SNCA-TARGETED siRNA COMPOSITIONS FOR TREATING SNCA-ASSOCIATED DISEASES - Patent application

Abstract

The present disclosure relates to double-stranded ribonucleic acid (dsRNAi) agents and compositions that target the SNCA gene, particularly in CNS tissues, and methods of using such dsRNAi agents and compositions to inhibit expression of the SNCA gene and to treat subjects with an SNCA-related neurodegenerative disease or disorder, such as Parkinson's disease (PD), multiple system atrophy (MSA), dementia with Lewy bodies (LBD), among other synucleinopathies.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is related to and claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 326,813, entitled "SNCA-Targeting siRNA Compositions for Treating SNCA-Associated Disease," filed April 2, 2022, and U.S. Provisional Patent Application No. 63 / 326,770, entitled "SNCA-Targeting siRNA Compositions for Treating SNCA-Associated Disease," filed April 1, 2022. The entire contents of the foregoing patent applications are incorporated herein by reference.

[0002] FIELD OF THE INVENTION The present disclosure relates generally to SNCA-targeting RNAi agents and methods.

[0003] Sequence Listing This application contains a Sequence Listing that has been filed electronically in eXtensible Markup Language (XML) format, which is incorporated herein by reference in its entirety. The XML copy, created on March 29, 2023, is named ALN-461 WO.xml and is 3242 KB in size. [Background technology]

[0004] The SNCA gene encodes the presynaptic neuronal protein α-synuclein (also referred to herein as alpha-synuclein or synuclein alpha), which is genetically and neuropathologically associated with Parkinson's disease (PD) (Stefanis, L. Cold Spring Harb Perspect Med. 2:a009399). While α-synuclein is thought to contribute to the pathogenesis of PD in various ways, it is generally believed that abnormal soluble oligomeric conformations of α-synuclein, called protofibrils, are toxic species that mediate disruption of cellular homeostasis and neuronal death through effects on various intracellular targets, including synaptic function. Furthermore, secreted α-synuclein is thought to exert deleterious effects on neighboring cells, potentially contributing to disease propagation. Although the extent to which α-synuclein is involved in all PD cases is unclear, targeting the toxic functions conferred by this protein when dysregulated may be a valuable therapeutic strategy not only for PD but also for other neurodegenerative conditions called synucleinopathies, all of which share a common neuropathological hallmark as a result of α-synuclein accumulations called Lewy bodies (LBs) and Lewy neurites (LNs). In addition to PD, such documented or suspected SNCA-related synucleinopathies include, but are not limited to, multiple system atrophy ganglioglioma (MSA), dementia with Lewy bodies (LBD), pure autonomic failure (PAF), Pick's disease, progressive supranuclear palsy, dementia pugilistica, chromosome 17-linked parkinsonism, Richko-Bodig disease, tangle-predominant 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's syndrome, psychosis, schizophrenia, and Creutzfeldt-Jakob disease.

[0005] PD, LBD, and MSA are the three most common neurodegenerative disorders associated with SNCA brain pathology. PD is the most common movement disorder and is characterized by rigidity, hypokinesia, tremor, and postural instability. PD is thought to affect approximately 4 to 6 million people worldwide. LBD accounts for 5 to 15% of all dementias. In addition to memory loss and other often fluctuating dementia symptoms, LBD patients typically suffer from recurrent falls and visual hallucinations. MSA is a rapidly progressive, rare disorder that causes severe movement disorders in affected subjects within several years. The prevalence of MSA is reported to be 3.4 to 4.9 cases per 100,000 population.

[0006] Apart from the neuropathological changes observed in α-synucleinopathies, levels of α-synuclein protein are generally increased in affected brain regions ( Klucken et al., 2006 ).

[0007] Monomers, tetramers, and fibrillar aggregates of α-synuclein are major components of Lewy body (LB)-like intraneuronal inclusions, glial inclusions, and axonal spheroids in neurodegeneration accompanied by brain iron accumulation. Lewy-related pathology (LRP), primarily composed of α-synuclein, is present in the majority of autopsy cases of Alzheimer's disease, 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, 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], and triploidy and duplication, among others, have been identified in families affected by related neurodegenerative disorders. These findings suggest that not only pathogenic mutations in SNCA but also increased levels of alpha-synuclein protein affect disease outcome.

[0008] Although the role of SNCA mutations in disease pathogenesis is not fully understood, evidence points to a deleterious gain-of-function inherent in normal α-synuclein protein when it exceeds certain levels [Stefanis et al. (2012) Cold Spring Harb Perspect Med.] and / or when it abnormally interacts with cellular lipids and vesicles [reviewed in Kiechler et al. (2020) Front. Cell Dev. Biol.]. Consistent with this, SNCA-deficient mice, in contrast to transgenic overexpressing mice, did not exhibit overt neuropathological or behavioral phenotypes [Abeliovich et al. (2000) Neuron]. Posttranscriptional regulation of SNCA has also been shown to occur via 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 demonstrated that expression is suppressed, particularly in cases where long-term disease develops [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 overexpression of even wild-type SNCA messenger RNA (mRNA) was responsible for disease development. These data indicate that suppressing SNCA levels reduces α-synuclein-induced toxicity.

[0009] There are no disease-modifying treatments for synucleinopathies, including PD, multiple system atrophy, and dementia with Lewy bodies, and treatment options are limited, e.g., palliative. For example, currently, only symptomatic treatments are available for PD patients (by compensating for the loss of active dopamine in the brain) and AD patients (i.e., cholinesterase inhibitors). None of the existing treatment strategies for α-synucleinopathies target the underlying disease process.

[0010] Thus, bearing in mind that the involvement of SNCA in several neurodegenerative disorders (synucleinopathies) has been described, there remains a need for agents that are both highly biologically active and stable in vivo, and that can selectively and efficiently silence the SNCA gene (e.g., eliminate or reduce the effects of harmful α-synuclein species) using the cell's own RNAi machinery, which can effectively inhibit the expression of the target SNCA gene, particularly for agents that target the central nervous system (CNS), given the described functionality of α-synuclein in CNS tissues. Summary of the Invention [Problem to be solved by the invention]

[0011] The present disclosure provides an RNAi agent composition that affects the RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of synuclein alpha (SNCA) gene.SNCA gene can be in a cell, for example, in a cell of a subject such as a human.In certain embodiments, the RNAi agent is designed and targeted to knock down SNCA in the cell and / or tissue of CNS. The present disclosure also provides methods of using the RNAi agent compositions of the present disclosure to inhibit expression of the SNCA gene, or to treat a subject who would benefit from inhibiting or reducing expression of the SNCA gene, for example, a subject suffering from or prone to suffering from an SNCA-associated neurodegenerative disease or disorder, such as PD, multiple system atrophy (MSA), dementia with Lewy bodies (LBD), pure autonomic failure (PAF), Pick's disease, progressive supranuclear palsy, dementia pugilistica, Parkinsonism linked to chromosome 17, Richko-Bodig disease, tangle-predominant 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. [Means for solving the problem]

[0012] Thus, in one aspect, the disclosure provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of SNCA, the dsRNA agent comprising 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, the sense strand having 0 or 1 mismatches, the dsRNA agent comprising 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; the sense strand of the dsRNA agent having 0 or 1 mismatches from the 5' end of the sense strand. and a lipophilic moiety attached at position 6 or 16 in addition to the nucleotide sequence of SEQ ID NO: 193, SEQ ID NO: 200, SEQ ID NO: 207, or SEQ ID NOs: 185-192, 194-199, 201-206, or 208-227 of Table 3, with 0 or 1 mismatches; the dsRNA agent does not contain GalNAc modifications; and the dsRNA agent includes eight phosphorothioate internucleotide linkages located at the penultimate and final internucleotide linkages at the 3'- and 5'-ends of each of the sense and antisense strands of the dsRNA agent.

[0013] Thus, in one aspect, the disclosure provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of SNCA, the dsRNA agent comprising 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, and the sense strand has 0 or 1 mismatches, the dsRNA agent comprising a nucleotide sequence of Table 3 (SEQ ID NOS:142-184); the sense strand of the dsRNA agent comprising a lipophilic amino acid attached at position 6 or 16, counting from the 5' end of the sense strand. the antisense strand comprises a nucleotide sequence of Table 3 (SEQ ID NOS:185-227) with 0 or 1 mismatches; the dsRNA agent does not comprise a GalNAc modification; and the dsRNA agent comprises six phosphorothioate internucleotide linkages, the six phosphorothioate internucleotide linkages being 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.

[0014] In certain embodiments, the lipophilic moiety is an aliphatic compound, an alicyclic compound, or a polyalicyclic compound.

[0015] 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-propanediol, a heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenoic acid, dimethoxytrityl, or phenoxazine.

[0016] In one embodiment, the lipophilic moiety is a saturated or unsaturated C-C 30 It contains a hydrocarbon chain and an optional functional group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, or alkyne.

[0017] In some embodiments, the lipophilic moiety is a saturated or unsaturated C-C 18 Contains a hydrocarbon chain. The lipophilic part can be a saturated or unsaturated C 16 It may contain a hydrocarbon chain.

[0018] In certain embodiments, the lipophilic moiety is

[0019] [ka] where B is a nucleotide base or a nucleotide base analog. B may be adenine, guanine, cytosine, thymine, or uracil.

[0020] In certain embodiments, the lipophilic moiety is linked via a linker or a carrier. The lipophilic moiety is conjugated via a carrier that replaces the nucleotide at position 6 or 16 of the sense strand (counting the 5'-end of the sense strand as position 1). In related embodiments, the carrier is a cyclic group (e.g., pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, or decalinyl), or an acyclic moiety based on a serinol backbone or a diethanolamine backbone.

[0021] In some embodiments, the lipophilic moiety is conjugated to the dsRNA agent via a linker that contains an ether, a thioether, a urea, a carbonate, an amine, an amide, a maleimide-thioether, a disulfide, a phosphodiester, a sulfonamide linkage, a product of a click reaction, or a carbamate.

[0022] In one embodiment, the lipophilic moiety is conjugated via a biocleavable linker that is DNA, RNA, disulfide, amide, and functionalized mono- or oligosaccharides of galactosamine, glucosamine, glucose, galactose, and / or mannose, and combinations thereof.

[0023] In certain embodiments, substantially all of the nucleotides in the sense strand, or the antisense strand, or both, are modified nucleotides.

[0024] In one embodiment, all of the nucleotides in the sense strand are modified nucleotides.

[0025] In another embodiment, all of the antisense strand are modified nucleotides.

[0026] In additional embodiments, all of the nucleotides in the sense strand and all of the nucleotides in the antisense strand are modified nucleotides.

[0027] In certain embodiments, an sdRNA agent has one or more modified nucleotides, wherein at least one of the one or more modified nucleotides is a deoxy-nucleotide, 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 nucleotide containing a non-natural base, a tetranucleotide, a tetracarboxylic acid, a ... These nucleotides include hydropyran-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 mimics, nucleotides containing vinyl phosphate, nucleotides containing adenosine-glycol nucleic acid (GNA), nucleotides containing thymidine-glycol nucleic acid (GNA) S-isomers, nucleotides containing 2-hydroxymethyl-tetrahydrofuran-5-phosphate, nucleotides containing 2'-deoxythymidine-3' phosphate, nucleotides containing 2'-deoxyguanosine-3'-phosphate, and terminal nucleotides linked to cholesteryl derivatives and dodecanoic acid bisdecylamide groups.

[0028] 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 containing vinyl phosphate. The dsRNA agent comprises at least one of each of the following modifications: a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-C16 (2'-O-hexadecyl) modified nucleotide, or a nucleotide containing vinyl phosphate.

[0029] In certain embodiments, the dsRNA agent further comprises a phosphate or a phosphate mimic at the 5'-end of the antisense strand. The phosphate mimic may be 5'-vinylphosphonate (VP).

[0030] In some embodiments, a dsRNA agent comprises a pattern of modified nucleotides as shown in Table 2 (wherein the locations of the 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 depicted dsRNA agent).

[0031] In one embodiment, the dsRNA agent is 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-1747 AD-1804720, AD-1804721, AD-1804722, AD-1804723, AD-1804724, AD-1804725, or AD-1804726. The dsRNA agent can have the sense strand nucleotide sequence of AD-1747580, AD-1747583, or AD-1747585.

[0032] In certain embodiments, the sense strand of a dsRNA agent has one of the following modification patterns: 5'-nsnsnnn(Nhd)NfnNfNfNfnnnnnnnnsnsn-3', 5'-nsnsnnnnnnNfNfNfnnnn(Nhd)nnnsnsn-3', or 5'-nsnsnnn(Nhd)nnNfNfNfnnnnnnnnsnsn-3', where 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.

[0033] In embodiments, the sense strand of a dsRNA agent has the following modification pattern: 5'-(L1)(inv)snnnnnnnnNfNfNfnnnnnnnnns(inv)(L2)-3', where each (inv) is an inverted nucleotide (e.g., a reverse abasic nucleotide such as a reverse abasic ribonucleotide), at least one of (L1) and (L2) is a ligand that includes a lipophilic group (e.g., including a C16 alkyl or C16 alkenyl group), and the other of (L1) and (L2) is absent or is hydrogen.

[0034] In some embodiments, the antisense strand of a dsRNA agent has one of the following modification patterns: 5'-VPnsdNsnndNndNnnnndNnNfnnnnnnnsnsn-3', 5'-VPnsNfsnndNn(Ngn)nnnnnnNfnNfnnnnnsnsn-3', or 5'-VPnsNfsnndNn(N2p)nnnnnnNfnNfnnnnnsnsn-3', where 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, (Ngn) is a glycol nucleic acid, S-isomer, and N2p is a 2'-phosphate nucleotide.

[0035] In embodiments, the antisense strand of a dsRNA agent has the following modification patterns: 5'-ZnsdNsnndNndNnnnndNnNfnnnnnsnsn-3', 5'-ZnsNfsnndNn(Ngn)nnnnnnNfnNfnnnsnsn-3', 5'-ZnsNfsnNfnNfnNfnNfnNfnNfnNfnNfnsNfsn-3', 5'-ZnsNfsnNfnNfnNfnNfnNfnNfnNfnNfnsnsn-3', 5'-ZnsNfsnNfnNfnNfnNfnNfnNfnNfnNfnsnsn-3', 5'-ZnsNfsnNfnNfnNfn nnnNfnNfnNfnNfnsNfsn-3', 5'-ZnsNfsnsNfnNfnNfnNfnNfnNfnNfnNfnNfnNfsn-3', 5'-ZnsNfsnsNfnNfnNfnNfnNfnNfnNfnNfsn-3', 5'-ZnsNfsnsNfnNfnnnnNfnNfnNfnNfnNfnNfsn-3', or 5'-ZnsNfsnsNfnNfnNfnNfnNfnNfnNfnNfnNfnnsn-3', where Z is a 5'-phosphate mimetic, e.g., 5'-cyclopropylphosphonate (5'-CP). (The structure of 5'-CP, as used herein, is

[0036] [ka] )

[0037] In certain embodiments, the sense strand of a dsRNA agent comprises the modification pattern: 5'-nsnsnnn(Nhd)NfnNfNfNfnnnnnnnnsnsn-3' and the antisense strand comprises the modification pattern: 5'-VPnsdNsnndNndNnnnndNnNfnnnnnnnsnsn-3', where VP is vinyl-phosphonate, 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 a dsRNA agent comprises the modification pattern: 5'-nsnsnnn(Nhd)NfnNfNfNfnnnnnnnnsnsn-3' and the antisense strand comprises the modification pattern: 5'-VPnsNfsnndNn(Ngn)nnnnnnNfnNfnnnnnsnsn-3', where VP is vinyl-phosphonate, 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 glycol nucleic acid, S-isomer.

[0039] In certain embodiments, the sense strand of a dsRNA agent comprises the modification pattern: 5'-nsnsnnn(Nhd)NfnNfNfNfnnnnnnnnsnsn-3' and the antisense strand comprises the modification pattern: 5'-VPnsNfsnndNn(N2p)nnnnnnNfnNfnnnnnsnsn-3', where VP is vinyl-phosphonate, 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 glycol nucleic acid, S-isomer, and N2p is a 2'-phosphate nucleotide.

[0040] In certain embodiments, the sense strand of a dsRNA agent comprises the modification pattern: 5'-nsnsnnnnnnNfNfNfnnnn(Nhd)nnnsnsn-3' and the antisense strand comprises the modification pattern: 5'-VPnsdNsnndNndNnnnndNnNfnnnnnnnsnsn-3', where VP is vinyl-phosphonate, 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 a dsRNA agent comprises the modification pattern: 5'-nsnsnnnnnnNfNfNfnnnn(Nhd)nnnsnsn-3' and the antisense strand comprises the modification pattern: 5'-VPnsNfsnndNn(Ngn)nnnnnnNfnNfnnnnnsnsn-3', where VP is vinyl-phosphonate, 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 glycol nucleic acid, S-isomer.

[0042] In certain embodiments, the sense strand of a dsRNA agent comprises the modification pattern: 5'-nsnsnnnnnnNfNfNfnnnn(Nhd)nnnsnsn-3' and the antisense strand comprises the modification pattern: 5'-VPnsNfsnndNn(N2p)nnnnnnNfnNfnnnnnsnsn-3', where VP is vinyl-phosphonate, 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 glycol nucleic acid, S-isomer, and N2p is a 2'-phosphate nucleotide.

[0043] In certain embodiments, the sense strand of a dsRNA agent comprises the modification pattern: 5'-nsnsnnn(Nhd)nnNfNfNfnnnnnnnnsnsn-3' and the antisense strand comprises the modification pattern: 5'-VPnsdNsnndNndNnnnndNnNfnnnnnnnsnsn-3', where VP is vinyl-phosphonate, 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 glycol nucleic acid, S-isomer.

[0044] In certain embodiments, the sense strand of a dsRNA agent comprises the modification pattern: 5'-nsnsnnn(Nhd)nnNfNfNfnnnnnnnnsnsn-3' and the antisense strand comprises the modification pattern: 5'-VPnsNfsnndNn(Ngn)nnnnnnNfnNfnnnnnsnsn-3', where VP is vinyl-phosphonate, 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 glycol nucleic acid, S-isomer.

[0045] In certain embodiments, the sense strand of a dsRNA agent comprises the modification pattern: 5'-nsnsnnn(Nhd)nnNfNfNfnnnnnnnnsnsn-3' and the antisense strand comprises the modification pattern: 5'-VPnsNfsnndNn(N2p)nnnnnnNfnNfnnnnnsnsn-3', where VP is vinyl-phosphonate, 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 glycol nucleic acid, S-isomer, and N2p is a 2'-phosphate nucleotide.

[0046] Another embodiment of the present disclosure provides a dsRNA agent for inhibiting expression of SNCA, the dsRNA agent comprising 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 and modification of Table 2 (SEQ ID NOS: 13-55) with 0 or 1 mismatch; and the antisense strand comprises a nucleotide sequence and modification of Table 2 (SEQ ID NOS: 56-98) with 0 or 1 mismatch.

[0047] In one embodiment, the dsRNA agent has 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: 261), or 5'-uscsaug(Ahd)aaGfGfAfcuuucaaasgsa-3' (SEQ ID NO: 19). where a is 2'-O-methyl adenosine-3'-phosphate, u is 2'-O-methyl uridine-3'-phosphate, g is 2'-O-methyl guanosine-3'-phosphate, c is 2'-O-methyl cytidine-3'-phosphate, s is a phosphorothioate internucleotide linkage, Af is 2'-fluoroadenosine-3'-phosphate, Gf is 2'-fluoro (Ahd) is 2'-O-hexadecyl adenosine-3'-phosphate, (Uhd) is 2'-O-hexadecyl uridine-3'-phosphate, (Chd) is 2'-O-hexadecyl cytidine-3'-phosphate, (Chd) is 2'-O-hexadecyl adenosine-3'-phosphate, (Chd) is 2'-O-hexadecyl cytidine-3'-phosphate, (Chd) is 2'-O-hexadecyl adenosine-3'-phosphate, (Chd) is 2'-O-hexadecyl uridine-3'-phosphate, (Chd) is 2'-O-hexadecyl cyt ... adenosine-3'-phosphate, (Chd) is 2'-O-hexadecyl

[0048] In another embodiment, the dsRNA agent has 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)cuga gcAfcUfuguacsasg-3' (SEQ ID NO: 77), 5'-VPusdAscudTcdAaucadCuGfcugauggsasa-3' (SEQ ID NO: 86), 5'-VPusdCsagdAudCucaadGaAfacugggasgsc-3' (SEQ ID NO: 262), or 5'-VPusdCsuudTgdAaagudCcUfuucaugasasu-3' (SEQ ID NO: 62), where VP is vinyl-phosphonate. a is 2'-O-methyl adenosine-3'-phosphate, u is 2'-O-methyl uridine-3'-phosphate, g is 2'-O-methyl guanosine-3'-phosphate, c is 2'-O-methyl cytidine-3'-phosphate, s is a phosphorothioate internucleotide linkage, Af is 2'-fluoro adenosine-3'-phosphate, Gf is 2'-fluoro guanosine-3'-phosphate, and 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 glycol nucleic acid (GNA), S-isomer.

[0049] In further embodiments, the dsRNA agent has the following duplex pair 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) a sense strand 5'-uscsaug(Ahd)aaGfGfAfcuuucaaasgsa-3' (SEQ ID NO: 19) and an antisense strand: 5'-VPusdCsuudTgdAaagudCcUfuucaugasasu-3' (SEQ ID NO: 62), where 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 linkage, Af is 2'-fluoroadenosine-3'-phosphate, and Gf is 2'-fluoroguanosine-3'-phosphate. 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, (Agn) is adenosine glycol nucleic acid (GNA), S-isomer, (Ahd) is 2'-O-hexadecyl adenosine-3'-phosphate, (Uhd) is 2'-O-hexadecyluridine-3'-phosphate, and (Chd) is 2'-O-hexadecylcytidine-3'-phosphate;

[0050] An additional embodiment of the present disclosure provides a dsRNA agent for inhibiting expression of SNCA having the sense strand sequence 5'-asasgug(Chd)ucAfGfUfuccaaugusgsa-3' (SEQ ID NO: 35) and the antisense strand sequence 5'-VPusdCsacdAudTggaadCuGfagcacuusgsu-3' (SEQ ID NO: 78), where 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, and c is 2'-O-methylguanosine-3'-phosphate. 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 embodiment of the disclosure provides a dsRNA agent for inhibiting expression of SNCA having the sense strand sequence 5'-uscsuuugcuCfCfCfaguu(Uhd)cuusgsa-3' (SEQ ID NO: 28) and the antisense strand sequence 5'-VPusdCsaadGadAacugdGgAfgcaaagasusa-3' (SEQ ID NO: 71), where VP is vinyl-phosphonate, a is 2'-O-methyladenosine-3'-phosphate, u is 2'-O-methyluridine-3'-phosphate, and 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 embodiment of the disclosure is a dsRNA agent for inhibiting expression of SNCA, comprising the sense strand sequence 5'-gsasgca(Ahd)guGfAfCfaaauguugsgsa-3' (SEQ ID NO: 21) and the antisense strand sequence 5'-VPusdCscadAcdAuuugdTcAfcuugcucsusu-3' (SEQ ID NO: 64), where 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, and c is 2'-O-methylcysteine. is 2'-fluoroadenosine-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 dsRNA agent is its pharmaceutically acceptable salt. The "pharmaceutically acceptable salt" of each RNAi agent herein includes, but is not limited to, sodium salt, calcium salt, lithium salt, potassium salt, ammonium salt, magnesium salt, and mixtures thereof. Those skilled in the art will understand that the RNAi agent can be provided as a polycationic salt with one cation per free acid group of the appropriately modified phosphodiester backbone and / or any other acidic modification (e.g., 5'-terminal phosphonate group). For example, an oligonucleotide of "n" nucleotides in length contains n-1 appropriately modified phosphodiesters, and therefore, a 21nt-long oligonucleotide can be provided as a salt with up to 20 cations (e.g., 20 sodium cations). Similarly, an RNAi agent with a 21nt-long sense strand and a 23nt-long antisense strand can be provided as a salt with up to 42 cations (e.g., 42 sodium cations). In the foregoing example, where the RNAi agent also includes a 5'-terminal phosphate or 5'-terminal vinylphosphonate group, the RNAi agent can be provided as a salt with up to 44 cations (eg, 44 sodium cations).

[0054] Another aspect of the disclosure provides a cell containing a dsRNA agent disclosed herein.

[0055] An additional aspect of the present disclosure provides a pharmaceutical composition for use in inhibiting the expression of alpha-synuclein, comprising a dsRNA agent disclosed herein.

[0056] In certain embodiments, the dsRNA agent and / or pharmaceutical composition is administered in an unbuffered solution. The unbuffered solution may be saline or water.

[0057] In some embodiments, dsRNA agent is administered with buffer solution.Buffer solution can comprise acetate, citrate, prolamin, carbonate or phosphate or their combination.In certain embodiments, buffer solution is phosphate buffered saline (PBS).

[0058] A further aspect of the 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 for inhibiting expression of the alpha-synuclein (SNCA) gene in a cell and / or preventing the formation of alpha-synuclein aggregates in a cell or a subject, the method comprising: (a) contacting the cell or subject with a dsRNA 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 mRNA transcripts of the SNCA gene, thereby inhibiting expression of the SNCA gene in the cell or subject and / or preventing the formation of alpha-synuclein aggregates in the cell or subject.

[0061] In one embodiment, the cell is in a subject. The subject may be 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. The SNCA-associated disease may be a synucleinopathy. The synucleinopathy may be PD, multiple system atrophy (MSA), dementia with Lewy bodies (LBD), pure autonomic failure (PAF), Pick's disease, progressive supranuclear palsy, dementia pugilistica, chromosome 17-linked parkinsonism, Richtko-Bodig disease, tangle-predominant dementia, argyrophilic grain disease, ganglioglioma, ganglioneuroma, 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's syndrome, psychosis, schizophrenia, and / or Creutzfeldt-Jakob disease.

[0063] In some embodiments, SNCA expression in a cell or subject is inhibited 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] An additional aspect of the present disclosure provides a method of treating a subject diagnosed with an SNCA-associated neurodegenerative disease, comprising administering to the subject a therapeutically effective amount of a dsRNA agent or pharmaceutical composition disclosed herein, wherein the subject can optionally be re-administered 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 an SNCA-associated neurodegenerative disease.

[0066] In some embodiments, treatment includes prevention of disease progression.

[0067] In one embodiment, an SNCA-associated neurodegenerative disease is characterized by the following symptoms: tremors, slowed movements (bradykinesia), muscle rigidity, impaired posture and balance, loss of automatic movements, changes in speech, changes in writing, visual, auditory, olfactory, or tactile hallucinations, dizziness, falls, dysregulation of bodily functions (autonomic nervous system) such as bowel problems, cognitive problems such as confusion, decreased attention, visuospatial problems and memory loss, sleep disorders such as rapid eye movement (REM) sleep behavior disorder (dreams are physically acted out while asleep), fluctuations in attention including episodes of drowsiness, prolonged spatial gaze, long daytime naps or uncontrolled waking. It is characterized by one or more of the following: speechlessness, depression, and lethargy; orthostatic hypotension (a sudden drop in blood pressure that occurs when standing up, making the person feel dizzy or unsteady and requiring them to sit, squat, or lie down to prevent fainting); clumsiness and lack of coordination; bladder control problems; contractures of the hands and limbs (chronic shortening of the muscles or tendons around the joints, preventing the joints from moving freely); Pisa syndrome (an abnormal posture in which the body appears to lean to one side); cervical flexion (the neck bends forward and the head drops down); and / or involuntary and uncontrollable sighing or gasping.

[0068] In certain embodiments, the dsRNA agent is administered to the 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 to the subject intrathecally.

[0070] In one embodiment, the method further involves administering to the subject an additional agent or therapy suitable for the treatment or prevention of the 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 an additional therapeutic agent to the subject.

[0075] In certain embodiments, the method reduces the expression of SNCA in brain or spinal cord tissue, the brain or spinal cord tissue being the 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 expression of SNCA in a subject, the method comprising administering to the subject a therapeutically effective amount of a dsRNA agent or pharmaceutical composition disclosed herein, thereby inhibiting expression of SNCA in the subject.

[0077] An additional aspect of the present disclosure provides a method of inhibiting 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 expression of SNCA in the subject.

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

[0079] Another aspect of the present disclosure provides a kit for carrying out the method disclosed herein, said kit comprising the dsRNA agent disclosed herein and the instruction manual for using it.Kit can also comprise the means for administering dsRNA agent to subject.

[0080] definition In order that this disclosure may be more readily understood, certain terms are first defined. Additionally, whenever a value or range of values ​​for a parameter is listed, it is intended that values ​​and ranges intermediate to the listed values ​​are also intended to be part of this disclosure.

[0081] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element, e.g., a plurality of elements.

[0082] The term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to."

[0083] The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless context clearly indicates otherwise.

[0084] The term "about" is used herein to mean within a range that is typical 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 is present before a series of numbers or ranges, it will be understood that "about" can modify each number or range in the series.

[0085] The term "at least" before a number or a series of numbers is understood to include the number adjacent to the term "at least," as well as all numbers or integers that can be logically included if the context is clear. 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 the term "at least" is present before a series of numbers or a range, it is understood that "at least" can modify each of the numbers and ranges in the series.

[0086] As used herein, "less than" or "less than" shall be understood as meaning from the value adjacent to the phrase and its logically smaller value or integer, if logical from the context, to zero.For example, a duplex with an overhang of "2 nucleotides or less" has an overhang of 2, 1, or 0 nucleotides.When "less than" is present before a series of numbers or ranges, it shall be understood that "less than" can modify each number or range in the series.As used herein, a range includes both upper and lower limits.

[0087] As used herein, a method of detection can include determining that the amount of analyte present is below the detection level of the method.

[0088] In the event of a discrepancy between the nucleotide sequence for a given target site and the sense or antisense strand, the given sequence prevails.

[0089] If the chemical structure and chemical name do not agree, the chemical structure takes precedence.

[0090] The terms "SNCA," "α-synuclein," "synuclein alpha," or "alpha-synuclein" refer to the gene and the protein encoded by that gene that are associated with neurodegenerative diseases termed "synucleinopathies." The human SNCA gene region encompasses approximately 114 kb. The SNCA transcript contains 13 exons, and 15 mRNA isoforms have been identified or are otherwise predicted to occur. The nucleotide and amino acid sequences of SNCA may be found, for example, in GenBank Accession No. NM_007308.3 [human (Homo sapiens) SNCA, SEQ ID NO: 1, reverse complement, SEQ ID NO: 2]; GenBank Accession No. XM_005555421 [cynomolgus monkey (Macaca fascicularis) SNCA, SEQ ID NO: 3, reverse complement, SEQ ID NO: 4]; GenBank Accession No. NM_009221 [house mouse (Mus musculus) SNCA, SEQ ID NO: 5, reverse complement, SEQ ID NO: 6]; GenBank Accession No. NM_019169.2 [rat (Rattus norvegicus) SNCA, SEQ ID NO: 7, reverse complement, SEQ ID NO: 8]; and GenBank Accession No. XM_535656.7 [gray wolf (Canis lupus familiaris) SNCA, SEQ ID NO: 228, reverse complement, SEQ ID NO: 229].

[0091] The term "SNCA" as used herein also refers to, for example, the isoform 1 transcript NM_000345.4 (SEQ ID NO: 232) encoding the polypeptide NP_000336.1; the isoform 2 transcript NM_001146054.2 (SEQ ID NO: 230) encoding the polypeptide NP_001139526.1; the isoform 3 transcript NM_001146055.2 (SEQ ID NO: 231) encoding the polypeptide NP_001139527.1; the isoform 4 transcript NM_001146055.2 (SEQ ID NO: 231) encoding the polypeptide NP_009292.1; isoform 4 transcript NM_007308.3 (SEQ ID NO: 1); isoform 5 transcript NM_001375285.1 (SEQ ID NO: 233) encoding polypeptide NP_001362214.1; isoform 6 transcript NM_001375286.1 (SEQ ID NO: 234) encoding polypeptide NP_001362215.1; isoform 7 transcript NM_001375287.1 (SEQ ID NO: 235) encoding polypeptide NP_001362216.1; isoform 8 transcript NM_001375287.1 (SEQ ID NO: 235) encoding polypeptide NP_001362217.1 isoform 8 transcript NM_001375288.1 (SEQ ID NO: 236); isoform 9 transcript NM_001375290.1 ​​(SEQ ID NO: 237), encoding polypeptide NP_001362219.1; and predicted isoform X1 transcript XM_011532203.1 (SEQ ID NO: 238), encoding polypeptide XP_011530505.1; predicted isoform X2 transcript XM_011532204.3 (SEQ ID NO: 239), encoding polypeptide XP_011530506.1; predicted isoform X3 transcript XM_011532205.2 (SEQ ID NO: 240) encoding polypeptide XP_011530507.1; predicted isoform X4 transcript XM_011532206.1 (SEQ ID NO: 241) encoding polypeptide XP_011530508.1; predicted isoform X5 transcript XM_011532207.1 (SEQ ID NO: 242) encoding polypeptide XP_011530509.1; and predicted isoform X8 transcript XM_017008563 encoding polypeptide XP_016864052.1.SNCA refers to variants of the SNCA gene, including naturally occurring sequence variants providing SEQ ID NO: 1 (SEQ ID NO: 243) (the unique sequences associated with each of the foregoing accession numbers are incorporated herein by reference in the form available as of the filing date of this application). Further examples of SNCA sequences can be found in publicly available databases, such as GenBank, OMIM, UniProt, NCBI dbSNP (see, e.g., www.ncbi.nlm.nih.gov / gene / 6622), and the Macaca Genome Project website (macaque.genomics.org.cn / page / species / index.jsp). Further information regarding SNCA can be found, for example, at www.ncbi.nlm.nih.gov / gene / 6622. The entire contents of each of the foregoing GenBank accession numbers and Gene database numbers are incorporated herein by reference as of the filing date of this application.

[0092] Three protein isoforms of α-synuclein are listed in UniProt. The longest α-synuclein isoform is an approximately 14 kDa protein (isoform 1, UniProt, 140 amino acids P37840). Other α-synuclein isoforms in UniProt include isoforms 2-4, 112 amino acids P37840-2; and isoforms 2-5, 126 amino acids P37840-3. The 140 amino acid α-synuclein protein is encoded by five exon pairs mapped to chromosomal locus 4q21.3-q22. The α-synuclein protein has an N-terminal region consisting of an incomplete KXKEGV (SEQ ID NO: 244) motif, a highly hydrophobic NAC domain, and a highly acidic C-terminal domain. Under physiological conditions, SNCA is thought to be an intrinsically disordered monomer or a helically folded tetramer. Alpha-synuclein accounts for 1% of the total protein in the cytoplasm of brain cells and is predominantly expressed in the neocortex, hippocampus, substantia nigra, thalamus, and cerebellum. Alpha-synuclein is also expressed in lower amounts in the heart, skeletal muscle, pancreas, lymphocytes, and blood cells. The function of SNCA is poorly understood, but evidence suggests that it plays an important role in maintaining an adequate supply of synaptic vesicles at presynaptic terminals. Alpha-synuclein is involved in regulating dopamine release and transport, the fibrillization of the microtubule-associated protein tau, and a neuroprotective phenotype in nondopaminergic neurons by regulating both p53 expression and the transcriptional activation of proapoptotic genes, thereby reducing caspase-3 activation. The primary mechanism by which alpha-synuclein induces neurodegenerative diseases such as Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy appears to be through increased levels of alpha-synuclein protein and the formation of alpha-synuclein fibrillar aggregates.

[0093] As used herein, "target sequence" refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of SNCA gene, such as the mRNA that is the product of RNA processing of a primary transcript.In one embodiment, the target portion of the sequence will be at least long enough to serve as a substrate for RNAi-dependent cleavage at or near a portion of the nucleotide sequence of an mRNA molecule formed during the transcription of SNCA gene.In one embodiment, the target sequence is located within the protein coding region of SNCA gene.In another embodiment, the target sequence is located within the 3'UTR of SNCA gene.

[0094] 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, 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 The target sequence may be 9-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. In 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 the above-listed ranges and lengths are also contemplated as part of this disclosure.

[0095] As used herein, the term "strand comprising a sequence" means an oligonucleotide comprising a chain of nucleotides described by a sequence referenced using standard nucleotide nomenclature.

[0096] " G ", " C ", " A ", " T " and " U " each generally refer to the nucleotide that contains guanine, cytosine, adenine, thymidine and uracil as a base, respectively, in relation to modified or unmodified nucleotide. However, it should be understood that the term "ribonucleotide" or "nucleotide" can also refer to modified nucleotides, as described in more detail below, or substitute replacement moieties (see, for example, Table 1). Those skilled in the art are well aware that guanine, cytosine, adenine, thymidine and uracil can be replaced with other moieties without substantially changing the base pairing properties of the oligonucleotide that contains the nucleotide with such replacement moieties. For example, but not limited to, the nucleotide that contains inosine as its base can form base pairs with the nucleotide that contains adenine, cytosine or uracil. Thus, the nucleotide that contains uracil, guanine or adenine can be replaced with the nucleotide that contains inosine, for example, in the nucleotide sequence of the dsRNA that is featured in the present disclosure. In another example, adenine and cytosine in any of the oligonucleotides can be substituted with guanine and uracil, respectively, to form G-U Wobble base pairs with the target mRNA. Sequences containing such substituted moieties are suitable for the compositions and methods featured in this disclosure.

[0097] The terms " iRNA ", " RNAi agent ", " iRNA agent ", " RNA interference agent ", used interchangeably herein, refer to the agent that contains the RNA as defined herein and mediates targeted cleavage in RNA transcription via the RNA-induced silencing complex (RISC) pathway.RNA interference (RNAi) is the process that directs the sequence-specific degradation of mRNA.RNAi regulates, for example, inhibits, the expression of SNCA in cells, for example, cells in a subject, such as a mammalian subject.

[0098] In one embodiment, the RNAi agent of the present disclosure comprises a single-stranded RNAi that interacts with a target RNA sequence, such as an SNCA target mRNA sequence, to direct 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 into double-stranded small interfering RNAs (siRNAs) containing a sense strand and an antisense strand by a type III endonuclease known as Dicer [Sharp et al. (2001) Genes Dev. 15:485]. Dicer, an RNase III-like enzyme, processes these dsRNAs into 19-23 base pair small interfering RNAs with a characteristic two-base 3' overhang [Bernstein, et al., (2001) Nature 409:363]. These siRNAs are then introduced into RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, allowing complementary antisense strands to induce target recognition [Nykanen, et al., (2001) Cell 107:309]. Upon binding to the appropriate target mRNA, one or more endonucleases in RISC cleave the target to induce silencing [Elbashir, et al., (2001) Genes Dev. 15:188]. Thus, in one aspect, the present disclosure relates to a single-stranded RNA (ssRNA) (the antisense strand of the siRNA duplex) that is produced in cells, promotes the formation of RISC complex, and thereby silences the target gene, i.e., the SNCA gene. Therefore, the term "siRNA" is used herein to also mean the RNAi described above.

[0099] In another embodiment, the RNAi agent can be a single-stranded RNA introduced into a cell or organism to inhibit target mRNA. The single-stranded RNAi agent binds to the RISC endonuclease Argonaute 2 and then cleaves the target mRNA. The single-stranded siRNA is generally 15-30 nucleotides and chemically modified. The design and testing of single-stranded RNA is described in U.S. Patent No. 8,101,348 and Lima et al., (2012) Cell 150:883-894, the entire contents of each of which are incorporated herein by reference. Any of the antisense nucleotide sequences described herein can be used as the single-stranded siRNA described herein or as the single-stranded siRNA chemically modified by the method described in Lima et al., (2012) Cell 150:883-894.

[0100] In another embodiment, the "RNAi agent" for use in the compositions and methods of the present disclosure is double-stranded RNA, and is referred to herein as a "double-stranded RNAi agent," a "double-stranded RNA (dsRNA) molecule," a "dsRNA agent," or a "dsRNA." The term "dsRNA" refers to a complex of ribonucleic acid molecules having a duplex structure comprising two antiparallel, substantially complementary nucleic acid strands, said to have a "sense" or "antisense" orientation with respect to the target RNA, i.e., the SNCA gene. In some embodiments of the present disclosure, the double-stranded RNA (dsRNA) induces the degradation of the target RNA, for example, mRNA, by a post-transcriptional gene silencing mechanism, referred to herein as RNA interference or RNAi.

[0101] Generally, dsRNA molecule can comprise ribonucleotide, but as described in detail herein, each strand or both strands can also comprise one or more ribonucleotides, for example, deoxyribonucleotide, modified nucleotide.In addition, as used herein, " RNAi agent " can comprise the ribonucleotide with chemical modification; RNAi agent can comprise the substantial modification in multiple nucleotides.

[0102] As used herein, the term "modified nucleotide" refers to a nucleotide that has independently modified sugar moiety, modified internucleotide linkage, or modified nucleobase.Therefore, the term modified nucleotide encompasses the substitution, addition, or removal of, for example, functional group or atom, etc., to internucleoside linkage, sugar moiety, or nucleobase.The modifications suitable for use in the agent of the present disclosure include all types of modifications disclosed herein or known in the art.Any such modifications used in siRNA type molecules are encompassed by "RNAi agent" for the purpose of this specification and claims.

[0103] In certain embodiments of the present disclosure, the inclusion of deoxy-nucleotides, which are recognized as naturally occurring forms of nucleotides, when present in an RNAi agent can be considered to constitute modified nucleotides.

[0104] The duplex region can be any length that allows for specific degradation of the desired target RNA by the RISC pathway, and can be about 9 to 36 base pairs in length, e.g., about 15-30 base pairs in length, e.g., 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, e.g., about 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, The length may range from 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. Ranges and lengths intermediate to the above-listed ranges and lengths are also contemplated as part of the invention.

[0105] The two strands forming the duplex structure can be different portions of a larger RNA molecule, or they can be separate RNA molecules. When the two strands are part of a larger molecule and are therefore connected by an uninterrupted chain of nucleotides between the 3' end of one strand and the 5' end of the other strand forming the duplex structure, the connecting RNA strands are called "hairpin loops." A hairpin loop can contain at least one unpaired nucleotide. In some embodiments, a hairpin loop can contain 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 not targeted to the target site of the dsRNA. In some embodiments, a hairpin loop can be 10 or fewer nucleotides. In some embodiments, a hairpin loop can be 8 or fewer unpaired nucleotides. In some embodiments, a hairpin loop can be 4 to 10 unpaired nucleotides. In some embodiments, a hairpin loop can be 4 to 8 unpaired nucleotides.

[0106] In certain embodiments, the two strands of a double-stranded oligomeric compound may be linked together. The two strands may be linked to each other at both ends or only at one end. Linked at one end means that the 5'-end of the first strand is linked to the 3'-end of the second strand, or the 3'-end of the first strand is linked to the 5'-end of the second strand. When the two strands are linked to each other at both ends, the 5'-end of the first strand is linked to the 3'-end of the second strand, and the 3'-end of the first strand is linked to the 5'-end of the second strand. The two strands may be linked together by an oligonucleotide linker, including, but not limited to, (N)n, where N is independently a modified or unmodified nucleotide, and n is 3 to 23. In some embodiments, n is 3 to 10, for example, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the oligonucleotide linker is selected from the group consisting of GNRA, (G)4, (U)4, and (dT)4, where N is a modified or unmodified nucleotide, and R is a modified or unmodified purine nucleotide. Some of the 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. Those skilled in the art will recognize that any chemical modification or variation of the oligonucleotides described herein can be used in the oligonucleotide linker.

[0107] Hairpin and dumbbell-shaped oligomeric compounds will have a duplex region equal to or at least equal to 14, 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The duplex region can be 200, 100, or 50 nucleotide pairs or less in length. In some embodiments, the duplex region ranges from 15 to 30, 17 to 23, 19 to 23, and 19 to 21 nucleotide pairs in length.

[0108] In some embodiments, hairpin oligomeric compounds may have a single-stranded overhang or terminal unpaired region at the 3' end, and in some embodiments, at the antisense end of the hairpin. In some embodiments, the overhang is 1 to 4, more typically 2 to 3, nucleotides in length. Hairpin oligomeric compounds capable of inducing RNA interference are also referred to herein as "shRNAs."

[0109] The two substantially complementary strands of dsRNA are contained in separate RNA molecules, and these molecules can be, but are not necessarily, connected by covalent bonds.The two strands are covalently connected between the 3'-end of one strand and the 5'-end of each other strand that form a duplex structure by means other than an uninterrupted nucleotide chain, and in this case, the connecting structure is called "linker".The RNA strands can have the same or different number of nucleotides.The maximum number of base pairs is the number of nucleotides in the shortest strand of dsRNA minus all the overhangs that exist in the duplex.In addition to the duplex structure, RNAi can also contain one or more nucleotide overhangs.

[0110] In one embodiment, the RNAi agent of the present disclosure is a dsRNA, each strand of which is 24-30 nucleotides in length and interacts with a target RNA sequence, such as an SNCA target mRNA sequence, to induce RNA cleavage. Without wishing to be bound by theory, long double-stranded RNA introduced into cells is degraded into siRNAs by a type III endonuclease known as Dicer [Sharp et al. (2001) Genes Dev. 15: 485]. Dicer, an RNase III-like enzyme, processes dsRNA into 19-23 base pair small interfering RNAs with characteristic two-base 3' overhangs [Bernstein, et al., (2001) Nature 409: 363]. The siRNA is then introduced into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, allowing the complementary antisense strand to induce target recognition [Nykanen, et al., (2001) Cell 107: 309]. Upon binding to the appropriate target mRNA, one or more endonucleases within the 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 that interacts with the SNCA RNA sequence to induce cleavage of the target RNA.

[0111] In one embodiment, the RNAi agent of the present disclosure is a dsRNA agent, each strand of which contains 19-23 nucleotides that interact with the SNCA RNA sequence to induce cleavage of the target RNA. Without wishing to be bound by theory, long double-stranded RNA introduced into cells is degraded into siRNAs by a type III endonuclease known as Dicer [Sharp et al. (2001) Genes Dev. 15: 485]. Dicer, an RNase III-like enzyme, processes dsRNA into 19-23 base pair small interfering RNAs with characteristic two-base 3' overhangs [Bernstein, et al., (2001) Nature 409: 363]. The siRNA is then introduced into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, allowing the complementary antisense strand to induce target recognition [Nykanen, et al., (2001) Cell 107: 309]. Upon binding to the appropriate target mRNA, one or more endonucleases within the 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 19-23 nucleotide dsRNA that interacts with the SNCA RNA sequence to induce target RNA cleavage.

[0112] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the double-stranded structure of an RNAi agent, such as dsRNA.For example, when the 3'-end of one strand of dsRNA extends beyond the 5'-end of the other strand, and vice versa, there is a nucleotide overhang.DsRNA can comprise at least one nucleotide overhang; alternatively, the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more nucleotides.The nucleotide overhang can comprise or consist of nucleotide / nucleoside analogs, such as deoxynucleotides / nucleosides.The overhang can be on the sense strand, antisense strand, or any combination thereof.In addition, the nucleotide of the overhang can be present at the 5'-end, 3'-end, or both of the antisense strand or sense strand of dsRNA.

[0113] In one embodiment of 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 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' and 5' ends of one strand of RNAi agent comprise an overhang of at least 1 nucleotide.

[0114] In one embodiment, the antisense strand of dsRNA has an overhang of 1 to 10 nucleotides at the 3'-end, 5'-end, both ends, or either end, for example, 0 to 3, 1 to 3, 2 to 4, 2 to 5, 4 to 10, 5 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In one embodiment, the sense strand of dsRNA has an overhang of 1 to 10 nucleotides at the 3'-end, 5'-end, both ends, or either end, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In another embodiment, one or more of the nucleotides in the overhang are replaced with nucleoside thiophosphate.

[0115] In certain embodiments, the overhang on the sense strand or the antisense strand, or both, can comprise an extended length greater than 10 nucleotides, e.g., 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 at the 3'-end of the sense strand of the duplex. In certain embodiments, the extended overhang is 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 at the 3'-end of the antisense strand of the duplex. In certain embodiments, the extended overhang is at the 5'-end of the antisense strand of the duplex. In certain embodiments, one or more of the nucleotides in the overhang are replaced with a nucleoside thiophosphate. In certain embodiments, the overhang comprises a self-complementary portion such that the overhang can form a stable hairpin structure under physiological conditions.

[0116] The term "blunt" or "blunt-ended" as used herein in reference to 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 the dsRNA can be blunt. When both ends of the dsRNA are blunt, the dsRNA is said to be blunt-ended. For clarity, a "blunt-ended" dsRNA is a dsRNA that is blunt at both ends, i.e., there are no nucleotide overhangs at either end of the molecule. In most cases, such a molecule will be double-stranded throughout its entire length.

[0117] In some embodiments, the dsRNA comprises a 3' overhang. In some embodiments, the dsRNA comprises a 1-nucleotide 3' overhang. In some embodiments, the dsRNA comprises a 2-nucleotide 3' overhang. In some embodiments, the dsRNA comprises a 5' overhang. In some embodiments, the dsRNA comprises a 1-nucleotide 5' overhang. In some embodiments, the dsRNA comprises a 2-nucleotide 5' overhang.

[0118] In some embodiments, the dsRNA has one end with an overhang and the other end with a blunt end. The overhang can be a sense strand 3' overhang, a sense strand 5' overhang, an antisense strand 3' overhang, or an 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 on both ends. The overhangs on each end are independently a sense strand 3' overhang, a sense strand 5' overhang, an antisense strand 3' overhang, or an 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 overhanging nucleotides can be modified nucleotides, reverse nucleotides, abasic nucleotides, or reverse abasic nucleotides. The inverted nucleotides may be linked via a 3'-3' phosphodiester linkage.

[0119] 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 3' and 5' terminal inverted abasic residues of the sense strand may be overhangs.

[0120] 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.

[0121] The term "antisense strand" or "guide strand" refers to the strand of an RNAi agent, eg, a dsRNA, which includes a region that is substantially complementary to a target sequence, eg, SNCA mRNA.

[0122] As used herein, term " complementary region " refers to the region on antisense strand that is substantially complementary to sequence, for example, target sequence, for example, SNCA nucleotide sequence, as defined herein.If complementary region is not completely complementary to target sequence, its mismatch can be in the internal region or terminal region of molecule.Generally, the most tolerable mismatch is in terminal region, for example, within 5, 4, 3 or 2 nucleotides of the 5'-end or 3'-end of RNAi agent.

[0123] In some embodiments, a double-stranded RNA agent of the present disclosure includes a nucleotide mismatch in the antisense strand.

[0124] In some embodiments, the antisense strand of a double-stranded RNA agent of the present disclosure contains four or fewer mismatches with the target mRNA, e.g., the antisense strand contains four, three, two, one, or zero mismatches with the target mRNA. In some embodiments, the antisense strand of a double-stranded RNA agent of the present disclosure contains four or fewer mismatches with the sense strand, e.g., the antisense strand contains four, three, two, one, or zero mismatches with the sense strand. In some embodiments, a double-stranded RNA agent of the present disclosure contains a nucleotide mismatch in the sense strand. In some embodiments, the sense strand of a double-stranded RNA agent of the present disclosure contains four or fewer mismatches with the antisense strand, e.g., the sense strand contains four, three, two, one, or zero mismatches with the antisense strand. In some embodiments, the nucleotide mismatch is, for example, within 5, 4, or 3 nucleotides from the 3' end of the iRNA. In another embodiment, the nucleotide mismatch is, for example, at the 3' terminal nucleotide of the iRNA agent. In some embodiments, the mismatch is not in the seed region.

[0125] Thus, the RNAi agents described herein can contain one or more mismatches to the target sequence. In one embodiment, the RNAi agents described herein contain three or fewer mismatches (i.e., three, two, one, or zero mismatches). In one embodiment, the RNAi agents described herein contain two or fewer mismatches. In one embodiment, the RNAi agents described herein contain one or fewer mismatches. In one embodiment, the RNAi agents described herein contain zero mismatches. In certain embodiments, when the antisense strand of an RNAi agent contains a mismatch to the target sequence, the mismatch can also be limited to within the last five nucleotides from the 5' or 3' end of the region of complementarity, as appropriate. For example, in such an embodiment, for a 23-nucleotide RNAi agent, the strand complementary to a region of the SNCA gene generally does not contain any mismatches within the central 13 nucleotides. Using methods described herein or known in the art, it can be determined whether an RNAi agent containing a mismatch to the target sequence is effective in inhibiting expression of the SNCA gene. It is important to consider the efficacy of mismatched RNAi agents to inhibit SNCA expression, especially when the specific region of complementarity in the SNCA gene is known to have polymorphic sequence variation within the population.

[0126] The term "sense strand" or "passenger strand," as used herein, refers to the strand of an RNAi agent that includes a region that is substantially complementary to a region of the antisense strand, as those terms are defined herein.

[0127] As used herein, "substantially all of the nucleotides are modified" means that most, but not all, of the nucleotides are modified and may include 5, 4, 3, 2, or 1 unmodified nucleotides.

[0128] As used herein, the term "cleavage region" refers to the region located directly adjacent to the cleavage site.Cleavage site is the site on the target where cleavage occurs.In some embodiments, the cleavage region comprises three bases that are directly adjacent to either end of the cleavage site.In some embodiments, the cleavage region comprises two bases that are directly adjacent to either end of the cleavage site.In some embodiments, specifically, the cleavage site occurs at the site that is bound by nucleotides 10 and 11 of the antisense strand, and the cleavage region comprises nucleotides 11, 12, and 13.

[0129] As used herein, unless otherwise specified, the term "complementary," when used to describe a first nucleotide sequence in the context of a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising a first nucleotide sequence to hybridize to form a duplex with an oligonucleotide or polynucleotide comprising the second nucleotide sequence under certain conditions, as understood by those skilled in the art. Such conditions may be, for example, stringent conditions, where stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, at 50°C or 70°C for 12-16 hours, followed by washing [see, e.g., "Molecular Cloning: A Laboratory Manual," Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press]. Other conditions, such as physiologically relevant conditions that may be encountered in an organism, may be applied. Those skilled in the art will be able to determine the most appropriate set of conditions for testing the complementarity of two sequences depending on the ultimate use of the hybridized nucleotides.

[0130] The complementary sequence in an RNAi agent, for example, in the dsRNA described herein, comprises the base pairing of the oligonucleotide or polynucleotide comprising the first nucleotide sequence with the oligonucleotide or polynucleotide comprising the second nucleotide sequence throughout the entire length of one or both nucleotide sequences.Such sequences can be referred to herein as " completely complementary " with respect to each other.However, in the present specification, when a first sequence is considered to be " substantially complementary " with a second sequence, the two sequences can be completely complementary, or they can form one or more, but generally not more than 5, 4, 3, or 2 mismatched base pairs during hybridization, while maintaining the ability to hybridize under the conditions most suitable for their final use, for example, the inhibition of gene expression by the RISC pathway, in the case of a duplex of up to 30 base pairs.However, when two oligonucleotides are designed to form one or more single-stranded overhangs during hybridization, these overhangs are not considered as mismatches when determining complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, where the longer oligonucleotide comprises a 21-nucleotide sequence that is perfectly complementary to the shorter oligonucleotide, can still be considered "perfectly complementary" for the purposes described herein.

[0131] "Complementary" sequences, as used herein, may also include or be formed entirely of non-Watson-Crick base pairs, or base pairs formed from non-naturally occurring modified nucleotides, so long as the above requirements regarding their ability to hybridize are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U Wobble or Hoogstein base pairing.

[0132] The terms "complementary," "fully complementary," and "substantially complementary" can be used herein in reference 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 in connection with their use.

[0133] As used herein, a polynucleotide that is "substantially complementary to at least a portion of" a messenger RNA (mRNA) means a polynucleotide that is substantially complementary to a continuous 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 SNCA mRNA if the sequence is substantially complementary to an uninterrupted portion of the mRNA encoding SNCA.

[0134] Thus, in some embodiments, the antisense polynucleotides disclosed herein are perfectly complementary to the target SNCA sequence.

[0135] In certain embodiments, the antisense strand polynucleotides disclosed herein are substantially complementary to a target SNCA sequence and comprise a contiguous nucleotide sequence that is at least 80% complementary, e.g., 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 to the equivalent region of the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, or 228, or a fragment of SEQ ID NO: 1, 3, 5, 7, or 228, over its entire length.

[0136] In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a target SNCA sequence and comprise a contiguous nucleotide sequence that is at least 80% complementary, e.g., 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 to any one of the sense strand nucleotide sequences in Table 2 or 3, or a fragment of any one of the sense strand nucleotide sequences in Table 2 or 3, throughout its length.

[0137] In one embodiment, an RNAi agent of the present disclosure comprises a sense strand that is substantially complementary to an antisense polynucleotide that is the same as a target SNCA sequence, wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least about 80% complementary, e.g., 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 to the nucleotide sequence of SEQ ID NO: 2, 4, 6, 8, or 229, or an equivalent region of a fragment of any one of SEQ ID NOs: 2, 4, 6, 8, or 229, throughout its entire length.

[0138] In some embodiments, an iRNA of the present disclosure comprises a sense strand that is substantially complementary to an antisense polynucleotide that is complementary to a target SNCA sequence, wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least 80%, e.g., 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 to any one of the antisense strand nucleotide sequences in Table 2 or 3, or a fragment of any one of the antisense strand nucleotide sequences in Table 2 or 3, throughout its length.

[0139] 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, 24, 25, 26, 27, 28, 29, 30 or more nucleotide pairs in length.

[0140] In some embodiments, the antisense strand of a double-stranded iRNA agent is at least equal to or is 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0141] In some embodiments, the sense strand of a double-stranded iRNA agent is at least equal to or is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0142] In one embodiment, the sense and antisense strands of the double-stranded iRNA agent are each 15 to 30 nucleotides in length.

[0143] In one embodiment, the sense and antisense strands of the double-stranded iRNA agent are each 19 to 25 nucleotides in length.

[0144] In one embodiment, the sense and antisense strands of the double-stranded iRNA agent are each 21 to 23 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 23 to 21 nucleotides in length.

[0147] In one embodiment, the sense strand of the iRNA agent is 21 nucleotides in length and the antisense strand is 23 nucleotides in length, and the strands form a double-stranded region of 21 contiguous base pairs with a 2-nucleotide long single-stranded overhang at the 3'-end.

[0148] In one embodiment, the sense strand of the iRNA agent is 23 nucleotides in length and the antisense strand is 21 nucleotides in length, and the strands form a double-stranded region of 21 contiguous base pairs with 1-nucleotide-long single-stranded overhangs at the 3'- and 5'-ends.

[0149] In some embodiments, the majority of the nucleotides in each strand are ribonucleotides; however, as described in detail herein, each or both strands can also contain one or more non-ribonucleotides, such as deoxyribonucleotides or modified nucleotides. Furthermore, an "iRNA" can contain ribonucleotides with chemical modifications. Such modifications can include all types of modifications disclosed herein or known in the art. All such modifications used in iRNA molecules are encompassed by "iRNA" for purposes of this specification and claims.

[0150] In one embodiment of the present disclosure, the agent for use in the methods and compositions of the present disclosure is a single-stranded antisense nucleic acid molecule that inhibits target mRNA through antisense inhibition mechanism.Single-stranded antisense RNA molecules are complementary to the sequence in target mRNA.Single-stranded antisense oligonucleotides can inhibit translation stoichiometrically by base pairing with mRNA and physically interfering with the translation mechanism.See Dias, N. et al., (2002) Mol Cancer Ther 1: 347-355.Single-stranded antisense RNA molecules can be about 15 to about 30 nucleotides in length and have a sequence that is complementary to the target sequence.For example, single-stranded antisense RNA molecules can comprise a sequence that is at least about 15, 16, 17, 18, 19, 20 or more consecutive nucleotides from any one of the antisense sequences described herein.

[0151] In one embodiment, at least partial suppression of expression of the SNCA gene is assessed by a decrease in the amount of SNCA mRNA that can be isolated from or detected in a first cell or group of cells in which the SNCA gene is transcribed and that has been treated or has been treated such that expression of the SNCA gene is inhibited 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 treated as the first. The degree of inhibition can be expressed in terms of the following formula:

[0152]

number

[0153] As used herein, the phrase " contacting cell with RNAi agent " such as dsRNA includes contacting cell by any possible means.Contacting cell with RNAi agent includes contacting cell with RNAi agent in vitro or contacting cell with RNAi agent in vivo.Contacting can be carried out directly or indirectly.Therefore, for example, RNAi agent can be physically contacted with cell by carrying out a method separately, or RNAi agent can be placed in a situation that can allow or cause it to contact cell afterwards.

[0154] Contacting cells in vitro can be achieved, for example, by incubating cells with an RNAi agent. Contacting cells in vivo can be achieved, for example, by injecting an RNAi agent into or near the tissue where the cells are located, or by injecting the RNAi agent into another region, for example, the central nervous system (CNS), by intrathecal injection, intravitreal injection, or other injection, as appropriate, or by injecting the RNAi agent into the bloodstream or subcutaneous space so that the agent subsequently reaches the tissue where the cells to be contacted are located. For example, the RNAi agent can contain or be coupled to a ligand that directs or stabilizes the RNAi agent to the desired site, for example, the CNS, such as a lipophilic moiety, as described below and further detailed in, for example, PCT / US2019 / 031170, which is incorporated herein by reference. In some embodiments, the RNAi agent can optionally contain or be coupled to a lipophilic moiety(s) in the absence of a GalNAc derivative. A combination of in vitro and in vivo contacting methods is also possible. For example, cells may be contacted with an RNAi agent in vitro and then transferred into a subject.

[0155] In one embodiment, contacting a cell with an RNAi agent includes "introducing" or "delivering an RNAi agent into a cell" by promoting or performing uptake or absorption into the cell. The absorption or uptake of the RNAi agent can occur by spontaneous diffusive or active cellular processes, or by auxiliary agents or devices. The introduction of an RNAi agent into a cell can be in vitro or in vivo. For example, in the case of in vivo introduction, the RNAi agent can be injected into a tissue site or administered systemically. In vitro introduction into a cell includes methods known in the art, such as electroporation and lipofection. Further approaches are described herein below or known in the art.

[0156] The terms "lipophilic" or "lipophilic moiety" refer 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 In this case, K ow is the ratio of the concentration of a chemical in the octanol phase to the concentration of the chemical in the aqueous phase in a two-phase system at equilibrium. The octanol-water partition coefficient is a laboratory-measured property of a substance. However, it can also be predicted by using coefficients attributable to the structural components of a chemical calculated using first principles or empirical methods [see, e.g., Tetko et al., J. Chem. Inf. Comput. Sci. 41:1407-21 (2001), incorporated herein by reference in its entirety]. It provides a thermodynamic measure of a substance's tendency to prefer a non-aqueous or oily environment rather than water (i.e., hydrophilic / lipophilic balance). In principle, a chemical can be expressed as a function of the log K ow is greater than 0, it is lipophilic in nature. Typically, a lipophilic moiety has a log K ow For example, the log K of 6-aminohexanol owis expected to be approximately 0.7. Using the same method, the log K ow is expected to be 10.7.

[0157] The lipophilicity of a molecule can be modified with respect to the functional groups it carries. For example, adding a hydroxyl or amine group to the end of the lipophilic moiety can increase the partition coefficient (e.g., logK ow ) value can be increased or decreased.

[0158] Alternatively, the hydrophobicity of the double-stranded RNAi agent that is conjugated with one or more lipophilic moieties can be measured by its protein binding properties.For example, in certain embodiments, the unbound fraction of the plasma protein binding assay of double-stranded RNAi agent can be determined to be positively correlated with the relative hydrophobicity of double-stranded RNAi agent, which can be positively correlated with the silencing activity of double-stranded RNAi agent.

[0159] In one embodiment, the plasma protein binding assay that is determined is electrophoretic mobility shift assay (EMSA) using human serum albumin protein.The exemplary protocol of this binding assay is described in detail in, for example, PCT / US2019 / 031170.The hydrophobicity of double-stranded RNAi agent measured by the fraction of unbound siRNA in 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.

[0160] Thus, conjugating a lipophilic moiety to an internal position of a double-stranded RNAi agent provides optimal hydrophobicity in siRNA for enhanced in vivo delivery.

[0161] The term "lipid nanoparticle" or "LNP" refers to a vesicle that comprises a lipid layer that encapsulates a pharmaceutically active molecule, such as a nucleic acid molecule, for example, an RNAi agent or the plasmid from which the RNAi agent is transcribed.LNP is described in, for example, U.S. Patent No. 6,858,225, U.S. Patent No. 6,815,432, U.S. Patent No. 8,158,601 and U.S. Patent No. 8,058,069, the entire contents of which are incorporated herein by reference.

[0162] As used herein, a "subject" is an animal, e.g., a mammal, e.g., a primate (e.g., a human, a non-human primate, e.g., a monkey or chimpanzee), or a non-primate (e.g., a rat or a mouse). In a preferred embodiment, the subject is a human, e.g., a human being treated or evaluated for a disease, disorder, or condition that would benefit from reduced SNCA expression; a human being at risk for a disease, disorder, or condition that would benefit from reduced SNCA expression; a human being with a disease, disorder, or condition that would benefit from reduced SNCA expression; or a human being treated for a disease, disorder, or condition that would benefit from reduced SNCA expression as described herein.

[0163] As used herein, the term "treating" or "treatment" refers to the alleviation of one or more signs or symptoms associated with SNCA gene expression or SNCA protein production, e.g., an SNCA-associated neurodegenerative disease, e.g., in a subject having such a neurodegenerative disease, SNCA expression or activity is decreased in areas of increased neuronal dysfunction or death, a synucleinopathy, e.g., PD, multiple system atrophy (MSA), dementia with Lewy bodies (LBD), pure autonomic failure (PAF), Pick's disease, progressive supranuclear palsy, "Treatment" refers to a beneficial or desired result, including, but not limited to, alleviating or ameliorating dementia pugilistica, chromosome 17-linked Parkinsonism, Rytschko-Bodig disease, tangle-predominant 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. "Treatment" can also mean prolonging survival as compared to expected survival if treatment is not performed.

[0164] The term "lower" in relation to the level of SNCA or a disease marker or symptom in a subject refers to a statistically significant decrease in such level. The decrease can be, for example, at least 10%, 15%, 20%, 25%, 30%, 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% of the disease marker, e.g., protein level or gene expression level. When referring to the level of SNCA in a subject, "lower" refers to a reduction to a level that is accepted as within the normal range in individuals without such disorder, as appropriate. In certain embodiments, "lower" refers to a decrease in the difference between the level of a marker or symptom in a subject suffering from a disease and a level that is accepted within the normal range for an individual, e.g., the level of decreased movement speed (bradykinesia) and ability to regulate posture and balance in an individual with Parkinson's disease versus an individual who does not have Parkinson's disease or whose symptoms are within the normal range.

[0165] As used herein, "prevention" or "preventing" refers to a disease or disorder that would benefit from a reduction in the expression of the SNCA gene or the production of SNCA protein in a subject who is predisposed to an SNCA-associated disorder, e.g., due to genetic factors or age, and in which the subject has not yet met the diagnostic criteria for an SNCA-associated disorder. As used herein, prevention can be understood as administering an agent to a subject who has not yet met the diagnostic criteria for an SNCA-associated disorder to delay or reduce the likelihood that the subject will develop an SNCA-associated disorder. Because the agent is a pharmaceutical, it is understood that administration is typically under the direction of a medical professional who is able to identify subjects who have not yet met the diagnostic criteria for an SNCA-associated disorder as being predisposed to developing an SNCA-associated disorder.

[0166] The term "synucleinopathy" refers to a group of neurodegenerative disorders characterized by fibrillar aggregates of α-synuclein protein, which tend to accumulate in the cytoplasm of select populations of neurons and glia. Synucleinopathy is therefore a classification of SNCA-associated neurodegenerative diseases and disorders, including Parkinson's disease (PD), dementia with Lewy bodies (LBD), pure autonomic failure (PAF), and multiple system atrophy (MSA), among other neurodegenerative disorders. Clinically, synucleinopathies are characterized by a chronic and progressive decline in motor, cognitive, behavioral, and autonomic function, depending on the distribution of intracerebral lesions. Due to clinical overlap, differential diagnosis can sometimes be very difficult. Parkinsonism is a cardinal symptom of PD, but it may be indistinguishable from parkinsonism in LBD and MSA. Autonomic dysfunction, an isolated finding in PAF, can also be present in PD and LBD, and is usually more pronounced and appears earlier in MSA. LBD is the same disease as PD, but may involve widespread cortical pathology, leading to dementia, cognitive fluctuations, and characteristic visual hallucinations.

[0167] The likelihood of developing a synucleinopathy, such as PD, LBD, etc., is reduced, for example, if an individual with one or more 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 compared to a population with the same risk factors and not receiving the treatment described herein. Not developing an SNCA-associated disorder, such as PD or LBD (or other synucleinopathy), or delaying the onset of PD or LBD (or other synucleinopathy) by several months or years is considered effective prevention. Prevention may require the administration of two or more doses of an iRNA agent. Suitable methods for identifying subjects at risk of developing any of the above-mentioned SNCA-associated diseases are provided, and the iRNA agents provided herein can be used as pharmaceuticals for or in methods of preventing SNCA-associated diseases. Risk factors for various SNCA-associated diseases are discussed herein.

[0168] As used herein, the terms "Parkinson's disease" or "PD" refer to a progressive nervous system disorder that affects movement. The primary pathological features of PD are cell death in the basal ganglia of the brain (by the end of the disease, up to 70% of the dopamine-secreting neurons in the substantia nigra are affected) and the presence of Lewy bodies (accumulation of SNCA-encoded α-synuclein protein) in many of the remaining neurons. Symptoms begin gradually and may range from barely noticeable tremor in one hand to rigidity or slowed movements. Other early symptoms include a blank expression, lack of arm movement while walking, and slurred speech. Parkinson's disease symptoms worsen over time. The average age at onset of PD is 60 years, and the later the onset, the greater the severity of the symptoms. Clinical features include, but are not limited to, more severe tremors, slowed movements (bradykinesia), muscle rigidity, impaired posture and balance, loss of motor control, changes in speech, and eventually dementia, hallucinations, and wheelchair confinement.

[0169] As used herein, the term "Lewy body dementia (LBD)" refers to a type of progressive dementia that results in a decline in thinking, reasoning, and independent function due to the aggregation of α-synuclein protein within affected brain neurons, known as Lewy bodies and Lewy neurites. The aggregation of α-synuclein protein causes affected nerve cells to function suboptimally and ultimately die. Symptoms include visual, auditory, olfactory, or tactile hallucinations; Parkinson's disease signs (parkinsonism); dysregulation of bodily functions (autonomic nervous system) such as dizziness, falls, and bowel problems; cognitive problems such as confusion; decreased attention, visual-spatial problems, and memory loss; sleep disorders such as rapid eye movement (REM) sleep behavior disorder (dreams are physically acted out while asleep); fluctuations in attention, including episodes of drowsiness; prolonged periods of spatial gaze; extended daytime napping or disorganized speech; depression; and lethargy.

[0170] 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, olivocerebellar atrophy, or striatocerebellar degeneration, MSA shares many Parkinson's disease-like symptoms, such as slowed movements, muscle stiffness, and poor balance. Treatment for MSA includes medications 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 contains nerve cells (neurons) containing abnormal amounts of alpha-synuclein. MSA subjects typically survive approximately 7 to 10 years after MSA symptoms first appear. However, survival rates for MSA vary widely. Occasionally, MSA subjects live for more than 15 years with the disease. Respiratory problems are often the cause of death. MSA progresses slowly, eventually leading to death. It affects many parts of the body. Symptoms of MSA typically begin in adulthood, usually in the 50s or 60s. MSA is classified into two types: Parkinsonian and cerebellar. The types differ based on the symptoms present at the time of diagnosis. Parkinsonian MSA is the most common type of MSA and has signs and symptoms similar to those of Parkinson's disease, including muscle stiffness, difficulty bending and straightening the limbs, slowed movements (bradykinesia), tremors (which are rare in MSA compared to classic Parkinson's disease), and problems with posture and balance. The signs and symptoms of cerebellar MSA include problems with muscle coordination (ataxia), movement and coordination disorders such as unsteady gait and loss of balance, slurred, slow, or faint speech (dysarthria), visual disturbances such as blurred or double vision, difficulty focusing, difficulty swallowing (dysphagia) or chewing, and general signs and symptoms.In addition, the main sign of multiple system atrophy is postural (orthostatic) hypotension, which is a form of hypotension that causes dizziness, lightheadedness, or fainting when the patient stands up from a sitting or lying position.In addition, MSA patients may develop dangerously high blood pressure levels while lying down (supine hypertension).Other difficulties associated with MSA include involuntary (autonomic) bodily functions such as bladder and bowel problems, constipation, and loss of bladder or bowel control (incontinence); sweating abnormalities, reduced sweat, tear, and saliva production; heat intolerance due to reduced sweating; impaired temperature regulation (often resulting in cold hands and feet); sleep disturbances; agitated sleep due to "acting out" dreams; abnormal nighttime breathing; sexual dysfunction, inability to get or maintain an erection (impotence); decreased libido; cardiovascular problems; changes in the color of the hands and feet caused by blood pooling; cold hands and feet; psychiatric problems; and difficulty controlling emotions such as inappropriate laughing or crying. Possible complications of MSA include abnormal breathing during sleep, injuries from falls caused by poor balance or fainting, progressive immobility that can lead to secondary problems such as skin wasting, loss of ability to care for daily activities, vocal cord paralysis that makes speaking and breathing difficult, and increasing difficulty swallowing.

[0171] 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).

[0172] As used herein, a "therapeutically effective amount" is intended to include an amount of an RNAi agent that, when administered to a subject with an SNCA-associated disease, is sufficient to treat the disease (e.g., by reducing, ameliorating, or maintaining an existing disease or one or more symptoms of the disease). A "therapeutically effective amount" may vary depending on the RNAi agent, how the agent is administered, the disease and its severity, as well as medical history, age, weight, family history, genetic makeup, type of prior or concurrent treatment, if any, and other individual characteristics of the subject being treated.

[0173] As used herein, a "prophylactically effective amount" is intended to include an amount of an RNAi agent sufficient to prevent or ameliorate the disease or one or more symptoms of the disease when administered to a subject with an SNCA-associated disease. Amelioration of the disease includes slowing the progression of the disease or reducing the severity of subsequent disease. A "prophylactically effective amount" may vary depending on the RNAi agent, how the agent is administered, the degree of risk of the disease, as well as the patient's medical history, age, weight, family history, genetic makeup, type of previous or concurrent treatment, if any, and other individual characteristics of the patient being treated.

[0174] A "therapeutically effective amount" or a "prophylactically effective amount" also encompasses the amount of an RNAi agent that produces some desired local or systemic effect at a reasonable benefit / risk ratio applicable to any treatment. The RNAi agent used in the methods of the present disclosure can be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0175] The phrase "pharmaceutically acceptable" is used herein to mean compounds, materials, compositions, or dosage forms that are suitable for use in contact with the tissues of human and animal subjects, within the scope of sound medical judgment, without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0176] The phrase "pharmaceutically acceptable carrier," as used herein, refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, magnesium talc, calcium or zinc stearate, or stearic acid), or solvent encapsulating material, involved in the transport or transportation of a compound of interest from one organ or part of the body to another organ, e.g., part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the subject being treated. Some examples of materials that can function 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, ethyl cellulose, 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 wax; and (9) oils, such as peanut oil, cottonseed oil, safflower oil, and sesame oil. , olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, 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) ethyl alcohol; (20) pH buffer 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 affinity substances used in pharmaceutical formulations.

[0177] The term "sample," as used herein, encompasses similar bodily fluids, cells, or tissues isolated from a subject, as well as collections of bodily fluids, cells, or tissues present within a subject. Examples of biological fluids include blood, serum, and serous fluid, plasma, cerebrospinal fluid, ocular fluid, lymphatic fluid, urine, saliva, and the like. Tissue samples can include samples from tissues, organs, or localized regions. For example, samples can be obtained from specific organs, parts of organs, or bodily fluids or cells within those organs. In certain embodiments, samples can be obtained from the brain (e.g., the entire brain or a segment in the brain, e.g., the striatum, or a type of cell in the brain, e.g., neurons and glial cells (astrocytes, oligodendrocytes, microglia)). In other embodiments, "a sample obtained from a subject" refers to blood obtained from the subject or plasma or serum obtained therefrom. In further embodiments, "a sample obtained from a subject" refers to brain tissue (or a subcomponent thereof) obtained from the subject. ) or retinal tissue (or a subcomponent thereof).

[0178] Although the sequences in Table 2 are described as modified or conjugated sequences, it will be understood that in certain embodiments, the RNA of an RNAi agent of the present disclosure, e.g., a dsRNA of the present disclosure, can comprise any one of the sequences set forth in Tables 2 or 3 that is modified or conjugated differently than described herein. Except where a specific position / location is specified, a lipophilic ligand can be included in any of the positions provided herein. [Brief explanation of the drawings]

[0179] [Figure 1]Figures 1A and 1B show the effects of selected SNCA-targeting RNAi agents on SNCA levels in human BE(2)-C neuroblastoma cells, a dual-luciferase reporter system in COS-7 African green monkey fibroblast-like cells, and human SNCA-AAV-overexpressing mice. Figure 1A shows the results of SNCA mRNA knockdown obtained with each siRNA for the SNCA-targeting 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 (the "parent" siRNAs carrying the triantennary GalNAc modification relative to the CNS-directed siRNAs of the present disclosure) using a dual luciferase reporter system in BE(2)-C cells and COS-7 cells, respectively. Figure 1B shows the results of in vivo liver SNCA knockdown of the indicated duplexes in human SNCA-AAV-overexpressing mice. To determine the RNA in vivo efficacy of RNAi compounds in mice, full-length human SNCA was first transduced by AAV. Seven days after AAV administration, the following selected duplexes were delivered: duplexes targeting the 3'UTR of human SNCA AD-464778, AD-464782, AD-464694, AD-464634, and AD-464779; and duplexes targeting the coding sequences of SNCA AD-464314, AD-464313, AD-464590, AD-464585, AD-464229, AD-464586, and AD-464592. Data were normalized to samples treated with PBS. [Figure 2] Figure 2 shows the SNCA knockdown results of the 40 most potent SNCA-targeting siRNAs (liver-targeting duplexes) based on a combination of hotspot and structure-activity relationship (SAR) evaluations for a total of 360 duplexes. The 40 siRNAs shown showed the highest SNCA knockdown results at a duplex concentration of 0.1 nM, demonstrating a clear linear dose response. [Figure 3]FIG. 3 shows a schematic 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). [Figure 4] Figure 4 shows the results of an in vivo AAV titration study in which human SNCA was administered to mouse liver using AAV, followed by evaluation of the extent of delivery and expression. On the left, the cycle threshold for PCR-mediated detection of human SNCA, used to assess human SNCA expression in mouse liver, was shown to decrease in a dose-responsive manner when human SNCA was 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), were demonstrated to robustly inhibit SNCA expression in mouse liver in vivo when administered at either 3 mg / kg (mpk) or 10 mpk to huSNCA-expressing mice. [Figure 5]Figures 5A and 5B show the results of in vivo SNCA knockdown in mouse liver and a schematic representation of the duplex sequence modification patterns for all 17 SNCA-targeting siRNAs evaluated in the in vivo AAV study. Figure 5A shows the results of in vivo human SNCA knockdown observed in mice administered human SNCA via AAV. The 17 newly evaluated SCNA-targeting siRNAs shown were compared to the previously identified SNCA-targeting duplex AD-464634.2 and a PBS control.Figure 5B shows the potent 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: 249), and AD-1746465 (sense strand: 5'-uscsuuugcuCfCfCfaguu(Uhd)cuugaL96-3' SEQ ID NO: 249; antisense strand: 5'-VPusdCsaadGadAacugdGgAfgcaaagasusa-3' SEQ ID NO: 249), which were selected for potential use as parent siRNAs for non-human primate (NHP) studies. SEQ ID NO: 250), AD-1571188 (sense strand: 5'-gsusaca(Ahd)GfuGfCfUfcaguuccaaaL96-3' SEQ ID 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' 25A-25C show schematic diagrams 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). [Figures 6A-6C]Figures 6A-6C show a comparison of mouse in vivo knockdown, in vitro knockdown, and location information for the seven SNCA-targeting siRNAs identified above that yielded three lead candidate parent siRNAs for NHP studies, with structures also shown. Figure 6A shows a comparison of mouse in vivo knockdown and in vitro knockdown for the seven SNCA-targeting siRNAs identified above: AD-1549333, AD-1746465, AD-1571188, AD-1549401, AD-1549054, AD-1746466, and AD-1548886. Figure 6B shows the location of the target SNCA mRNA for each of the candidate lead parent siRNAs AD-1549333, AD-1746465, AD-1571188, AD-1549401, AD-1549054, AD-1746466, and AD-1548886. Figure 6C shows, from top to bottom, 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). These parent duplexes were adapted to generate lead SNCA-targeting duplexes AD-1747585, AD-1747583, and AD-1747580, respectively, with the sequences and modification patterns shown in Table 2, for CNS administration in NHP studies. [Figure 7] Figure 7 presents a schematic diagram showing the three duplexes selected for the NHP study: AD-1747585 (with sense strand SEQ ID NO: 35 and antisense strand SEQ ID NO: 78), AD-1747583 (with sense strand SEQ ID NO: 28 and antisense strand SEQ ID NO: 71), and AD-1747580 (with sense strand SEQ ID NO: 21 and antisense strand SEQ ID NO: 64). [Figure 8]Figures 8A and 8B show the initial selection and pharmacokinetic distribution of three candidate lead duplexes that were progressed to testing in non-human primate (NHP) animals. Figure 8A shows the results of initial in vivo efficacy and ED50 values of three candidate lead duplexes selected for progression to NHP animal studies. Figure 8B shows that each of the three candidate lead SNCA-targeted duplexes administered by intrathecal injection of a single 60 mg dose of siRNA to non-human primate (NHP) animals showed similar pharmacokinetics among the animals administered for 24 hours after injection. [Figure 9] Figures 9A and 9B show pharmacokinetic (PK) data and pharmacodynamic (PD) data obtained from NHP animal studies of three candidate lead SNCA-targeted duplexes administered by intrathecal injection. Figure 9A shows that on day 29 after injection ("D29"), each of the three SNCA-targeted duplexes tested showed a high-potency reduction of SNCA mRNA in NHP tissues of both the prefrontal cortex and the midbrain (left panel, PD results). A strong correlation was observed between tissue PK (tissue concentration of the duplexes tested) and tissue SNCA mRNA PD across the prefrontal cortex and midbrain tissues of the NHP tested (right panel, PK / PD results), and the three duplexes tested showed similar potencies (no statistically significant differences were observed between the three leads at the 29-day time point based on the results of mRNA knockdown). Nominally, the following IC50 rankings were observed in NHP on day 29: AD-1747583 (0.532 μg / g) < AD-1747580 (1.050 μg / g) < AD-1747585 (1.300 μg / g). Figure 9B shows that on day 29 after injection ("D29"), α-synuclein protein levels were decreased in the brains and spinal cords of NHPs that received intrathecal administration of SNCA-targeted duplexes, and little difference was observed among the three candidate lead agents tested. [Figure 10] Figure 10 shows an overview of the NHP data on day 29. [Figure 11]Figure 11 shows that at day 84 ("D84") after intrathecal administration of SNCA-targeting duplexes or control molecules, significant levels of SNCA mRNA knockdown were observed in both prefrontal cortex and midbrain tissues across all three candidate lead duplexes tested (left panel). Consistent with the results observed at day 29 post-administration, the day 84 samples showed good correlation between tissue PK and tissue mRNA PD, with similar efficacy observed for each of the three candidate lead duplexes investigated (right panel). Notably, only one animal in the AD-1747585-treated cohort met the dosing criteria, resulting in the remainder of the AD-1747585-treated cohort being re-administered. [Figures 12A-12E]Figures 12A-12E show that both SNCA mRNA and α-synuclein protein levels were reduced in different brain regions at 84 days post-administration with little differentiation between the three candidate lead duplexes tested (AD-1747580, AD-1747583, and AD-1747585). Also shown are SNCA mRNA / α-synuclein protein correlation and re-administration data. Figure 12A shows that SNCA mRNA was reduced in NHPs at 84 days post-administration of 60 mg of the duplex via the intrathecal route. Figure 12B shows that α-synuclein protein levels were also reduced in the brain and spinal cord (across all regions examined) at 84 days post-administration with little differentiation between the three candidate lead duplexes. Figure 12C shows that α-synuclein protein levels were highly correlated with SNCA mRNA levels across the different brain regions examined at 84 days post-administration. Figure 12D shows that certain NHPs identified as mis-dosed upon initial dosing were re-administered and brain and spinal cord tissues examined beyond day 84. SNCA NHP tissue protein pharmacodynamics (PD) demonstrated robust knockdown across various brain regions, particularly in AD-1747580-readministered NHPs. Figure 12E shows α-synuclein protein levels in cerebrospinal fluid (CSF) over an extended time course, including re-administration and monitoring of certain originally mis-administered NHPs beyond day 84. In such re-administered NHP animals, CSF α-synuclein protein levels showed a reduction of up to 75% for animals (re-)dosed with the dual-chain AD-1747585, while CSF α-synuclein protein levels showed a reduction of up to 50% for animals (re-)dosed with the dual-chain AD-1747580 and AD-1747583. [Figure 13]Figure 13 shows that at 84 days after duplex administration, animals treated with duplexes AD-1747580 and AD-1747583 showed a maximum 90% reduction in the observed levels of α-synuclein protein in NHP CSF (PD effect). While duplex AD-1747585 showed approximately 75% knockdown of CSF α-synuclein protein, notably duplex AD-1747585 also showed variable knockdown of CSF α-synuclein protein, which may be due to blood contamination due to the high expression of SNCA in blood cells. [Figures 14A-14D]Figures 14A-14D show the discrepancy observed between knockdown tissue SNCA mRNA levels and knockdown α-synuclein protein levels in the same tissues at day 29 (the "early" time point), with such discrepancy resolving at day 84, indicating that such effects are due to the long half-life of α-synuclein protein in the tissues examined. Figure 14A shows that at day 29 post-administration, α-synuclein protein (PD effect of duplexes) in the cerebral cortex and midbrain of treated NHPs showed modest (<60%) knockdown for all three lead duplexes tested (left panel). Such modest reductions in α-synuclein protein levels in the cerebral cortex and midbrain indicated an extended half-life of α-synuclein protein in those tissues. Tissue protein PK / PD was observed to be disparate for all three duplexes tested (center panel); however, tissue α-synuclein protein levels were highly correlated with tissue SNCA mRNA levels (right panel). Figure 14B shows that α-synuclein protein knockdown in CSF correlated with α-synuclein protein knockdown in the cerebral cortex (left panel) and striatum (center panel) at 29 days post-administration. In contrast, α-synuclein protein knockdown in midbrain tissue correlated poorly with CSF α-synuclein protein knockdown at 29 days post-administration (right panel). Figure 14C shows that SNCA mRNA levels were reduced in prefrontal cortex and midbrain tissues at 29 days post-administration (here, for the SNCA-targeting duplex AD-1747580), while α-synuclein protein levels were only slightly reduced, indicating that the long protein half-life of α-synuclein protein likely explains this discrepancy. Figure 14D shows that at 84 days post-administration, α-synuclein protein levels in cerebral cortex and midbrain tissues of treated NHP animals showed robust knockdown for all three lead duplexes (PD effect, left panel).Robust α-synuclein protein knockdown observed in cerebral cortex and midbrain NHP tissues at day 84 confirmed the long protein half-life of α-synuclein. Tissue protein PK / PD curves showed similar profiles for all three duplexes (middle panel; AD-1747585 cohort showed only one animal met dosing criteria, with the remainder of the cohort being re-dosed). α-synuclein protein levels were highly correlated with mRNA levels in both cerebral cortex and midbrain tissues (right panel). [Figure 15] FIG. 15 shows that α-synuclein protein knockdown in the CSF of NHP animals also correlates with α-synuclein protein knockdown in the prefrontal cortex and midbrain at 84 days after duplex administration. [Figure 16] Figure 16 shows the tissue exposure of siRNA across various tested brain regions in NHP animals at 84 days after duplex administration. With few exceptions, tissue penetration of the siRNA agents was good, even though the tissues most exposed to the siRNA duplexes (e.g., prefrontal cortex) did not necessarily show the greatest observed levels of knockdown of SNCA mRNA and / or α-synuclein protein. [Figure 17] Figure 17 shows that none of the three candidate lead duplexes administered to NHP animals had a significant effect on body weight at either 29 or 84 days post-administration compared to control NHP animals administered vehicle alone in parallel. [Figure 18] Figure 18 shows the results of detecting neurofilament light chain (NfL) in the CSF of NHP animal cohorts administered with candidate lead duplexes compared to control NHP animal cohorts administered with artificial CSF (aCSF) only. Although transient spikes in NfL were observed after administration of all three duplexes, these NfL spikes were largely persistent beyond day 29 post-administration, indicating that the three SNCA-targeting candidate duplexes did not appear to have an NfL profile indicative of duplex toxicity. [Figures 19A-19D]Figures 19A-19D show RNA-Seq data for each of the three candidate lead duplexes investigated. Figure 19A shows a schematic of the AD-1747580 duplex (sense strand: SEQ ID NO: 21; antisense strand: SEQ ID NO: 64), and the RNA-Seq data show that in cells administered the AD-1747580 duplex and subjected to RNA-Seq analysis, 93% knockdown of SNCA was achieved, while no other loci across the genome showed greater than 50% knockdown (or robust, significant increases) in the presence of the AD-1747580 duplex compared to the appropriate controls. Figure 19B shows the potency-matched RNA-Seq profile of the candidate lead duplex AD-1747580, where the RNA-Seq results for the parent duplex AD-1549054 are also shown. On the left, a schematic diagram of the GalNAc-modified AD-1549054 parent duplex is shown (sense strand: 5'-gsasgca(Ahd)guGfAfCfaaauguuggaL96-3' SEQ ID NO: 255; antisense strand: 5'-VPusdCscadAcdAuuugdTcAfcuugcucsusu-3' SEQ ID NO: 256); the RNA-Seq data for this duplex demonstrated approximately 74% SNCA knockdown in treated cells (treated with a 10 nM dose of the AD-1549054 parent duplex). In the middle and right, schematic diagrams of the AD-1747580 duplex (sense strand: SEQ ID NO: 21; antisense strand: SEQ ID NO: 64) over RNA-Seq results for the duplex at 1 nM (middle, showing approximately 78% SNCA knockdown) and 10 nM (right, showing approximately 93% SNCA knockdown) doses, respectively. Figure 19C shows the potency-matched RNA-Seq profile of the candidate lead duplex AD-1747583, where the RNA-Seq results of the parent duplex AD-1549283 are also shown.On the left, a schematic diagram of the GalNAc-modified AD-1549283 parent duplex is shown (sense strand: 5'-uscsuuu(Ghd)cuCfCfCfaguuucuugaL96-3' SEQ ID NO: 268; antisense strand: 5'-VPusdCsaadGadAacugdGgAfgcaaagasusa-3' SEQ ID NO: 269); the RNA-Seq data for this duplex demonstrated approximately 79% SNCA knockdown in treated cells (treated with a 10 nM dose of the AD-1549283 parent duplex). In the middle and right, schematic diagrams of the AD-1747583 duplex (sense strand: SEQ ID NO: 28; antisense strand: SEQ ID NO: 71) are shown over the RNA-Seq results for the duplex at 1 nM (middle, showing approximately 67% SNCA knockdown) and 10 nM (right, showing approximately 92% SNCA knockdown) doses, respectively. Notably, three off-target loci were identified as significantly affected (>50% reduction) by administration of the AD-1747583 duplex: PYGB, NREP, and LCLAT1. Figure 19D shows the potency-matched RNA-Seq profile of the candidate lead duplex AD-1747585, where the RNA-Seq results of the parent duplex AD-1549333 are also shown. On the left, a schematic of the GalNAc-modified AD-1549333 parent duplex is shown (sense strand: 5'-asasgug(Chd)ucAfGfUfuccaaugugaL96-3' SEQ ID NO: 247; antisense strand: 5'-VPusdCsacdAudTggaadCuGfagcacuusgsu-3' SEQ ID NO: 248), and the RNA-Seq data above for this duplex demonstrated approximately 81% SNCA knockdown in treated cells (treated with a 10 nM dose of the AD-1549333 parent duplex). Center and right are schematic diagrams of AD-1747585 duplexes (sense strand: SEQ ID NO: 35; antisense strand: SEQ ID NO: 78) over RNA-Seq results of duplexes at doses of 0.1 nM (center left, indicating approximately 68% SNCA knockdown), 1 nM (center right, indicating approximately 93% SNCA knockdown), and 10 nM (right, indicating approximately 96% SNCA knockdown), respectively.Of note, one off-target locus was identified as being significantly affected (>50% reduction) by administration of the AD-1747585 duplex: HMGB2. [Figure 20] FIG. 20 shows a table summarizing the knockdown and RNA-Seq data for all three candidate lead SNCA-targeting duplexes administered to NHP animals. DETAILED DESCRIPTION OF THE INVENTION

[0180] Throughout the figures, the term "2-C16" refers to a 2'-O-hexadecyl modification. The present invention is further illustrated by the following detailed description.

[0181] Detailed Description of the Invention The present disclosure provides RNAi compositions that perform RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of the SNCA gene. The SNCA gene can be in a cell, e.g., a cell in a subject, such as a human. The present disclosure also provides methods for inhibiting expression of the SNCA gene or for treating disorders that would benefit from inhibiting or reducing expression of the SNCA gene, such as SNCA-associated diseases, e.g., synucleinopathies, e.g., PD, multiple system atrophy (MSA), dementia with Lewy bodies (LBD), pure autonomic failure (PAF), Pick's disease, progressive supranuclear palsy, dementia pugilistica, chromosome 17-linked parkinsonism, and Ricciko-Bodig. Also provided are methods of using the RNAi agent compositions of the present disclosure to treat subjects with Alzheimer's disease, tangle-predominant 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's syndrome, psychosis, schizophrenia, and Creutzfeldt-Jakob disease.

[0182] RNAi agents of the present disclosure may be about 30 nucleotides in length or less, e.g., 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24 , 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, which region is substantially complementary to at least a portion of an mRNA transcript of the SNCA gene. In certain embodiments, the RNAi agents of the present disclosure comprise an RNA strand (antisense strand) having a region that is about 21-23 nucleotides in length, which region is substantially complementary to at least a portion of an mRNA transcript of the SNCA gene.

[0183] In certain embodiments, RNAi agents of the present disclosure comprise an RNA strand (antisense strand) having a region of at least 19 contiguous nucleotides that is substantially complementary to at least a portion of an mRNA transcript of an SNCA gene, and can comprise a longer length, e.g., up to 66 nucleotides, e.g., 36-66, 26-36, 25-36, 31-60, 22-43, 27-53 nucleotides in length. These RNAi agents with longer antisense strand lengths may also comprise a second RNA strand (sense strand) of 20-60 nucleotides in length, in which case the sense and antisense strands form a duplex of 18-30 contiguous nucleotides.

[0184] The use of these RNAi agents allows for the targeted degradation of the mRNA of the SNCA gene in mammals. Thus, methods and compositions comprising these RNAi agents are useful for treating subjects who would benefit from a decrease in the level or activity of SNCA protein, such as subjects with an SNCA-associated neurodegenerative disease, e.g., a synucleinopathy, e.g., PD, multiple system atrophy (MSA), dementia with Lewy bodies (LBD), pure autonomic failure (PAF), Pick's disease, progressive supranuclear palsy, dementia pugilistica, parkinsonism linked to chromosome 17, Richko-Bodig disease, tangle-predominant 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's syndrome, psychosis, schizophrenia, and Creutzfeldt-Jakob disease.

[0185] The accumulation of α-synuclein within neurons is thought to lead to the formation of Lewy bodies (round, eosinophilic hyaline inclusions measuring 10–20 pm) or Lewy neurites (elongated, filamentous, dystrophic axons and dendrites). While not wishing to be bound by theory, Lewy body and Lewy neurite deposition is widespread in PD brains, with the substantia nigra being the most common area for such deposition. Because PD manifests with both motor (substantia nigra-based) and non-motor (other parts of the brain, such as the cerebral cortex) symptoms, the pathology is widespread. Substantia nigra cells are crucial for the execution of movement and postural functions, explaining the nature of PD's motor symptoms, while the impact on other cell types explains PD's non-motor symptoms, such as dementia, mood changes, and depression. In LBD brains, widespread deposition of Lewy bodies and Lewy neurites is seen in both the midbrain and cerebral cortex.

[0186] Alpha-synuclein is a protein found primarily in neurons. Within neurons, it is primarily located presynaptically and is speculated to play a role in regulating synaptic activity. Three major isoforms of alpha-synuclein have been identified, the longest and most common of which contains 140 amino acids.

[0187] Oxidative stress is involved in several neurodegenerative diseases 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 can also lead to the generation of highly reactive aldehydes from polyunsaturated fatty acids (Yoritaka et al., 1996).

[0188] Brain pathology indicative of Alzheimer's disease (AD), i.e., amyloid plaques and neurofibrillary tangles, is present in approximately 50% of LBD cases. It is unclear whether the coexistence of the pathologies represents two distinct diseases or simply variants of each disorder. Cases with the coexisting pathologies are sometimes described as the Lewy body variant of AD (Hansen et al., 1990).

[0189] Research has also suggested a role for SNCA in AD and Down syndrome, as it has been demonstrated that in these disorders, α-synuclein protein accumulates in the limbic system ( Crews et al., 2009 ).

[0190] Rare, dominantly inherited forms of PD and LBD can be caused by point mutations or duplications in the SNCA gene. Pathogenic mutations in A30P and A53T (Kruger et al., 1998) (Polymeropoulos et al., 1998) and gene duplications (Chartier-Harlin et al., 2004) have been described to cause familial PD, whereas other α-synuclein mutations, E46K (Zarranz et al., 2004) and α-synuclein gene triplications (Singleton et al., 2003), have been reported to cause PD or LBD.

[0191] The pathogenic consequences of α-synuclein mutations are only partially understood. However, in vitro data indicate that A30P and A53T mutations increase the rate of aggregation (Conway et al., 2000). A wide range of differently organized α-synuclein species (monomers, dimers, and oligomers, including protofibrils) are involved in the aggregation process, all of which may have different toxicities. It is unclear which molecular species exerts toxicity in the brain. However, studies have shown that oligomeric forms of α-synuclein are particularly neurotoxic. Further evidence for the role of oligomers comes from the observation that certain α-synuclein mutations (A30P and A53T) that cause inherited Parkinson's disease increase the rate of oligomerization.

[0192] How the α-synuclein aggregation cascade begins is not fully understood. Conformational changes in monomeric α-synuclein likely initiate the formation of dimers and trimers, and these intermediate-sized species continue to form more soluble oligomers, including protofibrils, before being deposited as insoluble fibrils in Lewy bodies. Once formed, α-synuclein oligomers may associate with new α-synuclein monomers and / or smaller multimers, thus accelerating the fibrillation process. Since some evidence suggests that α-synuclein pathology can spread from neuron to neuron in affected brains, such seeding may also occur in the extracellular space.

[0193] The following detailed description discloses methods for making and using compositions containing RNAi agents to inhibit expression of the SNCA gene, as well as compositions or methods for treating subjects with diseases and disorders that would benefit from inhibiting or reducing expression of the gene.

[0194] I. RNAi Agents of the Disclosure Described herein are RNAi agents that inhibit the expression of the SNCA gene. In one embodiment, the RNAi agent is administered to a subject, e.g., a mammal, for example, a subject suffering from an SNCA-associated neurodegenerative disease, e.g., a synucleinopathy, e.g., PD, multiple system atrophy (MSA), dementia with Lewy bodies (LBD), pure autonomic failure (PAF), Pick's disease, progressive supranuclear palsy, dementia pugilistica, parkinsonism linked to chromosome 17, Rytzko-Bodig disease, tangle-predominant dementia, argyrophilic grain disease, ganglioglioma, ganglion cell carcinoma, or the like. The present invention also includes double-stranded ribonucleic acid (dsRNA) molecules for inhibiting expression of the SNCA gene in cells, such as cells of humans with encephalopathy, 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 region of complementarity that is complementary to at least a portion of the mRNA formed upon expression of the SNCA gene. In embodiments, the region of complementarity is about 15-30 nucleotides in length or less. Upon contact with a cell expressing the SNCA gene, the RNAi agent inhibits expression of the SNCA gene (e.g., human gene, primate gene, non-primate gene) by at least 50%, as assessed, for example, by PCR or branched DNA (bDNA)-based methods, or by protein-based methods, such as immunofluorescence analysis using Western blotting or flow cytometry techniques.

[0195] dsRNA comprises two RNA strands, which are complementary and hybridize to form a duplex structure under the conditions that dsRNA is used.One strand of dsRNA (antisense strand) comprises a complementary region that is substantially complementary, and generally completely complementary to the target sequence.The target sequence can be obtained from the sequence of mRNA formed during the expression of SNCA gene.The other strand (sense strand) comprises a region that is complementary to the antisense strand, so that when the two strands are combined under suitable conditions, they hybridize to form a duplex structure.As described elsewhere herein and known in the art, the complementary sequence of dsRNA can also be contained as a self-complementary region of a single nucleic acid molecule, so that they are opposite each other on separate oligonucleotides.

[0196] Generally, the duplex structure is 15 to 30 base pairs in length, e.g., 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-22, 19-23, 19-24, 19-25, 19-26, 19-27, 19-28, 19-29, 19-30, 19-31, 19-32, 19-33, 19-34, 19-35, 19-36, 19-37, 19-38, 19-39, 19-40, 19-41, 19-42, 19-43, 19-44, 19-45, 19-46, 19-47, 19-48, 19-49, 19-50, 19-51, 19-52, 19-53, 19-54, 19-55, 19-56, 19-57, 19-58, 19-59, 19-60, 19-61, 19-62, 19-63, 19-64, 19-65, 19- 9, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. In certain preferred embodiments, the duplex structure is 18 to 25 base pairs in length, e.g., 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-25, 20-24, 20-23, 20-22, 20-21, 21-25, 21-24, 21-23, 21-22, 22-25, 22-24, 22-23, 23-25, 23-24, or 24-25 base pairs in length, e.g., 19-21 base pairs in length. Ranges and lengths intermediate to the above-listed ranges and lengths are also contemplated as part of the present disclosure.

[0197] Similarly, the region of complementarity to the target sequence may be 15 to 30 nucleotides in length, e.g., 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19- 27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, e.g., 19-23 nucleotides in length or 21-23 nucleotides in length. Ranges and lengths intermediate to the above-listed ranges and lengths are also contemplated as part of this disclosure.

[0198] In some embodiments, the dsRNA is 15 to 23 nucleotides long, 24 to 23 nucleotides long (optionally 25 to 30 nucleotides long). Generally, the dsRNA may be long enough to serve as a substrate for the Dicer enzyme. For example, it is well known in the art that dsRNA longer than about 21-23 nucleotides can serve as a substrate for Dicer. As those skilled in the art will recognize, the region of RNA targeted for cleavage is most often a portion of a longer RNA molecule, often an mRNA molecule. In relevant cases, a "portion" of the mRNA target is a continuous sequence of the mRNA target long enough to be a substrate for RNAi-dependent cleavage (i.e., cleavage by the RISC pathway).

[0199] Those skilled in the art will appreciate that the duplex region may be a primary functional portion of a dsRNA, e.g., 15 to 36 base pairs, e.g., 15-36, 15-35, 15-34, 15-33, 15-32, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-22, 18-23, 18-24, 18-25, 18-24, 18-23, 18-22, 18-21, 18-25, 18-26, 18-27, 18-28, 18-29, 18-30, 18-31, 18-32, 18-33, 18-34, 18-35, 18-36, 18-37, 18-38, 18-39, 18-40, 18-41, 18-42, 18-43, 18-44, 18-45, 18-46, 18-47, 18-48, 18-49, 18-50, 18-51, 18-52, 18- It will also be appreciated that the duplex region may be 8-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs, e.g., 19-21 base pairs. Therefore, in one embodiment, an RNA molecule or a complex of RNA molecules with a duplex region of more than 30 base pairs is dsRNA, as long as it is processed into a functional duplex of, for example, 15-30 base pairs, which targets the desired RNA for cleavage.Therefore, those skilled in the art will recognize that in one embodiment, miRNA is dsRNA.In another embodiment, dsRNA is not naturally occurring miRNA.In another embodiment, the RNAi agent useful for targeting SNCA expression is not produced in target cells by cleavage of larger dsRNA.

[0200] The dsRNA described herein can further comprise one or more single-stranded nucleotide overhangs, for example, 1, 2, 3 or 4 nucleotides.Nucleotide overhangs can comprise or consist of nucleotide / nucleoside analogs such as deoxynucleotides / nucleosides.Overhangs can be on sense strand, antisense strand, or any combination thereof.In addition, the nucleotide of overhang can be on the 5' end, 3' end, or both of the antisense strand or sense strand of dsRNA.In certain embodiments, longer and extended overhangs are possible.

[0201] 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 Biosearch, Applied Biosystems, Inc., as further described below.

[0202] The iRNA compounds of the present disclosure may be prepared using a two-step procedure. First, the individual strands of the double-stranded RNA molecule are prepared separately. Then, the component strands are annealed. The individual strands of the siRNA compounds can be prepared using solution phase or solid phase organic synthesis, or both. Organic synthesis offers the advantage that oligonucleotide strands containing unnatural nucleotides or modified nucleotides can be easily prepared. The single-stranded oligonucleotides of the present disclosure can be prepared using solution phase or solid phase organic synthesis, or both.

[0203] siRNAs can be produced by a variety of methods, e.g., in bulk. Exemplary methods include organic synthesis and RNA cleavage, e.g., in vitro cleavage.

[0204] siRNAs can be produced by separately synthesizing a single-stranded RNA molecule, or each strand of a double-stranded RNA molecule, and then annealing the constituent strands.

[0205] Large bioreactors, such as OligoPilot II from Pharmacia Biotec AB (Uppsala, Sweden), can be used to produce large quantities of specific RNA strands for a given siRNA. The OligoPilot II reactor can efficiently couple nucleotides using only a 1.5 molar excess of phosphoramidite nucleotides. Ribonucleotide amidites are used to generate RNA strands. Standard cycles of monomer addition can be used to synthesize 21- to 23-nucleotide strands of siRNA. Typically, the two complementary strands are generated separately and then annealed, for example, after release from the solid support and deprotection.

[0206] Organic synthesis can be used to generate distinct siRNA species.The complementarity of this species to SNCA gene can be precisely specified.For example, this species can be complementary to the region that contains polymorphism, for example, single nucleotide polymorphism.Furthermore, the location of polymorphism can be precisely defined.In some embodiments, polymorphism is located in internal region, for example, at least 4, 5, 7 or 9 nucleotides from one or both ends.

[0207] In one embodiment, the generated RNA is carefully purified to remove ends.iRNA is cut into siRNA in vitro, for example, by using Dicer or equivalent RNAse III-based activity.For example, dsiRNA can be incubated in an in vitro extract from Drosophila, or can be incubated with purified components, for example, purified RNAse or RISC (RNA-induced silencing complex).For example, see Ketting et al. Genes Dev 2001 Oct 15;15(20):2654-9 and Hammond Science 2001 Aug 10;293(5532):1146-50.

[0208] Cleavage of a dsiRNA generally generates multiple siRNA species, each a specific 21- to 23-nt fragment of the source dsiRNA molecule. For example, there may be siRNAs that contain sequences complementary to overlapping and adjacent regions of the source dsiRNA molecule.

[0209] Regardless of synthesis method, siRNA preparation can be prepared in a solution (for example, aqueous solution or organic solution) suitable for formulation.For example, siRNA preparation can be precipitated, redissolved in pure double distilled water, and lyophilized.The dried siRNA can then be resuspended in a solution suitable for the intended formulation process.

[0210] In one embodiment, the dsRNA of the present disclosure comprises at least two nucleotide sequences, i.e., a sense strand and an antisense strand.The sense strand sequence for SNCA can be selected from the group of sequences provided in Table 2 or 3, and the corresponding nucleotides of the sense strand and the antisense strand can be selected from the group of sequences in Table 2 or 3. In this embodiment, one of the two sequences is complementary to the other of the two sequences, and in this case, one of the sequences is substantially complementary to the sequence of the mRNA generated during the expression of the SNCA gene.Therefore, in this embodiment, the dsRNA will comprise two oligonucleotides, one oligonucleotide being described as the sense strand (passenger strand) in Table 2 or 3, and the second oligonucleotide being described as the antisense strand (guide strand) corresponding to the sense strand in Table 2 or 3 for SNCA.

[0211] In one embodiment, the sequences substantially complementary to the dsRNA are contained in separate oligonucleotides, hi another embodiment, the sequences substantially complementary to the dsRNA are contained in a single oligonucleotide.

[0212] Although the sequences provided herein are described as modified or conjugated sequences, it will be understood that the RNA of the RNAi agents of the present disclosure, e.g., the dsRNA of the present disclosure, can comprise any one of the sequences set forth in Tables 2 or 3 that is modified or conjugated differently than described herein. One or more lipophilic ligands can be included at any of the positions of the RNAi agents provided herein.

[0213] Those skilled in the art are well aware that dsRNAs with duplex structures of about 20 to 23 base pairs, for example, 21 base pairs, are hailed 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-described embodiment, due to the nature of the oligonucleotide sequences provided herein, the dsRNAs described herein can comprise at least one strand with a minimum length of 21 nucleotides. It can be reasonably expected that shorter duplexes, minus a few nucleotides at one or both ends, can be similarly effective compared to the dsRNAs described above. Thus, dsRNAs having a sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides derived from one of the sequences provided herein and that differ in their ability to inhibit expression of the SNCA gene by no more than 10, 15, 20, 25, or 30% inhibition from dsRNAs containing the complete sequence using in vitro assays with Be(2)-C cells and an RNA agent at a 10 nM concentration and the PCR assays provided in the Examples herein are contemplated to be within the scope of this disclosure.

[0214] One benchmark assay for inhibition of SNCA involves contacting human Be(2)-C cells with a dsRNA agent disclosed herein, and sufficient or effective SNCA inhibition is identified if at least a 5%, at least a 10%, at least a 15%, at least a 20%, at least a 25%, at least a 30%, at least a 35%, at least a 40%, at least a 45%, at least a 50%, at least a 55%, at least a 60%, at least a 65%, at least a 70%, at least a 75%, at least a 80%, at least a 85%, at least a 90%, at least a 95%, at least a 97%, at least a 98%, at least a 99%, or more reduction in SNCA transcript or protein is observed in the contacted cells compared to an appropriate control (e.g., cells not contacted with the SNCA-targeting dsRNA). A dsRNA agent of the disclosure may be administered at a 10 nM concentration, and a PCR assay may be performed as provided in the Examples herein (eg, Example 2 below).

[0215] In addition, the RNA described herein specifies the site of SNCA transcript that is susceptible to RISC-mediated cleavage.Therefore, the present disclosure further features the RNAi agent that targets within this site.As used herein, if an RNAi agent promotes the cleavage of the transcript at any of the specific sites, it is said to target within the specific site of RNA transcript.Such RNAi agent will generally comprise at least about 15 nucleotides, optionally at least 19 nucleotides, from one of the sequences provided herein, coupled with additional nucleotide sequences taken from the region adjacent to the selected sequence in SNCA gene.

[0216] The RNAi agents described herein may contain one or more mismatches to the target sequence. In one embodiment, the RNAi agents described herein contain three or fewer mismatches (i.e., three, two, one, or zero mismatches). In one embodiment, the RNAi agents described herein contain two or fewer mismatches. In one embodiment, the RNAi agents described herein contain one or fewer mismatches. In one embodiment, the RNAi agents described herein contain zero mismatches. In certain embodiments, when the antisense strand of an RNAi agent contains a mismatch to the target sequence, the mismatch can also be limited to within the last five nucleotides from the 5'-end or 3'-end of the region of complementarity, as appropriate. For example, in such an embodiment, for a 23-nucleotide RNAi agent, the strand complementary to a region of the SNCA gene generally does not contain any mismatches within the central 13 nucleotides. Using the methods described herein or known in the art, it can be determined whether an RNAi agent containing a mismatch to the target sequence is effective in inhibiting expression of the SNCA gene. It is important to consider the efficacy of mismatched RNAi agents in inhibiting SNCA gene expression, especially when the specific region of complementarity in the SNCA gene is known to have polymorphic sequence variation within the population.

[0217] II. Modified RNAi Agents of the Present Disclosure In a preferred embodiment, the RNA of the RNAi agent of the present disclosure, for example, dsRNA, is chemically modified to enhance stability or other beneficial characteristics.In a specific embodiment of the present disclosure, substantially all of the nucleotides of the RNAi agent of the present disclosure are modified.In other embodiments of the present disclosure, all of the nucleotides of the RNAi agent of the present disclosure are modified.The RNAi agent of the present disclosure in which "substantially all of the nucleotides are modified" is mostly, but not entirely, modified, and can include 5, 4, 3, 2 or unmodified nucleotides.In still other embodiments of the present disclosure, the RNAi agent of the present disclosure can include 5, 4, 3, 2 or 1 modified nucleotide.

[0218] Nucleic acids featured in this disclosure can be synthesized or modified by methods well established in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, S.L. et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, incorporated herein by reference. Modifications include, for example, terminal modifications, such as 5'-end modifications (phosphorylation, conjugation, reverse ligation) or 3'-end modifications (conjugation, DNA nucleotides, reverse ligation, etc.), base modifications, such as replacement with a stabilizing base, a destabilizing base, or a base that base-pairs with an expanded repertoire partner, removal of a base (abasic nucleotide) or a conjugated base, sugar modifications (e.g., at the 2' or 4' position) or sugar replacement, or backbone modifications, including modification or replacement of a phosphodiester bond. Specific examples of RNAi agents useful in the embodiments described herein include, but are not limited to, RNAs containing modified backbones or that do not contain natural internucleoside linkages. Among the RNAs with modified backbone, those that do not have phosphorus atom in backbone include.For the purpose of this specification, as sometimes referred to in the art, the modified RNA that does not have phosphorus atom in its internucleoside backbone can also be considered as oligonucleoside.In some embodiments, modified RNAi agent has phosphorus atom in its internucleoside backbone.

[0219] Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methylphosphonates, and other alkyl phosphonates, including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates with normal 3'-5' linkages, their analogs with 2'-5' linkages, and those with reverse polarity, where adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'.Various salts, such as sodium salts, mixed salts, and free acid forms are also included.

[0220] Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Patent Nos. 3,687,808, 4,469,863, 4,476,301, 5,023,243, 5,177,195, 5,188,897, 5,264,423, 5,276,019, 5,278,302, 5,286,711, and 5,286,712. No. 7, No. 5,321,131, No. 5,399,676, No. 5,405,939, No. 5,453,496, No. 5,455,233, No. 5,466,677, No. 5,476 , No. 925, No. 5,519,126, No. 5,536,821, No. 5,541,316, No. 5,550,111, No. 5,563,253, No. 5,571,799, No. 5,5 87,361, 5,625,050, 6,028,188, 6,124,445, 6,160,109, 6,169,170, 6,172,209, No. 6,239,265, No. 6,277,603, No. 6,326,199, No. 6,346,614, No. 6,444,423, No. 6,531,590, No. 6,534,639 , 6,608,035, 6,683,167, 6,858,715, 6,867,294, 6,878,805, 7,015,315, 7,041,816, 7,273,933, 7,321,029 and U.S. Reissue Patent No. RE39464, the entire contents of each of which are incorporated herein by reference.

[0221] Modified RNA backbones that do not contain phosphorus atoms have backbones formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages. These include morpholino linkages (formed in part from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and others with mixed N, O, S, and CH2 constituent moieties.

[0222] Representative United States 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, Nos. 5,489,677, 5,541,307, 5,561,225, 5,596,086, 5,602,240, 5,608,046, 5,610,289, 5,618,704, 5,623,070, 5,663,312, 5,633,360, 5,677,437 and 5,677,439, the entire contents of each of which are incorporated herein by reference.

[0223] In other embodiments, RNA mimetics suitable for use in RNAi agents are contemplated, in which both the sugar and internucleoside linkage, i.e., the backbone of nucleotide units, are replaced with novel groups. Base units are maintained for hybridization with appropriate nucleic acid target compounds. One such oligomeric compound, an RNA mimetic that has been found to have excellent hybridization properties, is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. Nucleic acid bases are retained and are directly or indirectly linked to the aza nitrogen atom of the amide portion of the backbone. Representative US patents that teach the preparation of PNA compounds include, but are not limited to, US Patent Nos. 5,539,082, 5,714,331 and 5,719,262, the entire contents of each of which are incorporated herein by reference. Additional PNA compounds suitable for use in the RNAi agents of the present disclosure are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0224] Some embodiments featured in this disclosure include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, particularly --CH2--NH--CH2-, --CH2--N(CH3)--O--CH2-- [known as the methylene(methylimino) or MMI backbone], --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2-- and --N(CH3)--CH2--CH2-- [the natural phosphodiester backbone is represented as --O--P--O--CH2--] of the above-referenced U.S. Pat. No. 5,489,677, and amide backbones of the above-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAs featured herein have the morpholino backbone structures of the above-referenced US Pat. No. 5,034,506.

[0225] Modified RNAs may also contain one or more substituted sugar moieties. The RNAi agents, e.g., dsRNAs, featured herein may contain 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, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C6. 10 Alkyl or C2-C 10 It can be alkenyl or alkynyl. Exemplary suitable modifications include O[(CH) 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, where n and m are from 1 to about 10. In other embodiments, the dsRNA contains at the 2' position: C1 to C 10The modification may include one of the following: lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH, OCN, Cl, Br, CN, CF, OCF, SOCH, SOCH, ONO, NO, N, NH, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving group, reporter group, interfering substance, group for improving the pharmacokinetic properties of RNAi agents or group for improving the pharmacodynamic properties of RNAi agents, and other substituents with similar properties. In some embodiments, the modification includes 2'-methoxyethoxy (2'-O--CHCHOCH, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., an alkoxy-alkoxy group. Another exemplary modification is the 2'-dimethylaminooxyethoxy, i.e., O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described herein below in the Examples, 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. Further 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, and 2'-NMA (N-methylacetamide).

[0226] Other modifications include 2'-methoxy (2'-OCH), 2'-aminopropoxy (2'-OCHCHNH), 2'-O-hexadecyl, and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of an RNAi agent, particularly at the 3'-position of the sugar on the 3'-terminal nucleotide or in a 2'-5'-linked dsRNA and at the 5'-position of the 5'-terminal nucleotide. RNAi agents can also have sugar mimetics, such as cyclobutyl moieties instead of pentofuranosyl sugars. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Patent 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, and 5,56 Nos. 7,811, 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, certain of which are commonly owned with the present application, the entire contents of each of which are incorporated herein by reference.

[0227] The RNAi agents of the present disclosure may also include nucleobase (often simply referred to in the art as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), 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 ... These include 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and adenine, 8-azaguanine and adenine, 7-deazaguanine and adenine, and 3-deazaguanine and adenine.Additional nucleobases include those disclosed in U.S. Patent 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, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., (1991) Angewandte Chemie, International Edition, 30:613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the present disclosure. 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 0.6-1.2°C (Sanghvi, YS, Crooke, ST, and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), and are an exemplary base substitution, even more particularly when combined with a 2'-O-methoxyethyl sugar modification.

[0228] Representative United States patents that teach the preparation of certain of the above-mentioned modified nucleobases, as well as other modified nucleobases, include, but are not limited to, the above-mentioned U.S. Patent 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, Nos. 5,594,121, 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 incorporated herein by reference.

[0229] The RNAi agent of the present disclosure can also be modified to include one or more locked nucleic acids (LNA).Locked nucleic acids are nucleotides with modified ribose moieties, which contain an additional bridge connecting the 2' and 4' carbons of the ribose moiety.This structure effectively "locks" the ribose in a 3'-endo conformation.The addition of locked nucleic acids to siRNA has been shown to increase 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].

[0230] The RNAi agents of the present disclosure can also be modified to include one or more bicyclic sugar moieties. A "bicyclic sugar" is a furanosyl ring modified by a two-atom bridge. A "bicyclic nucleoside" ("BNA") is a nucleoside having a sugar moiety that includes a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the bridge connects the 4'-carbon and 2'-carbon of the sugar ring. Thus, in some embodiments, the agents of the present disclosure can include one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide with a modified ribose moiety in which the ribose moiety includes an additional bridge connecting the 2' and 4' carbons. In other words, an LNA is a nucleotide containing a bicyclic sugar moiety that includes a 4'-CH2-O-2' bridge. This structure effectively "locks" the ribose into a 3'-endo conformation. The addition of locked nucleic acids to siRNA has been shown to increase 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 containing a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, the antisense polynucleotide agent of the present disclosure contains one or more bicyclic nucleosides containing 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"). ethyl) or "cEt") and 4'-CH(CHOCH)-O-2' (and its analogs; see, e.g., U.S. Pat. No. 7,399,845); 4'-C(CH)(CH)-O-2' (and its analogs; see, e.g., U.S. Pat. No. 8,278,283); 4'-CH-N(OCH)-2' (and its analogs; see, e.g., U.S. Pat. No. 8,278,425); 4'-CH-ON(CH)-2' (see, e.g., U.S. Patent Application Publication No. 2004 / 0171570); 4'-CH-N(R)-O-2', where R is H, C, or C. 12 alkyl, or a protecting group (see, e.g., U.S. Pat. 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. Pat. No. 8,278,426). The entire contents of each of the foregoing documents are incorporated herein by reference.

[0231] Additional representative U.S. patents and U.S. patent application 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, 7,399,808, and 7,409,824. Nos. 45, 7,427,672, 7,569,686, 7,741,457, 8,022,193, 8,030,467, 8,278,425, 8,278,426, 8,278,283, U.S. Patent Application Publication No. 2008 / 0039618 and U.S. Patent Application Publication No. 2009 / 0012281, the entire contents of each of which are incorporated herein by reference.

[0232] For example, any of the above bicyclic nucleosides can be prepared with one or more stereochemical sugar configurations including α-L-ribofuranose and β-D-ribofuranose (see WO 99 / 14226).

[0233] The RNAi agent of the present disclosure can also be modified to include one or more constrained ethyl nucleotides. As used herein, a "constrained ethyl nucleotide" or "cEt" is a locked nucleic acid that includes a bicyclic sugar moiety that includes a 4'-CH(CH3)-0-2' bridge. In one embodiment, the constrained ethyl nucleotide is in the S conformation, referred to herein as "S-cEt."

[0234] The RNAi agents of the present disclosure may also include one or more "conformationally restricted nucleotides" ("CRNs"). A CRN is a nucleotide analogue with a linker connecting the C2' and C4' carbons of ribose or the C3 and C5' carbons of ribose. The CRN locks the ribose ring into a stable conformation and increases hybridization affinity to mRNA. The linker is of sufficient length to position the oxygen in an optimal position for stability and affinity, resulting in less ribose ring puckering.

[0235] Representative publications that teach the preparation of certain of the above CRNs include, but are not limited to, U.S. Patent Application Publication No. 2013 / 0190383 and WO 2013 / 036868, the entire contents of each of which are incorporated herein by reference.

[0236] In some embodiments, the RNAi agent of the present disclosure comprises one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNA is an unlocked acyclic nucleic acid, and any sugar bond has been removed to form an unlocked "sugar" residue. In one example, UNA also encompasses a monomer in which the bond between C1'-C4' (i.e., the covalent carbon-oxygen-carbon bond between the C1' and C4' carbons) has been removed. In another example, the C2'-C3' bond of the sugar (i.e., the covalent carbon-carbon bond between the C2' and C3' carbons) has been removed [see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039, which are incorporated herein by reference].

[0237] Representative U.S. publications that teach the preparation of UNAs include, but are not limited to, U.S. Patent No. 8,314,227 and U.S. Patent Application Publication Nos. 2013 / 0096289, 2013 / 0011922, and 2011 / 0313020, the entire contents of each of which are incorporated herein by reference.

[0238] Potential stabilizing modifications to the ends of RNA molecules can include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3'-phosphate, inverted 2'-deoxy-modified ribonucleotides, such as inverted dT (idT), inverted dA (idA), and inverted abasic 2'-deoxyribonucleotides (iAb), as well as others. Disclosure of this modification can be found in WO 2011 / 005861.

[0239] In one example, the 3' and 5' ends of the oligonucleotide are linked to inverted 2'-deoxy-modified ribonucleotides, such as inverted dT (idT), inverted dA (idA), or inverted abasic 2'-deoxyribonucleotides (iAb). In a particular example, the inverted 2'-deoxy-modified ribonucleotide is linked to the 3' end of the oligonucleotide, such as the 3' end of the sense strand described herein, in which case the linkage is via a 3'-3' phosphodiester bond or a 3'-3' phosphorothioate bond.

[0240] 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.

[0241] In one particular example, an inverted 2'-deoxy-modified ribonucleotide is linked to the 3' end of an oligonucleotide, e.g., the 3' end of the sense strand described herein, where the linkage is via a 3'-3' phosphodiester bond or a 3'-3'-phosphorothioate bond.

[0242] 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).

[0243] Other modifications of the RNAi agent of the present disclosure include 5' phosphate or 5' phosphate mimic, for example, 5'-terminal phosphate or phosphate mimic on the antisense strand of the RNAi agent.Suitable phosphate mimics are disclosed, for example, in US Patent Application Publication No. 2012 / 0157511, the entire contents of which are incorporated herein by reference.

[0244] A. Modified RNAi Agents Containing Motifs of the Disclosure In certain aspects of the present disclosure, the double-stranded RNAi agent of the present disclosure includes an agent having chemical modifications, for example, as disclosed in International Publication No. 2013 / 075035, the entire contents of which are incorporated herein by reference.As shown herein and in International Publication No. 2013 / 075035, excellent results can be obtained by introducing one or more motifs of three identical modifications on three consecutive nucleotides into the sense or antisense strand of the RNAi agent at or near the cleavage site.In some embodiments, the sense and antisense strands of the RNAi agent can be otherwise completely modified.The introduction of these motifs disrupts the modification pattern of the sense or antisense strand, if present.The RNAi agent can also be conjugated with a lipophilic ligand, for example, a C16 ligand on the sense strand.The RNAi agent can also be modified, for example, with an (S)-glycol nucleic acid (GNA) modification at one or more residues of the antisense strand.The resulting RNAi agent exhibits excellent gene silencing activity.

[0245] Thus, the present disclosure provides double-stranded RNAi agents capable of inhibiting the expression of a target gene (i.e., the SNCA gene) in vivo. The RNAi agent comprises a sense strand and an antisense strand. Each strand of the RNAi agent can be 15 to 30 nucleotides in length. For example, each strand can be 16 to 30 nucleotides in length, 17 to 30 nucleotides in length, 25 to 30 nucleotides in length, 27 to 30 nucleotides in length, 17 to 23 nucleotides in length, 17 to 19 nucleotides in length, 19 to 25 nucleotides in length, 19 to 23 nucleotides in length, 19 to 21 nucleotides in length, 21 to 25 nucleotides in length, or 21 to 23 nucleotides in length. In certain embodiments, each strand is 19 to 23 nucleotides in length.

[0246] The sense and antisense strands typically form a duplex, double-stranded RNA ("dsRNA"), also referred to herein as an "RNAi agent." The duplex region of an RNAi agent can be 15-30 nucleotide pairs in length. 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 duplex region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides in length. In a preferred embodiment, the duplex region is 19-21 nucleotide pairs in length.

[0247] In one embodiment, the RNAi agent may contain one or more overhang regions or capping groups at the 3', 5', or both ends of one or both strands. The overhangs may be 1 to 6 nucleotides in length, e.g., 2 to 6 nucleotides, 1 to 5 nucleotides, 2 to 5 nucleotides, 1 to 4 nucleotides, 2 to 4 nucleotides, 1 to 3 nucleotides, 2 to 3 nucleotides, or 1 to 2 nucleotides in length. In a preferred embodiment, the nucleotide overhang region is 2 nucleotides in length. The overhang may be the result of one strand being longer than the other or the result of two strands of the same length being staggered. The overhang may form a mismatch with the target mRNA, be complementary to the targeted gene sequence, or be a different sequence. The first and second strands may also be joined by additional bases, for example, to form a hairpin, or by other non-basic linkers.

[0248] In one embodiment, the nucleotides in the overhang region of an RNAi agent can each independently be a modified or unmodified nucleotide, including, but not limited to, a 2'-sugar modified, e.g., 2-F, 2'-O-methyl, thymidine (T), and any combination thereof.

[0249] For example, TT can be an overhang sequence at either end on either strand. The overhang can form a mismatch with the target mRNA, or can be complementary to the targeted gene sequence, or can be another sequence.

[0250] The 5'- or 3'-overhang of the sense strand, antisense strand, or both strands of the RNAi agent can be phosphorylated. In some embodiments, the overhang region(s) contain two nucleotides with phosphorothioate between them, and the two nucleotides can be the same or different. In one embodiment, the overhang is present at the 3'-end of the sense strand, antisense strand, or both strands. In one embodiment, the 3'-overhang is present in the antisense strand. In one embodiment, the 3'-overhang is present in the sense strand.

[0251] RNAi agent can contain only a single overhang, which can enhance the interference activity of RNAi without affecting its overall stability.For example, the single-stranded overhang can be located at the 3' end of the sense strand or at the 3' end of the antisense strand.RNAi can also have a blunt end located at the 5' end of the antisense strand (or the 3' end of the sense strand), or vice versa.Generally, the antisense strand of RNAi has a nucleotide overhang at the 3' end, and the 5' end is blunt.Without wishing to be bound by theory, the blunt end at the 5' end of the asymmetric antisense strand and the 3' end overhang of the antisense strand are favorable for the guide strand loading into the RISC process.

[0252] In one embodiment, the RNAi agent is a 19-nucleotide double-ended bluntmer, with the sense strand containing at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 7, 8, and 9 from the 5' end, and the antisense strand containing at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0253] In another embodiment, the RNAi agent is a 20-nucleotide double-ended bluntmer, and the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 8, 9, and 10 from the 5' end. The antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0254] In yet another embodiment, the RNAi agent is a 21-nucleotide double-ended bluntmer, and the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 9, 10, and 11 from the 5' end. The antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0255] In one embodiment, the RNAi agent comprises a 21-nucleotide sense strand and a 23-nucleotide antisense strand, wherein the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 9, 10, and 11 from the 5' end, and the antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end, and one end of the RNAi agent is blunt, and the other end contains a two-nucleotide overhang. Optionally, the two-nucleotide overhang is at the 3' end of the antisense strand. When the two-nucleotide overhang is at the 3' end of the antisense strand, there may be two phosphorothioate internucleotide linkages between the three terminal nucleotides, two of which are overhanging nucleotides, and the third nucleotide is the next paired nucleotide after the overhanging nucleotide. In one embodiment, the RNAi agent further comprises two phosphorothioate internucleotide linkages between the three terminal nucleotides at both the 5' end of the sense strand and the 5' end of the antisense strand.In one embodiment, every nucleotide in the sense strand and the antisense strand of the RNAi agent, including the nucleotide that is part of a motif, is a modified nucleotide.In one embodiment, each residue is independently modified with 2'-O-methyl or 3'-fluoro, for example, in the alternating motif.Optionally, the RNAi agent can further comprise a ligand (for example, a lipophilic ligand, optionally a C16 ligand).

[0256] In one embodiment, the RNAi agent comprises a sense and an antisense strand, wherein the sense strand is 25-30 nucleotide residues in length, and starting from the 5'-most nucleotide (position 1), positions 1-23 of the first strand comprise at least 8 ribonucleotides; and the antisense strand is 36-66 nucleotide residues in length, and starting from the 3'-most nucleotide, comprises at least 8 ribonucleotides at positions that pair with positions 1-23 of the sense strand to form a duplex, wherein at least the 3'-most nucleotide of the antisense strand is not paired with the sense strand, and up to six consecutive 3'-most nucleotides are not paired with the sense strand, thereby forming a 3' single-stranded overhang of 1-6 nucleotides. The double-stranded nucleic acid comprises at least 19 ribonucleotides of the sense strand, forming a 10-30 nucleotide single-stranded 5' overhang, at least the 5'- and 3'-terminal nucleotides of the sense strand being base-paired with nucleotides of the antisense strand when the sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially double-stranded region between the sense and antisense strands, the antisense strand being sufficiently complementary to the target RNA along at least 19 ribonucleotides of the length of the antisense strand, and reducing target gene expression when the double-stranded nucleic acid is introduced into a mammalian cell, the sense strand containing at least one motif of three 2'-F modifications on three consecutive nucleotides, at least one of the motifs occurring at or near the cleavage site, and the antisense strand containing at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.

[0257] In one embodiment, the RNAi agent comprises a sense and an antisense strand, the RNAi agent comprising a first strand having a length of at least 25 and at most 29 nucleotides, and a second strand having a length of at most 30 nucleotides and having at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end, the 3' end of the first strand and the 5' end of the second strand forming a blunt end, the second strand being 1-4 nucleotides longer at its 3' end than the first strand, the duplex region being at least 25 nucleotides long, the second strand being sufficiently complementary to a target mRNA along at least 19 nucleotides of the second strand length, the RNAi agent reducing target gene expression when introduced into a mammalian cell, and Dicer cleavage of the RNAi agent preferentially yielding siRNAs comprising the 3' end of the second strand, thereby reducing target gene expression in the mammal. Optionally, the RNAi agent may further comprise a ligand.

[0258] In one embodiment, the sense strand of the RNAi agent contains at least one motif of three identical modifications on three consecutive nucleotides, one of the motifs occurring at the cleavage site in the sense strand.

[0259] In one embodiment, the antisense strand of the RNAi agent can also contain at least one motif of three identical modifications on three consecutive nucleotides, one of the motifs occurring at or near the cleavage site in the antisense strand.

[0260] For RNAi agents with a duplex region 17-23 nucleotides long, the cleavage sites in the antisense strand are typically approximately positions 10, 11, and 12 from the 5' end. Thus, three identical modification motifs may occur in the antisense strand at positions 9, 10, and 11; positions 10, 11, and 12; positions 11, 12, and 13; positions 12, 13, and 14; or positions 13, 14, and 15, with the numbers starting from the first nucleotide from the 5' end of the antisense strand, or the numbers starting from the first paired nucleotide in the duplex region from the 5' end of the antisense strand. The cleavage site in the antisense strand may also vary depending on the length of the duplex region of the RNAi agent from the 5' end.

[0261] The sense strand of RNAi agent can contain at least one motif of three identical modifications on three consecutive nucleotides at the site of strand breakage, and antisense strand can have at least one motif of three identical modifications on three consecutive nucleotides at or near the site of strand breakage.When sense strand and antisense strand form dsRNA duplex, sense strand and antisense strand can be arranged so that one motif of three nucleotides on sense strand and one motif of three nucleotides on antisense strand have at least one nucleotide overlap, that is, at least one of the three nucleotides of the motif in sense strand and at least one of the three nucleotides of the motif in antisense strand form base pairs.Alternatively, at least two nucleotides can overlap, or all three nucleotides can overlap.

[0262] In one embodiment, the sense strand of an RNAi agent may contain two or more motifs of three identical modifications on three consecutive nucleotides. The first motif may occur at or near the cleavage site of the strand, and the other motifs may be wing modifications. As used herein, the term "wing modification" refers to a motif occurring in another part of the strand, away from the motif at or near the cleavage site of the same strand. The wing modifications may be adjacent to the first motif or separated by at least one or more nucleotides. When the motifs are immediately adjacent to each other, the chemistry of the motifs is distinct from each other, and when the motifs are separated by one or more nucleotides, the chemistry may be the same or different. Two or more wing modifications may be present. For example, when two wing modifications are present, each wing modification may occur at one end of the first motif at or near the cleavage site, or on either side of the lead motif.

[0263] Similar to the sense strand, the antisense strand of an RNAi agent may contain two or more motifs of three identical modifications on three consecutive nucleotides, with at least one of the motifs occurring at or near the site of strand cleavage.The antisense strand may also contain one or more wing modifications in the same sequence as the wing modifications that may be present on the sense strand.

[0264] In one embodiment, wing modifications on the sense or antisense strand of an RNAi agent typically do not include the first one or two terminal nucleotides at the 3' end, 5' end, or both ends of the strand.

[0265] In another embodiment, wing modifications on the sense or antisense strand of an RNAi agent typically do not include the first one or two paired nucleotides in the duplex region at the 3' end, 5' end, or both ends of the strand.

[0266] When the sense and antisense strands of an RNAi agent each contain at least one wing modification, the wing modifications can fall at the same end of the duplex region and have an overlap of 1, 2, or 3 nucleotides.

[0267] When the sense or antisense strand of an RNAi agent each contains at least two wing modifications, the sense and antisense strands can be arranged such that two modifications from one strand each occupy one end of the duplex region, with an overlap of 1, 2, or 3 nucleotides; two modifications from one strand each occupy the other end of the duplex region, with an overlap of 1, 2, or 3 nucleotides; and two modifications from one strand occupy either side of the lead motif, with an overlap of 1, 2, or 3 nucleotides in the duplex region.

[0268] In one embodiment, the RNAi agent contains mismatch(es) or combinations thereof within the double strand with the target. Mismatches can occur in overhang regions or duplex regions. Base pairs can be ranked based on their tendency to promote dissociation or melting (e.g., the free energy of association or dissociation of a particular pairing; the simplest approach is to examine pairs on an individual basis, but next-neighbor analysis or similar analysis can also be used). In terms of promoting dissociation, A:U is preferred over G:C, G:U is preferred over G:C, and I:C (I=inosine) is preferred over G:C. Mismatches, such as non-canonical pairings or those other than canonical pairings (as described elsewhere herein), are preferred over canonical (A:T, A:U, G:C) pairings, and pairings involving universal bases are preferred over canonical pairings.

[0269] In one embodiment, the RNAi agent comprises 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 independently selected from the group of A:U, G:U, I:C, and mismatch pairs, e.g., non-canonical pairings or other than canonical pairings or pairings including universal bases, to promote dissociation of the antisense strand at the 5' end of the duplex.

[0270] In one embodiment, the nucleotide at position 1 in the double-stranded region from the 5' end of the antisense strand 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 in the double-stranded region from the 5' end of the antisense strand is an AU base pair.For example, the first base pair in the double-stranded region from the 5' end of the antisense strand is an AU base pair.

[0271] In another embodiment, the nucleotide at the 3' end of the sense strand is deoxythymine (dT). In another embodiment, the nucleotide at the 3' end of the antisense strand is deoxythymine (dT). In one embodiment, there is a short sequence of deoxythymine nucleotides, for example, two dT nucleotides at the 3' end of the sense or antisense strand.

[0272] In one embodiment, the sense strand sequence has formula (I): 5'n p -N a -(XXX) i -N b -YYY -N b -(ZZZ) j -N a -n q 3' (I) [In the formula, i and j each independently represent 0 or 1; p and q each independently represent 0 to 6; each N a independently represent oligonucleotide sequences comprising 0 to 25 modified nucleotides, each sequence comprising at least two differently modified nucleotides; each N bindependently represent an oligonucleotide sequence comprising 0 to 10 modified nucleotides; each n p and n q independently represent an overhanging nucleotide; Nb and Y do not have the same modification, and XXX, YYY and ZZZ each independently represent one motif of three identical modifications on three consecutive nucleotides. Where appropriate, YYY are all 2'-F modified nucleotides.

[0273] In one embodiment, N a or N b includes alternating pattern modifications.

[0274] In one embodiment, the YYY motif occurs at or near the cleavage site of the sense strand. For example, if the RNAi agent has a duplex region 17-23 nucleotides in length, the YYY motif can occur at or near the cleavage site of the sense strand (e.g., can occur at positions 6, 7, 8, 7, 8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12, or 11, 12, 13), with the number starting from the first nucleotide from the 5' end, or, optionally, the number starting from the first paired nucleotide within the duplex region from the 5' end.

[0275] In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or i and j are both 1. Thus, the sense strand has the following formula: 5' n p -N a -YYY-N b -ZZZ-N a -n q 3' (Ib), 5' n p -N a -XXX-N b -YYY-N a -n q 3' (Ic), or 5' n p -N a-XXX-N b -YYY-N b -ZZZ-N a -n q 3' (Id) It can be expressed as:

[0276] When the sense strand is represented by formula (Ib), N b represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides.

[0277] each N a can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0278] When the sense strand is represented by formula (Ic), N b represents an oligonucleotide sequence containing 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. a may independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0279] When the sense strand is represented by formula (Id), each N b independently represent an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. b is 0, 1, 2, 3, 4, 5 or 6. Each N a may independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0280] Each of X, Y and Z may be the same as or different from one another.

[0281] In other embodiments, i is 0, j is 0, and the sense strand has the formula: 5' n p -N a -YYY-Na -n q 3' (Ia) It can be expressed as:

[0282] When the sense strand is represented by formula (Ia), each N a may independently comprise an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0283] In one embodiment, the antisense strand sequence of the RNAi has the formula (II): 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) [In the formula, k and l each independently represent 0 or 1; p' and q' each independently represent 0 to 6; each N a ' independently represent oligonucleotide sequences containing 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each N b independently represents an oligonucleotide sequence comprising 0 to 10 modified nucleotides, each n p ' and n q ' independently represent an overhanging nucleotide; N b ' and Y' do not have the same modification, X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides. It can be expressed as:

[0284] In one embodiment, N a ' or N b ' includes alternating pattern modifications.

[0285] The Y'Y'Y' motif occurs at or near the cleavage site of the sense strand. For example, if the RNAi agent has a duplex region 17 to 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, the numbers starting from the first nucleotide from the 5' end, or, optionally, the numbers starting from the first paired nucleotide in the duplex region from the 5' end. Optionally, the Y'Y'Y' motif occurs at positions 11, 12, 13.

[0286] In one embodiment, the Y'Y'Y' motif is all 2'-OMe modified nucleotides.

[0287] In one embodiment, k is 1 and l is 0, or k is 0 and l is 1, or k and l are both 1.

[0288] Thus, the antisense strand has the formula: 5' n q’ -N a '-Z'Z'Z'-N b '-Y'Y'Y'-N a '-n p’ 3' (IIb), 5' n q’ -N a '-Y'Y'Y'-N b '-X'X'X'-n p’ 3' (IIc), or 5' n q’ -N a '-Z'Z'Z'-N b '-Y'Y'Y'-N b '-X'X'X'-N a '-n p’ 3' (IId) It can be expressed as:

[0289] When the antisense strand is represented by formula (IIb), N b ’represents an oligonucleotide sequence containing 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. a ' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0290] When the antisense strand is represented by formula (IIc), N b ' represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. a ' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0291] When the antisense strand is represented by formula (IId), each N b Each N' independently represents an oligonucleotide sequence containing 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. a ' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. b is 0, 1, 2, 3, 4, 5 or 6.

[0292] In other embodiments, k is 0, l is 0, and the antisense strand has the formula: 5' n p’ -N a’ -Y'Y'Y'- N a’ -n q’ 3' (Ia) It can be expressed as:

[0293] When the antisense strand is represented by formula (IIa), each N a ' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0294] Each of X', Y' and Z' may be the same as or different from one another.

[0295] Each nucleotide of sense strand and antisense strand 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 of sense strand and antisense strand is independently modified with 2'-O-methyl or 2'-fluoro.Each X, Y, Z, X', Y' and Z' can specifically represent 2'-O-methyl modification or 2'-fluoro modification.

[0296] In one embodiment, the sense strand of the RNAi agent may contain a YYY motif occurring at positions 9, 10, and 11 of the strand when the duplex region is 21 nt, the numbers starting from the first nucleotide from the 5' end, or, where appropriate, the numbers may start from the 5' end with the first paired nucleotide in the duplex region, and Y represents a 2'-F modification. The sense strand may further contain a XXX motif or a ZZZ motif as a wing modification at the opposite end of the duplex region, where XXX and ZZZ each independently represent a 2'-OMe modification or a 2'-F modification.

[0297] In one embodiment, the antisense strand may contain a Y'Y'Y' motif occurring at positions 11, 12, 13 of the strand, where the number starts from the first nucleotide from the 5' end, or, where appropriate, the number may start from the 5' end with the first paired nucleotide in the duplex region, and Y' represents a 2'-O-methyl modification. The antisense strand may further contain an X'X'X' motif or a Z'Z'Z' motif as a wing modification at the opposite end of the duplex region, where X'X'X' and Z'Z'Z' each independently represent a 2'-OMe modification or a 2'-F modification.

[0298] The sense strand represented by any one of the above formulas (Ia), (Ib), (Ic) and (Id) forms a duplex with the antisense strand represented by any one of the formulas (IIa), (IIb), (IIc) and (IId), respectively.

[0299] Thus, an RNAi agent for use in the methods of the disclosure can include a sense strand and an antisense strand, each strand having 14-30 nucleotides, and the RNAi duplex can have the formula (III): Sense: 5' n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3' Antisense: 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' (III) [In the formula, i, j, k, and l each independently represent 0 or 1; p, p', q and q' each independently represent 0 to 6; each N a and N a ’ independently represent oligonucleotide sequences comprising 0 to 25 modified nucleotides, each sequence comprising at least two differently modified nucleotides; each N b and N b ’ independently represent an oligonucleotide sequence comprising 0 to 10 modified nucleotides; each n p ',n p , n q ' and n q independently represent overhanging nucleotides, each of which may or may not be present; XXX, YYY, ZZZ, X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides. is expressed by

[0300] 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 l is 0, or k is 1 and l is 0, or k is 0 and l is 1, or k and l are both 0, or k and l are both 1.

[0301] An exemplary combination of sense and antisense strands that form an RNAi duplex has the following formula: 5' n p - N a -YYY -N a -n q 3' 3' n p ’ -N a ’ -Y'Y'Y' -N a ’ n q ’ 5' (IIIa) 5' n p -N a -YYY -N b -ZZZ -N a -n q 3' 3' n p ’ -N a ’ -Y'Y'Y'-N b ’ -Z'Z'Z'-N a ’ n q ’ 5' (IIIb) 5' n p -N a -XXX-Nb -YYY-N a -n q 3' 3' n p ’ -N a ’ -X'X'X'-N b ’ -Y'Y'Y'-N a ’ -n q ’ 5' (IIIc) 5' n p -N a -XXX -N b -YYY -N b - ZZZ -N a -n q 3' 3' n p ’ -N a ’ -X'X'X'-N b ’ -Y'Y'Y'-N b ’ -Z'Z'Z'-N a -n q ’ 5' (IIId) Includes:

[0302] When the RNAi agent is represented by formula (IIIa), each N a independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0303] When the RNAi agent is represented by formula (IIIb), each N b independently represent an oligonucleotide sequence containing 1 to 10, 1 to 7, 1 to 5, or 1 to 4 modified nucleotides. a independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0304] When the RNAi agent is represented by formula (IIIc), each Nb , N b Each N' independently represents an oligonucleotide sequence containing 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. a independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0305] When the RNAi agent is represented by formula (IIId), each N b , N b Each N' independently represents an oligonucleotide sequence containing 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. a , N a ’ independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. a , N a ', N b and N b ’ each independently comprises an alternating pattern of modifications.

[0306] In one embodiment, when the RNAi agent is represented by formula (IIId), N a The modification is a 2'-O-methyl or a 2'-fluoro modification. In another embodiment, when the RNAi agent is represented by formula (IIId), N a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n p In yet another embodiment, when the RNAi agent is represented by formula (IIId), N' is linked to the adjacent nucleotide via a phosphorothioate linkage. a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n pIn another embodiment, when the RNAi agent is represented by formula (IIId), N' is linked to adjacent nucleotides via phosphorothioate linkages, and the sense strand is conjugated to one or more C16 (or related) moieties attached by a bivalent or trivalent branched linker (described below). a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n p ' is linked to adjacent nucleotides via phosphorothioate linkages, and the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more lipophilic, e.g., C16 (or related) moieties, which may optionally be attached by a linker.

[0307] In one embodiment, when the RNAi agent is represented by formula (IIIa), N a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n p ' is linked to adjacent nucleotides via phosphorothioate linkages, and the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more lipophilic, e.g., C16 (or related) moieties attached by a linker.

[0308] In one embodiment, the RNAi agent is a multimer that contains at least two double strands represented by formula (III), (IIIa), (IIIb), (IIIc) and (IIId), and the double strands are connected by a linker. The linker may be cleavable or non-cleavable. The multimer may further comprise a ligand. Each double strand may target the same gene, or may target two different genes, or each double strand may target the same gene at two different target sites.

[0309] In one embodiment, the RNAi agent is a multimer that contains 3, 4, 5, 6 or more double strands represented by formula (III), (IIIa), (IIIb), (IIIc) and (IIId), and the double strands are connected by a linker. The linker may be cleavable or non-cleavable. The multimer may further comprise a ligand. Each double strand may target the same gene, or may target two different genes, or each double strand may target the same gene at two different target sites.

[0310] 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 of the 3' ends may be conjugated to a ligand. Each of the agents may target the same gene, or may target two different genes, or each of the agents may target the same gene at two different target sites.

[0311] Various publications describe the multimeric RNAi agent that can be used in the method of the present disclosure.Such publications include International Publication No. 2007 / 091269, International Publication No. 2010 / 141511, International Publication No. 2007 / 117686, International Publication No. 2009 / 014887 and International Publication No. 2011 / 031520 and United States Patent No. 7858769, the entire contents of each of which are incorporated herein by reference.

[0312] In certain embodiments, the compositions and methods of the present disclosure include a 5'-phosphate mimic, such as a vinyl phosphonate (VP) modification of an RNAi agent as described herein. In an exemplary embodiment, a vinyl phosphonate of the present disclosure has the following structure:

[0313] [ka] The vinyl phosphonate of the present disclosure can be attached to either the antisense strand or the sense strand of the dsRNA of the present disclosure.In certain preferred embodiments, the vinyl phosphonate of the present disclosure is attached to the antisense strand of the dsRNA at the 5' end of the antisense strand of the dsRNA, as appropriate.

[0314] In an exemplary embodiment, a 5' vinylphosphonate modified nucleotide of the disclosure has the following structure:

[0315] [ka] and wherein X is O or S; R is hydrogen, hydroxy, fluoro, or C 1~20 alkoxy (e.g., methoxy, or n-hexadecyloxy); R 5’ is =C(H)-P(O)(OH)2, and the C5' carbon and R 5’ and the double bond between is in the E or Z orientation (e.g., E orientation); B is a nucleobase or a modified nucleobase, and B may be adenine, guanine, cytosine, thymine, or uracil.

[0316] In one embodiment, R 5’ is ═C(H)—P(O)(OH) and the double bond between the C5′ carbon and R5′ is in the E orientation. In another embodiment, R is methoxy and R 5’ is ═C(H)—P(O)(OH) and the double bond between the C5′ carbon and R5′ is in the 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.

[0317] Vinyl phosphate modifications are also contemplated for the compositions and methods of the present disclosure. An exemplary vinyl phosphate structure is:

[0318] [ka] is.

[0319] Further exemplary vinyl phosphate structures include those described above, where R5' is =C(H)-O-P(O)(OH)2 and the double bond between the C5' carbon and R5' is in the E or Z orientation (e.g., E orientation).

[0320] In some embodiments, the 5-phosphate mimetic is

[0321] [ka] or a salt thereof (e.g., a sodium salt), where the cleaved bond is a covalent bond to the 4'-carbon of the 5'-terminal nucleotide. In some embodiments, the 5'-terminal nucleotide is

[0322] [ka] or a salt thereof (e.g., sodium salt), where B is a modified or unmodified nucleobase (e.g., uracil or 5-methyluracil).

[0323] B. Thermal destabilization modification In certain embodiments, dsRNA molecules can be optimized for RNA interference by incorporating a thermally destabilizing modification into the seed region of the antisense strand (i.e., positions 2-9 of the 5'-end of the antisense strand) to reduce or inhibit off-target gene silencing. It has been discovered that dsRNAs having an antisense strand containing at least one thermally destabilizing duplex modification within the first 9 nucleotide positions counting from the 5' end of the antisense strand have reduced off-target gene silencing activity. Thus, in some embodiments, the antisense strand contains at least one (e.g., 1, 2, 3, 4, 5, or more) thermally destabilizing duplex modification within the first 9 nucleotide positions of the 5' region of the antisense strand. In some embodiments, one or more thermally destabilizing duplex modifications are located in positions 2-9, or optionally, positions 4-8, from the 5' end of the antisense strand. In some further embodiments, the thermally destabilizing duplex modification(s) are located at positions 6, 7, or 8 from the 5' end of the antisense strand. In still some further embodiments, the thermally destabilizing modification of the duplex is located at position 7 from the 5' end of the antisense strand. The term "thermally destabilizing modification(s)" includes modification(s) that will result in a dsRNA having a lower overall melting temperature (Tm) (optionally 1, 2, 3, or 4 degrees lower than the Tm of a dsRNA without such modification(s). In some embodiments, the thermally destabilizing modification of the duplex is located at position 2, 3, 4, 5, or 9 from the 5' end of the antisense strand.

[0324] 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 or acyclic nucleotides, such as unlocked nucleic acids (UNA) or glycol nucleic acids (GNA), and 2'-5' linked ribonucleotides ("3'-RNA").

[0325] Exemplary abasic modifications include, but are not limited to, the following:

[0326] [ka] wherein R=H, Me, Et, or OMe; R'=H, Me, Et, or OMe; and R"=H, Me, Et, or OMe.

[0327] [ka] wherein B is a modified or unmodified nucleobase.

[0328] Exemplary sugar modifications include, but are not limited to, the following:

[0329] [ka] wherein B is a modified or unmodified nucleobase.

[0330] In some embodiments, the thermally destabilizing modification of the duplex is selected from the group consisting of:

[0331] [ka] where B is a modified or unmodified nucleobase, and the asterisk in each structure represents either R, S, or racemic.

[0332] In some embodiments, the thermally destabilizing modification of the duplex is selected from the group consisting of:

[0333] [ka] wherein B is a modified or unmodified nucleobase and the asterisk represents either R, S, or racemic (e.g., S).

[0334] The term "acyclic nucleotide" refers to any nucleotide having an acyclic ribose sugar, e.g., in which any of the bonds 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 from the nucleotide. In some embodiments, an acyclic nucleotide is

[0335] [ka] wherein B is a modified or unmodified nucleobase and R 1 and R 2 are independently H, halogen, OR3, or alkyl; and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar. The term "UNA" refers to an unlocked acyclic nucleic acid in which one of the sugar bonds has been removed to form an unlocked "sugar" residue. In one example, UNA also encompasses a monomer in which the C1'-C4' bond has been removed (i.e., a covalent carbon-oxygen-carbon bond between the C1' and C4' carbons). In another example, the C2'-C3' bond of the sugar (i.e., a covalent carbon-carbon bond between the C2' and C3' carbons) has been removed [see Mikhailov et al., Tetrahedron Letters, 26 (17): 2059 (1985) and Fluiter et al., Mol. Biosyst., 10: 1039 (2009), which are incorporated herein by reference in their entireties]. Acyclic derivatives offer greater backbone flexibility without affecting Watson-Crick pairing. Acyclic nucleotides can be linked via 2'-5' or 3'-5' linkages.

[0336] The term "GNA" refers to glycol nucleic acid, a polymer similar to DNA or RNA, but differing in the composition of its "backbone" in that it is made up of repeating glycerol units linked by phosphodiester bonds:

[0337] [ka]

[0338] The thermally destabilizing modification of the double strand can be a mismatch (i.e., non-complementary base pair) between the thermally destabilizing nucleotide and the opposite nucleotide in the opposite strand of 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 combinations thereof.Other mismatch base pairings known in the art are also suitable for the present disclosure.Mismatches can occur between nucleotides that are either naturally occurring or modified nucleotides, that is, mismatch base pairing can occur between the nucleobases derived from each nucleotide independently of the modification on the ribose sugar of the nucleotide.In certain embodiments, the dsRNA molecule contains at least one nucleobase in mismatch pairing that is a 2'-deoxynucleobase, for example, the 2'-deoxynucleobase is in the sense strand.

[0339] In some embodiments, the duplex thermally destabilizing modification in the seed region of the antisense strand is a nucleotide that has impaired WCH bonding with the complementary base on the target mRNA, such as:

[0340] [ka] Includes:

[0341] Many 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 by reference in its entirety.

[0342] Thermally destabilizing modifications can also include universal base and phosphate modifications that have reduced or eliminated ability to form hydrogen bonds with opposing bases.

[0343] In some embodiments, the thermal destabilizing modification of duplex comprises the nucleotide with non-canonical base, for example but not limited to, the nucleobase modification that the ability to form hydrogen bond with the base in opposite strand is impaired or completely lost.These nucleobase modifications have been evaluated for the destabilization of the central region of dsRNA duplex, as described in International Publication No. 2010 / 0011895, the entire contents of which are incorporated herein by reference.Exemplary nucleobase modifications are:

[0344] [ka]

[0345] In some embodiments, the duplex thermally destabilizing modifications in the seed region of the antisense strand include one or more α-nucleotides that are complementary to bases on the target mRNA, such as the following:

[0346] [ka] wherein R is H, OH, OCH, F, NH, NHMe, NMe, or O-alkyl. Includes:

[0347] Exemplary phosphate modifications known to decrease the thermal stability of dsRNA duplexes compared to native phosphodiester linkages are as follows:

[0348] [ka]

[0349] The alkyl R group can be a C1-C6 alkyl. Specific alkyl R groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, pentyl, and hexyl. As those skilled in the art will recognize, considering that the functional role of nucleobase defines the specificity of the RNAi agent of the present disclosure, nucleobase modification can be carried out in various ways as described herein, for example, for the purpose of enhancing on-target effect against off-target effect, for example, for introducing destabilizing modifications into the RNAi agent of the present disclosure, but the range of modifications that can be used and generally exist on the RNAi agent of the present disclosure tends to be greater for non-nucleobase modifications, for example, modifications to the sugar group or phosphate backbone of polyribonucleotide.Such modifications are described in more detail in other sections of this disclosure, and are expressly intended for the RNAi agent of the present disclosure that has either natural nucleobase or modified nucleobase, as described above or elsewhere herein.

[0350] In addition to the antisense strand that contains thermal destabilizing modifications, dsRNA can also contain one or more stabilizing modifications.For example, dsRNA can contain at least two (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) stabilizing modifications.Without being limited, all stabilizing modifications can be present in one strand.In some embodiments, both sense and antisense strands contain at least two stabilizing modifications.Stabilizing modifications can occur at any nucleotide of sense strand or antisense strand.For example, stabilizing modifications can occur at any nucleotide on sense strand or antisense strand, and each stabilizing modification can occur in an alternating pattern on sense strand or antisense strand, or both sense strand and antisense strand contain stabilizing modifications in an alternating pattern.The alternating pattern of stabilizing modifications on sense strand can be the same or different from that of antisense strand, and the alternating pattern of stabilizing modifications on sense strand can have a shift compared to the alternating pattern of stabilizing modifications on antisense strand.

[0351] In some embodiments, the antisense strand comprises at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) stabilizing modifications. Without limitation, the stabilizing modifications in the antisense strand can be located at any position. In some embodiments, the antisense strand comprises stabilizing modifications at positions 2, 6, 8, 9, 14, and 16 from the 5' end. In some other embodiments, the antisense strand comprises stabilizing modifications at positions 2, 6, 14, and 16 from the 5' end. In yet some other embodiments, the antisense strand comprises stabilizing modifications at positions 2, 14, and 16 from the 5' end.

[0352] In some embodiments, antisense strand comprises at least one stabilizing modification adjacent to destabilizing modification.For example, stabilizing modification can be at the 5'-end or 3'-end of destabilizing modification, that is, at the nucleotide of position -1 or +1 from the position of destabilizing modification.In some embodiments, antisense strand comprises stabilizing modification at each of the 5'-end and 3'-end of destabilizing modification, that is, at the nucleotide of position -1 and +1 from the position of destabilizing modification.

[0353] In some embodiments, the antisense strand comprises at least two stabilizing modifications at the 3' end of the destabilizing modification, ie, at positions +1 and +2 from the position of the destabilizing modification.

[0354] In some embodiments, the sense strand comprises at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) stabilizing modifications. Without limitation, the stabilizing modifications in the sense strand can be located at any position. In some embodiments, the sense strand comprises stabilizing modifications at positions 7, 10, and 11 from the 5' end. In some other embodiments, the sense strand comprises stabilizing modifications at positions 7, 9, 10, and 11 from 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, counting from the 5' end of the antisense strand. 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, counting from the 5' end of the antisense strand. In some embodiments, the sense strand comprises blocks of two, three, or four stabilizing modifications.

[0355] In some embodiments, the sense strand does not contain a stabilizing modification at a position opposite or complementary to a thermally destabilizing modification of the duplex in the antisense strand.

[0356] Exemplary thermally stabilizing modifications include, but are not limited to, 2'-fluoro modifications. Other thermally stabilizing modifications include, but are not limited to, LNA.

[0357] In some embodiments, the dsRNA of the present disclosure comprises at least four (for example, 4, 5, 6, 7, 8, 9, 10 or more) 2'-fluoro nucleotides. Without being limited thereto, all 2'-fluoro nucleotides may be present in one strand. In some embodiments, both sense and antisense strands comprise at least two 2'-fluoro nucleotides. 2'-fluoro modification can occur at any nucleotide of sense strand or antisense strand. For example, 2'-fluoro modification can occur at any nucleotide on sense strand or antisense strand, and each 2'-fluoro modification can occur in an alternating pattern on sense strand or antisense strand, or both sense strand and antisense strand contain 2'-fluoro modification in an alternating pattern. The alternating pattern of 2'-fluoro modification on sense strand can be the same or different from that of antisense strand, and the alternating pattern of 2'-fluoro modification on sense strand can have a shift compared to the alternating pattern of 2'-fluoro modification on antisense strand.

[0358] In some embodiments, the antisense strand comprises at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-fluoro nucleotides. Without limitation, the 2'-fluoro modifications in the antisense strand can be present at any position. In some embodiments, the antisense strand comprises 2'-fluoro nucleotides at positions 2, 6, 8, 9, 14 and 16 from the 5' end. In some other embodiments, the antisense strand comprises 2'-fluoro nucleotides at positions 2, 6, 14 and 16 from the 5' end. In yet some other embodiments, the antisense strand comprises 2'-fluoro nucleotides at positions 2, 14 and 16 from the 5' end.

[0359] In some embodiments, antisense strand comprises at least one 2'-fluoro nucleotide adjacent to destabilizing modification.For example, 2'-fluoro nucleotide can be at the 5' end or 3' end of destabilizing modification, that is, at the nucleotide of position -1 or +1 from the position of destabilizing modification.In some embodiments, antisense strand comprises 2'-fluoro nucleotide at each of the 5' end and 3' end of destabilizing modification, that is, at the position -1 and +1 from the position of destabilizing modification.

[0360] In some embodiments, the antisense strand comprises at least two 2'-fluoro nucleotides at the 3' end of the destabilizing modification, ie, at positions +1 and +2 from the position of the destabilizing modification.

[0361] In some embodiments, the sense strand comprises at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-fluoro nucleotides. Without limitation, the 2'-fluoro modifications in the sense strand can be present at any position. In some embodiments, the antisense strand comprises 2'-fluoro nucleotides at positions 7, 10 and 11 from the 5' end. In some other embodiments, the sense strand comprises 2'-fluoro nucleotides at positions 7, 9, 10 and 11 from 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 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 2, 3 or 4 blocks of 2'-fluoro nucleotides.

[0362] In some embodiments, the sense strand does not contain 2'-fluoro nucleotides at positions opposite or complementary to thermally destabilizing modifications of the duplex in the antisense strand.

[0363] In some embodiments, a dsRNA molecule of the present disclosure comprises a 21-nucleotide (nt) sense strand and a 23-nucleotide (nt) antisense strand, wherein the antisense strand contains at least one thermally destabilized nucleotide, wherein the at least one thermally destabilized nucleotide occurs in the seed region of the antisense strand (i.e., positions 2-9 at the 5'-end of the antisense strand), one end of the dsRNA is blunt and the other end comprises a 2-nt overhang, and the dsRNA further has at least one (e.g., 1, 2, 3, 4, 5, 6, or all 7) of the following features: The dsRNA may comprise: (i) the antisense strand comprises two, three, four, five, or six 2'-fluoro modifications, (ii) the antisense strand 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 at the 3' end of the antisense strand.

[0364] In some embodiments, a dsRNA molecule of the present disclosure comprises a sense strand and an antisense strand, wherein: the sense strand is 25-30 nucleotides in length, and starting from the 5'-terminal nucleotide (position 1), positions 1-23 of the sense strand comprise at least 8 ribonucleotides; the antisense strand is 36-66 nucleotides in length, and starting from the 3'-terminal nucleotide, at least 8 ribonucleotides at those positions are paired with positions 1-23 of the sense strand to form a duplex; at least the 3'-terminal nucleotide of the antisense strand is unpaired with the sense strand, and up to six of the 3'-terminal nucleotides are unpaired with the sense strand, thereby forming a 1-6 nucleotide 3' single-stranded overhang; and the 5' end of the antisense strand is free of 10-30 contiguous nucleotides that are unpaired with the sense strand. the antisense strand comprises at least one ribonucleotide at the 5'-end of the sense strand, thereby forming a 10-30 nucleotide single-stranded 5' overhang; at least the 5'- and 3'-terminal nucleotides of the sense strand are base-paired with nucleotides in the antisense strand when the sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially duplexed 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 antisense strand length so as to reduce expression of the target gene when the double-stranded nucleic acid is introduced into a mammalian cell; the antisense strand comprises at least one thermally destabilized nucleotide, where the at least one thermally destabilized nucleotide is located in the seed region of the antisense strand (i.e., positions 2-9 of the 5'-end of the antisense strand).For example, the thermally destabilizing nucleotide occurs between positions 14-17 at the 5' end of the sense strand and the positions opposite or complementary to them, and the dsRNA may further have at least one (e.g., one, two, three, four, five, six, or all seven) of the following features: (i) the antisense strand contains two, three, four, five, or six 2'-fluoro modifications; (ii) the antisense strand contains one, two, three, four, or five phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated to a ligand; (iv) the sense strand contains two, three, four, or five 2'-fluoro modifications; (v) the sense strand contains one, two, three, four, or five phosphorothioate internucleotide linkages; (vi) the dsRNA contains at least four 2'-fluoro modifications; and (vii) the dsRNA contains a duplex region 12-30 nucleotide pairs in length.

[0365] In some embodiments, a dsRNA molecule of the present disclosure comprises a sense and an antisense strand, wherein the dsRNA molecule comprises a sense strand having a length of at least 25 and at most 29 nucleotides and an antisense strand having a length of at most 30 nucleotides, wherein the sense strand comprises a modified nucleotide at position 11 from its 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, wherein the antisense strand is 1-4 nucleotides longer at its 3' end than the sense strand, wherein the duplex region is at least 25 nucleotides in length, and wherein the antisense strand is sufficiently complementary to a target mRNA along at least 19 nucleotides of the length of the antisense strand, wherein the dsRNA molecule reduces target gene expression when introduced into a mammalian cell, wherein Dicer cleavage of the dsRNA preferentially yields siRNA comprising the 3' end of the antisense strand, thereby reducing target gene expression in the mammal, and wherein the antisense strand comprises at least one The dsRNA contains thermally destabilized nucleotides, with at least one thermally destabilized nucleotide in the seed region of the antisense strand (i.e., positions 2-9 at the 5' end of the antisense strand), and may further have at least one (e.g., one, two, three, four, five, six, or all seven) of the following features: (i) the antisense contains two, three, four, five, or six 2'-fluoro modifications; (ii) the antisense contains two, three, four, five, or six 2'-fluoro modifications; (i) the antisense strand contains 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated to a ligand; (iv) the sense strand contains 2, 3, 4, or 5 2'-fluoro modifications; (v) the sense strand contains 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (vi) the dsRNA contains at least four 2'-fluoro modifications; and (vii) the dsRNA has a duplex region 12 to 29 nucleotide pairs in length.

[0366] In some embodiments, any nucleotide in the sense strand and antisense strand of dsRNA molecule can be modified.Each nucleotide can be modified with the same or different modifications, which can include one or more of non-linked phosphate oxygen or one or more of linking phosphate oxygen, one or both of the modifications of ribose sugar components, for example, the 2' hydroxyl on ribose sugar, the large-scale replacement of phosphate moiety with " dephosphorylation " linker, the modification or replacement of naturally occurring base, and the replacement or modification of ribose-phosphate backbone.

[0367] Because nucleic acids are polymers of subunits, many modifications occur at positions that are repeated within nucleic acids, such as modifications of bases or phosphate moieties or non-linked Os at phosphate moieties. In some cases, modifications occur at all target positions in nucleic acids, but in many cases, they do not occur. For example, modifications can occur only at the 3' or 5' terminal position, or only in the terminal region, for example, at the terminal nucleotide position of the chain, or at the last 2, 3, 4, 5, or 10 nucleotides. Modifications can occur in double-stranded regions, single-stranded regions, or both. Modifications can occur only in the double-stranded region of RNA, or only in the single-stranded region of RNA. For example, phosphorothioate modifications at non-linked O positions can occur only at one or both ends, or only in the terminal region, for example, at the terminal nucleotide position of the chain, or at the last 2, 3, 4, 5, or 10 nucleotides, or in double-stranded and single-stranded regions, especially at the ends. The 5' end or both ends can be phosphorylated.

[0368] For example, it may be possible to enhance stability, include specific bases in the overhang, or include modified nucleotides or nucleotide substitutes in the single-stranded overhang, for example, in the 5' or 3' overhang, or both.For example, it may be desirable to include purine nucleotides in the overhang.In some embodiments, all or part of the bases in the 3' or 5' overhang can be modified, for example, with the modifications described herein.Modifications can include, for example, the use of modifications at the 2' position of the ribose sugar with modifications known in the art, for example, the use of modified deoxyribonucleotides, 2'-deoxy-2'-fluoro (2'-F) or 2'-O-methyl, instead of the ribosugar of the nucleic acid base, and modifications at the phosphate group, for example, phosphorothioate modifications.The overhang does not need to be homologous to the target sequence.

[0369] In some embodiments, each residue of sense strand and antisense strand is independently modified with LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy or 2'-fluoro.Strands can contain two or more modifications.In some embodiments, each residue of sense strand and antisense strand is independently modified with 2'-O-methyl or 2'-fluoro.It should be understood that these modifications are in addition to at least one thermally destabilizing modification of the double strand that exists in the antisense strand.

[0370] At least two different modifications are usually present on the sense strand and the antisense strand. These two modifications can be 2'-deoxy, 2'-O-methyl or 2'-fluoro modifications, acyclic nucleotides, etc. In some embodiments, the sense strand and the antisense strand each contain 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'-ON-methylacetamide (2'-O-NMA) nucleotides, 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) nucleotides, 2'-O-aminopropyl (2'-O-AP) nucleotides, or 2'-ara-F nucleotides. It should be understood that these modifications are in addition to at least one thermally destabilizing modification of the duplex present in the antisense strand.

[0371] In some embodiments, the dsRNA molecules of the present disclosure comprise alternating pattern modifications, particularly in the B1, B2, B3, B1', B2', B3', and B4' regions. The term "alternating motif" or "alternating pattern" as used herein refers to a motif with one or more modifications, where each modification occurs at alternating nucleotides in a single strand. Alternating nucleotides can refer to one every other nucleotide or one every third nucleotide, or similar patterns. For example, if A, B, and C each represent one type of modification to a nucleotide, the alternating motif can be "ABABABABABAB...", "AABBAABBAABB...", "AABAABAABAAB...", "AAABAAABAAAB...", "AABBBAAABBB...", or "ABCABCABCABC...", etc.

[0372] The types of modifications contained within the alternating motifs can be the same or different. For example, if A, B, C, and D each represent one type of modification on a nucleotide, the alternating turns, i.e., the modifications on every other nucleotide, can be the same, but each of the sense or antisense strands can be selected from several possibilities for modifications within the alternating motif, such as "ABABAB...", "ACACAC...", "BDBDBD..." or "CDCDCD...".

[0373] In some embodiments, the dsRNA molecules of the present disclosure comprise an alternating motif modification pattern on the sense strand that is shifted relative to the alternating motif modification pattern on the antisense strand. The shift can be such that the modified groups of the nucleotides of the sense strand correspond to the differently modified groups of the nucleotides of the antisense strand, or vice versa. For example, when the sense strand is paired with the antisense strand in a dsRNA duplex, the alternating motif in the sense strand may begin with "ABABAB" from the 5'-3' end of the strand, and the alternating motif in the antisense strand may begin with "BABABA" from the 3'-5' end of the strand within the duplex region. As another example, the alternating motif in the sense strand may begin with "AABBAABB" from the 5'-3' end of the strand, and the alternating motif in the antisense strand may begin with "BBAABBAA" from the 3'-5' end of the strand within the duplex region, resulting in a complete or partial shift in the modification pattern between the sense strand and the antisense strand.

[0374] In one particular 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.

[0375] In one particular 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.

[0376] In another particular 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.

[0377] In one particular 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.

[0378] In one particular 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 a 2'-deoxy-2'-fluoro modified nucleotide and each B is a 2'-O methyl modified nucleotide.

[0379] The dsRNA molecule of the present disclosure can further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage.Phosphorothioate or methylphosphonate internucleotide linkage modification can occur at any nucleotide of sense strand or antisense strand or both at any position of chain.For example, internucleotide linkage modification can occur at any nucleotide on sense strand or antisense strand, and each internucleotide linkage modification can occur in an alternating pattern on sense strand or antisense strand, or sense strand or antisense strand comprises two or more internucleotide linkage modifications in an alternating pattern.The alternating pattern of internucleotide linkage modification on sense strand can be the same or different from that of antisense strand, and the alternating pattern of internucleotide linkage modification on sense strand can have a shift with respect to the alternating pattern of internucleotide linkage modification on antisense strand.

[0380] In some embodiments, dsRNA molecule comprises phosphorothioate or methylphosphonate internucleotide linkage modification in overhang region.For example, overhang region comprises two nucleotides with phosphorothioate or methylphosphonate internucleotide linkage between the two nucleotides.Internucleotide linkage modification can also be performed to link overhang nucleotide with the terminal pairing nucleotide in duplex region.For example, at least 2, 3, 4 or all overhang nucleotides can be linked by phosphorothioate or methylphosphonate internucleotide linkage, and there can be additional phosphorothioate or methylphosphonate internucleotide linkage that connects overhang nucleotide with the pairing nucleotide adjacent to overhang nucleotide.For example, there can be at least two phosphorothioate internucleotide linkages between the terminal three nucleotides, two of which are overhang nucleotides, and the third is the pairing nucleotide adjacent to overhang nucleotide.Optionally, these terminal three nucleotides can be the 3' end of antisense strand.

[0381] In some embodiments, the sense strand of the dsRNA molecule comprises 1 to 10 blocks of 2 to 10 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 positioned 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 with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0382] 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 and an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0383] 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, one of the phosphorothioate or methylphosphonate internucleotide linkages being 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 with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0384] 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, one of the phosphorothioate or methylphosphonate internucleotide linkages being 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 with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0385] 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, one of the phosphorothioate or methylphosphonate internucleotide linkages being 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 with an antisense strand comprising either phosphorothioate, methylphosphonate, or phosphate linkages.

[0386] 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, one of the phosphorothioate or methylphosphonate internucleotide linkages being 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 with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0387] 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, one of the phosphorothioate or methylphosphonate internucleotide linkages being 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 with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0388] 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, one of the phosphorothioate or methylphosphonate internucleotide linkages being 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 with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0389] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of nine phosphorothioate or methylphosphonate internucleotide linkages separated by one, two, three, or four phosphate internucleotide linkages, one of the phosphorothioate or methylphosphonate internucleotide linkages being 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 with an antisense strand comprising either phosphorothioate, methylphosphonate, or phosphate linkages.

[0390] In some embodiments, the dsRNA molecules of this disclosure further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within the 1-10 terminal positions 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.

[0391] In some embodiments, dsRNA molecules of the present disclosure further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 1-10 of the internal region of the duplex 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 8-16 of the duplex region, counting from the 5' end of the sense strand, and the dsRNA molecule may further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within the terminal positions 1-10.

[0392] In some embodiments, dsRNA molecules of this disclosure further comprise one to five phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 1-5 of the sense strand and one to five phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) and one to five phosphorothioate or methylphosphonate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and one to five within positions 18-23 (counting from the 5' end).

[0393] In some embodiments, dsRNA molecules of this disclosure further comprise one phosphorothioate internucleotide linkage modification within positions 1-5 and one phosphorothioate or methylphosphonate internucleotide linkage modification within positions 18-23 (counting from the 5' end) of the sense strand and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) of the antisense strand.

[0394] In some embodiments, the dsRNA molecule of the present disclosure comprises a phosphorothioate internucleotide linkage modification between the first and second nucleotides and between the second and third nucleotides at the 5'-end of the antisense strand. In some embodiments, the dsRNA molecule of the present disclosure comprises a phosphorothioate internucleotide linkage modification between the first and second nucleotides and between the second and third nucleotides at the 3'-end of the antisense strand. In some embodiments, the dsRNA molecule of the present disclosure comprises a phosphorothioate internucleotide linkage modification between the first and second nucleotides and between the second and third nucleotides at the 5'-end and 3'-end of the antisense strand.

[0395] In some embodiments, dsRNA molecules of this disclosure comprise phosphorothioate internucleotide linkage modifications between the first and second nucleotide and between the second and third nucleotide at the 5'-end of the antisense strand.

[0396] In some embodiments, the dsRNA molecules of the present disclosure comprise phosphorothioate internucleotide linkage modifications between the first and second nucleotide and between the second and third nucleotide at the 5'-end of the sense strand, and between the first and second nucleotide and between the second and third nucleotide at both the 5'-end and 3'-end of the antisense strand.

[0397] In some embodiments, the dsRNA molecules of the present disclosure comprise phosphorothioate internucleotide linkages between the first and second nucleotides, between the second and third nucleotides, and between the third and fourth nucleotides at the 5'-end of the antisense strand, a phosphorothioate internucleotide linkage modification between the first and second nucleotides at the 3'-end of the antisense strand, and a phosphorothioate internucleotide linkage modification between the first and second nucleotides at the 5'- and 3'-ends of the sense strand.

[0398] In some embodiments, dsRNA molecules of this disclosure further comprise two phosphorothioate internucleotide linkage modifications within positions 1-5 and one phosphorothioate internucleotide linkage modification within positions 18-23 (counting from the 5' end) of the sense strand and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) of the antisense strand.

[0399] In some embodiments, dsRNA molecules of this disclosure further comprise two phosphorothioate internucleotide linkage modifications within positions 1-5 and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and one and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) of the antisense strand.

[0400] In some embodiments, dsRNA molecules of this disclosure further comprise two phosphorothioate internucleotide linkage modifications within positions 1-5 and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 (counting from the 5' end) of the antisense strand.

[0401] In some embodiments, dsRNA molecules of this disclosure further comprise one phosphorothioate internucleotide linkage modification within positions 1-5 and one phosphorothioate internucleotide linkage modification within positions 18-23 (counting from the 5' end) of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) of the antisense strand.

[0402] In some embodiments, dsRNA molecules of this disclosure further comprise one phosphorothioate internucleotide linkage modification within positions 1-5 and one within positions 18-23 (counting from the 5' end) of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 (counting from the 5' end) of the antisense strand.

[0403] In some embodiments, dsRNA molecules of this disclosure further comprise one phosphorothioate internucleotide linkage modification within positions 1-5 of the sense strand (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand (counting from the 5' end).

[0404] In some embodiments, dsRNA molecules of this disclosure further comprise two phosphorothioate internucleotide linkage modifications (counting from the 5' end) within positions 1-5 of the sense strand and one phosphorothioate internucleotide linkage modification at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications (counting from the 5' end) within positions 18-23 of the antisense strand.

[0405] In some embodiments, dsRNA molecules of this disclosure further comprise two phosphorothioate internucleotide linkage modifications within positions 1-5 and one within positions 18-23 (counting from the 5' end) of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 (counting from the 5' end) of the antisense strand.

[0406] In some embodiments, dsRNA molecules of this disclosure further comprise two phosphorothioate internucleotide linkage modifications within positions 1-5 and one phosphorothioate internucleotide linkage modification within positions 18-23 (counting from the 5' end) of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) of the antisense strand.

[0407] In some embodiments, dsRNA molecules of this disclosure further comprise two phosphorothioate internucleotide linkage modifications within positions 1-5 and one phosphorothioate internucleotide linkage modification within positions 18-23 (counting from the 5' end) of the sense strand and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) of the antisense strand.

[0408] In some embodiments, the dsRNA molecules of this disclosure further comprise two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 20 and 21 (counting from the 5' end) of the sense strand and one phosphorothioate internucleotide linkage modification at position 1 and one at position 21 (counting from the 5' end) of the antisense strand.

[0409] In some embodiments, 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 (counting from the 5' end) of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 20 and 21 (counting from the 5' end) of the antisense strand.

[0410] In some embodiments, 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 (counting from the 5' end) of the sense strand and one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the antisense strand.

[0411] In some embodiments, 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 (counting from the 5' end) of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 21 and 22 (counting from the 5' end) of the antisense strand.

[0412] In some embodiments, 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 (counting from the 5' end) of the sense strand and one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the antisense strand.

[0413] In some embodiments, 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 (counting from the 5' end) of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 23 and 23 (counting from the 5' end) of the antisense strand.

[0414] In some embodiments, the compounds of the present disclosure comprise a pattern of backbone chiral centers. In some embodiments, the general pattern of backbone chiral centers comprises at least 5 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 6 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 7 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 8 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 9 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 10 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 11 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 12 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 13 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 14 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 15 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 16 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 17 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 18 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 19 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises no more than 8 internucleotide linkages in the Rp configuration.In some embodiments, the general pattern of backbone chiral centers comprises seven or fewer internucleotide linkages in the Rp configuration. In some embodiments, the general pattern of backbone chiral centers comprises six or fewer internucleotide linkages in the Rp configuration. In some embodiments, the general pattern of backbone chiral centers comprises five or fewer internucleotide linkages in the Rp configuration. In some embodiments, the general pattern of backbone chiral centers comprises four or fewer internucleotide linkages in the Rp configuration. In some embodiments, the general pattern of backbone chiral centers comprises three or fewer internucleotide linkages in the Rp configuration. In some embodiments, the general pattern of backbone chiral centers comprises two or fewer internucleotide linkages in the Rp configuration. In some embodiments, the general pattern of backbone chiral centers comprises one or fewer internucleotide linkages in the Rp configuration. In some embodiments, the general pattern of backbone chiral centers comprises eight or fewer non-chiral internucleotide linkages (phosphodiesters as a non-limiting example). In some embodiments, the general pattern of backbone chiral centers comprises seven or fewer non-chiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises six or fewer non-chiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises five or fewer non-chiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises four or fewer non-chiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises three or fewer non-chiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises two or fewer non-chiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises one or fewer non-chiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises at least 10 internucleotide linkages in the Sp configuration and eight or fewer non-chiral internucleotide linkages.In some embodiments, the general pattern of backbone chiral centers comprises at least 11 internucleotide linkages in the Sp configuration and no more than 7 nonchiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises at least 12 internucleotide linkages in the Sp configuration and no more than 6 nonchiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises at least 13 internucleotide linkages in the Sp configuration and no more than 6 nonchiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises at least 14 internucleotide linkages in the Sp configuration and no more than 5 nonchiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises at least 15 internucleotide linkages in the Sp configuration and no more than 4 nonchiral internucleotide linkages. In some embodiments, the internucleotide linkages in the Sp configuration may be contiguous or noncontiguous. In some embodiments, the internucleotide linkages in the Rp configuration may be contiguous or noncontiguous. In some embodiments, the nonchiral internucleotide linkages may be contiguous or noncontiguous.

[0415] In some embodiments, compounds of the present disclosure include blocks that are stereochemical blocks. In some embodiments, the blocks are Rp blocks, in that each internucleotide linkage 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 blocks are Sp blocks, in that each internucleotide linkage 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, provided oligonucleotides include both Rp and Sp blocks. In some embodiments, provided oligonucleotides include one or more Rp blocks but do not include Sp blocks. In some embodiments, provided oligonucleotides include one or more Sp blocks but do not include Rp blocks. In some embodiments, provided oligonucleotides include one or more PO blocks, in which each internucleotide linkage is a natural phosphate linkage.

[0416] In some embodiments, compounds of the present disclosure include a 5'-block that is an Sp block in which each sugar moiety comprises a 2'-F modification. In some embodiments, the 5'-block is an Sp block in which each internucleotide linkage is a modified internucleotide linkage and each sugar moiety comprises a 2'-F modification. In some embodiments, the 5'-block is an Sp block in which each internucleotide linkage is a phosphorothioate linkage and each sugar moiety comprises a 2'-F modification. In some embodiments, the 5'-block comprises four or more nucleoside units. In some embodiments, the 5'-block comprises five or more nucleoside units. In some embodiments, the 5'-block comprises six or more nucleoside units. In some embodiments, the 5'-block comprises seven or more nucleoside units. In some embodiments, the 3'-block is an Sp block in which each sugar moiety comprises a 2'-F modification. In some embodiments, the 3'-block is an Sp block in which each internucleotide linkage is a modified internucleotide linkage and each sugar moiety comprises a 2'-F modification. In some embodiments, the 3'-block is an Sp block in which each of the internucleotide linkages is a phosphorothioate linkage 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.

[0417] In some embodiments, compounds of the disclosure include a certain type of nucleoside in a region, or an oligonucleotide is followed by a particular type of internucleotide linkage, e.g., a natural phosphate linkage, a modified internucleotide linkage, an Rp chiral internucleotide linkage, an Sp chiral internucleotide linkage, etc. 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 linkage (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 linkage (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 linkage (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 linkage (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 linkage (PO). In some embodiments, A and G are followed by Sp. In some embodiments, A and G are followed by Rp.

[0418] In some embodiments, the antisense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 21 and 22 and between nucleotide positions 22 and 23, the antisense strand contains at least one thermally destabilizing modification of the duplex located in the seed region of the antisense strand (i.e., at positions 2-9 at the 5' end of the antisense strand), and the dsRNA may further have 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 modifications; (ii) the antisense strand comprises two, three, four, five, or six 2'-fluoro modifications; (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; (vii) the dsRNA comprises a duplex region 12 to 40 nucleotide pairs in length; and (viii) the dsRNA has a blunt end at the 5' end of the antisense strand.

[0419] 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, the antisense strand contains at least one thermally destabilizing modification of the duplex located in the seed region of the antisense strand (i.e., at positions 2-9 at the 5' end of the antisense strand or at positions 2-8 at the 5' end of the antisense strand), and the dsRNA may further have at least one (e.g., 1, 2, 3, 4, 5, 6, 7, or all 8) of the following features: (i) the antisense strand has at least one thermally destabilizing modification of the seed region of the antisense strand (i.e., at positions 2-9 at the 5' end of the antisense strand or at positions 2-8 at the 5' end of the antisense strand); or six 2'-fluoro modifications, (ii) the sense strand is conjugated to a ligand and / or a lipophilic moiety, (iii) the sense strand contains two, three, four, or five 2'-fluoro modifications, (iv) the sense strand contains one, two, three, four, or five phosphorothioate internucleotide linkages, (v) the dsRNA contains at least four 2'-fluoro modifications, (vi) the dsRNA contains a duplex region 12 to 40 nucleotide pairs in length, (vii) the dsRNA contains a duplex region 12 to 40 nucleotide pairs in length, and (viii) the dsRNA has a blunt end at the 5' end of the antisense strand.

[0420] In some embodiments, the sense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3, the antisense strand contains at least one thermally destabilizing modification of the duplex located in the seed region of the antisense strand (i.e., at positions 2-9 at the 5' end of the antisense strand), and the dsRNA may further have 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 modifications; (ii) the antisense strand comprises one or more 2'-fluoro modifications; , (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 12 to 40 nucleotide pairs in length; and (viii) the dsRNA has a blunt end at the 5' end of the antisense strand.

[0421] In some embodiments, the sense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3, 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, the antisense strand contains at least one thermally destabilizing modification of the duplex located in the seed region of the antisense strand (i.e., at positions 2-9 at the 5' end of the antisense strand), and the dsRNA has at least one of the following features (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88 , 6, or all seven): (i) the antisense strand includes two, three, four, five, or six 2'-fluoro modifications; (ii) the sense strand is conjugated to a ligand and / or a lipophilic moiety; (iii) the sense strand includes two, three, four, or five 2'-fluoro modifications; (iv) the sense strand includes three, four, or five phosphorothioate internucleotide linkages; (v) the dsRNA includes at least four 2'-fluoro modifications; (vi) the dsRNA includes a duplex region 12 to 40 nucleotide pairs in length; and (vii) the dsRNA has a blunt end at the 5' end of the antisense strand.

[0422] In some embodiments, the dsRNA molecule of the present disclosure comprises a mismatch (or mismatches) or combinations thereof within the double strand with the target. Mismatches can occur in the overhang region or the double-stranded region. Base pairs can be ranked based on their tendency to promote dissociation or melting (for example, the free energy of association or dissociation of a particular pairing; the simplest approach is to examine pairs on an individual basis, but next-neighbor analysis or similar analysis can also be used). In terms of promoting dissociation, A:U is preferred over G:C, G:U is preferred over G:C, and I:C is preferred over G:C (I=inosine). Mismatches, such as non-canonical pairings or those other than canonical pairings (as described elsewhere herein), are preferred over canonical (A:T, A:U, G:C) pairings, and pairings involving universal bases are preferred over canonical pairings.

[0423] In some embodiments, the dsRNA molecules of the present disclosure comprise 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 the group of A:U, G:U, I:C, and mismatch pairs, e.g., non-canonical or other than canonical pairings or pairings including universal bases, to promote dissociation of the antisense strand at the 5' end of the duplex.

[0424] In some embodiments, the nucleotide at position 1 in the double-stranded region from the 5' end of the antisense strand 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 in the double-stranded region from the 5' end of the antisense strand is an AU base pair.For example, the first base pair in the double-stranded region from the 5' end of the antisense strand is an AU base pair.

[0425] It has been found that the introduction of a 4'- or 5'-modified nucleotide at the 3' end of a dinucleotide phosphodiester (PO), phosphorothioate (PS) or phosphorodithioate (PS2) linkage at any position in a single- or double-stranded oligonucleotide can exert a steric effect on the internucleotide linkage, thus protecting it from nucleases and stabilizing it.

[0426] In some embodiments, 5'-modified nucleoside is introduced at the 3' end of dinucleotide at any position in single-stranded or double-stranded siRNA.For example, 5'-alkylated nucleoside can be introduced at the 3' end of dinucleotide at any position in single-stranded or double-stranded siRNA.The alkyl group at the 5' position of ribose sugar can be racemic or chirally pure R or S isomer.Exemplary 5'-alkylated nucleoside is 5'-methyl nucleoside.5'-methyl can be racemic or chirally pure R or S isomer.

[0427] In some embodiments, 4'-modified nucleosides are introduced at the 3' end of dinucleotides at any position in single-stranded or double-stranded siRNA. For example, 4'-alkylated nucleosides can be introduced at the 3' end of dinucleotides at any position in single-stranded or double-stranded siRNA. The alkyl group at the 5' position of the ribose sugar can be racemic or chirally pure R or S isomer. An exemplary 4'-alkylated nucleoside is 4'-methyl nucleoside. 4'-methyl can be racemic or chirally pure R or S isomer. Alternatively, 4'-O-alkylated nucleosides can be introduced at the 3' end of dinucleotides at any position in single-stranded or double-stranded siRNA. The 4'-O-alkyl group at the ribose sugar can be racemic or chirally pure R or S isomer. Exemplary 4'-O-alkylated nucleosides include 4'-O-methyl nucleosides, which can be either racemic or chirally pure R or S isomers.

[0428] In some embodiments, 5'-alkylated nucleoside is introduced at any position of the sense strand or antisense strand of dsRNA, and this modification maintains or improves the efficacy of dsRNA.5'-alkyl can be either racemic or chirally pure R or S isomer.Exemplary 5'-alkylated nucleoside is 5'-methyl nucleoside.5'-methyl can be either racemic or chirally pure R or S isomer.

[0429] In some embodiments, 4'-alkylated nucleoside is introduced at any position of the sense strand or antisense strand of dsRNA, and this modification maintains or improves the efficacy of dsRNA.4'-alkyl can be either racemic or chirally pure R or S isomer.Exemplary 4'-alkylated nucleoside is 4'-methyl nucleoside.4'-methyl can be either racemic or chirally pure R or S isomer.

[0430] In some embodiments, 4'-O-alkylated nucleosides are introduced at any position of the sense strand or antisense strand of dsRNA, and such modifications maintain or improve the efficacy of dsRNA.5'-Alkyl can be either racemic or chirally pure R or S isomer.Exemplary 4'-O-alkylated nucleosides include 4'-O-methyl nucleosides.4'-O-methyl can be either racemic or chirally pure R or S isomer.

[0431] In some embodiments, the dsRNA molecules of the present disclosure may contain 2'-5' linkages (having 2'-H, 2'-OH, and 2'-OMe, with P=O or P=S). For example, 2'-5' linkage modifications can be used to promote nuclease resistance or inhibit binding of the sense strand to the antisense strand, or can be used at the 5' end of the sense strand to prevent sense strand activation by RISC.

[0432] In another embodiment, the dsRNA molecules of the present disclosure can contain 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 binding of the sense strand to the antisense strand, or can be used at the 5' end of the sense strand to prevent sense strand activation by RISC.

[0433] Multimeric siRNA is described in various publications, and all of them can be used with the dsRNA of the present disclosure.Such publications include International Publication No. 2007 / 091269, United States Patent No. 7858769, International Publication No. 2010 / 141511, International Publication No. 2007 / 117686, International Publication No. 2009 / 014887 and International Publication No. 2011 / 031520, all of which are incorporated herein in their entirety.

[0434] Ligands and / or lipophilic moieties can be attached to polynucleotides via carriers. Exemplary carriers include (i) at least one "backbone attachment point," optionally two "backbone attachment points," and (ii) at least one "tethering attachment point." As used herein, "backbone attachment point" refers to a functional group, e.g., a hydroxyl group, or generally a bond available and suitable for incorporation of the carrier into a backbone, e.g., a phosphate or modified phosphate, e.g., sulfur-containing backbone, of a ribonucleic acid. In some embodiments, a "tethering attachment point" (TAP) refers to a constituent ring atom, e.g., a carbon atom or heteroatom (other than the atom providing the backbone attachment point), of a cyclic carrier to which a selected moiety is attached. This moiety can be, for example, a lipophilic alkyl group, optionally a C16 lipophilic moiety. The selected moiety may be connected to the cyclic carrier by an intervening tether. Thus, the cyclic carrier may contain a functional group, e.g., an amino group, or generally provide a bond suitable for incorporating or tethering another chemical entity, e.g., a ligand and / or lipophilic moiety, to the constituent ring.

[0435] RNAi agent can be conjugated to ligand and / or lipophilic moiety via carrier, and carrier can be cyclic or acyclic group.Optionally, cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3] dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl and decalin, and optional acyclic group is selected from serinol skeleton and diethanolamine skeleton.

[0436] In certain specific embodiments, the RNAi agent for use in the methods of the present disclosure is an agent selected from the group of agents listed in Table 2 or 3. These agents may further comprise a ligand, e.g., one or more lipophilic moieties.

[0437] III. Ligand-Conjugated iRNAAnother modification of the iRNA of the present disclosure includes chemically linking the iRNA to one or more ligands, moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the iRNA, for example, into cells. Such moieties include, but are not limited to, lipid moieties such as cholesterol moieties (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, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), and the like. 20:533-538), fatty chains such as dodecanediol or undecyl residues (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-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), polyamine 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), palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0438] In certain embodiments, the ligand alters the distribution, targeting, or life span of the iRNA agent into which it is incorporated. In some embodiments, the ligand provides enhanced affinity for a selected target, for example, a molecule, a cell or cell type, a compartment, for example, a cellular or organ compartment, a tissue, an organ, or a region of the body, for example, compared to a species in which such a ligand is not present. Conventional ligands do not participate in duplex pairing in double-stranded nucleic acids.

[0439] Ligands can include naturally occurring substances such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL) or globulins), carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid), or lipids. Ligands can also be recombinant or synthetic molecules, such as synthetic polymers, e.g., synthetic polyamino acids. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolized) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylamide), N-isopropylacrylamide polymer, or polyphosphazine. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimeric polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or alpha-helical peptides.

[0440] Ligands can also include targeting groups, such as cell or tissue targeting agents, such as lectins, glycoproteins, lipids, or proteins, for example, antibodies that bind to specific cell types, such as kidney cells.Targeting groups can be thyroid-stimulating hormone, melanocyte-stimulating hormone, lectins, glycoproteins, surfactant protein A, mucin carbohydrates, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, multivalent fucose, glycosylated polyamino acids, multivalent galactose, transferrin, bisphosphonates, polyglutamic acid, polyaspartic acid, lipids, cholesterol steroids, bile acid, folic acid, vitamin B12, biotin, or RGD peptide or RGD peptide mimetics.In certain embodiments, the ligand is multivalent galactose, such as N-acetyl-galactosamine.

[0441] Other examples of ligands include dyes, intercalating agents (e.g., acridine), crosslinkers (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules such as cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithium, and the like. Examples of suitable cleavage inhibitors include cleavage inhibitors (e.g., oleic acid, O3-(oleoyl)cholenoic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, 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, bis-imidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

[0442] The ligand can be a protein, such as a glycoprotein or peptide, a molecule with specific affinity for the co-ligand, or an antibody, such as an antibody that binds to a specific cell type, such as cancer cells, endothelial cells, or bone cells. Ligands can also include hormones and hormone receptors. They can also include non-peptide species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent mannose, or multivalent fucose. Ligands can be, for example, lipopolysaccharides, p38 MAP kinase activators, or NF-κB activators.

[0443] The ligand can be, for example, a substance, e.g., a drug, that can increase cellular uptake of an iRNA agent by, for example, disrupting the cytoskeleton of a cell, e.g., by disrupting cellular microtubules, microfilaments, or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, jasplakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[0444] In some embodiments, the ligand attached to the iRNA described herein acts as a pharmacokinetic modulator (PK modulator). PK modulators include lipophilic substances, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, and the like. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, and the like. Oligonucleotides containing several phosphorothioate linkages are also known to bind to serum proteins. Therefore, short oligonucleotides, e.g., oligonucleotides of about 5, 10, 15, or 20 bases containing multiple phosphorothioate linkages in the backbone, are also suitable as ligands (e.g., as PK-modulating ligands) for the present disclosure. Furthermore, aptamers that bind to serum components (e.g., serum proteins) are also suitable for use as PK-modulating ligands in the embodiments described herein.

[0445] Ligand-conjugated iRNAs of the present disclosure can be synthesized by using oligonucleotides bearing pendant reactive functionalities, e.g., derived from the attachment of a linking molecule onto an oligonucleotide (described below). This reactive oligonucleotide can be reacted directly with commercially available ligands, ligands that have been synthesized with any of a variety of protecting groups, or ligands that have a linking moiety attached to them.

[0446] The oligonucleotides used in the conjugates of the present disclosure can be conveniently and routinely produced by known techniques of solid phase synthesis.The equipment for such synthesis is sold by several vendors, including, for example, Applied Biosystems® (Foster City, California).Any other means for such synthesis known in the art can also or alternatively be used.It is also known to use similar techniques to prepare other oligonucleotides, for example, phosphorothioates and alkylated derivatives.

[0447] For the ligand-conjugated oligonucleotides and molecules having ligand-sequence-specific 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 have a linking moiety, ligand-nucleotide or nucleoside conjugate precursors that already have a ligand molecule, or non-nucleoside ligand-containing building blocks.

[0448] When using a nucleotide-conjugate precursor that already has a linking moiety, the synthesis of the sequence-specific linked nucleoside is usually completed, and then a ligand molecule is reacted with the linking moiety to form a ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides of the present disclosure are synthesized by automated synthesizers using phosphoramidites derived from ligand-nucleoside conjugates, in addition to standard and non-standard phosphoramidites that are commercially available and routinely used in oligonucleotide synthesis.

[0449] A. Lipid Conjugates In certain embodiments, the ligand or conjugate is a lipid or lipid-based molecule.This lipid or lipid-based molecule can usually bind to serum protein, for example, human serum albumin (HSA).HSA-binding ligand allows the distribution of conjugate to target tissue in the body, for example, non-renal target tissue.For example, target tissue can be CNS tissue, for example, brain tissue.Other molecules that can bind to HSA can also be used as ligand.For example, naproxen or aspirin can be used.Lipid or lipid-based ligand can (a) increase the resistance of conjugate to degradation, (b) increase targeting or transport into target cell or cell membrane, or (c) adjust the binding to serum protein, for example, HSA.

[0450] The lipid-based ligand can be used to modulate, e.g., manage (e.g., inhibit), the binding of the conjugate to the target tissue. For example, a lipid or lipid-based ligand that binds more strongly to HSA is less likely to be targeted to the kidney and therefore less likely to be eliminated from the body. A lipid or lipid-based ligand that binds less strongly to HSA can be used to target the conjugate to the kidney.

[0451] In certain embodiments, lipid-based ligand binds to HSA.For example, the ligand can bind to HSA with sufficient affinity, so that the distribution of conjugate to non-renal tissue is enhanced.However, the affinity is usually not so strong that HSA-ligand binding cannot be reversed.

[0452] In certain embodiments, the lipid-based ligand binds weakly or not at all to HSA, thereby enhancing distribution of the conjugate to the kidney. Other moieties that target kidney cells can be used instead of or in addition to the lipid-based ligand.

[0453] In another embodiment, the ligand is a moiety, such as a vitamin, that is taken up by target cells, for example, proliferating cells. These are particularly useful for treating disorders characterized by unwanted cell proliferation, for example, malignant or non-malignant types, such as cancer cells. Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins include 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).

[0454] B. Cell-penetrating agents In another aspect, the ligand is a cell-penetrating agent, for example, a helical cell-penetrating agent. In certain embodiments, these cell-penetrating agents are amphipathic. Exemplary cell-penetrating agents include peptides, such as tat or antennopedia. When the agent is a peptide, it may be modified, including peptidyl mimetics, invertomers, non-peptide or pseudopeptide linkages, and the use of D-amino acids. Helical agents are usually α-helical agents and may have a lipophilic and lipophobic phase.

[0455] The ligand can be a peptide or peptidomimetic. Peptidomimetics (also referred to herein as oligopeptidomimetics) are molecules capable of folding into defined three-dimensional structures similar to natural peptides. Attachment of peptides and peptidomimetics to iRNA agents can affect the pharmacokinetic distribution of iRNAs, such as by enhancing cellular recognition and uptake. The peptide or peptidomimetic moiety can be about 5 to 50 amino acids in length, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0456] The peptide or peptidomimetic can be, for example, a cell-penetrating peptide, a cationic peptide, an amphipathic peptide, or a hydrophobic peptide (e.g., composed primarily of Tyr, Trp, or Phe). The peptide moiety can be a dendrimeric peptide, a constrained peptide, or a crosslinked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF, which has the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 9). An RFGF analog containing a hydrophobic MTS (e.g., the amino acid sequence AALLPVLLAAP (SEQ ID NO: 10)) can also be a targeting moiety. The peptide moiety can be a "delivery" peptide, capable of transporting large polar molecules, including peptides, oligonucleotides, and proteins, across cell membranes. For example, the sequence derived from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 11)) and the sequence derived from the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 12)) have been found to function as delivery peptides. Peptides or peptidomimetics can be encoded by random sequences of DNA, such as peptides identified from phage display libraries or one-bead-one-compound (OBOC) combinatorial libraries (Lam et al., Nature, 354:82-84, 1991). Typically, peptides or peptidomimetics tethered to dsRNA agents via incorporated monomer units include cell-targeting peptides, such as arginine-glycine-aspartic acid (RGD) peptides or RGD mimics. The peptide portion can range in length from about 5 amino acids to about 40 amino acids. The peptide portion can have structural modifications, for example, to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.

[0457] The RGD peptide for use in the compositions and methods of the present disclosure can be linear or cyclic, and can be modified, for example, glycosylated or methylated, to facilitate targeting to specific tissue(s).RGD-containing peptides and peptidomimetics can contain D-amino acids and synthetic RGD mimics.In addition to RGD, other moieties can be used to target integrin ligands.Preferred conjugates of this ligand target PECAM-1 or VEGF.

[0458] RGD peptide moieties can be used to target specific cell types, such as tumor cells, e.g., endothelial tumor cells or breast cancer tumor cells (Zitzmann et al., Cancer Res., 62:5139-43, 2002). RGD peptides can facilitate targeting of dsRNA agents to tumors in various other tissues, including the lung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001). Typically, RGD peptides facilitate targeting of iRNA agents to the kidney. RGD peptides can be linear or cyclic, and can be modified, e.g., glycosylated or methylated, to facilitate targeting to a specific tissue(s). For example, glycosylated RGD peptides can facilitate targeting of iRNA agents to α- V It can be delivered to tumor cells that express β3 (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001).

[0459] A "cell-penetrating peptide" is capable of penetrating cells, such as microbial cells, e.g., bacterial or fungal cells, or mammalian cells, e.g., human cells. Microbial cell-penetrating peptides can be, for example, α-helical linear peptides (e.g., LL-37 or seropin P1), disulfide bond-containing peptides (e.g., α-defensins, β-defensins, or bactenecins), or peptides containing only one or two dominant amino acids (e.g., PR-39 or indolicidin). Cell-penetrating peptides can also contai...

Claims

1. A double-stranded ribonucleic acid (dsRNA) agent or salt thereof for inhibiting SNCA expression, wherein the dsRNA agent or salt thereof comprises a sense strand and an antisense strand that form a double-stranded region, a) The nucleotide sequence of the sense strand differs from the nucleotide sequence 5'-gsasgca(Ahd)guGfAfCfaaaauguugsgsa-3' of SEQ ID NO: 21 by four or fewer modified or unmodified nucleotides, and the nucleotide sequence of the antisense strand differs from the nucleotide sequence 5'-VPusdCscadAcdAuuugdTcAfcuugcucsusu-3' of SEQ ID NO: 64 by four or fewer modified or unmodified nucleotides. a, g, c, and u are 2-O-methyl(2-OMe)A, G, C, and U; Af, Gf, and Cf are 2-fluoro(2-F)A, G, and C; dA, dT, and dC are 2-deoxyA, T, and C; Ahd is 2'-O-hexadecyladenosine-3'-phosphate; VP is vinyl-phosphonate; s is phosphorothioate linkage; b) The nucleotide sequence of the sense strand differs from the nucleotide sequence 5'-gsasgca(Ahd)guGfAfCfaaaauguugsgsa-3' of SEQ ID NO: 21 by three or fewer modified or unmodified nucleotides, and the nucleotide sequence of the antisense strand differs from the nucleotide sequence 5'-VPusdCscadAcdAuuugdTcAfcuugcucsusu-3' of SEQ ID NO: 64 by three or fewer modified or unmodified nucleotides. a, g, c, and u are 2-O-methyl(2-OMe)A, G, C, and U; Af, Gf, and Cf are 2-fluoro(2-F)A, G, and C; dA, dT, and dC are 2-deoxyA, T, and C; Ahd is 2'-O-hexadecyladenosine-3'-phosphate; VP is vinyl-phosphonate; s is phosphorothioate linkage; c) The nucleotide sequence of the sense strand differs from the nucleotide sequence 5'-gsasgca(Ahd)guGfAfCfaaaauguugsgsa-3' of SEQ ID NO: 21 by two or fewer modified or unmodified nucleotides, and the nucleotide sequence of the antisense strand differs from the nucleotide sequence 5'-VPusdCscadAcdAuuugdTcAfcuugcucsusu-3' of SEQ ID NO: 64 by two or fewer modified or unmodified nucleotides. a, g, c, and u are 2-O-methyl(2-OMe)A, G, C, and U; Af, Gf, and Cf are 2-fluoro(2-F)A, G, and C; dA, dT, and dC are 2-deoxyA, T, and C; Ahd is 2'-O-hexadecyladenosine-3'-phosphate; VP is vinyl-phosphonate; s is phosphorothioate linkage; d) The nucleotide sequence of the sense strand differs from the nucleotide sequence 5'-gsasgca(Ahd)guGfAfCfaaaauguugsgsa-3' of SEQ ID NO: 21 by one or fewer modified or unmodified nucleotides, and the nucleotide sequence of the antisense strand differs from the nucleotide sequence 5'-VPusdCscadAcdAuuugdTcAfcuugcucsusu-3' of SEQ ID NO: 64 by one or fewer modified or unmodified nucleotides. a, g, c, and u are 2-O-methyl(2-OMe)A, G, C, and U; Af, Gf, and Cf are 2-fluoro(2-F)A, G, and C; dA, dT, and dC are 2-deoxyA, T, and C; Ahd is 2'-O-hexadecyladenosine-3'-phosphate; VP is vinyl-phosphonate; s is phosphorothioate linkage; e) The sense strand contains the nucleotide sequence 5'-gsasgca(Ahd)guGfAfCfaaaauguugsgsa-3' of SEQ ID NO: 21, and the antisense strand contains the nucleotide sequence 5'-VPusdCscadAcdAuuugdTcAfcuugcucsusu-3' of SEQ ID NO: 64, a, g, c, and u are 2-O-methyl(2-OMe)A, G, C, and U; Af, Gf, and Cf are 2-fluoro(2-F)A, G, and C; dA, dT, and dC are 2-deoxyA, T, and C; Ahd is 2'-O-hexadecyladenosine-3'-phosphate; VP is vinyl-phosphonate; s is phosphorothioate linkage; f) The sense strand consists of the nucleotide sequence 5'-gsasgca(Ahd)guGfAfCfaaaauguugsgsa-3' of SEQ ID NO: 21, and the antisense strand consists of the nucleotide sequence 5'-VPusdCscadAcdAuuugdTcAfcuugcucsusu-3' of SEQ ID NO:

64. a, g, c, and u are 2-O-methyl(2-OMe)A, G, C, and U; Af, Gf, and Cf are 2-fluoro(2-F)A, G, and C; dA, dT, and dC are 2-deoxyA, T, and C; Ahd is 2'-O-hexadecyladenosine-3'-phosphate; VP is vinyl-phosphonate; s is phosphorothioate linkage; g) The sense strand is as shown in Tables 1-1 and 1-2 below. Table 1-1 Table 1-2 The nucleotide sequence includes one or fewer nucleotides that are different from the nucleotide sequence selected from the group consisting of SEQ ID NOs. 150, 157, 164, and SEQ ID NOs. 142-149, 151-156, 158-163, and 165-184, The sense strand of the dsRNA agent or a salt thereof includes a lipophilic moiety attached to position 6 or 16 counting from the 5' end of the sense strand. The antisense strand contains a nucleotide sequence that differs by one or fewer nucleotides from a nucleotide sequence selected from the group consisting of SEQ ID NOs: 193, 200, 207, and SEQ ID NOs: 185-192, 194-199, 201-206, and 208-227 in Tables 1-1 and 1-2. The dsRNA agent or its salt does not contain GalNAc modification. The dsRNA agent or a salt thereof contains eight phosphorothioate nucleotide links located at the second-to-last and last nucleotide links at the 3'- and 5'- ends of the sense and antisense strands, respectively, or h) The sense strand is shown in Tables 2-1, 2-2, and 2-3 below. Table 2-1 Table 2-2 Table 2-3 The nucleotide sequences and modifications (SEQ ID NOs. 13-55) are included, The antisense strand includes the nucleotide sequences and modifications (SEQ ID NOs. 56-98) shown in Tables 2-1, 2-2, and 2-3. dsRNA preparation or a salt thereof.

2. The lipophilic portion is saturated or unsaturated C ４ ~C ３０ A dsRNA agent or a salt thereof according to claim 1, having the characteristic (g) of containing a hydrocarbon chain and a suitable functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.

3. The lipophilic portion is saturated or unsaturated C ６ ~C １８ A dsRNA agent or a salt thereof according to claim 1, having the characteristic (g) of containing a hydrocarbon chain.

4. The lipophilic portion is saturated or unsaturated C １６ A dsRNA agent or a salt thereof according to claim 1, having the characteristic (g) of containing a hydrocarbon chain.

5. The lipophilic part, 【Chemistry 1】 And, A dsRNA agent or a salt thereof according to claim 1, characterized (g) in which B is a nucleotide base or a nucleotide base analog, and B may be selected from the group consisting of adenine, guanine, cytosine, thymine, and uracil.

6. A dsRNA agent or a salt thereof according to claim 1, characterized (g) by having a lipophilic portion bound via a linker or carrier.

7. A dsRNA agent or a salt thereof according to claim 1, characterized (g) by being conjugated via a carrier that replaces the lipophilic portion with a nucleotide at position 6 or 16 of the sense strand.

8. A dsRNA agent or a salt thereof according to claim 1, characterized (g) by all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand containing nucleotide modifications.

9. At least one of the nucleotide modifications is a deoxy-nucleotide modification, a 3'-terminal deoxythymidine (dT) nucleotide modification, a 2'-O-methyl nucleotide modification, a 2'-fluoro nucleotide modification, a 2'-deoxy-nucleotide modification, a locked nucleotide modification, an unlocked nucleotide modification, a conformation-restricted nucleotide modification, a restricted ethyl nucleotide modification, a debasalized nucleotide modification, a 2'-amino-nucleotide modification, a 2'-O-allyl-nucleotide modification, a 2'-C-alkyl-nucleotide modification, a 2'-hydroxy-nucleotide modification, a 2'-methoxyethyl nucleotide modification, a 2'-O-alkyl-nucleotide modification, a morpholino nucleotide modification, a phosphoramide modification, a nucleotide modification containing a non-natural base, a tetrahydropyran nucleotide modification, a 1,5-anhydrohexitol nucleotide modification, A dsRNA agent or a salt thereof according to claim 8, selected from the group consisting of cyclohexenyl nucleotide modification, nucleotide modification containing a 5'-phosphorothioate group, nucleotide modification containing a 5'-methylphosphonate group, nucleotide modification containing a 5'-phosphate or 5'-phosphate mimetic, nucleotide modification containing a vinyl phosphate, nucleotide modification containing adenosine glycol nucleic acid (GNA), nucleotide modification containing a thymidine glycol nucleic acid (GNA) S-isomer, nucleotide modification containing 2-hydroxymethyl-tetrahydrofuran-5-phosphate, nucleotide modification containing 2'-deoxythymidine-3' phosphate, nucleotide modification containing 2'-deoxyguanosine-3'-phosphate, and cholesteryl derivatives and terminal nucleotide modifications linked to a dodecanoic acid bisdecylamide group.

10. A dsRNA agent or salt thereof according to claim 1, further comprising a phosphate or phosphate mimetic at the 5' end of the antisense strand, characterized (g).

11. The dsRNA agent or a salt thereof according to claim 1, wherein a sense strand or an antisense strand is conjugated to a ligand.

12. The dsRNA agent or a salt thereof according to claim 11, wherein the ligand comprises an antibody.

13. A cell containing the dsRNA agent or a salt thereof according to any one of claims 1 to 12.

14. A pharmaceutical composition comprising a dsRNA agent or a salt thereof according to any one of claims 1 to 12 and a pharmaceutically acceptable carrier.

15. An in vitro method for inhibiting the expression of the alpha-synuclein (SNCA) gene in cells and / or preventing the formation of alpha-synuclein aggregates in cells, (a) Contacting cells with a dsRNA agent or a salt thereof according to any one of claims 1 to 12, (b) Maintain the cells produced in step (a) for a sufficient time to obtain degradation of the mRNA transcript of the SNCA gene, thereby inhibiting the expression of the SNCA gene in the cells and / or preventing the formation of alpha-synuclein aggregates in the cells. A method that includes this.

16. The pharmaceutical composition according to claim 14, for use in treating a subject diagnosed with SNCA-associated neurodegenerative disease.

17. The pharmaceutical composition according to claim 16, wherein the target is a human.

18. SNCA-associated neurodegenerative diseases include tremors, bradykinesia (slowness of movement), muscle rigidity, postural and balance disorders, loss of automatic movements, speech changes, writing changes, visual, auditory, olfactory, or tactile hallucinations, dysregulation of bodily functions (autonomic nervous system), dizziness, falls, bowel problems, cognitive problems, confusion, decreased attention, visuospatial problems, memory loss, sleep disorders, rapid eye movement (REM) sleep behavior disorder (dreams are physically acted out during sleep), fluctuations in attention including episodes of sleepiness, prolonged spatial gazing, prolonged daytime napping, disorganized speech, depression, lethargy, and orthostatic hypotension (standing upright). The pharmaceutical composition according to claim 16, characterized by one or more symptoms selected from the group consisting of: a sudden drop in blood pressure when rising, causing dizziness or lightheadedness, and requiring the person to sit, squat, or lie down to prevent fainting; clumsiness or lack of coordination; bladder control problems; contractures of the hands and limbs (chronic shortening of muscles or tendons around joints, resulting in restricted joint movement); Pisa syndrome (an abnormal posture in which the body appears to be tilted to one side); cervical flexion (bending the neck forward and lowering the head); and involuntary and uncontrollable sighing or gasping.

19. The pharmaceutical composition according to claim 16, wherein the SNCA-associated neurodegenerative disease is selected from the group consisting of synucleinopathy, Parkinson's disease (PD), multiple system atrophy (MSA), Lewy body dementia (LBD), pure autonomic neuropathy (PAF), Pick's disease, progressive supranuclear palsy, boxer's dementia, chromosome 17-linked parkinsonism, Lichko-Bodig disease, entangled dementia, argyrophilic granuloma, ganglioglioma, gangliocytoma, meningeal hemangioma, subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis, Hallerwolden-Spatz disease, lipofuscinosis, corticobasal degeneration, frontotemporal dementia, frontotemporal lobar degeneration, Alzheimer's disease, Huntington's disease, Down syndrome, psychosis, schizophrenia, and Creutzfeldt-Jakob disease.

20. The pharmaceutical composition according to claim 16, wherein the dsRNA agent is for intrathecal administration.

21. The pharmaceutical composition according to claim 16, to be used in combination with additional agents or treatments suitable for the treatment or prevention of SNCA-associated neurodegenerative diseases or disorders.

22. A kit comprising the pharmaceutical composition according to claim 16, instructions for use, and, optionally, means for administering the pharmaceutical composition to a subject.

23. A dsRNA agent or a salt thereof according to any one of claims 1 to 12, in the form of a sodium salt.

Images