SNCA-targeted siRNA compositions for treatment of SNCA-related diseases

Abstract

The present disclosure relates to double stranded ribonucleic acid (dsRNAi) agents and compositions that target SNCA genes, in particular, in CNS tissue, as well as methods of inhibiting expression of SNCA genes and methods of using such dsRNAi agents and compositions to treat subjects having SNCA-associated neurodegenerative diseases or disorders, such as Parkinson's disease (PD), multi-system atrophy (MSA), dementia with Lewis's bodies (LBD), and other synucleinopathies.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is related to and claims the benefit of U.S. Provisional Patent Application No. 63 / 326,813, filed on April 2, 2022, entitled “SNCA-Targeting siRNA Compositions for Treating SNCA-Associated Disease,” and U.S. Provisional Patent Application No. 63 / 326,770, filed on April 1, 2022, entitled “SNCA-Targeting siRNA Compositions for Treating SNCA-Associated Disease,” under 35 U.S.C. §119(e). The entire contents of the foregoing patent applications are incorporated herein by reference. Technical Field

[0003] The present disclosure generally relates to SNCA-targeted RNAi agents and methods.

[0004] Sequence Listing

[0005] This application contains a sequence listing, which is submitted electronically in Extensible Markup Language (XML) format and is incorporated herein by reference in its entirety. This XML copy was created on March 29, 2023, is named ALN-461WO.xml, and is 3242KB in size. Background Art

[0006] The SNCA gene encodes the presynaptic neuronal protein, α-synuclein (also referred to herein as alpha-synuclein or synuclein-α), which is genetically and neuropathologically linked to Parkinson's disease (PD) (Stefanis, L. Cold Spring Harb Perspect Med. 2: a009399). α-Synuclein is thought to contribute to PD pathogenesis in multiple ways, but abnormal soluble oligomeric forms of α-synuclein, called protofibrils, are generally considered toxic, mediating disruption of cellular homeostasis and neuronal death through effects on various intracellular targets, including synaptic function. Furthermore, secreted α-synuclein is believed to exert deleterious effects on neighboring cells, potentially contributing to the spread of the disease. Although the extent to which α-synuclein is implicated in all cases of PD is unclear, targeting the toxic functions conferred by this protein when it is dysregulated presents a potentially valuable therapeutic strategy, not only for PD but also for other neurodegenerative disorders known as synucleinopathies, all of which exhibit a common neuropathological hallmark due to the accumulation of α-synuclein, termed Lewy bodies (LBs) and Lewy neurites (LNs). In addition to PD, these documented or suspected SNCA-associated synucleinopathies include, but are not limited to, multiple system atrophy (MSA), dementia with Lewy bodies (LBD), pure autonomic failure (PAF), Pick's disease, progressive supranuclear palsy, dementia pugilistica, Parkinson's disease linked to chromosome 17, Lytico-Bodig disease, tangle-dominant dementia, argyrophilic grain disease, ganglioglioma, gangliocytoma, meningioangiomatosis, subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis, Hallervorden-Spatz disease, lipofuscinosis, corticobasal degeneration, frontotemporal dementia, frontotemporal lobar degeneration, Alzheimer's disease, Huntington's disease, Down syndrome, psychosis, schizophrenia, and Creutzfeldt-Jakob disease.

[0007] PD, LBD, and MSA are three of the most common examples of neurodegenerative disorders with SNCA brain pathology. PD is the most common movement disorder and is characterized by rigidity, hypokinesia, tremors, and postural instability. PD is believed to affect approximately 4 to 6 million people worldwide. LBD accounts for 5-15% of all dementias. In addition to forgetfulness and other dementia symptoms that often fluctuate, LBD patients often suffer from recurrent falls and visual hallucinations. MSA is a rapidly progressive orphan disease that causes severe movement disorders in affected subjects within a few years. The incidence of MSA is reported to be between 3.4 and 4.9 cases per 100,000 people.

[0008] In addition to the neuropathological changes observed in α-synucleinopathies, α-synuclein levels are often elevated in affected brain regions (Klucken et al., 2006).

[0009] α-Synuclein monomers, tetramers, and fibrillar aggregates are the major components of Lewy body (LB)-like neuronal inclusions, glial inclusions, and axonal spheroids in neurodegeneration with brain iron accumulation. Lewy-related pathology (LRP), composed primarily of α-synuclein, is present in most Alzheimer's disease biopsies, and elevated α-synuclein levels in patients are associated with cognitive decline (Twohig et al. (2019) Molecular Neurodegeneration). Autosomal dominant mutations in the SNCA gene, particularly including A53T, A30P, E46K, and H50Q (Zarranz et al. (2004) Ann. Neurol. 55, 164-173, Choi et al. (2004) FEBS Lett. 576, 363-368, and Tsigelny et al. (2015) ACS Chem. Neurosci. 6, 403-416), A53T (Polymeropoulos et al. (1997) Science), as well as triple and double mutations, have been identified as running in families with related neurodegenerative diseases. The foregoing indicates that not only pathogenic mutations in SNCA, but also increases in α-synuclein affect disease outcome.

[0010] The role of SNCA mutations in disease onset is not fully understood; however, there is evidence pointing to inherent gain-of-function toxicity of normal α-synuclein when levels exceed certain levels (Stefanis et al. (2012) Cold Spring Harb Perspect Med.) and / or when it interacts abnormally with cellular lipids and vesicles (reviewed in Kiechler et al. (2020) Front. Cell Dev. Biol.). Consistent with this, SNCA-null mice, in contrast to transgenic overexpressors, show no overt neuropathology or behavioral phenotype (Abeliovich et al. (2000) Neuron). Posttranscriptional regulation of SNCA has also been shown to occur through endogenous microRNAs that bind to the 3' end of the gene (Junn et al. (2009) PNAS 106:13052-13057; Doxakis (2010), JBC). Furthermore, studies of familial point mutations in SNCA have shown suppressed expression, particularly in cases with prolonged disease onset (Markopoulou et al. (1999) Ann Neurol. 46(3):374-81 and Kobayashi et al. (2003) Brain 126(Pt 1):32-42). Similarly, Voutsinas et al. (2010) Hum Mutat. 31(6):685-91) found that even overexpression of wild-type SNCA messenger RNA (mRNA) was causative of disease onset. These data suggest that suppression of total SNCA levels reduces α-synuclein-induced toxicity.

[0011] For synucleinopathies, including PD, multiple system atrophy, and Lewy body dementia, there are no disease-modifying treatments, and treatment options are limited, e.g., only palliative. For example, currently, only symptomatic treatments are available for PD patients (by replacing the loss of active dopamine in the brain) and AD patients (i.e., cholinesterase inhibitors). None of the existing treatment strategies for α-synucleinopathies targets the underlying disease process.

[0012] Thus, in view of the described involvement of SNCA in several neurodegenerative disorders (synucleinopathies), there remains a need for agents (particularly agents targeting the central nervous system (CNS) given the described functionality of α-synuclein in CNS tissues) that can selectively and effectively silence the SNCA gene (e.g., eliminate or reduce the effects of toxic α-synuclein substances) using the cell's own RNAi machinery, that have high biological activity and in vivo stability, and that can effectively inhibit the expression of the target SNCA gene. Summary of the Invention

[0013] The present disclosure provides RNAi agent compositions that achieve RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of the synuclein alpha (SNCA) gene. The SNCA gene can be intracellular, for example, in a subject such as a human. In certain embodiments, the RNAi agent is designed and directed against knockdown of SNCA in cells and / or tissues of the CNS. The present disclosure also provides methods of using the RNAi agent compositions of the present disclosure for inhibiting expression of a SNCA gene or for treating a subject who would benefit from inhibiting or reducing expression of a SNCA gene, e.g., a subject suffering from or susceptible to a SNCA-associated neurodegenerative disease or disorder, e.g., PD, multiple system atrophy (MSA), Lewy body dementia (LBD), pure autonomic failure (PAF), Pick's disease, progressive supranuclear palsy, dementia pugilistica, Parkinson's disease linked to chromosome 17, Lytico-Bodig's disease, tangle-dominant dementia, argyrophilic grain disease, ganglioglioma, gangliocytoma, meningioangiomatosis, subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis, Hallervorden-Spatz disease, lipofuscinosis, corticobasal degeneration, frontotemporal dementia, frontotemporal lobar degeneration, Alzheimer's disease, and Huntington's disease.

[0014] Thus, in one aspect, the present disclosure provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of SNCA, wherein the dsRNA agent comprises a sense strand having a 5'-end and a 3'-end and an antisense strand having a 5'-end and a 3'-end, wherein the sense strand and the antisense strand form a double-stranded region, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 150, SEQ ID NO: 157, SEQ ID NO: 164, or SEQ ID NOs: 142-149, 151-156, 158-163, or 165-184 of Table 3, having 0 or 1 mismatches; the sense strand of the dsRNA agent comprises a lipophilic moiety attached at position 6 or 16, counted from the 5'-end of the sense strand; the antisense strand comprises SEQ ID NO: 193, SEQ ID NO: 200, SEQ ID NO: 207, or SEQ ID NO: 194 of Table 3 ID NO: 185-192, 194-199, 201-206 or 208-227, having 0 or 1 mismatches; the dsRNA agent does not include a GalNAc modification; and the dsRNA agent includes eight phosphorothioate internucleotide linkages located at the penultimate and last internucleotide linkages at the respective 3'-end and 5'-end of the sense and antisense strands of the dsRNA agent.

[0015] Thus, in one aspect, the present disclosure provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of SNCA, wherein the dsRNA agent comprises a sense strand having a 5'-end and a 3'-end and an antisense strand having a 5'-end and a 3'-end, wherein the sense strand and the antisense strand form a double-stranded region, wherein: the sense strand comprises a nucleotide sequence of Table 3 (SEQ ID NOs: 142-184) with 0 or 1 mismatch; the sense strand of the dsRNA agent comprises a lipophilic moiety attached at position 6 or 16 counted from the 5'-end of the sense strand; the antisense strand comprises a nucleotide sequence of Table 3 (SEQ ID NOs: 142-184) with 0 or 1 mismatch; NO:185-227), with 0 or 1 mismatches; the dsRNA agent does not include a GalNAc modification; and the dsRNA agent includes six phosphorothioate internucleotide linkages, wherein the six phosphorothioate internucleotide linkages are located at the penultimate and last internucleotide linkages at the 5' and 3' ends of the antisense strand and the penultimate and last internucleotide linkages at the 5' end of the sense strand.

[0016] In certain embodiments, the lipophilic moiety is an aliphatic, alicyclic, or polyalicyclic compound.

[0017] In some embodiments, the lipophilic moiety is a lipid, cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexanol, hexadecylglycerol, borneol, menthol, 1,3-propylene glycol, heptadecyl, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.

[0018] In one embodiment, the lipophilic moiety contains a saturated or unsaturated C4-C 30 A hydrocarbon chain and an optional functional group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide or alkyne.

[0019] In some embodiments, the lipophilic portion contains saturated or unsaturated C6-C 18 Optionally, the lipophilic portion contains a saturated or unsaturated C 16 Hydrocarbon chain.

[0020] In one embodiment, the lipophilic moiety is wherein B is a nucleotide base or a nucleotide base analog. Optionally, B is adenine, guanine, cytosine, thymine or uracil.

[0021] In certain embodiments, the lipophilic moiety is attached via a linker or a carrier. Optionally, the lipophilic moiety is conjugated to a carrier that replaces a nucleotide at position 6 or 16 of the sense strand (counted as position 1 from the 5'-terminal nucleotide of the sense strand). In related embodiments, the carrier is a cyclic group (e.g., pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3] dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinone, tetrahydrofuranyl or decahydronaphthyl) or a non-cyclic portion based on a serine backbone or a diethanolamine backbone.

[0022] In some embodiments, the lipophilic moiety is conjugated to the dsRNA agent via a linker comprising an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide linkage, the product of a click reaction, or a carbamate.

[0023] In one embodiment, the lipophilic moiety is conjugated to the dsRNA agent via a biocleavable linker that is a DNA, RNA, disulfide, amide, galactosamine, glucosamine, galactose and / or mannose functionalized monosaccharide or oligosaccharide, or a combination thereof.

[0024] In certain embodiments, substantially all of the nucleotides of the sense strand or the antisense strand, or both, are modified nucleotides.

[0025] In one embodiment, all nucleotides of the sense strand are modified nucleotides.

[0026] In another embodiment, all nucleotides of the antisense strand are modified nucleotides.

[0027] In further embodiments, all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides.

[0028] In certain embodiments, the dsRNA agent has one or more modified nucleotides, wherein at least one of the one or more modified nucleotides is a deoxynucleotide, a 3'-terminal deoxythymidine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-hydroxy modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non- Natural base nucleotides, tetrahydropyran modified nucleotides, 1,5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides containing 5'-phosphorothioate groups, nucleotides containing 5'-methylphosphonate groups, nucleotides containing 5' phosphate or 5' phosphate mimetics, nucleotides containing vinyl phosphate, nucleotides containing adenosine-diol nucleic acid (GNA), nucleotides containing thymidine-diol nucleoside (GNA) S-isomers, nucleotides containing 2-hydroxymethyl-tetrahydrofuran-5-phosphate, nucleotides containing 2'-deoxythymidine-3'-phosphate, nucleotides containing 2'-deoxyguanosine-3'-phosphate, or terminal nucleotides linked to a cholesterol derivative and a dodecylamide group.

[0029] In another embodiment, the dsRNA agent comprises at least one modified nucleotide that is a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-C16 (2'-O-hexadecyl) modified nucleotide, or a nucleotide comprising vinyl phosphate. Optionally, the dsRNA agent comprises at least one of the following modifications: a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-C16 (2'-O-hexadecyl) modified nucleotide, and a nucleotide comprising vinyl phosphate.

[0030] In certain embodiments, the dsRNA agent further comprises a phosphate or a phosphate mimetic at the 5'-end of the antisense strand. Optionally, the phosphate mimetic is 5'-vinylphosphonate (VP).

[0031] In some embodiments, the dsRNA agent comprises a pattern of modified nucleotides as shown in Table 2 (wherein the positions of 2'-C16, 2'-O-methyl, 2'-deoxy, GNA, phosphorothioate, vinylphosphonate, and 2'-fluoro modifications are as shown in Table 2, regardless of the individual nucleotide base sequence of the dsRNA agent shown).

[0032] In one embodiment, the dsRNA agent has AD-1804698, AD-1804699, AD-1747575, AD-1747576, AD-1804700, AD-1747577, AD-1747578, AD-1747579, AD-1747580, AD-1747581, AD-1804701, AD-1747582, AD-1804702, AD-1804703, AD-1804704, AD-1747583, AD-1804705, AD-1804706, AD-1804707, AD-1804708, AD-1804709, AD-1804698, AD-1804699, AD-1804691, AD-1804692, AD-1804693, AD-1804694, AD-1804695, AD-1804696, AD-1804697, AD-1804698, AD-1804699, AD-1804699 or the sense strand nucleotide sequence of AD-1747591, AD-1747585, AD-1804710, AD-1804711, AD-1747586, AD-1804712, AD-1747587, AD-1804713, AD-1804714, AD-1747588, AD-1804715, AD-1804716, AD-1804717, AD-1804718, AD-1804719, AD-1804720, AD-1804721, AD-1804722, AD-1804723, AD-1804724, AD-1804725, or AD-1804726. Optionally, the dsRNA agent has the sense strand nucleotide sequence of AD-1747580, AD-1747583, or AD-1747585.

[0033] In certain embodiments, the sense strand of the dsRNA agent has one of the following modification patterns: 5'-nsnsnnn(Nhd)NfnNfNfNfnnnnnnsnsn-3', 5'-nsnsnnnnnnNfNfNfnnnn(Nhd)nnnsnsn-3', or 5'-nsnsnnn(Nhd)nnNfNfNfnnnnnnnnsnsn-3', wherein n is a 2'-O-methyl-nucleotide, s is a phosphorothioate internucleotide linkage, Nf is a 2'-fluoro-nucleotide, and (Nhd) is a 2'-O-hexadecyl-nucleotide.

[0034] In embodiments, the sense strand of the dsRNA agent has the following modification pattern: 5'-(L1)(inv)snnnnnnnnNfNfNfnnnnnnnnns(inv)(L2)-3', wherein each (inv) is an inverted nucleotide (e.g., an inverted abasic nucleotide, such as an inverted abasic ribonucleotide), and at least one of (L1) and (L2) is a ligand comprising a lipophilic group (e.g., comprising a C16 alkyl or C16 alkenyl group), and the other of (L1) and (L2) is absent or is hydrogen.

[0035] In some embodiments, the antisense strand of the dsRNA agent has one of the following modification patterns: 5'-VPnsdNsnndNndNnnnndNnFnnnnnnnsnsn-3', 5'-VPnsNfsnndNn(Ngn)nnnnnnNfnNfnnnnnsnsn-3', or 5'-VPnsNfsnndNn(N2p)nnnnnnNfnNfnnnnnsnsn-3', wherein VP is vinyl-phosphonate, n is a 2'-O-methyl-nucleotide, s is a phosphorothioate internucleotide linkage, dN is a 2'-deoxy-nucleotide, Nf is a 2'-fluoro-nucleotide, and (Ngn) is a diol nucleic acid S-isomer, and N2p is a 2'-phosphate nucleotide.

[0036] In embodiments, the antisense strand of the dsRNA agent has one of the following modification patterns: 5'-ZnsdNsnndNndNnnnndNnFnnnnnsnsn-3', 5'-ZnsNfsnndNn(Ngn)nnnnnnNfnNfnnnsnsn-3', 5'-ZnsNfsnNfnNfnNfnNfnNfnNfnNfnNfsn-3', 5'-ZnsNfsnNfnNfnNfnNfnNfnNfnNfnsnsn-3', 5'-ZnsNfsnNfnNfnNfnNfnNfnNfnNfnsnsn-3', 5'-ZnsNfsnNfnNfnNfnNfnNfnNfnNfnsnsn-3', 5'-ZnsNfsnNfnNfnNfnNfnNfnNfnNfnsnsn-3', 5'-ZnsNfsn -ZnsNfsnNfnNfnnnnnfnNfnNfnNfnNfsn-3', 5'-ZnsNfsnsNfnNfnNfnNfnNfnNfnNfnNfsn-3', 5'-ZnsNfsnsNfnNfnnnnnNfnNfnNfnNfsn-3', or 5'-ZnsNfsnsNfnNfnNfnNfnNfnNfnNfnNfn-3', wherein Z is a 5'-phosphate mimetic, such as 5'-cyclopropylphosphonate (5'-CP). (As used herein, the structure of 5'-CP is )

[0037] In certain embodiments, the sense strand of the dsRNA agent comprises the modification pattern: 5'-nsnsnnn(Nhd)NfnNfNfNfnnnnnnnnsnsn-3'; and the antisense strand comprises the modification pattern: 5'-VPnsdNsnndNndNnnnndNnFnnnnnnnsnsn-3', wherein VP is vinylphosphonate, n is a 2'-O-methyl-nucleotide, s is a phosphorothioate internucleotide linkage, Nf is a 2'-fluoro-nucleotide, dN is a 2'-deoxy-nucleotide, and (Nhd) is a 2'-O-hexadecyl-nucleotide.

[0038] In certain embodiments, the sense strand of the dsRNA agent comprises the modification pattern: 5'-nsnsnnn(Nhd)NfnNfNfNfnnnnnnsnsn-3'; and the antisense strand comprises the modification pattern: 5'-VPnsNfsnndNn(Ngn)nnnnnnNfnNfnnnnnsnsn-3', wherein VP is vinylphosphonate, n is a 2'-O-methyl-nucleotide, s is a phosphorothioate internucleotide linkage, Nf is a 2'-fluoro-nucleotide, dN is a 2'-deoxy-nucleotide, (Nhd) is a 2'-O-hexadecyl-nucleotide, and (Ngn) is a diol nucleic acid S-isomer.

[0039] In certain embodiments, the sense strand of the dsRNA agent comprises the modification pattern: 5'-nsnsnnn(Nhd)NfnNfNfNfnnnnnnsnsn-3'; and the antisense strand comprises the modification pattern: 5'-VPnsNfsnndNn(N2p)nnnnnnNfnNfnnnnnsnsn-3', wherein VP is vinylphosphonate, n is a 2'-O-methyl-nucleotide, s is a phosphorothioate internucleotide linkage, Nf is a 2'-fluoro-nucleotide, dN is a 2'-deoxy-nucleotide, (Nhd) is a 2'-O-hexadecyl-nucleotide, (Ngn) is a diol nucleic acid S-isomer, and N2p is a 2'-phosphate nucleotide.

[0040] In certain embodiments, the sense strand of the dsRNA agent comprises the modification pattern: 5'-nsnsnnnnnnNfNfNfnnnn(Nhd)nnnsnsn-3'; and the antisense strand comprises the modification pattern: 5'-VPnsdNsnndNndNnnnndNnFnnnnnnnsnsn-3', wherein VP is vinylphosphonate, n is a 2'-O-methyl-nucleotide, s is a phosphorothioate internucleotide linkage, Nf is a 2'-fluoro-nucleotide, dN is a 2'-deoxy-nucleotide, and (Nhd) is a 2'-O-hexadecyl-nucleotide.

[0041] In certain embodiments, the sense strand of the dsRNA agent comprises the modification pattern: 5'-nsnsnnnnnnNfNfNfnnnn(Nhd)nnnsnsn-3'; and the antisense strand comprises the modification pattern: 5'-VPnsNfsnndNn(Ngn)nnnnnnNfnNfnnnnnsnsn-3', wherein VP is vinylphosphonate, n is a 2'-O-methyl-nucleotide, s is a phosphorothioate internucleotide linkage, Nf is a 2'-fluoro-nucleotide, dN is a 2'-deoxy-nucleotide, (Nhd) is a 2'-O-hexadecyl-nucleotide, and (Ngn) is a diol nucleic acid S-isomer.

[0042] In certain embodiments, the sense strand of the dsRNA agent comprises the modification pattern: 5'-nsnsnnnnnnNfNfNfnnnn(Nhd)nnnsnsn-3'; and the antisense strand comprises the modification pattern: 5'-VPnsNfsnndNn(N2p)nnnnnnNfnNfnnnnnsnsn-3', wherein VP is vinylphosphonate, n is a 2'-O-methyl-nucleotide, s is a phosphorothioate internucleotide linkage, Nf is a 2'-fluoro-nucleotide, dN is a 2'-deoxy-nucleotide, (Nhd) is a 2'-O-hexadecyl-nucleotide, (Ngn) is a diol nucleic acid S-isomer, and N2p is a 2'-phosphate nucleotide.

[0043] In certain embodiments, the sense strand of the dsRNA agent comprises the modification pattern: 5'-nsnsnnn(Nhd)nnNfNfNfnnnnnnnnsnsn-3'; and the antisense strand comprises the modification pattern: 5'-VPnsdNsnndNndNnnnndNnFnnnnnnnsnsn-3', wherein VP is vinylphosphonate, n is a 2'-O-methyl-nucleotide, s is a phosphorothioate internucleotide linkage, Nf is a 2'-fluoro-nucleotide, dN is a 2'-deoxy-nucleotide, (Nhd) is a 2'-O-hexadecyl-nucleotide, and (Ngn) is a diol nucleic acid S-isomer.

[0044] In certain embodiments, the sense strand of the dsRNA agent comprises the modification pattern: 5'-nsnsnnn(Nhd)nnNfNfNfnnnnnnnnsnsn-3'; and the antisense strand comprises the modification pattern: 5'-VPnsNfsnndNn(Ngn)nnnnnnNfnNfnnnnnsnsn-3', wherein VP is vinylphosphonate, n is a 2'-O-methyl-nucleotide, s is a phosphorothioate internucleotide linkage, Nf is a 2'-fluoro-nucleotide, dN is a 2'-deoxy-nucleotide, (Nhd) is a 2'-O-hexadecyl-nucleotide, and (Ngn) is a diol nucleic acid S-isomer.

[0045] In certain embodiments, the sense strand of the dsRNA agent comprises the modification pattern: 5'-nsnsnnn(Nhd)nnNfNfNfnnnnnnnnsnsn-3'; and the antisense strand comprises the modification pattern: 5'-VPnsNfsnndNn(N2p)nnnnnnNfnNfnnnnnsnsn-3', wherein VP is vinylphosphonate, n is a 2'-O-methyl-nucleotide, s is a phosphorothioate internucleotide linkage, Nf is a 2'-fluoro-nucleotide, dN is a 2'-deoxy-nucleotide, (Nhd) is a 2'-O-hexadecyl-nucleotide, (Ngn) is a diol nucleic acid S-isomer, and N2p is a 2'-phosphate nucleotide.

[0046] Another aspect of the present disclosure provides a dsRNA agent for inhibiting the expression of SNCA, wherein the dsRNA agent comprises a sense strand having a 5'-end and a 3'-end and an antisense strand having a 5'-end and a 3'-end, wherein the sense strand and the antisense strand form a double-stranded region, wherein: the sense strand comprises the nucleotide sequence and modifications of Table 2 (SEQ ID NOs: 13 to 55), with 0 or 1 mismatches; and the antisense strand comprises the nucleotide sequence and modifications of Table 2 (SEQ ID NOs: 56 to 98), with 0 or 1 mismatches.

[0047] In one embodiment, the dsRNA agent has one of the following sense strand nucleotide sequences: 5'-asasgug(Chd)ucAfGfUfuccaaugusgsa-3' (SEQ ID NO:35), 5'-uscsuuugcuCfCfCfaguu(Uhd)cuusgsa-3' (SEQ ID NO:28), 5'-gsasgca(Ahd)guGfAfCfaaauguugsgsa-3' (SEQ ID NO:21), 5'-gsusaca(Ahd)GfuGfCfUfcaguuccasasa-3' (SEQ ID NO:34), 5'-cscsauc(Ahd)gcAfGfUfgauugaagsusa-3' (SEQ ID NO:43), 5'-uscsccag(Uhd)uUfCfUfugagaucusgsa-3' (SEQ ID NO: NO:261) or 5'-uscsaug(Ahd)aaGfGfAfcuuucaaasgsa-3' (SEQ ID NO:19), wherein a is 2'-O-methyladenosine-3'-phosphate, u is 2'-O-methyluridine-3'-phosphate, g is 2'-O-methylguanosine-3'-phosphate, c is 2'-O-methylcytidine-3'-phosphate, s is a phosphorothioate internucleotide linkage, Af is 2'-fluoroadenosine-3'-phosphate, Gf is 2'-fluoroguanosine-3'-phosphate, Uf is 2'-fluorouridine-3'-phosphate, Cf is 2'-fluorocytidine-3'-phosphate, (Ahd) is 2'-O-hexadecyladenosine-3'-phosphate, (Uhd) is 2'-O-hexadecyluridine-3'-phosphate, and (Chd) is 2'-O-hexadecylcytidine-3'-phosphate.

[0048] In another embodiment, the dsRNA agent has one of the following antisense strand nucleotide sequences: 5'-VPusdCsacdAudTggaadCuGfagcacuusgsu-3' (SEQ ID NO:78), 5'-VPusdCsaadGadAacugdGgAfgcaaagasusa-3' (SEQ ID NO:71), 5'-VPusdCscadAcdAuuugdTcAfcuugcucsusu-3' (SEQ ID NO:64), 5'-VPusUfsugdGa(Agn)cugagcAfcUfuguacsasg-3' (SEQ ID NO:77), 5'-VPusdAscudTcdAaucadCuGfcugauggsasa-3' (SEQ ID NO:86), 5'-VPusdCsagdAudCucaadGaAfacugggasgsc-3' (SEQ ID NO: NO: 262) or 5'-VPusdCsuudTgdAaagudCcUfuucaugasasu-3' (SEQ ID NO:62), wherein VP is vinylphosphonate, a is 2'-O-methyladenosine-3'-phosphate, u is 2'-O-methyluridine-3'-phosphate, g is 2'-O-methylguanosine-3'-phosphate, c is 2'-O-methylcytidine-3'-phosphate, s is a phosphorothioate internucleotide linkage, Af is 2'-fluoroadenosine-3'-phosphate, Gf is 2'-fluoroguanosine-3'-phosphate, Uf is 2'-fluorouridine-3'-phosphate, Cf is 2'-fluorocytidine-3'-phosphate, dA is 2'-deoxyadenosine-3'-phosphate, dC is 2'-deoxycytidine-3'-phosphate, dG is 2'-deoxyguanosine-3'-phosphate, dT is 2'-deoxythymidine-3'-phosphate, and (Agn) is adenosine-diol nucleic acid (GNA) S-isomer.

[0049] In further embodiments, the dsRNA agent has one of the following duplex pairs of nucleotide sequences: (i) sense strand: 5'-asasgug(Chd)ucAfGfUfuccaaugusgsa-3' (SEQ ID NO:35) and antisense strand: 5'-VPusdCsacdAudTggaadCuGfagcacuusgsu-3' (SEQ ID NO:78); (ii) sense strand: 5'-uscsuuugcuCfCfCfaguu(Uhd)cuusgsa-3' (SEQ ID NO:28) and antisense strand: 5'-VPusdCsaadGadAacugdGgAfgcaaagasusa-3' (SEQ ID NO:71); (iii) sense strand: 5'-gsasgca(Ahd)guGfAfCfaaauguugsgsa-3' (SEQ ID NO:21) and antisense strand: 5'-VPusdCscadAcdAuuugdTcAfcuugcucsusu-3' (SEQ ID NO:64); (iv) sense strand: 5'-gsusaca(Ahd)GfuGfCfUfcaguuccasasa-3' (SEQ ID NO:34) and antisense strand: 5'-VPusUfsugdGa(Agn)cugagcAfcUfuguacsasg-3' (SEQ ID NO:77); (v) sense strand: 5'-cscsauc(Ahd)gcAfGfUfgauugaagsusa-3' (SEQ ID NO:43) and antisense strand: 5'-VPusdAscudTcdAaucadCuGfcugauggsasa-3' (SEQ ID NO:86); (vi) sense strand: 5'-uscsccag(Uhd)uUfCfUfugagaucusgsa-3' (SEQ ID NO:261) and antisense strand: 5'-VPusdCsagdAudCucaadGaAfacugggasgsc-3' (SEQ ID NO:262);or (vii) sense strand: 5'-uscsaug(Ahd)aaGfGfAfcuuucaaasgsa-3' (SEQ ID NO: 19) and antisense strand: 5'-VPusdCsuudTgdAaagudCcUfuucaugasasu-3' (SEQ ID NO: 62), wherein VP is vinylphosphonate, a is 2'-O-methyladenosine-3'-phosphate, u is 2'-O-methyluridine-3'-phosphate, g is 2'-O-methylguanosine-3'-phosphate, c is 2'-O-methylcytidine-3'-phosphate, s is a phosphorothioate internucleotide linkage, Af is 2'-fluoroadenosine-3'-phosphate, Gf is 2'-fluoroguanosine-3'-phosphate, Uf is 2'-fluorouridine-3'-phosphate, and Cf is 2'-fluorouridine-3'-phosphate. , dA is 2'-deoxyadenosine-3'-phosphate, dC is 2'-deoxycytidine-3'-phosphate, dG is 2'-deoxyguanosine-3'-phosphate, dT is 2'-deoxythymidine-3'-phosphate, (Agn) is adenosine-diol nucleic acid (GNA) S-isomer, (Ahd) is 2'-O-hexadecyladenosine-3'-phosphate, (Uhd) is 2'-O-hexadecyluridine-3'-phosphate, and (Chd) is 2'-O-hexadecylcytidine-3'-phosphate. ;

[0050] Another aspect of the present disclosure provides a dsRNA agent for inhibiting the expression of SNCA, the dsRNA agent having a sense strand sequence 5'-asasgug(Chd)ucAfGfUfuccaaugusgsa-3' (SEQ ID NO: 35) and an antisense strand sequence 5'-VPusdCsacdAudTggaadCuGfagcacuusgsu-3' (SEQ ID NO:78), wherein VP is vinylphosphonate, a is 2'-O-methyladenosine-3'-phosphate, u is 2'-O-methyluridine-3'-phosphate, g is 2'-O-methylguanosine-3'-phosphate, c is 2'-O-methylcytidine-3'-phosphate, s is a phosphorothioate internucleotide linkage, Af is 2'-fluoroadenosine-3'-phosphate, Gf is 2'-fluoroguanosine-3'-phosphate, Uf is 2'-fluorouridine-3'-phosphate, dA is 2'-deoxyadenosine-3'-phosphate, dC is 2'-deoxycytidine-3'-phosphate, dT is 2'-deoxythymidine-3'-phosphate, and (Chd) is 2'-O-hexadecylcytidine-3'-phosphate.

[0051] Another aspect of the present disclosure provides a dsRNA agent for inhibiting the expression of SNCA, the dsRNA agent having a sense strand sequence 5'-uscsuuugcuCfCfCfaguu(Uhd)cuusgsa-3' (SEQ ID NO: 28) and an antisense strand sequence 5'-VPusdCsaadGadAacugdGgAfgcaaagasusa-3' (SEQ ID NO:71), wherein VP is vinylphosphonate, a is 2'-O-methyladenosine-3'-phosphate, u is 2'-O-methyluridine-3'-phosphate, g is 2'-O-methylguanosine-3'-phosphate, c is 2'-O-methylcytidine-3'-phosphate, s is a phosphorothioate internucleotide linkage, Af is 2'-fluoroadenosine-3'-phosphate, Cf is 2'-fluorocytidine-3'-phosphate, dA is 2'-deoxyadenosine-3'-phosphate, dC is 2'-deoxycytidine-3'-phosphate, dG is 2'-deoxyguanosine-3'-phosphate, and (Uhd) is 2'-O-hexadecyluridine-3'-phosphate.

[0052] A further aspect of the present disclosure provides a dsRNA agent for inhibiting the expression of SNCA, the dsRNA agent having a sense strand sequence 5'-gsasgca(Ahd)guGfAfCfaaauguugsgsa-3' (SEQ ID NO: 21) and an antisense strand sequence 5'-VPusdCscadAcdAuuugdTcAfcuugcucsusu-3' (SEQ ID NO: NO:64), wherein VP is vinylphosphonate, a is 2'-O-methyladenosine-3'-phosphate, u is 2'-O-methyluridine-3'-phosphate, g is 2'-O-methylguanosine-3'-phosphate, c is 2'-O-methylcytidine-3'-phosphate, s is a phosphorothioate internucleotide linkage, Af is 2'-fluoroadenosine-3'-phosphate, Gf is 2'-fluoroguanosine-3'-phosphate, Cf is 2'-fluorocytidine-3'-phosphate, dA is 2'-deoxyadenosine-3'-phosphate, dC is 2'-deoxycytidine-3'-phosphate, dT is 2'-deoxythymidine-3'-phosphate, and (Ahd) is 2'-O-hexadecyladenosine-3'-phosphate.

[0053] In another embodiment, the RNAi agent is a pharmaceutically acceptable salt thereof. "Pharmaceutically acceptable salts " of each RNAi agent herein include, but are not limited to, sodium salts, calcium salts, lithium salts, potassium salts, ammonium salts, magnesium salts and mixtures thereof. It will be understood by those skilled in the art that RNAi agents can be provided as polycationic salts having a cation of each free acid group (e.g., 5'-terminal phosphate group) of an optionally modified phosphodiester backbone and / or any other acidic modification. For example, an oligonucleotide of "n" nucleotide length contains n-1 optionally modified phosphodiester such that an oligonucleotide of 21nt length can be provided as a salt with up to 20 cations (e.g., 20 sodium cations). Similarly, an RNAi agent with a sense strand of 21nt length and an antisense strand of 23nt length can be provided as a salt with up to 42 cations (e.g., 42 sodium ions). In the foregoing examples, where the RNAi agent further comprises a 5'-terminal phosphate or 5'-terminal vinylphosphonate group, the RNAi agent can be provided as a salt having up to 44 cations (eg, 44 sodium cations).

[0054] Another aspect of the disclosure provides cells containing the dsRNA agents disclosed herein.

[0055] Additional aspects of the present disclosure provide a pharmaceutical composition for inhibiting the expression of α-synuclein, the pharmaceutical composition comprising a dsRNA agent disclosed herein.

[0056] In certain embodiments, the dsRNA agent and / or pharmaceutical composition is administered in a non-buffered solution. Optionally, the non-buffered solution is saline or water.

[0057] In some embodiments, the dsRNA agent is administered with a buffer solution. Optionally, the buffer solution comprises acetate, citrate, prolamin, carbonate or phosphate, or a combination thereof. In certain embodiments, the buffer solution is phosphate buffered saline (PBS).

[0058] A further aspect of the present disclosure provides a pharmaceutical composition comprising a dsRNA agent disclosed herein and a lipid formulation.

[0059] In one embodiment, the lipid formulation comprises or is a lipid nanoparticle (LNP).

[0060] Another aspect of the present disclosure provides a method of inhibiting the expression of an α-synuclein (SNCA) gene in a cell and / or preventing the formation of α-synuclein aggregates in a cell or a subject, the method comprising: (a) contacting a cell or a subject with a dRNA agent or pharmaceutical composition disclosed herein; and (b) maintaining the cell or subject produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the SNCA gene, thereby inhibiting the expression of the SNCA gene in the cell and / or preventing the formation of α-synuclein aggregates in the cell or the subject.

[0061] In one embodiment, the cell is in a subject. Optionally, the subject is a human. Alternatively, the subject is a rhesus monkey, a cynomolgus monkey, a mouse, or a rat.

[0062] In certain embodiments, the human subject suffers from an SNCA-associated disease. Optionally, the SNCA-associated disease is a synucleinopathy. Optionally, the synucleinopathy is PD, multiple system atrophy (MSA), Lewy body dementia (LBD), pure autonomic failure (PAF), Pick's disease, progressive supranuclear palsy, dementia pugilistica, Parkinson's disease linked to chromosome 17, Lytico-Bodig's disease, tangle-dominant dementia, argyrophilic grain disease, ganglioglioma, gangliocytoma, meningioangiomatosis, subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis, Hallervorden-Spatz disease, lipofuscinosis, corticobasal degeneration, frontotemporal dementia, frontotemporal lobar degeneration, Alzheimer's disease, Huntington's disease, Down syndrome, psychosis, schizophrenia and / or Creutzfeldt-Jakob disease.

[0063] In some embodiments, SNCA expression in the cell or subject is inhibited by the dsRNA agent by at least about 50%, at least about 40%, at least about 30%, at least about 20%, or at least about 10% compared to a control cell or control subject.

[0064] Additional aspects of the present disclosure provide a method of treating a subject diagnosed with a SNCA-associated neurodegenerative disease, the method involving administering to the subject a therapeutically effective amount of a dsRNA agent or pharmaceutical composition disclosed herein, optionally wherein the subject is re-administered with a therapeutically effective amount of a dsRNA agent or pharmaceutical composition disclosed herein, thereby treating the subject.

[0065] In certain embodiments, treatment comprises amelioration of at least one sign or symptom of a SNCA-associated neurodegenerative disease.

[0066] In some embodiments, treating includes preventing the progression of the disease.

[0067] In one embodiment, an SNCA-associated neurodegenerative disease is characterized by one or more of the following symptoms: tremor, slow movements (bradykinesia), muscle stiffness, impaired posture and balance, loss of spontaneous movement, changes in speech, changes in handwriting, visual hallucinations, auditory hallucinations, olfactory hallucinations, tactile hallucinations, poor regulation of bodily functions (autonomic nervous system) such as dizziness, falls, and bowel problems, cognitive problems such as confusion, difficulty concentrating, visual-spatial problems, and memory loss, sleep difficulties such as rapid eye movement (REM) sleep behavior disorder (in which dreams are physically acted out during sleep), fluctuations in attention (including cravings), and Examples include sleep apnea, prolonged staring into space, long daytime naps or slurred speech, depression, and apathy; orthostatic hypotension (a sudden drop in blood pressure when a person stands up, causing the person to feel dizzy and lightheaded and need to sit, squat, or lie down to prevent passing out; clumsy or uncoordinated movements; bladder control problems; contractures of the hands or limbs (chronic shortening of the muscles or tendons around a joint that prevents the joint from moving freely); Pisa syndrome (an abnormal posture in which the body leans to one side); nuchal flexion (in which the neck bends forward and the head drops); and involuntary, uncontrollable sighing or gasping.

[0068] In certain embodiments, the dsRNA agent is administered to a subject at a dose of about 0.01 mg / kg to about 50 mg / kg.

[0069] In some embodiments, the dsRNA agent or pharmaceutical composition is administered intrathecally to the subject.

[0070] In one embodiment, the method further involves administering to the subject an additional agent or therapy useful for treating or preventing a SNCA-associated neurodegenerative disease or disorder.

[0071] In certain embodiments, the SNCA-associated neurodegenerative disease is Parkinson's disease (PD).

[0072] In one embodiment, the SNCA-associated neurodegenerative disease is Lewy body dementia (LBD) or multiple system atrophy (MSA).

[0073] In some embodiments, SNCA expression is inhibited by at least about 30%.

[0074] In another embodiment, the method further involves administering to the subject an additional therapeutic agent.

[0075] In certain embodiments, the method reduces SNCA expression in brain or spinal cord tissue. Optionally, the brain or spinal cord tissue is cerebral cortex, cerebellum, basal ganglia, hippocampus, amygdala, thalamus, brainstem, cervical spinal cord, lumbar spinal cord and / or thoracic spinal cord.

[0076] Another aspect of the present disclosure provides a method of inhibiting the expression of SNCA in a subject, the method involving administering to the subject a therapeutically effective amount of a dsRNA agent or pharmaceutical composition disclosed herein, thereby inhibiting the expression of SNCA in the subject.

[0077] Additional aspects of the present disclosure provide a method for treating or preventing a SNCA-associated disease in a subject, the method involving administering to the subject a therapeutically effective amount of a dsRNA agent or pharmaceutical composition disclosed herein, thereby treating or preventing the SNCA-associated disease in the subject.

[0078] In certain embodiments, the administering step produces at least 60% knockdown of SNCA mRNA or α-synuclein in one or more tissues of the subject. Optionally, the one or more tissues of the subject include CSF, prefrontal cortex, midbrain, thoracic spine, hippocampus, medulla pons, caudate nucleus of the striatum, and / or cerebellum.

[0079] A further aspect of the present disclosure provides a kit for performing the methods disclosed herein, the kit comprising a dsRNA agent disclosed herein and instructions for use thereof. Optionally, the kit further comprises a device for administering the dsRNA agent to a subject.

[0080] definition

[0081] In order to more readily understand the present disclosure, certain terms are first defined. Additionally, it should be noted that whenever a numerical value or numerical range of a parameter is stated, values ​​and ranges intermediate to the stated values ​​are also intended to be part of the present disclosure.

[0082] The articles "a" and "an" are used herein to refer to one or to more than one (ie, to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element, for example, a plurality of elements.

[0083] The term "including" is used herein to mean, and is used interchangeably with, the phrase "including, but not limited to."

[0084] The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless the context clearly dictates otherwise.

[0085] The term "about" is used herein to refer to within the typical tolerance range in the art. For example, "about" can be understood as about 2 standard deviations from the mean. In certain embodiments, "about" means + 10%. In certain embodiments, "about" means + 5%. When "about" precedes a series of numbers or a range, it should be understood that "about" can modify each number in the series or range.

[0086] The term "at least" preceding a number or a range of numbers should be understood to include the number adjacent to the term "at least," as well as all subsequent numbers or integers that can logically be included, as clear from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleotides of a 21-nucleotide nucleic acid molecule" means that 18, 19, 20, or 21 nucleotides have the specified property. When "at least" precedes a range of numbers or a range, it should be understood that "at least" can modify each number in the range or series.

[0087] As used herein, "no more than" or "less than" is understood to refer to the value adjacent to the phrase and logically lower values ​​or integers, as understood logically from the context, up to zero. For example, a duplex having an overhang of "no more than 2 nucleotides" has an overhang of 2, 1, or 0 nucleotides. When "no more than" precedes a series of numbers or a range, it is understood that "no more than" can modify each number in the series or range. As used herein, a range includes both an upper limit and a lower limit.

[0088] As used herein, a detection method may include determining that an analyte is present in an amount below the level of detection of the method.

[0089] In the event of a conflict between the indicated target site and the nucleotide sequence of either the sense or antisense strand, the indicated sequence takes precedence.

[0090] In the event of a conflict between a chemical structure and a chemical name, the chemical structure shall prevail.

[0091] The terms "SNCA," "α-synuclein," "synuclein α," or "alpha-synuclein" refer to a gene and the protein encoded by it that is associated with neurodegenerative diseases known as synucleinopathies. The human SNCA gene region spans approximately 114 kb. The SNCA transcript contains 13 exons, and 15 mRNA isoforms have been identified or are otherwise predicted to be produced. The nucleotide and amino acid sequences of SNCA can be found, for example, in GenBank Accession No. NM_007308.3 (human (Homo sapiens) SNCA, SEQ ID NO: 1, reverse complement sequence SEQ ID NO: 2); GenBank Accession No. XM_005555421 (cynomolgus monkey (Macaca fascicularis) SNCA, SEQ ID NO: 3, reverse complement sequence SEQ ID NO: 4); GenBank Accession No. NM_009221 (mouse (Mus musculus) SNCA, SEQ ID NO: 5, reverse complement sequence SEQ ID NO: 6); GenBank Accession No. NM_019169.2 (rat (Rattus norvegicus) SNCA, SEQ ID NO: 7, reverse complement sequence SEQ ID NO: 8); and GenBank Accession No. XM_535656.7 (domestic dog (Canis lupus familiaris) SNCA, SEQ ID NO: 228, reverse complement sequence SEQ ID NO: 10). NO:229).

[0092] As used herein, the term "SNCA" also refers to variants of the SNCA gene, including naturally occurring sequence variants as provided, for example, isoform 1 transcript NM_000345.4 (SEQ ID NO: 232), which encodes polypeptide NP_000336.1; isoform 2 transcript NM_001146054.2 (SEQ ID NO: 230), which encodes polypeptide NP_001139526.1; isoform 3 transcript NM_001146055.2 (SEQ ID NO: 231), which encodes polypeptide NP_001139527.1; isoform 4 transcript NM_007308.3 (SEQ ID NO: 1), which encodes polypeptide NP_009292.1, as described above; isoform 5 transcript NM_001375285.1 (SEQ ID NO: 232), which encodes polypeptide NP_001139526.1; NO: 233), which encodes the polypeptide NP_001362214.1; the subtype 6 transcript NM_001375286.1 (SEQ ID NO: 234), which encodes the polypeptide NP_001362215.1; the subtype 7 transcript NM_001375287.1 (SEQ ID NO: 235), which encodes the polypeptide NP_001362216.1; the subtype 8 transcript NM_001375288.1 (SEQ ID NO: 236), which encodes the polypeptide NP_001362217.1; the subtype 9 transcript NM_001375290.1 ​​(SEQ ID NO: 237), which encodes the polypeptide NP_001362219.1; and the predicted subtype X1 transcript XM_011532203.1 (SEQ ID NO: 238), which encodes polypeptide XP_011530505.1; predicted subtype X2 transcript XM_011532204.3 (SEQ ID NO: 239), which encodes polypeptide XP_011530506.1; predicted subtype X3 transcript XM_011532205.2 (SEQ ID NO: 240), which encodes polypeptide XP_011530507.1; predicted subtype X4 transcript XM_011532206.1 (SEQ ID NO: 241), which encodes polypeptide XP_011530508.1; predicted subtype X5 transcript XM_011532207.1 (SEQ ID NO: 242), encoding polypeptide XP_011530509.1; and predicted subtype X8 transcript XM_017008563.1 (SEQ ID NO: 243), encoding polypeptide XP_016864052.1 (the unique sequences associated with each of the aforementioned accession numbers are incorporated herein by reference in the form available as of the filing date of this application).Additional examples of SNCA sequences can be found in publicly available databases, such as GenBank, OMIM, UniProt, NCBI dbSNP (see, for example, www.ncbi.nlm.nih.gov / gene / 6622), and the Macaca Genome Project website (macaque.genomics.org.cn / page / species / index.jsp). Additional information about SNCA can be found, for example, at www.ncbi.nlm.nih.gov / gene / 6622. As of the date of filing this application, the entire contents of each of the aforementioned GenBank accession numbers and gene database numbers are incorporated herein by reference.

[0093] Three protein isoforms of α-synuclein are described in UniProt. The longest α-synuclein isoform is a protein of approximately 14 kDa (isoform 1 UniProt, P37840 of 140 amino acids). Other α-synuclein isoforms in UniProt include: isoforms 2-4, P37840-2 of 112 amino acids; and isoforms 2-5, P37840-3 of 126 amino acids. The 140 amino acid α-synuclein is encoded by 5 exon pairs mapped to the chromosome site 4q21.3-q22. α-synuclein has an N-terminal region composed of an incomplete KXKEGV (SEQ ID NO: 244) motif, an extremely hydrophobic NAC domain, and a highly acidic C-terminal domain. Under physiological conditions, SNCA is believed to be an intrinsically disordered monomer or a helically folded tetramer. α-Synuclein accounts for 1% of all proteins in the cytosol of brain cells and is primarily expressed in the neocortex, hippocampus, substantia nigra, thalamus, and cerebellum. α-Synuclein is also expressed in lower amounts in heart, skeletal muscle, pancreas, lymphocytes, and blood cells. Although the function of SNCA is not fully understood, evidence suggests that it plays an important role in maintaining an adequate supply of synaptic vesicles in presynaptic terminals. α-Synuclein is implicated in the regulation of dopamine release and transport, the fibrillization of the microtubule-associated protein tau, and the modulation of a neuroprotective phenotype in non-dopaminergic neurons through both regulation of p53 expression and inhibition of transactivation of pro-apoptotic genes, resulting in reduced caspase-3 activation. The primary mechanism by which α-synuclein induces neurodegenerative diseases such as Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy is the elevated levels of α-synuclein, resulting in the formation of α-synuclein fibrillary aggregates.

[0094] As used herein, "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of the SNCA gene, including mRNA that is a product of RNA processing as a product of the primary transcript. In one embodiment, the target portion of the sequence is at least long enough to serve as a substrate for RNAi-directed cleavage at or near that portion of the nucleotide sequence of the mRNA molecule formed during transcription of the SNCA gene. In one embodiment, the target sequence is within the protein-coding region of the SNCA gene. In another embodiment, the target sequence is within the 3'-UTR of the SNCA gene.

[0095] The target sequence can be about 9 to 36 nucleotides in length, for example, about 15 to 30 nucleotides in length. For example, the target sequence can be about 15 to 30 nucleotides in length, such as 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 2 In some embodiments, the target sequence is about 19 to about 30 nucleotides in length. In other embodiments, the target sequence is about 19 to about 25 nucleotides in length. In still other embodiments, the target sequence is about 19 to about 23 nucleotides in length. In some embodiments, the target sequence is about 21 to about 23 nucleotides in length. Ranges and lengths intermediate to those recited above are also contemplated as being part of this disclosure.

[0096] As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising a chain of nucleotides described in the order mentioned using standard nucleotide nomenclature.

[0097] In the case of modified or unmodified nucleotides, "G", "C", "A", "T" and "U" each generally represent a nucleotide containing guanine, cytosine, adenine, thymine and uracil as a base, respectively. However, it will be understood that the term "ribonucleotide" or "nucleotide" can also refer to a modified nucleotide, or a surrogate substitution moiety (see, e.g., Table 1), as further described in detail below. It will be well understood by those skilled in the art that guanine, cytosine, adenine, thymine and uracil can be replaced by other moieties without substantially altering the base pairing properties of the oligonucleotide comprising the nucleotide carrying such a substitution moiety. For example, and not by way of limitation, a nucleotide comprising inosine as its base can base pair with a nucleotide containing adenine, cytosine or uracil. Thus, a nucleotide containing uracil, guanine or adenine can be replaced in the nucleotide sequence of a dsRNA described in the present disclosure by a nucleotide containing, for example, inosine. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced by guanine and uracil, respectively, to form GU wobble base pairing with the target mRNA.Sequences containing such replacement moieties are suitable for use in the compositions and methods described in the present disclosure.

[0098] The terms "iRNA," "RNAi agent," "iRNA agent," and "RNA interfering agent," as used interchangeably herein, refer to agents that contain RNA, as that term is defined herein, and that mediate targeted cleavage of RNA transcripts through the RNA-induced silencing complex (RISC) pathway. RNA interference (RNAi) is a process that directs sequence-specific degradation of mRNA. RNAi modulates (e.g., inhibits) expression of SNCA in cells (e.g., cells in a subject, such as a mammalian subject).

[0099] In one embodiment, the RNAi agents of the present disclosure include single-stranded RNAi that interact with a target RNA sequence, such as a SNCA target mRNA sequence, to guide the cleavage of the target RNA. Without wishing to be bound by theory, it is believed that long double-stranded RNA introduced into cells is degraded by a type III endonuclease called Dicer into double-stranded short interfering RNAs (siRNAs) comprising a sense strand and an antisense strand (Sharp et al. (2001) Genes Dev. 15:485). Dicer (an RNase III-like enzyme) processes these dsRNAs into 19-23 base pair short interfering RNAs with characteristic two-base 3' overhangs (Bernstein et al. (2001) Nature 409:363). These siRNAs are then incorporated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen et al. (2001) Cell 107:309). When bound to the appropriate target mRNA, one or more endonucleases within RISC cleave the target to induce silencing (Elbashir et al. (2001) Genes Dev. 15:188). Thus, in one aspect, the present disclosure relates to single-stranded RNA (ssRNA) produced within the cell (the antisense strand of the siRNA duplex) and the single-stranded RNA promotes the formation of the RISC complex to achieve silencing of the target gene (i.e., the SNCA gene). Thus, the term "siRNA" is also used herein to refer to RNAi as described above.

[0100] In another embodiment, RNAi agent can be a single-stranded RNA introduced into a cell or organism to suppress target mRNA. Single-stranded RNAi agent combines RISC endonuclease (Argonaute 2), and then it cuts the target mRNA. Single-stranded siRNA is typically 15-30 nucleotides and is chemically modified. The design and testing of single-stranded RNA are described in U.S. Patent number 8,101,348 and Lima et al. (2012) Cell 150:883-894, and the entire contents of each of these references are hereby incorporated herein by reference. Any antisense nucleotide sequence described herein can be used as described herein or as described in Lima et al. (2012) Cell 150:883-894 method chemically modified single-stranded siRNA.

[0101] In another embodiment, the "RNAi agent" used in the compositions and methods of the present disclosure is double-stranded RNA, and is referred to herein as a "double-stranded RNAi agent," "double-stranded RNA (dsRNA) molecule," "dsRNA agent," or "dsRNA." The term "dsRNA" refers to a complex of ribonucleic acid molecules having a duplex structure comprising two antiparallel and substantially complementary nucleic acid strands, referred to as having "sense" and "antisense" orientations relative to the target RNA (i.e., the SNCA gene). In some embodiments of the present disclosure, double-stranded RNA (dsRNA) triggers the degradation of a target RNA (e.g., mRNA) through a post-transcriptional gene silencing mechanism referred to herein as RNA interference or RNAi.

[0102] Typically, dsRNA molecules can include ribonucleotides, but as described in detail herein, each strand or both strands can also include one or more non-ribonucleotides, for example, deoxyribonucleotides and / or modified nucleotides. In addition, as used in this specification, "RNAi agent" can include ribonucleotides with chemical modifications; RNAi agents can include substantial modifications at multiple nucleotides.

[0103] As used herein, the term "modified nucleotides" refers to nucleotides that independently have a modified sugar moiety, modified internucleotide bond or modified nucleobase. Thus, the term "modified nucleotides" encompasses substitution, addition or removal of, for example, a functional group or atom of an internucleoside bond, a sugar moiety or a nucleobase. Modifications suitable for use in the disclosed medicaments include all types of modifications disclosed herein or known in the art. For purposes of this specification and claims, "RNAi agents" encompass any such modification used in siRNA-type molecules.

[0104] In certain embodiments of the present disclosure, the inclusion of deoxynucleotides, which are recognized as naturally occurring forms of nucleotides, can be considered to constitute modified nucleotides if present within a RNAi agent.

[0105] The duplex region can be of any length that allows for specific degradation of the desired target RNA by the RISC pathway, and can range from about 9 to 36 base pairs in length, such as about 15 to 30 base pairs in length, for example, about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length, such as about 15 to 30, 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 21, 15 to 22, 15 to 23. 8 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 1 21 to 22, 20 to 21, 21 to 30, 21 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, or 21 to 22 base pairs in length. Ranges and lengths intermediate to those recited above are also contemplated as part of the invention.

[0106] The two chains forming the duplex structure can be different parts of a larger RNA molecule, or they can be independent RNA molecules. In the case where the two chains are parts of a larger molecule, and therefore are connected by an uninterrupted nucleotide chain between the 3'-end of a chain forming the duplex structure and the 5'-end of the corresponding other chain, the connecting RNA chain is called a "hairpin loop". The hairpin loop can include at least one unpaired nucleotide. In some embodiments, the hairpin loop can include at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides or nucleotides that do not point to the target site of the dsRNA. In some embodiments, the hairpin loop can be 10 or less nucleotides. In some embodiments, the hairpin loop can be 8 or less unpaired nucleotides. In some embodiments, the hairpin loop can be 4-10 unpaired nucleotides. In some embodiments, the hairpin loop can be 4-8 nucleotides.

[0107] In certain embodiments, the two chains of the double-stranded oligomeric compound can be connected together. The two chains can be connected to each other at both ends or only at one end. Connection at one end refers to the 5'-end of the first chain being connected to the 3'-end of the second chain or the 3'-end of the first chain being connected to the 5'-end of the second chain. When the two chains are connected to each other at both ends, the 5'-end of the first chain is connected to the 3'-end of the second chain and the 3'-end of the first chain is connected to the 5'-end of the second chain. The two chains can be connected together by an oligonucleotide linker, which includes but is not limited to (N)n; wherein N is independently a modified or unmodified nucleotide and n is 3-23. In some embodiments, n is 3-10, such as 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, the oligonucleotide linker is selected from GNR A, (G) 4, (U) 4 and (dT) 4, wherein N is a modified or unmodified nucleotide and R is a modified or unmodified purine nucleotide. Some nucleotides in the linker can participate in base pairing interactions with other nucleotides in the linker. The two chains can also be linked together by a non-nucleoside linker, such as the linkers described herein.One skilled in the art will appreciate that any of the oligonucleotide chemical modifications or variations described herein can be used in an oligonucleotide linker.

[0108] Hairpin and dumbbell oligomeric compounds have a duplex region of equal to or at least 14, 15, 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The length of the duplex region can be equal to or less than 200, 100, or 50. In some embodiments, the length of the duplex region ranges from 15-30, 17-23, 19-23, and 19-21 nucleotide pairs.

[0109] Hairpin oligomeric compounds may have single-stranded overhangs or terminal unpaired regions, in some embodiments on the 3' side, and in some embodiments, on the antisense side of the hairpin. In some embodiments, the overhangs are 1-4, more typically 2-3 nucleotides in length. Hairpin oligomeric compounds that can induce RNA interference are also referred to herein as "shRNAs."

[0110] When the two substantially complementary chains of a dsRNA are composed of separate RNA molecules, these molecules are not necessarily, but can be covalently linked. When the two chains are covalently linked by means other than an uninterrupted nucleotide chain between the 3'-end of one chain and the 5'-end of the corresponding other chain forming a duplex structure, the connecting structure is called a "linker". The RNA chains can have the same or different numbers of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest chain of the dsRNA minus any overhangs present in the duplex. In addition to the duplex structure, RNAi can include one or more nucleotide overhangs.

[0111] In one embodiment, the RNAi agent of the present disclosure is a dsRNA in which each strand is 24 to 30 nucleotides in length that interacts with a target RNA sequence (e.g., a SNCA target mRNA sequence) to guide cleavage of the target RNA. Without wishing to be bound by theory, long double-stranded RNA introduced into cells is degraded into siRNA by a type III endonuclease called Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a nuclease III-like enzyme, processes dsRNA into short interfering RNAs of 19 to 23 base pairs with a characteristic two-base 3' overhang (Bernstein, et al., (2001) Nature 409:363). Subsequently, the siRNA is incorporated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, allowing the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). When bound to an appropriate target mRNA, one or more endonucleases within RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). In one embodiment, the RNAi agent of the present disclosure is a 24-30 nucleotide dsRNA agent that interacts with the SNCA RNA sequence to guide the cleavage of the target RNA.

[0112] In one embodiment, the RNAi agent of the present disclosure is a dsRNA agent wherein each strand comprises 19 to 23 nucleotides, which interacts with the SNCA RNA sequence to guide the cleavage of the target RNA. Without wishing to be bound by theory, long double-stranded RNA introduced into cells is degraded into siRNA by a type III endonuclease called Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a nuclease III-like enzyme, processes dsRNA into short interfering RNAs of 19 to 23 base pairs with a characteristic two-base 3' overhang (Bernstein, et al., (2001) Nature 409:363). Subsequently, the siRNA is incorporated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, allowing the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). When bound to an appropriate target mRNA, one or more endonucleases within RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). In one embodiment, the RNAi agent of the present disclosure is a dsRNA of 19 to 23 nucleotides that interacts with the SNCA RNA sequence to guide the cleavage of the target RNA.

[0113] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the duplex structure of an RNAi agent (e.g., dsRNA). For example, a nucleotide overhang is present when the 3' end of one strand of a dsRNA extends beyond the 5' end of the other strand, or vice versa. The dsRNA can comprise an overhang having at least one nucleotide; alternatively, the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more. The nucleotide overhang can comprise or consist of nucleotide / nucleoside analogs (including deoxynucleotides / nucleosides). The overhang can be on the sense strand, the antisense strand, or any combination thereof. In addition, the nucleotides of the overhang can be present on the 5'-end, 3'-end, or both ends of the antisense strand or the sense strand of the dsRNA.

[0114] In one embodiment of the dsRNA, at least one strand comprises a 3' overhang of at least 1 nucleotide. In another embodiment, at least one strand comprises a 3' overhang of at least 2 nucleotides, for example, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, at least one strand of the RNAi agent comprises a 5' overhang of at least 1 nucleotide. In certain embodiments, at least one strand comprises a 5' overhang of at least 2 nucleotides, for example, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In still other embodiments, both the 3' end and the 5' end of one strand of the RNAi agent comprise an overhang of at least 1 nucleotide.

[0115] In one embodiment, the antisense strand of the dsRNA has an overhang of 1 to 10 nucleotides, e.g., 0 to 3, 1 to 3, 2 to 4, 2 to 5, 4 to 10, 5 to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, at the 3' end, the 5' end, both ends, or either end. In one embodiment, the sense strand of the dsRNA has an overhang of 1 to 10 nucleotides, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, at the 3' end, the 5' end, both ends, or either end. In another embodiment, one or more nucleotides in the overhang are replaced with nucleoside phosphorothioates.

[0116] In certain embodiments, the overhang on the sense strand or antisense strand or both may include an extended length longer than 10 nucleotides, such as a length of 1 to 30 nucleotides, 2 to 30 nucleotides, 10 to 30 nucleotides, or 10 to 15 nucleotides. In certain embodiments, the extended overhang is on the sense strand of the duplex. In certain embodiments, the extended overhang is present at the 3' end of the sense strand of the duplex. In certain embodiments, the extended overhang is present at the 5' end of the sense strand of the duplex. In certain embodiments, the extended overhang is on the antisense strand of the duplex. In certain embodiments, the extended overhang is present at the 3' end of the antisense strand of the duplex. In certain embodiments, the extended overhang is present at the 5' end of the antisense strand of the duplex. In certain embodiments, one or more nucleotides in the overhang are replaced by nucleoside thiophosphates. In certain embodiments, the overhang includes a self-complementary portion so that the overhang can form a hairpin structure that is stable under physiological conditions.

[0117] As used herein, the term "blunt-ended" or "flat-ended" in reference to a dsRNA means that there are no unpaired nucleotides or nucleotide analogs at a given end of the dsRNA, i.e., there are no nucleotide overhangs. One or both ends of a dsRNA may be blunt-ended. In cases where both ends of a dsRNA are blunt-ended, the dsRNA is referred to as blunt-ended. For clarity, a "blunt-ended" dsRNA is a dsRNA that has blunt ends at both ends, i.e., there are no nucleotide overhangs at either end of the molecule. Most typically, such molecules are double-stranded throughout their entire length.

[0118] In some embodiments, the dsRNA comprises a 3' overhang. In some embodiments, the dsRNA comprises a 3' overhang of 1 nucleotide. In some embodiments, the dsRNA comprises a 3' overhang of 2 nucleotides. In some embodiments, the dsRNA comprises a 5' overhang. In some embodiments, the dsRNA comprises a 5' overhang of 1 nucleotide. In some embodiments, the dsRNA comprises a 5' overhang of 2 nucleotides.

[0119] In some embodiments, one end of the dsRNA has an overhang and one end has a blunt end. The overhang can be the sense strand 3' overhang, the sense strand 5' overhang, the antisense strand 3' overhang, or the antisense strand 5' overhang. In some embodiments, the overhang is a 1-nucleotide overhang. In some embodiments, the overhang is a 2-nucleotide overhang. In some embodiments, the dsRNA has two blunt ends. In some embodiments, the dsRNA has overhangs at both ends. The overhang at each end can independently be the sense strand 3' overhang, the sense strand 5' overhang, the antisense strand 3' overhang, or the antisense strand 5' overhang. In some embodiments, the overhang is a 1-nucleotide overhang. In some embodiments, the overhang is a 2-nucleotide overhang. One or more of the overhanging nucleotides can be modified nucleotides, inverted nucleotides, abasic nucleotides, or inverted abasic nucleotides. Inverted nucleotides can be linked via a 3'-3' phosphodiester bond.

[0120] In some embodiments, the sense strand is 23 nucleotides in length and the antisense strand is 21 nucleotides in length. In some embodiments, the sense strand is 23 nucleotides in length and the antisense strand is 21 nucleotides in length, wherein the 3' and 5' terminal nucleotide positions of the sense strand are inverted abasic residues. The inverted abasic residues at the 3' and 5' terminal ends of the sense strand can be overhangs.

[0121] In some embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length, wherein the antisense strand contains a 2 nucleobase 3' overhang.

[0122] The term "antisense strand" or "guide strand" refers to the strand of a RNAi agent (eg, dsRNA) that comprises a region that is substantially complementary to a target sequence (eg, SNCA mRNA).

[0123] As used herein, the term "complementarity region" refers to a region on the antisense strand that is substantially complementary to a sequence as defined herein (e.g., a target sequence, such as an SNCA nucleotide sequence). Where the complementary region is not completely complementary to the target sequence, mismatches may be in the interior or terminal regions of the molecule. Typically, the most tolerated mismatches are in the terminal regions, for example, within 5, 4, 3, or 2 nucleotides of the 5'- or 3'-end of the RNAi agent.

[0124] In some embodiments, double-stranded RNA agents of the present disclosure include nucleotide mismatches in the antisense strand.

[0125] In some embodiments, the antisense strand of a double-stranded RNA agent of the present invention comprises no more than 4 mismatches with the target mRNA, for example, the antisense strand comprises 4, 3, 2, 1 or 0 mismatches with the target mRNA. In some embodiments, the antisense strand of a double-stranded RNA agent of the present invention comprises no more than 4 mismatches with the sense strand, for example, the antisense strand comprises 4, 3, 2, 1 or 0 mismatches with the sense strand. In some embodiments, the double-stranded RNA agent of the present invention comprises nucleotide mismatches in the sense strand. In some embodiments, the sense strand of a double-stranded RNA agent of the present invention comprises no more than 4 mismatches with the antisense strand, for example, the sense strand comprises 4, 3, 2, 1 or 0 mismatches with the antisense strand. In some embodiments, the nucleotide mismatch is within 5, 4, or 3 nucleotides of the 3' end of the iRNA. In another embodiment, the nucleotide mismatch is in the 3'-terminal nucleotide of the iRNA agent, for example. In some embodiments, the mismatch is not in the seed region.

[0126] Thus, the RNAi agents described herein may contain one or more mismatches with the target sequence. In one embodiment, the RNAi agents described herein contain no more than 3 mismatches (i.e., 3, 2, 1, or 0 mismatches). In one embodiment, the RNAi agents described herein contain no more than 2 mismatches. In one embodiment, the RNAi agents described herein contain no more than 1 mismatch. In one embodiment, the RNAi agents described herein contain 0 mismatches. In certain embodiments, if the antisense strand of the RNAi agent contains a mismatch with the target sequence, the mismatch can optionally be limited to the last 5 nucleotides at the 5'- or 3'-end of the complementary region. For example, in such embodiments, for a 23 nucleotide RNAi agent, the strand complementary to a region of the SNCA gene typically does not contain any mismatches within the central 13 nucleotides. The methods described herein or methods known in the art can be used to determine whether a RNAi agent containing a mismatch with the target sequence effectively inhibits the expression of the SNCA gene. It is important to consider the efficacy of RNAi agents with mismatches in inhibiting SNCA gene expression, particularly if the specific complementary region in the SNCA gene is known to have polymorphic sequence variation within a population.

[0127] As used herein, the term "sense strand" or "follower strand" refers to the strand of a RNAi agent that comprises a region that is substantially complementary to a region of the antisense strand, as that term is defined herein.

[0128] As used herein, "substantially all nucleotides are modified" is modified to a large extent, but not completely, and may include no more than 5, 4, 3, 2, or 1 unmodified nucleotides.

[0129] As used herein, the term "cleavage zone" refers to the region adjacent to the cleavage site. The cleavage site is the site on the target where cleavage occurs. In some embodiments, the cleavage zone comprises three bases on either end of the cleavage site and adjacent to the cleavage site. In some embodiments, the cleavage zone comprises two bases on either end of the cleavage site and adjacent to the cleavage site. In some embodiments, the cleavage site specifically occurs at the site where nucleotides 10 and 11 of the antisense strand bind, and the cleavage zone comprises nucleotides 11, 12, and 13.

[0130] As used herein, and unless otherwise indicated, the term "complementary" when used to describe a first nucleotide sequence relative to a second nucleotide sequence, refers to an oligonucleotide or polynucleotide comprising the first nucleotide sequence that hybridizes to an oligonucleotide or polynucleotide comprising the second nucleotide sequence under specific conditions and forms the ability of a duplex structure, as understood by the skilled person. For example, such conditions can be stringent conditions, wherein stringent conditions can include: 400mM NaCl, 40mMPIPES, pH 6.4, 1mM EDTA, 50°C or 70°C, 12 hours to 16 hours, followed by washing (see, for example, Molecular Cloning:A Laboratory Manual, Sambrook et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions can be applied, such as the physiologically relevant conditions that may be encountered inside an organism. The skilled person will be able to determine the condition set that is most suitable for the complementarity test of two sequences according to the final application of the hybridization nucleotides.

[0131] As described herein, the complementary sequence in RNAi agent (for example, in dsRNA) includes the oligonucleotide comprising the first nucleotide sequence or the polynucleotide and the oligonucleotide comprising the second nucleotide sequence or the polynucleotide base pairing on the total length of one or two nucleotide sequences.Such sequences can be referred to as "completely complementary" to each other in this article.However, when the first sequence is referred to as "substantially complementary" relative to the second sequence in this article, the two sequences can be completely complementary, or for the duplex of up to 30 base pairs, it can form one or more, but generally no more than 5,4,3 or 2 base pairs of mispairing during hybridization, while retaining the ability to hybridize under the conditions most relevant to its final application, such as by RISC approach to inhibit gene expression.However, in the case where two oligonucleotides are designed to form one or more single-stranded overhangs when hybridization, such overhangs should not be considered as mispairing for the determination of complementarity.For example, comprising an oligonucleotide having a length of 21 nucleotides and a length of another oligonucleotide of 23 nucleotides, wherein the longer oligonucleotide comprises the sequence of 21 nucleotides completely complementary to the shorter oligonucleotides, still can be referred to as "completely complementary" for purposes described herein.

[0132] As used herein, a "complementary" sequence may also comprise or be entirely composed of non-Watson-Crick base pairs or base pairs formed by non-natural and modified nucleotides, as long as the above requirements for their hybridization ability are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble base pairing or Hoogstein base pairing.

[0133] The terms "complementary," "fully complementary," and "substantially complementary" herein can be used with respect to base matching between the sense and antisense strands of a dsRNA, or between the antisense strand of an RNAi agent and a target sequence, as understood from the context of their use.

[0134] As used herein, a polynucleotide that is "substantially complementary to at least a portion of a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of an mRNA of interest (e.g., an mRNA encoding SNCA). For example, a polynucleotide is complementary to at least a portion of an SCNA mRNA if the sequence is substantially complementary to an uninterrupted portion of an mRNA encoding SCNA.

[0135] Thus, in some embodiments, the antisense strand polynucleotides disclosed herein are fully complementary to the target SNCA sequence.

[0136] In certain embodiments, the antisense strand polynucleotides disclosed herein are substantially complementary to the target SNCA sequence and comprise a contiguous nucleotide sequence that is at least about 80% complementary over the entire length to the equivalent region of the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7 or 228 (for SNCA), or a fragment of SEQ ID NO: 1, 3, 5, 7 or 228, such as about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

[0137] In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target SNCA sequence and comprise a contiguous nucleotide sequence that is at least about 80% complementary over its entire length to any one of the sense strand nucleotide sequences in Tables 2 or 3, or a fragment of any one of the sense strand nucleotide sequences in Tables 2 or 3, such as about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

[0138] In one embodiment, the RNAi agent of the present disclosure includes a sense strand that is substantially complementary to an antisense polynucleotide that is, in turn, identical to the target SNCA sequence, and wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least about 80% complementary over its entire length to the nucleotide sequence of SEQ ID NO: 2, 4, 6, 8 or 229, or the equivalent region of the nucleotide sequence of a fragment of any of SEQ ID NO: 2, 4, 6, 8 or 229, such as about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

[0139] In some embodiments, the iRNA of the present disclosure includes a sense strand that is substantially complementary to an antisense polynucleotide that is in turn complementary to a target SNCA sequence, and wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least about 80% complementary over its entire length to any one of the antisense strand nucleotide sequences in Tables 2 or 3, or a fragment of any one of the antisense strand nucleotide sequences in Tables 2 or 3. For example, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% complementary.

[0140] In some embodiments, the double-stranded region of a double-stranded iRNA agent is equal to or at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotide pairs in length.

[0141] In some embodiments, the antisense strand of a double-stranded iRNA agent is equal to or at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0142] In some embodiments, the sense strand of a double-stranded iRNA agent is equal to or at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0143] In one embodiment, the sense and antisense strands of the double-stranded iRNA agent are each 15 to 30 nucleotides in length.

[0144] In one embodiment, the sense and antisense strands of the double-stranded iRNA agent are each 19 to 25 nucleotides in length.

[0145] In one embodiment, the sense and antisense strands of the double-stranded iRNA agent are each 21 to 23 nucleotides in length.

[0146] In one embodiment, the sense and antisense strands of the double-stranded iRNA agent are each 21 to 23 nucleotides in length.

[0147] In one embodiment, the sense and antisense strands of the double-stranded iRNA agent are each 23 to 21 nucleotides in length.

[0148] In one embodiment, the sense strand of the iRNA agent is 21 nucleotides in length and the antisense strand is 23 nucleotides in length, wherein the strands form a double-stranded region of 21 consecutive base pairs with a 2 nucleotide long single-stranded overhang at the 3' end.

[0149] In one embodiment, the sense strand of the iRNA agent is 23 nucleotides in length and the antisense strand is 21 nucleotides in length, wherein the strands form a double-stranded region of 21 consecutive base pairs with 1 nucleotide long single-stranded overhangs at the 3' and 5' ends.

[0150] In some embodiments, the majority of nucleotides in each chain are ribonucleotides, but as described in detail herein, each chain or both chains may also include one or more non-ribonucleotides, e.g., deoxyribonucleotides and / or modified nucleotides. In addition, "iRNA" may include ribonucleotides with chemical modifications. Such modifications may include all types of modifications disclosed herein or known in the art. For purposes of this specification and claims, any such modification, as used in an iRNA molecule, is encompassed by "iRNA."

[0151] In one aspect of the present disclosure, the agent used in the methods and compositions of the present disclosure is a single-stranded antisense nucleic acid molecule that inhibits the target mRNA via an antisense inhibition mechanism. The single-stranded antisense RNA molecule is complementary to the sequence in the target mRNA. The single-stranded antisense oligonucleotide can inhibit translation in a stoichiometric manner by base pairing with the mRNA and physically hindering the translation mechanism, see Dias, N. et al., (2002) Mol Cancer Ther 1: 347-355. The single-stranded antisense RNA molecule can be a length of about 15 to about 30 nucleotides and have a sequence complementary to the target sequence. For example, the single-stranded antisense RNA molecule can include a sequence of at least about 14, 15, 16, 17, 18, 19, 20 or more consecutive nucleotides from any antisense sequence described herein.

[0152] In one embodiment, at least partial inhibition of SNCA gene expression is assessed by a decrease in the amount of SNCA mRNA that can be isolated or detected from a first cell or group of cells in which SNCA is transcribed and that has been treated such that expression of the SNCA gene is inhibited, as compared to a second cell or group of cells (control cells) that is substantially identical to the first cell or group of cells but has not been so treated. The degree of inhibition can be represented by the following formula:

[0153]

[0154] As used herein, the phrase "contacting a cell with an RNAi agent (such as a dsRNA)" includes contacting the cell by any possible means. Contacting a cell with an RNAi agent includes contacting the cell with the RNAi agent in vitro or contacting the cell with the RNAi agent in vivo. Contact can be performed directly or indirectly. Thus, for example, the RNAi agent can be brought into physical contact with the cell by the individual performing the method, or alternatively, the RNAi agent can be placed in a situation that allows or allows it to subsequently contact the cell.

[0155] In vitro contact cells can be carried out by incubating cells with RNAi agents. In vivo contact cells can be carried out by injecting RNAi agents into or near the tissue where the cells are located, or by optionally injecting RNAi agents into another region (such as central nervous system (CNS)) via intrathecal, intravitreal or other injections, or injected into the bloodstream or subcutaneous space so that the subsequent arrival of the agent at the tissue where the cells to be contacted are located is carried out. For example, RNAi agents can contain or be coupled to ligands, such as described below and further described in one or more lipophilic parts in PCT / US2019 / 031170, for example, incorporated herein by reference, which guide RNAi agents to target sites such as CNS or otherwise stabilize at the target site. In some embodiments, RNAi agents can contain or be coupled to one or more lipophilic parts, optionally in the absence of GalNAc derivatives. A combination of in vitro and in vivo contact methods is also possible. For example, cells can be contacted with RNAi agents in vitro and subsequently transplanted into a subject.

[0156] In one embodiment, contacting a cell with a RNAi agent includes "introducing or delivering the RNAi agent into a cell" by promoting or enabling uptake or absorption into the cell. Absorption or uptake of the RNAi agent can occur by unassisted diffusion or active cellular processes, or by an adjuvant or device. Introduction of the RNAi agent into the cell can be in vitro and / or in vivo. For example, for in vivo introduction, the RNAi agent can be injected into a tissue site or administered systemically. In vitro introduction into cells includes methods known in the art, such as electroporation and lipofection. Further approaches are described below and / or are known in the art.

[0157] The term "lipophilic" or "lipophilic moiety" refers broadly to any compound or chemical moiety that has an affinity for lipids. One way to characterize the lipophilicity of a lipophilic moiety is by the octanol-water partition coefficient log K ow , where K ow Is the ratio of the concentration of a chemical in the octanol phase of a two-phase system in equilibrium to its concentration in the aqueous phase. The octanol-water partition coefficient is a laboratory-measured property of a substance. However, it can also be predicted by using coefficients attributed to the structural components of the chemical, which are calculated using first principles or empirical methods (see, for example, Tetko et al., J. Chem. Inf. Comput. Sci. 41: 1407-21 (2001), which is incorporated herein by reference in its entirety). It provides a thermodynamic measure of the tendency of a substance to prefer a non-aqueous or oily environment rather than water (i.e., its hydrophilic / lipophilic balance). In principle, when the logK of a chemical substance is ow When it is greater than 0, it is lipophilic. Typically, a lipophilic moiety has a logK greater than 1, greater than 1.5, greater than 2, greater than 3, greater than 4, greater than 5, or greater than 10. ow For example, the logK of 6-aminohexanol is ow Using the same method, the log K of cholesterol N-(hexan-6-ol)carbamate is predicted to be approximately 0.7. ow The forecast is 10.7.

[0158] The lipophilicity of a molecule can be altered depending on the functional groups it carries. For example, adding a hydroxyl or amine group to the end of a lipophilic moiety can increase or decrease the partition coefficient (e.g., log K) of the lipophilic moiety. ow )value.

[0159] Alternatively, the hydrophobicity of a double-stranded RNAi agent conjugated to one or more lipophilic moieties can be measured by its protein binding characteristics. For example, in certain embodiments, the unbound fraction in a plasma protein binding assay of a double-stranded RNAi agent can be determined to be positively correlated with the relative hydrophobicity of the double-stranded RNAi agent, which may be positively correlated with the silencing activity of the double-stranded RNAi agent.

[0160] In one embodiment, the determined plasma protein binding assay is an electrophoretic mobility shift assay (EMSA) using human serum albumin. Exemplary protocols for this binding assay are described in detail, for example, in PCT / US2019 / 031170. The hydrophobicity of the double-stranded RNAi agent measured by the fraction of unbound siRNA in the binding assay is greater than 0.15, greater than 0.2, greater than 0.25, greater than 0.3, greater than 0.35, greater than 0.4, greater than 0.45, or greater than 0.5 for enhanced in vivo delivery of siRNA.

[0161] Therefore, conjugating a lipophilic moiety to an internal position of a double-stranded RNAi agent provides optimal hydrophobicity for enhanced in vivo delivery of siRNA.

[0162] The term "lipid nanoparticle" or "LNP" refers to a vesicle comprising a lipid layer that encapsulates a pharmaceutically active molecule, such as a nucleic acid molecule, e.g., an RNAi agent or a plasmid from which an RNAi agent is transcribed. LNPs are described, e.g., in U.S. Patent Nos. 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are hereby incorporated by reference herein.

[0163] As used herein, a "subject" is an animal, such as a mammal, including a primate (such as a human, a non-human primate, such as a monkey and a chimpanzee) or a non-primate (such as a rat or a mouse). In preferred embodiments, the subject is a human, such as a human being treated or evaluated for a disease, disorder, or condition that would benefit from reduced expression of SNCA; a human at risk for a disease, disorder, or condition that would benefit from reduced expression of SNCA; a human having a disease, disorder, or condition that would benefit from reduced expression of SNCA; or a human being treated for a disease, disorder, or condition that would benefit from reduced expression of SNCA, as described herein.

[0164] As used herein, the terms "treat" or "treatment" refer to beneficial or desired results in a subject suffering from a neurodegenerative disease, including but not limited to alleviation or improvement of one or more signs or symptoms associated with SNCA gene expression or SNCA protein production, e.g., SNCA-associated neurodegenerative diseases, such as synucleinopathies, such as PD, multiple system atrophy (MSA), Lewy body dementia (LBD), pure autonomic failure (PAF), Pick's disease, progressive supranuclear palsy, dementia pugilistica, Parkinson's disease linked to chromosome 17, , Lytico-Bodig's disease, tangle-dominant dementia, argyrophilic grain disease, ganglioglioma, gangliocytoma, meningioangiomatosis, subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis, Hallervorden-Spatz disease, lipofuscinosis, corticobasal degeneration, frontotemporal dementia, frontotemporal lobar degeneration, Alzheimer's disease, Huntington's disease, Down syndrome, psychosis, schizophrenia, and Creutzfeldt-Jakob disease; decreased SNCA expression or activity in areas of increased neuronal dysfunction or death. "Treatment" can also mean prolonging survival as compared to expected survival in the absence of treatment.

[0165] In the context of SNCA levels or disease markers or symptoms in a subject, the term "reduction" refers to a statistically significant decrease in such levels. The decrease can be, for example, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more. In certain embodiments, the decrease is at least 20%. In certain embodiments, the decrease is at least 50% decrease in disease markers (e.g., protein or gene expression levels). "Reduction" in the context of SNCA levels in a subject is optionally a decrease to a level generally recognized as being within the normal range for individuals without such a condition. In certain embodiments, "reduction" is a decrease in the difference between the level of a marker or symptom in a subject with a disease and a level generally accepted to be within the normal range for that individual, for example, reduced levels of motor speed (bradykinesia) and the ability to regulate posture and balance in individuals with Parkinson's disease versus individuals without Parkinson's disease or with symptoms that are within the normal range.

[0166] As used herein, "prevention" or "preventing," when used in reference to a disease or condition that would benefit from a reduction in SNCA gene expression or SNCA protein production, for example, in a subject susceptible to a SNCA-associated disorder due to, for example, genetic factors or age, means wherein the subject does not yet meet the diagnostic criteria for a SNCA-associated disorder. As used herein, prevention is understood to mean administering an agent to a subject who does not yet meet the diagnostic criteria for a SNCA-associated disorder to delay or reduce the likelihood that the subject will develop a SNCA-associated disorder. Because the agent is a pharmaceutical agent, it is understood that administration is typically under the guidance of a healthcare professional who can identify a subject who does not yet meet the diagnostic criteria for a SNCA-associated disorder as susceptible to developing a SNCA-associated disorder.

[0167] The term "synucleinopathy" refers to a group of neurodegenerative disorders characterized by fibrillary aggregates of α-synuclein that tend to accumulate in the cytoplasm of selective neuronal and glial cell populations. Thus, synucleinopathies are a class of SNCA-associated neurodegenerative diseases and disorders that include Parkinson's disease (PD), Lewy body dementia (LBD), pure autonomic failure (PAF), and multiple system atrophy (MSA), among other neurodegenerative diseases. Clinically, synucleinopathies are characterized by chronic and progressive decline in motor, cognitive, behavioral, and autonomic function, depending on the distribution of lesions in the brain. Because of clinical overlap, differential diagnosis is sometimes very difficult. Parkinsonism is the cardinal symptom of PD, but it can be difficult to distinguish from the parkinsonism of LBD and MSA. Autonomic dysfunction, which is an isolated finding in PAF, may be present in PD and LBD, but is generally more prominent and presents earlier in MSA. LBD can be the same disorder as PD, but with widespread cortical pathology leading to dementia, cognitive fluctuations, and characteristic visual hallucinations.

[0168] The likelihood of developing a synucleinopathy (e.g., PD, LBD, etc.) is reduced, for example, when an individual with risk factors for PD or LBD (or other synucleinopathy) does not develop PD or LBD (or other synucleinopathy) or develops PD or LBD (or other synucleinopathy) with less severity relative to a population with the same risk factors who did not receive the treatment described herein. The absence of a SNCA-related disorder, such as PD or LBD (or other synucleinopathy), or the delay of developing PD or LBD (or other synucleinopathy) by several months or years is considered effective prevention. Prevention may require administration of more than one dose of an iRNA agent. Provided suitable methods are provided for identifying subjects at risk for developing any of the aforementioned SNCA-related diseases, the iRNA agents provided herein can be used as pharmaceutical agents for or in methods of preventing SNCA-related diseases. Risk factors for various SNCA-related diseases are discussed herein.

[0169] As used herein, the term "Parkinson's disease" or "PD" refers to a progressive neurological disorder that affects movement. The main pathological features of PD are cell death in the basal ganglia of the brain (affecting up to 70% of dopamine-secreting neurons in the substantia nigra pars compacta by the end of life) and the presence of Lewy bodies (accumulations of α-synuclein encoded by SNCA) in many of the remaining neurons. Symptoms begin gradually, sometimes with a barely noticeable tremor or stiffness or slow movement of just one hand. Other early symptoms include lack of facial expression, lack of arm movement when walking, and slurred speech. Parkinson's disease symptoms worsen over time. The average age of onset of PD is 60 years, and later onset is associated with greater symptom severity. Clinical features include, but are not limited to, more severe tremors, slow movements (bradykinesia), muscle rigidity, impaired posture and balance, loss of voluntary movement, speech changes, and ultimately dementia, hallucinations, and confinement to a wheelchair.

[0170] As used herein, "Lewy body dementia (LBD)" refers to a type of progressive dementia in which a decline in thinking, reasoning, and independent function is caused by the accumulation of alpha-synuclein in diseased brain neurons (called Lewy bodies and Lewy neurites). Aggregates of alpha-synuclein lead to suboptimal functionality and eventual death of affected neurons. Symptoms include visual, auditory, olfactory, or tactile hallucinations; signs of Parkinson's disease (Parkinson-like signs); poor regulation of body functions (autonomic nervous system) such as confusion, falls, and bowel problems; cognitive problems such as confusion, difficulty concentrating, visual-spatial problems, and memory loss; sleep difficulties such as rapid eye movement (REM) sleep behavior disorder (in which dreams are physically expressed during sleep); fluctuations in attention, including episodes of narcolepsy, prolonged staring into space, long daytime naps, or disorganized speech, depression, and apathy.

[0171] As used herein, the term "multiple system atrophy (MSA)" refers to a rare degenerative neurological disorder that affects a subject's involuntary (autonomic) functions, including blood pressure, breathing, bladder function, and motor control. Previously known as Shy-Drager syndrome, olivopontocerebellar atrophy, or striatonigral degeneration, MSA shares many Parkinson's-like symptoms, such as slow movements, muscle stiffness, and poor balance. MSA treatment includes medication and lifestyle changes to help manage symptoms, but there is no cure. MSA causes deterioration and shrinkage (atrophy) of parts of the brain (cerebellum, basal ganglia, and brainstem) that regulate internal body functions, digestion, and motor control. Damaged brain tissue in MSA subjects reveals nerve cells (neurons) containing abnormal amounts of α-synuclein. MSA subjects typically survive about 7 to 10 years after MSA symptoms first appear. However, survival rates for MSA vary widely. Occasionally, MSA subjects can live with the disease for 15 years or longer. Death is usually due to breathing problems. MSA progresses gradually and ultimately leads to death. It affects many parts of the body. MSA symptoms typically develop in adulthood, often in the fifties or sixties. MSA is classified into two types: parkinsonian and cerebellar. The type depends on the symptoms observed at the time of diagnosis. Parkinsonian MSA is the most common type of MSA, with signs and symptoms similar to those of Parkinson's disease, such as muscle stiffness, difficulty bending the arms and legs, slow movements (bradykinesia), tremors (rare in MSA compared to classic Parkinson's disease), and problems with posture and balance. Cerebellar MSA signs and symptoms include problems with muscle coordination (ataxia), and may also include impaired movement and coordination, such as unsteady gait and loss of balance, slurred speech, slow or low speech (stuttering), visual disturbances such as blurred vision or double vision and difficulty focusing the eyes, difficulty swallowing (dysphagia) or difficulty chewing, as well as general signs and symptoms. In addition, the cardinal sign of multiple system atrophy is postural (orthostatic) hypotension, a form of hypotension in which the subject feels dizzy or lightheaded, or even passes out, when standing from a sitting or lying position. MSA subjects may also develop dangerously high blood pressure levels when lying down (supine hypertension). Other difficulties associated with MSA include unconscious (autonomic) body functions, such as urinary and bowel dysfunction, constipation, loss of bladder or bowel control (incontinence), abnormal sweating, reduced sweat, tear and saliva production, heat intolerance due to reduced sweating, impaired temperature control (often causing cold hands and feet), sleep disorders, restless sleep due to "acting out" dreams, abnormal nighttime breathing, sexual dysfunction, inability to achieve or maintain an erection (impotence), loss of libido, cardiovascular problems, changes in color of the hands and feet due to blood pooling, cold hands and feet, mental problems and difficulty controlling emotions, such as inappropriate laughing or crying.Possible complications of MSA include abnormal breathing during sleep; injuries from falls due to poor balance or coma; and progressive immobility, which can lead to secondary problems such as skin breakdown, loss of self-care ability in daily activities, vocal cord paralysis leading to speech and breathing difficulties, and increased difficulty swallowing.

[0172] In one embodiment, the SNCA-associated disease or disorder (synucleinopathy) is one of Parkinson's disease, dementia with Lewy bodies, multiple system atrophy (MSA), and pure autonomic failure (PAF).

[0173] As used herein, a "therapeutically effective amount" is intended to include an amount of a RNAi agent that, when administered to a subject with an SNCA-related disease, is sufficient to affect treatment of the disease (e.g., by reducing, ameliorating, or maintaining existing disease or one or more disease symptoms). A "therapeutically effective amount" may vary depending on the RNAi agent, how the agent is administered, the disease and its severity, and the medical history, age, weight, family history, genetic makeup, type of previous or concomitant treatment (if any), and other individual characteristics of the subject to be treated.

[0174] As used herein, a "prophylactically effective amount" is intended to include an amount of a RNAi agent that, when administered to a subject with a SNCA-related disorder, is sufficient to prevent or ameliorate the disease or one or more symptoms of the disease. Amelioration of the disease includes slowing the progression of the disease or reducing the severity of the disease if it later occurs. A "prophylactically effective amount" may vary depending on the RNAi agent, how the agent is administered, the risk of the disease, and the patient's medical history, age, weight, family history, genetic makeup, type of previous or concomitant treatment (if any), and other individual characteristics of the patient to be treated.

[0175] "Therapeutically effective amount" or "prophylactically effective amount" also includes the amount of RNAi agent that produces some desired local or systemic effect at a reasonable benefit / risk ratio applicable to any treatment. The RNAi agent employed in the methods of the present disclosure can be administered in sufficient amount to produce a reasonable benefit / risk ratio applicable to such treatment.

[0176] The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human and animal subjects without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0177] As used herein, the phrase "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc, magnesium, calcium, or zinc stearate, or stearic acid), or solvent encapsulating material, that is involved in carrying or transporting the subject compound from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject being treated. Some examples of materials that can be used as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants such as magnesium stearate, sodium lauryl sulfate, and talc; (8) excipients such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols such as propylene glycol; (11) 1) polyols, such as glycerol, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) pH buffered solutions; (21) polyesters, polycarbonates, or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids; (23) serum components, such as serum albumin, HDL, and LDL; and (22) other non-toxic compatible substances used in pharmaceutical formulations.

[0178] As used herein, the term " sample " comprises the collection of similar fluids, cells or tissues separated from the subject, and the fluid, cells or tissues present in the subject's body. The example of biological fluid includes blood, serum and serosal fluid, blood plasma, cerebrospinal fluid, eye fluid, lymph, urine, saliva etc. Tissue sample can include the sample from tissue, organ or local area. For example, sample can be derived from the fluid or cell in specific organ, the part of organ or these organs. In certain embodiments, sample can be derived from brain (for example, some sections of whole brain or brain, for example striatum, or some types of cells in brain, for example neurons and glial cells (astroglia, oligodendrocyte, microglia)). In other embodiments, " sample derived from subject " refers to the blood extracted from subject or the blood plasma or serum derived therefrom. In further embodiments, " sample derived from subject " refers to brain tissue (or its subcomponent) or retinal tissue (or its subcomponent) derived from subject.

[0179] It should be understood that although the sequences in Table 2 are described as modified or conjugated sequences, in certain embodiments, the RNA of the RNAi agent of the present disclosure (e.g., the dsRNA of the present disclosure) may comprise any of the sequences shown in Tables 2 or 3, which are modified or conjugated differently than described in the table. The lipophilic ligand may be included in any position provided in this application, unless a specific position / location is indicated. BRIEF DESCRIPTION OF THE DRAWINGS

[0180] Figure 1A and Figure 1B Shown are the effects of selected SNCA-targeted RNAi agents on SNCA levels in a dual-luciferase reporter system in human BE(2)-C neuroblastoma cells, COS-7 African green monkey fibroblast-like cells, and in mice overexpressing human SNCA-AAV. Figure 1A Shown are the SNCA mRNA knockdown results for SNCA-targeted RNAi agents AD-464778, AD-464782, AD-464694, AD-464634, AD-464779, AD-464314, AD-464313, AD-464590, AD-464585, AD-464229, AD-464586, and AD-464592 ("parental" siRNAs carrying triantennary GalNAc modifications, relative to the CNS-directed siRNAs of the present disclosure) in BE(2)-C cells and for each siRNA using a dual luciferase reporter system in COS-7 cells. Figure 1B In vivo results for hepatic SNCA knockdown of the indicated duplexes in mice overexpressing human SNCA-AAV are shown. To characterize the in vivo efficacy of RNAi compounds in mice, full-length human SNCA was first transduced via AAV. Seven days after AAV administration, the following selected duplexes were delivered: duplexes AD-464778, AD-464782, AD-464694, AD-464634, and AD-464779 targeting the 3'UTR of human SNCA; and duplexes AD-464314, AD-464313, AD-464590, AD-464585, AD-464229, AD-464586, and AD-464592 targeting the coding sequence of SNCA. Data were normalized to PBS-treated samples.

[0181] Figure 2Shown are the SNCA knockdown results of the forty most potent SNCA-targeting siRNAs (liver-targeting duplexes) in a combined hotspot and structure-activity relationship (SAR) evaluation of a total of 360 duplexes. The forty indicated siRNAs showed the highest SNCA knockdown results at 0.1 nM duplex concentration and exhibited a clear linear dose-response.

[0182] Figure 3 Shown is a schematic diagram of an exemplary AD-1549290 duplex having the sense strand sequence 5'-uscscca(Ghd)uuUfCfUfugagaucugaL96-3' (SEQ ID NO: 245) and the antisense strand sequence 5'-VPusdCsagdAudCucaadGaAfacugggasgsc-3' (SEQ ID NO: 246).

[0183] Figure 4 Shown are the results of an in vivo AAV titration study in which AAV was used to administer human SNCA to mouse livers, followed by assessment of the extent of delivery and expression. On the left, the cycle threshold for PCR-mediated human SNCA detection, used to assess human SNCA expression in mouse livers, is shown to decrease in a dose-responsive manner when human SNCA is administered using AAV. On the right, previously characterized SNCA-targeting siRNAs AD-464634 (targeting the SNCA 3'UTR sequence) and AD-464314 (targeting the SNCA coding sequence) demonstrated robust in vivo inhibition of SNCA expression in mouse livers when administered at 3 mg / kg (mpk) or 10 mpk to huSNCA-expressing mice.

[0184] Figure 5A and Figure 5B Shown are the in vivo SNCA knockdown results in mouse liver for all 17 SNCA-targeting siRNAs evaluated in the in vivo AAV studies, along with a schematic illustration of the duplex sequence modification patterns. Figure 5A Shown are the in vivo human SNCA knockdown results observed in mice administered human SNCA via AAV. The 17 indicated newly evaluated SCNA-targeting siRNAs were compared with the previously identified SNCA-targeting duplex AD-464634.2 and with a PBS control. Figure 5BThe liver-targeting duplexes AD-1549333 (sense strand: 5'-asasgug(Chd)ucAfGfUfuccaaugugaL96-3' SEQ ID NO: 247; antisense strand: 5'-VPusdCsacdAudTggaadCuGfagcacuusgsu-3' SEQ ID NO: 248), AD-1746465 (sense strand: 5'-uscsuuugcuCfCfCfaguu(Uhd)cuugaL96-3' SEQ ID NO: 249; antisense strand: 5'-VPusdCsaadGadAacugdGgAfgcaaagasusa-3' SEQ ID NO: 250), and AD-1571188 (sense strand: 5'-gsusaca(Ahd)GfuGfCfUfcaguuccaaaL96-3' SEQ ID NO: NO: 251; antisense strand: 5'-VPusUfsugdGa(Agn)cugagcAfcUfuguacsasg-3' SEQ ID NO: 252), AD-1549401 (sense strand: 5'-cscsauc(Ahd)gcAfGfUfgauugaaguaL96-3' SEQ ID NO: 253; antisense strand: 5'-VPusdAscudTcdAaucadCuGfcugauggsasa-3' SEQ ID NO: 254), AD-1549054 (sense strand: 5'-gsasgca(Ahd)guGfAfCfaaauguuggaL96-3' SEQ ID NO: 255; antisense strand: 5'-VPusdCscadAcdAuuugdTcAfcuugcucsusu-3' SEQ ID Schematic illustration of the sequences and modification patterns of AD-1746466 (sense strand: 5'-uscsccag(Uhd)uUfCfUfugagaucugaL96-3' SEQ ID NO:257; antisense strand: 5'-VPusdCsagdAudCucaadGaAfacugggasgsc-3' SEQ ID NO:258), and AD-1548886 (sense strand: 5'-uscsaug(Ahd)aaGfGfAfcuuucaaagaL96-3' SEQ ID NO:259; antisense strand: 5'-VPusdCsuudTgdAaagudCcUfuucaugasasu-3' SEQ ID NO:260), which duplexes were selected as potential parental siRNAs for non-human primate (NHP) studies.

[0185] Figures 6A to 6CShown is a comparison of in vivo knockdown in mice, in vitro knockdown, and positional information among the seven SNCA-targeting siRNAs identified above, which resulted in three lead candidate parental siRNAs for NHP studies; structures are also shown. Figure 6A Shown is a comparison of in vivo and in vitro knockdown in mice of the seven SNCA-targeting siRNAs AD-1549333, AD-1746465, AD-1571188, AD-1549401, AD-1549054, AD-1746466, and AD-1548886 identified above. Figure 6B The target SNCA mRNA locations of each of the candidate lead parent siRNAs AD-1549333, AD-1746465, AD-1571188, AD-1549401, AD-1549054, AD-1746466, and AD-1548886 are shown. Figure 6C The sequences and modification patterns of the selected lead candidate parent duplexes AD-1549333 (sense strand: SEQ ID NO: 247; antisense strand: SEQ ID NO: 248), AD-1746465 (sense strand: SEQ ID NO: 249; antisense strand: SEQ ID NO: 250), and AD-1549054 (sense strand: SEQ ID NO: 255; antisense strand: SEQ ID NO: 256) are shown from top to bottom. These parent duplexes were adapted accordingly to generate the lead SNCA-targeting duplexes AD-1747585, AD-1747583, and AD-1747580, which have the sequences and modification patterns shown in Table 2 for CNS administration in NHP studies.

[0186] Figure 7 Schematic diagrams are presented showing three duplexes selected for NHP studies: AD-1747585 (having a sense strand of SEQ ID NO:35 and an antisense strand of SEQ ID NO:78), AD-1747583 (having a sense strand of SEQ ID NO:28 and an antisense strand of SEQ ID NO:71), and AD-1747580 (having a sense strand of SEQ ID NO:21 and an antisense strand of SEQ ID NO:64).

[0187] Figure 8A and Figure 8B Shown are the initial selection and pharmacokinetic profiles of three candidate lead duplexes that were advanced to testing in non-human primates (NHPs). Figure 8A Showing initial in vivo efficacy results and ED for three lead candidate duplexes selected for NHP animal studies 50 value. Figure 8BEach of the three candidate lead SNCA-targeting duplexes administered via intrathecal injection of a single 60 mg dose of siRNA to non-human primates (NHPs) was shown to exhibit similar pharmacokinetics in the dosed animals during the first 24 hours after injection.

[0188] Figure 9A and Figure 9B Shown are pharmacokinetic (PK) and pharmacodynamic (PD) data obtained in NHP animal studies of three candidate lead SNCA-targeting duplexes administered via intrathecal injection. Figure 9A It is shown that at day 29 post-injection ("D29"), each of the three tested SNCA-targeting duplexes exhibited highly potent reduction of SNCA mRNA in NHP tissues in both the prefrontal cortex and midbrain (left panel, PD results). A strong correlation between tissue PK (tissue concentration of the tested duplexes) and tissue SNCA mRNA PD was observed across the tested NHP prefrontal cortex and midbrain tissues (right panel, PK / PD results), with each of the three tested duplexes showing similar potency (no statistically significant differences were observed between the three leads at day 29 based on mRNA knockdown results). Nominally, the following IC values ​​were observed in NHPs at day 29: 50 Ranking: AD-1747583 (0.532 μg / g) <AD-1747580(1.050μg / g)<AD-1747585(1.300μg / g)。 Figure 9B It was shown that α-synuclein levels were reduced in the brain and spinal cord of NHPs receiving an intrathecal dose of the SNCA-targeting duplex at day 29 post-injection ("D29"), with little differentiation observed among the three candidate lead agents tested.

[0189] Figure 10 A summary of the Day 29 NHP data is shown.

[0190] Figure 11 Shown is that at day 84 ("D84") following intrathecal administration of SNCA-targeting duplexes or control molecules, significant levels of SNCA mRNA knockdown were observed in both prefrontal cortex and midbrain tissues for all three candidate lead duplexes tested (left panel). Consistent with the results observed above at day 29 post-administration, day 84 samples demonstrated a good correlation between tissue PK and tissue mRNA PD, with similar potency observed for each of the three candidate lead duplexes examined (right panel). Notably, only one animal in the group administered AD-1747585 met dosing criteria, which resulted in re-dosing of the remainder of the group administered AD-1747585.

[0191] 12A to 12EIt is shown that at day 84 post-administration, both SNCA mRNA levels and α-synuclein levels were reduced in different brain regions, with little observed difference between the three lead candidates tested (AD-1747580, AD-1747583, and AD-1747585 duplexes), and SNCA mRNA / α-synuclein correlation and re-dosing data are also shown. Figure 12A It was shown that SNCA mRNA was decreased in NHPs on day 84 after administration of 60 mg of the duplex dose via the intrathecal route. Figure 12B It was shown that α-synuclein levels were also reduced in the brain and spinal cord (all regions examined) at day 84, again with little observed difference between the three candidate lead duplexes. Figure 12C It was shown that at day 84 after administration, α-synuclein levels in the different brain regions examined showed a high correlation with SNCA mRNA levels. Figure 12D We showed that in some NHPs that were confirmed to have been misadministered upon initial administration, re-dosing and examination of brain and spinal cord tissue beyond day 84 revealed robust knockdown of SNCA NHP tissue protein pharmacodynamics (PD) in various brain regions, particularly in NHPs re-dosed with AD-1747580. Figure 12E Cerebrospinal fluid (CSF) α-synuclein levels were shown over an extended time course, including re-dosing of certain NHPs that were initially mis-dosed and monitoring at time points beyond day 84. In such re-dosed NHP animals, CSF α-synuclein levels exhibited up to a 75% reduction for animals (re)dosed with duplex AD-1747585, and up to a 50% reduction for animals (re)dosed with duplexes AD-1747580 and AD-1747583.

[0192] Figure 13 It was shown that at day 84 after duplex administration, up to 90% reduction in α-synuclein levels in NHP CSF was observed for animals dosed with duplexes AD-1747580 and AD-1747583 (PD effect). Meanwhile, duplex AD-1747585 showed approximately 75% CSF α-synuclein knockdown, and it is noteworthy that duplex AD-1747585 also exhibited variable CSF α-synuclein knockdown, likely due to blood contamination, as SNCA is highly expressed in blood cells.

[0193] 14A to 14DWe show that at day 29 (as an “early” time point), there is a divergence between knockdown tissue SNCA mRNA levels and knockdown α-synuclein levels observed in the same tissues, and this difference has resolved by the day 84 time point, suggesting that such effects are attributable to the long half-life of α-synuclein in the tissues examined. Figure 14A Shown, at day 29 after administration, for all three lead duplexes tested, only a moderate (<60%) knockdown of α-synuclein (PD effect of the duplex) was observed in the cortex and midbrain of treated NHPs (left panel). This modest reduction in α-synuclein in the cortex and midbrain indicates an extended half-life of α-synuclein in these tissues. For all three duplexes tested, a scattering of tissue protein PK / PD was observed (middle panel); however, tissue α-synuclein levels were highly correlated with tissue SNCA mRNA levels (right panel). Figure 14B Figure 2 shows that α-synuclein knockdown in CSF correlates with α-synuclein knockdown in the cortex (left panel) and striatum (center panel) at day 29 after administration. In contrast, α-synuclein knockdown in midbrain tissue did not correlate well with α-synuclein knockdown in CSF at day 29 after administration (right panel). Figure 14C showed that, while SNCA mRNA levels were reduced in prefrontal cortex and midbrain tissues at day 29 after administration (here, of the SNCA-targeting duplex AD-1747580), α-synuclein levels were only modestly reduced, indicating that the long protein half-life of α-synuclein likely explains this differentiation. Figure 14D Shown that at day 84 after administration, for all three lead duplexes, alpha-synuclein levels in the cortex and midbrain tissues of treated NHP animals showed a strong knockdown (PD effect, left panel). The strong alpha-synuclein knockdown observed in cortex and midbrain NHP tissues at day 84 confirmed the long protein half-life of alpha-synuclein. For all three duplexes, the tissue protein PK / PD curves showed a similar distribution (middle panel; the AD-1747585 group was marked as having only one animal meeting the dosing criteria, while the remainder of the group was re-dosed). alpha-synuclein levels were highly correlated with mRNA levels in both cortex and midbrain tissues (right panel).

[0194] Figure 15 showed that α-synuclein knockdown in the CSF of NHP animals was also observed to be associated with α-synuclein knockdown in the prefrontal cortex and midbrain on day 84 after duplex administration.

[0195] Figure 16The siRNA tissue exposure in various tested brain regions of NHP animals is shown at day 84 after duplex administration. With a few exceptions, tissue penetration of the siRNA agent was good, and even tissues with the greatest exposure to the siRNA duplex (e.g., prefrontal cortex) did not necessarily show the greatest observed levels of SNCA mRNA and / or α-synuclein knockdown.

[0196] Figure 17 It was shown that none of the three candidate lead duplexes administered to NHP animals exhibited any significant effect on body weight compared to control NHP animals dosed in parallel with vehicle alone, either on day 2 or day 84 after administration.

[0197] Figure 18 Shown are the results obtained when detecting neurofilament light chain (NfL) in the CSF of a group of NHP animals dosed with candidate lead duplexes, as compared to control NHP animals administered only artificial CSF (aCSF). Although a transient surge in NfL was observed after dosing for all three duplexes, most of these NfL surges did not persist beyond day 29 after administration, indicating that the three SNCA-targeted candidate lead duplexes did not exhibit an NfL profile indicative of duplex toxicity.

[0198] 19A to 19D RNA-Seq data are shown for each of the three candidate lead duplexes examined. Figure 19A A schematic diagram of the AD-1747580 duplex (sense strand: SEQ ID NO: 21; antisense strand SEQ ID NO: 64) is shown, with RNA-Seq data indicating that 93% knockdown of SNCA was achieved in cells administered the AD-1747580 duplex and subjected to RNA-Seq analysis compared to appropriate controls, while no other loci on the genome showed greater than 50% knockdown (or robust and significant elevation) in the presence of the AD-1747580 duplex. Figure 19BShown are potency-matched RNA-Seq profiles for candidate lead duplex AD-1747580, along with RNA-Seq results for parental duplex AD-1549054. On the left, a schematic illustration of the GalNAc-modified AD-1549054 parental duplex (sense strand: 5'-gsasgca(Ahd)guGfAfCfaaauguuggaL96-3' SEQ ID NO: 255; antisense strand: 5'-VPusdCscadAcdAuuugdTcAfcuugcucsusu-3' SEQ ID NO: 256) is shown, with RNA-Seq data for the duplex below, demonstrating approximately 74% SNCA knockdown in treated cells (administered a 10 nM dose of the AD-1549054 parental duplex). Schematic diagrams of the AD-1747580 duplex (sense strand: SEQ ID NO: 21; antisense strand: SEQ ID NO: 64) are shown in the middle and right panels, and RNA-Seq results of the duplex at corresponding doses of 1 nM (middle, showing approximately 78% SNCA knockdown) and 10 nM (right, showing approximately 93% SNCA knockdown) are shown below. Figure 19C Shown are potency-matched RNA-Seq profiles for candidate lead duplex AD-1747583, along with RNA-Seq results for parental duplex AD-1549283. On the left, a schematic diagram of the GalNAc-modified AD-1549283 parental duplex (sense strand: 5'-uscsuuu(Ghd)cuCfCfCfaguuucuugaL96-3' SEQ ID NO: 268; antisense strand: 5'-VPusdCsaadGadAacugdGgAfgcaaagasusa-3' SEQ ID NO: 269) is shown, with RNA-Seq data for the duplex below, demonstrating approximately 79% SNCA knockdown in treated cells (administered a 10 nM dose of the AD-1549283 parental duplex). Schematic diagrams of the AD-1747583 duplex (sense strand: SEQ ID NO: 28; antisense strand: SEQ ID NO: 71) are shown in the center and right, with RNA-Seq results for the duplex at corresponding doses of 1 nM (center, showing approximately 67% SNCA knockdown) and 10 nM (right, showing approximately 92% SNCA knockdown) below. Notably, three off-target loci were identified as significantly affected (>50% reduction) by administration of the AD-1747583 duplex: PYGB, NREP, and LCLAT1. Figure 19DShown are potency-matched RNA-Seq profiles for candidate lead duplex AD-1747585, along with RNA-Seq results for parental duplex AD-1549333. On the left, a schematic diagram of the GalNAc-modified AD-1549333 parental duplex (sense strand: 5'-asasgug(Chd)ucAfGfUfuccaaugugaL96-3' SEQ ID NO: 247; antisense strand: 5'-VPusdCsacdAudTggaadCuGfagcacuusgsu-3' SEQ ID NO: 248) is shown, with RNA-Seq data for the duplex below, demonstrating approximately 81% SNCA knockdown in treated cells (administered a 10 nM dose of the AD-1549333 parental duplex). Schematic diagrams of the AD-1747586 duplex (sense strand: SEQ ID NO: 35; antisense strand: SEQ ID NO: 78) are shown in the center and right, with RNA-Seq results for the duplex at corresponding doses of 0.1 nM (center left, showing approximately 68% SNCA knockdown), 1 nM (center right, showing approximately 93% SNCA knockdown), and 10 nM (right, showing approximately 96% SNCA knockdown). Notably, one off-target locus was identified as significantly affected (>50% reduction) by administration of the AD-1747585 duplex: HMGB2.

[0199] Figure 20 The table shown summarizes the knockdown and RNA-Seq data for all three candidate lead SNCA-targeting duplexes administered to NHP animals.

[0200] Throughout the figures, the term "2-C16" refers to the 2'-O-hexadecyl modification. The present invention is further illustrated by the following detailed description. DETAILED DESCRIPTION

[0201] The present disclosure provides RNAi compositions that achieve RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of the SNCA gene. The SNCA gene can be within a cell, such as a cell in a subject such as a human. The present disclosure also provides methods of using the RNAi compositions of the present disclosure for inhibiting expression of a SNCA gene or for treating a subject having a disorder that would benefit from inhibition or reduction of SNCA gene expression, e.g., a SNCA-associated disease, e.g., a synucleinopathy, such as PD, multiple system atrophy (MSA), Lewy body dementia (LBD), pure autonomic failure (PAF), Pick's disease, progressive supranuclear palsy, dementia pugilistica, Parkinson's disease linked to chromosome 17, Lytico-Bodig's disease, tangle-dominant dementia, argyrophilic grain disease, ganglioglioma, gangliocytoma, meningioangiomatosis, subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis, Hallervorden-Spatz disease, lipofuscinosis, corticobasal degeneration, frontotemporal dementia, frontotemporal lobar degeneration, Alzheimer's disease, Huntington's disease, Down syndrome, psychosis, schizophrenia, and Creutzfeldt-Jakob disease.

[0202] The RNAi agents of the present invention include an RNA strand (antisense strand) having a region of about 30 nucleotides or less in length, such as 15 to 30, 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29, 1 21 to 22, which region is substantially complementary to at least a portion of the mRNA transcript of the SNCA gene. In certain embodiments, the RNAi agent of the present disclosure comprises an RNA strand (antisense strand) having a region of about 21 to 23 nucleotides in length that is substantially complementary to at least a portion of the mRNA transcript of the SNCA gene.

[0203] In certain embodiments, the RNAi agents of the present disclosure include an RNA strand (antisense strand) that may include a longer length, for example, up to 66 nucleotides, for example, 36 to 66, 26 to 36, 25 to 36, 31 to 60, 22 to 43, 27 to 53 nucleotides in length, having a region of at least 19 consecutive nucleotides that is substantially complementary to at least a portion of the mRNA transcript of the SNCA gene. These RNAi agents with longer antisense strands optionally include a second RNA strand (sense strand) of 20 to 60 nucleotides in length, wherein the sense strand forms a duplex with the antisense strand of 18 to 30 consecutive nucleotides.

[0204] The use of these RNAi agents enables targeted degradation of mRNA of the SNCA gene in mammals. Thus, methods and compositions comprising these RNAi agents can be used to treat subjects who would benefit from reduced levels or activity of SNCA proteins, such as subjects with SNCA-associated neurodegenerative diseases, for example, synucleinopathies such as PD, multiple system atrophy, Lewy body dementia (LBD), pure autonomic failure (PAF), Pick's disease, progressive supranuclear palsy, dementia pugilistica, Parkinson's disease linked to chromosome 17, Lytico-Bodig's disease, tangle-dominant dementia, argyrophilic grain disease, ganglioglioma, gangliocytoma, meningioangiomatosis, subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis, Hallervorden-Spatz disease, lipofuscinosis, corticobasal degeneration, frontotemporal dementia, frontotemporal lobar degeneration, Alzheimer's disease, Huntington's disease, Down syndrome, psychosis, schizophrenia, and Creutzfeldt-Jakob disease.

[0205] The neuron accumulation of alpha-synaptic nucleoprotein is described as causing the formation of Lewy bodies (round eosinophilic spheres transparent 10-20pm large inclusion bodies) or Lewy neurites (extended linear dystrophic axons and dendrites). Without wishing to be bound by theory, in PD brain, the deposition of Lewy bodies and Lewy neurites is extensive, although it is believed to be substantia nigra for the most susceptible region of this type of deposition. Pathology is extensive, because PD has motion (based on substantia nigra) and non-motion (other parts of brain, for example, cortex etc.) symptoms. Substantia nigra cells are most important for executing motion and posture function, have explained the characteristic of PD motor symptoms, and have explained PD non-motor symptoms such as dementia, mood changes and depression on the impact of other cell types. In LBD brain, the extensive deposition of Lewy bodies and Lewy neurites is seen in both midbrain and cortical areas.

[0206] Alpha-synuclein is a protein found primarily in neurons. Within neurons, alpha-synuclein is primarily localized presynaptically, and is therefore hypothesized to play a role in regulating synaptic activity. Three major isoforms of alpha-synuclein have been identified, the longest and most common form of which contains 140 amino acids.

[0207] Oxidative stress has been implicated in a number of neurodegenerative disorders characterized by the pathological accumulation of misfolded α-synuclein. Various reactive oxygen species can induce peroxidation of lipids such as cell membranes or lipoproteins and also lead to the generation of highly reactive aldehydes from polyunsaturated fatty acids (Yoritaka et al., 1996).

[0208] Brain pathology indicative of Alzheimer's disease (AD), namely amyloid plaques and neurofibrillary tangles, is found in approximately 50% of LBD cases. It is unclear whether the presence of parallel pathology signifies two distinct diseases or simply represents a variant of each condition. Sometimes, cases with shared pathology are described as having the Lewy body variant of AD (Hansen et al., 1990).

[0209] Studies have also implicated a role for SNCA in AD and Down syndrome, as α-synuclein has been shown to accumulate in limbic areas in these disorders (Crews et al., 2009).

[0210] Rare, dominantly inherited forms of PD and LBD can be caused by point mutations or duplications in the SNCA gene. Pathogenic mutations A30P and A53T (Kruger et al., 1998) (Polymeropoulos et al., 1998) and gene duplications (Chartier-Harlin et al., 2004) have been described as causing familial PD, while an additional α-synuclein mutation, E46K (Zarranz et al., 2004), and a triple duplication of the α-synuclein gene (Singleton et al., 2003) have been reported to cause PD or LBD.

[0211] The pathological consequences of α-synuclein mutations are only partially understood. However, in vitro data show that A30P and A53T mutations increase the rate of aggregation (Conway et al., 2000). A large number of α-synuclein species of different compositions (monomers, dimers, oligomers, including fibrils) are involved in the aggregation process, all of which may have different toxic properties. It is not clear which molecular species exert toxic effects in the brain. However, studies indicate that oligomeric forms of α-synuclein are particularly neurotoxic. Additional evidence for the role of oligomers is provided by the observation that certain α-synuclein mutations (A30P and A53T) that cause hereditary Parkinson's disease lead to increased oligomerization rates.

[0212] It is not yet fully known how the α-synuclein aggregation cascade begins. It is possible that the altered conformation of monomeric α-synuclein initiates the formation of dimers and trimers, which proceed to form more soluble oligomers, including fibrils, and these medium-sized substances are then deposited as insoluble fibrils in Lewy bodies. It is also conceivable that once α-synuclein oligomers are formed, they can bind to new monomers and / or smaller polymers of α-synuclein and therefore accelerate the fibrillation process. Such seeding effects may also occur in the extracellular space, as some evidence suggests that α-synuclein pathology may spread between neurons in the diseased brain.

[0213] The following detailed description discloses how to make and use compositions containing RNAi agents to inhibit expression of the SNCA gene, as well as compositions and methods for treating subjects suffering from diseases and conditions that would benefit from inhibition or reduction of gene expression.

[0214] I. RNAi Agents of the Disclosure

[0215] Described herein are RNAi agents that inhibit expression of the SNCA gene. In one embodiment, the RNAi agent comprises a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting expression of the SNCA gene in a cell, such as a cell in a subject, e.g., a mammal, such as a human with an SNCA-associated neurodegenerative disease, e.g., a synucleinopathy, such as PD, multiple system atrophy (MSA), Lewy body dementia (LBD), pure autonomic failure (PAF), Pick's disease, progressive supranuclear palsy, dementia pugilistica, Parkinson's disease linked to chromosome 17, Lytico-Bodig's disease, tangle-dominant dementia, argyrophilic grain disease, ganglioglioma, gangliocytoma, meningioangiomatosis, subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis, Hallervorden-Spatz disease, lipofuscinosis, corticobasal degeneration, frontotemporal dementia, frontotemporal lobar degeneration, Alzheimer's disease, Huntington's disease, Down syndrome, psychosis, schizophrenia, and Creutzfeldt-Jakob disease. The dsRNA comprises an antisense strand having a complementary region that is complementary to at least a portion of the mRNA formed during expression of the SNCA gene. In embodiments, the complementary region is about 15 to 30 nucleotides or less in length. When contacted with a cell expressing the SNCA gene, the RNAi agent inhibits expression of the SNCA gene (e.g., a human gene, a primate gene, a non-primate gene) by at least 50%, as determined by, for example, PCR or branched DNA (bDNA)-based methods, or by protein-based methods, such as immunofluorescence analysis using, for example, Western blotting or flow cytometry.

[0216] dsRNA comprises two complementary RNA strands that hybridize under the conditions under which the dsRNA is used to form a duplex structure. One strand of the dsRNA (the antisense strand) comprises a region of complementarity that is substantially complementary, and typically completely complementary, to a target sequence. The target sequence may be derived from the sequence of an mRNA formed during expression of the SNCA gene. The other strand (the sense strand) comprises a region complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under appropriate conditions. As described elsewhere herein and as is known in the art, the complementary sequence of a dsRNA may also be contained as a self-complementary region of a single nucleic acid molecule, as opposed to being located on separate oligonucleotides.

[0217] Typically, the duplex structure is 15 to 30 base pairs in length, e.g., 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, 20 to 22, 20 to 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, or 21 to 22 base pairs in length. In certain preferred embodiments, the duplex structure is 18 to 25 base pairs in length, e.g., 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 25, 21 to 24, 21 to 23, 21 to 22, 22 to 25, 22 to 24, 22 to 23, 23 to 25, 23 to 24, or 24 to 25 base pairs in length, e.g., 19 to 21. Ranges and lengths intermediate to the above-recited ranges and lengths are also considered part of this disclosure.

[0218] Similarly, the region of complementarity to the target sequence is 15 to 30 nucleotides in length, e.g., 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, or 21 to 22 nucleotides, e.g., a length of 19 to 23 nucleotides or a length of 21 to 23 nucleotides. Ranges and lengths intermediate to the ranges and lengths recited above are also considered part of this disclosure.

[0219] In some embodiments, the dsRNA is a length of 15 to 23 nucleotides or a length of 24 to 30 nucleotides (optionally, a length of 25 to 30 nucleotides). Typically, the dsRNA can be long enough to serve as a substrate for the Dicer enzyme. For example, as is well known in the art, dsRNAs having a length exceeding about 21 to 23 nucleotides can be used as substrates for the Dicer enzyme. As also recognized by those of ordinary skill in the art, the region of the RNA targeted for cutting is most typically a portion of a larger RNA molecule (typically an mRNA molecule). In related cases, a "portion" of an mRNA target is a continuous sequence of an mRNA target that is long enough to serve as a substrate for RNAi-guided cutting (i.e., cutting by the RISC pathway).

[0220] It is also recognized by those skilled in the art that the duplex region is the major functional portion of a dsRNA, e.g., about 15 to 36 base pairs, e.g., 15 to 36, 15 to 35, 15 to 34, 15 to 33, 15 to 32, 15 to 31, 15 to 30, 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 15 to 25, 15 to 24, 15 to 23, 18 to 2 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, 20 to 22, 20 to 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, or 21 to 22 base pairs, e.g., a duplex region of 19 to 21 base pairs. Thus, in one embodiment, an RNA molecule or complex of RNA molecules having a duplex region greater than 30 base pairs is a dsRNA to the extent that it is processed into a functional duplex of, for example, 15-30 base pairs that targets the desired RNA for cleavage. Thus, one of ordinary skill will recognize that, in one embodiment, a miRNA is a dsRNA. In another embodiment, the dsRNA is not a naturally occurring miRNA. In another embodiment, the RNAi agent useful for targeting SNCA expression is not produced in the target cell by cleaving a larger dsRNA.

[0221] The dsRNA as described herein may further comprise one or more single-stranded nucleotide overhangs, e.g., 1, 2, 3, or 4 nucleotides. The nucleotide overhangs may comprise or consist of nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides. The overhangs may be on the sense strand, the antisense strand, or any combination thereof. In addition, the nucleotides of the overhangs may be present on the 5' end, the 3' end, or both ends of the antisense strand or the sense strand of the dsRNA. In certain embodiments, longer, extended overhangs are possible.

[0222] As discussed further below, dsRNA can be synthesized by standard methods known in the art, for example, by using an automated DNA synthesizer, such as those commercially available from, for example, Biosearch, Applied Biosystems, Inc.

[0223] iRNA compounds of the present disclosure can be prepared using a two-step procedure. First, a single strand of a double-stranded RNA molecule is prepared separately. Then, the component strands are annealed. The single strand of the siRNA compound can be prepared using solution phase or solid phase organic synthesis or both. The advantage provided by organic synthesis is that oligonucleotide chains containing non-natural or modified nucleotides can be easily prepared. Single-stranded oligonucleotides of the present disclosure can be prepared using solution phase or solid phase organic synthesis or both.

[0224] siRNA can be produced by a variety of methods, such as in bulk. Exemplary methods include organic synthesis and RNA cleavage, e.g., in vitro cleavage.

[0225] siRNA can be prepared by independently synthesizing single-stranded RNA molecules or each corresponding strand of a double-stranded RNA molecule, after which the component strands can be annealed.

[0226] Large bioreactors, for example, the OligoPilot II from Pharmacia Biotec AB (Uppsala Sweden), can be used to produce large amounts of specific RNA chains for given siRNAs. The OligoPilot II reactor can effectively couple nucleotides using only 1.5 molar excesses of phosphoramidite nucleotides. To prepare RNA chains, ribonucleotide imidazolides are used. Standard cycles of monomer addition can be used to synthesize 21 to 23 nucleotides for siRNAs. Typically, two complementary chains are produced independently and then annealed, for example, after release and deprotection from a solid support.

[0227] Organic synthesis can be used to produce discrete siRNA species. The complementarity of the species to the SNCA gene can be precisely specified. For example, the species can be complementary to a region encompassing a polymorphism (e.g., a single nucleotide polymorphism). Furthermore, the location of the polymorphism can be precisely defined. In some embodiments, the polymorphism is located in an internal region, for example, at least 4, 5, 7, or 9 nucleotides from one or both termini.

[0228] In one embodiment, the generated RNA is carefully purified to remove the ends. iRNA is cleaved into siRNA in vitro, for example, using the enzyme Dicer or a comparable RNAse III-based activity. For example, dsiRNA can be incubated in an in vitro extract from Drosophila or using purified components (e.g., purified RNAse or RISC (RNA-induced silencing complex)). See, for example, Ketting et al. Genes Dev 2001 Oct 15; 15(20): 2654-9 and Hammond Science 2001 Aug 10; 293(5532): 1146-50.

[0229] DsiRNA cleavage typically produces multiple siRNA species, each a specific 21 or 23 nt fragment of the source dsiRNA molecule. For example, there can be siRNAs that include sequences complementary to overlapping and adjacent regions of the source dsiRNA molecule.

[0230] Regardless of the synthesis method, the siRNA preparation can be prepared in a solution suitable for formulation (e.g., an aqueous solution or an organic solution). For example, the siRNA preparation can be precipitated and redissolved in pure double-distilled water and lyophilized. The dried siRNA can then be resuspended in a solution suitable for the desired formulation process.

[0231] In one aspect, the dsRNA of the present disclosure comprises at least two nucleotide sequences, a sense sequence and an antisense sequence. The sense strand sequence of SNCA can be selected from the sequence groups provided in Table 2 or 3, and the corresponding nucleotide sequence of the antisense strand of the sense strand can be selected from the sequence groups provided in Table 2 or 3. In this aspect, one of the two sequences is complementary to the other of the two sequences, wherein one of the sequences is substantially complementary to the mRNA sequence produced upon expression of the SNCA gene. Thus, in this aspect, the dsRNA comprises two oligonucleotides, one of which is described as the sense strand (follower strand) in Table 2 or 3, and the second oligonucleotide is described as the corresponding antisense strand (guide strand) of the sense strand in Table 7 or 8.

[0232] In one embodiment, the substantially complementary sequences of the dsRNA are contained on separate oligonucleotides. In another embodiment, the substantially complementary sequences of the dsRNA are contained on a single oligonucleotide.

[0233] It should be understood that, for example, although the sequences provided herein are described as modified or conjugated sequences, the RNA of the RNAi agents of the present disclosure (e.g., dsRNA of the present disclosure) can comprise any of the sequences shown in Tables 2 or 3, which are modified or conjugated differently than described in the tables. One or more lipophilic ligands can be included at any position of the RNAi agents provided herein.

[0234] It is well known to those skilled in the art that dsRNAs having a duplex structure of about 20-23 base pairs (e.g., 21 base pairs) have been praised as being particularly effective in inducing RNA interference (Elbashir et al., (2001) EMBO J., 20:6877-6888). However, others have found that shorter or longer RNA duplex structures can also be effective (Chu and Rana (2007) RNA 14:1714-1719; Kim et al., (2005) Nat Biotech 23:222-226). In the above embodiments, due to the properties of the oligonucleotide sequences provided herein, the dsRNA described herein can comprise at least one strand of at least 21 nucleotides in length. It is reasonable to expect that shorter duplexes with only a few nucleotides less at one or both ends compared to the above dsRNAs can be similarly effective. Thus, dsRNAs having a sequence of at least 15, 16, 17, 18, 19, 20 or more consecutive nucleotides derived from one of the sequences provided herein, and whose ability to inhibit expression of the SNCA gene differs by no more than 10%, 15%, 20%, 25% or 30% inhibition (as determined using in vitro assays and PCR assays using Be(2)-C cells and a 10 nM concentration of the RNA agent provided in the Examples herein) as compared to a dsRNA comprising the entire sequence are considered to be within the scope of the present disclosure.

[0235] A benchmark assay for SNCA inhibition involves contacting human Be(2)-C cells with a dsRNA agent disclosed herein, wherein sufficient or effective SNCA inhibition is identified if at least a 5% decrease, at least a 10% decrease, at least a 15% decrease, at least a 20% decrease, at least a 25% decrease, at least a 30% decrease, at least a 35% decrease, at least a 40% decrease, at least a 45% decrease, at least a 50% decrease, at least a 55% decrease, at least a 60% decrease, at least a 65% decrease, at least a 70% decrease, at least a 75% decrease, at least a 80% decrease, at least a 85% decrease, at least a 90% decrease, at least a 95% decrease, at least a 97% decrease, at least a 98% decrease, at least a 99% decrease, or more, of SNCA transcript or protein is observed compared to an appropriate control (e.g., cells not contacted with a SNCA-targeting dsRNA). Optionally, the dsRNA agent of the present disclosure is administered at a concentration of 10 nM and the PCR assay is performed as provided in the Examples herein (e.g., Example 2 below).

[0236] In addition, the RNA described herein identifies a site in the SNCA transcript that is susceptible to RISC-mediated cleavage. Thus, the present disclosure further features RNAi agents that target this site. As used herein, an RNAi agent is said to target a specific site in an RNA transcript if it promotes cleavage of the transcript at any position within that specific site. Such RNAi agents typically comprise at least about 15 consecutive nucleotides, optionally at least 19 nucleotides, from one of the sequences provided herein, coupled to an additional nucleotide sequence taken from a region adjacent to the selected sequence in the SNCA gene.

[0237] The RNAi agents described herein may contain one or more mismatches with the target sequence. In one embodiment, the RNAi agents described herein contain no more than 3 mismatches (i.e., 3, 2, 1, or 0 mismatches). In one embodiment, the RNAi agents described herein contain no more than 2 mismatches. In one embodiment, the RNAi agents described herein contain no more than 1 mismatch. In one embodiment, the RNAi agents described herein contain 0 mismatches. In certain embodiments, if the antisense strand of the RNAi agent contains a mismatch with the target sequence, the mismatch may optionally be limited to the last 5 nucleotides of the 5' or 3' end of the complementary region. For example, in such embodiments, for a 23-nucleotide RNAi agent, the strand complementary to a region of the SNCA gene generally does not contain mismatches within the central 13 nucleotides. The methods described herein or methods known in the art can be used to determine whether a RNAi agent containing a mismatch with the target sequence is effective in inhibiting the expression of the SNCA gene. Considering the efficacy of RNAi agents with mismatches in inhibiting the expression of the SNCA gene is important, particularly if a particular complementary region in the SNCA gene is known to have polymorphic sequence variation within a population.

[0238] II. Modified RNAi Agents of the Disclosure

[0239] In preferred embodiments, the RNA of RNAi agent of the present disclosure (e.g., dsRNA) is chemically modified to enhance stability or other beneficial features. In certain embodiments of the present disclosure, substantially all nucleotides of RNAi agent of the present disclosure are modified. In other embodiments of the present disclosure, all nucleotides of RNAi agent of the present disclosure are modified. Wherein "substantially all nucleotides are modified" RNAi agent of the present disclosure is modified to a great extent but not completely, and may include no more than 5, 4, 3, 2 or 1 unmodified nucleotides. In other embodiments of the present disclosure, RNAi agent of the present disclosure may include no more than 5, 4, 3, 2 or 1 modified nucleotides.

[0240] The nucleic acids described in the present disclosure can be synthesized or modified by methods recognized in the art, such as those described in "Current protocols in nucleic acid chemistry", Beaucage, SL et al. (eds.), John Wiley & Sons, Inc., New York, NY, USA, which are incorporated herein by reference. Modifications include, for example, end modifications, such as 5'-end modifications (phosphorylation, conjugation, reverse connection, etc.) or 3'-end modifications (conjugation, DNA nucleotides, reverse connection, etc.); base modifications, for example, base replacement with stable bases, destabilizing bases, or base pairing with an extended partner library, base removal (abasic nucleotides), or conjugated bases; sugar modifications (e.g., at the 2'-position or 4'-position) or replacement of sugars; or backbone modifications, including modification or replacement of phosphodiester bonds. Specific examples of RNAi agents that can be used in the embodiments described herein include, but are not limited to, RNAs containing modified backbones or containing no natural internucleoside bonds. RNAs with modified backbones particularly include those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referred to in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbones can also be considered oligonucleosides. In some embodiments, the modified RNAi agent has a phosphorus atom in its internucleoside backbone.

[0241] The RNA backbone of modification comprises for example phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl and other alkylphosphonates (including 3'-alkylenephosphonates and chiral phosphonates), phosphinate, phosphoramidate (including 3'-aminophosphoramidate and aminoalkylphosphoramidate), thionophosphoramidate, thionoalkylphosphonates, thionoalkylphosphotriester and boranophosphate with normal 3'-5' bond, 2'-5' connected analogs of these, and those with reversed polarity, wherein adjacent nucleoside units are connected to 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts are also included, such as sodium salts, mixed salts and free acid forms.

[0242] Representative U.S. patents that teach the preparation of the above-mentioned phosphorus-containing bonds include, but are not limited to, the following: U.S. Patents 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5, 587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,5 Nos. 34,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Patent No. RE39464, the entire contents of each of which are hereby incorporated herein by reference.

[0243] The modified RNA backbones that do not contain a phosphorus atom have backbones formed from short-chain alkyl or cycloalkyl nucleoside bonds, mixed heteroatoms and alkyl or cycloalkyl nucleoside bonds, or one or more short-chain heteroatoms or heterocyclic nucleoside bonds. These include backbones with morpholino linkages (partially formed from the sugar portion of the nucleoside); siloxane backbones; sulfide, sulfoxide, and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and thioformacetyl backbones; olefin-containing backbones; sulfamic acid ester backbones; methyleneimino and methylenehydrazine backbones; sulfonic acid ester and sulfonamide backbones; amide backbones; and other backbones with mixed N, O, S, and CH2 component parts.

[0244] Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Patent Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5, Nos. 489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, the entire contents of each of which are hereby incorporated herein by reference.

[0245] In other embodiments, suitable RNA mimics are considered for RNAi agents, wherein the sugar and internucleoside bonds (i.e., main chains) of the nucleotide units are replaced by new groups. The base units are maintained to hybridize with appropriate nucleic acid target compounds. One such oligomeric compound (an RNA mimic with excellent hybridization properties) is referred to as peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced by an amide-containing main chain, especially an aminoethylglycine main chain. The core base is retained and directly or indirectly bound to the nitrogen-nitrogen atom of the amide portion of the main chain. Representative U.S. patents teaching the preparation of PNA compounds include, but are not limited to, U.S. Patent No. 5,539,082; No. 5,714,331; and No. 5,719,262, the entire contents of each of which are hereby incorporated herein by reference. Other PNA compounds suitable for RNAi agents of the present disclosure are described in, for example, Nielsen et al., Science, 1991, 254, 1497-1500.

[0246] Some embodiments described herein include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular -CH2-NH-CH2-, -CH2-N(CH3)-O-CH2- [referred to as methylene(methylimino) or MMI backbones], -CH2-ON(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2-, and -N(CH3)-CH2-CH2- [wherein the natural phosphodiester backbone is represented as -OPO-CH2-] of U.S. Pat. No. 5,489,677 cited above, as well as the amide backbones of U.S. Pat. No. 5,602,240 cited above. In some embodiments, the RNAs described herein have the morpholino backbone structures of U.S. Pat. No. 5,034,506 cited above.

[0247] The modified RNA may also contain one or more substituted sugar moieties. The RNAi agents described herein (e.g., dsRNA) may comprise one of the following at the 2'-position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, wherein alkyl, alkenyl, and alkynyl groups may be substituted or unsubstituted C1 to C 10 Alkyl or C2 to C 10 Exemplary suitable modifications include O[(CH2) n O] m CH3, O(CH2). n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2 and O(CH2) n ON[(CH2) n CH3)]2, wherein n and m are 1 to about 10. In other embodiments, the dsRNA comprises one of the following at the 2'-position: C1 to C 10 Lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, intercalator, group for improving the pharmacokinetic properties of RNAi agent or group for improving the pharmacodynamic properties of RNAi agent, and other substituents with similar properties. In some embodiments, modification includes 2'-methoxyethoxy (2'-O-CH2CH2OCH3), also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78: 486-504), i.e., alkoxy-alkoxy group. Another exemplary modification is 2'-dimethylaminooxyethoxy, i.e., O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described in the Examples below, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O-CH2-O-CH2-N(CH2)2. Additional exemplary modifications include: 5'-Me-2'-F nucleotides, 5'-Me-2'-OMe nucleotides, 5'-Me-2'-deoxynucleotides (both R and S isomers in these three families); 2'-alkoxyalkyl groups; and 2'-NMA (N-methylacetamide).

[0248] Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2), 2'-O-hexadecyl and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of the RNAi agent, particularly the 3' position of the sugar on the 3'-terminal nucleotide or 2'-5' linked dsRNA and the 5' position of the 5'-terminal nucleotide. The RNAi agent can also have a sugar mimetic, such as a cyclobutyl moiety in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, the following U.S. Patents Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, some of which are commonly owned by the present application. The entire contents of each of the foregoing are hereby incorporated herein by reference.

[0249] The RNAi agents of the present disclosure may also comprise modifications or substitutions of nucleobases (often referred to in the art as "bases"). As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine, and Thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-sulfanyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halo, specifically 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P., ed., Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pp. 858-859, Kroschwitz, JL, ed., John Wiley & Sons, 1990, those disclosed in Englisch et al., (1991) Angewandte Chemie, International Edition, 30:613, and those disclosed in Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pp. 289-302, Crooke, ST and Lebleu, B., eds., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds described herein. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine.5-Methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6°C to 1.2°C (Sanghvi, YS, Crooke, ST and Lebleu, B., eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with the 2'-O-methoxyethyl sugar modification.

[0250] Representative U.S. patents that teach the preparation of some of the above-mentioned modified nucleobases, as well as other modified nucleobases, include, but are not limited to, the aforementioned U.S. Patents Nos. 3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,59 Nos. 4,121, 5,596,091; 5,614,617; 5,681,941; 5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, the entire contents of each of which are hereby incorporated herein by reference.

[0251] The RNAi agents of the present disclosure may also be modified to include one or more locked nucleic acids (LNAs). Locked nucleic acids are nucleotides with modified ribose moieties, wherein the ribose moiety includes an additional bridge connecting 2' and 4' carbons. This structure effectively "locks" the ribose in a 3'-endo conformation. Adding locked nucleic acids to siRNA has been shown to improve siRNA stability in serum and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, OR. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).

[0252] RNAi agent of the present disclosure can also be modified to include one or more bicyclic sugar moieties." bicyclic sugar " is a furanosyl ring modified by two atom bridges." bicyclic nucleoside " (" BNA ") is a nucleoside with a sugar moiety comprising a bridge of two carbon atoms connecting a sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the bridge connects the 4' carbon and 2'-carbon of the sugar ring. Therefore, in some embodiments, the medicament of the present disclosure can include one or more locked nucleic acids (LNA). Locked nucleic acid is a nucleotide with a modified ribose moiety, wherein the ribose moiety includes an extra bridge connecting 2' and 4' carbon. In other words, LNA is a nucleotide comprising a bicyclic sugar moiety, which includes a 4'-CH2-O-2' bridge. This structure effectively "locks" ribose in a 3'-endostructural conformation. Adding locked nucleic acid to siRNA has been shown to improve siRNA stability in serum and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, OR. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). Examples of bicyclic nucleosides for use in the polynucleotides of the present disclosure include, but are not limited to, nucleosides comprising a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, the antisense polynucleotide agents of the present disclosure comprise one or more bicyclic nucleosides comprising a 4' to 2' bridge.Examples of such 4' to 2' bridged bicyclic nucleosides include, but are not limited to, 4'-(CH2)-O-2'(LNA); 4'-(CH2)-S-2'; 4'-(CH2)2-O-2'(ENA); 4'-CH(CH3)-O-2' (also known as "constrained ethyl" or "cEt") and 4'-CH(CH2OCH3)-O-2' (and analogs thereof; see, e.g., U.S. Patent No. 7,399,845); 4'-C(CH3)(CH3)-O-2' (and analogs thereof; see, e.g., U.S. Patent No. 8,278,283); 4'-CH2-N(OCH3)-2' (and analogs thereof; see, e.g., U.S. Patent No. 8,278,283); No. 8,278,425); 4'-CH2-ON(CH3)-2' (see, e.g., U.S. Patent Publication No. 2004 / 0171570); 4'-CH2-N(R)-O-2', wherein R is H, C1-C12 alkyl, or a protecting group (see, e.g., U.S. Patent No. 7,427,672); 4'-CH2-C(H)(CH3)-2' (see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH2-C(═CH2)-2' (and the like; see, e.g., U.S. Patent No. 8,278,426). The entire contents of each of the foregoing are hereby incorporated herein by reference.

[0253] Other representative U.S. patents and U.S. patent publications that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Patent Nos. 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133; 7,084,125; 7,399,845; 7,427,672; 7,569,686; 7,741,457; 8,022,193; 8,030,467; 8,278,425; 8,278,426; 8,278,283; US 2008 / 0039618 and US 2009 / 0012281, the entire contents of each of which are hereby incorporated herein by reference.

[0254] Any of the foregoing bicyclic nucleosides can be prepared with one or more stereochemical sugar configurations, including, for example, α-L-ribofuranose and β-D-ribofuranose (see WO 99 / 14226).

[0255] RNAi agents of the present disclosure may also be modified to include one or more constrained ethyl nucleotides. As used herein, "constrained ethyl nucleotides" or "cEt" are locked nucleic acids comprising a bicyclic sugar moiety comprising a 4'-CH(CH3)-O-2' bridge. In one embodiment, the constrained ethyl nucleotides are in an S conformation, referred to herein as "S-cEt."

[0256] The RNAi agents of the present disclosure may also comprise one or more "conformationally restricted nucleotides" ("CRNs"). CRNs are nucleotide analogs having a linker connecting the C2' and C4' carbons of ribose or the C3' and C5' carbons of ribose. CRNs lock the ribose ring into a stable conformation and increase hybridization affinity for mRNA. The linker is of sufficient length to place the oxygen in a position optimal for stability and affinity, resulting in less ribose ring wrinkling.

[0257] Representative publications teaching the preparation of certain of the aforementioned CRNs include, but are not limited to, US 2013 / 0190383; and WO 2013 / 036868, the entire contents of each of which are hereby incorporated herein by reference.

[0258] In some embodiments, the RNAi agent of the present disclosure includes one or more monomers that are UNA (unlocking nucleic acid) nucleotides. UNA is an unlocking acyclic nucleic acid in which any bond of a sugar has been removed, thereby forming an unlocking "sugar" residue. In one example, UNA also encompasses monomers in which the bond between C1'-C4' has been removed (i.e., a covalent carbon-oxygen-carbon bond between C1' and C4' carbon). In another example, the C2'-C3' bond of a sugar (i.e., a covalent carbon-carbon bond between C2' and C3' carbon) has been removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039, hereby incorporated by reference).

[0259] Representative U.S. publications that teach the preparation of UNA include, but are not limited to, US 8,314,227; and U.S. Patent Publication Nos. 2013 / 0096289; 2013 / 0011922; and 2011 / 0313020, the entire contents of each of which are hereby incorporated by reference herein.

[0260] Potential stabilizing modifications to the ends of RNA molecules may include N-(acetylaminohexanoyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(hexanoyl-4-hydroxyprolinol) (Hyp-C6), N-(acetyl-4-hydroxyprolinol) (Hyp-NHAc), thymidine-2'-O-deoxythymidine (ether), N-(aminohexanoyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosyl-uridine-3'-phosphate, inverted 2'-deoxy-modified ribonucleotides such as inverted dT (idT), inverted dA (idA), and inverted abasic 2'-deoxyribonucleotides (iAb), etc. Disclosure of such modifications can be found in WO 2011 / 005861.

[0261] In one example, the 3' or 5' end of the oligonucleotide is linked to a reverse 2'-deoxy-modified ribonucleotide, such as reverse dT (idT), reverse dA (idA), or reverse abasic 2'-deoxyribonucleotide (iAb). In a specific example, a reverse 2'-deoxy-modified ribonucleotide is linked to the 3' end of the oligonucleotide, such as the 3' end of the sense strand as described herein, wherein the connection is via a 3'-3' phosphodiester bond or a 3'-3'-phosphorothioate bond.

[0262] In another example, the 3' end of the sense strand is linked to an inverted abasic ribonucleotide (iAb) via a 3'-3'-phosphorothioate bond. In another example, the 3' end of the sense strand is linked to an inverted dA (idA) via a 3'-3'-phosphorothioate bond.

[0263] In a specific example, a reverse 2'-deoxy-modified ribonucleotide is linked to the 3' end of an oligonucleotide, such as the 3' end of the sense strand described herein, wherein the linkage is via a 3'-3' phosphodiester bond or a 3'-3'-phosphorothioate bond.

[0264] In another example, the 3'-terminal nucleotide of the sense strand is an inverted dA (idA) and is linked to the preceding nucleotide via a 3'-3'-linkage (eg, a 3'-3'-phosphorothioate linkage).

[0265] Other modifications of the RNAi agents of the present disclosure include 5' phosphates or 5' phosphate mimetics, for example, 5'-terminal phosphates or phosphate mimetics on the antisense strand of the RNAi agent. Suitable phosphate mimetics are disclosed, for example, in US 2012 / 0157511, the entire contents of which are incorporated herein by reference.

[0266] A. Modified RNAi Agents Comprising Motifs of the Disclosure

[0267] In some aspects of the present disclosure, double-stranded RNAi agents of the present disclosure include agents with chemical modifications, as disclosed in, for example, WO 2013 / 075035, the entire contents of which are incorporated herein by reference. As shown in this article and WO 2013 / 075035, by introducing one or more motifs of three identical modifications on three consecutive nucleotides into the sense strand or antisense strand of an RNAi agent, particularly at or near the cleavage site, excellent results can be obtained. In some embodiments, the sense strand and antisense strand of an RNAi agent can be modified completely in other ways. The introduction of these motifs interrupts the modification pattern (if any) of the sense or antisense strand. The RNAi agent can optionally be conjugated with a lipophilic ligand, such as a C16 ligand, for example, on the sense strand. The RNAi agent can optionally be modified with (S)-glycol nucleic acid (GNA) modifications, for example, on one or more residues of the antisense strand. The RNAi agent gained presents excellent gene silencing activity.

[0268] Therefore, the present disclosure provides double-stranded RNAi agents that can inhibit the expression of a target gene (i.e., SNCA gene) in vivo. The RNAi agent includes a sense strand and an antisense strand. Each strand of the RNAi agent can be 15-30 nucleotides in length. For example, each strand can be 16-30 nucleotides in length, 17-30 nucleotides in length, 25-30 nucleotides in length, 27-30 nucleotides in length, 17-23 nucleotides in length, 17-21 nucleotides in length, 17-19 nucleotides in length, 19-25 nucleotides in length, 19-23 nucleotides in length, 19-21 nucleotides in length, 21-25 nucleotides in length, or 21-23 nucleotides in length. In certain embodiments, each strand is 19-23 nucleotides in length.

[0269] The sense strand and the antisense strand typically form a duplex double-stranded RNA ("dsRNA"), also referred to herein as a "RNAi agent". The length of the duplex region of an RNAi agent can be 15-30 nucleotide pairs. For example, the duplex region can be 16-30 nucleotide pairs in length, 17-30 nucleotide pairs in length, 27-30 nucleotide pairs in length, 17-23 nucleotide pairs in length, 17-21 nucleotide pairs in length, 17-19 nucleotide pairs in length, 19-25 nucleotide pairs in length, 19-23 nucleotide pairs in length, 19-21 nucleotide pairs in length, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs in length. In another example, the length of the duplex region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides. In preferred embodiments, the duplex region is 19-21 nucleotide pairs in length.

[0270] In one embodiment, the RNAi agent can contain one or more overhang regions or blocking groups at the 3'-end, 5'-end or both ends of one or both chains. The length of the overhang can be 1-6 nucleotides, for example, 2-6 nucleotides in length, 1-5 nucleotides in length, 2-5 nucleotides in length, 1-4 nucleotides in length, 2-4 nucleotides in length, 1-3 nucleotides in length, 2-3 nucleotides in length or 1-2 nucleotides in length. In a preferred embodiment, the length of the nucleotide overhang region is 2 nucleotides. The overhang is the result of one chain being longer than the other, or the result of two chains of the same length being staggered. The overhang can form a mismatch with the target mRNA, or it can be complementary to the targeted gene sequence, or another sequence. The first and second chains can also be joined, for example, by additional bases to form a hairpin, or by other non-basic linkers.

[0271] In one embodiment, the nucleotides in the overhang region of the RNAi agent can each independently be a modified or unmodified nucleotide, including but not limited to 2'-sugar modifications such as 2-F, 2'-O-methyl, thymidine (T), and any combination thereof.

[0272] For example, TT can be an overhang sequence at either end of either strand. The overhang can form a mismatch with the target mRNA, or it can be complementary to the targeted gene sequence, or it can be another sequence.

[0273] The 5'-or 3'-overhang on the sense strand, antisense strand or two chains of the RNAi agent can be phosphorylated. In some embodiments, the overhang region contains two nucleotides with thiophosphate between two nucleotides, wherein the two nucleotides can be identical or different. In one embodiment, the overhang is present at the 3'-end of the sense strand, antisense strand or two chains. In one embodiment, this 3'-overhang is present in the antisense strand. In one embodiment, this 3'-overhang is present in the sense strand.

[0274] RNAi agent may contain only a single overhang, which can enhance the interference activity of RNAi without affecting its overall stability. For example, a single-stranded overhang can be located at the 3'-end of the sense strand, or alternatively, at the 3'-end of the antisense strand. RNAi may also have a blunt end, located at the 5'-end (or 3'-end of the sense strand) of the antisense strand, or vice versa. Typically, the antisense strand of RNAi has a nucleotide overhang at the 3'-end, and the 5'-end is a blunt end. Although it is not desired to be bound by theory, the asymmetric blunt end at the 5'-end of the antisense strand and the antisense strand 3'-end overhang are conducive to the guide strand being loaded into the RISC process.

[0275] In one embodiment, the RNAi agent is a 19-nucleotide-long double-ended bluntmer, wherein the sense strand contains at least one 2'-F modified motif on three consecutive nucleotides at positions 7, 8, and 9 from the 5'-end. The antisense strand contains at least one 2'-O-methyl modified motif on three consecutive nucleotides at positions 11, 12, and 13 from the 5'-end.

[0276] In another embodiment, the RNAi agent is a 20 nucleotide long double-ended blunt-end polymer, wherein the sense strand contains at least one 5'-terminal motif of three consecutive 2'-F modifications at three nucleotides at positions 8, 9, and 10. The antisense strand contains at least one 5'-terminal motif of three consecutive 2'-O-methyl modifications at three nucleotides at positions 11, 12, and 13.

[0277] In yet another embodiment, the RNAi agent is a double-ended blunt-end polymer of 21 nucleotides in length, wherein the sense strand contains at least one 2'-F modified motif at three consecutive nucleotides at positions 9, 10, and 11 at the 5'-end. The antisense strand contains at least one 2'-O-methyl modified motif at three consecutive nucleotides at positions 11, 12, and 13 at the 5'-end.

[0278] In one embodiment, the RNAi agent includes a sense strand of 21 nucleotides and an antisense strand of 23 nucleotides, wherein the sense strand contains at least one 2'-F modified motif on three consecutive nucleotides at positions 9, 10, and 11 at the 5'-end; the antisense strand contains at least one 2'-O-methyl modified motif on three consecutive nucleotides at positions 11, 12, and 13 at the 5'-end, wherein one end of the RNAi agent is blunt-ended, and the other end includes a 2-nucleotide overhang. Optionally, the 2-nucleotide overhang is located at the 3'-end of the antisense strand. When the 2-nucleotide overhang is located at the 3'-end of the antisense strand, there may be two phosphorothioate internucleotide bonds between the terminal three nucleotides, two of which are overhang nucleotides, and the third nucleotide is a paired nucleotide next to the overhang nucleotide. In one embodiment, the RNAi agent additionally has two phosphorothioate internucleotide bonds between the terminal three nucleotides of the 5'-end of the sense strand and the 5'-end of the antisense strand. In one embodiment, each nucleotide in the sense strand and antisense strand of the RNAi agent (including nucleotides as part of the motif) is a modified nucleotide. In one embodiment, each residue is independently modified with 2'-O-methyl or 3'-fluoro, for example, in an alternating motif. Optionally, the RNAi agent further comprises a ligand (e.g., a lipophilic ligand, optionally a C16 ligand).

[0279] In one embodiment, the RNAi agent comprises a sense strand and an antisense strand, wherein the sense strand is 25-30 nucleotide residues in length, wherein starting from the 5'-terminal nucleotide (position 1) position 1, positions 1-23 of the first strand include at least 8 ribonucleotides; the antisense strand is 36-66 nucleotide residues in length and, starting from the 3'-terminal nucleotide, includes at least 8 ribonucleotides in positions that pair with positions 1-23 of the sense strand to form a duplex; wherein at least the 3'-terminal nucleotide of the antisense strand is unpaired with the sense strand, and up to 6 consecutive 3'-terminal nucleotides are unpaired with the sense strand, thereby forming a 3'-single-stranded overhang of 1-6 nucleotides; wherein the 5'- The terminus comprises 10-30 consecutive nucleotides that are unpaired with the sense strand, thereby forming a single-stranded 5'-overhang of 10-30 nucleotides; wherein when the sense strand and the antisense strand are aligned for maximum complementarity, at least the 5'-terminal and 3'-terminal nucleotides of the sense strand base pair with nucleotides of the antisense strand, thereby forming a substantially double-stranded region between the sense strand and the antisense strand; and the antisense strand is sufficiently complementary to the target RNA along at least 19 ribonucleotides of the antisense strand length to reduce target gene expression when the double-stranded nucleic acid is introduced into a mammalian cell; and wherein the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides, wherein at least one of the motifs occurs at or near the cleavage site. The antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.

[0280] In one embodiment, the RNAi agent comprises a sense strand and an antisense strand, wherein the RNAi agent comprises a first strand of at least 25 and at most 29 nucleotides in length and a second strand of at most 30 nucleotides in length, having at least one 2'-O-methyl modified motif on three consecutive nucleotides at positions 11, 12, and 13 at the 5' end; wherein the 3'-end of the first strand and the 5'-end of the second strand form a blunt end, and the second strand is 1-4 nucleotides longer than the first strand at its 3'-end, wherein the length of the duplex region is at least 25 nucleotides, and the second strand is sufficiently complementary to the target mRNA along at least 19 nucleotides of the length of the second strand to reduce target gene expression when the RNAi agent is introduced into a mammalian cell, and wherein dicer cleavage of the RNAi agent preferentially produces an siRNA comprising the 3'-end of the second strand, thereby reducing target gene expression in mammals. Optionally, the RNAi agent further comprises a ligand.

[0281] In one embodiment, the sense strand of the RNAi agent contains at least one motif of three identical modifications on three consecutive nucleotides, wherein one of the motifs occurs at the cleavage site in the sense strand.

[0282] In one embodiment, the antisense strand of the RNAi agent may also contain at least one motif of three identical modifications on three consecutive nucleotides, wherein one of the motifs occurs at or near the cleavage site in the antisense strand.

[0283] For RNAi agents with duplex regions of 17-23 nucleotides in length, the cleavage sites of the antisense strand are typically near positions 10, 11, and 12 of the 5'-end. Thus, three identical modified motifs can appear at positions 9, 10, and 11 of the antisense strand; positions 10, 11, and 12; positions 11, 12, and 13; positions 12, 13, and 14; or positions 13, 14, and 15 of the antisense strand, with the count starting from the first nucleotide at the 5'-end of the antisense strand, or the count starting from the first paired nucleotide at the 5'-end of the antisense strand within the duplex region. The cleavage site in the antisense strand can also vary depending on the length of the duplex region of the RNAi from the 5' end.

[0284] The sense strand of the RNAi agent may contain at least one motif of three identical modifications on three consecutive nucleotides at the cleavage site of the strand; and the antisense strand may have at least one motif of three identical modifications on three consecutive nucleotides at or near the cleavage site of the strand. When the sense strand and the antisense strand form a dsRNA duplex, the sense strand and the antisense strand may be aligned so that one motif of three nucleotides on the sense strand overlaps with one motif of three nucleotides on the antisense strand, i.e., at least one of the three nucleotides of the motif in the sense strand forms a base pair with at least one of the three nucleotides of the motif in the antisense strand. Alternatively, at least two nucleotides may overlap, or all three nucleotides may overlap.

[0285] In one embodiment, the sense strand of an RNAi agent can contain more than one motif of three identical modifications on three consecutive nucleotides. The first motif can occur at or near the cleavage site of the chain, and other motifs can be wing modifications. The term "wing modification" herein refers to a motif that occurs at another part of the chain, which is separated from the motif at or near the cleavage site of the same chain. The wing modification is adjacent to the first motif or is separated by at least one or more nucleotides. When the motifs are adjacent to each other, the chemistry of the motif is different from each other, and when the motif is separated by one or more nucleotides, the chemistry can be the same or different. There may be two or more wing modifications. For example, when there are two wing modifications, each wing modification can occur at or near the cleavage site or at one end of the first motif on either side of the previous motif.

[0286] As with the sense strand, the antisense strand of the RNAi agent can contain more than one motif of three identical modifications on three consecutive nucleotides, wherein at least one motif occurs at or near the cleavage site of the strand. The antisense strand can also contain one or more wing modifications arranged similarly to those that may be present on the sense strand.

[0287] In one embodiment, wing modifications on the sense or antisense strand of the RNAi agent typically do not include the first one or two terminal nucleotides at the 3 '-end, the 5 '-end, or both ends of the strand.

[0288] In another embodiment, the wing modifications on the sense or antisense strand of the RNAi agent typically do not include the first one or two paired nucleotides within the duplex region at the 3 '-end, the 5 '-end, or both ends of the strand.

[0289] When the sense and antisense strands of the RNAi agent each contain at least one wing modification, the wing modifications can fall on the same end of the duplex region and have an overlap of one, two, or three nucleotides.

[0290] When the sense and antisense strands of the RNAi agent each contain at least two wing modifications, the sense and antisense strands can be aligned so that the two modifications, each from one strand, fall on one end of the duplex region with an overlap of one, two, or three nucleotides; the two modifications, each from one strand, fall on the other end of the duplex region with an overlap of one, two, or three nucleotides; and the two modifications of one strand fall on each side of the preceding motif with an overlap of one, two, or three nucleotides in the duplex region.

[0291] In one embodiment, the RNAi agent comprises a mismatch with the target, within the duplex, or a combination thereof. Mismatches may be present in the overhang region or the duplex region. Base pairs can be ranked based on their tendency to promote dissociation or melt (e.g., based on the free energy of association or dissociation of a particular pairing, the simplest way is to examine pairing on the basis of a single pairing, although neighbor or similar analysis can also be used). In terms of promoting dissociation: A: U is better than G: C; G: U is better than G: C; and I: C (I = inosine) is better than G: C. Mismatches, such as non-canonical or extra-canonical pairings (as described elsewhere herein) are better than canonical (A: T, A: U, G: C) pairings; and pairings comprising universal bases are better than canonical pairings.

[0292] In one embodiment, the RNAi agent comprises at least one of the first 1, 2, 3, 4 or 5 base pairs at the 5'-end of the antisense strand within the duplex region, independently selected from: A:U, G:U, I:C and mispairing, e.g., non-canonical or extra-canonical pairings or pairings comprising universal bases, to promote dissociation of the antisense strand at the 5'-end of the duplex.

[0293] In one embodiment, the nucleotide at position 1 of the 5'-end of the antisense strand in the duplex region is selected from the group consisting of: A, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2, or 3 base pairs at the 5'-end of the antisense strand in the duplex region is an AU base pair. For example, the first base pair at the 5'-end of the antisense strand in the duplex region is an AU base pair.

[0294] In another embodiment, the nucleotide at the 3'-end of the sense strand is deoxythymidine (dT). In another embodiment, the nucleotide at the 3'-end of the antisense strand is deoxythymidine (dT). In one embodiment, there is a short sequence of deoxythymidine nucleotides, for example, two dT nucleotides at the 3' end of the sense strand or the antisense strand.

[0295] In one embodiment, the sense strand sequence can be represented by formula (I):

[0296] 5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3'(I)

[0297] in:

[0298] i and j are each independently 0 or 1;

[0299] p and q are each independently 0 to 6;

[0300] Each N a independently represent an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;

[0301] Each N b independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;

[0302] Each n p and n q Overhang nucleotides are represented independently;

[0303] where N b and Y do not have the same modifications; and

[0304] XXX, YYY and ZZZ each independently represent a motif of three identical modifications on three consecutive nucleotides. Optionally, all YYY are 2'-F modified nucleotides.

[0305] In one embodiment, N a or N b Contains alternating patterns of modifications.

[0306] In one embodiment, the YYY motif appears at or near the cleavage site of the sense strand. For example, when the RNAi agent has a duplex region of 17-23 nucleotides in length, the YYY motif can appear at or near the cleavage site of the sense strand (e.g., can appear at positions 6, 7, 8; 7, 8, 9; 8, 9, 10; 9, 10, 11; 10, 11, 12 or 11, 12, 13), counting from the first nucleotide at the 5' end; or optionally, counting from the first paired nucleotide at the 5' end within the duplex region.

[0307] In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. Thus, the sense strand can be represented by the formula:

[0308] 5'n p -N a -YYY-N b -ZZZ-N a -n q 3'(Ib);

[0309] 5'n p -N a -XXX-N b -YYY-N a -n q 3'(Ic); or

[0310] 5'n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3'(Id).

[0311] When the sense strand is represented by formula (Ib), N b refers to an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides.

[0312] Each N a Oligonucleotide sequences comprising 2-20, 2-15 or 2-10 modified nucleotides may be independently represented.

[0313] When the sense strand is represented by formula (Ic), N b represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N a Oligonucleotide sequences comprising 2-20, 2-15 or 2-10 modified nucleotides may be independently represented.

[0314] When the sense strand is represented by formula (Id), each N b independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. b is 0, 1, 2, 3, 4, 5, or 6. Each N a An oligonucleotide sequence comprising 2-20, 2-15 or 2-10 modified nucleotides may independently be represented.

[0315] Each of X, Y, and Z may be the same as or different from each other.

[0316] In other embodiments, i is 0 and j is 0, and the sense strand can be represented by the formula:

[0317] 5'n p -N a -YYY-N a -n q 3'(Ia).

[0318] When the sense strand is represented by formula (Ia), each N a An oligonucleotide sequence comprising 2-20, 2-15 or 2-10 modified nucleotides may independently be represented.

[0319] In one embodiment, the antisense strand sequence of RNAi can be represented by formula (II):

[0320] 5'n q' -N a '-(Z'Z'Z') k -N b '-Y'Y'Y'-N b '-(X'X'X') l -N' a -n p '3'(II)

[0321] in:

[0322] k and l are each independently 0 or 1;

[0323] p' and q' are each independently 0 to 6;

[0324] Each N a ' independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;

[0325] Each N b ' independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;

[0326] Each n p ' and n q ' independently represent overhang nucleotides;

[0327] where N b ' and Y' do not have the same modification;

[0328] And X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent a motif of three identical modifications on three consecutive nucleotides.

[0329] In one embodiment, N a ' or N b 'Includes alternating pattern modifications.

[0330] The Y'Y'Y' motif occurs at or near the cleavage site of the antisense strand. For example, when the RNAi agent has a duplex region of 17-23 nucleotides in length, the Y'Y'Y' motif can occur at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, counting from the first nucleotide at the 5' end; or optionally, counting from the first paired nucleotide at the 5' end within the duplex region. Optionally, the Y'Y'Y' motif occurs at positions 11, 12, 13.

[0331] In one embodiment, the Y'Y'Y' motif is entirely 2'-OMe modified nucleotides.

[0332] In one embodiment, k is 1 and l is 0, or k is 0 and l is 1, or both k and l are 1.

[0333] Therefore, the antisense strand can be represented by the following formula:

[0334] 5'n q' -N a '-Z'Z'Z'-N b '-Y'Y'Y'-N a '-n p' 3'(IIb);

[0335] 5'n q' -N a '-Y'Y'Y'-N b '-X'X'X'-n p' 3'(IIc); or

[0336] 5'n q' -N a '-Z'Z'Z'-N b '-Y'Y'Y'-N b '-X'X'X'-Na '-n p' 3'(IId).

[0337] When the antisense strand is represented by formula (IIb), N b ' represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N a ' independently represents an oligonucleotide sequence comprising 2-20, 2-15 or 2-10 modified nucleotides.

[0338] When the antisense strand is represented by formula (IIc), N b ' represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N a ' independently represents an oligonucleotide sequence comprising 2-20, 2-15 or 2-10 modified nucleotides.

[0339] When the antisense strand is represented by formula (IId), each N b ' independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N a ' independently represents an oligonucleotide sequence comprising 2-20, 2-15 or 2-10 modified nucleotides. Optionally, N b is 0, 1, 2, 3, 4, 5, or 6.

[0340] In other embodiments, k is 0 and l is 0, and the antisense strand can be represented by the formula:

[0341] 5'n p' -N a' -Y'Y'Y'-N a' -n q' 3'(Ia).

[0342] When the antisense strand is represented by formula (IIa), each N a ' independently represents an oligonucleotide sequence comprising 2-20, 2-15 or 2-10 modified nucleotides.

[0343] X', Y' and Z' may be the same as or different from each other.

[0344] Each nucleotide in the sense and antisense strands can be independently modified with LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-hydroxyl, or 2'-fluoro. For example, each nucleotide in the sense and antisense strands can be independently modified with 2'-O-methyl or 2'-fluoro. In particular, each of X, Y, Z, X', Y', and Z' can represent a 2'-O-methyl modification or a 2'-fluoro modification.

[0345] In one embodiment, when the duplex region is 21 nt, the sense strand of the RNAi agent may contain a YYY motif appearing at positions 9, 10, and 11 of the strand, with the count starting from the first nucleotide at the 5' end, or optionally, the count starting from the first paired nucleotide at the 5' end within the duplex region; and Y represents a 2'-F modification. The sense strand may additionally contain a XXX motif or a ZZZ motif as wing modifications at opposite ends of the duplex region; and XXX and ZZZ each independently represent a 2'-OMe modification or a 2'-F modification.

[0346] In one embodiment, the antisense strand may contain a Y'Y'Y' motif appearing at positions 11, 12, or 13 of the strand, counting starting from the first nucleotide at the 5' end, or optionally, counting starting from the first paired nucleotide at the 5' end within the duplex region; and Y' represents a 2'-O-methyl modification. The antisense strand may additionally contain an X'X'X' motif or a Z'Z'Z' motif as wing modifications at opposite ends of the duplex region; and X'X'X' and Z'Z'Z' each independently represent a 2'-OMe modification or a 2'-F modification.

[0347] The sense strand represented by any one of the above formulae (Ia), (Ib), (Ic) and (Id) forms a duplex with the antisense strand represented by any one of the formulae (IIa), (IIb), (IIc) and (IId), respectively.

[0348] Thus, the RNAi agent used in the methods of the present disclosure can comprise a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the RNAi duplex being represented by formula (III):

[0349] Meaning: 5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3'

[0350] Antonym: 3'n p '-N a '-(X'X'X')k -N b '-Y'Y'Y'-N b '-(Z'Z'Z') l -N a '-n q '5'

[0351] (III)

[0352] in:

[0353] i, j, k and l are each independently 0 or 1;

[0354] p, p', q and q' are each independently 0 to 6;

[0355] Each N a and N a ' independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;

[0356] Each N b and N b ' independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;

[0357] in

[0358] Each may exist or not exist p '、n p 、n q ' and n q Each of independently represents an overhang nucleotide; and

[0359] XXX, YYY, ZZZ, X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent a motif of three identical modifications on three consecutive nucleotides.

[0360] In one embodiment, i is 0 and j is 0; or i is 1 and j is 0; or i is 0 and j is 1; or i and j are both 0; or i and j are both 1. In another embodiment, k is 0 and 1 is 0; or k is 1 and 1 is 0; k is 0 and 1 is 1; or k and 1 are both 0; or k and 1 are both 1.

[0361] Exemplary combinations of sense and antisense strands that form RNAi duplexes include the following:

[0362] 5'n p -N a -YYY-N a -n q 3'

[0363] 3'n p'-N a '-Y'Y'Y'-N a 'n q '5'

[0364] (IIIa)

[0365] 5'n p -N a -YYY-N b -ZZZ-N a -n q 3'

[0366] |3'n p '-N a '-Y'Y'Y'-N b '-Z'Z'Z'-N a 'n q '5'

[0367] (IIIb)

[0368] 5'n p -N a -XXX-N b -YYY-N a -n q 3'

[0369] 3'n p '-N a '-X'X'X'-N b '-Y'Y'Y'-N a '-n q '5'

[0370] (IIIc)

[0371] 5'n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3'

[0372] 3'n p '-N a '-X'X'X'-N b '-Y'Y'Y'-N b '-Z'Z'Z'-N a -n q '5'

[0373] (IIId)

[0374] When the RNAi agent is represented by the formula (IIIa), each N a It should be noted that there may be some inaccuracies in the translation due to the lack of clear context for some of these notations. If possible, it would be beneficial to have more background information to ensure a more precise translation.Independently refers to an oligonucleotide sequence comprising 2 to 20, 2 to 15 or 2-10 modified nucleotides.

[0375] When the RNAi agent is represented by formula (IIIb), each N b N independently represents an oligonucleotide sequence comprising 1 to 10, 1 to 7, 1 to 5, or 1-4 modified nucleotides. a Independently refers to an oligonucleotide sequence comprising 2 to 20, 2 to 15 or 2 to 10 modified nucleotides.

[0376] When the RNAi agent is represented by formula (IIIc), each N b 、N b ' independently represents an oligonucleotide sequence comprising 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. Each N a Independently refers to an oligonucleotide sequence comprising 2 to 20, 2 to 15 or 2 to 10 modified nucleotides.

[0377] When the RNAi agent is represented by formula (IIId), each N b 、N b ' independently represents an oligonucleotide sequence comprising 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. Each N a 、N a ' independently represents an oligonucleotide sequence comprising 2 to 20, 2 to 15 or 2 to 10 modified nucleotides. N a 、N a '、N b and N b 'Each independently contains alternating patterns of modifications.

[0378] In one embodiment, when the RNAi agent is represented by formula (IIId), N a In another embodiment, when the RNAi agent is represented by formula (IIId), N a The modification is 2'-O-methyl modification or 2'-fluoro modification, and n p '>0, and at least one n p ' is linked to the adjacent nucleotide via a phosphorothioate bond. In yet another embodiment, when the RNAi agent is represented by formula (IIId), N a Modification is 2'-O-methyl modification or 2'-fluoro modification, n p '>0, and at least one n p' is linked to adjacent nucleotides via phosphorothioate bonds, and the sense strand is conjugated to one or more C16 (or related) moieties attached via a divalent or trivalent branched linker (described below). In another embodiment, when the RNAi agent is represented by formula (IIId), N a Modification is 2'-O-methyl modification or 2'-fluoro modification, n p '>0, and at least one n p ' linked to an adjacent nucleotide via a phosphorothioate bond, the sense strand comprising at least one phosphorothioate bond, and conjugated to one or more lipophilic, eg, C16 (or related) moieties, optionally attached via a linker.

[0379] In one embodiment, when the RNAi agent is represented by formula (IIIa), N a Modification is 2'-O-methyl modification or 2'-fluoro modification, n p '>0, and at least one n p ' linked to adjacent nucleotides via phosphorothioate bonds, the sense strand comprising at least one phosphorothioate bond, and conjugated to one or more lipophilic, eg, C16 (or related) moieties attached via a linker.

[0380] In one embodiment, the RNAi agent is a polymer containing at least two duplexes represented by Formula (III), (IIIa), (IIIb), (IIIc) and (IIId), wherein the duplexes are connected by a linker. The linker can be cleavable or non-cleavable. Optionally, the polymer further comprises a ligand. Each duplex can target the same gene or two different genes; or each duplex can target the same gene at two different target sites.

[0381] In one embodiment, the RNAi agent is a polymer containing 3, 4, 5, 6 or more duplexes represented by Formula (III), (IIIa), (IIIb), (IIIc) and (IIId), wherein the duplexes are connected by a linker. The linker can be cleavable or non-cleavable. Optionally, the polymer further comprises a ligand. Each duplex can target the same gene or two different genes; or each duplex can target the same gene at two different target sites.

[0382] In one embodiment, two RNAi agents represented by Formula (III), (IIIa), (IIIb), (IIIc), and (IIId) are linked to each other at the 5' end and one or both 3' ends, and are optionally conjugated to a ligand. Each agent can target the same gene or two different genes; or each agent can target the same gene at two different target sites.

[0383] Various publications describe poly RNAi agents that can be used in the methods of the present disclosure. Such publications include WO2007 / 091269, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887, and WO2011 / 031520; and US7858769, the entire contents of each of which are hereby incorporated herein by reference.

[0384] In certain embodiments, the compositions and methods of the present disclosure include 5'-phosphate mimetics such as vinylphosphonate (VP) modifications of the RNAi agents as described herein. In exemplary embodiments, the vinylphosphonate of the present disclosure has the following structure:

[0385]

[0386] The vinylphosphonates of the present disclosure can be attached to the antisense strand or the sense strand of the dsRNA of the present disclosure. In certain preferred embodiments, the vinylphosphonates of the present disclosure are attached to the antisense strand of the dsRNA, optionally the 5'-end of the antisense strand of the dsRNA.

[0387] In an exemplary embodiment, a 5' vinylphosphonate modified nucleotide of the present disclosure has the structure:

[0388]

[0389] Where X is O or S;

[0390] R is hydrogen, hydroxy, fluorine or C 1-20 Alkoxy (e.g., methoxy or n-hexadecyloxy);

[0391] R 5 ' is =C(H)-P(O)(OH)2, and the double bond between the C5' carbon and R5' is in the E or Z orientation (e.g., the E orientation); and

[0392] B is a nucleobase or a modified nucleobase, optionally wherein B is adenine, guanine, cytosine, thymine or uracil.

[0393] In one embodiment, R 5 ' is =C(H)-P(O)(OH)2, and the double bond between the C5' carbon and R5' is in E orientation. In another embodiment, R is methoxy and R 5 ' is =C(H)-P(O)(OH)2, and the double bond between the C5' carbon and R5' is in E orientation. In another embodiment, X is S, R is methoxy and R 5 ' is =C(H)-P(O)(OH)2, and the double bond between the C5' carbon and R5' is in the E orientation.

[0394] Vinyl phosphate modifications are also contemplated for use in the compositions and methods of the present disclosure. An exemplary vinyl phosphate structure is:

[0395]

[0396] Additional exemplary vinyl phosphate structures include the preceding structures, wherein R5' is =C(H)-OP(O)(OH)2, and the double bond between the C5' carbon and R5' is in the E or Z orientation (eg, the E orientation).

[0397] In some embodiments, the 5-phosphate mimetic is selected from or a salt thereof (e.g., a sodium salt), wherein the bond that is broken is a covalent bond to the 4'-carbon of the 5'-terminal nucleotide. In some embodiments, the 5'-terminal nucleotide may be or a salt thereof (eg, sodium salt), wherein B is a modified or unmodified nucleobase (eg, uracil or 5-methyluracil).

[0398] B. Thermal Destabilization Modification

[0399] In certain embodiments, dsRNA molecules can be optimized RNA interference by incorporating heat destabilization modifications in the seed region of the antisense strand (i.e., position 2 to 9 at the 5'-end of the antisense strand) to reduce or inhibit off-target gene silencing. It has been found that dsRNA with an antisense strand (comprising at least one heat destabilization modification of a duplex within the first 9 nucleotide positions of the antisense strand counted from the 5' end) has reduced off-target gene silencing activity. Therefore, in some embodiments, the antisense strand is included in at least one (e.g., one, two, three, four, five or more) heat destabilization modification of a duplex within the first 9 nucleotide positions of the 5' district of the antisense strand. In some embodiments, one or more heat destabilization modifications of the duplex are located at positions 2 to 9 from the 5'-end of the antisense strand, or optionally positions 4 to 8. In some other embodiments, the heat destabilization modification of the duplex is located at positions 6, 7 or 8 from the 5'-end of the antisense strand. In some other embodiments, the heat destabilization modification of the duplex is located at positions 7 from the 5'-end of the antisense strand. The term "thermal destabilizing modification" includes modifications that result in a lower overall melting temperature (Tm) of the dsRNA (optionally, a Tm that is one, two, three, or four degrees lower than the Tm of the dsRNA without such modification). In some embodiments, the thermal destabilizing modification of the duplex is located at position 2, 3, 4, 5, or 9 from the 5' end of the antisense strand.

[0400] Thermally destabilizing modifications can include, but are not limited to, abasic modifications; mismatches with the opposite nucleotide in the opposite strand; and sugar modifications, such as 2'-deoxy modifications, acyclic nucleotides, e.g., unlocked nucleic acids (UNA) or glycol nucleic acids (GNA); and 2'-5'-linked ribonucleotides ("3'-RNA").

[0401] Exemplary abasic modifications include, but are not limited to, the following:

[0402]

[0403] wherein R=H, Me, Et or OMe; R'=H, Me, Et or OMe; R"=H, Me, Et or OMe

[0404]

[0405] wherein B is a modified or unmodified nucleobase.

[0406] Exemplary sugar modifications include, but are not limited to, the following:

[0407]

[0408] wherein B is a modified or unmodified nucleobase.

[0409] In some embodiments, the thermally destabilizing modification of the duplex is selected from the group consisting of:

[0410]

[0411] Where B is a modified or unmodified nucleobase, and the asterisk on each structure indicates R, S, or racemic.

[0412] In some embodiments, the thermally destabilizing modification of the duplex is selected from the group consisting of:

[0413]

[0414] wherein B is a modified or unmodified nucleobase and the asterisk indicates R, S, or racemic (eg, S).

[0415] The term "acyclic nucleotide" refers to any nucleotide having an acyclic ribose sugar, e.g., wherein any linkage between the ribose carbons (e.g., C1'-C2', C2'-C3', C3'-C4', C4'-O4', or C1'-O4') is absent or at least one of the ribose carbons or oxygens (e.g., C1', C2', C3', C4', or O4'), independently or in combination, is absent in the nucleotide. In some embodiments, an acyclic nucleotide is Wherein B is a modified or unmodified nucleobase, R 1 and R 2is independently H, halogen, OR3 or alkyl; and R3 is H, alkyl, cycloalkyl, aryl, heteroaryl or sugar. The term "UNA" refers to an unlocked acyclic nucleic acid in which any bond of a sugar is removed, thereby forming an unlocked "sugar" residue. In one example, UNA also encompasses monomers in which the bond between C1'-C4' (i.e., the carbon-oxygen-carbon covalent bond between C1' and C4' carbon) is removed. In another example, the C2'-C3' bond of the sugar (i.e., the carbon-carbon covalent bond between C2' and C3' carbon) is removed (see, Mikhailov et.al., Tetrahedron Letters, 26(17):2059(1985) and Fluiter et al., Mol. Biosyst., 10:1039(2009), incorporated herein by reference in their entirety). Acyclic derivatives provide greater backbone flexibility without affecting Watson-Crick pairing. Acyclic nucleotides can be linked via 2'-5' or 3'-5' linkages.

[0416] The term "GNA" refers to diol nucleic acid, a polymer similar to DNA or RNA, but with a different "backbone" composition, consisting of repeating glycerol units linked by phosphodiester bonds:

[0417]

[0418] The heat destabilization modification of the duplex can be a mismatch (i.e., non-complementary base pair) between a heat destabilizing nucleotide and the relative nucleotide in the relative chain within the dsRNA duplex. Exemplary mismatch base pairs include G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, U:T, or a combination thereof. Other mismatch base pairs known in the art are also applicable to the present disclosure. Mismatches can be present between nucleotides (naturally occurring nucleotides or modified nucleotides), that is, mismatch base pairing can be present between the core bases from the corresponding nucleotides, regardless of the modification on the ribose of the nucleotides. In certain embodiments, the dsRNA molecule contains at least one core base in the mismatch pairing that is a 2'-deoxynucleobase; for example, the 2'-deoxynucleobase is in the sense strand.

[0419] In some embodiments, the thermally destabilizing modification of the duplex in the seed region of the antisense strand comprises nucleotides with impaired WCH-bonding to complementary bases on the target mRNA, such as:

[0420]

[0421] Further examples of abasic nucleotides, acyclic nucleotide modifications (including UNA and GNA), and mismatch modifications are described in detail in WO 2011 / 133876, which is incorporated herein by reference in its entirety.

[0422] Thermally destabilizing modifications may also include universal base and phosphate modifications that have reduced or eliminated ability to form hydrogen bonds with opposing bases.

[0423] In some embodiments, the thermal destabilizing modifications of the duplex include nucleotides with non-canonical bases, such as, but not limited to, nucleobase modifications with impaired or completely abolished ability to form hydrogen bonds with bases in the opposite strand. These nucleobase modifications were evaluated for destabilization of the central region of the dsRNA duplex, as described in WO 2010 / 0011895, which is incorporated herein by reference in its entirety. Exemplary nucleobase modifications are:

[0424]

[0425] In some embodiments, the thermally destabilizing modification of the duplex in the seed region of the antisense strand includes one or more α-nucleotides that are complementary to bases on the target mRNA, such as:

[0426]

[0427] wherein R is H, OH, OCH3, F, NH2, NHMe, NMe2 or O-alkyl.

[0428] Exemplary phosphate modifications known to reduce the thermal stability of dsRNA duplexes compared to native phosphodiester bonds are:

[0429]

[0430] The alkyl group of the R group can be a C1-C6 alkyl group. Specific alkyl groups of the R group include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, pentyl, and hexyl.

[0431] As will be appreciated by those skilled in the art, given that the functional role of the nucleobase is to define the specificity of the RNAi agents of the present disclosure, and nucleobase modifications can be performed in various ways as described herein, such as by introducing destabilizing modifications into the RNAi agents of the present disclosure, for example for the purpose of enhancing on-target effects relative to off-target effects, for non-nucleobase modifications, such as modifications to the sugar groups or phosphate backbones of polyribonucleotides, the range of modifications available and typically present on the RNAi agents of the present disclosure is often much greater. Such modifications are described in more detail in other parts of the present disclosure, and are specifically contemplated for use in RNAi agents of the present disclosure having natural or modified nucleobases as described above or elsewhere herein.

[0432] In addition to including the antisense strand modified by thermal destabilization, dsRNA can also include one or more stable modifications. For example, dsRNA can include at least two (e.g., two, three, four, five, six, seven, eight, nine, ten or more) stable modifications. Without limitation, stable modifications can all be present in one chain. In some embodiments, both the sense strand and the antisense strand include at least two stable modifications. Stable modifications can be present on any nucleotide of the sense strand or the antisense strand. For example, a stable modification can be present on each nucleotide on the sense strand or the antisense strand; Each stable modification can exist in an alternating pattern on the sense strand or the antisense strand; Or the sense strand or the antisense strand both include a stable modification of an alternating pattern. The alternating pattern of the stable modification on the sense strand can be the same or different from the antisense strand, and the alternating pattern of the stable modification on the sense strand can have an offset relative to the alternating pattern of the stable modification on the antisense strand.

[0433] In some embodiments, the antisense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten or more) stabilizing modifications. Without limitation, the stabilizing modifications in the antisense strand can be present at any position. In some embodiments, the antisense comprises stabilizing modifications at positions 2, 6, 8, 9, 14, and 16 of the 5' end. In still other embodiments, the antisense comprises stabilizing modifications at positions 2, 6, 14, and 16 of the 5' end. In still other embodiments, the antisense comprises stabilizing modifications at positions 2, 14, and 16 of the 5' end.

[0434] In some embodiments, the antisense strand comprises at least one stabilizing modification adjacent to a destabilizing modification. For example, the stabilizing modification can be a nucleotide at the 5'-end or 3'-end of the unstable modification, i.e., at the -1 or +1 position relative to the position of the unstable modification. In some embodiments, the antisense strand comprises a stabilizing modification at each of the 5'-end and 3'-end of the unstable modification, i.e., at the -1 and +1 positions relative to the unstable modification position.

[0435] In some embodiments, the antisense strand comprises at least two stabilizing modifications 3'-terminus of the destabilizing modification, ie, at positions +1 or +2 to the position of the destabilizing modification.

[0436] In some embodiments, the sense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten or more) stabilizing modifications. Without limitation, the stabilizing modifications in the sense strand may be present at any position. In some embodiments, the sense strand comprises stabilizing modifications at positions 7, 10, and 11 of the 5'-end. In some other embodiments, the sense strand comprises stabilizing modifications at positions 7, 9, 10, and 11 of the 5'-end. In some embodiments, the sense strand comprises stabilizing modifications at positions opposite or complementary to positions 11, 12, and 15 of the antisense strand, counted from the 5'-end. In some other embodiments, the sense strand comprises stabilizing modifications at positions opposite or complementary to positions 11, 12, 13, and 15 of the antisense strand, counted from the 5'-end of the antisense strand. In some embodiments, the sense strand comprises two, three, or four blocks of stabilizing modifications.

[0437] In some embodiments, the sense strand does not comprise a stabilizing modification at a position opposite or complementary to a thermally destabilizing modification of the duplex in the antisense strand.

[0438] Exemplary thermostable modifications include, but are not limited to, 2'-fluoro modifications.Other thermostable modifications include, but are not limited to, LNA.

[0439] In some embodiments, the dsRNA of the present disclosure comprises at least four (e.g., four, five, six, seven, eight, nine, ten or more) 2'-fluoro nucleotides. Without limitation, 2'-fluoro nucleotides can all be present in one chain. In some embodiments, both the sense strand and the antisense strand comprise at least two 2'-fluoro nucleotides. The 2'-fluoro modification can be present on any nucleotide of the sense strand or the antisense strand. For example, the 2'-fluoro modification can be present on each nucleotide on the sense strand or the antisense strand; each 2'-fluoro modification can be present in an alternating pattern on the sense strand or the antisense strand; or the sense strand or the antisense strand both comprise 2'-fluoro modifications in an alternating pattern. The alternating pattern of the 2'-fluoro modifications on the sense strand can be the same or different from the antisense strand, and the alternating pattern of the 2'-fluoro modifications on the sense strand can have an offset relative to the alternating pattern of the 2'-fluoro modifications on the antisense strand.

[0440] In some embodiments, the antisense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten or more) 2'-fluoro nucleotides. Without limitation, the 2'-fluoro modification in the antisense strand can be present in any position. In some embodiments, the antisense comprises 2'-fluoro nucleotides at position 2, 6, 8, 9, 14 and 16 of the 5' end. In some other embodiments, the antisense comprises 2'-fluoro nucleotides at position 2, 6, 14 and 16 of the 5' end. In still some other embodiments, the antisense comprises 2'-fluoro nucleotides at position 2, 14 and 16 of the 5' end.

[0441] In some embodiments, the antisense strand comprises at least one 2'-fluoro nucleotide adjacent to a destabilizing modification. For example, the 2'-fluoro nucleotide can be a nucleotide at the 5' or 3' end of the destabilizing modification, i.e., at position -1 or +1 relative to the destabilizing modification position. In some embodiments, the antisense strand comprises a 2'-fluoro nucleotide at each of the 5' and 3' ends of the destabilizing modification, i.e., at positions -1 and +1 relative to the destabilizing modification position.

[0442] In some embodiments, the antisense strand comprises at least two 2'-fluoro nucleotides 3' to the destabilizing modification, ie, at positions +1 or +2 to the position of the destabilizing modification.

[0443] In some embodiments, the sense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten or more) 2'-fluoro nucleotides. Without limitation, the 2'-fluoro modification in the sense strand can be present at any position. In some embodiments, the antisense comprises 2'-fluoro nucleotides at positions 7, 10, and 11 of the 5' end. In some other embodiments, the sense strand comprises 2'-fluoro nucleotides at positions 7, 9, 10, and 11 of the 5' end. In some embodiments, the sense strand comprises 2'-fluoro nucleotides at positions opposite or complementary to positions 11, 12, and 15 of the antisense strand, counting from the 5' end of the antisense strand. In some other embodiments, the sense strand comprises 2'-fluoro nucleotides at positions opposite or complementary to positions 11, 12, 13, and 15 of the antisense strand, counting from the 5' end of the antisense strand. In some embodiments, the sense strand comprises blocks of two, three, or four 2'-fluoro nucleotides.

[0444] In some embodiments, the sense strand does not comprise a 2'-fluoro nucleotide at a position opposite or complementary to a thermally destabilizing modification of the duplex in the antisense strand.

[0445] In some embodiments, the dsRNA molecules of the present disclosure comprise a 21 nucleotide (nt) sense strand and a 23 nucleotide (nt) antisense strand, wherein the antisense strand contains at least one thermally destabilizing nucleotide, wherein the at least one thermally destabilizing nucleotide occurs in the seed region of the antisense strand (i.e., positions 2 to 9 of the 5' end of the antisense strand), wherein one end of the dsRNA is blunt and the other end comprises a 2 nt overhang, and wherein the dsRNA optionally further has at least one of the following features (e.g., one, two, three, four, five, six

[0015] In some embodiments, the dsRNA comprises at least one 2'-fluoro modification and at least seven 2'-fluoro modifications: (i) the antisense comprises two, three, four, five, or six 2'-fluoro modifications; (ii) the antisense comprises one, two, three, four, or five phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated to a ligand; (iv) the sense strand comprises two, three, four, or five 2'-fluoro modifications; (v) the sense strand comprises one, two, three, four, or five phosphorothioate internucleotide linkages; (vi) the dsRNA comprises at least four 2'-fluoro modifications; and (vii) the dsRNA comprises a blunt end at the 5' end of the antisense strand. Optionally, a 2 nt overhang is located at the 3' end of the antisense.

[0446] In some embodiments, the dsRNA molecules of the present disclosure include a sense strand and an antisense strand, wherein: the sense strand is 25-30 nucleotide residues in length, wherein starting from the 5'-terminal nucleotide (position 1), positions 1 to 23 of the sense strand include at least 8 ribonucleotides; the antisense strand is 36-66 nucleotide residues in length, and starting from the 3'-terminal nucleotide, at least 8 ribonucleotides in positions that pair with positions 1 to 23 of the sense strand to form a duplex; wherein at least the 3'-terminal nucleotide of the antisense strand is unpaired with the sense strand, and up to 6 consecutive 3'-terminal nucleotides are unpaired with the sense strand, thereby forming a 3' single-stranded overhang of 1-6 nucleotides; wherein the 5'- The terminus comprises 10-30 contiguous nucleotides that are unpaired with the sense strand, thereby forming a single-stranded 5' overhang of 10-30 nucleotides; wherein when the sense and antisense strands are aligned for maximum complementarity, at least the 5'-terminal and 3'-terminal nucleotides of the sense strand base pair with nucleotides of the antisense strand, thereby forming a substantially double-stranded region between the sense and antisense strands; and the antisense strand is sufficiently complementary to the target RNA along at least 19 ribonucleotides of the length of the antisense strand to reduce target gene expression when the double-stranded nucleic acid is introduced into a mammalian cell; and wherein the antisense strand contains at least one thermally destabilizing nucleotide, wherein the at least one thermally destabilizing nucleotide is in the seed region of the antisense strand (i.e., positions 2 to 9 at the 5'-end of the antisense strand). For example, the thermally destabilizing nucleotide occurs between positions opposite or complementary to positions 14 to 17 of the 5'-end of the sense strand, and wherein the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six, or all seven) of the following features: (i) the antisense comprises two, three, four, five, or six 2'-fluoro modifications; (ii) the antisense comprises one, two, three, four, or five phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated to a ligand; (iv) the sense strand comprises two, three, four, or five 2'-fluoro modifications; (v) the sense strand comprises one, two, three, four, or five phosphorothioate internucleotide linkages; and (vi) the dsRNA comprises at least four 2'-fluoro modifications; and (vii) the dsRNA comprises a duplex region that is 12-30 nucleotide pairs in length.

[0447] In some embodiments, a dsRNA molecule of the present disclosure comprises a sense strand and an antisense strand, wherein the dsRNA molecule comprises a sense strand of at least 25 and at most 29 nucleotides in length and an antisense strand of at most 30 nucleotides in length, wherein the sense strand comprises a modified nucleotide at position 11 of the 5' end that is susceptible to enzymatic degradation, wherein the 3'-end of the sense strand and the 5'-end of the antisense strand form a blunt end, and the antisense strand is 1-4 nucleotides longer at its 3'-end than the sense strand, wherein a duplex region of at least 25 nucleotides in length, and the antisense strand is sufficiently complementary to a target mRNA along at least 19 nt of the length of the antisense strand to reduce target gene expression when the dsRNA molecule is introduced into a mammalian cell, and wherein dicer cleavage of the dsRNA preferentially generates an siRNA comprising the 3'-end of the antisense strand, thereby reducing expression of the target gene in the mammal. , wherein the antisense strand contains at least one thermally destabilizing nucleotide, wherein the at least one thermally destabilizing nucleotide is in the seed region of the antisense strand (i.e., positions 2 to 9 at the 5' end of the antisense strand), and wherein the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six, or all seven) of the following features: (i) the antisense comprises 2, 3, 4, 5, or 6 2'-fluoro modifications; (ii) the antisense comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated to a ligand; (iv) the sense strand comprises 2, 3, 4, or 5 2'-fluoro modifications; (v) the sense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; and (vi) the dsRNA comprises at least four 2'-fluoro modifications; and (vii) the dsRNA has a duplex region of 12-29 nucleotide pairs in length.

[0448] In some embodiments, each nucleotide in the sense and antisense strands of the dsRNA molecule can be modified. Each nucleotide can be modified with the same or different modifications, which can include one or more changes in one or both of the non-linked phosphate oxygens or one or more of the linked phosphate oxygens; changes in the ribose composition, for example, changes in the 2' hydroxyl group on the ribose; wholesale replacement of phosphate moieties with "dephospho" linkers; modification or substitution of naturally occurring bases; and substitution or modification of the ribose-phosphate backbone.

[0449] Because nucleic acid is the polymer of subunit, many modifications are present in the position of repetition in nucleic acid, such as the modification of base, or phosphate moiety, or the non-connected O of phosphate moiety.In some cases, modification is present in all described positions in nucleic acid, but in many cases it is not.For example, modification can be only present in 3' or 5' terminal position, can be only present in terminal region, for example, position on terminal nucleotide or in the last 2, 3, 4, 5 or 10 nucleotides of chain.Modification can be present in double-stranded region, single-stranded region or both.Modification can be only present in the double-stranded region of RNA, or can be only present in the single-stranded region of RNA.For example, the phosphorothioate modification at the non-connected O position can be only present in one or two ends, can be only present in the terminal region, for example, position on terminal nucleotide or in the last 2, 3, 4, 5 or 10 nucleotides of chain, or can be present in double-stranded and single-stranded region, particularly at the end.One or more 5'-ends can be phosphorylated.

[0450] For example, for enhancing stability, specific bases can be included in the overhang, or in the single-stranded overhang, for example, in 5' or 3' overhang or in both, modified nucleotides or nucleotide substitutes. For example, comprising purine nucleotides in the overhang may be desirable. In some embodiments, all or some bases in 3' or 5' overhang can be modified, for example, by modification as described herein. Modification can include, for example, using modification known in the art at the 2' position of ribose, for example, with deoxyribonucleotides, 2'-deoxy-2'-fluoro (2'-F) or 2'-O-methyl modifications replacing the ribose of core bases, and the modification of phosphate groups, for example, phosphorothioate modifications. The overhang does not need to be homologous to the target sequence.

[0451] In some embodiments, each residue in the sense and antisense strands is independently modified with LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, or 2'-fluoro. A strand may contain more than one modification. In some embodiments, each residue in the sense and antisense strands is independently modified with 2'-O-methyl or 2'-fluoro. It is understood that these modifications are in addition to at least one thermal destabilizing modification of the duplex present in the antisense strand.

[0452] There are usually at least two different modifications on the sense strand and the antisense strand. These two modifications can be 2'-deoxy, 2'-O-methyl or 2'-fluoro modifications, acyclic nucleotides or other modifications. In some embodiments, the sense strand and the antisense strand each comprise two differently modified nucleotides selected from 2'-O-methyl or 2'-deoxy. In some embodiments, each residue of the sense strand and the antisense strand is independently modified with 2'-O-methyl nucleotides, 2'-deoxy nucleotides, 2'-deoxy-2'-fluoro nucleotides, 2'-O-N-methylacetamido (2'-O-NMA) nucleotides, 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) nucleotides, 2'-O-aminopropyl (2'-O-AP) nucleotides or 2'-ara-F nucleotides. Similarly, it should be understood that these modifications are in addition to at least one thermal destabilization modification of the duplex present in the antisense strand.

[0453] In some embodiments, the dsRNA molecules of the present disclosure comprise modifications of alternating patterns, particularly in the B1, B2, B3, B1 ', B2 ', B3 ', B4 ' regions. As used herein, the term "alternating motif" or "alternating pattern" refers to a motif having one or more modifications, each modification being present in alternating nucleotides of a chain. Alternating nucleotides may refer to one every other nucleotide or one every three nucleotides, or a similar pattern. For example, if A, B, and C each represent a type of modification to a nucleotide, the alternating motif may be "ABABABABABAB...", "AABBAABBAABB...", "AABAABAABAAB...", "AAABAAABAAAB...", "AAABBBAAABBB...", or "ABCABCABCABC...", etc.

[0454] The types of modifications contained in the alternating motif can be the same or different. For example, if A, B, C, and D each represent a type of modification on a nucleotide, the alternating pattern (i.e., the modification on every other nucleotide) can be the same, but the sense strand or the antisense strand can each be selected from several modification possibilities within the alternating motif, such as "ABABAB...", "ACACAC...", "BDBDBD...", or "CDCDCD...", etc.

[0455] In some embodiments, the dsRNA molecules of the present disclosure include a modification pattern relative to the alternating motif on the antisense strand, and the modification pattern of the alternating motif on the sense strand is offset. The offset can cause the modification groups of the nucleotides of the sense strand to correspond to the differently modified groups of the nucleotides of the antisense strand, and vice versa. For example, when the sense strand is paired with the antisense strand in the dsRNA duplex, the alternating motif in the sense strand can start with "ABABAB" from the 5'-3' of the strand, and the alternating motif in the antisense strand can start with "BABABA" from the 3'-5' of the strand within the duplex region. As another example, the alternating motif in the sense strand can start with "AABBAABB" from the 5'-3' of the strand, and the alternating motif in the antisense strand can start with "BBAABBAA" from the 3'-5' of the strand within the duplex region, so there is a complete or partial transfer of the modification pattern between the sense strand and the antisense strand.

[0456] In one specific example, the alternating motif in the sense strand is "ABABAB" from 5'-3' of the strand, where each A is an unmodified ribonucleotide and each B is a 2'-O-methyl modified nucleotide.

[0457] In one specific example, the alternating motif in the sense strand is "ABABAB" from 5'-3' of the strand, where each A is a 2'-deoxy-2'-fluoro modified nucleotide and each B is a 2'-O-methyl modified nucleotide.

[0458] In another specific example, the alternating motif in the antisense strand is "BABABA" from 3'-5' of the strand, where each A is a 2'-deoxy-2'-fluoro modified nucleotide and each B is a 2'-O-methyl modified nucleotide.

[0459] In one specific example, the alternating motif in the sense strand is "ABABAB" from 5'-3' of the strand and the alternating motif in the antisense strand is "BABABA" from 3'-5' of the strand, where each A is an unmodified ribonucleotide and each B is a 2'-O-methyl modified nucleotide.

[0460] In one specific example, the alternating motif in the sense strand is "ABABAB" from 5'-3' of the strand and the alternating motif of the antisense strand is "BABABA" from 3'-5' of the strand, wherein each A is a 2'-deoxy-2'-fluoro modified nucleotide and each B is a 2'-O-methyl modified nucleotide.

[0461] The dsRNA molecules of the present disclosure may further comprise at least one phosphorothioate or methylphosphonate internucleotide bond. The phosphorothioate or methylphosphonate internucleotide bond modification may be present on any nucleotide of the sense strand or the antisense strand or both strands at any position in the strand. For example, an internucleotide bond modification may be present on each nucleotide on the sense strand or the antisense strand; each internucleotide bond modification may be present in an alternating pattern on the sense strand or the antisense strand; or the sense strand or the antisense strand may comprise two or more internucleotide bond modifications in an alternating pattern. The alternating pattern of internucleotide bond modifications on the sense strand may be the same or different from that of the antisense strand, and the alternating pattern of internucleotide bond modifications on the sense strand may have an offset relative to the alternating pattern of internucleotide bond modifications on the antisense strand.

[0462] In some embodiments, the dsRNA molecule comprises a key between the phosphorothioate or methylphosphonate nucleotides in the overhang region.For example, the overhang region includes two nucleotides, with a key between the phosphorothioate or methylphosphonate nucleotides. It is also possible to carry out a key between the nucleotides to connect the overhang nucleotides to the terminal pairing nucleotides in the duplex region. For example, at least 2, 3, 4 or all overhang nucleotides can be connected by a key between the phosphorothioate or methylphosphonate nucleotides, and optionally, there may be a key between the other phosphorothioate or methylphosphonate nucleotides that connect the overhang nucleotides to the pairing nucleotides next to the overhang nucleotides. For example, there may be a key between at least two phosphorothioate nucleotides between the terminal three nucleotides, two of which are overhang nucleotides, and the third is the pairing nucleotides adjacent to the overhang nucleotides. Optionally, these terminal three nucleotides can be located at the 3'-end of the antisense strand.

[0463] In some embodiments, the sense strand of the dsRNA molecule comprises 1-10 blocks of two to ten phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is located at any position in the oligonucleotide sequence, and the sense strand is paired with an antisense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising a phosphorothioate or methylphosphonate or phosphate linkage.

[0464] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of two phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising phosphorothioate or methylphosphonate or phosphate linkages.

[0465] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of three phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising phosphorothioate or methylphosphonate or phosphate linkages.

[0466] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of four phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising phosphorothioate or methylphosphonate or phosphate linkages.

[0467] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of five phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising phosphorothioate or methylphosphonate or phosphate linkages.

[0468] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of six phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising phosphorothioate or methylphosphonate or phosphate linkages.

[0469] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of seven phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, or 8 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising phosphorothioate or methylphosphonate or phosphate linkages.

[0470] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of eight phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, or 6 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising phosphorothioate or methylphosphonate or phosphate linkages.

[0471] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of nine phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, or 4 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising phosphorothioate or methylphosphonate or phosphate linkages.

[0472] In some embodiments, the dsRNA molecules of the present disclosure further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within terminal positions 1 to 10 of the sense or antisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides can be linked by phosphorothioate or methylphosphonate internucleotide linkages at one or both ends of the sense or antisense strand.

[0473] In some embodiments, the dsRNA molecules of the present disclosure further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within 1 to 10 of the duplex internal region of each of the sense or antisense strands. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides may be linked by phosphorothioate methylphosphonate internucleotide linkages at positions 8 to 16 of the duplex region, counted from the 5'-end of the sense strand; the dsRNA molecules may optionally further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within terminal positions 1 to 10.

[0474] In some embodiments, the dsRNA molecules of the present disclosure further comprise one to five phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 1 to 5 and one to five phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 18 to 23 of the sense strand (counting from the 5'-end), and one to five phosphorothioate or methylphosphonate internucleotide linkage modifications at positions 1 and 2 and one to five within positions 18 to 23 of the antisense strand (counting from the 5'-end).

[0475] In some embodiments, the dsRNA molecules of the present disclosure further comprise one phosphorothioate internucleotide linkage modification within positions 1 to 5 and one phosphorothioate or methylphosphonate internucleotide linkage modification within positions 18 to 23 of the sense strand (counting from the 5' end), and one phosphorothioate internucleotide linkage modification at position 1 or 2 and two phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end).

[0476] In some embodiments, the dsRNA molecules of the present disclosure comprise a phosphorothioate internucleotide linkage modification between the 1st and 2nd nucleotides and between the 2nd and 3rd nucleotides at the 5' end of the antisense strand. In some embodiments, the dsRNA molecules of the present disclosure comprise a phosphorothioate internucleotide linkage modification between the 1st and 2nd nucleotides and between the 2nd and 3rd nucleotides at the 3' end of the antisense strand. In some embodiments, the dsRNA molecules of the present disclosure comprise a phosphorothioate internucleotide linkage modification between the 1st and 2nd nucleotides and between the 2nd and 3rd nucleotides at the 5' end and the 3' end of the antisense strand.

[0477] In some embodiments, the dsRNA molecules of the present disclosure comprise phosphorothioate internucleotide linkage modifications between the 1st and 2nd nucleotides and between the 2nd and 3rd nucleotides at the 5' end of the antisense strand.

[0478] In some embodiments, the dsRNA molecules of the present disclosure comprise phosphorothioate internucleotide linkage modifications between the 1st and 2nd nucleotides and between the 2nd and 3rd nucleotides of the 5' end of the sense strand, and between the 1st and 2nd nucleotides and between the 2nd and 3rd nucleotides at the 5' and 3' ends of the antisense strand.

[0479] In some embodiments, the dsRNA molecules of the present disclosure comprise a phosphorothioate internucleotide linkage modification between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, and between the 3rd and 4th nucleotides at the 5' end of the antisense strand, a phosphorothioate internucleotide linkage modification between the 1st and 2nd nucleotides at the 3' end of the antisense strand, and a phosphorothioate internucleotide linkage modification between the 1st and 2nd nucleotides at the 5' end and the 3' end of the sense strand.

[0480] In some embodiments, the dsRNA molecules of the present disclosure further comprise two phosphorothioate internucleotide linkage modifications within positions 1 to 5 and one phosphorothioate internucleotide linkage modification within positions 18 to 23 of the sense strand (counting from the 5' end), and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end).

[0481] In some embodiments, the dsRNA molecules of the present disclosure further comprise two phosphorothioate internucleotide linkage modifications within positions 1 to 5 and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the sense strand (counting from the 5' end), and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end).

[0482] In some embodiments, the dsRNA molecules of the present disclosure further comprise two phosphorothioate internucleotide linkage modifications within positions 1 to 5 and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the sense strand (counting from the 5' end), and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18 to 23 of the antisense strand (counting from the 5' end).

[0483] In some embodiments, the dsRNA molecules of the present disclosure further comprise one phosphorothioate internucleotide linkage modification within positions 1 to 5 and one phosphorothioate internucleotide linkage modification within positions 18 to 23 of the sense strand (counting from the 5' end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end).

[0484] In some embodiments, the dsRNA molecules of the present disclosure further comprise one phosphorothioate internucleotide linkage modification within positions 1 to 5 and one phosphorothioate internucleotide linkage modification within positions 18 to 23 of the sense strand (counting from the 5' end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18 to 23 of the antisense strand (counting from the 5' end).

[0485] In some embodiments, the dsRNA molecules of the present disclosure further comprise one phosphorothioate internucleotide linkage modification within positions 1 to 5 of the sense strand (counting from the 5' end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18 to 23 of the antisense strand (counting from the 5' end).

[0486] In some embodiments, the dsRNA molecules of the present disclosure further comprise two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end), and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end).

[0487] In some embodiments, the dsRNA molecules of the present disclosure further comprise two phosphorothioate internucleotide linkage modifications within positions 1 to 5 and one phosphorothioate internucleotide linkage modification within positions 18 to 23 of the sense strand (counting from the 5' end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18 to 23 of the antisense strand (counting from the 5' end).

[0488] In some embodiments, the dsRNA molecules of the present disclosure further comprise two phosphorothioate internucleotide linkage modifications within positions 1 to 5 and one phosphorothioate internucleotide linkage modification within positions 18 to 23 of the sense strand (counting from the 5' end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end).

[0489] In some embodiments, the dsRNA molecules of the present disclosure further comprise two phosphorothioate internucleotide linkage modifications within positions 1 to 5 and one phosphorothioate internucleotide linkage modification within positions 18 to 23 of the sense strand (counting from the 5' end), and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end).

[0490] In some embodiments, the dsRNA molecules of the present disclosure further comprise two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 20 and 21 of the sense strand (counting from the 5' end), and one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 of the antisense strand (counting from the 5' end).

[0491] In some embodiments, the dsRNA molecules of the present disclosure further comprise one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 of the sense strand (counting from the 5' end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 20 and 21 of the antisense strand (counting from the 5' end).

[0492] In some embodiments, the dsRNA molecules of the present disclosure further comprise two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 21 and 22 of the sense strand (counting from the 5' end), and one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 of the antisense strand (counting from the 5' end).

[0493] In some embodiments, the dsRNA molecules of the present disclosure further comprise one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 of the sense strand (counting from the 5' end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 21 and 22 of the antisense strand (counting from the 5' end).

[0494] In some embodiments, the dsRNA molecules of the present disclosure further comprise two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 22 and 23 of the sense strand (counting from the 5' end), and one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 of the antisense strand (counting from the 5' end).

[0495] In some embodiments, the dsRNA molecules of the present disclosure further comprise one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 of the sense strand (counting from the 5' end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 23 and 23 of the antisense strand (counting from the 5' end).

[0496] In some embodiments, the compounds of the present disclosure include a pattern of main chain chiral centers. In some embodiments, the common pattern of main chain chiral centers includes at least 5 internucleotide bonds of Sp configuration. In some embodiments, the common pattern of main chain chiral centers includes at least 6 internucleotide bonds of Sp configuration. In some embodiments, the common pattern of main chain chiral centers includes at least 7 internucleotide bonds of Sp configuration. In some embodiments, the common pattern of main chain chiral centers includes at least 8 internucleotide bonds of Sp configuration. In some embodiments, the common pattern of main chain chiral centers includes at least 9 internucleotide bonds of Sp configuration. In some embodiments, the common pattern of main chain chiral centers includes at least 10 internucleotide bonds of Sp configuration. In some embodiments, the common pattern of main chain chiral centers includes at least 11 internucleotide bonds of Sp configuration. In some embodiments, the common pattern of main chain chiral centers includes at least 12 internucleotide bonds of Sp configuration. In some embodiments, the common pattern of main chain chiral centers includes at least 13 internucleotide bonds of Sp configuration. In some embodiments, the common pattern of main-chain chiral centers includes at least 14 internucleotide bonds of the Sp configuration. In some embodiments, the common pattern of main-chain chiral centers includes at least 15 internucleotide bonds of the Sp configuration. In some embodiments, the common pattern of main-chain chiral centers includes at least 16 internucleotide bonds of the Sp configuration. In some embodiments, the common pattern of main-chain chiral centers includes at least 17 internucleotide bonds of the Sp configuration. In some embodiments, the common pattern of main-chain chiral centers includes at least 18 internucleotide bonds of the Sp configuration. In some embodiments, the common pattern of main-chain chiral centers includes at least 19 internucleotide bonds of the Sp configuration. In some embodiments, the common pattern of main-chain chiral centers includes no more than 8 internucleotide bonds of the Rp configuration. In some embodiments, the common pattern of main-chain chiral centers includes no more than 7 internucleotide bonds of the Rp configuration. In some embodiments, the common pattern of main-chain chiral centers includes no more than 6 internucleotide bonds of the Rp configuration. In some embodiments, the common pattern of main-chain chiral centers includes no more than 5 internucleotide bonds of the Rp configuration. In some embodiments, the common pattern of the main chain chiral center includes no more than 4 internucleotide bonds of the Rp configuration. In some embodiments, the common pattern of the main chain chiral center includes no more than 3 internucleotide bonds of the Rp configuration. In some embodiments, the common pattern of the main chain chiral center includes no more than 2 internucleotide bonds of the Rp configuration. In some embodiments, the common pattern of the main chain chiral center includes no more than 1 internucleotide bond of the Rp configuration. In some embodiments, the common pattern of the main chain chiral center includes no more than 8 achiral internucleotide bonds (as a non-limiting example, phosphodiester). In some embodiments, the common pattern of the main chain chiral center includes no more than 7 achiral internucleotide bonds.In some embodiments, the common pattern of backbone chiral centers includes no more than 6 achiral internucleotide bonds. In some embodiments, the common pattern of backbone chiral centers includes no more than 5 achiral internucleotide bonds. In some embodiments, the common pattern of backbone chiral centers includes no more than 4 achiral internucleotide bonds. In some embodiments, the common pattern of backbone chiral centers includes no more than 3 achiral internucleotide bonds. In some embodiments, the common pattern of backbone chiral centers includes no more than 2 achiral internucleotide bonds. In some embodiments, the common pattern of backbone chiral centers includes no more than 1 achiral internucleotide bond. In some embodiments, the common pattern of backbone chiral centers includes at least 10 internucleotide bonds in the Sp configuration and no more than 8 achiral internucleotide bonds. In some embodiments, the common pattern of backbone chiral centers includes at least 11 internucleotide bonds in the Sp configuration and no more than 7 achiral internucleotide bonds. In some embodiments, the common pattern of backbone chiral centers includes at least 12 internucleotide bonds in the Sp configuration and no more than 6 achiral internucleotide bonds. In some embodiments, the common pattern of backbone chiral centers includes at least 13 internucleotide bonds of Sp configuration and no more than 6 achiral internucleotide bonds. In some embodiments, the common pattern of backbone chiral centers includes at least 14 internucleotide bonds of Sp configuration and no more than 5 achiral internucleotide bonds. In some embodiments, the common pattern of backbone chiral centers includes at least 15 internucleotide bonds of Sp configuration and no more than 4 achiral internucleotide bonds. In some embodiments, the internucleotide bonds of Sp configuration are optionally continuous or discontinuous. In some embodiments, the internucleotide bonds of Rp configuration are optionally continuous or discontinuous. In some embodiments, the internucleotide bonds of achiral are optionally continuous or discontinuous.

[0497] In some embodiments, the compounds of the present disclosure include blocks that serve as stereochemical blocks. In some embodiments, the block is an Rp block, wherein each internucleotide bond of the block is Rp. In some embodiments, the 5'-block is an Rp block. In some embodiments, the 3'-block is an Rp block. In some embodiments, the block is an Sp block, wherein each internucleotide bond of the block is Sp. In some embodiments, the 5'-block is an Sp block. In some embodiments, the 3'-block is an Sp block. In some embodiments, the oligonucleotides provided include both Rp and Sp blocks. In some embodiments, the oligonucleotides provided include one or more Rp blocks but do not include an Sp block. In some embodiments, the oligonucleotides provided include one or more Sp blocks but do not include an Rp block. In some embodiments, the oligonucleotides provided include one or more PO blocks, wherein each internucleotide bond is a natural phosphate bond.

[0498] In some embodiments, the compounds of the present disclosure include a 5'-block as an Sp block, wherein each sugar moiety includes a 2'-F modification. In some embodiments, the 5'-block is an Sp block, wherein each internucleotide bond is a modified internucleotide bond, and each sugar moiety includes a 2'-F modification. In some embodiments, the 5'-block is an Sp block, wherein each internucleotide bond is a phosphorothioate bond, and each sugar moiety includes a 2'-F modification. In some embodiments, the 5'-block includes 4 or more nucleoside units. In some embodiments, the 5'-block includes 5 or more nucleoside units. In some embodiments, the 5'-block includes 6 or more nucleoside units. In some embodiments, the 5'-block includes 7 or more nucleoside units. In some embodiments, the 3'-block is an Sp block, wherein each sugar moiety includes a 2'-F modification. In some embodiments, the 3'-block is an Sp block, wherein each internucleotide bond is a modified internucleotide bond, and each sugar moiety includes a 2'-F modification. In some embodiments, the 3'-block is an Sp block, wherein each internucleotide bond is a phosphorothioate bond and each sugar moiety includes a 2'-F modification. In some embodiments, the 3'-block includes 4 or more nucleoside units. In some embodiments, the 3'-block includes 5 or more nucleoside units. In some embodiments, the 3'-block includes 6 or more nucleoside units. In some embodiments, the 3'-block includes 7 or more nucleoside units.

[0499] In some embodiments, the compounds of the present disclosure include one type of nucleoside or oligonucleotide in one region followed by a specific type of internucleotide bond, such as a natural phosphate bond, a modified internucleotide bond, an Rp chiral internucleotide bond, an Sp chiral internucleotide bond, and the like. In some embodiments, A is followed by Sp. In some embodiments, A is followed by Rp. In some embodiments, A is followed by a natural phosphate bond (PO). In some embodiments, U is followed by Sp. In some embodiments, U is followed by Rp. In some embodiments, U is followed by a natural phosphate bond (PO). In some embodiments, C is followed by Sp. In some embodiments, C is followed by Rp. In some embodiments, C is followed by a natural phosphate bond (PO). In some embodiments, G is followed by Sp. In some embodiments, G is followed by Rp. In some embodiments, G is followed by a natural phosphate bond (PO). In some embodiments, C and U are followed by Sp. In some embodiments, C and U are followed by Rp. In some embodiments, C and U are followed by a natural phosphate bond (PO). In some embodiments, A and G are followed by Sp. In some embodiments, A and G are followed by Rp.

[0500] In some embodiments, the antisense strand comprises a phosphorothioate internucleotide linkage between nucleotide positions 21 and 22 and between nucleotide positions 22 and 23, wherein the antisense strand contains at least one thermally destabilizing modification of the duplex located in the seed region of the antisense strand (i.e., positions 2 to 9 at the 5' end of the antisense strand), and wherein the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six, seven, or all eight) of the following features: (i) the antisense strand comprises two, three, four, five, or six 2'-fluoro modification; (ii) the antisense comprises 3, 4, or 5 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated to a ligand; (iv) the sense strand comprises 2, 3, 4, or 5 2'-fluoro modifications; (v) the sense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (vi) the dsRNA comprises at least four 2'-fluoro modifications; (vii) the dsRNA comprises a duplex region of 12-40 nucleotide pairs in length; and (viii) the dsRNA has a blunt end at the 5' end of the antisense strand.

[0501] In some embodiments, the antisense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23, wherein the antisense strand contains at least one thermally destabilizing modification of the duplex located in the seed region of the antisense strand (i.e., positions 2 to 9 at the 5' end of the antisense strand), and wherein the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six, seven, or all eight) of the following features: (i) the antisense strand comprises 2, 3, 4, 5, or six 2'-fluoro modifications; (ii) the sense strand is conjugated to a ligand and / or a lipophilic moiety; (iii) the sense strand comprises two, three, four, or five 2'-fluoro modifications; (iv) the sense strand comprises one, two, three, four, or five phosphorothioate internucleotide linkages; (v) the dsRNA comprises at least four 2'-fluoro modifications; (vi) the dsRNA comprises a duplex region that is 12-40 nucleotide pairs in length; (vii) the dsRNA comprises a duplex region that is 12-40 nucleotide pairs in length; and (viii) the dsRNA has a blunt end at the 5' end of the antisense strand.

[0502] In some embodiments, the sense strand comprises a phosphorothioate internucleotide linkage between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3, wherein the antisense strand contains at least one thermally destabilizing modification of the duplex located in the seed region of the antisense strand (i.e., positions 2 to 9 at the 5' end of the antisense strand), and wherein the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six, seven, or all eight) of the following features: (i) the antisense comprises two, three, four, five, or six 2'-fluoro modifications; (ii) ) the antisense comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated to a ligand and / or a lipophilic moiety; (iv) the sense strand comprises 2, 3, 4, or 5 2'-fluoro modifications; (v) the sense strand comprises 3, 4, or 5 phosphorothioate internucleotide linkages; (vi) the dsRNA comprises at least four 2'-fluoro modifications; (vii) the dsRNA comprises a duplex region of 12-40 nucleotide pairs in length; and (viii) the dsRNA has a blunt end at the 5' end of the antisense strand.

[0503] In some embodiments, the sense strand comprises a phosphorothioate internucleotide linkage between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3, the antisense strand comprises a phosphorothioate internucleotide linkage between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23, wherein the antisense strand contains at least one thermally destabilizing modification of the duplex located in the seed region of the antisense strand (i.e., positions 2 to 9 at the 5' end of the antisense strand), and wherein the dsRNA optionally further has at least one of the following features (e.g., one, two, three

[0014] In some embodiments, the dsRNA comprises at least four 2'-fluoro modifications; (vi) the dsRNA comprises a duplex region of 12-40 nucleotide pairs in length; and (vii) the dsRNA has a blunt end at the 5' end of the antisense strand.

[0504] In some embodiments, the dsRNA molecules of the present disclosure comprise mismatches with the target, within the duplex, or a combination thereof. Mismatches can be present in the overhang region or the duplex region. Base pairs can be ranked based on their tendency to promote dissociation or melt (e.g., based on the free energy of association or dissociation of a particular pairing, the simplest approach is to examine pairings individually, although neighbor or similar analyses can also be used). In terms of promoting dissociation: A:U is better than G:C; G:U is better than G:C; and I:C is better than G:C (I = inosine). Mismatches, such as non-canonical or extra-canonical pairings (as described elsewhere herein) are better than canonical (A:T, A:U, G:C) pairings; and pairings comprising universal bases are better than canonical pairings.

[0505] In some embodiments, the dsRNA molecules of the present disclosure include at least one of the first 1, 2, 3, 4, or 5 base pairs within the duplex region from the 5' end of the antisense strand, which can be independently selected from: A:U, G:U, I:C, and mispairing, such as non-canonical or extra-canonical pairings or pairings comprising universal bases, to promote dissociation of the antisense strand at the 5' end of the duplex.

[0506] In some embodiments, the nucleotide at position 1 from the 5' end of the duplex region of the antisense strand is selected from A, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2, or 3 base pairs in the duplex region of the 5' end of the antisense strand is an AU base pair. For example, the first base pair in the duplex region of the 5' end of the antisense strand is an AU base pair.

[0507] It was discovered that the introduction of a 4'-modified or 5'-modified nucleotide at the 3'-end of a phosphodiester (PO), phosphorothioate (PS) or phosphorodithioate (PS2) linkage of a dinucleotide at any position in a single-stranded or double-stranded oligonucleotide can exert a steric effect on the internucleotide linkage and thereby protect or stabilize it from the action of nucleases.

[0508] In some embodiments, a 5'-modified nucleoside is introduced at the 3' end of a dinucleotide at any position in a single-stranded or double-stranded siRNA. For example, a 5'-alkylated nucleoside can be introduced at the 3' end of a dinucleotide at any position in a single-stranded or double-stranded siRNA. The alkyl group at the 5' position of the ribose can be racemic or a chirally pure R or S isomer. An exemplary 5'-alkylated nucleoside is a 5'-methyl nucleoside. The 5'-methyl group can be racemic or a chirally pure R or S isomer.

[0509] In some embodiments, a 4'-modified nucleoside is introduced at the 3' end of a dinucleotide at any position in a single-stranded or double-stranded siRNA. For example, a 4'-alkylated nucleoside can be introduced at the 3' end of a dinucleotide at any position in a single-stranded or double-stranded siRNA. The alkyl group at the 4' position of the ribose can be racemic or a chirally pure R or S isomer. An exemplary 4'-alkylated nucleoside is a 4'-methyl nucleoside. The 4'-methyl group can be racemic or a chirally pure R or S isomer. Alternatively, a 4'-O-alkylated nucleoside can be introduced at the 3' end of a dinucleotide at any position in a single-stranded or double-stranded siRNA. The 4'-O-alkyl group of the ribose can be racemic or a chirally pure R or S isomer. An exemplary 4'-O-alkylated nucleoside is a 4'-O-methyl nucleoside. The 4'-O-methyl group can be racemic or a chirally pure R or S isomer.

[0510] In some embodiments, 5'-alkylated nucleosides are introduced at any position on the sense or antisense strand of a dsRNA, and such modifications maintain or improve the effectiveness of the dsRNA. The 5'-alkyl group can be a racemic or chirally pure R or S isomer. Exemplary 5'-alkylated nucleosides are 5'-methyl nucleosides. The 5'-methyl group can be a racemic or chirally pure R or S isomer.

[0511] In some embodiments, 4'-alkylated nucleosides are introduced at any position on the sense or antisense strand of a dsRNA, and such modifications maintain or improve the effectiveness of the dsRNA. The 4'-alkyl group can be a racemic or chirally pure R or S isomer. Exemplary 4'-alkylated nucleosides are 4'-methyl nucleosides. The 4'-methyl group can be a racemic or chirally pure R or S isomer.

[0512] In some embodiments, 4'-O-alkylated nucleosides are introduced at any position on the sense or antisense strand of a dsRNA, and such modifications maintain or improve the effectiveness of the dsRNA. The 5'-alkyl group can be a racemic or chirally pure R or S isomer. Exemplary 4'-O-alkylated nucleosides are 4'-O-methyl nucleosides. The 4'-O-methyl group can be a racemic or chirally pure R or S isomer.

[0513] In some embodiments, the dsRNA molecules of the present disclosure may include a 2'-5' bond (having 2'-H, 2'-OH, and 2'-OMe, and having P=O or P=S). For example, the 2'-5' bond modification may be used to promote nuclease resistance or inhibit the binding of the sense strand to the antisense strand, or may be used at the 5' end of the sense strand to prevent activation of the sense strand by RISC.

[0514] In another embodiment, the dsRNA molecules of the present disclosure can include L sugars (e.g., L-ribose, L-arabinose with 2'-H, 2'-OH, and 2'-OMe). For example, these L sugar modifications can be used to promote nuclease resistance or inhibit the binding of the sense strand to the antisense strand, or can be used at the 5' end of the sense strand to prevent activation of the sense strand by RISC.

[0515] Various publications describe multimeric siRNAs, all of which can be used in the dsRNA of the present disclosure. Such publications include WO2007 / 091269, US 7858769, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887, and WO2011 / 031520, which are hereby incorporated by reference in their entirety.

[0516] Part and / or lipophilic part can be attached to polynucleotide via carrier.Exemplary carrier includes (i) at least one " main chain attachment point ", preferably two " main chain attachment points " and (ii) at least one " tether attachment point ".As used herein, " main chain attachment point " refers to functional group, such as hydroxyl, or generally can be used for and be suitable for carrier to be incorporated into the key in main chain, such as phosphate ester of ribonucleic acid or modified phosphate ester (such as, sulfur-containing) main chain.In some embodiments, " tether attachment point " (TAP) refers to the composition ring atom of the cyclic carrier connecting selected part, such as carbon atom or heteroatom (different from the atom providing main chain attachment point).This part can be such as lipophilic alkyl group, optionally C16 lipophilic part.Optionally, the part selected is connected with cyclic carrier by inserting tether.Therefore, cyclic carrier can include functional group, such as amino, or generally provide and be suitable for being incorporated into or tethering another chemical entity (such as, to part and / or lipophilic part forming ring).

[0517] The RNAi agent can be conjugated to the ligand and / or lipophilic portion via a carrier, wherein the carrier can be a cyclic group or an acyclic group. Optionally, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3] dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinone, tetrahydrofuranyl and decahydronaphthyl. Optionally, the acyclic group is selected from serinol backbone or diethanolamine backbone.

[0518] In certain embodiments, the RNAi agent used in the methods of the present disclosure is an agent selected from the agents listed in Table 2 or 3. These agents may further comprise a ligand, such as one or more lipophilic moieties.

[0519] III. iRNA conjugated to ligand

[0520] Another modification of the RNA of the disclosed iRNAs involves chemically linking one or more ligands, moieties, or conjugates to the iRNA that enhance the activity, cellular distribution, or cellular uptake (e.g., into cells) of the iRNA. Such moieties include, but are not limited to, lipid moieties, such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86:6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), thioethers, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g., dodecanediol or an undecyl residue (Saison-Behmoaras et al., EMBO J, 1991, 10: 1111-1118; Kabanov et al., FEBS Lett., 1990, 259: 327-330; Svinarchuk et al., Biochimie, 1993, 75: 49-54), phospholipids, such as di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycerol-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36: 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18: 3777-3783), polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14: 969-973) or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0521] In certain embodiments, the ligand alters the distribution, targeting, or lifespan of the iRNA agent into which it is incorporated. In some embodiments, the ligand provides enhanced affinity for a selected target (e.g., a molecule, cell, or cell type), compartment (e.g., a cell or organ compartment, tissue, organ, or body region), for example, compared to a species in which such a ligand is not present. Typical ligands do not participate in duplex pairing in duplexed nucleic acids.

[0522] The ligand can include naturally occurring substances such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL) or globulin); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or lipids. The ligand can also be a recombinant or synthetic molecule, such as a synthetic polymer, for example, a synthetic polyamino acid. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly-(L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly-(2-ethyl acrylic acid), N-isopropylacrylamide polymer or polyphosphazene. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimer polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or alpha helical peptides.

[0523] Part can also include targeting group, such as cell or tissue targeting agent, such as lectin, glycoprotein, lipid or protein (for example, antibody), it is combined with specified cell type (such as nephrocyte).Targeting group can be thyrotropin, melanocyte stimulating hormone, lectin, glycoprotein, surfactant protein A, mucin sugar, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, multivalent fucose, glycosylated polyamino acids, multivalent galactose, transferrins, bisphosphonates, polyglutamic acid, polyaspartic acid, lipid, cholesterol, steroid, bile acid, folic acid, vitamin B12, biotin or RGD peptide or RGD peptide mimetic.In certain embodiments, part is multivalent galactose, for example, N-acetyl-galactosamine.

[0524] Other examples of ligands include dyes, intercalating agents (e.g., acridine), cross-linking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules (e.g., cholesterol, bile acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl, hexadecylglycerol, borneol, menthol, 1,3-propylene glycol, heptadecyl, palmitic acid, myristic acid, O3-(oleoyl) Lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl or phenoxazine, and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphates, amino groups, sulfhydryl groups, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP or AP.

[0525] The ligand can be a protein (e.g., a glycoprotein) or a peptide (e.g., a molecule with a specific affinity for a co-ligand) or an antibody (e.g., an antibody that binds to a specific cell type such as a cancer cell, an endothelial cell, or a bone cell). The ligand can also include hormones and hormone receptors. The ligand can also include non-peptide substances such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, or multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB.

[0526] The ligand can be a substance, such as a drug, that can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, for example, by disrupting the cell's microtubules, microfilaments, or intermediate filaments. The drug can be, for example, paclitaxel, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[0527] In some embodiments, the part connected to the iRNA as described herein is used as a pharmacokinetic regulator (PK regulator). PK regulators include lipophiles, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, etc. Exemplary PK regulators include but are not limited to cholesterol, fatty acids, cholic acid, lithocholic acid, dialkyl glycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin. It is also known that the oligonucleotides comprising multiple thiophosphate bonds are combined with serum proteins, so the short oligonucleotides comprising multiple thiophosphate bonds in the main chain (for example, oligonucleotides with about 5 bases, 10 bases, 15 bases or 20 bases) are also suitable for the present disclosure as a part (for example, as a PK regulating part). In addition, the aptamer in conjunction with serum components (for example, serum proteins) is also suitable for use as the PK regulating part in the embodiments described herein.

[0528] The ligand-conjugated iRNAs of the present disclosure can be synthesized using oligonucleotides carrying pendant reactive functional groups, such as oligonucleotides derived from linker molecules attached to the oligonucleotides (as described below). Such reactive oligonucleotides can be reacted directly with commercially available ligands, synthetic ligands with any of a variety of protecting groups, or ligands having a linker moiety attached thereto.

[0529] The oligonucleotides used in the conjugates of the present disclosure can be conveniently and routinely prepared by well-known solid phase synthesis techniques. Equipment for such synthesis is sold by several suppliers, including, for example, Applied (Foster City, Calif.). Any other means known in the art for such synthesis may additionally or alternatively be employed. It is also known to use similar techniques to prepare other oligonucleotides (such as phosphorothioate and alkylated derivatives).

[0530] In the ligand-conjugated oligonucleotides and ligand-molecules with sequence-specifically linked nucleosides of the present disclosure, the oligonucleotides and oligonucleosides can be assembled on a suitable DNA synthesizer using standard nucleotide or nucleoside precursors or nucleotide or nucleoside conjugate precursors that already carry a linking moiety, ligand-nucleotide or nucleoside conjugate precursors that already carry a ligand molecule, or building blocks with non-nucleoside ligands.

[0531] When using nucleotide conjugate precursors that already carry a linking moiety, the synthesis of sequence-specific linked nucleosides is typically completed, and the ligand molecule is then reacted with the linking moiety to form a ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides of the present disclosure or linked nucleosides are synthesized by an automated synthesizer using phosphoramidites derived from ligand-nucleoside conjugates and standard and non-standard phosphoramidites that are commercially available and routinely used for oligonucleotide synthesis.

[0532] A. Lipid Conjugates

[0533] In certain embodiments, the ligand or conjugate is a lipid or lipid-based molecule. Such lipids or lipid-based molecules can generally bind to serum proteins, such as human serum albumin (HSA). HSA binding ligands allow the conjugate to be distributed to target tissues, such as non-renal target tissues of the body. For example, the target tissue can be tissue of the CNS, such as brain tissue. Other molecules that can bind to HSA can also be used as ligands. For example, naproxen or aspirin can be used. The lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport to target cells or cell membranes, or (c) can be used to modulate binding to serum proteins, such as HSA.

[0534] Lipid-based ligands can be used to modulate, e.g., control (e.g., inhibit) the binding of the conjugate to the target tissue. For example, lipids or lipid-based ligands that bind more strongly to HSA are less likely to be targeted to the kidneys and, therefore, less likely to be cleared from the body. Lipids or lipid-based ligands that bind less strongly to HSA can be used to target the conjugate to the kidneys.

[0535] In certain embodiments, the lipid-based ligand binds to HSA. For example, the ligand may bind to HSA with sufficient affinity to enhance the distribution of the conjugate to non-renal tissues. However, the affinity is generally not so strong that HSA ligand binding cannot be reversed.

[0536] In certain embodiments, the lipid-based ligand binds weakly or not at all to HSA, resulting in enhanced distribution of the conjugate in the kidney.Other moieties that target renal cells may also be used in place of or in addition to the lipid-based ligand.

[0537] On the other hand, the ligand is a moiety that is taken up by the target cell (e.g., a proliferating cell), such as a vitamin. These are particularly useful for treating diseases characterized by unwanted cell proliferation, such as malignant or non-malignant types, such as cancer cells. Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins included are B vitamins, such as folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients taken up by cancer cells. Also included are HSA and low-density lipoprotein (LDL).

[0538] B. Cell Permeabilization Agents

[0539] On the other hand, the ligand is a cell permeating agent, such as a helical cell permeating agent. In certain embodiments, the agent is amphiphilic. Exemplary agents are peptides, such as tat or tentacles. If the agent is a peptide, it can be modified, including peptidyl mimetics, invertomers, non-peptide or pseudopeptide connections and the use of D-amino acids. Helical agents are typically α-helical agents and can have lipophilic and lipophobic phases.

[0540] The ligand can be a peptide or peptide mimetic. Peptide mimics (also referred to as oligopeptide mimics in this article) are molecules that can be folded into a defined three-dimensional structure similar to a natural peptide. The connection of peptide and peptide mimics to an iRNA agent may affect the pharmacokinetic distribution of iRNA, such as by enhancing cell recognition and absorption. The length of the peptide or peptide mimetic portion can be about 5-50 amino acids long, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids long.

[0541] The peptide or peptide mimetic can be, for example, a cell penetrating peptide, a cationic peptide, an amphipathic peptide or a hydrophobic peptide (for example, mainly composed of Tyr, Trp or Phe). The peptide portion can be a dendrimer peptide, a constrained peptide or a cross-linked peptide. In another alternative, the peptide portion can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF with the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 9). An RFGF analog containing hydrophobic MTS (for example, the amino acid sequence AALLPVLLAAP (SEQ ID NO: 10)) can also be a targeting moiety. The peptide portion can be a "delivery" peptide that can carry large polar molecules across the cell membrane including peptides, oligonucleotides and proteins. For example, sequences from HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 11)) and Drosophila antenna foot protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 12)) have been found to be able to play the role of delivery peptides. The peptide or peptide mimetic can be encoded by a random sequence of DNA, such as peptides identified from a phage display library or a one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). Typically, the peptide or peptide mimetic tethered to the dsRNA agent by an incorporated monomeric unit is a cell targeting peptide, such as an arginine-glycine-aspartic acid (RGD)-peptide or RGD mimetic. The length of the peptide portion can range from about 5 amino acids to about 40 amino acids. The peptide portion can have structural modifications, such as to improve stability or to direct conformational properties. Any of the structural modifications described below can be used.

[0542] The RGD peptides used in the compositions and methods of the present disclosure can be linear or cyclic and can be modified, for example, by glycosylation or methylation, to facilitate targeting to specific tissues. RGD-containing peptides and peptide mimetics can include D-amino acids as well as synthetic RGD mimetics. In addition to RGD, other moieties that target integrin ligands can also be used. Preferred conjugates of this ligand target PECAM-1 or VEGF.

[0543] The RGD peptide portion can be used to target specific cell types, such as tumor cells, such as endothelial tumor cells or breast cancer tumor cells (Zitzmann et al., Cancer Res., 62:5139-43, 2002). RGD peptides can promote the targeting of dsRNA agents to tumors in a variety of other tissues, including the lungs, kidneys, spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001). Typically, RGD peptides promote the targeting of iRNA agents to the kidneys. RGD peptides can be linear or cyclic and can be modified, such as glycosylated or methylated, to promote targeting to specific tissues. For example, glycosylated RGD peptides can deliver iRNA agents to cells expressing α-glucosidase. V β3 tumor cells (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001).

[0544] "Cell penetrating peptides" are capable of penetrating cells, such as microbial cells (such as bacteria or fungal cells) or mammalian cells (such as human cells). Microbial cell penetrating peptides can be, for example, α-helical linear peptides (e.g., LL-37 or Ceropin P1), disulfide bond-containing peptides (e.g., α-defensins, β-defensins or bovine antimicrobial peptides (bactenecin)), or peptides containing only one or two major amino acids (e.g., PR-39 or indolicidin). Cell penetrating peptides can also include a nuclear localization signal (NLS). For example, a cell penetrating peptide can be a dichotomous amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31: 2717-2724, 2003).

[0545] C.Connector

[0546] In some embodiments, the conjugates or ligands described herein can be linked to iRNA oligonucleotides using a variety of linkers, which can be cleavable or non-cleavable.

[0547] The term "linker" or "linking group" means an organic moiety that connects two parts of a compound, for example, covalently connects two parts of a compound. Linkers typically include a direct bond or an atom such as oxygen or sulfur; a unit such as NR8, C(O), C(O)NH, SO, SO2, SO2NH or a chain of atoms such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, aralkyl, aralkenyl, aralkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylaralkyl, alkylaralkenyl, alkylaralkynyl, alkenylaralkyl, alkenylaralkenyl, alkenylaralkynyl, alkynylaralkyl, alkynylaralkenyl, alkynylaralkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, wherein R is an alkyl, alkylheteroaryl, alkylheteroaryl, alkylheterocyclyl, alkylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylheteroaryl, the one or more methylene radicals being interrupted or terminated by O, S, S(O), SO2, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclyl; wherein R8 is hydrogen, acyl, aliphatic or substituted aliphatic. In certain embodiments, the linker is between about 1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17, 8-17, 6-16, 7-16, or 8-16 atoms in length.

[0548] A cleavable linking group is a linking group that is sufficiently stable outside of the cell, but which is cleaved after entering the target cell to release the two parts of the linker that hold it together. In preferred embodiments, the cleavage of the cleavable linking group in the target cell or under first reference conditions (which can, for example, be selected to simulate or represent intracellular conditions) is at least about 10 times, 20 times, 30 times, 40 times, 50...

Claims

1. A double-stranded nucleic acid (dsRNA) agent for inhibiting the expression of SNCA, wherein the dsRNA agent comprises a sense strand having a 5'-end and a 3'-end and an antisense strand having a 5'-end and a 3'-end, wherein the sense strand and the antisense strand form a double-stranded region, wherein: The sense strand comprises a nucleotide sequence selected from SEQ ID NO: 150, SEQ ID NO: 157, SEQ ID NO: 164, and SEQ ID NO: 142-149, 151-156, 158-163, and 165-184 of Table 3, having 0 or 1 mismatch; The sense strand of the dsRNA agent comprises a lipophilic moiety attached to position 6 or 16 counted from the 5 '-end of the sense strand; The antisense strand comprises a nucleotide sequence selected from SEQ ID NO: 193, SEQ ID NO: 200, SEQ ID NO: 207, and SEQ ID NO: 185-192, 194-199, 201-206, and 208-227 of Table 3, having 0 or 1 mismatch; The dsRNA agent does not comprise a GalNAc modification; and The dsRNA agent comprises eight phosphorothioate internucleotide linkages located at the penultimate and last internucleotide linkages at the respective 3 '-end and 5 '-end of each of the sense and antisense strands of the dsRNA agent.

2. The dsRNA agent of claim 1, wherein the lipophilic moiety is an aliphatic, alicyclic, or polyalicyclic compound.

3. The dsRNA agent of claim 1 or claim 2, wherein the lipophilic moiety is a lipid, cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O-(hexadecyl)glycerol, geranyloxyhexanol, hexadecylglycerol, borneol, menthol, 1,3-propylene glycol, heptadecyl, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.

4. The dsRNA agent of any one of the preceding claims, wherein the lipophilic portion contains a saturated or unsaturated C4-C 30 A hydrocarbon chain and an optional functional group selected from hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide and alkyne.

5. The dsRNA agent of any one of the preceding claims, wherein the lipophilic portion contains a saturated or unsaturated C6-C 18 Hydrocarbon chain.

6. The dsRNA agent of any one of the preceding claims, wherein the lipophilic portion contains a saturated or unsaturated C 16 Hydrocarbon chain.

7. The dsRNA agent of any one of the preceding claims, wherein the lipophilic portion is wherein B is a nucleotide base or a nucleotide base analog, optionally wherein B is selected from adenine, guanine, cytosine, thymine and uracil.

8. The dsRNA agent of any one of the preceding claims, wherein the lipophilic moiety is attached via a linker or a carrier.

9. The dsRNA agent of any one of the preceding claims, wherein the lipophilic moiety is conjugated via a carrier that replaces the nucleotide at position 6 or 16 of the sense strand.

10. The dsRNA agent of claim 9, wherein the carrier is a cyclic group selected from the group consisting of pyrrolidinyl, pyrazolyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinone, tetrahydrofuranyl, and decahydronaphthyl; or a non-cyclic moiety based on a selenoic acid backbone or a diethanolamine backbone.

11. The dsRNA agent of any one of claims 1 to 8, wherein the lipophilic moiety is conjugated to the dsRNA agent via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide linkage, the product of a click reaction, or a carbamate.

12. The dsRNA agent of any one of claims 1 to 8 and 11, wherein the lipophilic moiety is conjugated to the dsRNA agent via a biocleavable linker selected from the group consisting of DNA, RNA, disulfide, amide, functionalized monosaccharides or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.

13. The dsRNA agent of any one of the preceding claims, wherein substantially all of the nucleotides of the sense strand are modified nucleotides.

14. The dsRNA agent of any one of the preceding claims, wherein substantially all of the nucleotides of the antisense strand are modified nucleotides.

15. The dsRNA agent of any one of the preceding claims, wherein all nucleotides of the sense strand are modified nucleotides.

16. The dsRNA agent of any one of the preceding claims, wherein all nucleotides of the antisense strand are modified nucleotides.

17. The dsRNA agent of any one of the preceding claims, wherein all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides.

18. The dsRNA agent of any of the preceding claims, having one or more modified nucleotides, and wherein at least one of the one or more modified nucleotides is selected from the group consisting of deoxynucleotides, 3'-terminal deoxythymidine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, unlocked nucleotides, conformationally restricted nucleotides, constrained ethyl nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-alkyl modified nucleotides, 2'-hydroxy modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates , nucleotides comprising non-natural bases, tetrahydropyran-modified nucleotides, 1,5-anhydrohexitol-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides comprising 5'-phosphorothioate groups, nucleotides comprising 5'-methylphosphonate groups, nucleotides comprising 5'-phosphates or 5'-phosphate mimetics, nucleotides comprising vinyl phosphates, nucleotides comprising adenosine-diol nucleic acids (GNA), nucleotides comprising thymidine-diol nucleoside (GNA) S-isomers, nucleotides comprising 2-hydroxymethyl-tetrahydrofuran-5-phosphate, nucleotides comprising 2'-deoxythymidine-3'-phosphate, nucleotides comprising 2'-deoxyguanosine-3'-phosphate, and terminal nucleotides connected to cholesterol derivatives and dodecanoic acid didecylamide groups.

19. The dsRNA agent of any of the preceding claims, wherein the dsRNA agent comprises at least one modified nucleotide selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-C16 (2'-O-hexadecyl) modified nucleotides, and nucleotides comprising vinyl phosphate, optionally wherein the dsRNA agent comprises at least one of the following modifications: 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-C16 (2'-O-hexadecyl) modified nucleotides, and nucleotides comprising vinyl phosphate.

20. The dsRNA agent of any of the preceding claims, further comprising a phosphate or phosphate mimetic at the 5 '-end of the antisense strand.

21. The dsRNA agent of claim 20, wherein the phosphate mimetic is 5'-vinylphosphonate (VP).

22. The dsRNA agent of any of the preceding claims, comprising a pattern of modified nucleotides as shown in Table 2 (wherein the positions of 2'-C16, 2'-O-methyl, 2'-deoxy, GNA, phosphorothioate, vinylphosphonate, and 2'-fluoro modifications are as shown in Table 2, regardless of the individual nucleotide base sequences of the dsRNA agent shown).

23. A dsRNA agent as described in any of the preceding claims, which has a sense strand nucleotide sequence and an antisense strand nucleotide sequence of a single duplex selected from the group consisting of: AD-1804698, AD-1804699, AD-1747575, AD-1747576, AD-1804700, AD-1747577, AD-1747578, AD-1747579, AD-1 747580, AD-1747581, AD-1804701, AD-1747582, AD-1804702, AD-1804703, AD-1804704, A D-1747583, AD-1804705, AD-1804706, AD-1804707, AD-1804708, AD-1804709, AD-174759 1. AD-1747585, AD-1804710, AD-1804711, AD-1747586, AD-1804712, AD-1747587, AD-180 4713, AD-1804714, AD-1747588, AD-1804715, AD-1804716, AD-1804717, AD-1804718, AD- 1804719, AD-1804720, AD-1804721, AD-1804722, AD-1804723, AD-1804724, AD-1804725 and AD-1804726, optionally having a sense strand nucleotide sequence and an antisense strand nucleotide sequence of a single duplex selected from AD-1747580, AD-1747583 and AD-1747585.

24. The dsRNA agent of any of the preceding claims, wherein the sense strand of the dsRNA agent has a modification pattern selected from 5'-nsnsnnn(Nhd)NfnNfNfNfnnnnnnsnsn-3' and 5'-nsnsnnnnnnNfNfNfnnnn(Nhd)nnnsnsn-3', wherein n is a 2'-O-methyl nucleotide, s is a phosphorothioate internucleotide bond, Nf is a 2'-fluoro nucleotide, and (Nhd) is a 2'-O-hexadecyl nucleotide.

25. The dsRNA agent of any of the preceding claims, wherein the antisense strand of the dsRNA agent has a modification pattern selected from 5'-VPnsdNsnndNndNnnnndNnfnnnnnnnsnsn-3' and 5'-VPnsNfsnndNn(Ngn)nnnnnnNfnNfnnnnnsnsn-3', wherein VP is vinylphosphonate, n is a 2'-O-methyl nucleotide, s is a phosphorothioate internucleotide bond, dN is a 2'-deoxynucleotide, Nf is a 2'-fluoro nucleotide, and (Ngn) is a diol nucleic acid S-isomer.

26. A double-stranded nucleic acid (dsRNA) agent for inhibiting the expression of SNCA, wherein the dsRNA agent comprises a sense strand having a 5'-end and a 3'-end and an antisense strand having a 5'-end and a 3'-end, wherein the sense strand and the antisense strand form a double-stranded region, wherein: The sense strand comprises the nucleotide sequence of Table 2 (SEQ ID NOs: 13 to 55) and modifications, with 0 or 1 mismatches; and The antisense strand comprises the nucleotide sequence of Table 2 (SEQ ID NOs: 56 to 98) and modifications, with 0 or 1 mismatches.

27. The dsRNA agent of any of the preceding claims, having a sense strand nucleotide sequence selected from the group consisting of 5'-asasgug(Chd)ucAfGfUfuccaaugusgsa-3' (SEQ ID NO:35), 5'-uscsuuugcuCfCfCfaguu(Uhd)cuusgsa-3' (SEQ ID NO:28), 5'-gsasgca(Ahd)guGfAfCfaaauguugsgsa-3' (SEQ ID NO:21), 5'-gsusaca(Ahd)GfuGfCfUfcaguuccasa-3' (SEQ ID NO:34), 5'-cscsauc(Ahd)gcAfGfUfgauugaagsusa-3' (SEQ ID NO:43), 5'-uscsccag(Uhd)uUfCfUfugagaucusgsa-3' (SEQ ID NO: NO:261) and 5'-uscsaug(Ahd)aaGfGfAfcuuucaaasgsa-3' (SEQ ID NO:19), wherein a is 2'-O-methyladenosine-3'-phosphate, u is 2'-O-methyluridine-3'-phosphate, g is 2'-O-methylguanosine-3'-phosphate, c is 2'-O-methylcytidine-3'-phosphate, s is a phosphorothioate internucleotide bond, Af is 2'-fluoroadenosine-3'-phosphate, Gf is 2'-fluoroguanosine-3'-phosphate, Uf is 2'-fluorouridine-3'-phosphate, Cf is 2'-fluorocytidine-3'-phosphate, (Ahd) is 2'-O-hexadecyladenosine-3'-phosphate, (Uhd) is 2'-O-hexadecyluridine-3'-phosphate, and (Chd) is 2'-O-hexadecylcytidine-3'-phosphate.

28. The dsRNA agent of any of the preceding claims, having an antisense strand nucleotide sequence selected from the group consisting of 5'-VPusdCsacdAudTggaadCuGfagcacuusgsu-3' (SEQ ID NO:78), 5'-VPusdCsaadGadAacugdGgAfgcaaagasusa-3' (SEQ ID NO:71), 5'-VPusdCscadAcdAuuugdTcAfcuugcucsusu-3' (SEQ ID NO:64), 5'-VPusUfsugdGa(Agn)cugagcAfcUfuguacsasg-3' (SEQ ID NO:77), 5'-VPusdAscudTcdAaucadCuGfcugauggsasa-3' (SEQ ID NO:86), 5'-VPusdCsagdAudCucaadGaAfacugggasgsc-3' (SEQ ID NO:87). NO:262) and 5'-VPusdCsuudTgdAaagudCcUfuucaugasasu-3'(SEQID NO:62), wherein VP is vinylphosphonate, a is 2'-O-methyladenosine-3'-phosphate, u is 2'-O-methyluridine-3'-phosphate, g is 2'-O-methylguanosine-3'-phosphate, c is 2'-O-methylcytidine-3'-phosphate, s is a phosphorothioate internucleotide bond, Af is 2'-fluoroadenosine-3'-phosphate, Gf is 2'-fluoroguanosine-3'-phosphate, Uf is 2'-fluorouridine-3'-phosphate, Cf is 2'-fluorocytidine-3'-phosphate, dA is 2'-deoxyadenosine-3'-phosphate, dC is 2'-deoxycytidine-3'-phosphate, dG is 2'-deoxyguanosine-3'-phosphate, dT is 2'-deoxythymidine-3'-phosphate, and (Agn) is adenosine-diol nucleic acid (GNA) S-isomer.

29. The dsRNA agent of any of the preceding claims, having a duplex nucleotide sequence selected from the group consisting of: (i) sense strand: 5'-asasgug(Chd)ucAfGfUfuccaaugusgsa-3' (SEQ ID NO:35) and antisense strand: 5'-VPusdCsacdAudTggaadCuGfagcacuusgsu-3' (SEQ ID NO:78); (ii) sense strand: 5'-uscsuuugcuCfCfCfaguu(Uhd)cuusgsa-3' (SEQ ID NO:28) and antisense strand: 5'-VPusdCsaadGadAacugdGgAfgcaaagasusa-3' (SEQ ID NO:71); (iii) sense strand: 5'-gsasgca(Ahd)guGfAfCfaaauguugsgsa-3' (SEQ ID NO:21) and antisense strand: 5'-VPusdCscadAcdAuuugdTcAfcuugcucsusu-3' (SEQ ID NO:64); (iv) sense strand: 5'-gsusaca(Ahd)GfuGfCfUfcaguuccasasa-3' (SEQ ID NO:34) and antisense strand: 5'-VPusUfsugdGa(Agn)cugagcAfcUfuguacsasg-3' (SEQ ID NO:77); (v) sense strand: 5'-cscsauc(Ahd)gcAfGfUfgauugaagsusa-3' (SEQ ID NO:43) and antisense strand: 5'-VPusdAscudTcdAaucadCuGfcugauggsasa-3' (SEQ ID NO:86); (vi) sense strand: 5'-uscsccag(Uhd)uUfCfUfugagaucusgsa-3' (SEQ ID NO:261) and antisense strand: 5'-VPusdCsagdAudCucaadGaAfacugggasgsc-3' (SEQ ID NO:262);and (vii) sense strand: 5'-uscsaug(Ahd)aaGfGfAfcuuucaaasgsa-3' (SEQ ID NO: 19) and antisense strand: 5'-VPusdCsuudTgdAaagudCcUfuucaugasasu-3' (SEQ ID NO: 62), wherein VP is vinylphosphonate, a is 2'-O-methyladenosine-3'-phosphate, u is 2'-O-methyluridine-3'-phosphate, g is 2'-O-methylguanosine-3'-phosphate, c is 2'-O-methylcytidine-3'-phosphate, s is a phosphorothioate internucleotide bond, Af is 2'-fluoroadenosine-3'-phosphate, Gf is 2'-fluoroguanosine-3'-phosphate, Uf is 2'-fluorouridine-3'-phosphate, and Cf is 2'-fluorouridine-3'-phosphate. , dA is 2'-deoxyadenosine-3'-phosphate, dC is 2'-deoxycytidine-3'-phosphate, dG is 2'-deoxyguanosine-3'-phosphate, dT is 2'-deoxythymidine-3'-phosphate, (Agn) is adenosine-diol nucleic acid (GNA) S-isomer, (Ahd) is 2'-O-hexadecyladenosine-3'-phosphate, (Uhd) is 2'-O-hexadecyluridine-3'-phosphate, and (Chd) is 2'-O-hexadecylcytidine-3'-phosphate. ; 30. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of SNCA, which has a sense strand sequence 5'-asasgug(Chd)ucAfGfUfuccaaugusgsa-3' (SEQ ID NO: 35) and an antisense strand sequence 5'-VPusdCsacdAudTggaadCuGfagcacuusgsu-3' (SEQ ID NO:78), wherein VP is vinyl phosphonate, a is 2'-O-methyladenosine-3'-phosphate, u is 2'-O-methyluridine-3'-phosphate, g is 2'-O-methylguanosine-3'-phosphate, c is 2'-O-methylcytidine-3'-phosphate, s is a phosphorothioate internucleotide bond, Af is 2'-fluoroadenosine-3'-phosphate, Gf is 2'-fluoroguanosine-3'-phosphate, Uf is 2'-fluorouridine-3'-phosphate, dA is 2'-deoxyadenosine-3'-phosphate, dC is 2'-deoxycytidine-3'-phosphate, dT is 2'-deoxythymidine-3'-phosphate, and (Chd) is 2'-O-hexadecylcytidine-3'-phosphate.

31. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of SNCA, which has a sense strand sequence 5'-uscsuuugcuCfCfCfaguu(Uhd)cuusgsa-3' (SEQ ID NO: 28) and an antisense strand sequence 5'-VPusdCsaadGadAacugdGgAfgcaaagasusa-3' (SEQ ID NO:71), wherein VP is vinyl phosphonate, a is 2'-O-methyladenosine-3'-phosphate, u is 2'-O-methyluridine-3'-phosphate, g is 2'-O-methylguanosine-3'-phosphate, c is 2'-O-methylcytidine-3'-phosphate, s is a phosphorothioate internucleotide bond, Af is 2'-fluoroadenosine-3'-phosphate, Cf is 2'-fluorocytidine-3'-phosphate, dA is 2'-deoxyadenosine-3'-phosphate, dC is 2'-deoxycytidine-3'-phosphate, dG is 2'-deoxyguanosine-3'-phosphate, and (Uhd) is 2'-O-hexadecyluridine-3'-phosphate.

32. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of SNCA, which has a sense strand sequence 5'-gsasgca(Ahd)guGfAfCfaaauguugsgsa-3' (SEQ ID NO: 21) and an antisense strand sequence 5'-VPusdCscadAcdAuuugdTcAfcuugcucsusu-3' (SEQ ID NO:64), wherein VP is vinylphosphonate, a is 2'-O-methyladenosine-3'-phosphate, u is 2'-O-methyluridine-3'-phosphate, g is 2'-O-methylguanosine-3'-phosphate, c is 2'-O-methylcytidine-3'-phosphate, s is a phosphorothioate internucleotide bond, Af is 2'-fluoroadenosine-3'-phosphate, Gf is 2'-fluoroguanosine-3'-phosphate, Cf is 2'-fluorocytidine-3'-phosphate, dA is 2'-deoxyadenosine-3'-phosphate, dC is 2'-deoxycytidine-3'-phosphate, dT is 2'-deoxythymidine-3'-phosphate, and (Ahd) is 2'-O-hexadecyladenosine-3'-phosphate.

33. A cell containing the dsRNA agent of any one of claims 1 to 32.

34. A pharmaceutical composition for inhibiting the expression of alpha-synuclein, comprising the dsRNA agent of any one of claims 1 to 32.

35. The pharmaceutical composition of claim 34, wherein the dsRNA agent is administered in a non-buffered solution.

36. The pharmaceutical composition of claim 35, wherein the non-buffered solution is saline or water.

37. The pharmaceutical composition of claim 34, wherein the dsRNA agent is administered in a buffered solution.

38. The pharmaceutical composition of claim 37, wherein the buffer solution comprises acetate, citrate, prolamine, carbonate or phosphate, or any combination thereof.

39. The pharmaceutical composition of claim 37, wherein the buffer solution is phosphate buffered saline (PBS).

40. A pharmaceutical composition comprising the dsRNA agent of any one of claims 1 to 32 and a lipid formulation.

41. A pharmaceutical composition as described in claim 40, wherein the lipid formulation comprises LNP.

42. A method for inhibiting the expression of an α-synuclein (SNCA) gene in a cell and / or preventing the formation of α-synuclein aggregates in a cell or a subject, the method comprising: (a) contacting the cell or subject with a dsRNA agent as described in any one of claims 1 to 32 or a pharmaceutical composition as described in any one of claims 34 to 41; and (b) maintaining the cell or subject produced in step (a) for a period of time sufficient to obtain degradation of the mRNA transcript of the SNCA gene, thereby inhibiting the expression of the SNCA gene in the cell and / or preventing the formation of α-synuclein aggregates in the cell or subject.

43. The method of claim 42, wherein the cell is in a subject.

44. The method of claim 43, wherein the subject is a human.

45. The method of claim 43, wherein the subject is selected from the group consisting of rhesus monkeys, cynomolgus monkeys, mice, and rats.

46. ​​The method of claim 44, wherein the human subject suffers from a SNCA-associated disease.

47. The method of claim 46, wherein the SNCA-associated disease is a synucleinopathy, optionally selected from the group consisting of PD, multiple system atrophy (MSA), Lewy body dementia (LBD), pure autonomic failure (PAF), Pick's disease, progressive supranuclear palsy, dementia pugilistica, Parkinson's disease linked to chromosome 17, Lytico-Bodig's disease, tangle-dominant dementia, argyrophilic grain disease, ganglioglioma, gangliocytoma, meningioangiomatosis, subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis, Hallervorden-Spatz disease, lipofuscinosis, corticobasal degeneration, frontotemporal dementia, frontotemporal lobar degeneration, Alzheimer's disease, Huntington's disease, Down syndrome, psychosis, schizophrenia, and Creutzfeldt-Jakob disease.

48. The method of any one of claims 42 to 47, wherein SNCA expression in the cell or the subject is inhibited by the dsRNA agent by at least about 50%, at least about 40%, at least about 30%, at least about 20%, or at least about 10% compared to a control cell or a control subject.

49. A method of treating a subject diagnosed with a SNCA-associated neurodegenerative disease, the method comprising administering to the subject a therapeutically effective amount of a dsRNA agent as described in any one of claims 1 to 32 or a pharmaceutical composition as described in any one of claims 34 to 42, optionally wherein the subject is re-administered with a therapeutically effective amount of a dsRNA agent as described in any one of claims 1 to 32 or a pharmaceutical composition as described in any one of claims 34 to 42, thereby treating the subject.

50. The method of claim 49, wherein the subject is a human.

51. The method of claim 49 or claim 50, wherein treatment comprises amelioration of at least one sign or symptom of the SNCA-associated neurodegenerative disease.

52. The method of any one of claims 49 to 51, wherein treatment comprises preventing progression of the disease.

53. The method of any one of claims 49 to 52, wherein the SNCA-associated neurodegenerative disease is characterized by one or more symptoms selected from the group consisting of tremor, slow movements (bradykinesia), muscle rigidity, impaired posture and balance, loss of spontaneous movement, changes in speech, changes in handwriting, visual hallucinations, auditory hallucinations, olfactory hallucinations, tactile hallucinations, poor regulation of bodily functions (autonomic nervous system) such as dizziness, falls, and bowel problems, cognitive problems such as confusion, difficulty concentrating, visuospatial problems, and memory loss, sleep difficulties such as rapid eye movement (REM) sleep behavior disorder (in which dreams are physically manifested during sleep), Fluctuations in attention (including episodes of sleepiness, prolonged staring into space, long daytime naps, or disorganized speech), depression and apathy, orthostatic hypotension (a sudden drop in blood pressure that occurs when a person stands up, causing the person to feel dizzy and lightheaded, and the need to sit, squat, or lie down to prevent passing out), clumsy or uncoordinated movements, bladder control problems, contractures of the hands or limbs (chronic shortening of the muscles or tendons around a joint that prevents the joint from moving freely), Pisa syndrome (an abnormal posture in which the body assumes a leaning to one side), nuchal flexion (in which the neck is bent forward and the head is drooping), and involuntary and uncontrollable sighing or gasping.

54. The method of any one of claims 49 to 53, wherein the SNCA-associated neurodegenerative disease is selected from a synucleinopathy such as PD, multiple system atrophy (MSA), Lewy body dementia (LBD), pure autonomic failure (PAF), Pick's disease, progressive supranuclear palsy, dementia pugilistica, Parkinson's disease linked to chromosome 17, Lytico-Bodig's disease, tangle-dominant dementia, argyrophilic grain disease, ganglioglioma, gangliocytoma, meningioangiomatosis, subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis, Hallervorden-Spatz disease, lipofuscinosis, corticobasal degeneration, frontotemporal dementia, frontotemporal lobar degeneration, Alzheimer's disease, Huntington's disease, Down syndrome, psychosis, schizophrenia, and Creutzfeldt-Jakob disease.

55. The method of any one of claims 49 to 54, wherein the dsRNA agent is administered to the subject at a dose of about 0.01 mg / kg to about 50 mg / kg.

56. The method of any one of claims 49 to 55, wherein the dsRNA agent is administered intrathecally to the subject.

57. The method of any one of claims 49 to 56, further comprising administering to the subject an additional agent or therapy useful for treating or preventing a SNCA-associated neurodegenerative disease or disorder.

58. The method of any one of claims 49 to 57, wherein the SNCA-associated neurodegenerative disease is Parkinson's disease (PD).

59. The method of any one of claims 49 to 58, wherein the SNCA-associated neurodegenerative disease is selected from Lewy body dementia (LBD) and multiple system atrophy (MSA).

60. The method of any one of claims 49 to 59, wherein the SNCA expression is inhibited by at least about 30%.

61. The method of any one of claims 49 to 60, further comprising administering an additional therapeutic agent to the subject.

62. The method of any one of claims 49 to 61, wherein the dsRNA agent is administered intrathecally to the subject.

63. The method of claim 62, wherein the method reduces SNCA expression in brain or spinal cord tissue.

64. The method of claim 63, wherein the brain or spinal cord tissue is selected from the group consisting of cerebral cortex, cerebellum, basal ganglia, hippocampus, amygdala, thalamus, brainstem, cervical spinal cord, lumbar spinal cord, and thoracic spinal cord.

65. A method of inhibiting SNCA expression in a subject, the method comprising: Administering a therapeutically effective amount of the dsRNA agent of any one of claims 1 to 32 or the pharmaceutical composition of any one of claims 34 to 41 to the subject, thereby inhibiting SNCA expression in the subject.

66. A method for treating or preventing a SNCA-associated disease in a subject, the method comprising: Administering a therapeutically effective amount of the dsRNA agent of any one of claims 1 to 32 or the pharmaceutical composition of any one of claims 34 to 41 to the subject, thereby treating or preventing a SNCA-related disease in the subject.

67. The method of claim 66, wherein the SNCA-associated disease is selected from a synucleinopathy such as PD, multiple system atrophy (MSA), Lewy body dementia (LBD), pure autonomic failure (PAF), Pick's disease, progressive supranuclear palsy, dementia pugilistica, Parkinson's disease linked to chromosome 17, Lytico-Bodig's disease, tangle-dominant dementia, argyrophilic grain disease, ganglioglioma, gangliocytoma, meningioangiomatosis, subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis, Hallervorden-Spatz disease, lipofuscinosis, corticobasal degeneration, frontotemporal dementia, frontotemporal lobar degeneration, Alzheimer's disease, Huntington's disease, Down syndrome, psychosis, schizophrenia, and Creutzfeldt-Jakob disease.

68. The method of claim 66, wherein the administering step produces at least 60% knockdown of SNCA mRNA or α-synuclein in one or more tissues of the subject, optionally wherein the one or more tissues of the subject are selected from CSF, prefrontal cortex, midbrain, thoracic spine, hippocampus, medulla pons, striatum caudate, and cerebellum.

69. A kit for carrying out the method of any one of claims 42 to 67, comprising a) the dsRNA agent, b) Instructions for use, and c) optionally, a device for administering the dsRNA agent to a subject.

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