg(SNCAWT); Snca- / -. These PAC-Tg(SNCAWT); Snca- / - mice harbor a Snca knock-out allele and a transgene encoding the human SNCA.

[0375] Table 21. Dosing for mice of Example 8.

[0376] Each of the SNCA RNAi agents included modified nucleotides and were conjugated at the 5’ terminal end of the sense strand to a targeting ligand that included a lipid moiety or antigen binding moiety having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5, 6, 7, 8, 9, and 10 for specific modifications and structure information related to the SNCA RNAi agents, including the lipid and antigen binding moieties).

[0377] On Day 8, the mice were euthanized. From the mice, right half of the brain and thoracic spinal cord were harvested and collected for analysis. hSNCA expression was analyzed via qPCR in the cortex, thoracic spinal cord, cerebellum, and midbrain, with mPPIA as endogenous gene, normalized to Group 1 mice dosed with aCSF. The hSNCA expression data is shown in the following Table 22.

[0378] Table 22. SNCA expression in mice brain tissues of Example 8.

[0379] SNCA RNAi agents achieved inhibition of SNCA transcripts in SNCA transgenic mice out to at least Day 8. In the cortex, thoracic spinal cord, and midbrain, groups 2-8 showed reduction in SNCA transcripts, with group 5 showing particularly high reduction of hSNCA in the cortex and thoracic spinal cord. In the cerebellum, Groups 2-7 showed reduction in SNCA transcripts, with group 8 showing minimal reduction of hSNCA.Example 9. In Vivo Administration of SNCA RNAi Agents in SNCA Transgenic Mice.

[0380] SNCA RNAi agents were evaluated in vivo in mice. On Day 1, four (n=4) FBV-129S6- SncatalNbmTg(SNCA)lNbm / J mice (2 male, 2 female) for each group were dosed, via intracerebroventricular (ICV) injection, with SNCA RNAi agents formulated in artificial cerebrospinal fluid (aCSF) (at 30 pg or 60 pg total SNCA RNAi agent) or with aCSF. The SNCA RNAi agents were formulated at 3 mg / mL or 6 mg / mL at 10 pL total injection volume. The dosing was in accordance with the following Table 23.

[0381] FBV-129S6-SncatmlNbmTg(SNCA)lNbm / J mice can also be referred to as Snca'; PAC- Tg(SNCAWT), or PAC-Tg(SNCAWT); Snca- / -. These PAC-Tg(SNCAWT); Snca- / - mice harbor a Snca knock-out allele and a transgene encoding the human SNCA.

[0382] Table 23. Dosing for mice of Example 9.

[0383] *AC006654 is a duplex having the antisense strand sequence: vpusdCscadAcauuugdTcAfcuugcucsusu (SEQ ID NO: 505), and the sense strand sequence: gsasgcaaC16guGfAfCfaaauguugsgsa (SEQ ID NO: 729).

[0384] ** CA007436 is an antisense oligonucleotide (ASO) having the sequence: AMs,mCM,AMs,GMs,AMs,dTs,dAs,dTs,dTs,dTs,dTs,dTs,dGs,dTs,dTs,mCM,TM,GMs,mC Ms,mCM; wherein mC is a 5 ’-methyl cytosine nucleobase, M is a 2’ -MOE sugar moiety, dN is a deoxy nucleotide, N is a nucleobase (e.g., A adenine, C cytosine, G guanosine, T thymine, U uridine), and s is a phosphorothioate linkage.

[0385] Each of the SNCA RNAi agents included modified nucleotides and were conjugated at the 5’ terminal end of the sense strand to a targeting ligand that included a lipid moiety or antigen binding moiety having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5, 6, 7, 8, 9, and 10 for specific modifications and structure information related to the SNCA RNAi agents, including the lipid and antigen binding moieties).

[0386] On Day 8, the mice were euthanized. From the mice, right half of the brain and thoracic spinal cord were harvested and collected for analysis. hSNCA expression was analyzed via qPCR in the cortex, thoracic spinal cord, cerebellum, and midbrain, with mPPIA as endogenous gene, normalized to Group 1 mice dosed with aCSF. The hSNCA expression data is shown in the following Table 24.

[0387] Table 24. SNCA expression in mice brain tissues of Example 9.

[0388] SNCA RNAi agents achieved inhibition of SNC A transcripts in SNCA transgenic mice out to at least Day 8. In the cortex, thoracic spinal cord, and midbrain, Groups 2-14 showed reduction in SNCA transcripts. In the cerebellum, Groups 2-11, 13, and 14 showed reduction in SNCA transcripts; Group 12 showed negligible to no reduction in SNCA transcripts. Mostnotably, a single dose 60 pg AC009459 achieved -92% SNCA transcript inhibition (0.083) in the cortex.Example 10. In Vivo Administration of SNCA RNAi Agents in SNCA Transgenic Mice.

[0389] SNCA RNAi agents were evaluated in vivo in mice. On Days 1, 2, 3, and 4, five (n=5) FBV-129S6-SncatmlNbmTg(SNCA)lNbm / J mice (2 male, 3 female for groups 1-8, and 3 male, 2 female for groups 9 and 10) for each group were dosed, via subcutaneous (SC) injection, with SNCA RNAi agents formulated in PBS (at 2.5 mg / kg). The SNCA RNAi agent doses were adjusted for individual body weight. The SNCA RNAi agents were formulated at 0.25 mg / mL and the dose volume was 300 pL / 30 g (adjusted for individual animal body weight). The dosing was in accordance with the following Table 25.

[0390] FBV-129S6-SncatmlNbmTg(SNCA)lNbm / J mice can also be referred to as Snca'; PAC- Tg(SNCAWT), or PAC-Tg(SNCAWT); Snca- / -. These PAC-Tg(SNCAWT); Snca- / - mice harbor a Snca knock-out allele and a transgene encoding the human SNCA.

[0391] Table 25. Dosing for mice of Example 10.

[0392] Each of the SNCA RNAi agents included modified nucleotides and were conjugated at the 5’ terminal end of the sense strand to a targeting ligand that included a lipid moiety or antigen binding moiety having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5, 6, 7, 8, 9, and 10 for specific modifications and structure information related to the SNCA RNAi agents, including the lipid and antigen bindingmoieties).

[0393] On Day 36 (groups 1-5) or 64 (groups 6-10), the mice were euthanized. From the mice, right half of the brain and thoracic spinal cord were harvested and collected for analysis. hSNCA expression was analyzed via qPCR in the cortex, thoracic spinal cord, cerebellum, and midbrain, with mPPIA as endogenous gene, normalized to Group 1 (for groups 2-5) or Group 6 (for groups 7-10) mice dosed with PBS. The hSNCA expression data is shown in the following Table 26.

[0394] Table 26. SNCA expression in mice brain tissues of Example 10.

[0395] SNCA RNAi agents achieved inhibition of SNC A transcripts in SNCA transgenic mice out to at least Day 64. At day 36, AC010670 achieved the greatest knockdown in the thoracic spinal cord and the midbrain and ACO 10669 achieved the greatest knockdown in the cortex and the cerebellum. At day 64, ACO 10670 achieved the greatest knockdown in the thoracic spinal cord, the midbrain, and the cortex and ACO 10669 achieved the greatest knockdown in the cerebellum.Example 11. In Vivo Administration of SNCA RNAi Agents in SNCA Transgenic Mice.

[0396] SNCA RNAi agents were evaluated in vivo in mice. On Day 1, four (n=4) FBV-129S6- SncatmlNbmTg(SNCA)lNbm / J mice (2 male, 2 female) for each group were dosed, via intracerebroventricular (ICV) injection, with SNCA RNAi agents formulated in artificial cerebrospinal fluid (aCSF). The SNCA RNAi agents were formulated at 5 mg / mL at 10 pL total injection volume. The dosing was in accordance with the following Table 27.

[0397] FBV-129S6-SncatmlNbmTg(SNCA)lNbm / J mice can also be referred to as Snca'; PAC- Tg(SNCAWT), or PAC-Tg(SNCAWT); Snca- / -. These PAC-Tg(SNCAWT); Snca- / - mice harbor a Snca knock-out allele and a transgene encoding the human SNCA.

[0398] Table 27. Dosing for mice of Example 11.

[0399] Each of the SNCA RNAi agents included modified nucleotides and were conjugated at the 5’ terminal end of the sense strand to a targeting ligand that included a lipid moiety or antigen binding moiety having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5, 6, 7, 8, 9, and 10 for specific modifications and structure information related to the SNCA RNAi agents, including the lipid and antigen binding moieties).

[0400] On Day 15, the mice were euthanized. From the mice, right half of the brain and thoracic spinal cord were harvested and collected for analysis. hSNCA expression was analyzed via qPCR in the cortex, cerebellum, and midbrain, with mPPIA as endogenous gene, normalized to Group 1 mice dosed with aCSF. The hSNCA expression data is shown in the following Table 28.

[0401] Table 28. SNCA expression in mice brain tissues of Example 11.

[0402] SNCA RNAi agents achieved inhibition of SNC A transcripts in SNCA transgenic mice out to at least Day 15. ACO 10798 achieved the greatest knockdown in the cortex, ACO 10795 achieved the greatest knockdown in the cerebellum, and ACO 10800 achieved the greatest knockdown in the midbrain.Example 12. In Vivo Administration of SNCA RNAi Agents in SNCA Transgenic Mice.

[0403] SNCA RNAi agents were evaluated in vivo in mice. On Day 1, four (n=4) FBV-129S6- SncatmlNbmTg(SNCA)lNbm / J mice (2 male, 2 female for groups 1 and 3-12; 1 male, 3 femalefor group 2) for each group were dosed, via intracerebroventricular (ICV) injection, with SNCA RNAi agents formulated in artificial cerebrospinal fluid (aCSF) (at 45 mg / kg). The SNCA RNAi agents were formulated at 4.5 mg / mL. The dosing was in accordance with the following Table 29.

[0404] FBV-129S6-SncatmlNbmTg(SNCA)lNbm / J mice can also be referred to as Snca'; PAC- Tg(SNCAWT), or PAC-Tg(SNCAWT); Snca- / -. These PAC-Tg(SNCAWT); Snca- / - mice harbor a Snca knock-out allele and a transgene encoding the human SNCA.

[0405] Table 29. Dosing for mice of Example 12.

[0406] Each of the SNCA RNAi agents included modified nucleotides and were conjugated at the 5’ terminal end of the sense strand to a targeting ligand that included a lipid moiety or antigen binding moiety having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5, 6, 7, 8, 9, and 10 for specific modifications and structure information related to the SNCA RNAi agents, including the lipid and antigen binding moieties).

[0407] On Day 15, the mice were euthanized. From the mice, right half of the brain and thoracic spinal cord were harvested and collected for analysis. hSNCA expression was analyzed via qPCR in the cortex, thoracic spinal cord, cerebellum, and midbrain, with mPPIAas endogenous gene, normalized to Group 1 mice dosed with aCSF. The hSNCA expression data is shown in the following Table 30.

[0408] Table 30. SNCA expression in mice brain tissues of Example 12.

[0409] SNCA RNAi agents achieved inhibition of SNC A transcripts in SNCA transgenic mice out to at least Day 15. AC012817 achieved the greatest knockdown in the cortex, AC012816 achieved the greatest knockdown in the thoracic spinal cord, the cerebellum, and the midbrain.Example 13. In Vivo Testing of SNCA RNAi Agents in Cynomolgus Monkeys.

[0410] SNCA RNAi agents ACO 10745, AC010746, and AC010747 were evaluated in Cynomolgus monkeys (cynos) in vivo. On study days 1, 8, and 15, four cynos in each dosing group (n=4; 4 males) were administered a subcutaneous (SC) injection of 3 mg / kg (adjusted for individual animal body weight) of ACO 10745, ACO 10746, or ACO 10747 at a dose concentration of 1.5 mg / ml and an injection volume of 2.0 mL / kg, formulated in isotonic saline. The dosing was in accordance with Table 31 below.

[0411] Table 31. Dosing groups of Example 13.

[0412] The test animals were of non-human primate, Cynomolgus monkeys Macaca fascicularis), male, naive. The test animals were acclimated to laboratory housing, per facility and acclimation standard operating procedures, for at least 3 days prior to the initiation of dosing. The test animals were screened within 30 days prior to start of dosing, in accordance with the following criteria: 1) negative viral profile, 2) no history of diarrhea or no more than three (3) treatments for diarrhea in a 120-day period, and 3) measles vaccinated (or titer positive); vaccination not to be given within 28 days before TB test (TB tests were performed twice annually). The test animals were randomized and assigned to groups using a computer- based procedure prior to transfer into the study. Animals in poor health or at the extreme of the acceptable body weight range were not assigned to any test groups.

[0413] Each of the SNCA RNAi agents included modified nucleotides and were conjugated at the 5’ terminal end of the sense strand to a targeting ligand that included a lipid moiety or antigen binding moiety having the modified sequences as set forth in the duplex structures herein. (See Tables 3, 4, 5, 6, 7, 8, 9, and 10 for specific modifications and structureinformation related to the SNCA RNAi agents, including the lipid and antigen binding moieties).

[0414] Each cyno from Groups 1-4 were dosed on Day 1, 8, and 15. The RNAi agent test articles were administered via subcutaneous (SC) administration with a syringe and needle in the mid-scapular region. Dose sites were shaved before dosing and remarked as necessary throughout the study. Dose one (on Day 1) was delivered to the cynos’ left scapular region, dose two (on Day 8) was delivered to the right scapular region, and dose three (on Day 15) was delivered to the left scapular region. Each subcutaneous dose was delivered using a syringe with 23-25-gause needle.

[0415] On study day 29, animals from each group were euthanized and brain and spinal cord tissue was collected from each animal. Samples were analyzed by qPCR for SNCA mRNA knockdown. Samples were analyzed by JESS for protein knockdown. Average mRNA knockdown for frontal cortex, hippocampus, thoracic spinal cord, temporal cortex, caudate, and putamen for each group, relative to Group 1, are shown in Table 32 below:

[0416] Table 32. Relative expression of SNCA mRNA in various tissues analyzed by qPCR of Example 13.

[0417] As can be seen in Table 32, SNCA RNAi agents conjugated with an anti-Transferrin Fab achieved SNCA knockdown in CNS tissues when injected subcutaneously. SNCA mRNA inhibition was observed out to at least Day 29. ACO 10747 achieved the greatest knockdown in the hippocampus, frontal cortex, thoracic spinal cord, temporal cortex, caudate, putamen, visual cortex, midbrain, substantia nigra, hypothalamus, medulla, pons, cervical spinal cord, and lumbar spinal cord.Example 14. In Vivo Administration of SNCA RNAi Agents in SNCA Transgenic Mice.

[0418] SNCA RNAi agents were evaluated in vivo in mice. On Day 1, five (n=5) FBV-129S6- SncatalNbmTg(SNCA)lNbm / J mice (Group 1: 4 male, 1 female, Groups 2-8: 3 male, 2 female) for each group were dosed, via subcutaneous (SC) injection, with SNCA RNAi agents formulated in PBS at 3.0 mg / kg. The SNCA RNAi agent doses were adjusted for individual body weight. The SNCA RNAi agents were formulated at 0.3 mg / ml and a 10 mL / kg total injection volume. The dosing was in accordance with the following Table 33.

[0419] The SNCA RNAi agent used in this experiment comprises ACO 11922. ACO 11922 is a conjugate comprising Fab0183 conjugated to an siRNA comprising CA004779 and CS013988. Fab0183 comprises a heavy chain and a light chain. The Fab0183 heavy chain has the following sequence:EVQLVESGGGLVQPGNSLTLSCVASGFTFSNYGMHWIRQAPKKGLEWIAMIYYDSS KMNYADTVKGRFTISRDNSKNTLYLEMNSLRSEDTAMYYCAVPTSHYVVDVWGQ GVSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD (SEQ ID NO: 1010). The Fab0183 light chain has the following sequence:DIQMTQSPASLSASLEEIVTITCQASQDIGNWLAWYQQKPGKSPQLLIYGATSLADGV PSRFSGSRSGTQFSLKISRVQVEDIGIYYCLQAYNTPWTFGGGTKLELKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 1011).

[0420] FBV-129S6-SncatmlNbmTg(SNCA)lNbm / J mice can also be referred to as Snca'; PAC- Tg(SNCAWT), or PAC-Tg(SNCAWT); Snca- / -. These PAC-Tg(SNCAWT); Snca- / - mice harbor a Snca knock-out allele and a transgene encoding the human SNCA.

[0421] Table 33. Dosing for mice of Example 14.

[0422] On day 45 the mice from Groups 2 and 6 were euthanized, on day 53 the mice from Groups 3 and 7 were euthanized, and on day 74 the mice from Groups 4 and 8 were euthanized. From the mice, right half of the brain and thoracic spinal cord were harvested and collected for analysis. hSNCA expression was analyzed via qPCR in the thoracic spinal cord and brainstem with mPPIA as endogenous gene, normalized to Group 1 mice dosed with PBS. The hSNCA expression data is shown in the following Table 34.

[0423] Table 34. SNCA expression in mice brain tissues of Example 14.

[0424] AC011922 achieved inhibition of SNCA transcripts in SNCA transgenic mice out to at least day 123 in the brainstem and thoracic spinal cord.Example 15. In Vivo Administration of SNCA RNAi Agents in SNCA Transgenic Mice.

[0425] SNCA RNAi agents were evaluated in vivo in mice. On Day 1, five (n=5) FBV-129S6- SncatalNbmTg(SNCA)lNbm / J mice (2 male, 3 female) for each group were dosed, via subcutaneous (SC) injection, with SNCA RNAi agents formulated in PBS at 3.0 mg / kg. The SNCA RNAi agent doses were adjusted for individual body weight. The SNCA RNAi agents were formulated at 10 mL / 1 kg at 10 pL total injection volume. The dosing was in accordance with the following Table 35.

[0426] The SNCA RNAi agent used in this experiment comprises AC011922. AC011922 is a conjugate comprising Fab0183 and an siRNA comprising CA004779 and CS013988. Fab0183 comprises a heavy chain and a light chain. The Fab0183 heavy chain has the following sequence:EVQLVESGGGLVQPGNSLTLSCVASGFTFSNYGMHWIRQAPKKGLEWIAMIYYDSS KMNYADTVKGRFTISRDNSKNTLYLEMNSLRSEDTAMYYCAVPTSHYVVDVWGQ GVSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD (SEQ ID NO: 1010). The Fab0183 light chain has the following sequence: DIQMTQSPASLSASLEEIVTITCQASQDIGNWLAWYQQKPGKSPQLLIYGATSLADGV PSRFSGSRSGTQFSLKISRVQVEDIGIYYCLQAYNTPWTFGGGTKLELKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 1011).

[0427] FBV-129S6-SncatmlNbmTg(SNCA)lNbm / J mice can also be referred to as Snca'; PAC- Tg(SNCAWT), or PAC-Tg(SNCAWT); Snca- / -. These PAC-Tg(SNCAWT); Snca- / - mice harbor a Snca knock-out allele and a transgene encoding the human SNCA.

[0428] Table 35. Dosing for mice of Example 15.

[0429] On day 6, mice from Groups 2 and 5 were euthanized, on day 19, mice from Groups 3 and 6 were euthanized, and on day 34, mice from Groups 4 and 7 were euthanized. From the mice, right half of the brain and thoracic spinal cord were harvested and collected for analysis. hSNCA expression was analyzed via qPCR in the thoracic spinal cord and brainstem with mPPIA as endogenous gene, normalized to Group 1 mice dosed with PBS. The hSNCA expression data is shown in the following Table 36.

[0430] Table 36. SNCA expression in mice brain tissues of Example 15.

[0431] AC011922 achieved inhibition of SNCA transcripts in SNCA transgenic mice out to at least day 34 in the brainstem and thoracic spinal cord.OTHER EMBODIMENTS

[0432] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

CLAIMS:

1. An RNAi agent for inhibiting expression of a synuclein alpha (SNCA) gene, comprising: an antisense strand comprising at least 17 contiguous nucleotides differing by 0 or 1 nucleotides from any one of the sequences provided in Table 2 or Table 3; and a sense strand comprising a nucleotide sequence that is at least partially complementary to the antisense strand.

2. The RNAi agent of claim 1, wherein the antisense strand comprises nucleotides 2-18 of any one of the sequences provided in Table 2 or Table 3.

3. The RNAi agent of claim 1 or claim 2, wherein the sense strand comprises a nucleotide sequence of at least 17 contiguous nucleotides differing by 0 or 1 nucleotides from any one of the sequences provided in Table 2 or Table 4, and wherein the sense strand has a region of at least 85% complementarity over the 17 contiguous nucleotides to the antisense strand.

4. The RNAi agent of any one of claims 1-3, wherein at least one nucleotide of the SNCA RNAi agent is a modified nucleotide or includes a modified internucleoside linkage.

5. The RNAi agent of any one of claims 1-4, wherein all or substantially all of the nucleotides are modified nucleotides.

6. The RNAi agent of any one of claims 4-5, wherein the modified nucleotide is selected from the group consisting of: 2'-O-methyl nucleotide, 2'-fhioro nucleotide, 2'-deoxy nucleotide, 2', 3 '-seco nucleotide mimic, locked nucleotide, 2'-F-arabino nucleotide, 2'- methoxyethyl nucleotide, abasic nucleotide, ribitol, inverted nucleotide, inverted 2'-O- methyl nucleotide, inverted 2'-deoxy nucleotide, 2'-amino-modified nucleotide, 2'-alkyl- modified nucleotide, morpholino nucleotide, vinyl phosphonate-containing nucleotide, cyclopropyl phosphonate-containing nucleotide, and 3'-O-methyl nucleotide.

7. The RNAi agent of claim 5, wherein all or substantially all of the nucleotides are modified with 2'-O-methyl nucleotides, 2'-fluoro nucleotides, or combinations thereof.

8. The RNAi agent of any one of claims 1-7, wherein the antisense strand comprises the nucleotide sequence of any one of the modified sequences provided in Table 3.

9. The RNAi agent of any one of claims 1-8, wherein the sense strand comprises the nucleotide sequence of any one of the modified sequences provided in Table 4.

10. The RNAi agent of claim 1, wherein the antisense strand comprises the nucleotide sequence of any one of the modified sequences provided in Table 3 and the sense strandcomprises the nucleotide sequence of any one of the modified sequences provided in Table 4.

11. The RNAi agent of any one of claims 1-10, wherein the sense strand is between 18 and 30 nucleotides in length, and the antisense strand is between 18 and 30 nucleotides in length.

12. The RNAi agent of claim 11, wherein the sense strand and the antisense strand are each between 18 and 27 nucleotides in length.

13. The RNAi agent of claim 12, wherein the sense strand and the antisense strand are each between 18 and 24 nucleotides in length.

14. The RNAi agent of claim 13, wherein the sense strand and the antisense strand are each 21 nucleotides in length.

15. The RNAi agent of claim 14, wherein the RNAi agent has two blunt ends.

16. The RNAi agent of any one of claims 1-15, wherein the sense strand comprises one or two terminal caps.

17. The RNAi agent of any one of claims 1-16, wherein the sense strand comprises one or two inverted abasic residues.

18. The RNAi agent of claim 1, wherein the RNAi agent is comprised of a sense strand and an antisense strand that form a duplex having the structure of any one of the duplexes in Table 7, Table 8, or Table 9.

19. The RNAi agent of claim 18, wherein all or substantially all of the nucleotides are modified nucleotides.

20. The RNAi agent of claim 1, comprising an antisense strand that consists of, consists essentially of, or comprises a nucleotide sequence that differs by 0 or 1 nucleotides from any one of the following sequences (5’ - 3 '):UGAUACUUCAAUCACUGCU (SEQ ID NO: 217);UUAGAGAACACCCUCUUUU (SEQ ID NO: 110);UUUGAGAAAUGUCAUGACU (SEQ ID NO: 196);UGAUACUUCAAUCACUGCUGU (SEQ ID NO: 891);UUAGAGAACACCCUCUUUUGU (SEQ ID NO: 880); or UUUGAGAAAUGUCAUGACUGG (SEQ ID NO: 890).

21. The RNAi agent of claim 20, comprising an sense strand that consists of, consists essentially of, or comprises a nucleotide sequence that differs by 0 or 1 nucleotides from any one of the following sequences (5’ - 3 '):AGCAGUGAUUGAAGUAUCA (SEQ ID NO: 414) AAAAGAGGGUGUUCUCUAA (SEQ ID NO: 307) AGUCAUGACAUUUCUCAAA (SEQ ID NO: 393) AC AGCAGUGAUUGAAGUAUCA (SEQ ID NO: 958) AC AAAAGAGGGUGUUCUCUAA (SEQ ID NO: 974) CCAGUCAUGACAUUUCUCAAA (SEQ ID NO: 957).

22. The RNAi agent of claim 20 or 21, wherein all or substantially all of the nucleotides are modified nucleotides.

23. The RNAi agent of claim 1, comprising an antisense strand that comprises, consists of, or consists essentially of a modified nucleotide sequence that differs by 0 or 1 nucleotides from one of the following nucleotide sequences (5' 3'): cPrpusGfsauacUfucaaUfcAfcUfgcusgsu (SEQ ID NO: 572); cPrpusUfsagagAfacacCfcUfcUfuuusgsu (SEQ ID NO: 535); or cPrpusUfsugagAfaaugUfcAfuGfacugsg (SEQ ID NO: 495); wherein a represents 2'-O-methyl adenosine, c represents 2'-O-methyl cytidine, g represents 2'-O-methyl guanosine, u represents 2'-O-methyl uridine; Af represents 2'- fluoro adenosine, Cf represents 2'-fluoro cytidine, Gf represents 2'-fluoro guanosine, Uf represents 2'-fluoro uridine; cPrpu represents 5 ’-cyclopropyl phosphonate-2’-O-m ethyl uridine; s represents a phosphorothioate linkage; and wherein all or substantially all of the nucleotides on the sense strand are modified nucleotides.

24. The RNAi agent of claim 1, wherein the sense strand comprises, consists of, or consists essentially of a modified nucleotide sequence that differs by 0 or 1 nucleotides from one of the following nucleotide sequences (5' 3'): acagcaguGfAfUfugaaguauca (SEQ ID NO: 691); a_2NcaaaagaGfgGfUfguucucuaa (SEQ ID NO: 698); or ccagucauGfaCfAfuuucucaaa (SEQ ID NO: 631); wherein a represents 2'-O-methyl adenosine, c represents 2'-O-methyl cytidine, g represents 2'-O-methyl guanosine, u represents 2'-O-methyl uridine; Af represents 2'- fluoro adenosine, Cf represents 2'-fluoro cytidine, Gf represents 2'-fluoro guanosine, Uf represents 2'-fluoro uridine, and a_2N represents 2-aminoadenosine.

25. The RNAi agent of any one of claims 20-24, wherein the sense strand further includes inverted abasic residues at the 3’ terminal end of the nucleotide sequence, at the 5’ terminal end of the nucleotide sequence, or both.

26. The RNAi agent of any one of the preceding claims, wherein the RNAi agent is conjugated to a targeting ligand and has the duplex structure of any one of the duplex structures in Table 9.

27. The RNAi agent of any one of claims 1-25, wherein the RNAi agent is conjugated to an antigen binding protein.

28. The RNAi agent of claim 27, wherein the antigen binding protein is conjugated to the sense strand.

29. The RNAi agent of claim 27 or 28, wherein the antigen binding protein is an antibody fragment (Fab) that specifically binds to one or more epitopes on a transferrin receptor (TfRl).

30. The RNAi agent of claim 29, wherein the Fab comprises (i) 6 complementary determining regions (CDRs), (ii) 3 CDRs on the variable light chain (VL), and / or (iii) 3 CDRs on the variable heavy chain (VH).

31. The RNAi agent of claim 30, wherein the variable light chain has a VL CDR1 sequence selected from the group consisting of: RASDGLYSNLA (SEQ ID NO: 6), RASDNLYRNLA (SEQ ID NO: 7), and RASDKLYSNLA (SEQ ID NO: 8); a VL CDR2 sequence selected from the group consisting of: DATLLAS (SEQ ID NO: 9), DARNLAS (SEQ ID NO: 10), DAFNLAS (SEQ ID NO: 11), DATRLAS (SEQ ID NO: 12), DATKLAS (SEQ ID NO: 13), and DAKNLAS (SEQ ID NO: 14); and / or a VL CDR 3 sequence of QHFWGTPLT (SEQ ID NO: 15).

32. The RNAi agent of claim 30 or 31, wherein the variable light chain is selected from any one of the VL chains shown in Table A.

33. The RNAi agent of any one of claims 23-25, wherein the variable light chain comprises the sequence:DIQLTQSPSSLSASVGDRVTITCRASDKLYSNLAWYQQKPGKAPKLLIYDATLL ASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHFWGTPLTFGQGTKVEIK (SEQ ID NO: 32).

34. The RNAi agent of any one of claims 30-33, wherein the variable heavy chain has a VH CDR1 sequence selected from the group consisting of: GYTFNSYWMH (SEQ ID NO:16), GYTFKSYWMH (SEQ ID NO: 17), GFTFTSYWMH (SEQ ID NO: 18), GYTFTSYWVH (SEQ ID NO: 19), and GYTFTSYWMH (SEQ ID NO: 20), a VH CDR2 sequence selected from the group consisting of: EINPTNGRVNYIEKFKS (SEQ ID NO: 21), EINPTNGRFNYIEKFKS (SEQ ID NO: 22), EINPTNGRTNYIEKFKS (SEQ ID NO: 23), and EINPTNGRSNYIEKFKS (SEQ ID NO: 24); and / or a VH CDR3 sequence of: GTRAYHY (SEQ ID NO: 25).

35. The RNAi agent of any one of claims 30-34, wherein the variable heavy chain is selected from any one of the VH chains shown in Table B.

36. The RNAi agent of any one of claims 30-35, wherein the variable heavy chain comprises the sequence:EVQLVESGGGLVQPGGSLRLSCATSGFTFTSYWMHWVRQAPGKGLEWVAEIN PTNGRTNYIEKFKSRITLSVDKSKSTVYLQMNSLRAEDTAVYYCARGTRAYHY WGQGTLVTVSS (SEQ ID NO: 40).

37. The RNAi agent of any one of claims 29-36, wherein the Fab further comprises a light constant chain 1 (CL).

38. The RNAi agent of claim 37, wherein the light constant chain 1 (CL) sequence is: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 2).

39. The RNAi agent of any one of claims 29-38, wherein the Fab further comprises a heavy constant chain 1 (CH).

40. The RNAi agent of claim 39, wherein the heavy constant chain 1 (CH) sequence is: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH (SEQ ID NO: 4).

41. The RNAi agent of any one of claims 29-40, wherein the antibody fragment (Fab) binds TfRl with an affinity of at least 1 nM KD.

42. The RNAi agent of any one of claims 1-26, wherein the RNAi agent is conjugated to a lipid.

43. The RNAi agent of claim 42, wherein the lipid is selected from any one of the lipid moieties shown in Table 10.

44. The RNAi agent of claim 42 or 43, wherein the lipid moiety is conjugated to the sense strand.

45. The RNAi agent of claim 44, wherein the lipid moiety is conjugated to the 5’ terminal end of the sense strand.

46. A conjugate comprising the RNAi agent of any one of claims 1-26 conjugated to an antibody fragment (Fab) that specifically binds to one or more epitopes on a transferrin receptor (TfRl).

47. The conjugate of claim 46, wherein the Fab comprises (i) 6 complementary determining regions (CDRs), (ii) 3 CDRs on the variable light chain (VL), or (iii) 3 CDRs on the variable heavy chain (VH).

48. The conjugate of claim 47, wherein the variable light chain has a VL CDR1 sequence selected from the group consisting of: RASDGLYSNLA (SEQ ID NO: 6), RASDNLYRNLA (SEQ ID NO: 7), and RASDKLYSNLA (SEQ ID NO: 8); a VL CDR2 sequence selected from the group consisting of: DATLLAS (SEQ ID NO: 9), DARNLAS (SEQ ID NO: 10), DAFNLAS (SEQ ID NO: 11), DATRLAS (SEQ ID NO: 12), DATKLAS (SEQ ID NO: 13), and DAKNLAS (SEQ ID NO: 14); and / or a VL CDR 3 sequence of QHFWGTPLT (SEQ ID NO: 15).

49. The conjugate of claim 47 or 48, wherein the variable light chain is selected from any one of the VL chains shown in Table A.

50. The conjugate of any one of claims 47-49, wherein the variable light chain comprises the sequence:DIQLTQSPSSLSASVGDRVTITCRASDKLYSNLAWYQQKPGKAPKLLIYDATLL ASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHFWGTPLTFGQGTKVEIK (SEQ ID NO: 32).

51. The conjugate of any one of claims 47-50, wherein the variable heavy chain has a VH CDR1 sequence selected from the group consisting of: GYTFNSYWMH (SEQ ID NO: 16), GYTFKSYWMH (SEQ ID NO: 17), GFTFTSYWMH (SEQ ID NO: 18), GYTFTSYWVH (SEQ ID NO: 19), and GYTFTSYWMH (SEQ ID NO: 20), a VH CDR2 sequence selected from the group consisting of: EINPTNGRVNYIEKFKS (SEQ ID NO: 21), EINPTNGRFNYIEKFKS (SEQ ID NO: 22), EINPTNGRTNYIEKFKS (SEQ ID NO: 23), and EINPTNGRSNYIEKFKS (SEQ ID NO: 24); and / or a VH CDR3 sequence of: GTRAYHY (SEQ ID NO: 25).

52. The conjugate of any one of claims 47-51, wherein the variable heavy chain is selected from any one of the VH chains shown in Table B.

53. The conjugate of any one of claims 47-52, wherein the variable heavy chain comprises the sequence: EVQLVESGGGLVQPGGSLRLSCATSGFTFTSYWMHWVRQAPGKGLEWVAEIN PTNGRTNYIEKFKSRITLSVDKSKSTVYLQMNSLRAEDTAVYYCARGTRAYHY WGQGTLVTVSS (SEQ ID NO: 40).

54. The conjugate of any one of claims 46-53, wherein the Fab further comprises a light constant chain 1 (CL).

55. The conjugate of claim 54, wherein the light constant chain 1 (CL) sequence is: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 2).

56. The conjugate of any one of claims 46-55, wherein the Fab further comprises a heavy constant chain 1 (CH).

57. The conjugate of claim 56, wherein the heavy constant chain 1 (CH) sequence is: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH (SEQ ID NO: 4).

58. The conjugate of any one of claims 46-57, wherein the antibody fragment (Fab) binds TfRl with an affinity of at least 1 nM KD.

59. The conjugate of any one of claims 46-58, wherein the RNAi agent is conjugated to the Fab using a covalent or non-covalent bond, ionic bond, hydrogen bond, hydrophobic interaction, peptide, polymer, or a nucleic acid binding protein.

60. The conjugate of any one of claims 46-59, wherein the RNAi agent is conjugated to the Fab through a linker comprising a structure selected from the group consisting of:wherein5A represents a point of attachment to the Fab, andrepresents a point of attachment to the RNAi agent portion of the conjugate.

61. A composition comprising the RNAi agent of any one of claims 1-45, or the conjugate of any one of claims 46-60, wherein the composition further comprises a pharmaceutically acceptable excipient.

62. The composition of claim 61, further comprising a second RNAi agent capable of inhibiting the expression of SNCA gene expression.

63. The composition of any one of claims 61-62, further comprising one or more additional therapeutics.

64. The composition of any of claims 61-63, wherein the RNAi agent is a sodium salt.

65. The composition of any of claims 61-64, wherein the pharmaceutically acceptable excipient is water for injection.

66. The composition of any of claims 61-64, wherein the pharmaceutically acceptable excipient is a buffered saline solution.

67. A method for inhibiting expression of a SNCA gene in a cell, the method comprising introducing into a cell an effective amount of an RNAi agent of any one of claims 1-45, the conjugate of any one of claims 46-60, or the composition of any one of claims 61-66.

68. The method of claim 67, wherein the cell is within a subject.

69. The method of claim 68, wherein the subject is a human subject.

70. The method of any one of claims 67-69, wherein following the administration of the RNAi agent the SNCA gene expression is inhibited by at least about 30%.

71. A method of treating one or more symptoms or diseases associated with enhanced or elevated mutant SNCA activity levels, the method comprising administering to a human subject in need thereof a therapeutically effective amount of the RNAi agent of any one of claims 1-45, the conjugate of any one of claims 46-60, or the composition of any one of claims 61-66.

72. The method of claim 71, wherein the disease is a neurodegenerative disease.

73. The method of claim 72, wherein the neurodegenerative disease is a synucleinopathy.

74. The method of claim 73, wherein the disease is Parkinson’s Disease.

75. The method of any one of claims 67-74, wherein the RNAi agent is administered at a dose of about 0.01 mg / kg to about 5.0 mg / kg of body weight of the subject.

76. The method of any one of claims 67-74, wherein the RNAi agent is administered at a dose of about 0.03 mg / kg to about 2.0 mg / kg of body weight of the subject.

77. The method of any of claims 67-76, wherein the RNAi agent is administered in two or more doses.

78. Use of the RNAi agent of any one of claims 1-45, or the conjugate of any one of claims 46-60, for the treatment of a disease, disorder, or symptom that is mediated at least in part by mutant SNCA activity and / or SNCA gene expression.

79. Use of the composition according to any one of claims 61-66, for the treatment of a disease, disorder, or symptom that is mediated at least in part by SNCA activity and / or SNCA gene expression.

80. Use of the composition according to any one of claims 61-66, for the manufacture of a medicament for treatment of a disease, disorder, or symptom that is mediated at least in part by SNCA activity and / or SNCA gene expression.

81. The use of any one of claims 78-80, wherein the disease is a neurodegenerative disease.

82. A method of making an RNAi agent of any one of claims 1-45, comprising annealing a sense strand and an antisense strand to form a double-stranded ribonucleic acid molecule.

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