SNCA IRNA composition for treating or preventing SNCA-associated neurodegenerative diseases and method of using the same

Abstract

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

Description

[Technical Field]

[0001] Cross-reference of related applications This application relates to U.S. Provisional Patent Application No. 63 / 086,495, filed on 1 October 2020, entitled “SNCA iRNA Compositions and Methods of Use Thereof for Treating or Preventing SNCA-Associated Neurodegenerative Diseases,” claiming priority under 35 U.S. SC § 119(e). The entire contents of the aforementioned patent application are incorporated herein by reference.

[0002] This disclosure generally relates to SNCA-targeting RNAi agents and methods.

[0003] Sequence List This application includes a sequence listing filed electronically in ASCII format, the entirety of which is incorporated herein by reference. The ASCII copy, created on September 28, 2021, is named BN00007_0161_ALN_364WO_SL.txt and has a size of 687KB. [Background technology]

[0004] The SNCA gene encodes the presynaptic neuron protein α-synuclein (also referred to herein as α-synuclein or synuclein-α) and 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 many ways, it is generally believed that the abnormal soluble oligomeric conformation of α-synuclein, called protofibril, is a toxic species that mediates disruption of cellular homeostasis and neuronal death through its effects on various intracellular targets, including synaptic function. Furthermore, secreted α-synuclein is thought to exert harmful effects on neighboring cells, including seeding of aggregation, and therefore may contribute to disease transmission. While the extent to which α-synuclein is involved in all cases of Parkinson's disease (PD) is unclear, targeting the toxic function conferred by this protein when α-synuclein is dysregulated presents a potentially valuable therapeutic strategy not only for PD but also for other neurodegenerative conditions known as synuclein diseases, characterized by Lewy bodies (LBs) and Lewy neurites (LNs), which exhibit common neuropathological features as a result of α-synuclein accumulation. In addition to PD, recorded or suspected SNCA-associated synuclein disorders include, but are not limited to, multiple system atrophy, Lewy body dementia (LBD), pure autonomic dysplasia (PAF), Pick's disease, progressive supranuclear palsy, dementia pugilistica, parkinsonism associated with chromosome 17, Lytico-Bodig disease, neurofibrillary tangle dementia, argyrophilic granuloma, ganglioglioma, gangliocytoma, meningeal hemangioma, subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis, Hallerforden-Spatz disease, lipofuscinosis, corticobasal degeneration, frontotemporal dementia, frontotemporal lobar degeneration, Alzheimer's disease, Huntington's disease, Down syndrome, psychosis, schizophrenia, and Creutzfeldt-Jakob disease.

[0005] Parkinson's disease (PD) and left-brain dementia (LBD) are two of the most common examples of neurodegenerative disorders with SNCA (Synthetic Neuropathological Complication) brain pathology. PD is the most common type of motor disorder, characterized by rigidity, hypokinesia, tremor, and postural instability. PD is estimated to affect approximately 4 to 6 million people worldwide. LBD accounts for 5 to 15% of all dementia cases. In addition to memory loss and other often fluctuating cognitive symptoms, LBD patients typically experience recurrent falls and visual hallucinations.

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

[0007] Alpha-synuclein monomers, tetramers, and fibrillary aggregates are major components of Lewy body (LB)-like neuronal inclusions, glial inclusions, and axonal spheroids in neurodegeneration with cerebral iron accumulation. Lewy-associated lesions (LRPs), primarily composed of alpha-synuclein, are present in the majority of Alzheimer's autopsies, and higher levels of alpha-synuclein in patients are associated with cognitive decline (Twohig et al (2019) Molecular Neurodegeneration). In particular, autosomal dominant mutations of 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), and A53T (Polymeropoulos et al. (1997) Science), as well as triple duplication and duplication, have been identified as occurring in families affected by related neurodegenerative diseases. This indicates that not only pathogenic mutations in SNCA, but also increases in α-synuclein protein, influence disease outcomes.

[0008] Although the role of SNCA mutations in disease development is not fully understood, evidence points to the acquisition of toxic functions inherent to normal α-synuclein protein when normal α-synuclein protein exceeds a certain level (Stefanis et al. (2012) Cold Spring Harb Perspect Med.) and / or interacts abnormally with cellular lipids and vesicles (Kiechler et al. (2020) Front. Cell Dev. Biol, as outlined). Clearly consistent with this, SNCA null mice did not exhibit an obvious neuropathological or behavioral phenotype, in contrast to transgenic overexpression (Abeliovich et al. (2000) Neuron). Post-transcriptional 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 on familial point mutations in SNCA have demonstrated suppression of expression, particularly in cases of long-term disease onset (Markopoulou et al. (1999) Ann Neurol. 46(3):374-81 and Kobayashi et al. (2003) Brain 126(Pt 1):32-42). Similarly, Voutsinas et al. (2010) Hum Mutat. 31(6):685-91) found that even overexpression of wild-type SNCA messenger RNA (mRNA) can cause disease onset. These data suggest that suppression of total SNCA levels reduces α-synuclein-induced toxicity.

[0009] There are no disease-modifying treatments for synuclein diseases, including Parkinson's disease (PD), multiple system atrophy, and Lewy body dementia, and treatment options are limited, for example, merely palliative. For instance, currently, only symptomatic treatments are available for PD patients (by replacing the loss of active dopamine in the brain) and AD patients (i.e., cholinesterase inhibitors). None of the existing treatment strategies for alpha-synuclein diseases address the underlying disease process.

[0010] Therefore, given the documented involvement of SNCAs in several neurodegenerative disorders (synuclein diseases), there is still a need for drugs that possess both high biological activity and in vivo stability, and that can selectively and efficiently silence SNCA genes (e.g., eliminate or reduce the effects of toxic α-synuclein species) using the cell's own RNAi mechanisms that can effectively inhibit the expression of target SNCA genes. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] Stefanis,L.Cold Spring Harb Perspect Med.2:a009399 [Non-Patent Document 2] Klucken et al., 2006 [Non-Patent Document 3] Twohig et al.(2019)Molecular Neurodegeneration [Non-Patent Document 4] Zarranz et al.(2004)Ann.Neurol.55,164-173 [Non-Patent Document 5] Choi et al.(2004)FEBS Lett.576,363-368 [Non-Patent Document 6] Tsigelny et al.(2015)ACS Chem.Neurosci.6,403-416 [Non-Patent Document 7] Polymeropoulos et al.(1997)Science [Non-Patent Document 8] Stefanis et al. (2012) Cold Spring Harb Perspect Med. [Non-Patent Document 9] Kiechler et al.(2020)Front.Cell Dev.Biol [Non-Patent Document 10] Abeliovich et al. (2000) Neuron [Non-Patent Document 11] Junn et al. (2009) PNAS 106:13052-13057 [Non-Patent Document 12] Doxakis (2010), JBC [Non-Patent Document 13] Markopoulou et al. (1999) Ann Neurol. 46(3):374-81 [Non-Patent Document 14] Kobayashi et al. (2003)Brain 126(Pt 1):32-42 [Non-Patent Document 15] Voutsinas et al.(2010)Hum Mutat.31(6):685-91 [Overview of the project]

[0012] This disclosure provides an RNAi agent composition that affects RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the synuclein alpha (SNCA) gene. The SNCA gene may be present in cells, for example, in cells within a subject such as a human. The Disclosure also provides RNAi compositions of the Disclosure for inhibiting the expression of SNCA genes, or for treating subjects who benefit from inhibiting or reducing the expression of SNCA genes, for example, subjects suffering from or susceptible to SNCA-related neurodegenerative diseases or disorders, such as PD, multiple system atrophy, Lewy body disease (LBD), pure autonomic dysplasia (PAF), Pick's disease, progressive supranuclear palsy, boxer's dementia, chromosome 17-related parkinsonism, Ritiko-Bodig disease, neurofibrillary tangle-type senile dementia, argyrophilic granuloma, ganglioglioma, gangliocytoma, meningeal hemangioma, subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis, Hallerforden-Spatz disease, lipofuscinosis, corticobasal degeneration, frontotemporal dementia, frontotemporal lobar degeneration, Alzheimer's disease, and Huntington's disease.

[0013] Accordingly, in one embodiment, the present disclosure provides a double-stranded ribonucleic acid (RNAi) agent for inhibiting SNCA expression, the dsRNA agent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a nucleotide sequence containing at least 15 consecutive nucleotides having 0 or 1 mismatch of a portion of the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence having at least 90% nucleotide sequence identity with a portion of the nucleotide sequence of SEQ ID NO: 1, and the antisense strand comprises a nucleotide sequence containing at least 15 consecutive nucleotides having 0 or 1 mismatch of a corresponding portion of the nucleotide sequence of SEQ ID NO: 2, or a nucleotide sequence having at least 90% nucleotide sequence identity with a portion of the nucleotide sequence of SEQ ID NO: 2.

[0014] In another embodiment, the Disclosure provides a double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of an SNCA gene, wherein the RNAi agent comprises a sense strand and an antisense strand, the antisense strand comprising a complementary region containing at least 15 consecutive nucleotides that differ by three nucleotides or less (i.e., by 3, 2, 1, or 0 nucleotides) from any one of the antisense sequences listed in Table 2, Table 3, Table 12, or Table 13.

[0015] Optionally, the sense chain or antisense chain is conjugated to one or more lipophilic moieties.

[0016] In one embodiment, the sense strand has a nucleotide sequence comprising at least 17 consecutive nucleotides having zero or one mismatch in a portion of the nucleotide sequence of SEQ ID NO: 1, and the antisense strand has a nucleotide sequence comprising at least 17 consecutive nucleotides having zero or one mismatch in a corresponding portion of the nucleotide sequence of SEQ ID NO: 2, such that the sense strand is complementary to the at least 17 consecutive nucleotides of the antisense strand.

[0017] In some embodiments, the sense strand has a nucleotide sequence comprising at least 19 consecutive nucleotides having zero or one mismatch in a portion of the nucleotide sequence of SEQ ID NO: 1, and the antisense strand has a nucleotide sequence comprising at least 19 consecutive nucleotides having zero or one mismatch in a corresponding portion of the nucleotide sequence of SEQ ID NO: 2, such that the sense strand is complementary to the at least 19 consecutive nucleotides of the antisense strand.

[0018] In this embodiment, the sense strand has a nucleotide sequence comprising at least 21 consecutive nucleotides having zero or one mismatch in a portion of the nucleotide sequence of SEQ ID NO: 1, and the antisense strand has a nucleotide sequence comprising at least 21 consecutive nucleotides having zero or one mismatch in a corresponding portion of the nucleotide sequence of SEQ ID NO: 2, such that the sense strand is complementary to the at least 21 consecutive nucleotides of the antisense strand.

[0019] In some embodiments, the antisense strand includes a complementary region containing at least 15 consecutive nucleotides of any one of the antisense sequences listed in Table 2, Table 3, Table 12, or Table 13. In some embodiments, the antisense strand includes a complementary region containing at least 19 consecutive nucleotides that differ by three nucleotides or less (i.e., by 3, 2, 1, or 0 nucleotides) from any one of the antisense sequences listed in Table 2, Table 3, Table 12, or Table 13. In some embodiments, the antisense strand includes a complementary region containing at least 19 consecutive nucleotides of any one of the antisense sequences listed in Table 2, Table 3, Table 12, or Table 13. In some embodiments, thymine-uracil or uracil-thymine differences between aligned (compared) sequences are not counted as differing nucleotides between aligned (compared) sequences.

[0020] In some embodiments, the drug comprises one or more lipophilic moieties conjugated to one or more nucleotide positions (or possibly internal nucleotide positions) via a linker or carrier. In some embodiments, the lipophilic moieties are conjugated to one or more positions in the double-stranded region of the dsRNA drug. Optionally, one or more lipophilic moieties are conjugated to at least the sense strand. In some embodiments, one or more lipophilic moieties are conjugated to at least the antisense strand. In embodiments, one or more lipophilic moieties are conjugated to both strands.

[0021] In this embodiment, the lipophilicity of the lipophilic portion, as measured by logKow, is greater than 0.

[0022] In some embodiments, the hydrophobicity of the double-stranded RNAi agent, as measured by the unbound fraction in a plasma protein binding assay, is greater than 0.2. Optionally, the plasma protein binding assay is an electrophoretic mobility shift assay using human serum albumin protein.

[0023] Another aspect of the present disclosure provides a double-stranded RNAi agent for inhibiting the expression of an SNCA gene, the dsRNA agent comprising a sense strand and an antisense strand, wherein the sense strand comprises at least 15 consecutive nucleotides that differ by no more than 3 nucleotides (i.e., by 3, 2, 1, or 0 nucleotides) from any one of the sense strand sequences shown in Table 2, Table 3, Table 12, or Table 13, and the antisense strand comprises at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from any one of the antisense strand nucleotide sequences shown in Table 2, Table 3, Table 12, or Table 13. In one embodiment, the sense strand comprises at least 15 consecutive nucleotides from any one of the sense strand sequences shown in Table 2, Table 3, Table 12, or Table 13, and the antisense strand comprises at least 15 consecutive nucleotides from any one of the antisense strand nucleotide sequences shown in Table 2, Table 3, Table 12, or Table 13. In one embodiment, the sense strand comprises at least 19 consecutive nucleotides from any one of the sense strand sequences shown in Table 2, Table 3, Table 12, or Table 13, and the antisense strand comprises at least 19 consecutive nucleotides from any one of the antisense strand nucleotide sequences shown in Table 2, Table 3, Table 12, or Table 13 (i.e., differing by only 3, 2, 1, or 0 nucleotides).

[0024] Further aspects of this disclosure provide a double-stranded RNAi agent for inhibiting the expression of an SNCA gene, wherein the dsRNA agent comprises a sense strand and an antisense strand, the sense strand differing by only 3 nucleotides or less from any one of the nucleotide sequences of SEQ ID NOs. 1, 3, 5, or 7, or the entire nucleotide sequence of any one of SEQ ID NOs. 1, 3, 5, or 7, for example, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% from a nucleotide sequence (i.e., 3, 2, 1, or A sequence containing at least 15 consecutive nucleotides (different by only 0 nucleotides), where any substitution of uracil to thymine in SEQ ID NOs. 1, 3, 5, or 7 has at least 90% nucleotide sequence identity with any one of the nucleotide sequences of SEQ ID NOs. 1, 3, 5, or 7, or with the entire nucleotide sequence of any one of the nucleotide sequences of SEQ ID NOs. 1, 3, 5, or 7, for example, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% nucleotide sequence identity, and a sequence containing at least 15 consecutive nucleotides (different by only 0 nucleotides), where any substitution of uracil to thymine in SEQ ID NOs. 1, 3, 5, or 7 has at least 90% nucleotide sequence identity with any one of the nucleotide sequences of SEQ ID NOs. 1, 3, 5, or 7, and a sequence with 3 nucleotides. Differences that differ by only a few nucleotides (i.e., by only 3, 2, 1, or 0 nucleotides) are not counted as contributing differences, and the antisense strand contains at least 15 consecutive nucleotides that differ by only 3 nucleotides or less from any one of the nucleotide sequences of SEQ ID NOs. 2, 4, 6, or 8, or a nucleotide sequence having at least 90%, for example 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% nucleotide sequence identity with respect to the entire nucleotide sequence of any one of SEQ ID NOs. 2, 4, 6, or 8, and the sequence number A uracil substitution for thymine in any of sequence numbers 2, 4, 6, or 8 (when comparing aligned sequences) is not counted as a difference that contributes by only 3 nucleotides or less to any one nucleotide sequence of sequence number 2, 4, 6, or 8, or to the entire nucleotide sequence of any one of sequence numbers 2, 4, 6, or 8, and to a nucleotide sequence having 90%, e.g., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, where at least one of the sense strand and antisense strand isIt comprises one or more lipophilic moieties conjugated at the internal position of one or more nucleotides via an optional linker or carrier.

[0025] In one embodiment, a double-stranded RNAi agent targeting an SNCA includes a sense strand containing at least 15 consecutive nucleotides that differ by 3 nucleotides or less (i.e., by 3, 2, 1, or 0 nucleotides) from the nucleotide sequence of the double-stranded sense strand nucleotide sequence shown in Table 2, Table 3, Table 12, or Table 13.

[0026] In one embodiment, a double-stranded RNAi agent targeting an SNCA includes an antisense strand containing at least 15 consecutive nucleotides that differ by three nucleotides or less (i.e., by 3, 2, 1, or 0 nucleotides) from one of the double-stranded antisense nucleotide sequences in Tables 2, 3, 12, or 13.

[0027] Optionally, the double-stranded RNAi agent contains at least one modified nucleotide. In the embodiment, five or fewer sense strand nucleotides and five or fewer antisense strand nucleotides are unmodified.

[0028] In one embodiment, substantially all of the nucleotides in the sense strand are modified nucleotides. Optionally, all of the nucleotides in the sense strand are modified nucleotides.

[0029] In some embodiments, substantially all of the nucleotides in the sense strand are modified nucleotides. Optionally, all of the nucleotides in the antisense strand are modified nucleotides.

[0030] Optionally, all nucleotides in the sense strand and all nucleotides in the antisense strand are modified nucleotides.

[0031] In one embodiment, at least one of the modified nucleotides is a deoxy-nucleotide, a 3'-terminal deoxythymine (dT) nucleotide, a 2'-O-methyl-modified nucleotide, a 2'-fluoro-modified nucleotide, a 2'-deoxy-modified nucleotide, a lock nucleotide, an unlock nucleotide, a conformation-restricted nucleotide, a restricted ethyl nucleotide, a debasalized nucleotide, a 2'-amino-modified nucleotide, a 2'-O-allyl-modified nucleotide, a 2'-C-alkyl-modified nucleotide, a 2'-hydroxy-modified nucleotide, a 2'-methoxyethyl-modified nucleotide, a 2'-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base containing a nucleotide, a tetrahydropyran-modified nucleotide, or a 1,5-anhydride. These are lohexitol-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides containing a 5'-phosphorothioate group, nucleotides containing a 5'-methylphosphonate group, nucleotides containing 5'-phosphate or a 5'-phosphate mimetic, nucleotides containing vinylphosphonate, nucleotides containing adenosine glycol nucleic acid (GNA), nucleotides containing thymidine glycol nucleic acid (GNA) S-isomers, nucleotides containing 2-hydroxymethyltetrahydrofuran-5-phosphate, nucleotides containing 2'-deoxythymidine-3'-phosphate, nucleotides containing 2'-deoxyguanosine-3'-phosphate, or terminal nucleotides linked to a cholesteryl derivative or a dodecanoate bisdecylamide group.

[0032] In related embodiments, the modified nucleotide is a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a 3'-terminated deoxy-thymine nucleotide (dT), a loc nucleotide, a debasalized nucleotide, a 2'-amino modified nucleotide, a 2'-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, or a non-natural base containing a nucleotide.

[0033] In one embodiment, the modified nucleotide includes a short sequence of 3'-terminal deoxythymine nucleotides (dT).

[0034] In another embodiment, the nucleotide modifications are 2'-O-methyl, 2'-fluoro, and GNA modifications.

[0035] In further embodiments, the double-stranded RNAi agent comprises at least one phosphorothioate nucleotide interbonding. Optionally, the double-stranded RNAi agent comprises 6 to 8 (e.g., 6, 7, or 8) phosphorothioate nucleotide interbondings.

[0036] In one embodiment, the complementary region is at least 17 nucleotides long. Optionally, the complementary region is 19 to 23 nucleotides long. Optionally, the complementary region is 19 nucleotides long.

[0037] In one embodiment, each chain is 30 nucleotides or less in length.

[0038] In another embodiment, at least one strand includes a 3' overhang of at least one nucleotide. Optionally, at least one strand includes a 3' overhang of at least two nucleotides.

[0039] In this embodiment, the double-stranded region is 15 to 30 nucleotide pairs long.

[0040] The double-stranded region is of a length of 17 to 23 nucleotide pairs, chosen at random.

[0041] In some embodiments, the double-stranded region is 17 to 25 nucleotide pairs long.

[0042] In one embodiment, the double-stranded region is 23 to 27 nucleotide pairs long.

[0043] In this embodiment, the double-stranded region is 19 to 21 nucleotide pairs long.

[0044] In another embodiment, the double-stranded region is 21 to 23 nucleotide pairs long.

[0045] In one embodiment, each chain has 19 to 30 nucleotides. Optionally, each chain has 19 to 23 nucleotides. In another embodiment, each chain has 21 to 23 nucleotides.

[0046] In some embodiments, the double-stranded RNAi agent further comprises a lipophilic ligand, such as a C16 ligand, conjugated to the 3' end of the sense strand via a monovalent or branched divalent or trivalent linker.

[0047] In one embodiment, the ligand is [ka] [In the formula, B is a nucleotide base or a nucleotide base analogue, and B is optionally adenine, guanine, cytosine, thymine, or uracil.] That is the case.

[0048] In other embodiments, the drug further comprises a targeted ligand that targets liver tissue, for example, one or more GalNAc derivatives conjugated to a double-stranded RNAi drug, optionally via a linker or carrier.

[0049] In yet another embodiment, the agent further comprises a lipophilic ligand, such as a C16 ligand, conjugated to the 3' end of a sense chain via a monovalent or branched divalent or trivalent linker, and one or more GalNAc derivatives conjugated to the 3' end of a sense chain via a targeted ligand that targets liver tissue, such as a monovalent or branched divalent or trivalent linker.

[0050] In another embodiment, the complementary region to the SNCA includes one of the antisense sequences from Tables 2, 3, 12, or 13.

[0051] In further embodiments, the complementary region to the SNCA is one of the antisense sequences in Tables 2, 3, 12, or 13. In some embodiments, the nucleotide internal position includes all positions from each end of the chain except for the two terminal positions.

[0052] In the relevant embodiments, the internal position includes all positions from each end of the chain except for the three terminal positions. Optionally, the internal position excludes the cleavage region of the sense chain.

[0053] In some embodiments, the internal position excludes positions 9–12 counting from the 5' end of the sense strand. In one embodiment, the sense strand is 21 nucleotides long.

[0054] In other embodiments, the internal position excludes positions 11–13 counting from the 3' end of the sense strand. Optionally, the internal position excludes the cleavage region of the antisense strand. In one embodiment, the sense strand is 21 nucleotides long.

[0055] In some embodiments, the internal position excludes positions 12–14 counting from the 5' end of the antisense strand. In one embodiment, the antisense strand is 23 nucleotides long.

[0056] In another embodiment, internal positions excluding positions 11–13 on the sense strand counting from the 3' end and positions 12–14 on the antisense strand counting from the 5' end. In one embodiment, the sense strand is 21 nucleotides long and the antisense strand is 23 nucleotides long.

[0057] In further embodiments, one or more lipophilic moieties are conjugated to one or more of the following internal positions: positions 4–8 and 13–18 on the sense strand, and positions 6–10 and 15–18 on the antisense strand, counting from the 5' end of each strand. Optionally, one or more lipophilic moieties are conjugated to one or more of the following internal positions: positions 5, 6, 7, 15, and 17 on the sense strand, and positions 15 and 17 on the antisense strand, counting from the 5' end of each strand. In one embodiment, the sense strand is 21 nucleotides long and the antisense strand is 23 nucleotides long.

[0058] Optionally, the lipophilic portion is conjugated at position 21, 20, 15, 1, 7, 6, or 2 of the sense chain, or at position 16 of the antisense chain.

[0059] In one embodiment, the lipophilic portion is conjugated to the 21st, 20th, 15th, 1st, or 7th positions of the sense chain.

[0060] In this embodiment, the lipophilic portion is conjugated to the 21st, 20th, or 15th position of the sense chain.

[0061] In some embodiments, the lipophilic portion is conjugated to the 20th or 15th position of the sense chain.

[0062] In this embodiment, the lipophilic portion is conjugated at position 16 of the antisense chain.

[0063] In some embodiments, the lipophilic portion is an aliphatic, alicyclic, or polyalicyclic compound. Optionally, the lipophilic portion is a lipid, cholesterol, retinoic acid, cholic acid, adamantane acetate, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexianol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.

[0064] In some embodiments, the lipophilic portion is saturated or unsaturated C4-C 30 The material comprises a hydrocarbon chain and any selected functional group which is hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, or alkyne.

[0065] In one embodiment, the lipophilic portion is saturated or unsaturated C6-C 18 Contains hydrocarbon chains. Optionally, the lipophilic portion may be saturated or unsaturated C 16It contains a hydrocarbon chain. In the relevant embodiments, the lipophilic moiety is conjugated via a carrier that replaces one or more nucleotides at one or more internal positions. In some embodiments, the carrier is a cyclic group that is pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanil, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridadinyl, tetrahydrofuranyl, or dekalinyl, or an acyclic moiety based on a serinol skeleton or a diethanolamine skeleton.

[0066] In the embodiment, saturated or unsaturated C 16 The hydrocarbon chain is conjugated at the 6th position, counting from the 5' end of the chain.

[0067] In some embodiments, the lipophilic portion is conjugated to the double-stranded RNAi agent via a linker comprising an ether, thioether, urea, carbonate, amine, amide, maleimidothioether, disulfide, phosphodiester, sulfonamide bond, click reaction product, or carbamate.

[0068] In one embodiment, the lipophilic portion is conjugated to a nucleic acid base, a sugar portion, or an internucleoside bond.

[0069] In another embodiment, the double-stranded RNAi agent further comprises a phosphate or phosphate mimic at the 5' end of the antisense strand. In one embodiment, the phosphate mimic is 5'-vinylphosphonate (VP). In another embodiment, the phosphate mimic is 5'-cyclopropylphosphonate.

[0070] In one embodiment, the double-stranded RNAi agent further comprises a targeted ligand, such as a hydrophilic ligand, that targets a receptor that mediates delivery to CNS tissue. In one embodiment, the targeted ligand is a C16 ligand.

[0071] In some embodiments, the double-stranded RNAi agent further comprises a targeted ligand that targets brain tissue, such as the striatum.

[0072] In some embodiments, the double-stranded RNAi agent further comprises a targeted ligand that targets liver tissue, such as hepatocytes.

[0073] In one embodiment, the lipophilic moiety or targeted ligand is conjugated via a biocleavable linker, which is a functionalized monosaccharide or oligosaccharide of DNA, RNA, disulfide, amide, galactosamine, glucosamine, glucose, galactose, mannose, or a combination thereof.

[0074] In the relevant embodiments, the 3' end of the sense chain is protected via an end cap which is a cyclic group having an amine, and the cyclic group is pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanil, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridadinyl, tetrahydrofuranil, or dekalinyl.

[0075] In one embodiment, the RNAi agent comprises at least one modified nucleotide, which is a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a nucleotide containing glycol nucleic acid (GNA), or a nucleotide containing vinyl phosphonate. Optionally, the RNAi agent comprises at least one of each of the following modifications: a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a nucleotide containing glycol nucleic acid (GNA), and a nucleotide containing vinyl phosphonate.

[0076] In another embodiment, the RNAi agent comprises the modified nucleotide patterns shown in Table 2 or 12 below, and optionally, the positions of the 2'-C16, 2'-O-methyl, GNA, phosphorothioate, and 2'-fluoro modifications are independent of the individual nucleotide base sequences of the indicated RNAi agent.

[0077] In the embodiment, the dsRNA agent further includes a terminal chiral modification resulting from a first nucleotide bond at the 3' end of the antisense strand having an Sp-configured bound phosphorus atom, a terminal chiral modification resulting from a first nucleotide bond at the 5' end of the antisense strand having an Rp-configured bound phosphorus atom, or a terminal chiral modification resulting from a first nucleotide bond at the 5' end of the sense strand having either an Rp-configured or Sp-configured bound phosphorus atom.

[0078] In some embodiments, the dsRNA agent further includes a terminal chiral modification resulting from the first and second internucleotide bond at the 3' end of the antisense strand having an Sp-configured bound phosphorus atom, a terminal chiral modification resulting from the first internucleotide bond at the 5' end of the antisense strand having an Rp-configured bound phosphorus atom, or a terminal chiral modification resulting from the first internucleotide bond at the 5' end of the sense strand having either an Rp or Sp-configured bound phosphorus atom.

[0079] In one embodiment, the dsRNA agent further comprises a terminal chiral modification resulting from the first, second, and third internucleotide bondages at the 3' end of the antisense strand having an Sp-configured bound phosphorus atom, a terminal chiral modification resulting from the first internucleotide bondage at the 5' end of the antisense strand having an Rp-configured bound phosphorus atom, or a terminal chiral modification resulting from the first internucleotide bondage at the 5' end of the sense strand having either an Rp or Sp-configured bound phosphorus atom.

[0080] In an embodiment, the dsRNA agent further comprises a terminal chiral modification resulting from a nucleotide - nucleotide bond between the first and second nucleotides at the 3'-end of the antisense strand having a phosphorus atom in the Sp configuration, a terminal chiral modification resulting from a nucleotide - nucleotide bond between the third nucleotides at the 3'-end of the antisense strand having a phosphorus atom in the Rp configuration, a terminal chiral modification resulting from a nucleotide - nucleotide bond between the first nucleotides at the 5'-end of the antisense strand having a phosphorus atom in the Rp configuration, or a terminal chiral modification resulting from a nucleotide - nucleotide bond between the first nucleotides at the 5'-end of the sense strand having a phosphorus atom in either the Rp or Sp configuration.

[0081] In some embodiments, the dsRNA agent further comprises a terminal chiral modification resulting from a nucleotide - nucleotide bond between the first and second nucleotides at the 3'-end of the antisense strand having a phosphorus atom in the Sp configuration, a terminal chiral modification resulting from a nucleotide - nucleotide bond between the first and second nucleotides at the 5'-end of the antisense strand having a phosphorus atom in the Rp configuration, or a terminal chiral modification resulting from a nucleotide - nucleotide bond between the first nucleotides at the 5'-end of the sense strand having a phosphorus atom in either the Rp or Sp configuration.

[0082] In other embodiments, each of the duplexes of Tables 2, 9, and 12 may be particularly modified to provide another double - stranded iRNA agent of the present disclosure. In one example, the 3'-end of each sense strand can be modified by removing the 3'-end L96 ligand and exchanging the two phosphodiester nucleotide - nucleotide bonds between the three 3'-end nucleotides with phosphorothioate nucleotide - nucleotide bonds. That is, for the sense sequence of the following formula: 5’-N1-…-N n-2 N n-1 N n L96-3’ The three 3'-end nucleotides (N) of the sense sequence may be replaced by the following formula. 5’-N1-…-N n-2 s N n-1 s N n -3’。 That is, for example, regarding AD-1549052, the sense sequence is: asasgag(Chd)aaGfUfGfacaaauguuaL96 teeth, asasgag(Chd)aaGfUfGfacaaaugususa It can be replaced with, On the other hand, the antisense sequence remains unchanged to provide another double-stranded iRNA agent of the present disclosure. In other examples, the sense strand of each of the following double helixes is modified in accordance with the above description to provide the double helixes of the present disclosure: AD-596172, AD-596323, AD-596177, AD-596137, AD-596130, AD-596231, AD-595926, AD-596124, AD-596133, AD-595854, AD-596175, AD-596170, AD-596436, AD-596319, AD-596168, AD-596215, AD-596425, AD- 595769, AD-596171, AD-596392, AD-596402, AD-596144, AD-596396, AD-596517, AD-596426, AD-596169, AD-596391, AD-596320, AD-5962 83, AD-596362, AD-596431, AD-596515, AD-596128, AD-596235, AD-596322, AD-596427, AD-596127, AD-595855, AD-596129, and AD-595866. In other examples, each sense strand of the following double hedges is modified in accordance with the above description to provide the double hedges of the present disclosure: AD-596137.1, AD-596319.1, AD-596177.1, AD-596172.1, AD-596323.1, AD-596215.1, AD-596231.1, AD-596170.1, AD-596168.1, AD-596130.1, AD-595854.1, AD-595926.1, AD-596133.1, AD-596175.1, AD-596171.1, AD-595 769.1, AD-596392.1, AD-596425.1, AD-596515.1, AD-596144.1, AD-596436.1, AD-596124.1, AD-596402.1, AD-596517.1, AD-596391.1, AD-59616 9.1, AD-596396.1, AD-596427.1, AD-596426.1, AD-595866.1, AD-596431.1, AD-596362.1, AD-596320.1, AD-595855.1, AD-596235.1, AD-596283.1, AD-596129.1, AD-596390.1, AD-596131.1, AD-58643.17, AD-596322.1, AD-596128.1, and AD-596127.1. In other examples, each sense strand of the following double hedges is modified in accordance with the above description to provide the double hedges of this disclosure: AD-595769.2, AD-595770.1, AD-595773.1, AD-595774.1, AD-595926.2, AD-595933.1, AD-595935.1, AD-595937.1, AD-595938.1, AD-596099.1, AD-596215.2, AD-596217.1, AD-596276.1, AD-596328.1, AD- 596390.2, AD-596391.2, AD-596392.2, AD-596393.1, AD-596394.1, AD-596395.1, AD-596396.2, AD-596397.1, AD-596398.1, AD-59640 1.1, AD-596402.2, AD-596403.1, AD-596521.1, AD-596564.1, AD-689314.1, AD-689315.1, AD-689316.1, AD-689318.1, AD-689319.1, A D-689320.1, AD-689452.1, AD-689459.1, AD-689461.1, AD-689462.1, AD-689463.1, AD-689464.1, AD-689615.1, AD-689616.1, AD-689 747.1, AD-689748.1, AD-689753.1, AD-689755.1, AD-689786.1, AD-689787.1, AD-689788.1, AD-689835.1, AD-689907.1, AD-689925.1 , AD-689926.1, AD-689927.1, AD-689928.1, AD-689929.1, AD-689930.1, AD-689931.1, AD-689932.1, AD-689933.1, AD-689934.1, AD-6 89935.1, AD-689936.1, AD-689937.1, AD-689938.1, AD-689939.1, AD-690068.1, AD-690079.1, AD-690080.1, AD-690092.1, AD-691823.1, AD-691824.1, AD-691843.1, AD-691844.1, AD-691845.1, AD-691875.1, AD-691953.1, AD-ans 691954.1. In other examples, each sense strand of the following double hedges is qualified in accordance with the above description to provide the double hedges of this disclosure: AD-1549052.1, AD-1549359.1, AD-1549054.1, AD-1571262.1, AD-1549333.1, AD-1549407.1, AD-1548854.1, AD-1549403.1, AD-1549283.1, AD-1549641.1, AD-1549267.1, AD-1548851.1, AD-1548869.1, AD-1 549272.1, AD-1571164.1, AD-1549354.1, AD-1571188.1, AD-1549401.1, AD-1548886.1, AD-1571191.1, AD-1571193.1, AD-1548884.1, A D-1571187.1, AD-1549357.1, AD-1571194.1, AD-1549285.1, AD-1549266.1, AD-1549351.1, AD-1548870.1, AD-1549245.1, AD-1549334. 1, AD-1549397.1, AD-1549290.1, AD-1549525.1, AD-1549406.1, AD-1549284.1, AD-1549439.1, AD-1549269.1, AD-1549518.1, AD-1549 628.1, AD-1571199.1, AD-1549442.1, AD-1549596.1, AD-1549400.1, AD-1549280.1, AD-1549441.1, AD-1549556.1, AD-1571202.1, AD-1 549271.1, AD-1549517.1, AD-1549293.1, AD-1549639.1, AD-1549443.1, AD-1571195.1, AD-1549595.1, AD-1549546.1, AD-1549246.1, A D-1571192.1, AD-1571165.1, AD-1549270.1, AD-1549521.1, AD-1549541.1, AD-1549552.1, AD-1549522.1, AD-1549545.1, AD-1549519.1、AD-1549630.1、AD-1549353.1、AD-1549544.1、AD-1549642.1、AD-1549438.1、AD-1549412.1、AD-1571198.1、AD-1571258.1、AD-1571201.1、AD-1549640.1、AD-1571266.1、AD-1571172.1、AD-1549527.1、AD-1549547.1、AD-1549037.1、AD-1571205.1、AD-1549053.1、AD-1571264.1、AD-1571186.1、AD-1571204.1、AD-1549555.1、AD-1548887.1、AD-1549426.1、AD-1548844.1、AD-1549520.1、AD-1549543.1、AD-1549548.1、AD-1571206.1、AD-1549210.1、AD-1571200.1、AD-1571207.1、AD-1549542.1、AD-1549211.1、AD-1571263.1、AD-1549391.1、AD-1549212.1、AD-1549268.1、AD-1549352.1、AD-1571261.1、AD-1549044.1、AD-1549554.1、AD-1548975.1、AD-1549432.1、AD-1549524.1、AD-1549643.1、AD-1571196.1、AD-1571203.1、AD-1549425.1、AD-1549264.1、AD-1549249.1、AD-1571257.1、AD-1549265.1、AD-1548843.1、AD-1548845.1、AD-1571256.1、AD-1571255.1、AD-1571174.1、AD-1571173.1、AD-1548876.1、AD-1549615.1、AD-1571166.1、AD-1571269.1、AD-1548976.1、AD-1549038.1、AD-1571167.1、AD-1571170.1、AD-1548888.1、AD-1571189.1、AD-1571259.1、AD-1549224.1、AD-1571208.1、AD-1549222.1、AD-1571268.1、AD-1571270.1、AD-1549217.1、AD-1571184.1、AD-1571271.1. AD-1571272.1, AD-1571190.1, AD-1549055.1, AD-1571169.1, and AD-1571265.1.

[0083] Further aspects of this disclosure provide cells having the dsRNA agent of this disclosure.

[0084] One aspect of this disclosure provides a pharmaceutical composition for inhibiting the expression of a gene encoding an SNCA, comprising a dsRNA agent of this disclosure.

[0085] Further aspects of the present disclosure provide a method for inhibiting the expression of an SNCA gene in cells, the method comprising (a) contacting cells with a double-stranded RNAi agent of the present disclosure or a pharmaceutical composition of the present disclosure, and (b) maintaining the cells obtained in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the SNCA gene, thereby inhibiting the expression of the SNCA gene in the cells.

[0086] In one embodiment, cells are present within the target. The target is optionally human.

[0087] In one embodiment, the subject is a rhesus macaque, a crab-eating macaque, a mouse, or a rat.

[0088] In several embodiments, SNCA expression is inhibited by at least 50%.

[0089] In one embodiment, the subject meets at least one diagnostic criterion for SNCA-related disease.

[0090] In one embodiment, the human subject is diagnosed with or suffering from a synuclein disease such as SNCA-associated neurodegenerative disease, e.g., PD, multiple system atrophy, Lewy body disease (LBD), pure autonomic dysplasia (PAF), Pick's disease, progressive supranuclear palsy, boxer's dementia, chromosome 17-associated parkinsonism, Ritico Bodig disease, neurofibrillary tangle-type senile dementia, argyrophilic granuloma, ganglioglioma, gangliocytoma, meningeal hemangioma, subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis, Hallerforden-Spatz disease, lipofuscinosis, corticobasal degeneration, frontotemporal dementia, frontotemporal lobar degeneration, Alzheimer's disease, Huntington's disease, Down syndrome, psychosis, schizophrenia, and Creutzfeldt-Jakob disease.

[0091] In some embodiments, the method further comprises administering an additional therapeutic agent or treatment to the subject. Exemplary additional therapeutic agents and treatments include, for example, sedatives, antidepressants, clonazepam, sodium valproate, opioates, antiepileptics, cholinesterase inhibitors, memantine, benzodiazepines, levodopa, COMT inhibitors (e.g., tolcapone and entacapone), dopamine agonists (e.g., bromocriptine, pergolide, pramipexole, ropinirole, pyribezil, cabergoline, apomorphine and rislide), MAO-B inhibitors (e.g., This includes safinamides, selegiline and rasagiline), amantadine, anticholinergics, modafinil, pimavanserin, doxepin, rasagrine, antipsychotics, atypical antipsychotics (e.g., amisulpride, olanzapine, risperidone, and clozapine), riluzole, edaravone, deep brain stimulation, non-invasive ventilation (NIV), invasive ventilation, physiotherapy, occupational therapy, speech therapy, dietary therapy and swallowing techniques, feeding tubes, PEG tubes, probiotics, and psychotherapy.

[0092] In one embodiment, the double-stranded RNAi agent is administered in doses ranging from approximately 0.01 mg / kg to approximately 50 mg / kg.

[0093] In some embodiments, the double-stranded RNAi agent is administered intrathecally to the subject.

[0094] In one embodiment, the method reduces the expression of SNCA genes in brain (e.g., striatum) or spinal tissue. Optionally, the brain or spinal tissue may be the striatum, cortex, cerebellum, cervical vertebrae, lumbar vertebrae, or thoracic vertebrae.

[0095] In some embodiments, the double-stranded RNAi agent is administered subcutaneously to the subject.

[0096] In one embodiment, the method reduces the expression of the SNCA gene in the liver.

[0097] In other embodiments, the method reduces the expression of SNCA genes in the liver and brain.

[0098] Another aspect of the present disclosure provides a method for treating a subject diagnosed with an SNCA-associated neurodegenerative disease, the method comprising administering a therapeutically effective amount of the dsRNA agent or pharmaceutical composition of the present disclosure to the subject, thereby treating the subject.

[0099] In one embodiment, the treatment includes improvement of at least the signs or symptoms of the disease.

[0100] In one embodiment, treatment includes preventing the progression of the disease.

[0101] In embodiments, SNCA-related disorders include symptoms of Parkinson's disease (PD), such as tremors, slowing of movement (bradykinesia), muscle rigidity, postural and balance disorders, loss of automatic movement, changes in speech, and changes in writing; visual, auditory, olfactory, or tactile hallucinations; signs of Parkinson's disease (Parkinsonian signs); impaired control of bodily functions (autonomic nervous system), such as dizziness, falls, and 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 being physically acted out during sleep); fluctuating attention, including episodes of drowsiness; prolonged spatial gaze; long periods of daytime sleepiness or disorganized speech; depression and emotional blunting; and symptoms of pure autonomic dysfunction, such as orthostatic hypotension (blood pressure when standing up). It is characterized by symptoms of Lewy body dementia, such as a rapid decline in ability (requiring sitting, squatting, or lying down to prevent dizziness, lightheadedness, and loss of consciousness), symptoms of multiple system atrophy, such as delayed movement, tremor or rigidity, limping or incoordination, dysphonia, undulation, tremor, loss of consciousness or lightheadedness due to asthostatic hypotension, bladder control problems such as sudden urinary urgency or difficulty urinating, contractures of the hands or limbs (chronic shortening of the muscles or tendons around a joint that prevents free movement of the joint), Pisa syndrome (an abnormal posture in which the body appears to be tilted to one side), antecollis (a forward bend of the neck and downward drooping of the head), involuntary and uncontrolled sighing or gasping, and sleep disorders such as rapid eye movement (REM) sleep behavior disorder.

[0102] In one embodiment, the SNCA-related diseases are synuclein diseases such as PD, multiple system atrophy, Lewy body dementia (LBD), pure autonomic dysplasia (PAF), Pick's disease, progressive supranuclear palsy, boxer's dementia, chromosome 17-related parkinsonism, Ritico Bodig disease, neurofibrillary tangle-type senile dementia, argyrophilic granuloma, ganglioglioma, gangliocytoma, meningeal hemangioma, subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis, Hallerforden-Spatz disease, lipofuscinosis, corticobasal degeneration, frontotemporal dementia, frontotemporal lobar degeneration, Alzheimer's disease, Huntington's disease, Down syndrome, psychosis, schizophrenia, and Creutzfeldt-Jakob disease.

[0103] Further aspects of the present disclosure provide a method for preventing the development of SNCA-associated neurodegenerative disease in subjects meeting at least one diagnostic criterion for SNCA-associated neurodegenerative disease, the method comprising administering a therapeutically effective amount of the dsRNA agent or pharmaceutical composition of the present disclosure to a subject, thereby preventing the development of SNCA-associated neurodegenerative disease in a subject meeting at least one diagnostic criterion for SNCA-associated neurodegenerative disease.

[0104] In one embodiment, the method further includes administering an additional agent or therapy suitable for the treatment or prevention of SNCA-related diseases or disorders.

[0105] Another aspect of the present disclosure provides a method for inhibiting SNCA expression in a subject, the method comprising administering a therapeutically effective amount of the double-stranded RNAi agent or pharmaceutical composition of the present disclosure to the subject, thereby inhibiting SNCA expression in the subject.

[0106] Further aspects of the present disclosure provide methods for treating or preventing a subject disorder or SNCA-related neurodegenerative disease or disorder, the methods comprising administering a therapeutically effective amount of the double-stranded RNAi agent or pharmaceutical composition of the present disclosure to a subject to treat or prevent the subject SNCA-related neurodegenerative disease or disorder.

[0107] Another aspect of the present disclosure provides a double-stranded RNAi agent for inhibiting the expression of the SNCA gene, the double-stranded RNAi agent comprising an antisense strand and a sense strand complementary thereto, the antisense strand comprising a region complementary to a portion of the mRNA encoding SNCA, each strand being approximately 14 to approximately 30 nucleotides long, and the double-stranded RNAi agent is expressed by 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 [[End]]-N b ’-Y’Y’Y’-N b ’-(Z’Z’Z’) l [[End]]-N a ’-n q ’5’ (III) is represented by wherein i, j, k and l are each independently 0 or 1, p, p’, q and q’ are each independently 0 to 6, each N a and N a ’ represents an oligonucleotide sequence containing 0 to 25 nucleotides which are independently either modified or unmodified or a combination thereof, and each sequence contains at least two different modified nucleotides, each N b and N b ’ represents an oligonucleotide sequence containing 0 to 10 nucleotides which are independently either modified or unmodified or a combination thereof, each n which may or may not be present p , n p , n q and n q ’ independently represents an overhang nucleotide, 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, The modification of N b is different from the modification of Y, and the modification of N b ’ is different from the modification of Y’, where the sense strand is conjugated to at least one ligand.

[0108] In one embodiment, i is 0, j is 0, i is 1, j is 1, both i and j are 0, or both i and j are 1.

[0109] In another embodiment, k is 0, l is 0, k is 1, l is 1, both k and l are 0, or both k and l are 1.

[0110] In one embodiment, XXX is complementary to X’X’X’, YYY is complementary to Y’Y’Y’, and ZZZ is complementary to Z’Z’Z’.

[0111] In another embodiment, the YYY motif is present at or near the cleavage site of the sense strand.

[0112] In a further embodiment, the Y’Y’Y’ motif is present at positions 11, 12, and 13 of the antisense strand from the 5’ end. Optionally, Y’ is a ^{2’}-O-methyl.

[0113] In some embodiments, formula (III) is formula (IIIa): Sense: 5’n p -N a -YYY-N a -n q 3’ Antisense: 3’n p’ -N a’ -Y’Y’Y’-N a’ -n q’ 5’ (IIIa) represented by

[0114] In another embodiment, formula (III) is formula (IIIb): Sense: 5’n p -N a -YYY-N b -ZZZ-N a -n q ​​​​​​​​​​​​​​​​​​​​The '' independently represents an oligonucleotide sequence containing 1 to 5 modified nucleotides.

[0115] In a further embodiment, equation (III) is equation (IIIc): Sense: 5'n p -N a -XXX-N b -YYY-N a -n q 3' Antisense: 3'n p’ -N a’ -X'X'X'-N b’ -Y'Y'Y'-N a’ -n q’ 5' (IIIc) Represented by, In the formula, each N b and N b The '' independently represents an oligonucleotide sequence containing 1 to 5 modified nucleotides.

[0116] In one embodiment, equation (III) is equation (IIId): Sense: 5'n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3' Antisense: 3'n p’ -N a’ -X'X'X'-N b’ -Y'Y'Y'-N b’ -Z'Z'Z'-N a’ -n q’ 5' (IIId) Represented by, In the formula, each N b and N b ' independently represents an oligonucleotide sequence containing 1 to 5 modified nucleotides, each N a and N a The '' independently represents an oligonucleotide sequence containing 2 to 10 modified nucleotides.

[0117] In another embodiment, the double-stranded region is 15–30 nucleotide pairs long. Optionally, the double-stranded region is 17–23 nucleotide pairs long.

[0118] In one embodiment, the double-stranded region is 17–25 nucleotide pairs long. Optionally, the double-stranded region is 23–27 nucleotide pairs long.

[0119] In some embodiments, the double-stranded region is 19–21 nucleotide pairs long. Optionally, the double-stranded region is 21–23 nucleotide pairs long.

[0120] In one embodiment, each chain has 15 to 30 nucleotides. Optionally, each chain has 19 to 30 nucleotides. Optionally, each chain has 19 to 23 nucleotides.

[0121] In one embodiment, the double-stranded region is 19 to 21 nucleotide pairs long, and each strand has 19 to 23 nucleotides.

[0122] In another embodiment, nucleotide modifications of the RNAi agent include LNA, glycol nucleic acid (GNA), HNA, CeNA, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy or 2'-hydroxyl, and combinations thereof. Optionally, nucleotide modifications include 2'-O-methyl, 2'-fluoro or GNA, and combinations thereof. In related embodiments, the modification on the nucleotide is a 2'-O-methyl or 2'-fluoro modification.

[0123] In one embodiment, the RNAi agent comprises a ligand which is one or more lipophilic moieties, such as a C16 moiety, linked via a divalent or trivalent branched linker.

[0124] In other embodiments, the agent further comprises targeted ligands that target liver tissue, such as one or more GalNAc derivatives.

[0125] In yet other embodiments, the agent further comprises a lipophilic ligand conjugated to the 3' end of the sense strand via a monovalent or branched divalent or trivalent linker, such as a C16 ligand, and a targeting ligand that targets liver tissue, such as one or more GalNAc derivatives conjugated to the 3' end of the sense strand via a monovalent or branched divalent or trivalent linker.

[0126] In certain embodiments, the ligand is attached to the 3' end of the sense strand.

[0127] In some embodiments, the RNAi agent further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage. In related embodiments, the phosphorothioate or methylphosphonate internucleotide linkage is at the 3' end of one strand. Optionally, the strand is the antisense strand. In another embodiment, the strand is the sense strand. In related embodiments, the phosphorothioate or methylphosphonate internucleotide linkage is at the 5' end of one strand. Optionally, the strand is the antisense strand. In another embodiment, the strand is the sense strand.

[0128] In another embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is present at both the 5' and 3' ends of one strand. Optionally, the strand is the antisense strand. In another embodiment, the strand is the sense strand.

[0129] In a further embodiment, the base pair at position 1 at the 5' end of the antisense strand of the RNAi agent is an A:U base pair.

[0130] In certain embodiments, the Y nucleotide contains a 2'-fluoro modification.

[0131] In some embodiments, the Y' nucleotide contains a 2'-O-methyl modification.

[0132] In certain embodiments, p'>0. Optionally, p' = 2.

[0133] In some embodiments, q’ = 0, p = 0, q = 0, and the p’ overhang nucleotides are complementary to the target mRNA.

[0134] In one embodiment, q’ = 0, p = 0, q = 0 and the p’ overhang nucleotides are non-complementary to the target mRNA.

[0135] In one embodiment, the sense strand of the RNAi agent has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

[0136] In another embodiment, at least one n p ’ is linked to an adjacent nucleotide via a phosphorothioate bond. Optionally, all n p ’ are linked to an adjacent nucleotide via a phosphorothioate bond.

[0137] In certain embodiments, the SNCA RNAi agents of the present disclosure are one of those listed in Table 2, Table 3, Table 12 or Table 13. In some embodiments, all nucleotides of the sense strand and all nucleotides of the antisense strand contain modifications.

[0138] Another aspect of the present disclosure provides a double-stranded RNAi agent for inhibiting the expression of the SNCA gene in a cell, the double-stranded RNAi agent comprising a sense strand complementary to the antisense strand, the antisense strand comprising a region complementary to a portion of the mRNA encoding the SNCA gene, each strand being about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent being of 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 -Nb '-Y'Y'Y'-N b '-(Z'Z'Z') l -N a '-n q '5' (III) Represented by, During the ceremony, i, j, k, and l are each independently either 0 or 1. p, p', q, and q' are each independently between 0 and 6. each N a and N a ' independently represents an oligonucleotide sequence containing 0 to 25 nucleotides that are either modified, unmodified, or a combination thereof, and each sequence contains at least two different modified nucleotides. each N b and N b ' independently represents an oligonucleotide sequence containing 0 to 10 nucleotides that are either modified, unmodified, or a combination thereof. Each n may exist independently or not. p , n p ',n q and n q ' represents an overhanging nucleotide, 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, where the modifications are 2'-O-methyl or 2'-fluoro modifications. N b The modification of is different from the modification of Y, N b The modification of ' is different from the modification of Y', Here, the sense chain is conjugated to at least one ligand, optionally, the ligand being one or more lipophilic ligands, e.g., C16 ligands, or one or more GalNAc derivatives.

[0139] Further aspects of this disclosure provide a double-stranded RNAi agent for inhibiting the expression of the SNCA gene in cells, wherein the double-stranded RNAi agent comprises an antisense strand and a sense strand complementary thereto, the antisense strand comprising a region complementary to a portion of the mRNA encoding SNCA, and each strand being approximately 14 to approximately 30 nucleotides long, and the double-stranded RNAi agent is formulated as (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) Represented by, During the ceremony, i, j, k, and l are each independently either 0 or 1. Each n may or may not exist. p , n q and n q ' independently represents an overhang nucleotide, p, q, and q' are each independently between 0 and 6. n p '>0 and at least one n p ' is bound to the adjacent nucleotide via a phosphorothioate crosslink, each N a and N a ' independently represents an oligonucleotide sequence containing 0 to 25 nucleotides that are either modified, unmodified, or a combination thereof, and each sequence contains at least two different modified nucleotides. each N b and N b' independently represents an oligonucleotide sequence containing 0 to 10 nucleotides that are either modified, unmodified, or a combination thereof. 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, where the modifications are 2'-O-methyl, glycol nucleic acid (GNA), or 2'-fluoro modifications. N b The modification of is different from the modification of Y, N b The modification of ' is different from the modification of Y', Here, the sense chain is conjugated to at least one ligand, optionally, the ligand being one or more lipophilic ligands, e.g., C16 ligands, or one or more GalNAc derivatives.

[0140] Another aspect of the present disclosure provides a double-stranded RNAi agent for inhibiting the expression of the SNCA gene in cells, the double-stranded RNAi agent comprising an antisense strand and a sense strand complementary thereto, the antisense strand comprising a region complementary to a portion of the mRNA encoding SNCA (a nucleotide sequence having at least 90% nucleotide sequence identity to SEQ ID NO: 1, or the entire nucleotide sequence of SEQ ID NO: 1, e.g., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity), each strand being approximately 14 to approximately 30 nucleotides long, the double-stranded RNAi agent being of 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) Represented by, During the ceremony, i, j, k, and l are each independently either 0 or 1. Each n may or may not exist. p , n q and n q ' independently represents an overhang nucleotide, p, q, and q' are each independently between 0 and 6. n p '>0 and at least one n p ' is bound to the adjacent nucleotide via a phosphorothioate crosslink, each N a and N a ' independently represents an oligonucleotide sequence containing 0 to 25 nucleotides that are either modified, unmodified, or a combination thereof, and each sequence contains at least two different modified nucleotides. each N b and N b ' independently represents an oligonucleotide sequence containing 0 to 10 nucleotides that are either modified, unmodified, or a combination thereof. 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, where the modifications are 2'-O-methyl or 2'-fluoro modifications. N b The modification of is different from the modification of Y, N b The modification of ' is different from the modification of Y', Here, the sense chain is conjugated to at least one ligand, optionally, the ligand being one or more lipophilic ligands, e.g., C16 ligands, or one or more GalNAc derivatives.

[0141] Further aspects of the present disclosure provide a double-stranded RNAi agent for inhibiting the expression of the SNCA gene in cells, the double-stranded RNAi agent comprising an antisense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of the mRNA encoding SNCA (a nucleotide sequence having at least 90% nucleotide sequence identity to SEQ ID NO: 1, or the entire nucleotide sequence of SEQ ID NO: 1, e.g., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity), each strand being approximately 14 to approximately 30 nucleotides long, the double-stranded RNAi agent being 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) Represented by, During the ceremony, i, j, k, and l are each independently either 0 or 1. Each n may or may not exist. p , n q and n q ' independently represents an overhang nucleotide, p, q, and q' are each independently between 0 and 6. n p '>0 and at least one n p ' is bound to the adjacent nucleotide via a phosphorothioate crosslink, each N a and N a' independently represents an oligonucleotide sequence containing 0 to 25 nucleotides that are either modified, unmodified, or a combination thereof, and each sequence contains at least two different modified nucleotides. each N b and N b ' independently represents an oligonucleotide sequence containing 0 to 10 nucleotides that are either modified, unmodified, or a combination thereof. 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, where the modifications are 2'-O-methyl or 2'-fluoro modifications. N b The modification of is different from the modification of Y, N b The modification of ' is different from the modification of Y', The sense chain contains at least one phosphorothioate crosslink. Here, the sense chain is conjugated to at least one ligand, optionally, the ligand being one or more lipophilic ligands, e.g., C16 ligands, or one or more GalNAc derivatives.

[0142] Another aspect of the present disclosure provides a double-stranded RNAi agent for inhibiting the expression of the SNCA gene in cells, the double-stranded RNAi agent comprising an antisense strand and a sense strand complementary thereto, the antisense strand comprising a region complementary to a portion of the mRNA encoding SNCA (a nucleotide sequence having at least 90% nucleotide sequence identity to SEQ ID NO: 1, or the entire nucleotide sequence of SEQ ID NO: 1, e.g., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity), each strand being approximately 14 to approximately 30 nucleotides long, the double-stranded RNAi agent being of formula (III): Sense: 5'n p -N a -YYY-N a -n q 3' Antisense: 3'n p '-N a '-Y'Y'Y'-N a '-nq '5' (IIIa) Represented by, During the ceremony, Each n may or may not exist. p , n q and n q ' independently represents an overhang nucleotide, p, q, and q' are each independently between 0 and 6. n p '>0 and at least one n p ' is bound to the adjacent nucleotide via a phosphorothioate crosslink, each N a and N a ' independently represents an oligonucleotide sequence containing 0 to 25 nucleotides that are either modified, unmodified, or a combination thereof, and each sequence contains at least two different modified nucleotides. YYY and Y'Y'Y' each independently represent one motif of three identical modifications on three consecutive nucleotides, and the modifications are 2'-O-methyl or 2'-fluoro modifications. The sense chain contains at least one phosphorothioate crosslink. Here, the sense chain is conjugated to at least one ligand, optionally, the ligand being one or more lipophilic ligands, e.g., C16 ligands, or one or more GalNAc derivatives.

[0143] Further aspects of the present disclosure provide a double-stranded RNAi agent for inhibiting the expression of an SNCA gene, the SNCA-targeting double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, the sense strand comprising at least 15 consecutive nucleotides that differ by three nucleotides or less (i.e., by 3, 2, 1, or 0 nucleotides) from any one of the nucleotide sequences of SEQ ID NOs. 1, 3, 5, and 7, or at least 90% of the nucleotides from any one of the nucleotide sequences of SEQ ID NOs. 1, 3, 5, or 7 The antisense strand contains nucleotide sequences with sequence identity, e.g., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity, and the antisense strand contains at least 15 consecutive nucleotides that differ by only 3 nucleotides or less (i.e., 3, 2, 1, or 0 nucleotides) from any one of the nucleotide sequences of SEQ ID NOs. 2, 4, 6, and 8, or has at least 90% nucleotide sequence identity with respect to the entire nucleotide sequence of any one of SEQ ID NOs. 2, 4, 6, and 8, e.g., 90, 91, 92, The sequence contains nucleotide sequences with 93, 94, 95, 96, 97, 98, 99 or 100% identity, and any uracil substitution to thymine in the sequences provided in SEQ ID NOs. 1-8 (when comparing aligned sequences) is not counted as a difference contributing to a difference of 3 nucleotides or less with any one of the nucleotide sequences provided in SEQ ID NOs. 1-8, the sense strand contains modifications in which substantially all nucleotides are 2'-O-methyl, GNA, or 2'-fluoro modifications, and the sense strand has two phosphorothioethones at its 5' end. The antisense strand comprises a creotide interbonding, and substantially all of the nucleotides of the antisense strand comprise modifications selected from the group consisting of 2'-O-methyl modifications and 2'-fluoro modifications, wherein the antisense strand comprises two phosphorothioate internucleotide bonds at its 5' end and two phosphorothioate internucleotide bonds at its 3' end, and the sense strand is conjugated to one or more lipophilic ligands, e.g., a C16 ligand, and optionally further comprises a ligand comprising a liver-targeting ligand, e.g., one or more GalNAc derivatives.

[0144] Another aspect of the present disclosure provides a double-stranded RNAi agent for inhibiting the expression of an SNCA gene, the SNCA-targeting double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, the sense strand comprising at least 15 consecutive nucleotides that differ by only 3 nucleotides or less (i.e., 3, 2, 1, or 0 nucleotides) from any one of the nucleotide sequences of SEQ ID NOs. 1, 3, 5, and 7, or a nucleotide sequence having at least 90% nucleotide sequence identity, e.g., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with the entire nucleotide sequence of any one of SEQ ID NOs. 1, 3, 5, or 7, and the antisense strand comprising The sequence comprises at least 15 consecutive nucleotides that differ by only 3, 2, 1, or 0 nucleotides, or a nucleotide sequence having at least 90% nucleotide sequence identity with respect to the entire nucleotide sequence of any one of the nucleotide sequences of SEQ ID NOs: 2, 4, 6, and 8, e.g., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity, and any uracil substitution to thymine in any of the sequences provided in SEQ ID NOs: 1-8 (when comparing aligned sequences) is not counted as a difference that contributes to a difference of 3 nucleotides or less with respect to any one of the nucleotide sequences provided in SEQ ID NOs: 1-8, and the sense strand contains at least one 3'-terminal deoxythymine nucleotide (dT), and the antisense strand contains at least one 3'-terminal deoxythymine nucleotide (dT).

[0145] In one embodiment, all nucleotides in the sense strand and all nucleotides in the antisense strand are modified nucleotides.

[0146] In another embodiment, each chain has 19 to 30 nucleotides.

[0147] In one embodiment, the antisense strand of the RNAi agent includes at least one thermal destabilization modification of the double helix in the 5' region or the first 9 nucleotide positions of its precursor. Optionally, the thermal destabilization modification of the double helix is ​​one or more of the following: [ka] In the formula, B is a nucleic acid base.

[0148] Another aspect of the present disclosure provides cells containing the double-stranded RNAi agent of the present disclosure.

[0149] Further aspects of the present disclosure provide pharmaceutical compositions for inhibiting the expression of SNCA genes, comprising the double-stranded RNAi agent of the present disclosure.

[0150] In one embodiment, the double-stranded RNAi agent is administered in a non-buffered solution. Optionally, the non-buffered solution may be physiological saline or water.

[0151] In another embodiment, the double-stranded RNAi agent is administered with a buffer. Optionally, the buffer solution may include acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. In another embodiment, the buffer solution is phosphate-buffered saline (PBS).

[0152] Another aspect of the present disclosure provides a pharmaceutical composition comprising a double-stranded RNAi agent and a lipid formulation of the present disclosure.

[0153] In one embodiment, the lipid formulation includes lipid nanoparticles (LNPs).

[0154] Another aspect of the present disclosure provides a kit for carrying out the method of the present disclosure, the kit comprising a) a double-stranded RNAi agent of the present disclosure, b) instructions for use, and c) optionally, a device for administering the double-stranded RNAi agent to a target.

[0155] Further aspects of the present disclosure provide double-stranded ribonucleic acid (RNAi) agents for inhibiting the expression of SNCA genes, wherein the RNAi agent has a sense strand and an antisense strand, the antisense strand comprising a complementary region comprising at least 15 consecutive nucleotides that differ by three nucleotides or less (i.e., by 3, 2, 1, or 0 nucleotides) from any one of the antisense strand nucleic acid base sequences of Table 2, Table 3, Table 12, or Table 13, for example, at least 15 nucleotides (i.e., by 3, 2, 1, or 0 nucleotides) and at least 15 consecutive nucleotides that differ by at least 19 nucleotides (i.e., by 3, 2, 1, or 0 nucleotides). In one embodiment, the RNAi agent comprises one or more nucleotides including the following modifications: 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-C-alkyl modified nucleotides, glycol nucleic acids (GNAs), phosphorothioates (PSs), and vinyl phosphonates (VPs). Optionally, the RNAi agent comprises at least one of the following modifications: 2'-O-methyl modified nucleotide, 2'-fluoro modified nucleotide, 2'-C-alkyl modified nucleotide, glycol nucleic acid (GNA) nucleotide, phosphorothioate, and vinyl phosphonate (VP).

[0156] In another embodiment, the RNAi agent comprises four or more PS modifications, optionally six to ten PS modifications, and optionally eight PS modifications.

[0157] In a further embodiment, each of the sense and antisense strands of the RNAi agent has a 5' end and a 3' end, and the RNAi agent includes eight PS modifications located at the second-to-last and last internucleotide joints, respectively, from the 3' and 5' ends of the sense and antisense strands of the RNAi agent.

[0158] In another embodiment, each of the sense and antisense strands of the RNAi agent includes a 5' end and a 3' end, and the RNAi agent contains only one nucleotide containing a GNA. Optionally, the nucleotide containing the GNA is located on the antisense strand at the seventh nucleic acid base residue from the 5' end of the antisense strand.

[0159] In further embodiments, each of the sense and antisense strands of the RNAi agent includes a 5' end and a 3' end, and the RNAi agent contains 1 to 4 2'-C-alkyl-modified nucleotides. Optionally, the 2'-C-alkyl-modified nucleotides are 2'-C16-modified nucleotides. Optionally, the RNAi agent contains a single 2'-C-alkyl, e.g., a C16-modified nucleotide. Optionally, the single 2'-C-alkyl, e.g., a C16-modified nucleotide, is located on the sense strand at the 6th nucleic acid base position from the 5' end of the sense strand.

[0160] In another embodiment, each of the sense and antisense strands of the RNAi agent includes a 5' end and a 3' end, and the RNAi agent contains two or more 2'-fluoromodified nucleotides. Optionally, each of the sense and antisense strands of the RNAi agent contains two or more 2'-fluoromodified nucleotides. Optionally, the 2'-fluoromodified nucleotides are located on the sense strand at positions 7, 9, 10, and 11 from the 5' end of the sense strand, and on the antisense strand at positions 2, 14, and 16 from the 5' end of the antisense strand.

[0161] In further embodiments, each of the sense and antisense strands of the RNAi agent includes a 5' end and a 3' end, and the RNAi agent includes one or more VP modifications. Optionally, the RNAi agent includes a single VP modification at the 5' end of the antisense strand.

[0162] In another embodiment, each of the sense and antisense strands of the RNAi agent includes a 5' end and a 3' end, and the RNAi agent contains two or more 2'-O-methyl modified nucleotides. Optionally, the RNAi agent contains 2'-O-methyl modified nucleotides at all nucleic acid base positions not modified by 2'-fluoro, 2'-C-alkyl, or glycol nucleic acid (GNA). Optionally, two or more 2'-O-methyl modified nucleotides are located on the sense strand at positions 1, 2, 3, 4, 5, 8, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 from the 5' end of the sense strand, and on the antisense strand at positions 1, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, and 23 from the 5' end of the antisense strand.

[0163] definition Certain terms are defined first so that this disclosure may be more easily understood. Furthermore, whenever parameter values ​​or ranges of values ​​are enumerated, it should be noted that intermediate values ​​and ranges of the enumerated values ​​are also intended to be part of this disclosure.

[0164] The articles "a" and "an" are used herein to refer to one or more (i.e., at least one) grammatical objects of the article. For example, "one element" means one element or more elements, such as multiple elements.

[0165] The term “including” is used herein to mean “including, but not limited to,” and is used interchangeably with the term “including, but not limited to.”

[0166] The term "or" is used herein to mean and is interchangeable with the term "and / or" unless otherwise explicitly indicated in the context.

[0167] The term “approximately” is used herein to mean a typical range of tolerance in the art. For example, “approximately” can be understood as approximately 2 standard deviations from the mean. In some embodiments, “approximately” means ±10%. In some embodiments, “approximately” means ±5%. When “approximately” precedes a series of numbers or ranges, it is understood that “approximately” can modify each of the numbers in the series or range.

[0168] The term "at least" preceding a number or a series of numbers is understood, as is clear from the context, to include the number adjacent to the term "at least," and all subsequent numbers or integers that may logically be included. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For instance, "at least 18 nucleotides in a 21-nucleotide nucleic acid molecule" means that 18, 19, 20, or 21 nucleotides have the specified characteristic. When "at least" precedes a series of numbers or a range, it is understood that "at least" can modify each of the numbers in the series or range.

[0169] As used herein, “no more than” or “less than” is understood from the context to be a phrase logically to zero and a value adjacent to a logically lower value or integer. For example, a double helix with an overhang of “2 nucleotides or less” has an overhang of 2, 1, or 0 nucleotides. If “no more than” precedes a series of numbers or a range, it is understood that “no more than” can modify each of the numbers in the series or range. As used herein, a range includes both an upper and lower limit.

[0170] As used herein, the detection method may include a determination that the amount of analyte present is below the detection level of the method.

[0171] In the event of a discrepancy between the indicated target site and the nucleotide sequence of the sense or antisense strand, the indicated sequence takes precedence.

[0172] In cases of inconsistency between chemical structure and chemical name, the chemical structure takes precedence.

[0173] The terms "SNCA," "α-synuclein," "synuclein alpha," or "alpha-synuclein" refer to the gene associated with a neurodegenerative disease called "synuclein disease," as well as the protein encoded by that gene. The human SNCA gene region covers approximately 114 kb. The SNCA transcript contains 13 exons, and 15 mRNA isoforms have been identified or otherwise predicted. The nucleotide and amino acid sequences of SNCAs can be found, for example, under GenBank accession number NM_007308.3 (Homo sapiens SNCA, SEQ ID NO: 1, reverse complement, SEQ ID NO: 2), GenBank accession number XM_005555421 (Cynomolgus monkey SNCA, SEQ ID NO: 3, reverse complement, SEQ ID NO: 4); GenBank accession number NM_009221 (Mus musculus SNCA, SEQ ID NO: 5, reverse complement, SEQ ID NO: 6); GenBank accession number NM_019169.2 (Rat (Rattus norvegicus) SNCA, SEQ ID NO: 7, reverse complement, SEQ ID NO: 8); and GenBank accession number XM_535656.7 (Canis lupus familiaris SNCA, SEQ ID NO: 1806, reverse complement, SEQ ID NO: 3600).

[0174] As used herein, the term “SNCA” also refers to, for example, isoform 1 transcript NM_000345.4 (SEQ ID NO: 1809) encoding polypeptide NP_000336.1, isoform 2 transcript NM_001146054.2 (SEQ ID NO: 1807) encoding polypeptide NP_001139526.1, isoform 3 transcript NM_001146055.2 (SEQ ID NO: 1808) encoding polypeptide NP_001139527.1, and the above isoform 1 encoding polypeptide NP_009292.1. Isoform 4: Transcript NM_007308.3 (SEQ ID NO: 1); Isoform 5: Transcript NM_001375285.1 (SEQ ID NO: 1810); Isoform 6: Transcript NM_001375286.1 (SEQ ID NO: 1811); Isoform 7: Transcript NM_001375287.1 (SEQ ID NO: 1812); Isoform 7: Transcript NM_001375287.1 (SEQ ID NO: 1812); Isoform 8: Transcript NM_001375287.1 (SEQ ID NO: 1812); Isoform 18162217.1 Form 8 transcript NM_001375288.1 (SEQ ID NO: 1813); Isoform 9 transcript NM_001375290.1 ​​(SEQ ID NO: 1814) encoding polypeptide NP_001362219.1; Similarly, predictive isoform X1 transcript XM_011532203.1 (SEQ ID NO: 1815) encoding polypeptide XP_011530505.1; predictive isoform X2 transcript XM_011532204.3 (SEQ ID NO: 1816) encoding polypeptide XP_011530506.1; polypeptide XP_ Predictive isoform X3 transcript XM_011532205.2 (SEQ ID NO: 1817) encoding 011530507.1; predictive isoform X4 transcript XM_011532206.1 (SEQ ID NO: 1818) encoding polypeptide XP_011530508.1; predictive isoform X5 transcript XM_011532207.1 (SEQ ID NO: 1819) encoding polypeptide XP_011530509.1; and predictive isoform X8 transcript XM_017008563 encoding polypeptide XP_016864052.1.This refers to variants of the SNCA gene, including naturally occurring sequence variants provided in 1 (Sequence ID 1820) (the unique sequences associated with each of the preceding 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, e.g., 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). Additional information on SNCA can be found, e.g., www.ncbi.nlm.nih.gov / gene / 6622. The full contents of each of the above GenBank accession numbers and gene database numbers are incorporated herein by reference as of the filing date of this application.

[0175] Three protein isoforms of α-synuclein are described in UniProt. The longest α-synuclein isoform is a protein of approximately 14 kDa (isoform 1 UniProt, P37840 with 140 amino acids). Other α-synuclein isoforms in UniProt include isoforms 2-4, P37840-2, with 112 amino acids; and isoforms 2-5, P37840-3, with 126 amino acids. The 140-amino acid α-synuclein protein is encoded by five exon pairs that map to chromosomal loci 4q21.3-q22. The α-synuclein protein has an N-terminal region consisting of an incomplete KXKEGV motif, a highly hydrophobic NAC domain, and a highly acidic C-terminal domain. Under physiological conditions, SNCA is considered to be an essentially disordered monomer or a helically folded tetramer. Alpha-synuclein (α-synuclein) constitutes 1% of all proteins in the cytosol of brain cells and is primarily expressed in the neocortex, hippocampus, substantia nigra, thalamus, and cerebellum. α-synuclein is also expressed in smaller amounts in the heart, skeletal muscle, and pancreas. While the function of SNCAs is not fully understood, evidence suggests that SNCAs play a crucial role in maintaining a sufficient supply of synaptic vesicles at presynaptic terminals. α-synuclein is involved in regulating dopamine release and transport, microtubule-associated protein tau fibrillation, and neuroprotective phenotypes in non-dopaminergic neurons by modulating both p53 expression and the transactivation of pro-apoptotic genes, resulting in reduced caspase-3 activation. The primary mechanism by which α-synuclein induces neurodegenerative diseases such as Parkinson's disease, Lewy body dementia, and multiple system atrophy appears to be elevated levels of α-synuclein protein, leading to α-synuclein fibrillary aggregates.

[0176] As used herein, “target sequence” refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during transcription of an SNCA gene, including mRNA, which is the product of RNA processing of the primary transcript. In one embodiment, the target portion of the sequence is at least long enough to function as a substrate for RNAi-directed cleavage in that portion or near that portion of the nucleotide sequence of the mRNA molecule formed during transcription of the SNCA gene. In one embodiment, the target sequence is located within the protein-coding region of the SNCA gene. In another embodiment, the target sequence is located within the 3'UTR of the SNCA gene.

[0177] The target sequence can be approximately 9-36 nucleotides long, for example, approximately 15-30 nucleotides long. For example, the target sequence could be approximately 15-30 nucleotides, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 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 long. In some embodiments, the target sequence is about 19-30 nucleotides long. In other embodiments, the target sequence is about 19-25 nucleotides long. In yet another embodiment, the target sequence is about 19-23 nucleotides long. In some embodiments, the target sequence is about 21-23 nucleotides long. Intermediate ranges and lengths beyond those described above are also considered to be part of this disclosure.

[0178] As used herein, the term “sequence-containing chain” refers to an oligonucleotide containing a chain of nucleotides described by a sequence referred to using standard nucleotide nomenclature.

[0179] "G," "C," "A," "T," and "U" generally represent nucleotides containing guanine, cytosine, adenine, thymidine, and uracil as bases, respectively, in the context of modified or unmodified nucleotides. However, it will be understood that the terms "ribonucleotide" or "nucleotide" may also refer to modified nucleotides or substitutional moieties, as further detailed below (see, for example, Table 1). Those skilled in the art will be well aware that guanine, cytosine, adenine, thymidine, and uracil may be substituted by other moieties without substantially altering the base-pairing properties of oligonucleotides containing such substitutional moieties. For example, but not limited to, nucleotides containing inosine as their base can base-pair with nucleotides containing adenine, cytosine, or uracil. Thus, nucleotides containing uracil, guanine, or adenine may be replaced, for example, by nucleotides containing inosine in the nucleotide sequences of dsRNAs featured in this disclosure. In another example, adenine and cytosine at arbitrary locations in an oligonucleotide can be replaced with guanine and uracil, respectively, to form GU fluctuation base pairs with the target mRNA. Sequences containing such substitutions are suitable for the compositions and methods featured in this disclosure.

[0180] The terms “iRNA,” “RNAi agent,” “iRNA agent,” and “RNA interferant,” as used interchangeably herein, refer to agents containing RNA as defined herein and mediating targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway. RNA interference (RNAi) is a process that directs sequence-specific degradation of mRNA. RNAi modulates, for example, inhibits, the expression of SNCAs in cells within a subject, such as a mammalian subject.

[0181] 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. While we do not wish to be bound by theory, it is thought that long double-stranded RNA introduced into cells is degraded into short double-stranded interfering RNAs (siRNAs) containing sense and antisense strands by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease III-like enzyme, processes these dsRNAs into short interfering RNAs of 19-23 base pairs with a characteristic two-base 3' overhang (Bernstein, et al., (2001) Nature 409:363). These siRNAs are then incorporated into an RNA-induced silencing complex (RISC) in which one or more helicases unwind the siRNA double helix, allowing the complementary antisense strand to induce target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to a suitable target mRNA, one or more endonucleases within the RISC cleave the target, inducing silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in one embodiment, this disclosure relates to single-stranded RNA (ssRNA) (the antisense strand of the siRNA double helix) that is generated in cells and facilitates the formation of the RISC complex to result in the silencing of a target gene, namely an SNCA gene. Accordingly, the term "siRNA" is also used herein to refer to the above RNAi.

[0182] In another embodiment, the RNAi agent may be a single-stranded RNA introduced into a cell or organism to inhibit a target mRNA. The single-stranded RNAi agent binds to the RISC endonuclease Argonaut 2 and then cleaves the target mRNA. Single-stranded siRNAs are typically 15-30 nucleotides long and are chemically modified. Designs and tests of single-stranded RNAs are described in U.S. Patent No. 8,101,348 and Lima et al., (2012) Cell 150:883-894, the entire contents of each of these are incorporated herein by reference. Any of the antisense nucleotide sequences described herein may be used as single-stranded siRNAs described herein, or chemically modified by the methods described in Lima et al., (2012) Cell 150:883-894.

[0183] In another embodiment, the “RNAi agent” for use in the compositions and methods of the present disclosure is double-stranded RNA, and is referred herein to as “double-stranded RNAi agent,” “double-stranded RNA (dsRNA) molecule,” “dsRNA agent,” or “dsRNA.” The term “dsRNA” refers to a complex of ribonucleic acid molecules having a double-stranded structure containing two antiparallel and substantially complementary nucleic acid strands, which are said to have “sense” and “antisense” orientations with respect to the target RNA, i.e., the SNCA gene. In some embodiments of the present disclosure, double-stranded RNA (dsRNA) induces the degradation of the target RNA, e.g., mRNA, via a post-transcriptional gene silencing mechanism referred herein to as RNA interference or RNAi.

[0184] Generally, dsRNA molecules can contain ribonucleotides, but as described in detail herein, each or both strands may also contain one or more non-ribonucleotides, such as deoxyribonucleotides, modified nucleotides. Furthermore, as used herein, “RNAi agent” may contain chemically modified ribonucleotides, and RNAi agents may contain substantial modifications to multiple nucleotides.

[0185] As used herein, the term “modified nucleotide” independently refers to a nucleotide having a modified sugar moiety, a modified nucleotide-nucleotide bond, or a modified nucleic acid base. Therefore, the term “modified nucleotide” encompasses the substitution, addition, or removal of, for example, functional groups or atoms to the nucleoside bond, sugar moiety, or nucleic acid base. Modifications suitable for use in the agents of this disclosure include all types of modifications disclosed herein or known in the art. Any such modifications, such as those used in siRNA-type molecules, are encompassed within the “RNAi agents” for the purposes of this specification and the claims.

[0186] In some embodiments of this disclosure, the inclusion of a deoxyribonucleotide that, when present in an RNAi agent, is recognized as a naturally occurring form of nucleotide, can be considered to constitute a modified nucleotide.

[0187] The double-stranded region can be of any length that allows for the specific degradation of the desired target RNA via the RISC pathway, approximately 9–36 base pairs long, e.g., approximately 15–30 base pairs long, e.g., approximately 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, The base pair lengths may be in the range of approximately 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 pair lengths, such as 21-25, 21-24, 21-23, or 21-22. Intermediate ranges and lengths within the above ranges and lengths are also considered to be part of the present invention.

[0188] The two strands forming a double helix structure may be different parts of one large RNA molecule or may be separate RNA molecules. If the two strands are part of one large molecule and are therefore linked by a contiguous chain of nucleotides between the 3' end of one strand forming the double helix structure and the 5' end of the other strand, the linked RNA strands are called a “hairpin loop”. A hairpin loop may contain at least one unpaired nucleotide. In some embodiments, a hairpin loop may contain at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 20, or at least 23 or more unpaired nucleotides or nucleotides that are not directed toward the target site of the dsRNA. In some embodiments, a hairpin loop may contain 10 or fewer nucleotides. In some embodiments, a hairpin loop may contain 8 or fewer unpaired nucleotides. In some embodiments, a hairpin loop may contain 4 to 10 unpaired nucleotides. In some embodiments, a hairpin loop may contain 4 to 8 nucleotides.

[0189] In some embodiments, two strands of a double-stranded oligomeric compound can be linked together. The two strands can be linked at both ends or at only one end. Linking 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 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 can be linked together by an oligonucleotide linker (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 may be involved in base-pair interactions with other nucleotides in the linker. The two chains may also be joined together by a non-nucleoside linker, such as the linkers described herein. It will be understood by those skilled in the art that any oligonucleotide chemical modification or modification described herein can be used in oligonucleotide linkers.

[0190] Hairpin-type oligomeric compounds and dumbbell-type oligomeric compounds have a double-stranded region equal to or greater than 14, 15, 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The length of the double-stranded region may be 200, 100, or 50 or less. In some embodiments, the range of the double-stranded region is the length of 15-30, 17-23, 19-23, and 19-21 nucleotide pairs.

[0191] Hairpin oligomer compounds may have a single-stranded overhang or terminal unpairing region at 3' in some embodiments, and on the antisense side of the hairpin in some embodiments. In some embodiments, the overhang is 1 to 4 nucleotides long, more commonly 2 to 3 nucleotides long. Hairpin oligomer compounds capable of inducing RNA interference are also referred to herein as “shRNA”.

[0192] If the two substantially complementary strands of a dsRNA are composed of separate RNA molecules, these molecules can be covalently linked, although this is not always necessary. When the two strands are covalently linked by means other than a continuous chain of nucleotides between the 3' end of one strand and the 5' end of the other, the connecting structure is called a "linker." RNA strands can have the same or different numbers of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs present in the double helix. In addition to the double helix structure, RNAi can contain one or more nucleotide overhangs.

[0193] In one embodiment, the RNAi agent of this disclosure is a dsRNA having each strand length of 24-30 nucleotides that interacts with a target RNA sequence, such as an SNCA target mRNA sequence, to direct the cleavage of the target RNA. While we do not wish to be constrained by theory, long double-stranded RNA introduced into cells is degraded into siRNA by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease III-like enzyme, processes dsRNA into short, interfering RNAs of 19-23 base pairs with a characteristic two-base 3' overhang (Bernstein, et al., (2001) Nature 409:363). Next, the siRNA is incorporated into an RNA-induced silencing complex (RISC) in which one or more helicases unwind the siRNA double helix, 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, inducing silencing (Elbashir, et al., (2001) Genes Dev. 15:188).

[0194] In one embodiment, the RNAi agent of this disclosure is a dsRNA agent, each strand containing 19-23 nucleotides that interact with an SNCA RNA sequence to direct the cleavage of the target RNA. While we do not wish to be bound by theory, long double-stranded RNA introduced into cells is degraded into siRNA by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease III-like enzyme, processes dsRNA into short, interfering RNAs of 19-23 base pairs with a characteristic two-base 3' overhang (Bernstein, et al., (2001) Nature 409:363). The siRNA is then incorporated into an RNA-induced silencing complex (RISC) in which one or more helicases unwind the siRNA double helix, allowing a complementary antisense strand to induce target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to a suitable target mRNA, one or more endonucleases within the RISC cleave the target, inducing silencing (Elbashir, et al., (2001) Genes Dev. 15:188). In one embodiment, the RNAi agent of this disclosure is a 24-30 nucleotide dsRNA that interacts with an SNCA RNA sequence to direct cleavage of the target RNA.

[0195] As used herein, the term “nucleotide overhang” refers to at least one unpaired nucleotide protruding from the double-stranded structure of an RNAi agent, such as a dsRNA. For example, a nucleotide overhang exists if the 3' end of one strand of a dsRNA extends beyond the 5' end of the other strand, or vice versa. A dsRNA may contain an overhang of at least one nucleotide, or an overhang may contain at least two nucleotides, at least three nucleotides, at least four nucleotides, or at least five or more nucleotides. A nucleotide overhang may contain or consist of a nucleotide / nucleoside analog containing a deoxynucleotide / nucleoside. One or more overhangs may be on the sense strand, the antisense strand, or any combination thereof. Furthermore, one or more nucleotides of an overhang may be located at the 5' end, 3' end, or both ends of either the antisense strand or the sense strand of the dsRNA.

[0196] In one embodiment of dsRNA, at least one strand contains a 3' overhang of at least one nucleotide. In another embodiment, at least one strand contains a 3' overhang of at least two nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In yet another embodiment, at least one strand of the RNAi agent contains a 5' overhang of at least one nucleotide. In one embodiment, at least one strand contains a 5' overhang of at least two nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In yet another embodiment, both the 3' and 5' ends of one strand of the RNAi agent contain an overhang of at least one nucleotide.

[0197] In one embodiment, the antisense strand of the dsRNA has 1 to 10 nucleotides, e.g., 0 to 3, 1 to 3, 2 to 4, 2 to 5, 4 to 10, 5 to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, and has overhangs at the 3' end, 5' end, or both ends, or has no overhangs at any end. In another embodiment, one or more nucleotides in the overhangs are substituted with nucleoside thiophosphates.

[0198] In some embodiments, the overhang of the sense strand, the antisense strand, or both may be an elongated length longer than 10 nucleotides, for example, 1 to 30 nucleotides, 2 to 30 nucleotides, 10 to 30 nucleotides, or 10 to 15 nucleotides. In some embodiments, the elongated overhang is on the sense strand of the double helix. In some embodiments, the elongated overhang is located at the 3' end of the sense strand of the double helix. In some embodiments, the elongated overhang is located at the 5' end of the sense strand of the double helix. In some embodiments, the elongated overhang is on the antisense strand of the double helix. In some embodiments, the elongated overhang is located at the 3' end of the antisense strand of the double helix. In some embodiments, the elongated overhang is located at the 5' end of the antisense strand of the double helix. In some embodiments, one or more nucleotides in the overhang are substituted with nucleoside thiophosphates. In one embodiment, the overhang includes a self-complementary portion so that it can form a stable hairpin structure under physiological conditions.

[0199] As used herein with respect to dsRNA, the terms “blunt” or “blunt-ended” mean that there are no unpaired nucleotides or nucleotide analogs at a given end of the dsRNA, i.e., there are no nucleotide overhangs. One or both ends of a dsRNA may be blunt. If both ends of a dsRNA are blunt, it is said to be blunt-ended. To clarify, a “blunt-ended” dsRNA is one that is blunt at both ends, i.e., has no nucleotide overhangs at either end of the molecule. In most cases, such a molecule is double-stranded along its entire length.

[0200] The terms "antisense strand" or "guide strand" refer to a strand of RNAi agent, such as dsRNA, that contains a region substantially complementary to the target sequence, such as SNCA mRNA.

[0201] As used herein, the term “complementary region” refers to a region on an antisense strand that is substantially complementary to a sequence as defined herein, such as a target sequence, such as an SNCA nucleotide sequence. If the complementary region is not perfectly complementary to the target sequence, the mismatch may be in an internal or terminal region of the molecule. Generally, the most acceptable mismatch is in a terminal region, for example, within 5, 4, 3, or 2 nucleotides of the 5' or 3' end of an RNAi agent.

[0202] In some embodiments, the double-stranded RNA agent of the present disclosure contains a nucleotide mismatch in the antisense strand.

[0203] In some embodiments, the antisense strand of the double-stranded RNA agent of the Disclosure contains four or fewer mismatches with the target mRNA, for example, the antisense strand contains four, three, two, one, or zero mismatches with the target mRNA. In some embodiments, the antisense strand double-stranded RNA agent of the Disclosure contains four or fewer mismatches with the sense strand, for example, the antisense strand contains four, three, two, one, or zero mismatches with the sense strand. In some embodiments, the double-stranded RNA agent of the Disclosure contains nucleotide mismatches in the sense strand. In some embodiments, the sense strand of the double-stranded RNA agent of the Disclosure contains four or fewer mismatches with the antisense strand, for example, the sense strand contains four, three, two, one, or zero mismatches with the antisense strand. In some embodiments, the nucleotide mismatches are, for example, within five nucleotides, within four nucleotides, or within three nucleotides from the 3' end of the iRNA. In another embodiment, the nucleotide mismatches are, for example, at the 3' terminal nucleotide of the iRNA agent. In some embodiments, one or more mismatches are not in the seed region.

[0204] Therefore, the RNAi agents described herein may contain one or more mismatches to the target sequence. In one embodiment, the RNAi agent described herein contains three or fewer mismatches (i.e., 3, 2, 1, or 0 mismatches). In one embodiment, the RNAi agent described herein contains two or fewer mismatches. In one embodiment, the RNAi agent described herein contains one or fewer mismatches. In one embodiment, the RNAi agent described herein contains zero mismatches. In some embodiments, if the antisense strand of the RNAi agent contains a mismatch to the target sequence, the mismatch may be selectively restricted to the last five nucleotides from either the 5' or 3' end of the complementary region. For example, in such embodiments, for a 23-nucleotide RNAi agent, the strand complementary to the region of the SNCA gene generally does not contain any mismatches within the central 13 nucleotides. Using the methods described herein or methods known in the art, it is possible to determine whether an RNAi agent containing a mismatch to the target sequence is effective in inhibiting the expression of the SNCA gene. Considering the effectiveness of RNAi agents with mismatches when inhibiting SNCA gene expression is particularly important when specific complementary regions of the SNCA gene are known to have polymorphic sequence variations within the population.

[0205] As used herein, the terms “sense strand” or “passenger strand” refer to a strand of an RNAi agent that contains a region substantially complementary to the antisense strand region as defined herein.

[0206] As used herein, “substantially all nucleotides are modified” means that most of the nucleotides are modified but not completely modified, and may include 5, 4, 3, 2, or 1 or fewer unmodified nucleotides.

[0207] As used herein, the term “cleavage region” refers to a region located directly adjacent to a cleavage site. A cleavage site is a site on the target where cleavage occurs. In some embodiments, the cleavage region includes one of the ends of the cleavage site and three bases directly adjacent to the cleavage site. In some embodiments, the cleavage region includes one of the ends of the cleavage site and two bases directly adjacent to the cleavage site. In some embodiments, the cleavage site occurs specifically at a site bound by nucleotides 10 and 11 of the antisense strand, and the cleavage region includes nucleotides 11, 12, and 13.

[0208] Where used herein, unless otherwise specified, the term “complementary” means, as understood by those skilled in the art, the ability of an oligonucleotide or polynucleotide containing a first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide containing a second nucleotide sequence under specific conditions to form a double-stranded structure, when used to describe a first nucleotide sequence with respect to a second nucleotide sequence. Such conditions may be, for example, stringent conditions, which may include 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C for 12–16 hours, followed by washing (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 within living organisms, may be applied. Those skilled in the art will be able to determine the set of conditions best suited for testing the complementarity of the two sequences, depending on the final application of the hybridized nucleotides.

[0209] In RNAi agents, for example in dsRNA as described herein, complementary sequences include base pairings of an oligonucleotide or polynucleotide containing the first nucleotide sequence and an oligonucleotide or polynucleotide containing the second nucleotide sequence, over the full length of one or both nucleotide sequences. Such sequences may be referred to herein as “fully complementary.” However, where the first sequence is referred herein as “substantially complementary” with respect to the second sequence, the two sequences may be fully complementary, or they may form one or more, but generally five, four, three, or two or fewer, mismatched base pairs during hybridization to a double helix of up to 30 base pairs, while retaining their ability to hybridize under conditions most relevant to their ultimate use, e.g., inhibition of gene expression via the RISC pathway. However, if two oligonucleotides are designed to form one or more single-stranded overhangs during hybridization, such overhangs should not be considered mismatches for the purpose of determining complementarity. For example, a dsRNA comprising one 21-nucleotide oligonucleotide and another 23-nucleotide oligonucleotide, wherein the longer oligonucleotide contains a 21-nucleotide sequence that is perfectly complementary to the shorter oligonucleotide, can be referred to as “perfectly complementary” for the purposes described herein.

[0210] The “complementary” sequences used herein also include, or may be entirely formed from, non-Watson-Crick base pairs or base pairs formed from non-natural and modified nucleotides, provided that the above requirements regarding the ability to hybridize are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U fluctuations or Hoogstein base pairings.

[0211] The terms “complementary,” “fully complementary,” and “substantially complementary” as used herein can be used in reference to base matching between the sense strand and antisense strand of a dsRNA, or between the antisense strand and target sequence of an RNAi agent, as understood from the context of their use.

[0212] As used herein, a polynucleotide that is "substantially complementary to at least a portion" of messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a continuous portion of the mRNA of interest (e.g., mRNA encoding SNCA). For example, a polynucleotide is complementary to at least a portion of SNCA mRNA if its sequence is substantially complementary to an uninterrupted portion of the mRNA encoding SNCA.

[0213] Therefore, in some embodiments, the antisense strand polynucleotides disclosed herein are perfectly complementary to the target SNCA sequence.

[0214] In one embodiment, the antisense strand polynucleotides disclosed herein are substantially complementary to the target SNCA sequence and are at least about 80% complementary over their entire length to the equivalent region of the nucleotide sequence of SEQ ID NO: 1, 3, 5, or 7 of the SNCA, or fragments of SEQ ID NO: 1, 3, 5, or 7, for example, 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.

[0215] In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target SNCA sequence and are a continuous nucleotide sequence that is at least about 80% complementary over its entire length to any one of the sense strand nucleotide sequences in Tables 2, 3, 12, or 13, or a fragment of any one of the sense strand nucleotide sequences in Tables 2, 3, 12, or 13, for example, 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.

[0216] In one embodiment, the RNAi agent of the present disclosure comprises a sense strand substantially complementary to an antisense polynucleotide which is identical to the target SNCA sequence, wherein the sense strand polynucleotide comprises a continuous nucleotide sequence that is at least about 80% complementary over its entire length to the equivalent region of the nucleotide sequence of SEQ ID NO: 2, 4, 6, or 8, or a fragment of any one of SEQ ID NOs: 2, 4, 6, or 8, for example, fragments that are 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.

[0217] In some embodiments, the iRNA of the Disclosure then comprises a sense strand substantially complementary to an antisense polynucleotide complementary to a target SNCA sequence, the sense strand polynucleotide comprising a continuous nucleotide sequence that is at least about 80% complementary over its entire length to any one of the antisense nucleotide sequences of Tables 2, 3, 12, or 13, or a fragment of any one of the antisense nucleotide sequences of Tables 2, 3, 12, or 13, for example, fragments that are 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.

[0218] In some embodiments, the double-stranded region of the double-stranded iRNA agent is equal to, or at least equal to, the length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotide pairs.

[0219] In some embodiments, the antisense strand of the double-stranded iRNA agent is equal to or at least one of the following nucleotide lengths: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, or 30.

[0220] In some embodiments, the sense strand of the double-stranded iRNA agent is equal to or at least has a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.

[0221] In one embodiment, the sense strand and antisense strand of the double-stranded iRNA agent are each 15 to 30 nucleotides long.

[0222] In one embodiment, the sense strand and antisense strand of the double-stranded iRNA agent are each 19 to 25 nucleotides long.

[0223] In one embodiment, the sense strand and antisense strand of the double-stranded iRNA agent are each 21 to 23 nucleotides long.

[0224] In one embodiment, the sense strand of the iRNA agent is 21 nucleotides long, and the antisense strand is 23 nucleotides long, forming a double-stranded region of 21 consecutive base pairs with a 2-nucleotide single-stranded overhang at the 3' end.

[0225] In some embodiments, the majority of the nucleotides in each chain are ribonucleotides, but as described in detail herein, each or both chains may also contain one or more non-ribonucleotides, such as deoxyribonucleotides or modified nucleotides. Furthermore, “iRNA” may include ribonucleotides having chemical modifications. Such modifications may include all types of modifications disclosed herein or known in the art. Any such modifications used in an iRNA molecule are encompassed by “iRNA” for the purposes of this specification and the claims.

[0226] In one aspect of this disclosure, the agent for use in the methods and compositions of this disclosure is a single-stranded antisense nucleic acid molecule that inhibits target mRNA via an antisense inhibition mechanism. The single-stranded antisense RNA molecule is complementary to a sequence in the target mRNA. Single-stranded antisense oligonucleotides can inhibit translation in a stoichiometric manner by physically interfering with the base pairing and translation mechanisms to mRNA (see Dias, N. et al., (2002) Mol Cancer Ther 1:347-355). The single-stranded antisense RNA molecule may be about 15 to about 30 nucleotides long and may have a sequence complementary to the target sequence. For example, the single-stranded antisense RNA molecule may contain a sequence that is at least about 15, 16, 17, 18, 19, 20 or more consecutive nucleotides derived from any one of the antisense sequences described herein.

[0227] In one embodiment, at least partial repression of SNCA gene expression is assessed by a reduction in the amount of SNCA mRNA that can be isolated or detected from a first cell or cell population that is substantially identical to the first cell or cell population but has not been treated in such manner (control cells), compared to a second cell or cell population that is not treated in such manner. The degree of inhibition may be expressed in relation to the following:

number

[0228] As used herein, the phrase "bringing an RNAi agent, such as dsRNA, into contact with cells" includes bringing cells into contact by any possible means. Bringing cells into contact with an RNAi agent includes bringing cells into contact with an RNAi agent in vitro or in vivo. Contact may be direct or indirect. For example, an RNAi agent may be brought into physical contact with cells by an individual performing the method, or an RNAi agent may be placed in a situation that enables or causes subsequent contact with cells.

[0229] In vitro cell contact can be performed, for example, by incubating cells with an RNAi agent. In vivo cell contact can be performed, for example, by injecting the RNAi agent into or near the tissue in which the cells are located, or by injecting the RNAi agent into another region, such as the central nervous system (CNS), optionally via intrathecal, intravitreous, or other injection, or into the bloodstream or subcutaneous space, thereby the agent subsequently reaching the tissue in which the cells being contacted are located. For example, the RNAi agent may contain, or be coupled to, one or more lipophilic moieties, such as those described below and more specifically, as described in PCT / US2019 / 031170, which is incorporated herein by reference, etc., which induce or otherwise stabilize the RNAi agent at a site of interest, e.g., the CNS. In some embodiments, the RNAi agent may contain, or be coupled to, ligands, such as one or more GalNAc derivatives described below, which induce or otherwise stabilize the RNAi agent at a site of interest, e.g., the liver. In other embodiments, the RNAi agent may comprise one or more lipophilic moieties and one or more GalNAc derivatives, or may be coupled thereto. Combinations of in vitro and in vivo contact methods are also possible. For example, cells may be contacted with the RNAi agent in vitro and then transplanted into a target.

[0230] In one embodiment, contacting cells with an RNAi agent includes “introducing” or “delivering the RNAi agent to cells” by promoting or achieving uptake or absorption into the cells. Absorption or uptake of the RNAi agent may occur via unassisted diffusion or active cellular processes, or by auxiliaries or devices. Introduction of the RNAi agent into cells may be in vitro or in vivo. For example, for in vivo introduction, the RNAi agent may be injected into a tissue site or administered systemically. In vitro introduction into cells includes methods known in the art, such as electroporation and lipofection. Further approaches are described below herein or are known in the art.

[0231] The terms "lipophilic" or "lipophilic moiety" broadly refer to any compound or chemical part that has an affinity for lipids. One way to characterize the lipophilicity of a lipophilic moiety is by its octanol-water partition coefficient logK. ow This is due to K ow The octanol-water partition coefficient is the ratio of the concentration of a chemical in the octanol phase to the concentration in the aqueous phase of 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 derived from the structural components of the chemical, calculated using first-principles or empirical methods (see, for example, Tetko et al., J.Chem.Inf.Comput.Sci.41:1407-21(2001), the whole of which is incorporated herein by reference). This provides a thermodynamic measure of a substance's tendency to prefer non-aqueous or oily environments over water (i.e., its hydrophilic / lipophilic balance). In principle, a chemical substance has a logK ow If logK is greater than 0, it is characterized as lipophilic. Typically, the lipophilic portion is greater than 1, greater than 1.5, greater than 2, greater than 3, greater than 4, greater than 5, or greater than 10. ow It has, for example, the logK of 6-aminohexanol. owFor example, it is predicted to be approximately 0.7. Using the same method, the logK of cholesteryl N-(hexane-6-ol) carbamate ow It is predicted to be 10.7.

[0232] The lipophilicity of a molecule can change with respect to the functional groups it possesses. For example, by adding a hydroxyl group or an amine group to the end of the lipophilic portion, the partition coefficient (e.g., logK) of the lipophilic portion can be altered. ow The value can be increased or decreased.

[0233] Alternatively, the hydrophobicity of a double-stranded RNAi agent conjugated to one or more lipophilic moieties can be measured by its protein-binding properties. For example, in one embodiment, the unbound fraction in a plasma protein-binding assay of a double-stranded RNAi agent can be determined to be positively correlated with the relative hydrophobicity of the double-stranded RNAi agent, and subsequently, positively correlated with the silencing activity of the double-stranded RNAi agent.

[0234] In one embodiment, the plasma protein binding assay to be determined is an electrophoretic mobility shift assay (EMSA) using human serum albumin protein. An exemplary protocol for this binding assay is described in detail, for example, PCT / US2019 / 031170. The hydrophobicity of the double-stranded RNAi agent, as measured by the percentage of unbound siRNA in the binding assay, is greater than 0.15, greater than 0.2, greater than 0.25, greater than 0.3, greater than 0.35, greater than 0.4, greater than 0.45, or greater than 0.5 to enhance in vivo delivery of siRNA.

[0235] Therefore, by conjugating the lipophilic portion to one or more internal positions of the double-stranded RNAi agent, optimal hydrophobicity is provided to enhance the in vivo delivery of siRNA.

[0236] The terms “lipid nanoparticles” or “LNPs” refer to vesicles containing a lipid layer that encapsulate pharmaceutically active molecules such as nucleic acid molecules, for example, RNAi agents, or plasmids on which RNAi agents are transcribed. LNPs are described, for example, in U.S. Patents 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the full contents of which are incorporated herein by reference.

[0237] As used herein, “Subject” refers to animals such as mammals, including primates (e.g., humans, non-human primates, e.g., monkeys, and chimpanzees) or non-primates (e.g., rats, or mice). In preferred embodiments, the subject is a human being being treated or evaluated for a disease, disorder, or condition for which a reduction in SNCA expression would be beneficial; a human being at risk of a disease, disorder, or condition for which a reduction in SNCA expression would be beneficial; a human being having a disease, disorder, or condition for which a reduction in SNCA expression would be beneficial; or a human being being treated for a disease, disorder, or condition for which a reduction in SNCA expression would be beneficial as described herein.

[0238] As used herein, the terms “treat” or “cure” include, for example, SNCA-associated neurodegenerative diseases such as PD, multiple system atrophy, Lewy body disease (LBD), pure autonomic dysplasia (PAF), Pick’s disease, progressive supranuclear palsy, boxer’s dementia, chromosome 17-associated parkinsonism, Ritico Bodig disease, neurofibrillary tangle-type senile dementia, argyrophilic granuloma, ganglioglioma, gangliocytoma, meningeal hemangioma, subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis, Hallerforden-Spatz disease, and lipofuscinate. "Treatment" refers to the alleviation or improvement of one or more signs or symptoms associated with SNCA gene expression or SNCA protein production in synuclein diseases such as dysphagia, corticobasal degeneration, frontotemporal dementia, frontotemporal lobar degeneration, Alzheimer's disease, Huntington's disease, Down syndrome, psychosis, schizophrenia, and Creutzfeldt-Jakob disease, and includes, but is not limited to, a reduction in SNCA expression or activity in areas of increased neuronal dysfunction or death in controls with such neurodegenerative diseases, and any benefit or desired outcome. "Treatment" may also mean an extension of survival compared to expected survival in the absence of the treatment.

[0239] In the context of SNCA levels or disease marker or symptom levels in a subject, the term “lower” refers to a statistically significant decrease in such levels. The decrease may 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 some embodiments, the decrease is at least 20%. In some embodiments, the decrease is at least 50% of disease marker levels, such as protein or gene expression levels. “Lower” in the context of SNCA levels in a subject is, optionally, a decrease to a level acceptable as being within the normal range for individuals without such disorder. In one embodiment, “lower” refers to a reduction in the difference between the level of a marker or symptom for a subject suffering from the disease and a level that is acceptable within the normal range for that individual, for example, a reduction in the ability to adjust motor speed (bradykinesia) and posture and balance in individuals with Parkinson’s disease and individuals without Parkinson’s disease or with symptoms within the normal range.

[0240] As used herein, “prevention” or “preventing” means, for example, benefiting from reduced expression of the SNCA gene or production of the SNCA protein in a subject who is susceptible to SNCA-related disorders due to genetic factors or age, when used in relation to a disease or disorder, the subject has not yet met the diagnostic criteria for SNCA-related disorder. As used herein, prevention can be understood as the administration of a drug to a subject who has not yet met the diagnostic criteria for SNCA-related disorder, in order to delay or reduce the likelihood that the subject will develop SNCA-related disorder. Since the drug is a pharmaceutical product, it is understood that the administration is typically done under the direction of a healthcare professional who can identify a subject who has not yet met the diagnostic criteria for SNCA-related disorder as susceptible to developing SNCA-related disorder.

[0241] The term "synuclein disease" refers to a group of neurodegenerative disorders characterized by fibrillary aggregates of α-synuclein protein, which tend to accumulate in the cytoplasm of selective populations of neurons and glial cells. Therefore, synuclein disease is a class of SNCA-associated neurodegenerative diseases and disorders, which include Parkinson's disease (PD), Lewy body dementia (LBD), pure autonomic dysfunction (PAF), and multiple system atrophy (MSA), among other neurodegenerative diseases. Clinically, synuclein disease is characterized by a chronic and progressive decline in motor function, cognitive function, behavioral function, and autonomic function, depending on the distribution of lesions in the brain. Due to clinical overlap, differential diagnosis is sometimes very difficult. Parkinsonism is a major symptom of PD, but it can be indistinguishable from parkinsonism in LBD and MSA. Autonomic dysfunction is an isolated finding in PAF and can be present in PD and LBD, but is usually more pronounced and appears earlier in MSA. LBD can be the same disease as PD, but it has a widespread cortical pathological condition that leads to dementia, fluctuating cognition, and characteristic visual hallucinations.

[0242] The likelihood of developing a synuclein disease, such as PD or LBD, is reduced if, for example, an individual with one or more risk factors for PD or LBD (or other synuclein diseases) does not develop PD or LBD (or other synuclein diseases) with a lower severity compared to a population with the same risk factors but who has not received the treatments described herein, or if they do develop PD or LBD (or other synuclein diseases). Not developing an SNCA-related disorder, such as PD or LBD (or other synuclein diseases), or delaying the onset of PD or LBD (or other synuclein diseases) by several months or years, is considered effective prevention. Prevention may require administration of multiple doses of iRNA agents. If an appropriate method is provided for identifying subjects at risk of developing any of the above SNCA-related diseases, the iRNA agents provided herein can be used as pharmaceuticals for the prevention of or in methods of prevention of SNCA-related diseases. Various risk factors for SNCA-related diseases are discussed herein.

[0243] As used herein, the terms “Parkinson’s disease” or “PD” refer to a progressive neurological disorder affecting movement. The main pathological features of PD are cell death in the basal ganglia of the brain (affecting up to 70% of dopamine-secreting neurons in the substantia nigra pars compacta by the end of life) and the presence of Lewy bodies in many of the remaining neurons (accumulation of SNCA-coded α-synuclein protein). Symptoms begin gradually, sometimes with a barely noticeable tremor, rigidity, or slowing of movement in one hand. Other early symptoms include a lack of facial expression, a lack of arm movement while walking, and slurring during speech. The symptoms of Parkinson’s disease worsen over time. The mean age of onset for PD is 60 years, and later onset is associated with greater symptom severity. Clinical features include, but are not limited to, more severe tremors, bradykinesia (slowness of movement), muscle rigidity, postural and balance impairments, loss of automatism, speech changes, and eventually dementia, hallucinations, and wheelchair confinement.

[0244] As used herein, the term “Lewy body dementia (LBD)” refers to a type of progressive dementia caused by the aggregation of alpha-synuclein protein in diseased brain neurons, known as Lewy bodies and Lewy neurites, resulting in a decline in thinking, reasoning, and independent function. The alpha-synuclein protein aggregates lead to suboptimal function and eventual death of the affected neurons. Symptoms include visual, auditory, olfactory, or tactile hallucinations; signs of Parkinson's disease (Parkinsonian signs); impaired control 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 disturbances, such as rapid eye movement (REM) sleep behavior disorder (dreams being physically acted out during sleep); fluctuations of attention, including episodes of sleepiness; prolonged staring into space; long periods of daytime napping or disorganized speech; depression, and emotional blunting.

[0245] In one embodiment, SNCA-related disease or disorder (synuclein disease) is one of the following: Parkinson's disease, Lewy body dementia, multiple system atrophy (MSA), and pure autonomic dysregulation (PAF).

[0246] As used herein, “therapeutic dose” is intended to include an amount of RNAi agent sufficient to treat a disease (e.g., by reducing, improving, or maintaining one or more symptoms of an existing disease or disease) when administered to a subject with an SNCA-associated disease. The “therapeutic dose” may vary depending on the RNAi agent, the method of drug administration, the disease and its severity, as well as the medical history, age, weight, family history, genetic makeup, type of preceding or concomitant treatment, if any, and other individual characteristics of the subject being treated.

[0247] As used herein, “prophylactic effective dose” is intended to include an amount of RNAi agent sufficient to prevent or improve the disease or one or more symptoms of the disease when administered to a subject with SNCA-associated disorder. Improving the disease includes delaying the course of the disease or reducing the severity of the disease if it develops later. The “prophylactic effective dose” may vary depending on the RNAi agent, the method of drug administration, the degree of the disease risk, and the patient’s medical history, age, weight, family history, genetic makeup, the type of preceding or concomitant treatment, if any, and other individual characteristics.

[0248] The “therapeutic effective dose” or “preventive effective dose” also includes the amount of RNAi agent that produces any desired local or systemic effect with a reasonable benefit / risk ratio applicable to any treatment. The RNAi agent used in the method of this disclosure may be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0249] The term "pharmaceutically acceptable" is used herein to mean a compound, material, composition, or dosage form that is suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio, within the bounds of sound medical judgment.

[0250] When used herein, the term “pharmaceutically acceptable carrier” means a solvent encapsulation material involved in transporting or carrying a pharmaceutically acceptable material, liquid or solid filler, diluent, excipient, preparation agent, or other composition or vehicle (e.g., lubricant, magnesium talc, calcium stearate or zinc stearate, or stearic acid) or a compound of interest from one organ or part of the body to another organ or part of the body. Each carrier must be “acceptable” in the sense that it is compatible with the other components of the formulation and is not harmful to the subject being treated. Some examples of substances 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, ethylcellulose, and cellulose acetate; (4) tragacanth powder; (5) malt; (6) gelatin; (7) lubricants such as magnesium sulfate, sodium lauryl sulfate, and talc; (8) excipients such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycosides 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; (21) Polyesters, polycarbonates, or polyanhydrides; (22) Expanders such as polypeptides and amino acids; (23) Serum components such as serum albumin, HDL, and LDL; and (22) Other non-toxic, suitable substances used in pharmaceutical formulations.

[0251] As used herein, the term “sample” includes similar fluids, cells, or tissues isolated from a subject, as well as collections of fluids, cells, or tissues present within a subject. Examples of biological fluids include blood, serum and serous fluid, plasma, cerebrospinal fluid, ocular fluid, lymph, urine, saliva, etc. Tissue samples may include samples from tissues, organs, or local areas. For example, a sample may originate from a specific organ, a part of an organ, or fluids or cells within those organs. In some embodiments, a sample may originate from the brain (e.g., the whole brain or a specific segment of the brain, e.g., the striatum, or a specific type of cell in the brain, e.g., neurons and glial cells (astrocytes, oligodendrocytes, microglia)). In other embodiments, “sample derived from subject” refers to liver tissue (or its subcomponents) derived from the subject. In some embodiments, “sample derived from subject” refers to blood taken from a subject or plasma or serum derived therefrom. In further embodiments, “sample derived from subject” refers to brain tissue (or its subcomponents) or retinal tissue (or its subcomponents) derived from the subject.

[0252] Although the sequences in Table 2 or Table 12 are described as modified or conjugated sequences, it will be understood that the RNA of the RNAi agent of this disclosure, for example, the dsRNA of this disclosure, may contain any one of the sequences listed in Tables 2, 3, 12, or 13, and may be unmodified, unconjugated, or modified and conjugated in a different manner than those listed therein. That is, the modified sequences provided in Table 2 or 12 do not require an L96 ligand or any ligand. A lipophilic ligand may be included in any of the positions provided in this application. [Brief explanation of the drawing]

[0253] [Figure 1]Figure 1 shows the effect of selected SNCA-targeted RNAi agents on SNCA levels in human SNCA-AAV overexpressing mice. To identify 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, as well as duplexes targeting the coding sequences of SNCA AD-464590, AD-464313, AD-464314, AD-464585, AD-464586, AD-464592, and AD-464229. Data were normalized to PBS-treated samples. [Figure 2] Figure 2 shows schematic diagrams of the sequences and modification patterns of two selected SNCA-targeted RNAi duplexes: the sense (SEQ ID NO: 924) and antisense (SEQ ID NO: 1016) strands of AD-464634, and the sense (SEQ ID NO: 915) and antisense (SEQ ID NO: 1007) strands of AD-464314. Both duplexes were modified on the antisense strand with a vinyl phosphate group and on the sense strand with a triantennary GalNAc moiety (thereby promoting liver delivery). The shown residues were also 2'-fluoro-modified or 2'-O-methyl-modified, and phosphorothioate nucleoside interbonding was present in the last and second-to-last bonds (both 3' and 5' ends for the antisense strand, and only the 5' end for the sense strand), where shown. [Figure 3]Figure 3 shows the results of human SNCA knockdown obtained in the optimization of the in vivo activity of RNAi agents in huSNCA AAV-transformed mice (reliable data generated by AAV incubation / mice with 2e10 virus particles). Robust knockdown of human SNCA was observed at both day 7 and day 14 in mice treated with both the AD-464634 duplex targeting the huSNCA3'-UTR and the AD-464314 duplex targeting the sequence encoding huSNCA. A dose-response was observed for both duplexes tested, particularly at day 14. Since strong huSNCA knockdown was observed even at day 14, both duplexes were identified as suitable for further in vivo lead development studies. [Figure 4] Figure 4 shows the human SNCA expression levels observed in the liver tissue of huSNCA AAV transducer mice (huSNCA AAV transducer mice with either 2e10 or 2e11 virus particles, respectively), where huSNCA levels were measured on days 7, 14, and 21. [Figure 5] Figure 5 shows that mouse / rat cross-reactive double helices inhibited rat SNCA in vivo when administered to rat SNCA-AAV transducer mice. Selected RNAi agents included AD-476344, AD-475666, AD-476306, AD-476061, AD-464814, AD-475728, and AD-4644229. Data were normalized to PBS-treated samples. [Figure 6] Figure 6 shows the strong correlation observed between the SNCA knockdown levels measured in the hotspot walk in Table 14 and the calculated 1nM fitted values ​​used for the rank-ordered double helix in Table 14. [Modes for carrying out the invention]

[0254] The present invention will be further described by the following detailed description.

[0255] This disclosure provides an RNAi composition that induces RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of an SNCA gene. The SNCA gene may be present in cells, for example, in cells within a subject such as a human. The Disclosure also provides methods for using the RNAi compositions of the Disclosure to inhibit the expression of SNCA genes, or to treat controls having disorders that would benefit from inhibiting or reducing the expression of SNCA genes in, for example, SNCA-related diseases such as synuclein diseases including PD, multiple system atrophy, Lewy body disease (LBD), pure autonomic dysplasia (PAF), Pick's disease, progressive supranuclear palsy, boxer's dementia, chromosome 17-related parkinsonism, Ritiko-Bodig disease, neurofibrillary tangle-type senile dementia, argyrophilic granuloma, ganglioglioma, gangliocytoma, meningeal hemangioma, subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis, Haller-Vorden-Spatz disease, lipofuscinosis, corticobasal degeneration, frontotemporal dementia, frontotemporal lobar degeneration, Alzheimer's disease, Huntington's disease, Down syndrome, psychosis, schizophrenia, and Creutzfeldt-Jakob disease.

[0256] The RNAi agents of this disclosure are RNA strands (antisense strands) having a region of approximately 30 nucleotides or less in length, for example, 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 The region is 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides long, and this region is substantially complementary to at least a portion of the mRNA transcript of the SNCA gene. In one embodiment, the RNAi agent of the present disclosure comprises an RNA strand (antisense strand) having a region of about 21-23 nucleotides long, and this region is substantially complementary to at least a portion of the mRNA transcript of the SNCA gene.

[0257] In some embodiments, the RNAi agents of the present disclosure may include longer RNA strands (antisense strands) of at least 19 consecutive nucleotide regions substantially complementary to at least a portion of the mRNA transcript of an SNCA gene, 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 having longer antisense strands optionally include a second RNA strand (sense strand) of 20-60 nucleotides in length, and the sense and antisense strands form a double helix of 18-30 consecutive nucleotides.

[0258] The use of these RNAi agents enables targeted degradation of SNCA gene mRNA in mammals. Therefore, methods and compositions comprising these RNAi agents are useful for treating subjects who benefit from a reduction in the level or activity of SNCA proteins, such as subjects with SNCA-related neurodegenerative diseases, including PD, multiple system atrophy, Lewy body dementia (LBD), pure autonomic dysplasia (PAF), Pick's disease, progressive supranuclear palsy, boxer's dementia, chromosome 17-related parkinsonism, Ritiko-Bodig disease, neurofibrillary tangle-type senile dementia, argyrophilic granuloma, ganglioglioma, gangliocytoma, meningeal hemangioma, subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis, Hallerforden-Spatz disease, lipofuscinosis, corticobasal degeneration, frontotemporal dementia, frontotemporal lobar degeneration, Alzheimer's disease, Huntington's disease, Down syndrome, psychosis, schizophrenia, and synuclein diseases such as Creutzfeldt-Jakob disease.

[0259] Intraneuronal accumulation of α-synuclein has been described as leading to the formation of Lewy bodies, large inclusion bodies of round eosinophilic hyalin 10-20 pm, or Lewy neurites, elongated filiform dystrophy axons, and dendrites. In PD brains, the deposition of Lewy bodies and Lewy neurites is largely limited to neurons connecting the striatum and substantia nigra. These cells are crucial for the performance of motor and postural functions and explain the nature of PD symptoms. In LBD brains, widespread deposition of Lewy bodies and Lewy neurites is found in both midbrain and cortical regions.

[0260] Alpha-synuclein is a protein primarily found within neurons. Within neurons, alpha-synuclein is mainly located presynaptically and is therefore presumed 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.

[0261] Oxidative stress is involved in numerous neurodegenerative disorders characterized by the pathological accumulation of misfolded α-synuclein. Various reactive oxygen species can induce peroxidation of cell membranes or lipids such as lipoproteins, and can also lead to the formation of highly reactive aldehydes from polyunsaturated fatty acids (Yoritaka et al., 1996).

[0262] Brain pathology characteristic of Alzheimer's disease (AD), namely amyloid plaques and neurofibrillary tangles, is observed in approximately 50% of LBD cases. It is unclear whether the presence of these parallel pathologies signifies two distinct diseases or simply represents a variation of each disorder. Occasionally, cases with co-pathologies are described as having the Lewy body form of AD (Hansen et al., 1990).

[0263] The research also involves the role of SNCA in AD and Down syndrome, as evidenced by the demonstration that α-synuclein protein accumulates in the peripheral region in these disorders (Crews et al., 2009).

[0264] Rare dominant genotypes of PD and LBD can be caused by point mutations or duplications of the SNCA gene. Pathogenic mutations A30P and A53T (Kruger et al., 1998) (Polymeropoulos et al., 1998) and gene duplication (Chartier-Harlin et al., 2004) have been reported to cause familial PD, while one other α-synuclein mutation E46K (Zarranz et al., 2004) and α-synuclein gene triplication (Singleton et al., 2003) have been reported to cause either PD or LBD.

[0265] The pathogenic consequences of α-synuclein mutations are only partially understood. However, in vitro data show that A30P and A53T mutations increase the aggregation rate (Conway et al., 2000). A wide range of α-synuclein species with different compositions (monomers, dimers, and oligomers, including protofibrils) are involved in the aggregation process, and all of them may have different toxic properties. It is not clear which molecular species exert toxic effects in the brain. However, studies have shown that α-synuclein in oligomeric form is particularly neurotoxic. Further evidence for the role of oligomers is given by the observation that certain α-synuclein mutations (A30P and A53T) that cause hereditary Parkinson's disease result in an increased oligomerization rate.

[0266] The exact mechanism by which the α-synuclein aggregation cascade begins is not fully understood. It is likely that altered conformations of monomeric α-synuclein initiate the formation of dimers and trimers, which then continue to form higher soluble oligomers containing protofibrils before these intermediate-sized seeds deposit as insoluble fibrils in Lewy bodies. Once formed, α-synuclein oligomers can bind to new monomers and / or smaller polymers of α-synuclein, thus potentially accelerating the fibrillation process. Such a seeding effect may also occur in the extracellular space, as evidence suggests that the pathogenesis of α-synuclein can propagate from neuron to neuron in the affected brain.

[0267] The following detailed description discloses methods for preparing and using compositions containing RNAi agents for inhibiting the expression of SNCA genes, as well as compositions and methods for treating subjects with diseases and disorders that would benefit from the inhibition or reduction of gene expression.

[0268] I. RNAi agents of the present disclosure RNAi agents that inhibit the expression of SNCA genes are described herein. In one embodiment, the RNAi agent includes a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting the expression of SNCA genes in cells of mammals, such as humans, who have SNCA-related neurodegenerative diseases, such as PD, multiple system atrophy, Lewy body dementia (LBD), pure autonomic dysphysicosis (PAF), Pick's disease, progressive supranuclear palsy, boxer's dementia, chromosome 17-related parkinsonism, Ritico Bodig disease, neurofibrillary tangle-type senile dementia, argyrophilic granuloma, ganglioglioma, gangliocytoma, meningeal hemangioma, subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis, Hallerforden-Spatz disease, lipofuscinosis, corticobasal degeneration, frontotemporal dementia, frontotemporal lobar degeneration, Alzheimer's disease, Huntington's disease, Down syndrome, psychosis, schizophrenia, and synuclein diseases such as Creutzfeldt-Jakob disease. The dsRNA contains an antisense strand having a complementary region that is complementary to at least a portion of the mRNA formed during SNCA gene expression. In the embodiment, the complementary region is approximately 15 to 30 nucleotides in length or less. When in contact with cells expressing the SNCA gene, the RNAi agent inhibits the expression of the SNCA gene (e.g., human gene, primate gene, non-primate gene) by at least 50% when assayed by, for example, PCR or branched DNA (bDNA) based methods, or by protein-based methods such as immunofluorescence analysis using Western blotting or flow cytometry.

[0269] A dsRNA contains two complementary RNA strands that hybridize to form a double-stranded structure under the conditions in which the dsRNA is used. One strand of the dsRNA (the antisense strand) contains a complementary region that is substantially complementary to the target sequence and is generally fully complementary. The target sequence may be derived from the sequence of mRNA formed during the expression of an SNCA gene. The other strand (the sense strand) contains a region complementary to the antisense strand, thereby the two strands hybridize to form a double-stranded structure when combined under appropriate conditions. As described elsewhere in this specification and as known in the art, the complementary sequence of a dsRNA may also be contained as a self-complementary region of a single nucleic acid molecule, as opposed to being located on a separate oligonucleotide.

[0270] Generally, double structures are 15-30 base pairs long, for example, 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, 19-2 The lengths are 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 one preferred embodiment, the double-stranded structure is 18–25 base pairs long, for example, 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 long, for example, 19–21 base pairs long. Intermediate ranges and lengths between the above ranges and lengths are also considered to be part of this disclosure.

[0271] Similarly, the complementary region to the target sequence is 15-30 nucleotides long, for example, 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- The lengths are 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, for example, 19-23 nucleotides or 21-23 nucleotides. Intermediate ranges and lengths between the above ranges and lengths are also considered to be part of this disclosure.

[0272] In some embodiments, the dsRNA is 15–23 nucleotides long, or 24–30 nucleotides long (optionally, 25–30 nucleotides long). Generally, the dsRNA can be long enough to function as a substrate for the Dicer enzyme. For example, it is well known in the art that dsRNAs longer than about 21–23 nucleotides can serve as substrates for Dicer. As those skilled in the art will recognize, the RNA region targeted for cleavage is in most cases a larger RNA molecule, often a part of an mRNA molecule. Where applicable, the “part” of the mRNA target is a sequence of mRNA targets long enough to allow it to become a substrate for RNAi-directed cleavage (i.e., cleavage via the RISC pathway).

[0273] Those skilled in the art also know that the double-stranded region is the primary functional portion of dsRNA, for example, a double-stranded region of about 15-36 base pairs, for example, 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, 18-20, 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, for example, 19-21 base pairs. Therefore, in one embodiment, an RNA molecule or complex of RNA molecules having a double-stranded region greater than 30 base pairs to the extent that it is processed into a functional double helix of, for example, 15-30 base pairs, targeting the desired RNA for cleavage, is a dsRNA. Thus, those skilled in the art will recognize that in one embodiment, miRNA is a dsRNA. In another embodiment, dsRNA is not a naturally occurring miRNA. In another embodiment, RNAi agents useful for targeting SNCA expression are not generated in target cells by cleaving larger dsRNAs.

[0274] The dsRNA described herein may further comprise one or more single-stranded nucleotide overhangs, e.g., 1, 2, 3, or 4 nucleotides. The nucleotide overhangs may comprise or consist of nucleotide / nucleoside analogs containing deoxynucleotides / nucleosides. One or more overhangs may be on the sense strand, the antisense strand, or any combination thereof. Furthermore, one or more nucleotides of the overhang may be located at the 5' end, 3' end, or both ends of either the antisense strand or the sense strand of the dsRNA. In some embodiments, longer extended overhangs are possible.

[0275] dsRNA can be synthesized by standard methods known in the art, as further discussed below, for example, by using an automated DNA synthesizer, such as one commercially available from Biosearch, Applied Biosystems, Inc.

[0276] The iRNA compounds of this disclosure can be prepared using a two-step procedure. First, the individual strands of a double-stranded RNA molecule are prepared separately. Next, the constituent strands are annealed. The individual strands of the siRNA compound can be prepared using solution-phase or solid-phase organic synthesis, or both. Organic synthesis offers the advantage that oligonucleotide chains containing unnatural or modified nucleotides can be readily prepared. The single-stranded oligonucleotides of this disclosure can be prepared using solution-phase or solid-phase organic synthesis, or both.

[0277] siRNA can be produced in bulk, for example, by various methods. Exemplary methods include organic synthesis and RNA cleavage, such as in vitro cleavage.

[0278] siRNA can be prepared by separately synthesizing each strand of a single-stranded RNA molecule or a double-stranded RNA molecule, and then the constituent strands can be annealed.

[0279] Large bioreactors, such as the 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 conjugate nucleotides using only 1.5 molar excess phosphoramidite nucleotides. Ribonucleotide amidites are used to construct the RNA strands. A standard monomer addition cycle can be used to synthesize 21-23 nucleotide strands for siRNA. Typically, two complementary strands are produced separately and then annealed, for example, after release from a solid support and deprotection.

[0280] Organic synthesis can be used to produce individual siRNA species. The species complementarity to the SNCA gene can be precisely determined. For example, a species may be complementary to a region containing polymorphisms, such as single nucleotide polymorphisms. Furthermore, the location of the polymorphisms can be precisely defined. In some embodiments, the polymorphisms are located in an internal region, for example, at least 4, 5, 7, or 9 nucleotides from one or both ends.

[0281] In one embodiment, the generated RNA is carefully purified and the ends are removed. The iRNA is cleaved into siRNA in vitro using, for example, Dicer or an equivalent RNAseIII-based activity. For example, dsiRNA can be incubated in an in vitro extract derived from Drosophila or using purified components, such as purified RNAse or RISC (RNA-induced silencing complex). See, for example, Ketting et al. Genes Dev 2001 Oct 15;15(20):2654-9 and Hammond Science 2001 Aug 10;293(5532):1146-50.

[0282] dsiRNA cleavage generally generates multiple siRNA species, each being a specific 21-23nt fragment of the source dsiRNA molecule. For example, there may be siRNAs containing sequences complementary to the overlapping and adjacent regions of the source dsiRNA molecule.

[0283] Regardless of the synthesis method, siRNA preparations can be prepared in a solution suitable for formulation (e.g., an aqueous solution or an organic solution). For example, the siRNA preparation can be precipitated, redissolved in pure redistilled water, and freeze-dried. The dried siRNA can then be resuspended in a solution suitable for the intended formulation process.

[0284] In one embodiment, the dsRNA of the Disclosure comprises at least two nucleotide sequences, a sense sequence and an antisense sequence. The sense strand sequence for SNCA may be selected from the group of sequences provided in Table 2, Table 3, Table 12, or Table 13, and the corresponding nucleotide sequence of the antisense strand of the sense strand may be selected from the group of sequences in Table 2, Table 3, Table 12, or Table 13. In this embodiment, one of the two sequences is complementary to the other of the two sequences, and the one of the sequences is substantially complementary to the mRNA sequence produced in the expression of the SNCA gene. Thus, in this embodiment, the dsRNA comprises two oligonucleotides, one oligonucleotide described as the sense strand (passenger strand) in Table 2, Table 3, Table 12, or Table 13, and the second oligonucleotide described as the corresponding antisense strand (guide strand) of the sense strand for SNCA in Table 2, Table 3, Table 12, or Table 13.

[0285] In one embodiment, a substantially complementary sequence of the dsRNA is contained on a separate oligonucleotide. In another embodiment, a substantially complementary sequence of the dsRNA is contained on a single oligonucleotide.

[0286] Although the sequences provided herein are described as modified or conjugated sequences, it will be understood that the RNA of the RNAi agent of this disclosure, for example, the dsRNA of this disclosure, may include any of the sequences listed in Tables 2, 3, 12, or 13, which may be unmodified, unconjugated, or modified or conjugated in a manner different from those described herein. One or more lipophilic ligands or one or more GalNAc ligands may be included in any of the positions of the RNAi agent provided herein.

[0287] Those skilled in the art are well aware that dsRNAs having double-stranded structures of approximately 20 to 23 base pairs, for example, 21 base pairs, have been emphasized as particularly effective in inducing RNA interference (Elbashir et al., (2001) EMBO J., 20:6877-6888). However, other researchers have found that shorter or longer RNA double-stranded structures may also be effective (Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226). In the embodiments described above, due to the nature of the oligonucleotide sequences provided herein, the dsRNAs described herein may comprise at least one strand of a minimum length of 21 nucleotides. It can be reasonably expected that shorter double-stranded structures with only a few nucleotides subtracted from one or both ends may be equally effective compared to the dsRNAs described above. Therefore, dsRNAs having a sequence of at least 15, 16, 17, 18, 19, 20, or more consecutive nucleotides derived from one of the sequences provided herein, which have an ability to inhibit SNCA gene expression that differs from dsRNAs containing the complete sequence by at most 10, 15, 20, 25, or 30% of inhibition when used in in vitro assays and PCR assays with Be(2)-C cells and 10 nM concentration RNA agents provided in the examples herein, are intended to be within the scope of this disclosure.

[0288] One benchmark assay for SNCA inhibition comprises contacting human Be(2)-C cells with a dsRNA agent disclosed herein, where sufficient or effective SNCA inhibition is identified if, compared to a suitable control (e.g., cells not in contact with the SNCA-targeted dsRNA), a decrease of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or greater decreases are observed in the contacted cells. Optionally, the dsRNA agent of this disclosure is administered at a concentration of 10 nM, and a PCR assay is performed as provided in the examples of this specification (e.g., Example 2 below).

[0289] Furthermore, the RNAs described herein identify one or more sites in an SNCA transcript that are susceptible to RISC-mediated cleavage. Therefore, this disclosure further features RNAi agents that target these sites. As used herein, an RNAi agent is said to target a specific site within an RNA transcript if the RNAi agent promotes cleavage of the transcript somewhere within that specific site. Such an RNAi agent generally comprises at least about 15 consecutive nucleotides, optionally at least 19 nucleotides, from one of the sequences provided herein, ligated to an additional nucleotide sequence obtained from a region adjacent to a selected sequence of the SNCA gene.

[0290] The RNAi agents described herein may contain one or more mismatches to the target sequence. In one embodiment, the RNAi agent described herein contains three or fewer mismatches (i.e., 3, 2, 1, or 0 mismatches). In one embodiment, the RNAi agent described herein contains two or fewer mismatches. In one embodiment, the RNAi agent described herein contains one or fewer mismatches. In one embodiment, the RNAi agent described herein contains zero mismatches. In some embodiments, if the antisense strand of the RNAi agent contains a mismatch to the target sequence, the mismatch may be selectively restricted to the last 5 nucleotides from either the 5' or 3' end of the complementary region. For example, in such embodiments, for a 23-nucleotide RNAi agent, the strand complementary to the region of the SNCA gene generally does not contain any mismatches within the central 13 nucleotides. Using the methods described herein or methods known in the art, it is possible to determine whether an RNAi agent containing a mismatch to the target sequence is effective in inhibiting the expression of the SNCA gene. In particular, when specific complementary regions of the SNCA gene are known to have polymorphic sequence variations within a population, it becomes important to consider the effectiveness of RNAi agents with mismatches when inhibiting SNCA gene expression.

[0291] II. Modified RNAi agents of the present disclosure In one embodiment, the RNA of the RNAi agent of the Disclosure, e.g., dsRNA, is unmodified and does not contain any chemical modifications or conjugates known, for example, in the Art and described herein. In a preferred embodiment, the RNA of the RNAi agent of the Disclosure, e.g., dsRNA, is chemically modified to enhance stability or other beneficial characteristics. In one embodiment of the Disclosure, substantially all of the nucleotides of the RNAi agent of the Disclosure are modified. In another embodiment of the Disclosure, all of the nucleotides of the RNAi agent of the Disclosure are modified. The RNAi agent of the Disclosure that is "substantially all of its nucleotides modified" is mostly modified but not completely modified and may contain 5, 4, 3, 2, or 1 or fewer unmodified nucleotides. In yet another embodiment of the Disclosure, the RNAi agent of the Disclosure may contain 5, 4, 3, 2, or 1 or fewer modified nucleotides.

[0292] Nucleic acids featured in this disclosure may be synthesized or modified by methods well established in the art, such as those described herein by reference in “Current protocols in nucleic acid chemistry,” Beaucage, S. Let al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA. Modifications include, for example, terminal modifications, e.g., 5'-terminal modifications (phosphorylation, conjugation, reverse bond) or 3'-terminal modifications (conjugation, DNA nucleotide, reverse bond, etc.); base modifications, e.g., substitution with stabilizing bases, destabilizing bases, or bases that pair with partners in an expanded repertoire, removal of bases (debasalized nucleotides), or substitution with conjugate bases; sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions; or skeletal modifications (including modification or substitution of phosphodiester bonds). Specific examples of RNAi agents useful in the embodiments described herein include, but are not limited to, RNA containing a modified skeleton or RNA that does not contain natural nucleoside bonds. RNAs having a modified skeleton include, in particular, those that do not have a phosphorus atom in their skeleton. For the purposes of this specification, and as is sometimes referred to in the art, modified RNAs that do not have a phosphorus atom in their internucleoside skeleton can also be considered oligonucleosides. In some embodiments, the modified RNAi agent has a phosphorus atom in its internucleoside skeleton.

[0293] Examples of modified RNA backbones include phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotryesters, aminoalkylphosphotryesters, methyl and other alkylphosphonates including 3'-alkylenephosphonates and chiralphosphonates, phosphinates, phosphoramidates including 3'-aminophosphorumidates and aminoalkylphosphorumidates, thionophosphorumidates, thionoalkylphosphonates, thionoalkylphosphotryesters, and boranophosphates having the usual 3'-5' linkage, their 2'-5' linkage analogues, and those having reverse polarity where adjacent nucleoside unit pairs are linked from 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.

[0294] Representative U.S. patents teaching the preparation of the phosphorus-containing bond described above 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; and 5,286,717; Same No. 5,321,131; Same No. 5,399,676; Same No. 5,405,939; Same No. 5,453,496; Same No. 5,455,233; Same No. 5,466,677; Same 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 ,587,361; 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. 6,53 Examples include Patent Nos. 4,639; Nos. 6,608,035; Nos. 6,683,167; Nos. 6,858,715; Nos. 6,867,294; Nos. 6,878,805; Nos. 7,015,315; Nos. 7,041,816; Nos. 7,273,933; Nos. 7,321,029; and U.S. Patent RE39464 (the entire contents of each of these are incorporated herein by reference).

[0295] Modified RNA skeletons that do not contain phosphorus atoms have skeletons formed by short alkyl or cycloalkyl nucleoside bonds, mixed heteroatoms and alkyl or cycloalkyl nucleoside bonds, or one or more short heteroatoms or heterocyclic nucleoside bonds. These include those having morpholino bonds (partially formed from the sugar moiety of the nucleoside); siloxane skeletons; sulfide, sulfoxide, and sulfone skeletons; formacetyl and thioformacetyl skeletons; methyleneformacetyl and thioformacetyl skeletons; alkene-containing skeletons; sulfamate acid skeletons; methyleneimino and methylenehydrazino skeletons; sulfonate and sulfonamide skeletons; amide skeletons; and others having mixed N, O, S, and CH2 components.

[0296] Representative U.S. patents teaching the preparation of the above oligonucleotides include 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; and 5,489,677; Nos. 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 these are incorporated herein by reference.

[0297] In other embodiments, suitable RNA mimeographs are intended for use in RNAi agents in which both the sugar and the nucleoside bonds of the nucleotide units, i.e., the backbone, are replaced with novel groups. The base units are maintained for hybridization with suitable nucleic acid target compounds. One such oligomeric compound, an RNA mimeograph that has been shown to have excellent hybridization properties, is called a peptide nucleic acid (PNA). In PNA compounds, the RNA sugar backbone is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleic acid bases are retained and bond directly or indirectly to the aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents teaching the preparation of PNA compounds include, but are not limited to, U.S. Patents 5,539,082; 5,714,331; and 5,719,262, the entire contents of each of these are incorporated herein by reference. Additional PNA compounds suitable for use in the RNAi agents of this disclosure are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0298] Some embodiments featured in this disclosure include RNA having a phosphorothioate backbone and oligonucleotides having a heteroatom backbone, in particular --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--], and the amide backbone of U.S. Patent No. 5,602,240, as referenced above. In some embodiments, the RNA featured herein has the morpholino backbone structure of U.S. Patent No. 5,034,506, as referenced above.

[0299] Modified RNA may also contain one or more substituted sugar moieties. RNAi agents featured herein, such as dsRNA, may have 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-C 10 Alkyl or C2-C 10 They can be alkenyls and alkynyls. Exemplary suitable modifications include O[(CH2) n O] m CH3, O(CH2). n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2 and O(CH2) n ON[(CH2) n CH3)2 is an example, where n and m are 1 to approximately 10. In other embodiments, the dsRNA has one of the following at the 2' position: C1 to C 10Lower alkyl groups, substituted lower alkyl groups, alkali groups, aralkyl groups, O-alkaryl or O-aralkyl groups, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl groups, heterocycloalkaryl groups, aminoalkylamino groups, polyalkylamino groups, substituted silyl groups, RNA cleavage groups, reporter groups, intercalators, groups for improving the pharmacokinetic properties of RNAi agents, or groups for improving the pharmacodynamic properties of RNAi agents, and other substituents having similar properties. In some embodiments, the modification includes 2'-methoxyethoxy (2'-O--CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., an alkoxy-alkoxy group. Other exemplary modifications include 2'-dimethylaminooxyethoxy, i.e., the O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described in the following examples, and 2'-dimethylaminoethoxyethoxy (also known 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 of these three families), 2'-alkoxyalkyls; and 2'-NMA (N-methylacetamide).

[0300] Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2), 2'-O-hexadecyl, and 2'-fluoro (2'-F). Similar modifications can be made at other positions on the RNA of the RNAi agent, particularly at the 3' position of the sugar on the 3' terminal nucleotide or at the 5' position of the 2'-5' ligated dsRNA and the 5' position of the 5' terminal nucleotide. The RNAi agent may also have sugar mimes such as cyclobutyl moieties instead of pentofuranosyl sugars. Representative U.S. patents teaching 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 5,567. Examples include Nos. 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, of which certain are owned in common with this application. The entire contents of each of the above are incorporated herein by reference.

[0301] The RNAi agents of this disclosure may also include modifications or substitutions of nucleic acid bases (often simply referred to as “bases” in the art). As used herein, “unmodified” or “natural” nucleic acid bases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleic acid bases include other synthetic and natural nucleic acid bases, 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 Examples include thymine, 5-uracil (pseudolacil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyanal, other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine, and 3-deazaguanine and 3-deazaadenine.Further nucleic acid bases include those disclosed in U.S. Patent No. 3,687,808, those disclosed by Englisch et al., (1991) Angewandte Chemie, International Edition, 30:613, 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, JL, ed. John Wiley & Sons, 1990, 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. Some of these nucleic acid bases are particularly useful for enhancing the binding affinity of the oligomeric compounds featured in this disclosure. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-methylcytosine substitution has been shown to increase the stability of nucleic acid double helix 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 is an exemplary base substitution, more specifically in combination with 2'-O-methoxyethyl sugar modification.

[0302] Representative U.S. patents teaching the preparation of specific modified nucleic acid bases and other modified nucleic acid bases include U.S. Patents No. 3,687,808,4,845,205; No. 5,130,30; No. 5,134,066; No. 5,175,273; No. 5,367,066; No. 5,432,272; No. 5,457,187; No. 5,459,255; No. 5,484,908; No. 5,502,177; No. 5,525,711; No. 5,552,540; No. 5,587,469; No. 5,594,121,5,596, This includes, but is not limited to, Nos. 091; Nos. 5,614,617; Nos. 5,681,941; Nos. 5,750,692; Nos. 6,015,886; Nos. 6,147,200; Nos. 6,166,197; Nos. 6,222,025; Nos. 6,235,887; Nos. 6,380,368; Nos. 6,528,640; Nos. 6,639,062; Nos. 6,617,438; Nos. 7,045,610; Nos. 7,427,672; and Nos. 7,495,088, the entire contents of each of these are incorporated herein by reference.

[0303] The RNAi agents of this disclosure can also be modified to include one or more lock nucleic acids (LNAs). A lock nucleic acid is a nucleotide having a modified ribose moiety, the ribose moiety containing an extra crosslink connecting the 2' and 4' carbon atoms. This structure effectively "locks" the ribose in a 3'-end conformation. The addition of lock 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).

[0304] The RNAi agents of this disclosure may also be modified to include one or more bicyclic sugar moieties. A “bicyclic sugar” is a furanosyl ring modified by a bridge between two atoms. A “bicyclic nucleoside” (“BNA”) is a nucleoside having a sugar moiety that includes a bridge connecting two carbon atoms of a sugar ring, thereby forming a bicyclic system. In some embodiments, the bridge connects the 4'-carbon and 2'-carbon of the sugar ring. Thus, in some embodiments, the agents of this disclosure may include one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide having a modified ribose moiety, where the ribose moiety includes an extra bridge connecting the 2'-carbon and 4'-carbon. 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 in a 3'-end conformation. The addition of locking 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 this disclosure include, but are not limited to, nucleosides containing a bridge between the 4' and 2' ribosyl ring atoms. In some embodiments, the antisense polynucleotide agent of this disclosure comprises one or more bicyclic nucleosides containing a 4'-to-2' bridge.Examples of such 4'-to-2' bridged bicyclic nucleosides include 4'-(CH2)-O-2'(LNA); 4'-(CH2)-S-2'; 4'-(CH2)2-O-2'(ENA); 4'-CH(CH3)-O-2' (also known as "restricted ethyl" or "cEt") and 4'-CH(CH2OCH3)-O-2' (and its analogues; e.g., U.S. Patent No. 7,399,845); 4'-C(CH3)(CH3)-O-2' (and its analogues; e.g., U.S. Patent No. 8,278,283) See, for example, U.S. Patent No. 8,278,425); 4'-CH2-ON(CH3)-2' (see, for example, U.S. Patent Publication No. 2004 / 0171570); 4'-CH2-N(R)-O-2' (wherein R is H, C1-C12 alkyl, or a protecting group) (see, for example, No. 7,427,672); 4'-CH2-C(H)(CH3)-2' (see, for example, Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH2-C(=CH2)-2' (and its analogues; see, for example, U.S. Patent No. 8,278,426). All of the above contents are incorporated herein by reference.

[0305] Further representative U.S. patents and U.S. patent publications teaching the preparation of lock nucleic acid nucleotides include U.S. Patent Nos. 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133; 7,084,125; 7,399,845; 7, Nos. 427,672; 7,569,686; 7,741,457; 8,022,193; 8,030,467; 8,278,425; 8,278,426; 8,278,283; 2008 / 0039618; and 2009 / 0012281, among others, the entire contents of each of these are incorporated herein by reference.

[0306] For example, any of the aforementioned bicyclic nucleosides having one or more stereochemical sugar configurations, including α-L-ribofuranose and β-D-ribofuranose, can be prepared (see International Publication No. 99 / 14226).

[0307] The RNAi agents of this disclosure may also be modified to include one or more restricted ethyl nucleotides. As used herein, “restricted ethyl nucleotide” or “cEt” is a restricted nucleic acid comprising a bicyclic sugar moiety including a 4'-CH(CH3)-O-2' bridge. In one embodiment, the restricted ethyl nucleotide is in an S conformation, which is referred to herein as “S-cEt”.

[0308] The RNAi agents of this disclosure may also comprise one or more “contourally restricted nucleotides” (“CRNs”). A CRN is a nucleotide analog having a linker connecting the C2' and C4' carbons or the C3' and C5' carbons of ribose. CRNs fix the ribose ring to a stable conformation and increase hybridization affinity to mRNA. The linker is long enough to position oxygen in an optimal position for stability and affinity, resulting in less puckering of the ribose ring.

[0309] Representative publications teaching specific preparations of the above-mentioned CRNs include, but are not limited to, U.S. Patent No. 2013 / 0190383 and International Publication No. 2013 / 036868 (the entire contents of each of these are incorporated herein by reference).

[0310] In some embodiments, the RNAi agents of this disclosure comprise one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNA is an acyclic nucleic acid in which one of the sugar bonds has been removed, thereby forming an unlocked “sugar” residue. In one example, UNA also includes monomers 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 Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039, incorporated herein by reference).

[0311] Representative U.S. patent applications and publications teaching the preparation of UNA include, but are not limited to, U.S. Patent No. 8,314,227 and U.S. Patent Publications 2013 / 0096289; 2013 / 0011922; and 2011 / 0313020.

[0312] Potentially stabilizing modifications to the ends of RNA molecules 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-docosanoyluridine-3"-phosphate, and the reverse base dT (idT). Disclosure of these modifications can be found in International Publication No. 2011 / 005861.

[0313] Other modifications of the RNAi agents of this disclosure include 5'-phosphate or 5'-phosphate mimetic, for example, a 5'-terminal phosphate or phosphate mimetic on the antisense strand of the RNAi agent. Suitable phosphate mimetics are disclosed, for example, in U.S. Patent No. 2012 / 0157511, the entire contents of which are incorporated herein by reference.

[0314] A. Modified RNAi agents containing motifs of the present disclosure In certain embodiments of this disclosure, the double-stranded RNAi agents of this disclosure include agents having chemical modifications as disclosed, for example, in International Publication No. 2013 / 075035, the entirety of which is incorporated herein by reference. Excellent results can be obtained by introducing one or more motifs of three identical modifications on three consecutive nucleotides to the sense or antisense strand of the RNAi agent, particularly at or near the cleavage site, as shown herein and in International Publication No. 2013 / 075035. In some embodiments, the sense and antisense strands of the RNAi agent may otherwise be fully modified. The introduction of these motifs disrupts the modification pattern of the sense or antisense strand, if present. The RNAi agent may optionally be conjugated with a lipophilic ligand, for example, a C16 ligand, on the sense strand. The RNAi agent may optionally be modified with (S)-glycol nucleic acid (GNA) modifications at, for example, one or more residues on the antisense strand. The resulting RNAi agents exhibit excellent gene silencing activity.

[0315] Accordingly, this disclosure provides a double-stranded RNAi agent capable of inhibiting the expression of a target gene (i.e., an SNCA gene) in vivo. The RNAi agent comprises a sense strand and an antisense strand. Each strand of the RNAi agent may be 15 to 30 nucleotides long. For example, each strand may be 16 to 30 nucleotides long, 17 to 30 nucleotides long, 25 to 30 nucleotides long, 27 to 30 nucleotides long, 17 to 23 nucleotides long, 17 to 21 nucleotides long, 17 to 19 nucleotides long, 19 to 25 nucleotides long, 19 to 23 nucleotides long, 19 to 21 nucleotides long, 21 to 25 nucleotides long, or 21 to 23 nucleotides long. In one embodiment, each strand is 19 to 23 nucleotides long.

[0316] The sense strand and antisense strand typically form a double-stranded RNA ("dsRNA"), also referred to herein as the "RNAi agent." The double-stranded region of the RNAi agent may be 15–30 nucleotide pairs long. For example, the double-stranded region may be 16–30 nucleotide pairs long, 17–30 nucleotide pairs long, 27–30 nucleotide pairs long, 17–23 nucleotide pairs long, 17–21 nucleotide pairs long, 17–19 nucleotide pairs long, 19–25 nucleotide pairs long, 19–23 nucleotide pairs long, 19–21 nucleotide pairs long, 21–25 nucleotide pairs long, or 21–23 nucleotide pairs long. In another example, the double-stranded region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotide lengths. In a preferred embodiment, the double-stranded region is 19–21 nucleotide pairs long.

[0317] In one embodiment, the RNAi agent may include one or more overhang regions or cap groups at the 3' end, 5' end, or both ends of one or both strands. The overhangs may be 1 to 6 nucleotides long, 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 a preferred embodiment, the nucleotide overhang region is 2 nucleotides long. The overhangs may result from one strand being longer than the other, or from two strands of equal length being staggered. The overhangs may form a mismatch with the target mRNA, or may be complementary to the targeted gene sequence, or may be a different sequence. The first and second strands may also be joined, for example, by additional bases to form a hairpin, or by other non-base linkers.

[0318] In one embodiment, the nucleotides in the overhang region of the RNAi agent may independently be modified or unmodified nucleotides, including but not limited to 2'-sugar modifications such as 2'-F, 2'-O-methyl, thymidine (T), and any combination thereof.

[0319] For example, TT could be an overhang sequence to any end of either strand. The overhang could form a mismatch with the target mRNA, or it could be complementary to the targeted gene sequence, or it could be a different sequence altogether.

[0320] The 5' or 3' overhangs of the sense strand, antisense strand, or both strands of an RNAi agent can be phosphorylated. In some embodiments, one or more overhang regions include two nucleotides having a phosphorothioate between them, the two nucleotides may be the same or different. In one embodiment, the overhang is located at the 3' end of the sense strand, antisense strand, or both strands. In one embodiment, this 3'-overhang is located on the antisense strand. In one embodiment, this 3'-overhang is located on the sense strand.

[0321] RNAi agents may contain only a single overhang that can enhance the interference activity of RNAi without affecting its overall stability. For example, a single-stranded overhang may be located at the 3' end of the sense strand, or alternatively, at the 3' end of the antisense strand. RNAi may also have a blunt end located at the 5' end 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 a blunt end at the 5' end. While we do not wish to be bound by theory, asymmetric blunt ends at the 5' end of the antisense strand and 3' end overhangs of the antisense strand are favorable for guide strand loading into RISC processes.

[0322] In one embodiment, the RNAi agent is a 19-nucleotide double-ended blunt-ended, in which case the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides from the 5' end at positions 7, 8, and 9. The antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides from the 5' end at positions 11, 12, and 13.

[0323] In another embodiment, the RNAi agent is a 20-nucleotide double-ended blunt-ended, in which case the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides from the 5' end at positions 8, 9, and 10. The antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides from the 5' end at positions 11, 12, and 13.

[0324] In yet another embodiment, the RNAi agent is a 21-nucleotide-long double-ended blunt-ended, in which case 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.

[0325] In one embodiment, the RNAi agent comprises a 21-nucleotide sense strand and a 23-nucleotide antisense strand, in which case 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, with one end of the RNAi agent being a blunt end and the other end containing a 2-nucleotide overhang. Optionally, the 2-nucleotide overhang is located at the 3' end of the antisense strand. If the 2-nucleotide overhang is at the 3' end of the antisense strand, two phosphorothioate internucleotide bonds may exist between the three terminal nucleotides, in which case two of the three nucleotides are the overhang nucleotides and the third nucleotide is the paired nucleotide adjacent to the overhang nucleotides. In one embodiment, the RNAi agent further has two phosphorothioate internucleotide bonds 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, all nucleotides on the sense and antisense strands of the RNAi agent, including nucleotides that are part of a motif, are modified nucleotides. In one embodiment, each residue is independently modified with 2'-O-methyl or 3'-fluoro, for example, in an alternating motif. Optionally, the RNAi agent further comprises a ligand (e.g., a lipophilic ligand, possibly a C16 ligand).

[0326] In one embodiment, the RNAi agent comprises a sense strand and an antisense strand, the sense strand being 25-30 nucleotides long, in which position 1-23 of the first strand contains at least 8 ribonucleotides starting from the 5' terminal nucleotide (position 1), the antisense strand being 36-66 nucleotides long, and starting from the 3' terminal nucleotide, containing at least 8 ribonucleotides at positions that pair with position 1-23 of the sense strand to form a double helix, where at least the 3' terminal nucleotide of the antisense strand is not paired with the sense strand, and up to 6 consecutive 3' terminal nucleotides are not paired with the sense strand, thereby forming a 3' single-stranded overhang of 1-6 nucleotides, where the 5' end of the antisense strand contains 10-30 consecutive ribonucleotides that are not paired with the sense strand. The sense strand contains nucleotides, thereby forming a single-stranded 5' overhang of 10 to 30 nucleotides, where at least the 5' and 3' terminal nucleotides of the sense strand base-pair with the nucleotides of the antisense strand when the sense strand and antisense strand are aligned for maximum complementarity, thereby forming a substantially double-stranded region between the sense strand and the antisense strand, and the antisense strand is sufficiently complementary to the target RNA along at least 19 ribonucleotides of the antisense strand length to reduce target gene expression when the double-stranded nucleic acid is introduced into mammalian cells, wherein the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides, at least one of which is located at or near the cleavage site. The antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.

[0327] In one embodiment, the RNAi agent comprises a sense strand and an antisense strand, in which the RNAi agent comprises a first strand having a length of at least 25 and a maximum of 29 nucleotides, and a second strand having a length of up to 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, wherein the 3' end of the first strand and the 5' end of the second strand form blunt ends, the second strand has a double-stranded region that is 1 to 4 nucleotides longer at its 3' end than the first strand, here, at least 25 nucleotides long, and the second strand is sufficiently complementary to the target mRNA along at least 19 nucleotides of the length of the second strand in order to reduce the expression of the target gene when the RNAi agent is introduced into mammalian cells, and Dicer cleavage of the RNAi agent preferentially yields an siRNA including the 3' end of the second strand, thereby reducing the expression of the target gene in the mammal. Optionally, the RNAi agent further comprises a ligand.

[0328] In one embodiment, the sense strand of the RNAi agent contains at least one motif of three identical modifications on three consecutive nucleotides, in which case one of the motifs is located at a cleavage site of the sense strand.

[0329] In one embodiment, the antisense strand of the RNAi agent may also contain at least one motif of three identical modifications on three consecutive nucleotides, in which case one of the motifs is located at or near the cleavage site of the antisense strand.

[0330] For RNAi agents with a double-stranded region of 17–23 nucleotides in length, the cleavage sites on the antisense strand are typically around positions 10, 11, and 12 from the 5' end. Therefore, the three identical modification motifs can occur at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, with the count starting from the first nucleotide from the 5' end of the antisense strand, or from the first paired nucleotide within the double-stranded region from the 5' end of the antisense strand. The cleavage sites on the antisense strand can also vary depending on the length of the double-stranded region of the RNAi from the 5' end.

[0331] The sense strand of an RNAi agent may contain at least one motif from three identical modifications on three consecutive nucleotides at its cleavage site, and the antisense strand may have at least one motif from three identical modifications on three consecutive nucleotides at or near its cleavage site. When the sense strand and antisense strand form a dsRNA double helix, the sense strand and antisense strand may be aligned such that one motif of three nucleotides on the sense strand and one motif of three nucleotides on the antisense strand have at least one nucleotide duplication, i.e., at least one of the three nucleotides of that motif in the sense strand forms a base pair with at least one of the three nucleotides of that motif in the antisense strand. Alternatively, at least two nucleotides may be duplicated, or all three nucleotides may be duplicated.

[0332] 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 be located at or near a cleavage site on the strand, and the other motifs may be wing modifications. The term “wing modification” as used herein refers to a motif located on a different part of the strand separated from a motif at or near a cleavage site on the same strand. Wing modifications are either adjacent to the first motif or separated by at least one nucleotide. If the motifs are directly adjacent to each other, their chemical properties are different from each other; if the motifs are separated by one or more nucleotides, their chemical properties may be the same or different. There may be two or more wing modifications. For example, if there are two wing modifications, each wing modification may occur at or near a cleavage site, or at one end to the first motif located on both sides of the read motif.

[0333] 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 these motifs located at or near a cleavage site on the strand. This antisense strand may also contain one or more wing modifications in an alignment similar to that of the wing modifications that may be present on the sense strand.

[0334] In one embodiment, the wing modification on the sense or antisense strand of the RNAi agent typically does not include the first one or two terminal nucleotides at the 3' end, 5' end, or both ends of the strand.

[0335] In another embodiment, the wing modification on the sense or antisense strand of the RNAi agent typically does not include the first one or two pairs of nucleotides in the double-stranded region at the 3' end, 5' end, or both ends of the strand.

[0336] If the sense strand and antisense strand of an RNAi agent each contain at least one wing modification, the wing modification may be located at the same end of the double-stranded region and may have a duplication of 1, 2, or 3 nucleotides.

[0337] If the sense strand and antisense strand of the RNAi agent each contain at least two wing modifications, the sense strand and antisense strand can be aligned such that two modifications from one strand each enter one end of a double-stranded region having 1, 2, or 3 nucleotide overlaps, and two modifications from one strand each enter the other end of the double-stranded region having 1, 2, or 3 nucleotide overlaps, and two modifications, one strand dropping to both sides of the read motif, having 1, 2, or 3 nucleotide overlaps within the double-stranded region.

[0338] In one embodiment, the RNAi agent includes one or more mismatches with the target, within the double helix, or in combination thereof. Mismatches may occur in overhang regions or duplex regions. Base pairs can be ranked based on their tendency to promote dissociation or dissolution (for example, by the free energy of association or dissociation of a particular pairing; the simplest approach is to examine each pair individually, but the following adjacent or similar analyses can also be used). From the viewpoint 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, e.g., non-standard or non-standard pairings (as described elsewhere in this specification) are preferred over standard (A:T, A:U, G:C) pairings, and pairings including a universal base are preferred over standard pairings.

[0339] In one embodiment, the RNAi agent comprises the first 1, 2, 3, 4, or 5 base pairs in the double-stranded region from the 5' end of the antisense strand, independently selected from the groups A:U, G:U, and I:C, and at least one pair other than standard pairs, such as mismatch pairs, which include non-standard or universal bases to facilitate the dissociation of the antisense strand at the 5' end of the double helix.

[0340] 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.

[0341] 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, a short sequence of deoxythymine nucleotides, for example, two dT nucleotides, are present at the 3' end of the sense strand or antisense strand.

[0342] In one embodiment, the sense chain sequence may be represented by formula (I), 5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3' (I) During the ceremony, i and j are independently either 0 or 1. p and q are independently between 0 and 6. each N a Each independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, and each sequence contains at least two different modified nucleotides. each N b This independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides. each n p and n q These independently represent overhang nucleotides, In the formula, Nb and Y do not have the same modifications. XXX, YYY, and ZZZ each independently represent a single motif of three identical modifications on three consecutive nucleotides. Optionally, all YYY nucleotides are 2'-F modified nucleotides.

[0343] In one embodiment, N a or N b This includes alternating modification patterns.

[0344] In one embodiment, the YYY motif is located at or near a cleavage site on the sense strand. For example, if the RNAi agent has a double-stranded region of 17-23 nucleotides in length, the YYY motif may occur at or near a cleavage site on the sense strand (e.g., at positions 6, 7, 8, 7, 8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12 or 11, 12, 13), and its counting may begin from the first nucleotide counting from the 5' end, or optionally, from the first pair of nucleotides in the double-stranded region from the 5' end.

[0345] In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. Therefore, the sense chain can be expressed by 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).

[0346] If the sense chain is represented by formula (Ib), then N bThis represents an oligonucleotide sequence containing 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides.

[0347] each N a This can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0348] If the sense chain is expressed as equation (Ic), then N b This represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. a This can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0349] When the sense chain is expressed as formula (Id), each N b Each independently represents an oligonucleotide sequence containing 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Optionally, N b n is 0, 1, 2, 3, 4, 5, or 6. a This can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0350] X, Y, and Z may be the same as or different from each other.

[0351] In other embodiments, i is 0 and j is 0, and the sense chain can be represented by the following equation: 5'n p -N a -YYY-N a -n q 3' (Ia)

[0352] If the sense chain is represented by equation (Ia), then each N a This can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0353] In one embodiment, the antisense strand sequence of RNAi may be represented by 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) During the ceremony, k and l are independently either 0 or 1. p' and q' are each independently between 0 and 6. each N a ' independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, and each sequence contains at least two different modified nucleotides. each N b ' independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides. each n p 'and n q ' independently represents an overhang nucleotide, In the formula, N b 'and Y' do not have the same modifiers. X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent a single motif of three identical modifications on three consecutive nucleotides.

[0354] In one embodiment, N a 'or N b ' includes alternating modification patterns.

[0355] The Y'Y'Y' motif is located at or near the cleavage site of the antisense strand. For example, if an RNAi agent has a double-stranded region of 17-23 nucleotides in length, the Y'Y'Y' motif may occur at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, where the count begins from the first nucleotide from the 5' end, or optionally, from the first paired nucleotide within the double-stranded region from the 5' end. Optionally, the Y'Y'Y' motif is located at positions 11, 12, and 13.

[0356] In one embodiment, all Y'Y'Y' motifs are 2'-OMe modified nucleotides.

[0357] In one embodiment, k is 1 and l is 0, or k is 0 and l is 1, or both k and l are 1.

[0358] Therefore, the antisense chain can be expressed by the following 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).

[0359] If the antisense chain is represented by equation (IIb), then 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.

[0360] If the antisense chain is expressed as equation (IIc), then 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.

[0361] If the antisense chain is expressed as equation (IId), then each N b ' independently 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. Optionally, N b is 0, 1, 2, 3, 4, 5, or 6.

[0362] In other embodiments, k is 0, l is 0, and the antisense chain can be represented by the following equation: 5'n p’ -N a’ -Y'Y'Y'-N a’ -n q’ 3' (Ia).

[0363] If the antisense chain is expressed as equation (IIa), then each N a The ' independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0364] X', Y', and Z' may be the same as or different from each other.

[0365] Each nucleotide in the sense and antisense strands can be independently modified with LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-hydroxyl, or 2'-fluoro. For example, each nucleotide in the sense and antisense strands can be independently modified with 2'-O-methyl or 2'-fluoro. Each X, Y, Z, X', Y', and Z' may, in particular, represent a 2'-O-methyl modification or a 2'-fluoro modification.

[0366] In one embodiment, the sense strand of the RNAi agent may contain YYY motifs occurring at positions 9, 10, and 11 of the strand when the double-stranded region is 21nt, with the count starting from the first nucleotide from the 5' end, or optionally, the count starting from the first paired nucleotide in the double-stranded region from the 5' end, where Y represents a 2'-F modification. The sense strand may further contain an XXX motif or a ZZZ motif as a wing modification at the opposite end of the double-stranded region, where XXX and ZZZ independently represent a 2'-OMe modification or a 2'-F modification.

[0367] In one embodiment, the antisense strand may contain a Y'Y'Y' motif located at positions 11, 12, and 13 of the strand, where the count starts from the first nucleotide from the 5' end, or optionally, the count starts from the first pair nucleotide in the double-stranded region from the 5' end, 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 double-stranded region, where X'X'X' and Z'Z'Z' independently represent a 2'-OMe modification or a 2'-F modification.

[0368] A sense strand represented by any of the above formulas (Ia), (Ib), (Ic), and (Id) forms a double helix with an antisense strand represented by any of the above formulas (IIa), (IIb), (IIc), and (IId).

[0369] Therefore, the RNAi agent for use in the method of this disclosure may include a sense strand and an antisense strand, each having 14 to 30 nucleotides, and the RNAi double helix is ​​represented by 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) During the ceremony, i, j, k, and l are each independently either 0 or 1. p, p', q, and q' are each independently between 0 and 6. each N a and N a ' independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, and each sequence contains at least two different modified nucleotides. each N b and N b ' independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides. Each n may or may not exist. p ',n p , n q 'and n q This independently represents an overhang nucleotide,

[0370] XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one of three identical modification motifs on three consecutive nucleotides.

[0371] 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 both i and j are 0, or both i and j are 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 both k and l are 0, or both k and l are 1.

[0372] Exemplary combinations of sense and antisense strands that form an RNAi double helix include the following formulas: 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-N b -YYY-N a -n q3' 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)

[0373] If an RNAi agent is represented by formula (IIIa), then each N a Each independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0374] When an RNAi agent is represented by formula (IIIb), each N b Each N independently represents an oligonucleotide sequence containing 1-10, 1-7, 1-5, or 1-4 modified nucleotides. a Each independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0375] When an RNAi agent is represented by formula (IIIc), each N b , N b' independently represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. a Each independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0376] When an RNAi agent is represented by formula (IIId), each N b , N b ' independently represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. a ', N a ' independently represents 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 of these independently includes alternating modification patterns.

[0377] In one embodiment, when the RNAi agent is represented by formula (IIId), N a The modification is a 2'-O-methyl or 2'-fluoro modification. In another embodiment, if the RNAi agent is represented by formula (IIId), then N a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n p ' is bound to the adjacent nucleotide a via a phosphorothioate bond. In yet another embodiment, if 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 ' is bound to an adjacent nucleotide via a phosphorothioate bond, and the sense strand is conjugated to one or more C16 (or related) portions bound via a divalent or trivalent branched linker (described below). In another embodiment, if the RNAi agent is represented by formula (IIId), N aThe modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n p The nucleotide is linked to an adjacent nucleotide via a phosphorothioate bond, the sense strand contains at least one phosphorothioate bond, and the sense strand is conjugated to one or more lipophilic, e.g., C16 (or related) moieties, optionally linked via a divalent or trivalent branched linker.

[0378] 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 The nucleotide is linked to an adjacent nucleotide via a phosphorothioate bond, the sense strand contains at least one phosphorothioate bond, and the sense strand is conjugated to one or more lipophilic, e.g., C16 (or related) moieties, linked via a divalent or trivalent branched linker.

[0379] In one embodiment, the RNAi agent is a multimer containing at least two double helixes represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), the double helixes linked by a linker. The linker may be cleavable or incleavable. Optionally, the multimer further contains ligands. Each double helix may target the same gene or two different genes, or each double helix may target the same gene at two different target sites.

[0380] In one embodiment, the RNAi agent is a multimer containing three, four, five, six or more double helixes represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), where the double helixes are linked by linkers. The linkers may be cleavable or incleavable. Optionally, the multimer further contains ligands. Each double helix may target the same gene or two different genes, or each double helix may target the same gene at two different target sites.

[0381] In one embodiment, two RNAi agents represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId) are ligated together at their 5' ends, with one or both of their 3' ends optionally conjugated to a ligand. Each agent may target the same gene or two different genes, or each agent may target the same gene at two different target sites.

[0382] Various publications describe multimeric RNAi agents that can be used in the manner of this 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 U.S. Patent No. 7,858,769, the entire contents of each of these are incorporated herein by reference.

[0383] In some embodiments, the compositions and methods of the Disclosure include vinyl phosphonate (VP) modification of the RNAi agent described herein. In exemplary embodiments, the vinyl phosphonate of the Disclosure has the following structure: [ka]

[0384] The vinyl phosphonates of the Disclosure may be bound to either the antisense or sense strand of the dsRNA of the Disclosure. In certain preferred embodiments, the vinyl phosphonates of the Disclosure are bound to the antisense strand of the dsRNA, and optionally to the 5' end of the antisense strand of the dsRNA. Vinyl phosphate modification is also intended for the compositions and methods of this disclosure. An exemplary vinyl phosphate structure is as follows: [ka]

[0385] B. thermally destabilizing modification In some embodiments, a dsRNA molecule can be optimized for RNA interference by incorporating a thermal destabilization modification in the seed region of the antisense strand (i.e., at positions 2–9 of the 5' end of the antisense strand) to reduce or inhibit off-target gene silencing. dsRNAs having an antisense strand containing at least one thermal destabilization modification of the double helix within the first nine nucleotide positions from the 5' end of the antisense strand have been found to exhibit reduced off-target gene silencing activity. Therefore, in some embodiments, the antisense strand contains at least one (e.g., 1, 2, 3, 4, 5 or more) thermal destabilization modification of the double helix within the first nine nucleotide positions of the 5' region of the antisense strand. In some embodiments, one or more thermal destabilization modifications of the double helix are located at positions 2–9, or optionally 4–8, from the 5' end of the antisense strand. In some further embodiments, one or more thermal destabilization modifications of the double helix are located at positions 6, 7, or 8 from the 5' end of the antisense strand. In some further embodiments, the double-strand thermal destabilization modification is located at position 7 from the 5' end of the antisense strand. The term “thermal destabilization modification” includes modifications that result in a dsRNA with a lower overall melting temperature (Tm) (optionally, a Tm 1, 2, 3, or 4 degrees lower than the Tm of dsRNA without such modification). In some embodiments, the double-strand thermal destabilization modification is located at position 2, 3, 4, 5, or 9 from the 5' end of the antisense strand.

[0386] Examples of thermally destabilizing modifications include, but are not limited to, abasic modification; mismatch with opposing nucleotides in opposing chains; and sugar modification, such as 2'-deoxy modification or acyclic nucleotides, such as unlocked nucleic acids (UNAs) or glycolic acids (GNAs).

[0387] Examples of debasement modifications include, but are not limited to, the following: [ka] Here, R is H, Me, Et or OMe; R' is H, Me, Et or OMe; R'' is H, Me, Et or OMe, [ka] Here, B is a modified or unmodified nucleic acid base.

[0388] Examples of sugar modifications include, but are not limited to, the following: [ka] Here, B is a modified or unmodified nucleic acid base.

[0389] In some embodiments, the thermal destabilization modification of the double chain is selected from the group consisting of the following: [ka] Here, B is a modified or unmodified nucleic acid base, and the asterisk in each structure represents either R, S, or a racemic mixture.

[0390] The term "acyclic nucleotide" refers to any nucleotide having an acyclic ribose sugar in which, for example, one of the bonds between 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 oxygen atoms (e.g., C1', C2', C3', C4', or O4') is absent independently or in combination in the nucleotide. In some embodiments, an acyclic nucleotide is, [ka] Here, B is a modified or unmodified nucleic acid base, and R 1 and R 2(R3 is 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, a UNA also includes 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 entirety). Acyclic derivatives offer greater skeletal flexibility without affecting Watson-Crick pairing. Acyclic nucleotides can be linked via 2'-5' or 3'-5' bonds.

[0391] The term "GNA" refers to glycol nucleic acids, which are polymers similar to DNA or RNA, but differ in their "backbone" composition in that they are composed of repeating glycerol units linked by phosphodiester bonds. [ka]

[0392] Double-strand thermal destabilization modifications can be mismatches (i.e., non-complementary base pairs) between thermally destabilized nucleotides and opposing nucleotides in the opposite strand of a dsRNA double helix. 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 this disclosure. Mismatches can occur between nucleotides that are either naturally occurring or modified nucleotides; that is, mismatch base pairings can occur between nucleic acid bases from each nucleotide, independently of modifications on the ribose sugar of the nucleotides. In some embodiments, a dsRNA molecule contains at least one nucleic acid base in the mismatch pairing that is a 2'-deoxynucleotide, for example, the 2'-deoxynucleotide is in the sense strand.

[0393] In some embodiments, double-strand thermal destabilization modifications in the seed region of the antisense strand include nucleotides in which the W-CH bond to the complementary base on the target mRNA is impaired, for example: [ka]

[0394] Further examples of debasic nucleotides, acyclic nucleotide modifications (including UNAs and GNAs), and mismatch modifications are described in detail in International Publication No. 2011 / 133876, which is incorporated herein by reference in its entirety.

[0395] Thermal destabilization modifications may include universal bases in which the ability to form hydrogen bonds with opposing bases is reduced or abolished, as well as phosphate modifications.

[0396] In some embodiments, double-strand thermal destabilization modifications include nucleic acid base modifications in which nucleotides have non-standard bases, for example but not limited to, but whose ability to form hydrogen bonds with bases of the opposite strand is impaired or completely lost. These nucleic acid base modifications have been evaluated for destabilization of the central region of the dsRNA double helix, as described in International Publication No. 2010 / 0011895, which is incorporated in its entirety herein by reference. Exemplary nucleic acid base modifications are as follows: [ka]

[0397] In some embodiments, the double-strand thermal destabilization modification in the seed region of the antisense strand is one or more α-nucleotides complementary to the base on the target mRNA, for example, [ka] Here, R is H, OH, OCH3, F, NH2, NHMe, NMe2, or O-alkyl.

[0398] Exemplary phosphate modifications known to reduce the thermal stability of dsRNA double helix compared to natural phosphodiester bonds are as follows: [ka]

[0399] The alkyl group of the R group can be C1-C6 alkyl. Specific examples of alkyl groups of the R group include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, pentyl, and hexyl.

[0400] Those skilled in the art will recognize that while the specificity of the RNAi agents of this disclosure is defined by considering the functional roles of nucleic acid bases, nucleic acid base modifications can be carried out in various ways as described herein, for example, to introduce destabilizing modifications to the RNAi agents of this disclosure for the purpose of enhancing on-target effects compared to off-target effects, and that the range of available modifications, and the modifications generally present in the RNAi agents of this disclosure, tend to be far greater than non-nucleonucleotide modifications, such as modifications to the sugar groups or phosphate backbone of polyribonucleotides. Such modifications are described in more detail in other sections of this disclosure and are explicitly intended for the RNAi agents of this disclosure having either natural or modified nucleic acid bases as described above or elsewhere in this disclosure.

[0401] In addition to the antisense strand containing thermal destabilization modifications, the dsRNA may also contain one or more stabilization modifications. For example, the dsRNA may contain at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) stabilization modifications. Without limitation, all stabilization modifications may be present on a single strand. In some embodiments, both the sense strand and the antisense strand contain at least two stabilization modifications. Stabilization modifications may occur on any nucleotide of the sense strand or antisense strand. For example, stabilization modifications may occur on all nucleotides on the sense strand or antisense strand, each stabilization modification may occur in an alternating pattern on the sense strand or antisense strand, or the sense strand or antisense strand may contain both stabilization modifications in an alternating pattern. The alternating pattern of stabilization modifications on the sense strand may be the same as or different from that on the antisense strand, and the alternating pattern of stabilization modifications on the sense strand may have a shift relative to the alternating pattern of stabilization modifications on the antisense strand.

[0402] In some embodiments, the antisense chain includes at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) stabilization modifications. Stabilization modifications of the antisense chain may be present at any position, but are not limited to these. In some embodiments, the antisense includes stabilization modifications at positions 2, 6, 8, 9, 14, and 16 from the 5' end. In some other embodiments, the antisense includes stabilization modifications at positions 2, 6, 14, and 16 from the 5' end. In even more other embodiments, the antisense includes stabilization modifications at positions 2, 14, and 16 from the 5' end.

[0403] In some embodiments, the antisense strand includes at least one stabilizing modification adjacent to the destabilizing modification. For example, the stabilizing modification may be a nucleotide at the 5' or 3' end of the destabilizing modification, i.e., at a position -1 or +1 from the position of the destabilizing modification. In some embodiments, the antisense strand includes stabilizing modifications at the 5' and 3' ends of the destabilizing modification, respectively, i.e., at positions -1 and +1 from the position of the destabilizing modification.

[0404] In some embodiments, the antisense chain includes at least two stabilizing modifications at the 3' end of the destabilizing modification, i.e., at positions +1 and +2 from the position of the destabilizing modification.

[0405] In some embodiments, the sense strand includes at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) stabilization modifications. Stabilization modifications on the sense strand may be present at any position, but are not limited to these. In some embodiments, the sense strand includes stabilization modifications at positions 7, 10, and 11 from the 5' end. In some other embodiments, the sense strand includes stabilization modifications at positions 7, 9, 10, and 11 from the 5' end. In some embodiments, the sense strand includes stabilization 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 includes stabilization 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 includes blocks of two, three, or four stabilization modifications.

[0406] In some embodiments, the sense chain does not contain a stabilizing modification in the antisense chain at a position opposite or complementary to the thermal destabilizing modification of the double chain.

[0407] Exemplary thermal stabilization modifications include, but are not limited to, 2'-fluoro modifications. Other thermal stabilization modifications include, but are not limited to, LNAs.

[0408] In some embodiments, the dsRNA of this disclosure contains at least four (e.g., 4, 5, 6, 7, 8, 9, 10 or more) 2'-fluoronucleotides. However, all 2'-fluoronucleotides may be present on a single strand. In some embodiments, both the sense strand and the antisense strand contain at least two 2'-fluoronucleotides. 2'-fluoro modifications may occur on any nucleotide of the sense strand or antisense strand. For example, 2'-fluoro modifications may occur on all nucleotides on the sense strand or antisense strand, each 2'-fluoro modification may occur in an alternating pattern on the sense strand or antisense strand, or the sense strand or antisense strand may contain both 2'-fluoro modifications in an alternating pattern. The alternating pattern of 2'-fluoro modifications on the sense strand may be the same as or different from that on the antisense strand, and the alternating pattern of 2'-fluoro modifications on the sense strand may have a shift relative to the alternating pattern of 2'-fluoro modifications on the antisense strand.

[0409] In some embodiments, the antisense chain contains at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-fluoronucleotides. The 2'-fluoro modifications in the antisense chain may be present at any position, but are not limited to these. In some embodiments, the antisense contains 2'-fluoronucleotides at positions 2, 6, 8, 9, 14, and 16 from the 5' end. In some other embodiments, the antisense contains 2'-fluoronucleotides at positions 2, 6, 14, and 16 from the 5' end. In even more other embodiments, the antisense contains 2'-fluoronucleotides at positions 2, 14, and 16 from the 5' end.

[0410] In some embodiments, the antisense strand includes at least one 2'-fluoronucleotide adjacent to the destabilization modification. For example, the 2'-fluoronucleotide may be a nucleotide at the 5' or 3' end of the destabilization modification, i.e., at a position -1 or +1 from the position of the destabilization modification. In some embodiments, the antisense strand includes 2'-fluoronucleotides at the 5' and 3' ends of the destabilization modification, i.e., at positions -1 and +1 from the position of the destabilization modification.

[0411] In some embodiments, the antisense strand includes at least two 2'-fluoronucleotides at the 3' end of the destabilization modification, i.e., at positions +1 and +2 from the position of the destabilization modification.

[0412] In some embodiments, the sense strand contains at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-fluoronucleotides. The 2'-fluoro modifications in the sense strand may be present at any position, but are not limited to these. In some embodiments, the antisense strand contains 2'-fluoronucleotides at positions 7, 10, and 11 from the 5' end. In some other embodiments, the sense strand contains 2'-fluoronucleotides at positions 7, 9, 10, and 11 from the 5' end. In some embodiments, the sense strand contains 2'-fluoronucleotides 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 contains 2'-fluoronucleotides 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 contains blocks of two, three, or four 2'-fluoronucleotides.

[0413] In some embodiments, the sense strand does not contain a 2'-fluoronucleotide in the position opposite or complementary to the thermal destabilization modification of the double helix in the antisense strand.

[0414] In some embodiments, the dsRNA molecule of the present disclosure comprises a sense strand of 21 nucleotides (nt) and an antisense strand of 23 nucleotides (nt), wherein the antisense strand comprises at least one thermally destabilized nucleotide, the at least one of which occurs in the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), where one end of the dsRNA is blunt and the other end comprises a 2nt overhang, and the dsRNA optionally comprises at least one of the following features (e.g., 1, 2, 3, 4, 5, 6, or (All seven) further have: (i) the antisense contains 2, 3, 4, 5 or 6 2'-fluoro modifications; (ii) the antisense contains 1, 2, 3, 4 or 5 phosphorothioate nucleotide interbonds; (iii) the sense strand is conjugated with 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 nucleotide interbonds; (vi) the dsRNA contains at least 4 2'-fluoro modifications; (vii) the dsRNA has a blunt end at the 5' end of the antisense strand. Optionally, a 2nt overhang is at the 3' end of the antisense.

[0415] In some embodiments, a dsRNA molecule of the present disclosure comprising a sense strand and an antisense strand, wherein: the sense strand is 25–30 nucleotides long and starting from the 5' terminal nucleotide (position 1), positions 1–23 of the sense strand contain at least 8 ribonucleotides; the antisense strand is 36–66 nucleotides long and starting from the 3' terminal nucleotide, contains at least 8 ribonucleotides at positions that pair with positions 1–23 of the sense strand to form a double helix; wherein at least the 3' terminal nucleotide of the antisense strand is not paired with the sense strand, and up to 6 consecutive 3' terminal nucleotides are not paired with the sense strand, thereby forming a 3' single-stranded overhang of 1–6 nucleotides; wherein the 5' end of the antisense strand contains 10–30 consecutive nucleotides that are not paired with the sense strand The antisense strand contains an otinode, thereby forming a single-stranded 5' overhang of 10 to 30 nucleotides; where at least the 5' and 3' terminal nucleotides of the sense strand base-pair with the nucleotides of the antisense strand when the sense strand and antisense strand are aligned for maximum complementarity, thereby forming a substantially double-stranded region between the sense strand and the antisense strand, and when the double-stranded nucleic acid is introduced into a mammalian cell, the antisense strand is sufficiently complementary to the target RNA along at least 19 antisense-strand-length ribonucleotides, thereby reducing target gene expression; wherein the antisense strand contains at least one thermally destabilized nucleotide, the at least one thermally destabilized nucleotide located in the seed region of the antisense strand (i.e., at positions 2 to 9 of the 5' end of the antisense strand).For example, the thermally destabilized nucleotide is located between positions 14 and 17 at the 5' end of the sense strand, either opposite or complementary, and the dsRNA optionally further has at least one of the following features (e.g., one, two, three, four, five, six, or seven): (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 bonds; (iii) the sense strand is conjugated with 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 bonds; (vi) the dsRNA contains at least four 2'-fluoro modifications; (vii) the dsRNA contains a double-stranded region of 12 to 30 nucleotide pairs in length.

[0416] In some embodiments, the dsRNA molecule of the present disclosure comprises a sense strand and an antisense strand, wherein the dsRNA molecule comprises a sense strand having a length of at least 25 nucleotides and up to 29 nucleotides, and the antisense strand having the sense strand and having a length of up to 30 nucleotides comprises modified nucleotides that are susceptible to enzymatic degradation at positions 11 from the 5' end, the 3' end of the sense strand and the 5' end of the antisense strand form blunt ends, the antisense strand having a 3' end that is 1 to 4 nucleotides longer than the sense strand, and the double-stranded region having a length of at least 25 nucleotides is sufficiently complementary to the target mRNA along at least 19 nt of the antisense strand length to reduce target gene expression when the dsRNA molecule is introduced into mammalian cells, wherein dicer cleavage of the dsRNA preferentially results in siRNA including the 3' end of the antisense strand, thereby reducing the expression of the target gene in mammals. In this case, the antisense strand contains at least one thermally destabilized nucleotide, where at least one thermally destabilized nucleotide is located in the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), and the dsRNA optionally further has at least one of the following features (e.g., one, two, three, four, five, six, or all seven): (i) the antisense strand contains two, three, four, five, or six 2'-fluoromodifications; (ii) the antisense strand contains (iii) the sense strand contains 1, 2, 3, 4, or 5 phosphorothioate nucleotide interbonds; (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 nucleotide interbonds; (vi) the dsRNA contains at least 4 2'-fluoro modifications; and (vii) the dsRNA has a double-stranded region of 12 to 29 nucleotide pairs in length.

[0417] In some embodiments, all nucleotides in the sense and antisense strands of a dsRNA molecule may be modified. Each nucleotide may be modified by the same or different modifications, which may include: one or both of the unbound phosphate oxygens or one or more of the bound phosphate oxygens; changes in the components of the ribose sugar, e.g., changes in the 2' hydroxyl on the ribose sugar; large-scale exchange of the phosphate moiety by a "dephospho" linker; modifications or substitutions of naturally occurring bases; and substitutions or modifications of the ribose-phosphate backbone.

[0418] Since nucleic acids are polymers of subunits, most modifications occur at repeating positions within the nucleic acid, such as modifications of bases or phosphate groups, or at unbound oxygen atoms of phosphate groups. In some cases, modifications occur at all target positions in the nucleic acid, but often this does not happen. For example, modifications may occur only at the 3' or 5' end, in terminal regions, such as on terminal nucleotides, or only at the last 2, 3, 4, 5, or 10 nucleotides of the strand. Modifications can occur in double-stranded regions, single-stranded regions, or both. Modifications may occur only in double-stranded regions of RNA, or only in single-stranded regions of RNA. For example, phosphorothioate modifications at unbound oxygen atoms may occur only at one or both ends, in terminal regions, such as on terminal nucleotides, or only at the last 2, 3, 4, 5, or 10 nucleotides of the strand, or in both double-stranded and single-stranded regions, especially at the ends. One or more 5' ends can be phosphorylated.

[0419] In single-stranded overhangs, such as 5' or 3' overhangs, or both, it may be possible to, for example, enhance stability, include specific bases in the overhang, or include modified nucleotides or nucleotide substitutes. For example, it may be desirable to include purine nucleotides in the overhang. In some embodiments, all or some of the bases in the 3' or 5' overhang may be modified, for example, by modifications described herein. Examples of modifications include modifications known in the art, such as the use of deoxyribonucleotides, 2'-deoxy-2'-fluoro(2'-F) or 2'-O-methyl modified in place of ribosaccharides in nucleic acid bases, and phosphate group modifications, such as the use of modifications at the 2' position of ribose sugars having phosphorothioate modifications. The overhang does not need to be homologous to the target sequence.

[0420] In some embodiments, each residue in the sense and antisense chains is independently modified with LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, or 2'-fluoro. The chains may contain two or more modifications. In some embodiments, each residue in the sense and antisense chains is independently modified with 2'-O-methyl or 2'-fluoro. It should be understood that these modifications are in addition to at least one thermal destabilization modification of the double helix present in the antisense chain.

[0421] At least two distinct modifications are typically present in the sense and antisense strands. These two modifications may include 2'-deoxy, 2'-O-methyl, or 2'-fluoro modifications, acyclic nucleotides, etc. In some embodiments, the sense and antisense strands each contain two distinct modified nucleotides selected from 2'-O-methyl or 2'-deoxy. In some embodiments, each residue in the sense and antisense strands is independently modified with 2'-O-methyl nucleotide, 2'-deoxy nucleotide, 2'-deoxy-2'-fluoro nucleotide, 2'-ON-methylacetamide (2'-O-NMA) nucleotide, 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) nucleotide, 2'-O-aminopropyl (2'-O-AP) nucleotide, or 2'-ara-F nucleotide. Again, it should be understood that these modifications are in addition to at least one thermal destabilization modification of the double helix present in the antisense strand.

[0422] In some embodiments, the dsRNA molecules of this disclosure include alternating pattern modifications, particularly in the B1, B2, B3, B1', B2', B3', and B4' regions. As used herein, the terms “alternating motif” or “alternating pattern” refer to a motif having one or more modifications, each modification occurring on alternating nucleotides on a single strand. Alternating nucleotides may refer to one modification every other nucleotide, or one modification every three nucleotides, or similar patterns. For example, if A, B, and C each represent one type of modification to a nucleotide, the alternating motif may be “ABABABABABAB…”, “AABBAABBAABB…”, “AABAABAABAAB…”, “AAABAAABAAAB…”, “AAABBBAAABBB…”, or “ABCABCABCABC…”, etc.

[0423] The types of modifications included in the alternating motif may be the same or different. For example, if A, B, C, and D each represent one type of modification on a nucleotide, the alternating pattern, i.e., the modifications on every other nucleotide, may be the same, but each of the sense strand or antisense strand can be selected from several possible modifications within the alternating motif, such as "ABABAB...", "ACACAC...", "BDBDBD...", or "CDCDCD...".

[0424] In some embodiments, the dsRNA molecules of this disclosure include a modification pattern on the sense strand, compared to a modification pattern on the antisense strand in which the alternating motif is shifted. The shift may be such that a modified group of nucleotides on the sense strand corresponds to a different modified group of nucleotides on the antisense strand, and vice versa. For example, when the sense strand is paired with the antisense strand in a dsRNA double helix, the alternating motif on the sense strand may begin with "ABABAB" from 5'-3' of the strand in the double helix region, and the alternating motif on the antisense strand may begin with "BABABA" from 3'-5' of the strand. As another example, the alternating motif on the sense strand may begin with "AABBAABB" from 5'-3' of the strand, and the alternating motif on the antisense strand may begin with "BBAABBAA" from 3'-5' of the strand in the double helix region, resulting in a complete or partial shift of the modification patterns between the sense and antisense strands.

[0425] The dsRNA molecules of this disclosure may further include at least one phosphorothioate or methylphosphonate internucleotide bond. The phosphorothioate or methylphosphonate internucleotide bond modification may occur on any nucleotide of the sense strand or antisense strand, or at any position on the strand. For example, the internucleotide bond modification may occur on all nucleotides on the sense strand or antisense strand, each internucleotide bridge modification may occur in an alternating pattern on the sense strand or antisense strand, or the sense strand or antisense strand may contain both internucleotide bond modifications in an alternating pattern. The alternating pattern of internucleotide bond modifications on the sense strand may be the same as or different from that on the antisense strand, and the alternating pattern of internucleotide bond modifications on the sense strand may have a shift relative to the alternating pattern of internucleotide bond modifications on the antisense strand.

[0426] In some embodiments, the dsRNA molecule includes phosphorothioate or methylphosphonate internucleotide bond modifications in the overhang region. For example, the overhang region includes two nucleotides having a phosphorothioate or methylphosphonate internucleotide bond between the two nucleotides. Internucleotide bond modifications may also be made to bond the overhang nucleotides to terminal pair nucleotides in the double-stranded region. For example, at least 2, 3, 4, or all of the overhang nucleotides may be bonded via phosphorothioate or methylphosphonate internucleotide bonds, and optionally there may be additional phosphorothioate or methylphosphonate internucleotide bonds that bond the overhang nucleotides to the pair nucleotides adjacent to the overhang nucleotides. For example, at least two phosphorothioate internucleotide bonds may exist between three terminal nucleotides, two of which are overhang nucleotides and the third is the pair nucleotide adjacent to the overhang nucleotide. Optionally, these three terminal nucleotides may be at the 3' end of the antisense strand.

[0427] In some embodiments, the sense strand of the dsRNA molecule comprises 1 to 10 blocks of 2 to 10 phosphorothioate or methylphosphonate internucleotide bonds separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate nucleotide internucleotide bonds, one of which is positioned at any position in the oligonucleotide sequence, and the sense strand is paired with an antisense strand containing any combination of phosphorothioate, methylphosphonate, and phosphate nucleotide internucleotide bonds, or with an antisense strand containing either phosphorothioate, methylphosphonate, or phosphate bonds.

[0428] In some embodiments, the antisense strand of a dsRNA molecule comprises two blocks of two phosphorothioate or methylphosphonate internucleotide bonds separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphate nucleotide internucleotide bonds, one of which is positioned 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 nucleotide internucleotide bonds, or with an antisense strand comprising either phosphorothioate, methylphosphonate, or phosphate bonds.

[0429] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of three phosphorothioate or methylphosphonate internucleotide bonds separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate nucleotide internucleotide bonds, one of which is positioned at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand containing any combination of phosphorothioate, methylphosphonate, and phosphate nucleotide internucleotide bonds, or with an antisense strand containing either phosphorothioate, methylphosphonate, or phosphate bonds.

[0430] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of four phosphorothioate or methylphosphonate internucleotide bonds separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 phosphate nucleotide internucleotide bonds, one of which is positioned at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand containing any combination of phosphorothioate, methylphosphonate, and phosphate nucleotide internucleotide bonds, or with an antisense strand containing either phosphorothioate, methylphosphonate, or phosphate bonds.

[0431] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of five phosphorothioate or methylphosphonate internucleotide bonds separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 phosphate nucleotide internucleotide bonds, one of which is positioned at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand containing any combination of phosphorothioate, methylphosphonate, and phosphate nucleotide internucleotide bonds, or with an antisense strand containing either phosphorothioate, methylphosphonate, or phosphate bonds.

[0432] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of six phosphorothioate or methylphosphonate internucleotide bonds separated by one, two, three, four, five, six, seven, eight, nine, or ten phosphate nucleotide internucleotide bonds, one of which is positioned 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 nucleotide internucleotide bonds, or with an antisense strand comprising either phosphorothioate, methylphosphonate, or phosphate bonds.

[0433] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of seven phosphorothioate or methylphosphonate internucleotide bonds separated by one, two, three, four, five, six, seven, or eight phosphate nucleotide internucleotide bonds, one of which is positioned 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 nucleotide internucleotide bonds, or with an antisense strand comprising either phosphorothioate, methylphosphonate, or phosphate bonds.

[0434] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of eight phosphorothioate or methylphosphonate internucleotide bonds separated by one, two, three, four, five, or six phosphate nucleotide internucleotide bonds, one of which is positioned at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand containing any combination of phosphorothioate, methylphosphonate, and phosphate nucleotide internucleotide bonds, or with an antisense strand containing either phosphorothioate, methylphosphonate, or phosphate bonds.

[0435] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of nine phosphorothioate or methylphosphonate internucleotide bonds separated by one, two, three, or four phosphate nucleotide internucleotide bonds, one of which is positioned at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand containing any combination of phosphorothioate, methylphosphonate, and phosphate nucleotide internucleotide bonds, or with an antisense strand containing either phosphorothioate, methylphosphonate, or phosphate bonds.

[0436] In some embodiments, the dsRNA molecules of this disclosure further include one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within the 1-10 position of one or more terminal positions of the sense or antisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides may be linked via phosphorothioate or methylphosphonate internucleotide linkages at one or both ends of the sense or antisense strand.

[0437] In some embodiments, the dsRNA molecules of the present disclosure further include one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 1 to 10 of the internal region of each duplex of the sense strand or antisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides are linked via phosphorothioate-methylphosphonate internucleotide linkages at positions 8 to 16 of the duplex region, counting from the 5' end of the sense strand, and the dsRNA molecules may optionally further include one or more phosphorothioate or methylphosphonate internucleotide crosslinkage modifications within positions 1 to 10 of the terminal position.

[0438] In some embodiments, the dsRNA molecule of the present disclosure further comprises 1 to 5 phosphorothioate or methylphosphonate internucleotide binding modifications (may be multiple) within positions 1 to 5 of the sense strand and 1 to 5 phosphorothioate or methylphosphonate internucleotide binding modifications (counting from the 5' end) within positions 18 to 23 of the sense strand, and 1 to 5 phosphorothioate or methylphosphonate internucleotide binding modifications (counting from the 5' end) within positions 1 and 2 and within positions 18 to 23 of the antisense strand.

[0439] In some embodiments, the dsRNA molecule of the present disclosure further comprises one phosphorothioate nucleotide linkage modification within positions 1 to 5 of the sense strand and one phosphorothioate or methylphosphonate nucleotide linkage modification within positions 18 to 23 (counting from the 5' end), and one phosphorothioate nucleotide linkage modification at positions 1 and 2 of the antisense strand and two phosphorothioate or methylphosphonate nucleotide linkage modifications within positions 18 to 23 (counting from the 5' end).

[0440] In some embodiments, the dsRNA molecule of the present disclosure further comprises two phosphorothioate nucleotide linkage modifications within positions 1 to 5 of the sense strand and one phosphorothioate nucleotide linkage modification within positions 18 to 23 (counting from the 5' end), as well as one phosphorothioate nucleotide linkage modification at positions 1 and 2 of the antisense strand and two phosphorothioate nucleotide linkage modifications within positions 18 to 23 (counting from the 5' end).

[0441] In some embodiments, the dsRNA molecule of the present disclosure further comprises two phosphorothioate nucleotide linkage modifications within positions 1 to 5 of the sense strand and two phosphorothioate nucleotide linkage modifications within positions 18 to 23 (counting from the 5' end), as well as one phosphorothioate nucleotide linkage modification at positions 1 and 2 of the antisense strand and two phosphorothioate nucleotide linkage modifications within positions 18 to 23 (counting from the 5' end).

[0442] In some embodiments, the dsRNA molecule of the present disclosure further comprises two phosphorothioate nucleotide linkage modifications within positions 1 to 5 of the sense strand and two phosphorothioate nucleotide linkage modifications within positions 18 to 23 (counting from the 5' end), as well as one phosphorothioate nucleotide linkage modification at positions 1 and 2 of the antisense strand and one phosphorothioate nucleotide linkage modification within positions 18 to 23 (counting from the 5' end).

[0443] In some embodiments, the dsRNA molecule of the present disclosure further comprises one phosphorothioate nucleotide linkage modification within positions 1 to 5 of the sense strand and one phosphorothioate nucleotide linkage modification within positions 18 to 23 (counting from the 5' end), as well as two phosphorothioate nucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate nucleotide linkage modifications within positions 18 to 23 (counting from the 5' end).

[0444] In some embodiments, the dsRNA molecule of the present disclosure further comprises one phosphorothioate nucleotide linkage modification (counting from the 5' end) within positions 1-5 and 18-23 of the sense strand, and two phosphorothioate nucleotide linkage modifications at positions 1 and 2 of the antisense strand and one phosphorothioate nucleotide linkage modification (counting from the 5' end) within positions 18-23.

[0445] In some embodiments, the dsRNA molecule of the present disclosure further comprises one phosphorothioate nucleotide linkage modification (counting from the 5' end) within positions 1 to 5 of the sense strand, and two phosphorothioate nucleotide linkage modifications (counting from the 5' end) within positions 1 and 2 of the antisense strand, and one phosphorothioate nucleotide linkage modification (counting from the 5' end) within positions 18 to 23.

[0446] In some embodiments, the dsRNA molecule of the present disclosure further comprises two phosphorothioate nucleotide linkage modifications (counting from the 5' end) within positions 1 to 5 of the sense strand, and one phosphorothioate nucleotide linkage modification at positions 1 and 2 of the antisense strand and two phosphorothioate nucleotide linkage modifications (counting from the 5' end) within positions 18 to 23.

[0447] In some embodiments, the dsRNA molecule of the present disclosure further comprises two phosphorothioate nucleotide linkage modifications (counting from the 5' end) within positions 1-5 and 18-23 of the sense strand, and two phosphorothioate nucleotide linkage modifications (counting from the 5' end) within positions 1 and 2 of the antisense strand and one phosphorothioate nucleotide linkage modification (counting from the 5' end) within positions 18-23.

[0448] In some embodiments, the dsRNA molecule of the present disclosure further comprises two phosphorothioate nucleotide linkage modifications within positions 1 to 5 of the sense strand and one phosphorothioate nucleotide linkage modification within positions 18 to 23 (counting from the 5' end), as well as two phosphorothioate nucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate nucleotide linkage modifications within positions 18 to 23 (counting from the 5' end).

[0449] In some embodiments, the dsRNA molecule of the present disclosure further comprises two phosphorothioate nucleotide linkage modifications within positions 1 to 5 of the sense strand and one phosphorothioate nucleotide linkage modification within positions 18 to 23 (counting from the 5' end), as well as one phosphorothioate nucleotide linkage modification at positions 1 and 2 of the antisense strand and two phosphorothioate nucleotide linkage modifications within positions 18 to 23 (counting from the 5' end).

[0450] In some embodiments, the dsRNA molecule of the present disclosure further comprises two phosphorothioate nucleotide linkage modifications at positions 1 and 2 of the sense strand, and two phosphorothioate nucleotide linkage modifications at positions 20 and 21 (counting from the 5' end), and one phosphorothioate nucleotide linkage modification at positions 1 and 21 of the antisense strand (counting from the 5' end).

[0451] In some embodiments, the dsRNA molecule of the present disclosure further comprises one phosphorothioate nucleotide linkage modification at position 1 and one phosphorothioate nucleotide linkage modification at position 21 (counting from the 5' end) of the sense strand, and two phosphorothioate nucleotide linkage modifications at positions 1 and 2 of the antisense strand, as well as two phosphorothioate nucleotide linkage modifications within positions 20 and 21 (counting from the 5' end).

[0452] In some embodiments, the dsRNA molecule of the present disclosure further comprises two phosphorothioate nucleotide linkage modifications at positions 1 and 2 of the sense strand and two phosphorothioate nucleotide linkage modifications at positions 21 and 22 (counting from the 5' end), and one phosphorothioate nucleotide linkage modification at position 1 of the antisense strand and one phosphorothioate nucleotide linkage modification at position 21 (counting from the 5' end).

[0453] In some embodiments, the dsRNA molecule of the present disclosure further comprises one phosphorothioate nucleotide linkage modification at position 1 and one phosphorothioate nucleotide linkage modification at position 21 (counting from the 5' end) of the sense strand, and two phosphorothioate nucleotide linkage modifications at positions 1 and 2 of the antisense strand, and two phosphorothioate nucleotide linkage modifications at positions 21 and 22 (counting from the 5' end).

[0454] In some embodiments, the dsRNA molecule of the present disclosure further comprises two phosphorothioate nucleotide linkage modifications at positions 1 and 2 of the sense strand and two phosphorothioate nucleotide linkage modifications at positions 22 and 23 (counting from the 5' end), as well as one phosphorothioate nucleotide linkage modification at position 1 of the antisense strand and one phosphorothioate nucleotide linkage modification at position 21 (counting from the 5' end).

[0455] In some embodiments, the dsRNA molecule of the present disclosure further comprises one phosphorothioate nucleotide linkage modification at position 1 and one phosphorothioate nucleotide linkage modification at position 21 (counting from the 5' end), as well as two phosphorothioate nucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate nucleotide linkage modifications at positions 23 and 23 (counting from the 5' end).

[0456] In some embodiments, the compounds of the present disclosure include a pattern of skeletal chiral centers. In some embodiments, a common pattern of skeletal chiral centers includes at least five nucleotide internucleotide bonds in an Sp configuration. In some embodiments, a common pattern of skeletal chiral centers includes at least six nucleotide internucleotide bonds in an Sp configuration. In some embodiments, a common pattern of skeletal chiral centers includes at least seven nucleotide internucleotide bonds in an Sp configuration. In some embodiments, a common pattern of skeletal chiral centers includes at least eight nucleotide internucleotide bonds in an Sp configuration. In some embodiments, a common pattern of skeletal chiral centers includes at least nine nucleotide internucleotide bonds in an Sp configuration. In some embodiments, a common pattern of skeletal chiral centers includes at least ten nucleotide internucleotide bonds in an Sp configuration. In some embodiments, a common pattern of skeletal chiral centers includes at least eleven nucleotide internucleotide bonds in an Sp configuration. In some embodiments, a common pattern of skeletal chiral centers includes at least twelve nucleotide internucleotide bonds in an Sp configuration. In some embodiments, a common pattern of skeletal chiral centers includes at least thirteen nucleotide internucleotide bonds in an Sp configuration. In some embodiments, a common pattern of the skeletal chiral center includes at least 14 nucleotide internucleotide bonds in the Sp configuration. In some embodiments, a common pattern of the skeletal chiral center includes at least 15 nucleotide internucleotide bonds in the Sp configuration. In some embodiments, a common pattern of the skeletal chiral center includes at least 16 nucleotide internucleotide bonds in the Sp configuration. In some embodiments, a common pattern of the skeletal chiral center includes at least 17 nucleotide internucleotide bonds in the Sp configuration. In some embodiments, a common pattern of the skeletal chiral center includes at least 18 nucleotide internucleotide bonds in the Sp configuration. In some embodiments, a common pattern of the skeletal chiral center includes at least 19 nucleotide internucleotide bonds in the Sp configuration. In some embodiments, a common pattern of the skeletal chiral center includes 8 or fewer nucleotide internucleotide bonds in the Rp configuration. In some embodiments, a common pattern of the skeletal chiral center includes 7 or fewer nucleotide internucleotide bonds in the Rp configuration.In some embodiments, a common pattern of the skeletal chiral center includes six or fewer nucleotide internucleotide bonds in the Rp configuration. In some embodiments, a common pattern of the skeletal chiral center includes five or fewer nucleotide internucleotide bonds in the Rp configuration. In some embodiments, a common pattern of the skeletal chiral center includes four or fewer nucleotide internucleotide bonds in the Rp configuration. In some embodiments, a common pattern of the skeletal chiral center includes three or fewer nucleotide internucleotide bonds in the Rp configuration. In some embodiments, a common pattern of the skeletal chiral center includes two or fewer nucleotide internucleotide bonds in the Rp configuration. In some embodiments, a common pattern of the skeletal chiral center includes one or fewer nucleotide internucleotide bonds in the Rp configuration. In some embodiments, a common pattern of the skeletal chiral center includes eight or fewer non-chiral nucleotide internucleotide bonds (phosphodiesters as an unrestricted example). In some embodiments, a common pattern of the skeletal chiral center includes seven or fewer non-chiral nucleotide internucleotide bonds. In some embodiments, a common pattern of the skeletal chiral center includes six or fewer non-chiral nucleotide internucleotide bonds. In some embodiments, a common pattern of the skeletal chiral center includes five or fewer non-chiral nucleotide internucleotide bonds. In some embodiments, a common pattern of the skeletal chiral center includes four or fewer non-chiral nucleotide bonds. In some embodiments, a common pattern of the skeletal chiral center includes three or fewer non-chiral nucleotide bonds. In some embodiments, a common pattern of the skeletal chiral center includes two or fewer non-chiral nucleotide bonds. In some embodiments, a common pattern of the skeletal chiral center includes one or fewer non-chiral nucleotide bonds. In some embodiments, a common pattern of the skeletal chiral center includes at least ten nucleotide bonds in an Sp configuration and eight or fewer non-chiral nucleotide bonds. In some embodiments, a common pattern of the skeletal chiral center includes at least eleven nucleotide bonds in an Sp configuration and seven or fewer non-chiral nucleotide bonds. In some embodiments, a common pattern of the skeletal chiral center includes at least twelve nucleotide bonds in an Sp configuration and six or fewer non-chiral nucleotide bonds.In some embodiments, a common pattern of the skeletal chiral center includes at least 13 nucleotide internucleotide bonds in Sp configuration and 6 or fewer non-chiral nucleotide internucleotide bonds. In some embodiments, a common pattern of the skeletal chiral center includes at least 14 nucleotide internucleotide bonds in Sp configuration and 5 or fewer non-chiral nucleotide internucleotide bonds. In some embodiments, a common pattern of the skeletal chiral center includes at least 15 nucleotide internucleotide bonds in Sp configuration and 4 or fewer non-chiral nucleotide internucleotide bonds. In some embodiments, the Sp-configured nucleotide internucleotide bonds may be continuous or non-contiguous. In some embodiments, the Rp-configured nucleotide internucleotide bonds may be continuous or non-contiguous. In some embodiments, the non-chiral nucleotide internucleotide bonds may be continuous or non-contiguous.

[0457] In some embodiments, the compounds of the present disclosure comprise blocks, which are stereochemical blocks. In some embodiments, a block is an Rp block in that each internucleotide bond in the block is Rp. In some embodiments, a 5'-block is an Rp block. In some embodiments, a 3'-block is an Rp block. In some embodiments, a block is an Sp block in that each internucleotide bond in the block is Sp. In some embodiments, a 5'-block is an Sp block. In some embodiments, a 3'-block is an Sp block. In some embodiments, the oligonucleotides provided comprise both Rp blocks and Sp blocks. In some embodiments, the oligonucleotides provided comprise one or more Rp but no Sp blocks. In some embodiments, the oligonucleotides provided comprise one or more Sp but no Rp blocks. In some embodiments, the oligonucleotides provided comprise one or more PO blocks in which each internucleotide bond is in a native phosphate crosslink.

[0458] In some embodiments, the compounds of the present disclosure include a 5'-block in which each sugar moiety is an Sp block containing a 2'-F modification. In some embodiments, the 5'-block is an Sp block in which each nucleotide bond is a modified nucleotide bond and each sugar moiety is an Sp block containing a 2'-F modification. In some embodiments, the 5'-block is an Sp block in which each nucleotide bond is a phosphorothioate crosslink and each sugar moiety is an Sp block containing a 2'-F modification. In some embodiments, the 5'-block contains four or more nucleoside units. In some embodiments, the 5'-block contains five or more nucleoside units. In some embodiments, the 5'-block contains six or more nucleoside units. In some embodiments, the 5'-block contains seven or more nucleoside units. In some embodiments, the 3'-block is an Sp block in which each sugar moiety is an Sp block containing a 2'-F modification. In some embodiments, the 3'-block is an Sp block in which each nucleotide bond is a modified nucleotide bond and each sugar moiety is an Sp block containing a 2'-F modification. In some embodiments, the 3'-block is an Sp-block in which each nucleotide bond is a phosphorothioate bridge and each sugar moiety contains a 2'-F modification. In some embodiments, the 3'-block contains four or more nucleoside units. In some embodiments, the 3'-block contains five or more nucleoside units. In some embodiments, the 3'-block contains six or more nucleoside units. In some embodiments, the 3'-block contains seven or more nucleoside units.

[0459] In some embodiments, the compounds of the Disclosure contain a region of a certain type of nucleoside, or an oligonucleotide followed by a specific type of internucleotide bond, such as a natural phosphate crosslink, a modified internucleotide bond, an Rp chiral internucleotide bond, an Sp chiral internucleotide bond, 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 crosslink (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 crosslink (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 crosslink (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 crosslink (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 natural phosphate crosslinks (PO). In some embodiments, A and G are followed by Sp. In some embodiments, A and G are followed by Rp.

[0460] In some embodiments, the antisense strand includes phosphorothioate internucleotide bonds between nucleotides 21 and 22, and between nucleotides 22 and 23; the antisense strand includes at least one double-stranded thermal destabilization modification located in the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand); and the dsRNA optionally features: (i) the antisense strand includes 2, 3, 4, 5, or 6 2'-fluoro modifications; (ii) the antisense strand includes 3, 4, or 5 phosphorothioate internucleotide bonds; (iii) the sense strand is conjugated with 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 bonds; (vi) the dsRNA contains at least 4 2'-fluoro modifications; (vii) the dsRNA contains a double-stranded region 12 to 40 nucleotide pairs long; (viii) the dsRNA has a blunt end at the 5' end of the antisense strand; further comprising at least one of the above (e.g., 1, 2, 3, 4, 5, 6, 7 or all 8).

[0461] In some embodiments, the antisense strand includes phosphorothioate internucleotide bonds 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 includes at least one double-stranded thermal destabilization modification located in the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), and the dsRNA optionally has the following characteristics: (i) the antisense strand includes 2, 3, 4, 5, or 6 2'-fluoro modifications; (ii) the sense strand is conjugated with a ligand. (iii) the sense strand contains 2, 3, 4 or 5 2'-fluoro modifications; (iv) the sense strand contains 1, 2, 3, 4 or 5 phosphorothioate internucleotide bonds; (v) the dsRNA contains at least 4 2'-fluoro modifications; (vi) the dsRNA contains a double-stranded region of 12 to 40 nucleotide pairs length; (vii) the dsRNA contains a double-stranded region of 12 to 40 nucleotide pairs length; (viii) the dsRNA has a blunt end at the 5' end of the antisense strand; further comprising at least one of the above (e.g., 1, 2, 3, 4, 5, 6, 7 or all 8).

[0462] In some embodiments, the sense strand includes phosphorothioate internucleotide bonds between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3; the antisense strand includes at least one double-stranded thermal destabilization modification located in the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand); and the dsRNA optionally features: (i) the antisense strand includes 2, 3, 4, 5, or 6 2'-fluoro modifications; (ii) the antisense strand includes 1, 2, 3, 4, or 5 phosphorothioate internucleotide bonds; (iii) the sense strand is conjugated with a ligand; (iv) the sense strand contains two, three, four or five 2'-fluoro modifications; (v) the sense strand contains three, four or five phosphorothioate internucleotide bonds; (vi) the dsRNA contains at least four 2'-fluoro modifications; (vii) the dsRNA contains a double-stranded region of 12 to 40 nucleotide pairs length; (viii) the dsRNA has a blunt end at the 5' end of the antisense strand; further comprising at least one (e.g., one, two, three, four, five, six, seven, or all eight).

[0463] In some embodiments, the sense strand includes phosphorothioate internucleotide bonds between nucleotides 1 and 2, and between nucleotides 2 and 3; the antisense strand includes phosphorothioate internucleotide bonds between nucleotides 1 and 2, between nucleotides 2 and 3, between nucleotides 21 and 22, and between nucleotides 22 and 23; the antisense strand includes at least one double-stranded thermal destabilization modification located in the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand); and the dsRNA optionally has the following features: (i) antisense (ii) the sense strand contains 2, 3, 4, 5, or 6 2'-fluoro modifications; (iii) the sense strand contains 2, 3, 4, or 5 2'-fluoro modifications; (iv) the sense strand contains 3, 4, or 5 phosphorothioate internucleotide bonds; (v) the dsRNA contains at least 4 2'-fluoro modifications; (vi) the dsRNA contains a double-stranded region 12 to 40 nucleotide pairs long; (vii) the dsRNA has a blunt end at the 5' end of the antisense strand; and further comprises at least one of (e.g., 1, 2, 3, 4, 5, 6, or all 7) of the above.

[0464] In some embodiments, the dsRNA molecules of this disclosure contain one or more mismatches with targets, within the double helix, or combinations thereof. Mismatches may occur in overhang regions or duplex regions. Base pairs can be ranked based on their tendency to promote dissociation or dissolution (for example, by the free energy of association or dissociation of a particular pairing; the simplest approach is to examine each pair individually, but the following adjacent or similar analyses can also be used). From the viewpoint 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-standard pairings or non-standard pairings (as described elsewhere in this specification), are preferred over standard (A:T, A:U, G:C) pairings, and pairings containing a universal base are preferred over standard pairings.

[0465] In some embodiments, the dsRNA molecule of the present disclosure comprises at least one of the first 1, 2, 3, 4, or 5 base pairs in the double-stranded region from the 5' end of the antisense strand, and can be independently selected from the group of A:U, G:U, I:C, and mismatch pairs, such as non-standard or non-standard pairs or pairs containing universal bases, to facilitate the dissociation of the antisense strand at the 5' end of the double helix.

[0466] 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.

[0467] It has been found that introducing a 4'-modified or 5'-modified nucleotide to the 3' end of a phosphodiester (PO), phosphorothioate (PS), or phosphorodithioate (PS2) bond of a dinucleotide at any position on a single-stranded or double-stranded oligonucleotide exerts a steric effect on the nucleotide bond, thereby protecting or stabilizing it from nucleases.

[0468] In some embodiments, the 5'-modified nucleoside is introduced at the 3' end of a dinucleotide at any position in a single-stranded or double-stranded siRNA. For example, a 5'-alkylated nucleoside can be introduced at the 3' end of a dinucleotide at any position in a single-stranded or double-stranded siRNA. The alkyl group at the 5' position of the ribose sugar can be racemic or have pure R or S isomers. An exemplary 5'-alkylated nucleoside is the 5'-methyl nucleoside. The 5'-methyl can be either racemic or have pure R or S isomers.

[0469] In some embodiments, the 4'-modified nucleoside is introduced at the 3' end of a dinucleotide at any position in a single-stranded or double-stranded siRNA. For example, a 4'-alkylated nucleoside can be introduced at the 3' end of a dinucleotide at any position in a single-stranded or double-stranded siRNA. The alkyl group at the 4' position of the ribose sugar can be racemic or have pure R or S isomers. An exemplary 4'-alkylated nucleoside is the 4'-methyl nucleoside. The 4'-methyl group can be racemic or have pure R or S isomers. Alternatively, a 4'-O-alkylated nucleoside can be introduced at the 3' end of a dinucleotide at any position in a single-stranded or double-stranded siRNA. The 4'-O-alkyl group of the ribose sugar can be racemic or have pure R or S isomers. An exemplary 4'-O-alkylated nucleoside is the 4'-O-methyl nucleoside. The 4'-O-methyl nucleoside may be either racemic or have pure R or S isomers of chirality.

[0470] In some embodiments, the 5'-alkylated nucleoside is introduced at any position on the sense or antisense strand of the dsRNA, and such modification maintains or improves the potency of the dsRNA. The 5'-alkyl can be either racemic or have pure R or S isomers. An exemplary 5'-alkylated nucleoside is the 5'-methyl nucleoside. The 5'-methyl can be either racemic or have pure R or S isomers.

[0471] In some embodiments, the 4'-alkylated nucleoside is introduced at any position on the sense or antisense strand of the dsRNA, and such modification maintains or improves the potency of the dsRNA. The 4'-alkyl can be either racemic or have pure R or S isomers. An exemplary 4'-alkylated nucleoside is the 4'-methyl nucleoside. The 4'-methyl can be either racemic or have pure R or S isomers.

[0472] In some embodiments, the 4'-O-alkylated nucleoside is introduced at any position on the sense or antisense strand of the dsRNA, and such modification maintains or improves the potency of the dsRNA. The 5'-alkyl can be either racemic or have pure R or S isomers of chirality. An exemplary 4'-O-alkylated nucleoside is the 4'-O-methyl nucleoside. The 4'-O-methyl can be either racemic or have pure R or S isomers of chirality.

[0473] In some embodiments, the dsRNA molecules of this disclosure may include 2'-5' linkages (by 2'-H, 2'-OH, and 2'-OMe, and by P=O or P=S). For example, the 2'-5' linkage modification can be used to promote nuclease resistance, to inhibit sense binding to the antisense strand, or to avoid sense strand activation by RISC at the 5' end of the sense strand.

[0474] In another embodiment, the dsRNA molecule of this disclosure may contain L-sugars (e.g., L-ribose, L-arabinose having 2'-H, 2'-OH, and 2'-OMe). For example, these L-sugar modifications may be used to promote nuclease resistance, to inhibit sense binding to the antisense strand, or to avoid sense strand activation by RISC at the 5' end of the sense strand.

[0475] Various publications describe multimeric siRNAs that can be used with the dsRNAs of this disclosure. Such publications include International Publication No. 2007 / 091269, U.S. 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 by reference.

[0476] As described in more detail below, RNAi agents containing conjugations of one or more carbohydrate moieties can optimize one or more properties of the RNAi agent. Often, the carbohydrate moiety is bound to a modified subunit of the RNAi agent. For example, the ribose sugar of one or more ribonucleotide subunits of a dsRNA agent may be replaced by another moiety, such as a non-carbohydrate (and possibly cyclic) carrier to which a carbohydrate ligand is bound. Ribonucleotide subunits in which the ribose sugar of a subunit is thus substituted are referred to herein as ribose-substituted modified subunits (RRMS). The cyclic carrier may be a carbocyclic system, i.e., all ring atoms are carbon atoms, or it may be a heterocyclic system, i.e., one or more ring atoms may be heteroatoms, such as nitrogen, oxygen, and sulfur. The cyclic carrier may be a monocyclic system, or it may contain two or more rings, such as a fused ring. The cyclic carrier may be a fully saturated cyclic system, or it may contain one or more double bonds.

[0477] Ligands can be bound to polynucleotides via a carrier. The carrier comprises (i) at least one “skeletal linkage site,” optionally two “skeletal linkage sites,” and (ii) at least one “tethering linkage site.” As used herein, “skeletal linkage site” refers to a functional group such as a hydroxyl group, or generally a linkage that is available for and suitable for incorporating the carrier into the ribonucleic acid skeleton, such as a phosphate or modified phosphate, such as a sulfur-containing skeleton. In some embodiments, a “tethering linkage site” (TAP) refers to a constituent ring atom of the cyclic carrier that connects a selected moiety, such as a carbon atom or heteroatom (different from the atom providing the skeletal linkage site). The moiety may be, for example, a carbohydrate, such as a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide. Optionally, the selected moiety is linked to the cyclic carrier by an intervening tether. Thus, the cyclic carrier often contains, or generally provides, another chemical entity, such as a functional group suitable for incorporating a ligand into a constituent ring or for tethering, such as an amino group.

[0478] RNAi agents can be conjugated to ligands via a carrier, which may be a cyclic or acyclic group. Optionally, cyclic groups are selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridadinyl, tetrahydrofuryl, and decalin. Optionally, acyclic groups are selected from selinol skeletons and diethanolamine skeletons.

[0479] 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, Table 3, Table 12, or Table 13. These agents may further comprise ligands, for example, one or more lipophilic moieties, one or more GalNAc derivatives, or both one or more lipophilic moieties and one or more GalNAc derivatives.

[0480] III. iRNA conjugated to a ligand Another modification of the iRNA of this disclosure includes chemically linking one or more ligands, moieties, or conjugates to the iRNA that enhance the activity, cell distribution, or, for example, cellular uptake into the cell.Such parts include lipid portions, for example, cholesterol portions (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, for example, beryl-S-tritylthiol (Manoharan et al., Ann. NYAcad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), and thiocholesterol (Oberhauser et al., Nucl. Acids Res.,1992,20:533-538), aliphatic chains, e.g., 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, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al.,Tetrahedron Lett.,1995,36:3651-3654; Shea et al.,Nucl.Acids This includes, but is not limited to, Res., 1990, 18:3777-3783, polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), adamantane acetate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), palmityl moieties (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or octadecylamine or hexylamino-carbonyloxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0481] In some embodiments, ligands alter the distribution, targeting, or lifespan of the iRNA agent into which they are incorporated. In some embodiments, ligands provide enhanced affinity to selected targets, such as molecules, cells, or cell types, compartments, such as cell or organ compartments, tissues, organs, or body regions, compared to species lacking such ligands. Typical ligands would not be involved in double-strand pair formation in double-stranded nucleic acids.

[0482] Ligands are naturally occurring substances, such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin), carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid); or lipids. Ligands may also be synthetic polymers, such as recombinant or synthetic molecules including 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-coglycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphatidine. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptide-mimicking polyamines, dendrimer polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or α-helical peptides.

[0483] The ligand may also include a targeting group that binds to a specific cell type, such as kidney cells, for example, a cell or tissue targeting agent, such as a lectin, glycoprotein, lipid or protein, or an antibody. The targeting group may be tyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, polyhydric lactose, polyhydric galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyhydric mannose, polyhydric fucose, glycosylated polyamino acids, polyhydric galactose, transferrin, bisphosphonate, polyglutamic acid, polyaspartic acid, lipid, cholesterol, steroid, bile acid, folic acid, vitamin B12, biotin, or RGD peptide or RGD peptide mimetic. In one embodiment, the ligand is polyhydric galactose, for example, N-acetyl-galactosamine.

[0484] Other examples of ligands include dyes, intercalating agents (e.g., acridine), crosslinking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphylline, saffrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules (e.g., cholesterol, cholic acid, adamantane acetate, 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)lithocholic acid, O3-(o Examples include leoyl (cholenic acid, dimethoxytrityl or phenoxazine) and peptide conjugates (e.g., Antennapedia peptide, Tat peptide), alkylating agents, phosphates, amino acids, mercaptos, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino acids, alkyls, substituted alkyls, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

[0485] Ligands can be proteins, such as glycoproteins, or peptides, such as molecules with specific affinity for colligands, or antibodies, such as antibodies that bind to specific cell types, such as cancer cells, endothelial cells, or osteocytes. Ligands can also include hormones and hormone receptors. They can also include lipids, lectins, carbohydrates, vitamins, cofactors, and non-peptide species such as polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, or polyvalent fucose. Ligands can be, for example, lipopolysaccharides, p38MAP kinase activators, or NF-κB activators.

[0486] A ligand can be a substance, such as a drug, that can increase the uptake of an iRNA agent into a cell, for example, by disrupting the cytoskeleton of a cell, for example, by disrupting the microtubules, microfilaments, or intermediate filaments of a cell. Drugs may be, for example, taxone, vincristine, vinblastine, cytochalasin, nocodazole, japlaquinolide, latruncrine A, phalloidin, swinolide A, indanosine, or myoserbine.

[0487] In some embodiments, the ligands bound to iRNAs described herein act as pharmacokinetic modulators (PK modulators). PK modulators include lipophilic substances, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEGs, 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, and biotin. Since oligonucleotides containing multiple phosphorothioate bonds are also known to bind to serum proteins, short oligonucleotides, such as oligonucleotides of about 5, 10, 15, or 20 bases with multiple phosphorothioate bonds in their backbone, are also suitable for use as ligands (e.g., as PK-modulating ligands) in this disclosure. In addition, aptamers that bind to serum components (e.g., serum proteins) are also suitable for use as PK-modulating ligands in embodiments described herein.

[0488] iRNAs conjugated to the ligands of this disclosure may be synthesized using oligonucleotides having pendant-reactive functional groups (e.g., those derived from the attachment of linking molecules to the oligonucleotide (as described below)). These reactive oligonucleotides can be reacted directly with commercially available ligands, synthetic ligands having any of the various protecting groups, or ligands having a linking portion bound thereto.

[0489] The oligonucleotides used in the conjugates of this disclosure can be conveniently and routinely prepared by well-known solid-phase synthesis techniques. Apparatus for such synthesis is available from several distributors, including, for example, Applied Biosystems® (Foster City, Calif.). Additionally or alternatively, any other means known in the art for such synthesis can be used. Similar techniques are also known to be used to prepare other oligonucleotides, such as phosphorothioates and alkylated derivatives.

[0490] In the ligand-binding oligonucleotides and ligand molecule-supported sequence-specific ligated nucleosides of the present disclosure, the oligonucleotide(s) may be constructed in a suitable DNA synthesizer using a standard nucleotide or nucleoside precursor, a nucleotide or nucleoside conjugate precursor already supporting a ligation portion, a ligand-nucleotide or nucleoside-conjugate precursor already supporting a ligand molecule, or a construction block supporting a non-nucleoside ligand.

[0491] When using nucleotide-conjugate precursors that already have a linking portion, the synthesis of sequence-specifically linked nucleosides is usually completed, and then a ligand molecule is reacted with the linking portion to form a ligand-conjugate oligonucleotide. In some embodiments, the oligonucleotides or linked nucl...

Claims

1. A double-stranded ribonucleic acid (dsRNA) agent or a salt thereof for inhibiting SNCA expression, The dsRNA agent or a salt thereof comprises a sense strand and an antisense strand that form a double-stranded region. Each chain is independently 19 to 25 nucleotides long. The sense strand comprises at least 19 consecutive nucleotides of the nucleotide sequence 5'-GAGCAAGUGACAAAAUGUGGA-3' of SEQ ID NO: 2910, and the antisense strand comprises at least 19 consecutive nucleotides of the nucleotide sequence 5'-UCCAACAUUUGTCCACUUGCUCUU-3' of SEQ ID NO: 3265. All nucleotides of the sense strand and all nucleotides of the antisense strand include nucleotide modifications selected from the group consisting of 2'-O-methyl nucleotide modifications, 2'-fluoro nucleotide modifications, and deoxy nucleotide modifications. The dsRNA agent or a salt thereof further comprises 6 to 8 phosphorothioate nucleotide interbonds, One or more lipophilic moieties comprising one or more saturated or unsaturated C6-C18 hydrocarbon chains are conjugated to one or more internal positions selected from the group consisting of positions 4-8 and 13-18 on the sense chain, counting from the 5' end of the sense chain. Double-stranded ribonucleic acid (dsRNA) preparations or salts thereof.

2. The double-stranded ribonucleic acid (dsRNA) agent or a salt thereof according to Claim 1, wherein the sense strand comprises the nucleotide sequence 5'-GAGCAAGUGACAAAAUGUGGA-3' of SEQ ID NO: 2910, and the antisense strand comprises the nucleotide sequence 5'-UCCAACAUUUGTCCACUUGCUCUU-3' of SEQ ID NO: 3265.

3. The dsRNA agent or a salt thereof according to claim 1 or 2, wherein the lipophilic portion is conjugated via a linker or carrier.

4. A dsRNA agent or a salt thereof according to any one of claims 1 to 3, wherein each chain is independently 21 to 23 nucleotides long.

5. The dsRNA agent or a salt thereof according to claim 4, wherein the sense strand is 21 nucleotides long.

6. The dsRNA agent or a salt thereof according to claim 4, wherein the antisense strand is 23 nucleotides long.

7. A dsRNA agent or a salt thereof according to any one of claims 1 to 6, wherein at least one strand comprises a 3' overhang of at least one nucleotide, or at least one strand comprises a 3' overhang of at least two nucleotides.

8. The dsRNA agent or a salt thereof according to any one of claims 1 to 7, wherein the double-stranded region has a length of 19 to 25 nucleotide pairs.

9. The dsRNA agent or a salt thereof according to any one of claims 1 to 8, wherein the one or more lipophilic portions are conjugated to one or more internal positions selected from the group consisting of positions 5, 6, 7, 15, and 17 on the sense strand, counting from the 5' end of the sense strand.

10. The dsRNA agent or a salt thereof according to any one of claims 1 to 9, wherein the lipophilic portion contains a functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.

11. The aforementioned lipophilic portion is saturated or unsaturated C １６ A dsRNA agent or a salt thereof according to any one of claims 1 to 10, comprising a hydrocarbon chain.

12. The aforementioned saturated or unsaturated C １６ The dsRNA agent or a salt thereof according to claim 11, wherein a hydrocarbon chain is conjugated at the 6th position, counting from the 5' end of the chain.

13. The dsRNA agent or a salt thereof according to any one of claims 1 to 12, wherein the lipophilic portion is conjugated via a carrier that substitutes one or more nucleotides at the one or more internal positions or the double-stranded region.

14. The dsRNA agent or a salt thereof according to any one of claims 1 to 12, wherein the lipophilic portion is conjugated to a nucleic acid base, a sugar portion, or an internucleoside bond.

15. The dsRNA agent or a salt thereof according to any one of claims 1 to 14, further comprising a phosphate or phosphate mimetic at the 5' end of the antisense strand.

16. The dsRNA agent or a salt thereof according to claim 15, wherein the phosphate mimetic is 5'-vinylphosphonate (VP).

17. Cells containing the dsRNA agent or a salt thereof according to any one of claims 1 to 16.

18. A pharmaceutical composition for inhibiting the expression of a gene encoding SNCA, comprising a dsRNA agent or a salt thereof according to any one of claims 1 to 16.

19. An in vitro method for suppressing the expression of the SNCA gene in cells, (a) Contacting the cells with the dsRNA agent according to any one of claims 1 to 16 or the pharmaceutical composition according to claim 18, (b) Maintain the cells obtained 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. An in vitro method for suppressing the expression of the SNCA gene in cells, including the method described above.

20. A pharmaceutical composition for treating a subject diagnosed with SNCA-associated neurodegenerative disease, comprising a therapeutically effective amount of a dsRNA agent or a salt thereof according to any one of claims 1 to 16.

21. The aforementioned SNCA-related disorders include: tremors, bradykinesia (slow movement), muscle rigidity, postural and balance disorders, loss of automatic movement, speech changes, writing changes, visual, auditory, olfactory or tactile hallucinations, dizziness, falls and bowel problems, cognitive problems such as confusion, decreased attention, visuospatial problems and memory loss, sleep difficulties such as rapid eye movement (REM) sleep behavior disorder (dreams being physically acted out during sleep), variability in attention including episodes of sleepiness, prolonged staring into space, long periods of daytime sleepiness or disorganized speech, depression, and emotional blunting, and orthostatic hypotension (human The pharmaceutical composition according to claim 20, characterized by one or more symptoms selected from the group consisting of: a sudden drop in blood pressure upon standing, causing dizziness or lightheadedness, requiring the person to sit, squat, or lie down to prevent loss of consciousness; clumsiness or incoordination; bladder control problems; contracture of the hands or limbs (chronic shortening of the muscles or tendons around a joint that prevents the joint from moving freely); Pisa syndrome (an abnormal posture in which the body appears to be leaning to one side); anterior cervix (a forward bend of the neck and a downward drooping head); and involuntary, uncontrolled sighing or gasping.

22. The pharmaceutical composition according to claim 20, wherein the SNCA-related neurodegenerative disease is selected from the group consisting of PD, multiple system atrophy, Lewy body dementia (LBD), pure autonomic neuropathy (PAF), Pick's disease, progressive supranuclear palsy, boxer's dementia, chromosome 17-related parkinsonism, Ritico Bodig disease, neurofibrillary tangle-type senile dementia, argyrophilic granuloma, ganglioglioma, gangliocytoma, meningeal hemangioma, subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis, Haller-Vorden-Spatz disease, lipofuscinosis, corticobasal degeneration, frontotemporal dementia, frontotemporal lobar degeneration, Alzheimer's disease, Huntington's disease, Down syndrome, psychosis, schizophrenia, and synuclein diseases such as Creutzfeldt-Jakob disease.

23. The pharmaceutical composition according to any one of claims 20 to 22, wherein the subject is a human.

24. The pharmaceutical composition according to any one of claims 20 to 23, wherein the pharmaceutical composition is intended to be administered intrathecally to the subject.

25. The pharmaceutical composition according to any one of claims 20 to 24, further comprising an additional agent suitable for the treatment or prevention of SNCA-related diseases or disorders.

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