RNAi reagents with modified nucleotides

Abstract

Provided herein are novel compounds and RNAi agents comprising modified nucleotides, compositions comprising such compounds or RNAi agents, and methods of using such compounds or RNAi agents.

Description

[0001] Sequence Listing

[0002] This application is filed with a sequence listing in ST.26 XML format. The sequence listing is provided as a file named "30457_WO" created on October 30, 2023, and is 1,380 kilobytes in size. The sequence listing information in ST.26 XML format is incorporated herein by reference in its entirety. Background of the Invention

[0004] RNA interference (RNAi) is a highly conserved regulatory mechanism in which sequence-specific gene silencing is achieved by double-stranded RNA molecules (dsRNA) (Fire et al., Nature 391:806-811, 1998). Physiologically, RNAi is initiated by the Dicer enzyme, which cuts long dsRNA molecules into short double-stranded siRNA fragments of about 21 to 23 nucleotides. After siRNA unwinds, the antisense strand is loaded into the RNA-induced silencing complex (RISC) and hybridizes with the complementary sequence in the target mRNA, while the sense strand is degraded (Nakanishi, Wiley Interdiscip. Rev. RNA, Vol. 7:637-660, 2016). The silencing of the target mRNA is then mediated by the catalytic component Ago2 of RISC (Bobbin and Rossi, Annu. Rev. Pharmacol. Toxicol., Vol. 56:103-122, 2016).

[0005] RNAi reagents are susceptible to nuclease degradation. One of the challenges of RNAi-based therapies is the ability to deliver intact RNAi reagents to target tissues and cells. Chemical modification and / or ligand conjugation can be used to improve the stability of RNAi reagents and to deliver them to target tissues and cells. However, some chemical modifications and / or ligand conjugation are not well tolerated and generate safety concerns in human patients (Chi et al., Drug Discov. Today. May 2017; 22(5): 823-833).

[0006] There remains a need for safe and effective RNAi agents suitable for therapeutic use, eg, for treating human disease. SUMMARY OF THE INVENTION

[0008] Provided herein are novel compounds and RNAi agents comprising modified nucleotides that have good tolerability, efficacy, and tissue distribution profiles in animal models, compositions comprising such compounds or RNAi agents, and methods of using such compounds or RNAi agents.

[0009] In one aspect, provided herein are compounds comprising any of the following formulae:

[0010]

[0011] Where n is an integer from 1 to 4,

[0012]

[0013] Where n is an integer from 0 to 2,

[0014] and

[0015] wherein B is a nucleobase selected from adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), or a derivative thereof. In some embodiments, n in Formula Ic is 1. In some embodiments, n in Formula Ic is 2. In some embodiments, n in Formula Ic is 3. In some embodiments, n in Formula Ic is 4. In some embodiments, n in Formula IV is 0. In some embodiments, n in Formula IV is 1. In some embodiments, n in Formula IV is 2.

[0016] In some embodiments, the compound comprising any of Formula Ia, Ib, Ic, II-IV, or XXI is a nucleoside, nucleotide, or an analog thereof.

[0017] In another aspect, provided herein are RNAi agents comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a duplex region, wherein the sense strand or the antisense strand comprises a modified nucleotide of any one of the following formulae:

[0018]

[0019] Where n is an integer from 1 to 4,

[0020]

[0021] Where n is an integer from 0 to 2,

[0022] and

[0023] wherein B is a nucleobase selected from adenine, cytosine, guanine, thymine, uracil, or a derivative thereof. In some embodiments, n in Formula Ic is 1. In some embodiments, n in Formula Ic is 2. In some embodiments, n in Formula Ic is 3. In some embodiments, n in Formula Ic is 4. In some embodiments, n in Formula IV is 0. In some embodiments, n in Formula IV is 1. In some embodiments, n in Formula IV is 2.

[0024] In some embodiments, the sense strand is 15 to 50 nucleotides in length. In some embodiments, the antisense strand is 15 to 30 nucleotides in length.

[0025] In some embodiments, the sense strand comprises a modified nucleotide of any of Formulas Ia, Ib, Ic, II-IV, or XXI, for example, at any one of positions 1-6 or 12-21 from the 5' end. In some embodiments, the antisense strand comprises a modified nucleotide of any of Formulas Ia, Ib, Ic, II-IV, or XXI, for example, at any one of positions 6-10 or 15-18 from the 5' end.

[0026] In some embodiments, the sense strand and the antisense strand further comprise one or more 2'-fluoro modified nucleotides and 2'-O-methyl modified nucleotides. In some embodiments, the sense strand and the antisense strand comprise one or more modified internucleotide linkages, such as phosphorothioate linkages.

[0027] In some embodiments, the antisense strand comprises a phosphate analog at the 5' end (eg, 5'-vinylphosphonate). In some embodiments, the sense strand comprises an abasic portion or an inverted abasic portion.

[0028] In some embodiments, the antisense strand is complementary to a target mRNA selected from SNCA, MAPT, APP, ATXN2, ATXN3, SARM1, APOE, BACE1, FMR1, LRRK2, HTT, SOD1, SCN10A, SCN9A, or CACNA1B mRNA. In some embodiments, the antisense strand is complementary to SNCA mRNA. Exemplary RNAi agents targeting human SNCA mRNA are provided in Table 1. In some embodiments, the antisense strand is complementary to MAPT mRNA. Exemplary RNAi agents targeting human MAPT mRNA are provided in Table 2.

[0029] In another aspect, provided herein are pharmaceutical compositions comprising a compound or RNAi agent described herein and a pharmaceutically acceptable carrier.

[0030] In a further aspect, provided herein are methods for treating a neurodegenerative disease (e.g., synuclein disease or tau disease) in a patient in need thereof; such methods include administering to the patient an effective amount of a compound, RNAi agent, or pharmaceutical composition as described herein. In some embodiments, the compound, RNAi agent, or pharmaceutical composition is administered to the patient intrathecally, intraventricularly, or via intracisternal magna injection.

[0031] Also provided herein are methods of inhibiting or reducing a target mRNA in a cell, the method comprising contacting the cell comprising the target mRNA with a compound, RNAi agent, or pharmaceutical composition described herein.

[0032] In another aspect, provided herein are compounds, RNAi agents, or pharmaceutical compositions for use in treatment. Also provided herein are compounds, RNAi agents, or pharmaceutical compositions for use in treating neurodegenerative diseases, such as synuclein diseases or tauopathies. Also provided herein are uses of compounds or RNAi agents in the preparation of medicaments for treating neurodegenerative diseases, such as synuclein diseases or tauopathies. Detailed Description of the Invention

[0034] Provided herein are novel compounds and RNAi agents comprising modified nucleotides that have good tolerability, efficacy, and tissue distribution profiles in animal models, compositions comprising such compounds or RNAi agents, and methods of using such compounds or RNAi agents.

[0035] In one aspect, provided herein are compounds comprising any of the following formulae:

[0036]

[0037] Where n is an integer from 1 to 4,

[0038]

[0039] Where n is an integer from 0 to 2,

[0040] and

[0041] Wherein B is a nucleobase selected from adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U) or derivatives thereof. In some embodiments, B is a nucleobase selected from A, C, G, T, U. In some embodiments, B is a nucleobase selected from 5-methylcytosine, 2-thiouridine, 4-thiouridine, C5 modified pyrimidine, C2 modified purine, N8 modified purine, pseudouracil, isocytosine, isoguanine, 2,6-diaminopurine, pseudocytosine, 2-aminopurine, xanthine, hypoxanthine, 7-methylguanine, 5-hydroxymethylcytosine, 5,6-dihydrouracil, 5-carboxycytidine, phenoxazine, N6-alkyl-A or O6-alkyl-G nucleobase derivatives. In some embodiments, n in Formula Ic is 1. In some embodiments, n in Formula Ic is 2. In some embodiments, n in Formula Ic is 3. In some embodiments, n in Formula Ic is 4. In some embodiments, n in Formula IV is 0. In some embodiments, n in Formula IV is 1. In some embodiments, n in Formula IV is 2.

[0042] In some embodiments, the compound comprising any of Formula Ia, Ib, Ic, II-IV, or XXI is a nucleoside, nucleotide, or an analog thereof.

[0043] In some embodiments, provided herein are compounds comprising Formula Ia

[0044]

[0045] Wherein B is a nucleobase selected from adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U) or a derivative thereof. In some embodiments, B is a nucleobase selected from A, C, G, T, U. In some embodiments, B is a nucleobase derivative selected from 5-methylcytosine, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, C2-modified purine, N8-modified purine, pseudouracil, isocytosine, isoguanine, 2,6-diaminopurine, pseudocytosine, 2-aminopurine, xanthine, hypoxanthine, 7-methylguanine, 5-hydroxymethylcytosine, 5,6-dihydrouracil, 5-carboxycytidine, phenoxazine, N6-alkyl-A or O6-alkyl-G.

[0046] In some embodiments, provided herein are compounds comprising Formula Ib

[0047]

[0048] Wherein B is a nucleobase selected from adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U) or a derivative thereof. In some embodiments, B is a nucleobase selected from A, C, G, T, U. In some embodiments, B is a nucleobase derivative selected from 5-methylcytosine, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, C2-modified purine, N8-modified purine, pseudouracil, isocytosine, isoguanine, 2,6-diaminopurine, pseudocytosine, 2-aminopurine, xanthine, hypoxanthine, 7-methylguanine, 5-hydroxymethylcytosine, 5,6-dihydrouracil, 5-carboxycytidine, phenoxazine, N6-alkyl-A or O6-alkyl-G.

[0049] In some embodiments, provided herein are compounds comprising Formula Ic

[0050] where n is an integer from 1 to 4, and

[0051] Wherein B is a nucleobase selected from adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U) or derivatives thereof. In some embodiments, B is a nucleobase selected from A, C, G, T, U. In some embodiments, B is a nucleobase selected from 5-methylcytosine, 2-thiouridine, 4-thiouridine, C5 modified pyrimidine, C2 modified purine, N8 modified purine, pseudouracil, isocytosine, isoguanine, 2,6-diaminopurine, pseudocytosine, 2-aminopurine, xanthine, hypoxanthine, 7-methylguanine, 5-hydroxymethylcytosine, 5,6-dihydrouracil, 5-carboxycytidine, phenoxazine, N6-alkyl-A or O6-alkyl-G nucleobase derivatives. In some embodiments, n in Formula Ic is 1. In some embodiments, n in Formula Ic is 2. In some embodiments, n in Formula Ic is 3. In some embodiments, n in Formula Ic is 4.

[0052] In some embodiments, provided herein are compounds comprising Formula II

[0053]

[0054] Wherein B is a nucleobase selected from adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U) or a derivative thereof. In some embodiments, B is a nucleobase selected from A, C, G, T, U. In some embodiments, B is a nucleobase derivative selected from 5-methylcytosine, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, C2-modified purine, N8-modified purine, pseudouracil, isocytosine, isoguanine, 2,6-diaminopurine, pseudocytosine, 2-aminopurine, xanthine, hypoxanthine, 7-methylguanine, 5-hydroxymethylcytosine, 5,6-dihydrouracil, 5-carboxycytidine, phenoxazine, N6-alkyl-A or O6-alkyl-G.

[0055] In some embodiments, provided herein are compounds comprising Formula III

[0056]

[0057] Wherein B is a nucleobase selected from adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U) or a derivative thereof. In some embodiments, B is a nucleobase selected from A, C, G, T, U. In some embodiments, B is a nucleobase derivative selected from 5-methylcytosine, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, C2-modified purine, N8-modified purine, pseudouracil, isocytosine, isoguanine, 2,6-diaminopurine, pseudocytosine, 2-aminopurine, xanthine, hypoxanthine, 7-methylguanine, 5-hydroxymethylcytosine, 5,6-dihydrouracil, 5-carboxycytidine, phenoxazine, N6-alkyl-A or O6-alkyl-G.

[0058] In some embodiments, provided herein are compositions comprising Formula IV A compound wherein n is an integer from 0 to 2, and

[0059] Wherein B is a nucleobase selected from adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U) or a derivative thereof. In some embodiments, B is a nucleobase selected from A, C, G, T, U. In some embodiments, B is a nucleobase selected from 5-methylcytosine, 2-thiouridine, 4-thiouridine, C5 modified pyrimidine, C2 modified purine, N8 modified purine, pseudouracil, isocytosine, isoguanine, 2,6-diaminopurine, pseudocytosine, 2-aminopurine, xanthine, hypoxanthine, 7-methylguanine, 5-hydroxymethylcytosine, 5,6-dihydrouracil, 5-carboxycytidine, phenoxazine, N6-alkyl-A or O6-alkyl-G nucleobase derivatives. In some embodiments, n in Formula IV is 0. In some embodiments, n in Formula IV is 1. In some embodiments, n in Formula IV is 2.

[0060] In some embodiments, provided herein are compounds comprising Formula XXI

[0061]

[0062] Wherein B is a nucleobase selected from adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U) or a derivative thereof. In some embodiments, B is a nucleobase selected from A, C, G, T, U. In some embodiments, B is a nucleobase derivative selected from 5-methylcytosine, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, C2-modified purine, N8-modified purine, pseudouracil, isocytosine, isoguanine, 2,6-diaminopurine, pseudocytosine, 2-aminopurine, xanthine, hypoxanthine, 7-methylguanine, 5-hydroxymethylcytosine, 5,6-dihydrouracil, 5-carboxycytidine, phenoxazine, N6-alkyl-A or O6-alkyl-G.

[0063] In some embodiments, provided herein are compounds comprising any one of Formulae Va-VIIIa:

[0064]

[0065] In some embodiments, the compound comprising any of Formulae Va-VIIIa is a nucleoside, nucleotide, or an analog thereof.

[0066] In some embodiments, provided herein are compounds comprising any one of Formulas Vb-VIIIb:

[0067]

[0068] In some embodiments, the compound comprising any one of Formulae Vb-VIIIb is a nucleoside, nucleotide, or an analog thereof.

[0069] In some embodiments, provided herein are compounds comprising any one of Formulas Vc-VIIIc:

[0070] Where n is an integer from 1 to 4,

[0071] Where n is an integer from 1 to 4,

[0072] Where n is an integer from 1 to 4

[0073] Where n is an integer from 1 to 4

[0074] In some embodiments, n in Formula Vc-VIIIc is 1. In some embodiments, n in Formula Vc-VIIIc is 2. In some embodiments, n in Formula Vc-VIIIc is 3. In some embodiments, n in Formula Vc-VIIIc is 4.

[0075] In some embodiments, the compound comprising any one of Formulae Vc-VIIIc is a nucleoside, nucleotide, or an analog thereof.

[0076] In some embodiments, provided herein are compounds comprising any one of Formulas IX-XII:

[0077]

[0078] In some embodiments, the compound comprising any one of Formulae IX-XII is a nucleoside, nucleotide, or an analog thereof.

[0079] In some embodiments, provided herein are compounds comprising any one of Formulae XIII-XVI:

[0080]

[0081] In some embodiments, the compound comprising any one of Formulae XIII-XVI is a nucleoside, nucleotide, or an analog thereof.

[0082] In some embodiments, provided herein are compounds comprising any one of Formulae XVII-XX:

[0083] Where n is an integer from 0 to 2,

[0084] Where n is an integer from 0 to 2,

[0085] Where n is an integer from 0 to 2,

[0086] Where n is an integer from 0 to 2.

[0087] In some embodiments, n in Formula XVII-XX is 0. In some embodiments, n in Formula XVII-XX is 1. In some embodiments, n in Formula XVII-XX is 2. In some embodiments, the compound comprising any one of Formulas XVII-XX is a nucleoside, a nucleotide, or an analog thereof.

[0088] In some embodiments, provided herein are compounds comprising any one of Formulae XXII-XXV:

[0089]

[0090] In some embodiments, the compound comprising any one of Formulae XXII-XXV is a nucleoside, nucleotide, or an analog thereof.

[0091] In another aspect, provided herein are RNAi agents comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a duplex region, wherein the sense strand or the antisense strand comprises a modified nucleotide of any one of Formulas Ia, Ib, Ic, II-IV, or XXI:

[0092]

[0093] Where n is an integer from 1 to 4,

[0094]

[0095] where n is an integer from 0 to 2, and

[0096]

[0097] wherein B is a nucleobase selected from adenine, cytosine, guanine, thymine, uracil, or a derivative thereof. In some embodiments, B is a nucleobase selected from A, C, G, T, U. In some embodiments, B is a nucleobase derivative selected from 5-methylcytosine, 2-thiouridine, 4-thiouridine, a C5-modified pyrimidine, a C2-modified purine, an N8-modified purine, pseudouracil, isocytosine, isoguanine, 2,6-diaminopurine, pseudocytosine, 2-aminopurine, xanthine, hypoxanthine, 7-methylguanine, 5-hydroxymethylcytosine, 5,6-dihydrouracil, 5-carboxycytidine, phenoxazine, N6-alkyl-A, or O6-alkyl-G. In some embodiments, n in Formula Ic is 1. In some embodiments, n in Formula Ic is 2. In some embodiments, n in Formula Ic is 3. In some embodiments, n in Formula Ic is 4. In some embodiments, n in Formula IV is 0. In some embodiments, n in Formula IV is 1. In some embodiments, n in Formula IV is 2.

[0098] In some embodiments, provided herein are RNAi agents comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a duplex region, wherein the sense strand or the antisense strand comprises a modified nucleotide sequence of Formula Ia.

[0099]

[0100] Wherein B is a nucleobase selected from adenine, cytosine, guanine, thymine, uracil or a derivative thereof. In some embodiments, B is a nucleobase selected from A, C, G, T, U. In some embodiments, B is a nucleobase derivative selected from 5-methylcytosine, 2-thiouridine, 4-thiouridine, C5 modified pyrimidine, C2 modified purine, N8 modified purine, pseudouracil, isocytosine, isoguanine, 2,6-diaminopurine, pseudocytosine, 2-aminopurine, xanthine, hypoxanthine, 7-methylguanine, 5-hydroxymethylcytosine, 5,6-dihydrouracil, 5-carboxycytidine, phenoxazine, N6-alkyl-A or O6-alkyl-G.

[0101] In some embodiments, provided herein are RNAi agents comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a duplex region, wherein the sense strand or the antisense strand comprises a modified nucleotide sequence of Formula Ib.

[0102]

[0103] Wherein B is a nucleobase selected from adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U) or a derivative thereof. In some embodiments, B is a nucleobase selected from A, C, G, T, U. In some embodiments, B is a nucleobase derivative selected from 5-methylcytosine, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, C2-modified purine, N8-modified purine, pseudouracil, isocytosine, isoguanine, 2,6-diaminopurine, pseudocytosine, 2-aminopurine, xanthine, hypoxanthine, 7-methylguanine, 5-hydroxymethylcytosine, 5,6-dihydrouracil, 5-carboxycytidine, phenoxazine, N6-alkyl-A or O6-alkyl-G.

[0104] In some embodiments, provided herein are RNAi agents comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a duplex region, wherein the sense strand or the antisense strand comprises a modified nucleotide sequence of Formula Ic.

[0105] Where n is an integer from 1 to 4,

[0106] Wherein B is a nucleobase selected from adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U) or a derivative thereof. In some embodiments, B is a nucleobase selected from A, C, G, T, U. In some embodiments, B is a nucleobase selected from 5-methylcytosine, 2-thiouridine, 4-thiouridine, C5 modified pyrimidine, C2 modified purine, N8 modified purine, pseudouracil, isocytosine, isoguanine, 2,6-diaminopurine, pseudocytosine, 2-aminopurine, xanthine, hypoxanthine, 7-methylguanine, 5-hydroxymethylcytosine, 5,6-dihydrouracil, 5-carboxycytidine, phenoxazine, N6-alkyl-A or O6-alkyl-G nucleobase derivatives. In some embodiments, n in Formula I(c) is 1. In some embodiments, n in Formula I(c) is 2. In some embodiments, n in Formula I(c) is 3. In some embodiments, n in Formula I(c) is 4.

[0107] In some embodiments, provided herein are RNAi agents comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a duplex region, wherein the sense strand or the antisense strand comprises a modified nucleotide sequence of Formula II

[0108]

[0109] Wherein B is a nucleobase selected from adenine, cytosine, guanine, thymine, uracil or a derivative thereof. In some embodiments, B is a nucleobase selected from A, C, G, T, U. In some embodiments, B is a nucleobase derivative selected from 5-methylcytosine, 2-thiouridine, 4-thiouridine, C5 modified pyrimidine, C2 modified purine, N8 modified purine, pseudouracil, isocytosine, isoguanine, 2,6-diaminopurine, pseudocytosine, 2-aminopurine, xanthine, hypoxanthine, 7-methylguanine, 5-hydroxymethylcytosine, 5,6-dihydrouracil, 5-carboxycytidine, phenoxazine, N6-alkyl-A or O6-alkyl-G.

[0110] In some embodiments, provided herein are RNAi agents comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a duplex region, wherein the sense strand or the antisense strand comprises a modified nucleotide sequence of Formula III.

[0111]

[0112] Wherein B is a nucleobase selected from adenine, cytosine, guanine, thymine, uracil or a derivative thereof. In some embodiments, B is a nucleobase selected from A, C, G, T, U. In some embodiments, B is a nucleobase derivative selected from 5-methylcytosine, 2-thiouridine, 4-thiouridine, C5 modified pyrimidine, C2 modified purine, N8 modified purine, pseudouracil, isocytosine, isoguanine, 2,6-diaminopurine, pseudocytosine, 2-aminopurine, xanthine, hypoxanthine, 7-methylguanine, 5-hydroxymethylcytosine, 5,6-dihydrouracil, 5-carboxycytidine, phenoxazine, N6-alkyl-A or O6-alkyl-G.

[0113] In some embodiments, provided herein are RNAi agents comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a duplex region, wherein the sense strand or the antisense strand comprises a modified nucleotide sequence of Formula IV.

[0114] where n is an integer from 0 to 2, and

[0115] Wherein B is a nucleobase selected from adenine, cytosine, guanine, thymine, uracil, or a derivative thereof. In some embodiments, B is a nucleobase selected from A, C, G, T, U. In some embodiments, B is a nucleobase derivative selected from 5-methylcytosine, 2-thiouridine, 4-thiouridine, a C5-modified pyrimidine, a C2-modified purine, an N8-modified purine, pseudouracil, isocytosine, isoguanine, 2,6-diaminopurine, pseudocytosine, 2-aminopurine, xanthine, hypoxanthine, 7-methylguanine, 5-hydroxymethylcytosine, 5,6-dihydrouracil, 5-carboxycytidine, phenoxazine, N6-alkyl-A, or O6-alkyl-G. In some embodiments, n in Formula IV is 0. In some embodiments, n in Formula IV is 1. In some embodiments, n in Formula IV is 2.

[0116] In some embodiments, provided herein are RNAi agents comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a duplex region, wherein the sense strand or the antisense strand comprises a modified nucleotide sequence of Formula XXI.

[0117]

[0118] Wherein B is a nucleobase selected from adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U) or a derivative thereof. In some embodiments, B is a nucleobase selected from A, C, G, T, U. In some embodiments, B is a nucleobase derivative selected from 5-methylcytosine, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, C2-modified purine, N8-modified purine, pseudouracil, isocytosine, isoguanine, 2,6-diaminopurine, pseudocytosine, 2-aminopurine, xanthine, hypoxanthine, 7-methylguanine, 5-hydroxymethylcytosine, 5,6-dihydrouracil, 5-carboxycytidine, phenoxazine, N6-alkyl-A or O6-alkyl-G.

[0119] In some embodiments, provided herein are RNAi agents comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a duplex region, wherein the sense strand or the antisense strand comprises a modified nucleotide of any one of Formulas Va-VIIIa:

[0120]

[0121]

[0122] In some embodiments, provided herein are RNAi agents comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a duplex region, wherein the sense strand or the antisense strand comprises a modified nucleotide of any one of Formulas Vb-VIIIb:

[0123]

[0124]

[0125] In some embodiments, provided herein are RNAi agents comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a duplex region, wherein the sense strand or the antisense strand comprises a modified nucleotide of any one of Formulas Vc-VIIIc:

[0126] Where n is an integer from 1 to 4,

[0127] Where n is an integer from 1 to 4,

[0128] Where n is an integer from 1 to 4,

[0129] Where n is an integer from 1 to 4.

[0130] In some embodiments, n in Formula Vc-VIIIc is 1. In some embodiments, n in Formula Vc-VIIIc is 2. In some embodiments, n in Formula Vc-VIIIc is 3. In some embodiments, n in Formula Vc-VIIIc is 4.

[0131] In some embodiments, provided herein are RNAi agents comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a duplex region, wherein the sense strand or the antisense strand comprises a modified nucleotide of any one of Formulas IX-XII:

[0132]

[0133] In some embodiments, provided herein are RNAi agents comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a duplex region, wherein the sense strand or the antisense strand comprises a modified nucleotide of any one of Formulas XIII-XVI:

[0134]

[0135] In some embodiments, provided herein are RNAi agents comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a duplex region, wherein the sense strand or the antisense strand comprises a modified nucleotide of any one of Formulae XVII-XX:

[0136] Where n is an integer from 0 to 2,

[0137] Where n is an integer from 0 to 2,

[0138] Where n is an integer from 0 to 2,

[0139] Where n is an integer from 0 to 2.

[0140] In some embodiments, n in Formula XVII-XX is 0. In some embodiments, n in Formula XVII-XX is 1. In some embodiments, n in Formula XVII-XX is 2.

[0141] In some embodiments, provided herein are RNAi agents comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a duplex region, wherein the sense strand or the antisense strand comprises a modified nucleotide of any one of Formulae XXII-XXV:

[0142]

[0143]

[0144] In some embodiments, the length of the sense strand is 15 to 50 nucleotides. In some embodiments, the length of the antisense strand is 15 to 30 nucleotides. In some embodiments, both the sense strand and the antisense strand are 15 to 30 nucleotides in length, for example, 20 to 25 nucleotides in length. In some embodiments, the length of the sense strand is 21 nucleotides. In some embodiments, the length of the antisense strand is 23 nucleotides. In some embodiments, the length of the sense strand is 21 nucleotides, and the length of the antisense strand is 23 nucleotides. In some embodiments, the length of the duplex region is 15 to 21 nucleotides. In some embodiments, the length of the duplex region is 21 nucleotides. In some embodiments, the sense strand and the antisense strand may have an overhang at the 5' end or 3' end (i.e., a 5' overhang or a 3' overhang). For example, the sense strand and the antisense strand may have a 5' or 3' overhang of 1 to 5 nucleotides or 1 to 3 nucleotides. In some embodiments, the antisense strand comprises a 3' overhang of two nucleotides.

[0145] In some embodiments, the sense strand comprises a modified nucleotide of any one of Formulas Ia, Ib, Ic, II-IV, Va, Vb, Vc, VIa, VIb, VIc, VIIa, VIIb, VIIc, VIIIa, VIIIb, VIIIc, IX-XXV. In some embodiments, the sense strand comprises a modified nucleotide of any one of Formulas Ia, Ib, Ic, II-IV, Va, Vb, Vc, VIa, VIb, VIc, VIIa, VIIb, VIIc, VIIIa, VIIIb, VIIIc, IX-XXV, e.g., at any one of positions 1-6 or 12-21 from the 5' end. In some embodiments, the sense strand comprises a modified nucleotide of any one of Formulas Ia, Ib, Ic, II-IV, Va, Vb, Vc, VIa, VIb, VIc, VIIa, VIIb, VIIc, VIIIa, VIIIb, VIIIc, IX-XXV at position 13 from the 5' end.

[0146] In some embodiments, the antisense strand comprises a modified nucleotide of any one of Formulas Ia, Ib, Ic, II-IV, Va, Vb, Vc, VIa, VIb, VIc, VIIa, VIIb, VIIc, VIIIa, VIIIb, VIIIc, IX-XXV. In some embodiments, the antisense strand comprises a modified nucleotide of any one of Formulas Ia, Ib, Ic, II-IV, Va, Vb, Vc, VIa, VIb, VIc, VIIa, VIIb, VIIc, VIIIa, VIIIb, VIIIc, IX-XXV, e.g., at any one of positions 6-10 or 15-18 from the 5' end.

[0147] In some embodiments, the sense strand and the antisense strand further comprise one or more 2'-fluoro modified nucleotides and 2'-O-methyl modified nucleotides. In some embodiments, the sense strand comprises four 2'-fluoro modified nucleotides at positions 7, 9, 10, and 11 from the 5' end of the sense strand. In some embodiments, the sense strand comprises four and only four 2'-fluoro modified nucleotides at positions 7, 9, 10, and 11 from the 5' end of the sense strand. In some embodiments, the nucleotides at other positions of the sense strand are 2'-O-methyl modified nucleotides.

[0148] In some embodiments, the antisense strand comprises four 2'-fluoro modified nucleotides at positions 2, 6, 14, and 16 from the 5' end of the antisense strand. In some embodiments, the antisense strand comprises four and only four 2'-fluoro modified nucleotides at positions 2, 6, 14, and 16 from the 5' end of the antisense strand. In some embodiments, nucleotides at other positions of the antisense strand are 2'-O-methyl modified nucleotides.

[0149] In some embodiments, the sense strand comprises three 2'-fluoro modified nucleotides at positions 9, 10, and 11 from the 5' end of the sense strand. In some embodiments, the sense strand comprises three and only three 2'-fluoro modified nucleotides at positions 9, 10, and 11 from the 5' end of the sense strand. In some embodiments, the nucleotides at other positions of the sense strand are 2'-O-methyl modified nucleotides.

[0150] In some embodiments, the antisense strand comprises five 2'-fluoro modified nucleotides at positions 2, 5, 7, 14, and 16 from the 5' end of the antisense strand. In some embodiments, the antisense strand comprises five and only five 2'-fluoro modified nucleotides at positions 2, 5, 7, 14, and 16 from the 5' end of the antisense strand. In some embodiments, nucleotides at other positions of the antisense strand are 2'-O-methyl modified nucleotides.

[0151] In some embodiments, the antisense strand comprises five 2'-fluoro modified nucleotides at positions 2, 5, 8, 14, and 16 from the 5' end of the antisense strand. In some embodiments, the antisense strand comprises five and only five 2'-fluoro modified nucleotides at positions 2, 5, 8, 14, and 16 from the 5' end of the antisense strand. In some embodiments, nucleotides at other positions of the antisense strand are 2'-O-methyl modified nucleotides.

[0152] In some embodiments, the antisense strand comprises five 2'-fluoro modified nucleotides at positions 2, 3, 7, 14, and 16 from the 5' end of the antisense strand. In some embodiments, the antisense strand comprises five and only five 2'-fluoro modified nucleotides at positions 2, 3, 7, 14, and 16 from the 5' end of the antisense strand. In some embodiments, nucleotides at other positions of the antisense strand are 2'-O-methyl modified nucleotides.

[0153] In some embodiments, the sense strand and the antisense strand comprise one or more modified internucleotide linkages, such as phosphorothioate linkages. In some embodiments, the sense strand comprises four or five phosphorothioate linkages. In some embodiments, the antisense strand comprises four or five phosphorothioate linkages.

[0154] In some embodiments, the antisense strand comprises a phosphate analog at the 5' end. In some embodiments, the antisense strand comprises a 5'-vinylphosphonate at the 5' end.

[0155] In some embodiments, the sense strand comprises an abasic portion or an inverted abasic portion, such as an abasic portion or an inverted abasic portion from Table 3.

[0156] In some embodiments, the antisense strand is complementary to a target mRNA selected from SNCA, MAPT, APP, ATXN2, ATXN3, SARM1, APOE, BACE1, FMR1, LRRK2, HTT, SOD1, SCN10A, SCN9A, or CACNA1B mRNA. In some embodiments, the antisense strand is complementary to SNCA mRNA. In some embodiments, the antisense strand is complementary to MAPT mRNA.

[0157] Exemplary sense and antisense strand sequences of RNAi agents targeting human SNCA mRNA (SNCA RNAi agents) are provided in Table 1.

[0158] Table 1. Nucleic acid sequences of exemplary SNCA RNAi agents

[0159]

[0160]

[0161]

[0162]

[0163]

[0164]

[0165] Abbreviations—“m” indicates 2′-OMe; “f” indicates 2′-fluoro; “*” indicates phosphorothioate linkage; “VP” indicates 5′-vinylphosphonate; “ads” indicates Formula I(a); “ss” indicates Formula II; “L1” indicates Formula III; “L2” indicates Formula IV, wherein n is 0; “L3” indicates Formula XXI; “adsII” indicates Formula I(b); “iAb” indicates the inverted abasic residue in Table 3.

[0166] In some embodiments, provided herein are SNCA RNAi agents comprising a sense strand and an antisense strand comprising a pair of nucleic acid sequences selected from the group consisting of:

[0167] (a) the sense strand comprises a first nucleic acid sequence that has at least 90% (e.g., about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) sequence identity to SEQ ID NO: 1, and the antisense strand comprises a second nucleic acid sequence that has at least 90% (e.g., about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) sequence identity to SEQ ID NO: 2;

[0168] (b) the sense strand comprises a first nucleic acid sequence that has at least 90% (e.g., about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) sequence identity to SEQ ID NO: 83, and the antisense strand comprises a second nucleic acid sequence that has at least 90% (e.g., about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) sequence identity to SEQ ID NO: 84;

[0169] (c) the sense strand comprises a first nucleic acid sequence that has at least 90% (e.g., about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) sequence identity to SEQ ID NO: 85, and the antisense strand comprises a second nucleic acid sequence that has at least 90% (e.g., about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) sequence identity to SEQ ID NO: 86;

[0170] (d) the sense strand comprises a first nucleic acid sequence that has at least 90% (e.g., about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) sequence identity to SEQ ID NO: 94, and the antisense strand comprises a second nucleic acid sequence that has at least 90% (e.g., about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) sequence identity to SEQ ID NO: 95;

[0171] (e) the sense strand comprises a first nucleic acid sequence that has at least 90% (e.g., about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) sequence identity to SEQ ID NO: 96, and the antisense strand comprises a second nucleic acid sequence that has at least 90% (e.g., about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) sequence identity to SEQ ID NO: 97;

[0172] (f) the sense strand comprises a first nucleic acid sequence that has at least 90% (e.g., about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) sequence identity to SEQ ID NO: 98, and the antisense strand comprises a second nucleic acid sequence that has at least 90% (e.g., about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) sequence identity to SEQ ID NO: 99;

[0173] (g) the sense strand comprises a first nucleic acid sequence that has at least 90% (e.g., about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) sequence identity to SEQ ID NO: 100, and the antisense strand comprises a second nucleic acid sequence that has at least 90% (e.g., about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) sequence identity to SEQ ID NO: 101;

[0174] (h) the sense strand comprises a first nucleic acid sequence that has at least 90% (e.g., about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) sequence identity to SEQ ID NO: 102, and the antisense strand comprises a second nucleic acid sequence that has at least 90% (e.g., about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) sequence identity to SEQ ID NO: 103; and

[0175] (i) the sense strand comprises a first nucleic acid sequence that has at least 90% (e.g., about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) sequence identity to SEQ ID NO: 104, and the antisense strand comprises a second nucleic acid sequence that has at least 90% (e.g., about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) sequence identity to SEQ ID NO: 105.

[0176] In some embodiments, provided herein are SNCA RNAi agents comprising a sense strand and an antisense strand comprising a pair of nucleic acid sequences selected from the group consisting of:

[0177] (a) the sense strand comprises SEQ ID NO: 1, and the antisense strand comprises SEQ ID NO: 2;

[0178] (b) the sense strand comprises any one of SEQ ID NO: 3, 5, or 20, and the antisense strand comprises SEQ ID NO: 4;

[0179] (c) the sense strand comprises any one of SEQ ID NOs: 6, 8-19, 37, 38, or 67-81, and the antisense strand comprises SEQ ID NO: 7;

[0180] (d) the sense strand comprises SEQ ID NO: 19, and the antisense strand comprises SEQ ID NO: 66;

[0181] (e) the sense strand comprises SEQ ID NO: 9 or 16, and the antisense strand comprises SEQ ID NO: 82;

[0182] (f) the sense strand comprises SEQ ID NO: 83, and the antisense strand comprises SEQ ID NO: 84;

[0183] (g) the sense strand comprises SEQ ID NO: 85, and the antisense strand comprises SEQ ID NO: 86;

[0184] (h) the sense strand comprises SEQ ID NO: 87, and the antisense strand comprises SEQ ID NO: 88;

[0185] (i) the sense strand comprises SEQ ID NO: 89, and the antisense strand comprises SEQ ID NO: 90;

[0186] (j) the sense strand comprises SEQ ID NO: 91, and the antisense strand comprises SEQ ID NO: 92 or 93;

[0187] (k) the sense strand comprises SEQ ID NO: 94, and the antisense strand comprises SEQ ID NO: 95;

[0188] (1) the sense strand comprises SEQ ID NO: 96, and the antisense strand comprises SEQ ID NO: 97;

[0189] (m) the sense strand comprises SEQ ID NO: 98, and the antisense strand comprises SEQ ID NO: 99;

[0190] (n) the sense strand comprises SEQ ID NO: 100, and the antisense strand comprises SEQ ID NO: 101;

[0191] (o) the sense strand comprises SEQ ID NO: 102, and the antisense strand comprises SEQ ID NO: 103;

[0192] (p) the sense strand comprises SEQ ID NO: 104, and the antisense strand comprises SEQ ID NO: 105;

[0193] (q) the sense strand comprises SEQ ID NO: 106, and the antisense strand comprises SEQ ID NO: 107;

[0194] (r) the sense strand comprises SEQ ID NO: 108, and the antisense strand comprises SEQ ID NO: 109 or 122;

[0195] (s) the sense strand comprises SEQ ID NO: 110, and the antisense strand comprises SEQ ID NO: 111;

[0196] (t) the sense strand comprises SEQ ID NO: 112, and the antisense strand comprises SEQ ID NO: 113;

[0197] (u) the sense strand comprises SEQ ID NO: 114, and the antisense strand comprises SEQ ID NO: 115;

[0198] (v) the sense strand comprises SEQ ID NO: 116, and the antisense strand comprises SEQ ID NO: 117;

[0199] (w) the sense strand comprises SEQ ID NO: 118, and the antisense strand comprises SEQ ID NO: 119;

[0200] (x) the sense strand comprises SEQ ID NO: 120, and the antisense strand comprises SEQ ID NO: 121; and

[0201] (y) the sense strand comprises SEQ ID NO: 123, and the antisense strand comprises SEQ ID NO: 124.

[0202] In some embodiments, provided herein are SNCA RNAi agents comprising a sense strand comprising SEQ ID NO: 3 and an antisense strand comprising SEQ ID NO: 4. In some embodiments, provided herein are SNCA RNAi agents comprising a sense strand comprising SEQ ID NO: 19 and an antisense strand comprising SEQ ID NO: 7. In some embodiments, provided herein are SNCA RNAi agents comprising a sense strand comprising SEQ ID NO: 87 and an antisense strand comprising SEQ ID NO: 88. In some embodiments, provided herein are SNCA RNAi agents comprising a sense strand comprising SEQ ID NO: 89 and an antisense strand comprising SEQ ID NO: 90.

[0203] In some embodiments, provided herein are SNCA RNAi agents comprising a sense strand and an antisense strand consisting of a pair of nucleic acid sequences selected from the group consisting of:

[0204] (a) the sense strand consists of any one of SEQ ID NO: 3, 5, or 20, and the antisense strand consists of SEQ ID NO: 4;

[0205] (b) the sense strand consists of any one of SEQ ID NOs: 6, 8-19, 37, 38, or 67-81, and the antisense strand consists of SEQ ID NO: 7;

[0206] (c) the sense strand consists of SEQ ID NO: 19, and the antisense strand consists of SEQ ID NO: 66;

[0207] (d) the sense strand consists of SEQ ID NO: 9 or 16, and the antisense strand consists of SEQ ID NO: 82;

[0208] (e) the sense strand consists of SEQ ID NO: 87, and the antisense strand consists of SEQ ID NO: 88;

[0209] (f) the sense strand consists of SEQ ID NO: 89, and the antisense strand consists of SEQ ID NO: 90;

[0210] (g) the sense strand consists of SEQ ID NO: 91, and the antisense strand consists of SEQ ID NO: 92 or 93;

[0211] (h) the sense strand consists of SEQ ID NO: 106, and the antisense strand consists of SEQ ID NO: 107;

[0212] (i) the sense strand consists of SEQ ID NO: 108, and the antisense strand consists of SEQ ID NO: 109 or 122;

[0213] (j) the sense strand consists of SEQ ID NO: 110, and the antisense strand consists of SEQ ID NO: 111;

[0214] (k) the sense strand consists of SEQ ID NO: 112, and the antisense strand consists of SEQ ID NO: 113;

[0215] (1) the sense strand consists of SEQ ID NO: 114, and the antisense strand consists of SEQ ID NO: 115;

[0216] (m) the sense strand consists of SEQ ID NO: 116, and the antisense strand consists of SEQ ID NO: 117;

[0217] (n) the sense strand consists of SEQ ID NO: 118, and the antisense strand consists of SEQ ID NO: 119;

[0218] (o) the sense strand consists of SEQ ID NO: 120, and the antisense strand consists of SEQ ID NO: 121; and

[0219] (p) The sense strand consists of SEQ ID NO: 123, and the antisense strand consists of SEQ ID NO: 124.

[0220] In some embodiments, provided herein are SNCA RNAi agents comprising a sense strand consisting of SEQ ID NO: 3 and an antisense strand consisting of SEQ ID NO: 4. In some embodiments, provided herein are SNCA RNAi agents comprising a sense strand consisting of SEQ ID NO: 19 and an antisense strand consisting of SEQ ID NO: 7. In some embodiments, provided herein are SNCA RNAi agents comprising a sense strand consisting of SEQ ID NO: 87 and an antisense strand consisting of SEQ ID NO: 88. In some embodiments, provided herein are SNCA RNAi agents comprising a sense strand consisting of SEQ ID NO: 89 and an antisense strand consisting of SEQ ID NO: 90.

[0221] Table 2 provides exemplary sense and antisense strand sequences of RNAi agents targeting human MAPT mRNA (MAPT RNAi agents).

[0222] Table 2. Nucleic acid sequences of exemplary MAPT RNAi agents

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229] *The last nucleotide does not match the transcript.

[0230] Abbreviations – “m” indicates 2′-OMe; “f” indicates 2′-fluoro; “*” indicates phosphorothioate linkage; “VP” indicates 5′-vinylphosphonate; “n” indicates an abasic nucleotide; “ads” indicates Formula I(a); “ss” indicates Formula II; “L3” indicates Formula XXI; and “adsII” indicates Formula I(b).

[0231] Table 3. Abasic or Inverted Abasic (iAb) Moieties

[0232]

[0233] "5'" and "3'" indicate the 5' to 3' direction of the sequence.

[0234] In some embodiments, provided herein are MAPT RNAi agents comprising a sense strand and an antisense strand comprising a pair of nucleic acid sequences selected from the group consisting of:

[0235] (a) the sense strand comprises a first nucleic acid sequence that has at least 90% (e.g., about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) sequence identity to SEQ ID NO: 21, and the antisense strand comprises a second nucleic acid sequence that has at least 90% (e.g., about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) sequence identity to SEQ ID NO: 22;

[0236] (b) the sense strand comprises a first nucleic acid sequence that has at least 90% (e.g., about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) sequence identity to SEQ ID NO: 23, and the antisense strand comprises a second nucleic acid sequence that has at least 90% (e.g., about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) sequence identity to SEQ ID NO: 24;

[0237] (c) the sense strand comprises a first nucleic acid sequence that has at least 90% (e.g., about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) sequence identity to SEQ ID NO: 25, and the antisense strand comprises a second nucleic acid sequence that has at least 90% (e.g., about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) sequence identity to SEQ ID NO: 26;

[0238] (d) the sense strand comprises a first nucleic acid sequence that has at least 90% (e.g., about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) sequence identity to SEQ ID NO: 56, and the antisense strand comprises a second nucleic acid sequence that has at least 90% (e.g., about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) sequence identity to SEQ ID NO: 57;

[0239] (e) the sense strand comprises a first nucleic acid sequence that has at least 90% (e.g., about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) sequence identity to SEQ ID NO: 125, and the antisense strand comprises a second nucleic acid sequence that has at least 90% (e.g., about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) sequence identity to SEQ ID NO: 126;

[0240] (f) the sense strand comprises a first nucleic acid sequence that has at least 90% (e.g., about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) sequence identity to SEQ ID NO: 127, and the antisense strand comprises a second nucleic acid sequence that has at least 90% (e.g., about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) sequence identity to SEQ ID NO: 128;

[0241] (g) the sense strand comprises a first nucleic acid sequence that has at least 90% (e.g., about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) sequence identity to SEQ ID NO: 129, and the antisense strand comprises a second nucleic acid sequence that has at least 90% (e.g., about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) sequence identity to SEQ ID NO: 130;

[0242] (h) the sense strand comprises a first nucleic acid sequence that has at least 90% (e.g., about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) sequence identity to SEQ ID NO: 131, and the antisense strand comprises a second nucleic acid sequence that has at least 90% (e.g., about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) sequence identity to SEQ ID NO: 132;

[0243] (i) the sense strand comprises a first nucleic acid sequence that has at least 90% (e.g., about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) sequence identity to SEQ ID NO: 133, and the antisense strand comprises a second nucleic acid sequence that has at least 90% (e.g., about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) sequence identity to SEQ ID NO: 134; and

[0244] (j) the sense strand comprises a first nucleic acid sequence that has at least 90% (e.g., about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) sequence identity to SEQ ID NO: 135, and the antisense strand comprises a second nucleic acid sequence that has at least 90% (e.g., about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) sequence identity to SEQ ID NO: 136.

[0245] In some embodiments, provided herein are MAPT RNAi agents comprising a sense strand and an antisense strand comprising a pair of nucleic acid sequences selected from the group consisting of:

[0246] (a) the sense strand comprises SEQ ID NO: 21, and the antisense strand comprises SEQ ID NO: 22;

[0247] (b) the sense strand comprises SEQ ID NO: 23, and the antisense strand comprises SEQ ID NO: 24;

[0248] (c) the sense strand comprises SEQ ID NO: 25, and the antisense strand comprises SEQ ID NO: 26;

[0249] (d) the sense strand comprises any one of SEQ ID NOs: 27, 33, 39, 40, 47-49, and the antisense strand comprises SEQ ID NO: 28;

[0250] (e) the sense strand comprises any one of SEQ ID NOs: 29, 34, 35, 42, 50-51, 53, and the antisense strand comprises SEQ ID NO: 30;

[0251] (f) the sense strand comprises SEQ ID NOs: 31, 36, 43, 52, 154, 155, 161-163, and the antisense strand comprises SEQ ID NO: 32;

[0252] (g) the sense strand comprises SEQ ID NO: 39 or 40, and the antisense strand comprises SEQ ID NO: 41;

[0253] (h) the sense strand comprises SEQ ID NO: 44 or 46, and the antisense strand comprises SEQ ID NO: 45;

[0254] (i) the sense strand comprises SEQ ID NO: 53, and the antisense strand comprises SEQ ID NO: 54 or 55;

[0255] (j) the sense strand comprises SEQ ID NO: 56, and the antisense strand comprises SEQ ID NO: 57;

[0256] (k) the sense strand comprises SEQ ID NO: 125, and the antisense strand comprises SEQ ID NO: 126;

[0257] (1) the sense strand comprises SEQ ID NO: 127, and the antisense strand comprises SEQ ID NO: 128;

[0258] (m) the sense strand comprises SEQ ID NO: 129, and the antisense strand comprises SEQ ID NO: 130;

[0259] (n) the sense strand comprises SEQ ID NO: 131, and the antisense strand comprises SEQ ID NO: 132;

[0260] (o) the sense strand comprises SEQ ID NO: 133, and the antisense strand comprises SEQ ID NO: 134;

[0261] (p) the sense strand comprises SEQ ID NO: 135, and the antisense strand comprises SEQ ID NO: 136;

[0262] (q) the sense strand comprises SEQ ID NO: 137, and the antisense strand comprises SEQ ID NO: 138;

[0263] (r) the sense strand comprises SEQ ID NO: 139, and the antisense strand comprises SEQ ID NO: 140;

[0264] (s) the sense strand comprises SEQ ID NO: 141, and the antisense strand comprises SEQ ID NO: 142;

[0265] (t) the sense strand comprises SEQ ID NO: 143, and the antisense strand comprises SEQ ID NO: 144;

[0266] (u) the sense strand comprises SEQ ID NO: 145, and the antisense strand comprises SEQ ID NO: 146;

[0267] (v) the sense strand comprises SEQ ID NO: 147, and the antisense strand comprises SEQ ID NO: 148;

[0268] (w) the sense strand comprises SEQ ID NO: 34, and the antisense strand comprises any one of SEQ ID NOs: 149, 150, 151;

[0269] (x) the sense strand comprises SEQ ID NO: 31, and the antisense strand comprises any one of SEQ ID NOs: 152, 153, 156-159, 164, 165;

[0270] (y) the sense strand comprises SEQ ID NO: 160, and the antisense strand comprises SEQ ID NO: 152; and

[0271] (z) the sense strand comprises SEQ ID NO: 43 or 166, and the antisense strand comprises SEQ ID NO: 156.

[0272] In some embodiments, provided herein are MAPT RNAi agents comprising a sense strand and an antisense strand consisting of a pair of nucleic acid sequences selected from the group consisting of:

[0273] (a) the sense strand consists of any one of SEQ ID NOs: 27, 33, 39, 40, 47-49, and the antisense strand consists of SEQ ID NO: 28;

[0274] (b) the sense strand consists of any one of SEQ ID NOs: 29, 34, 35, 42, 50-51, 53, and the antisense strand consists of SEQ ID NO: 30;

[0275] (c) the sense strand consists of SEQ ID NOs: 31, 36, 43, 52, 154, 155, 161-163, and the antisense strand consists of SEQ ID NO: 32;

[0276] (d) the sense strand consists of SEQ ID NO: 39 or 40, and the antisense strand consists of SEQ ID NO: 41;

[0277] (e) the sense strand consists of SEQ ID NO: 44 or 46, and the antisense strand consists of SEQ ID NO: 45;

[0278] (f) the sense strand consists of SEQ ID NO: 53, and the antisense strand consists of SEQ ID NO: 54 or 55;

[0279] (g) the sense strand consists of SEQ ID NO: 137, and the antisense strand consists of SEQ ID NO: 138;

[0280] (h) the sense strand consists of SEQ ID NO: 139, and the antisense strand consists of SEQ ID NO: 140;

[0281] (i) the sense strand consists of SEQ ID NO: 141, and the antisense strand consists of SEQ ID NO: 142;

[0282] (j) the sense strand consists of SEQ ID NO: 143, and the antisense strand consists of SEQ ID NO: 144;

[0283] (k) the sense strand consists of SEQ ID NO: 145, and the antisense strand consists of SEQ ID NO: 146;

[0284] (1) the sense strand consists of SEQ ID NO: 147, and the antisense strand consists of SEQ ID NO: 148;

[0285] (m) the sense strand consists of SEQ ID NO: 34, and the antisense strand consists of any one of SEQ ID NOs: 149, 150, 151;

[0286] (n) the sense strand consists of SEQ ID NO: 31, and the antisense strand consists of any one of SEQ ID NOs: 152, 153, 156-159, 164, 165;

[0287] (o) the sense strand consists of SEQ ID NO: 160, and the antisense strand consists of SEQ ID NO: 152; and

[0288] (p) The sense strand consists of SEQ ID NO: 43 or 166, and the antisense strand consists of SEQ ID NO: 156.

[0289] The sense and antisense strands of the RNAi agents can be synthesized using any nucleic acid polymerization method known in the art, such as by solid phase synthesis using phosphoramidite chemistry (e.g., Current Protocols in Nucleic Acid Chemistry, Beaucage, SL et al. (eds.), John Wiley & Sons, Inc., New York, NY, USA), H-phosphonate, phosphotriester chemistry, or enzymatic synthesis. Automated commercial synthesizers, such as the MerMade from LGC Biosearch Technologies, can be used. TM 12, or other synthesizers from BioAutomation or Applied Biosystems. Sulfurizing agents such as phenylacetyl disulfide or DDTT (((dimethylaminomethylene) amino)-3H-1,2,4-dithiazoline-3-thione) can be used to introduce thiophosphate linkages. It is well known that oligonucleotides synthesized by similar techniques and commercially available modified phosphoramidites and controlled pore glass (CPG) products are modified or put together.

[0290] Purification process can be used for eliminating unwanted impurities from final oligonucleotide product. Common purification techniques for single-stranded oligonucleotide include reversed-phase ion-pair high performance liquid chromatography (RP-IP-HPLC), capillary gel electrophoresis (CGE), anion exchange HPLC (AX-HPLC) and size exclusion chromatography (SEC). After purification, oligonucleotide can be analyzed by mass spectrometry, and quantitatively at a wavelength of 260nm by spectrophotometry. Sense strand and antisense strand can then be annealed to form a duplex.

[0291] In another aspect, provided herein are pharmaceutical compositions comprising a compound as described herein or an RNAi reagent and a pharmaceutically acceptable carrier. Such pharmaceutical compositions may also include one or more pharmaceutically acceptable excipients, diluents or carriers. Pharmaceutical compositions can be prepared by methods well known in the art (e.g., Remington: The Science and Practice of Pharmacy, 23rd edition (2020), A. Loyd et al., Academic Press).

[0292] In a further aspect, provided herein are methods of treating a neurodegenerative disease in a patient in need thereof; such methods comprise administering to the patient an effective amount of a compound, RNAi agent, or pharmaceutical composition described herein.

[0293] In some embodiments, the neurodegenerative disease is a synucleinopathy selected from Parkinson's disease, Alzheimer's disease, multiple system atrophy, or dementia with Lewy bodies.

[0294] In some embodiments, the neurodegenerative disease is a tauopathy selected from the group consisting of Alzheimer's disease, frontotemporal dementia (FTD), frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), frontotemporal lobar degeneration (FTLD), behavioral variant frontotemporal dementia (bvFTD), nonfluent variant primary progressive aphasia (nfvPPA), Parkinson's disease, Pick's disease (PiD), primary progressive aphasia - semantic (PPA-S), primary progressive aphasia - pauci-verbal (PPA-L), multisystem tauopathy with Alzheimer's disease (MSTD), neurofibrillary tangles (NFT) dementia, FTD with motor neuron disease, progressive supranuclear palsy (PSP), amyotrophic lateral sclerosis / parkinsonism-dementia complex (ALS-PDC), argyrophilic grain dementia (AGD), British amyloid angiopathy, cerebral amyloid angiopathy, chronic traumatic encephalopathy (CTE), corticobasal degeneration (CBD), Creutzfeldt-Jakob disease (CJD), dementia pugilistica, diffuse neurofibrillary tangles with calcifications, Down syndrome, epilepsy, Gerstmann-Straussler-Scheinker disease, Hallervorden-Spatz disease disease), Huntington's disease, inclusion body myositis, lead encephalopathy, Lytico-Bodig disease, meningioangiomatosis, multiple system atrophy, myotonic dystrophy, Niemann-Pick disease type C (NP-C), non-Guam motor neuron disease with neurofibrillary tangles, postencephalitic Parkinson's disease, prion amyloid angiopathy, progressive subcortical gliosis, dementia with tangles, dementia with predominant tangles, ganglioglioma, gangliocytoma, subacute sclerosing encephalitis, tuberous sclerosis, lipofuscinosis, primary age-related tauopathy (PART), or globular glial tauopathy (GGT).

[0295] In some embodiments, the compound, RNAi agent, or pharmaceutical composition is administered to the patient intrathecally, intracerebroventricularly, or via intracisterna magna injection.

[0296] Also provided herein are methods of inhibiting or reducing a target mRNA in a cell, the method comprising contacting a cell comprising the target mRNA with a compound, RNAi agent, or pharmaceutical composition as described herein. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is in a subject. In some embodiments, the subject is a human subject.

[0297] Dosage regimens may be adjusted to provide the optimal desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.

[0298] Dosage values ​​may vary with the type and severity of the condition to be alleviated.It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions.

[0299] In another aspect, provided herein are compounds, RNAi agents, or pharmaceutical compositions for use in treatment. Also provided herein are compounds, RNAi agents, or pharmaceutical compositions for use in treating neurodegenerative diseases, such as synuclein diseases or tauopathies. Also provided herein are uses of compounds or RNAi agents in the preparation of medicaments for treating neurodegenerative diseases, such as synuclein diseases or tauopathies.

[0300] As used herein, the terms “a,” “an,” “the,” and similar terms used in the context of this disclosure (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0301] As used herein, the term "alkyl" means a saturated linear or branched monovalent hydrocarbon radical containing the indicated number of carbon atoms. For example, "C1-C 20 "Alkyl" means a radical having 1 to 20 carbon atoms in a linear or branched arrangement.

[0302] As used herein, "antisense strand" means an oligonucleotide that is complementary to a region of a target sequence. Likewise, and as used herein, "sense strand" means an oligonucleotide that is complementary to a region of the antisense strand.

[0303] As used herein, " complementary " means the structural relationship between two nucleotides (for example, on two opposite nucleic acids or on the relative region of a single nucleic acid chain), which allows two nucleotides to form base pairs with each other. For example, the purine nucleotides of a nucleic acid complementary to the pyrimidine nucleotides of the relative nucleic acid may base pair together by forming hydrogen bonds with each other. Complementary nucleotides can base pair in a Watson-Crick manner or in any other manner that allows the formation of a stable duplex. Similarly, two nucleic acids may have a multiple nucleotide region that is complementary to each other to form a complementary region, as described herein.

[0304] As used herein, "delivery moiety" refers to a chemical moiety that facilitates the entry of an oligonucleotide or RNAi agent into a cell. The delivery moiety can be a lipid, cholesterol, vitamin E, a carbohydrate, an amino sugar, a polypeptide, or a protein.

[0305] As used herein, reference to a "duplex" of a nucleic acid or oligonucleotide refers to the structure formed by complementary base pairing of two antiparallel nucleotide sequences (i.e., in opposite orientations), whether formed by two separate nucleic acid strands or by a single folded strand (e.g., via a hairpin).

[0306] An "effective amount" refers to the amount necessary to achieve the desired therapeutic result (for a period of time and for the means of administration). The effective amount of an RNA agent may vary depending on factors such as the disease state, age, sex, and weight of the individual, as well as the ability of the RNA agent to elicit a desired response in the individual. An effective amount is also an amount in which any toxic or deleterious effects of the RNA agent are outweighed by the therapeutically beneficial effects.

[0307] The term "knockdown" or "expression knockdown" refers to a decrease in mRNA or protein expression of a gene following treatment with an agent, such as an RNA agent.

[0308] As used herein, "modified internucleotide linkage" means an internucleotide linkage having one or more chemical modifications when compared to a reference internucleotide linkage having a phosphodiester linkage. Typically, the modified internucleotide linkage confers one or more desired properties to the nucleic acid in which the modified internucleotide linkage is present. For example, the modified nucleotide may improve thermal stability, resistance to degradation, nuclease resistance, solubility, bioavailability, biological activity, reduced immunogenicity, etc. In some embodiments, the modified internucleotide linkage is a phosphorothioate linkage.

[0309] As used herein, "modified nucleotides" refer to nucleotides having one or more chemical modifications when compared to a corresponding reference nucleotide selected from the group consisting of adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, adenine deoxyribonucleotides, guanine deoxyribonucleotides, cytosine deoxyribonucleotides, and thymidine deoxyribonucleotides. The modified nucleotides may have, for example, one or more chemical modifications in their sugar, core base, and / or phosphate groups. Additionally or alternatively, the modified nucleotides may have one or more chemical moieties conjugated to the corresponding reference nucleotides. In some embodiments, the modified nucleotides are 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, or 2'-O-alkyl-modified nucleotides, such as 2'-O-C16 alkyl-modified nucleotides. In some embodiments, the modified nucleotides have phosphate analogs, such as 5'-vinylphosphonate. In some embodiments, the modified nucleotides are abasic moieties or reverse abasic moieties.

[0310] As used herein, the term "synucleinopathy" refers to a disease characterized by fibrillar aggregates of alpha-synuclein protein in the cytoplasm of selective neuronal and glial cell populations in the central and / or peripheral nervous systems.

[0311] As used herein, the term "tauopathy" refers to a disease associated with abnormal tau protein expression, secretion, phosphorylation, cleavage and / or aggregation.

[0312] As used herein, "nucleotide" means an organic compound having a nucleoside (a nucleobase, such as adenine, cytosine, guanine, thymine, or uracil, and a pentose, such as ribose or 2'-deoxyribose) linked to a phosphate group, which can serve as the monomeric unit of nucleic acid polymers such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0313] As used herein, "oligonucleotide" means a polymer of linked nucleotides, each of which may be modified or unmodified. An oligonucleotide is typically less than about 100 nucleotides in length.

[0314] As used herein, "overhang" means one or more unpaired nucleotides that protrude from the duplex structure of a double-stranded oligonucleotide. An overhang may include one or more unpaired nucleotides that extend from the duplex region at the 5' end or 3' end of a double-stranded oligonucleotide. An overhang can be a 3' or 5' overhang on the antisense strand or sense strand of a double-stranded oligonucleotide.

[0315] As used herein, the term "patient" refers to a human patient.

[0316] As used herein, "phosphate analogue" means a chemical moiety that mimics the electrostatic and / or steric properties of a phosphate group. In some embodiments, a phosphate analogue is positioned at the 5' terminal nucleotide of an oligonucleotide, replacing the 5'-phosphate that is typically susceptible to enzymatic removal. 5' phosphate analogues can include phosphatase-resistant linkages. Examples of phosphate analogues include 5' methylenephosphonate (5'-MP) and 5'-(E)-vinylphosphonate (5'-VP). In some embodiments, the phosphate analogue is 5'-VP.

[0317] The term "% sequence identity" or "percent sequence identity" with respect to a reference nucleic acid sequence is defined as the percentage of nucleotides, nucleosides or nucleobases identical with those in the reference nucleic acid sequence, in the candidate sequence, after the optimal alignment sequence and, when necessary, introducing room or overhang to achieve maximum percent sequence identity. The comparison for determining the purpose of percent nucleic acid sequence identity can be achieved in the various ways within the art, for example, using publicly available computer software programs, such as those described in Current Protocols in Molecular Biology (Ausubel et al., ed., 1987, Supplement 30, section 7.7.18, Table 7.7.1), and including BLAST, BLAST-2, ALIGN, ClustalW2.0, Clustal X2.0 or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for measuring the comparison, including any algorithm for achieving maximum comparison on the full length of sequences to be compared. The percentage of "sequence identity" can be determined by comparing two optimally aligned sequences over a comparison window, wherein the nucleic acid sequence fragment in the comparison window may comprise an addition or deletion (e.g., a room or overhang) compared to the reference sequence (which does not comprise an addition or deletion) for optimal alignment of the two sequences. Percentage can be calculated by determining the number of positions at which identical nucleotides, nucleosides, or core bases occur in the two sequences to derive the matching position number, dividing the matching position number by the total number of positions in the comparison window, and multiplying the result by 100 to derive the percentage of sequence identity. The output is the percentage identity of the subject sequence relative to the query sequence.

[0318] As used herein, "RNAi," "RNAi agent," "iRNA," "iRNA agent," and "RNA interference agent" refer to agents that mediate sequence-specific degradation of a target mRNA by RNA interference, e.g., via the RNA-induced silencing complex (RISC) pathway. In some embodiments, the RNAi agent has a sense strand and an antisense strand, and the sense strand and the antisense strand form a duplex. In some embodiments, the sense strand has a delivery moiety, e.g., a delivery moiety conjugated to the 5' or 3' end of the sense strand or to nucleotides of the sense strand.

[0319] As used herein, a "strand" refers to a single contiguous sequence of nucleotides linked together by internucleotide linkages (eg, phosphodiester or phosphorothioate linkages). A strand may have two free ends (eg, a 5' end and a 3' end).

[0320] As used herein, "SNCA" refers to the α-synuclein (SNCA) mRNA transcript. The nucleic acid sequence of the human SNCA mRNA transcript can be found at NM_000345.4:

[0321]

[0322] The amino acid sequence of human SNCA protein can be found at NP_000336.1:

[0323]

[0324] The nucleic acid sequence of the mouse SNCA mRNA transcript can be found at NM_001042451.2; and the amino acid sequence of the mouse SNCA protein can be found at NP_001035916.1. The nucleic acid sequence of the rat SNCA mRNA transcript can be found at NM_019169.3; and the amino acid sequence of the rat SNCA protein can be found at NP_062042.1. The nucleic acid sequence of the monkey SNCA mRNA transcript can be found at XM_005555422.2; and the amino acid sequence of the monkey SNCA protein can be found at XP_005555479.1.

[0325] As used herein, "MAPT" refers to the human MAPT mRNA transcript encoding the microtubule-associated protein Tau. The nucleotide sequences of human MAPT transcript variants and the amino acid sequences of human Tau protein isoforms can be found at:

[0326] i. MAPT transcript variant 1 → Tau protein isoform 1: NM_016835.5 (nucleotide sequence) → NP_058519.3 (amino acid sequence);

[0327] ii. MAPT transcript variant 2 → Tau protein isoform 2: NM_005910.6 (nucleotide sequence) → NP_005901.2 (amino acid sequence);

[0328] iii. MAPT transcript variant 3 → Tau protein isoform 3: NM_016834.5 (nucleotide sequence) → NP_058518.1 (amino acid sequence);

[0329] iv. MAPT transcript variant 4 → Tau protein isoform 4: NM_016841.5 (nucleotide sequence) → NP_058525.1 (amino acid sequence);

[0330] v. MAPT transcript variant 5 → Tau protein isoform 5: NM_001123067.4 (nucleotide sequence) → NP_001116539.1 (amino acid sequence);

[0331] vi. MAPT transcript variant 6 → Tau protein isoform 6: NM_001123066.4 (nucleotide sequence) → NP_001116538.2 (amino acid sequence);

[0332] vii. MAPT transcript variant 7 → Tau protein isoform 7: NM_001203251.2 (nucleotide sequence) → NP_001190180.1 (amino acid sequence);

[0333] viii. MAPT transcript variant 8 → Tau protein isoform 8: NM_001203252.2 (nucleotide sequence) → NP_001190181.1 (amino acid sequence);

[0334] ix. MAPT transcript variant 9 → Tau protein isoform 9: NM_001377265.1 (nucleotide sequence) → NP_001364194.1 (amino acid sequence);

[0335] x. MAPT transcript variant 10 → Tau protein isoform 10: NM_001377266.1 (nucleotide sequence) → NP_001364195.1 (amino acid sequence);

[0336] xi. MAPT transcript variant 11 → Tau protein isoform 11: NM_001377267.1 (nucleotide sequence) → NP_001364196.1 (amino acid sequence);

[0337] xii. MAPT transcript variant 12 → Tau protein isoform 4: NM_001377268.1 (nucleotide sequence) → NP_001364197.1 (amino acid sequence).

[0338] The nucleotide sequence of human MAPT transcript variant 6 (encoding 2N4R Tau) can be found at NM_001123066.4:

[0339]

[0340]

[0341]

[0342] The corresponding amino acid sequence of human Tau protein isoform 6 can be found at NP_001116538.2:

[0343]

[0344] The nucleotide sequence of human MAPT transcript variant 5 (encoding 1N4R Tau) can be found at NM_001123067.4:

[0345]

[0346]

[0347]

[0348] The corresponding amino acid sequence of human Tau protein isoform 5 can be found at NP_001116539.1:

[0349]

[0350] The nucleotide sequence of human MAPT transcript variant 4 (encoding ON3R Tau) can be found at NM_016841.5:

[0351]

[0352]

[0353]

[0354] The corresponding amino acid sequence of human Tau protein isoform 4 can be found at NP_058525.1:

[0355]

[0356] As used herein, "subject" means mammals, including cats, dogs, mice, rats, chimpanzees, apes, monkeys and humans. Preferably, the subject is a human.

[0357] As used herein, "treatment" or "treating" refers to all processes in which there may be a slowing, control, delay or cessation of the progression of a condition or disease disclosed herein, or an amelioration of a condition or disease symptom, but does not necessarily indicate the complete elimination of all conditions or disease symptoms. Treatment includes the administration of a protein or nucleic acid or vector or composition for the treatment of a disease or condition in a patient, particularly a human. Example

[0358] Example 1. Synthesis of compounds and RNAi reagents

[0359] Certain abbreviations are defined as follows: “ACN” refers to acetonitrile; “AEX” refers to anion exchange; “C / D” refers to cleavage and deprotection; “CPG” refers to controlled pore glass; “aCSF” refers to artificial cerebrospinal fluid; “DCM” refers to dichloromethane; “DEA” refers to diethylamine; “DIPEA” refers to N,N-diisopropylethylamine; “DMA” refers to dimethylacetamide; “DMAP” refers to 4-dimethylaminopyridine; “DMF” refers to dimethylformamide; “DMSO” refers to dimethyl sulfoxide; “D "MT" refers to 4,4'-dimethoxytrityl; "EDCI" refers to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; "ES / MS" refers to electrospray ionization mass spectrometry; "EtOAc" refers to ethyl acetate; "EtOH" refers to ethanol and ethyl alcohol; "IP-RP" refers to ion-pair reversed phase; "LC / MS" refers to liquid chromatography-mass spectrometry; "MeOH" refers to methanol and methyl alcohol. alcohol); “MPA” refers to mobile phase A; “MPB” refers to mobile phase B; “MWCO” refers to molecular weight cutoff; “NaOAc” refers to sodium acetate; “NHS” refers to N-hydroxysuccinimide; “NMR” refers to nuclear magnetic resonance; “PBS” refers to phosphate-buffered saline; “PVDF” refers to polyvinylidene fluoride; “RP” refers to reverse phase; “siRNA” refers to small interfering ribonucleic acid; “TCEP” refers to tris(2-carboxyethyl)phosphine; “TEA” refers to triethylamine; “TFA” refers to trifluoroacetic acid; “THF” refers to tetrahydrofuran; “UPLC” refers to ultra performance liquid chromatography; and “UV” refers to ultraviolet light.

[0360] Solution 1

[0361]

[0362] Scheme 1, step A depicts the reaction of compound (1) with 2,2'-disulfide dipyridine in a solvent system such as MeOH and THF to give compound (2). Step B shows the reaction of compound (2) with 3-sulfanylpropionic acid in a solvent such as MeOH to give compound (3). Step C shows the addition of NHS to compound (3) using a coupling agent such as EDCI and a catalyst such as DMAP in a solvent such as DCM to give compound (4). Step D shows the addition of compound (4) to an appropriately modified sense strand in the presence of borate buffer to give compound (5).

[0363] Option 2

[0364]

[0365] Scheme 2, Step A depicts the ring-opening addition of an appropriately substituted (disulfanyl)alcohol reagent to compound (6) using borontrifluoride diethyl etherate in a solvent such as DMA to give compound (7). Step B shows the protection of compound (7) with dimethoxytrityl chloride using a base such as TEA and a catalyst such as DMAP in a solvent such as pyridine to give compound (8). Step C depicts the addition of 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite to compound (8) using a base such as DIPEA in a solvent such as DCM to give compound (9).

[0366] Option 3

[0367]

[0368] Scheme 3, Steps AC show the conversion of compound (6) to compound (12) and is essentially analogous to the process found in Scheme 2, Steps AC.

[0369] Option 4

[0370]

[0371] Scheme 4, step A depicts the tosylation of compound (13) using p-toluenesulfonyl chloride and a base such as pyridine in a solvent such as DCM to give compound (14).

[0372] Option 5

[0373]

[0374] Scheme 5, step A shows the alkylation of compound (15) by (4R,8R)-1-iodo-4,8,12-trimethyltridecane using a base such as potassium carbonate in a solvent such as DMF to give compound (16). Step B shows the coupling of compounds (14) and (16) using a base such as cesium carbonate in a solvent such as DMF to give compound (17). Step C depicts the deprotection of compound (17) using TFA and triethylsilane in a solvent such as DCM to give compound (18). Step D shows the coupling of compound (18) with an appropriately modified sense strand partner in the presence of TCEP to give compound (19).

[0375] Option 6

[0376]

[0377] Scheme 6, step A depicts the reaction of compound (20) with an appropriate thiol, such as 2-((3r,5r,7r)-adamantan-1-yl)ethane-1-thiol or dodecan-1-thiol, in the presence of borate buffer to give compound (21). Step B shows the addition of compound (21) to an appropriate modified sense strand partner in the presence of AMA solution to give compound (22).

[0378] Option 7

[0379]

[0380] Scheme 7, Step A depicts the conversion of compound (8) to compound (23) by first adding trimethylsilyl chloride in a solvent such as pyridine, followed by treatment with 1,2,4-triazole, TEA, and phosphorus oxychloride, and finally the addition of ammonia to give compound (23). Step B shows the acylation of compound (23) using acetic anhydride in a solvent such as DMF to give compound (24). Step C shows the conversion of compound (24) to compound (25) and is essentially similar to the process found in Scheme 2, Step C.

[0381] Preparation 1

[0382] 2-(Dodecyldisulfaneyl)pyridine

[0383]

[0384] 1-Dodecanethiol (12.7 g, 61.4 mmol) was added to a solution of 2,2'-disulfide dipyridine (20.5 g, 92.1 mmol) in MeOH (90 mL) and THF (5 mL). The mixture was stirred at ambient temperature for 16 hours and then concentrated in vacuo. The resulting residue was purified by flash chromatography on silica gel eluting with 0-15% EtOAc in hexanes to give the title compound (14.35 g, 75%) as a colorless oil. ES / MS (m / z): 312 (M+H).

[0385] The compounds in Table 15 were prepared in a manner substantially similar to that found in Preparation 1.

[0386] Table 15

[0387]

[0388] Preparation 2

[0389] 3-(Dodecyldisulfanyl)propionic acid

[0390]

[0391] 3-Sulfanylpropionic acid (7.58 g, 71.44 mmol) was added to a solution of 2-(dodecyldisulfanyl)pyridine (18.55 g, 59.5 mmol) in MeOH (60 mL). The reaction was stirred at ambient temperature for 1 hour and then concentrated in vacuo. The resulting residue was purified via flash chromatography on silica gel eluting with 5-30% EtOAc in hexanes to give the title compound (14 g, 76%) as a colorless oil. 1 HNMR (DMSO-d6) δ2.86 (t, 2H, J = 7.0Hz), 2.71 (t, 2H, J = 7.0Hz), 2.62 (t, 2H, J = 7.0Hz), 1.61 (quint, 2H), 1.33 (q, 2H), 1.28 (s, 16H), 0.90 (t, 3H, J = 6.8Hz).

[0392] The compounds in Table 16 were prepared in a manner substantially similar to that found in Preparation 2.

[0393] Table 16

[0394]

[0395] Preparation 3

[0396] 2,5-Dioxopyrrolidin-1-yl 3-(dodecyldisulfanyl)propionate

[0397]

[0398] NHS (1.35 g, 11.7 mmol) was added to a solution of 3-(dodecyldisulfanyl)propanoic acid (3.0 g, 9.8 mmol), EDCI (2.25 g, 11.7 mmol) and DMAP (0.24 g, 2 mmol) in DCM (39 mL). The mixture was stirred at ambient temperature for 3 hours and then concentrated in vacuo. The resulting residue was purified via flash chromatography on silica gel eluting with 0-40% EtOAc in hexanes to give the title compound (3.2 g, 81%) as a white solid. 1 H NMR(DMSO-d6)δ3.10(t,2H,J=6.3Hz),2.99(t,2H,J=6.3Hz),2.80(s,4H),2.75(t, 2H, J=7.0Hz), 1.61 (quint, 2H), 1.33 (q, 2H), 1.28 (s, 16H), 0.90 (t, 3H, J=6.8Hz).

[0399] The compounds in Table 17 were prepared in a manner substantially similar to that found in Preparation 3.

[0400] Table 17

[0401]

[0402] Preparation 4

[0403] 1-((2R,3R,4R,5R)-3-(2-(tert-butyldisulfanyl)ethoxy)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione

[0404]

[0405] To a suspension of 2,2'-anhydro-1-(β-D-arabinofuranosyl)uracil (4.80 g, 20.8 mmol), 2-(tert-butyldisulfanyl)ethanol (3.80 g, 22.9 mmol) and DMA (21 mL) was added boron trifluoride etherate (4.0 mL, 31.2 mmol). The mixture was heated to 130 ° C for 24 hours, then cooled to ambient temperature and diluted with EtOAc (150 mL). The solution was washed with saturated sodium chloride solution (4x50 mL). Silica gel (10 g) was added to organics, then concentrated to a dry powder in vacuo, and purified by flash chromatography on silica gel eluted with a hexane solution of 50-100% (5% MeOH / EtOAc) to give the title compound (2.10 g, 25%) as a thick, colorless oil. 1 H NMR(CD3CN) δ7.89(d,1H),5.86(d,1H),5.63(d,1H),4.19(q,1H),4.03-3.67(m,6H),3.31(t,1H),3.22(d,1H),2.95(t,2H),1.35(s,9H).

[0406] Preparation 5

[0407] 1-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-(2-(tert-butyldisulfanyl)ethoxy)-4-hydroxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione

[0408]

[0409] A solution of 1-((2R,3R,4R,5R)-3-(2-(tert-butyldisulfanyl)ethoxy)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (1.95 g, 5.0 mmol), 4,4'-bismethoxytrityl chloride (2.23 g, 6.5 mmol), TEA (0.91 mL, 6.5 mmol), DMAP (123 mg, 1.0 mmol) and pyridine (14 mL) was stirred at ambient temperature for 5 hours. The reaction was then quenched with MeOH (10 mL) and concentrated in vacuo. The residue was suspended in DCM (25 mL), added to silica gel (10 g), concentrated in vacuo to a dry powder, and purified via flash chromatography on silica gel eluting with 20-70% EtOAc in hexanes to give the title compound (2.70 g, 78%) as a white foam. 1 H NMR(CD3CN)δ7.76(d,1H),7.46(d,2H),7.40-7.25(m,7H),6.92(d,4H),5.86(d,1H),5.28(d,1H),4. 36(q,1H),4.05-3.87(m,4H),3.80(s,6H),3.45-3.35(m,2H),3.23(d,1H),2.98(t,2H),1.35(s,9H).

[0410] Preparation 6

[0411] (2R,3R,4R,5R)-5-(4-acetylamino-2-oxopyrimidin-1(2H)-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(2-(tert-butyldisulfanyl)ethoxy)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite

[0412]

[0413] The title compound was synthesized starting from 1-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-(2-(tert-butyldisulfanyl)ethoxy)-4-hydroxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione using methods similar to those described in WO2019 / 217459.

[0414] Step 1: A mixture of 1-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-(2-(tert-butyldisulfanyl)ethoxy)-4-hydroxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (2.928 g, 4.21 mmol), pyridine (32.5 mL, 401.6 mmol) and trimethylsilyl chloride (2.14 mL, 16.85 mmol) was stirred at ambient temperature for 30 minutes. After this, 1,2,4-triazole (3.26 g, 47.19 mmol) and triethylamine (8.7 mL, 62.36 mmol) were added, and the mixture was stirred for 10 minutes and then cooled to 0°C. Phosphorus oxychloride (0.98 mL, 10.53 mmol) was added, and the reaction mixture was stirred at 0°C for 2 hours. To 4-amino-1-[(2R, 3R, 4R, 5R)-5-[[bis(4-methoxyphenyl)-phenyl-methoxy] methyl]-3-[2-(tert-butyldisulfanyl) ethoxy]-4-hydroxy-tetrahydrofuran-2-yl] pyrimidine-2-one (2.26g, 77%). ES / MS(m / z): 692(MH).

[0415] Step 2: Acetic anhydride (0.62 mL, 6.51 mmol) was added to a solution of 4-amino-1-[(2R,3R,4R,5R)-5-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-3-[2-(tert-butyldisulfanyl)ethoxy]-4-hydroxy-tetrahydrofuran-2-yl]pyrimidin-2-one (2.26 g, 3.26 mmol) in DMF (20 mL) and stirred at ambient temperature for 22 hours. The reaction was then quenched with water and extracted with DCM (3x). The combined organics were washed with water and saturated aqueous sodium chloride solution, dried over sodium sulfate, filtered, and concentrated in vacuo. The resulting residue was purified by flash chromatography on silica gel eluting with 0-100% MeOH in EtOAc to give N-[1-[(2R,3R,4R,5R)-5-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-3-[2-(tert-butyldisulfanyl)ethoxy]-4-hydroxy-tetrahydrofuran-2-yl]-2-oxo-pyrimidin-4-yl]acetamide (837 mg, 35%). ES / MS (m / z): 734 (MH).

[0416] Step 3: N-[1-[(2R,3R,4R,5R)-5-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-3-[2-(tert-butyldisulfanyl)ethoxy]-4-hydroxy-tetrahydrofuran-2-yl]-2-oxo-pyrimidin-4-yl]acetamide (0.8374 g, 1.138 mmol), DCM (7.539 g, 0.2 M), DIPEA (0.450 g, 3.414 mmol) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.472 g, 1.934 mmol) were added together and stirred at ambient temperature. After one hour, additional 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.11 mL, 0.455 mmol) was added to the mixture. After one hour at ambient temperature, DCM (25 mL) was added. The mixture was washed with saturated aqueous sodium bicarbonate solution (3x), dried over sodium sulfate, filtered, and concentrated in vacuo. The resulting residue was purified by flash chromatography on silica gel eluting with 50-100% EtOAc in hexanes to give the title compound (717 mg, 67%). 1 H NMR(CD3CN)8.45(d,0.5H),8.36(d,0.5H),7.52-7.44(m,3H),7.41-7.26(m,6H),6.97-6.87(m,5H),5.91-5.86(m,1H),4.61 -4.53(m,0.5H),4.48-4.41(m,0.5H),4.23-3.40(m,19H),3.05-2.95(m,2H),2.66(t,1H),2.53(t,1H),1.37-1.03(m,21H). 31 P NMR (CD3CN) 149.7, 148.7.

[0417] Preparation 7

[0418] (2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(2-(tert-butyldisulfanyl)ethoxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite

[0419]

[0420] A solution of 1-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-(2-(tert-butyldisulfanyl)ethoxy)-4-hydroxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (2.70 g, 3.90 mmol), 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (1.52 mL, 6.6 mmol), DIPEA (2.05 mL, 11.7 mmol) and DCM (20 mL) was stirred at ambient temperature. After 1 hour, additional 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (0.36 mL, 1.6 mmol) was added. After 1 h, the crude reaction was poured into 20 mL of a 1% TEA / DCM slurry of silica gel (10 g), concentrated to dryness in vacuo, and purified via flash chromatography on silica gel eluting with 20-70% EtOAc in hexanes containing 1% TEA to give the title compound (2.60 g, 75%) as a white foam. 1 HNMR(CD3CN)δ7.84(d,0.5H),7.76(d,0.5H),7.52-7.25(m,9H),6.96-6.86(m,4H),5.91-5.85(m,1H),5.27-5.21(m,1H),4.5 6-4.41(m,1H),4.21-3.35(m,17H),2.98-2.91(m,2H),2.73-2.67(m,1H),2.58-2.52(m,1H),1.34(d,9H),1.26-0.97(m,12H). 31 P NMR (CD3CN) δ 149.7, 149.1.

[0421] Preparation 8

[0422] 1-((2R,3R,4R,5R)-3-(2-(((3S,5S,7S)-adamantan-1-yl)disulfanyl)ethoxy)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione

[0423]

[0424] To a suspension of 2,2'-anhydro-1-(β-D-arabinofuranosyl)uracil (1.70 g, 7.37 mmol), 2-(1-adamantyldisulfanyl)ethanol (2.70 g, 11.0 mmol) and DMA (8 mL) was added boron trifluoride etherate (1.4 mL, 11.0 mmol). The mixture was heated to 130°C for 12 hours and then cooled to ambient temperature. The mixture was diluted with EtOAc (50 mL) and washed with saturated aqueous sodium chloride solution (4 x 20 mL). Silica gel (10 g) was added to the organics, concentrated in vacuo to a dry powder, and purified via flash chromatography on silica gel eluted with 50-100% (5% MeOH / EtOAc) in hexane to give the title compound (0.76 g, 22%) as a thick, light brown oil. 1 H NMR(CD3CN)δ7.89(d,1H),5.86(d,1H),5.64(d,1H),4.23-4.15(m,1H),4.03-3.67(m,6H),3.29(br s,1H),3.21(br s,1H),2.91(t,2H),2.11-2.05(m,3H),1.90-1.85(m,6H),1.78-1.67(m,6H).

[0425] Preparation 9

[0426] 1-((2R,3R,4R,5R)-3-(2-(((3S,5S,7S)-adamantan-1-yl)disulfanyl)ethoxy)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione

[0427]

[0428] A solution of 1-((2R,3R,4R,5R)-3-(2-(((3S,5S,7S)-adamantan-1-yl)disulfanyl)ethoxy)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (0.76 g, 1.6 mmol), 4,4'-bismethoxytrityl chloride (0.73 g, 2.1 mmol), TEA (0.30 mL, 2.1 mmol), DMAP (40 mg, 0.32 mmol) and pyridine (5 mL) was stirred at ambient temperature for 16 hours. The reaction was then quenched with MeOH (1 mL) and concentrated in vacuo. The residue was suspended in DCM (5 mL), added to silica gel (5 g), concentrated to dryness in vacuo, and purified via flash chromatography on silica gel eluting with 20-70% EtOAc in hexanes to give the title compound as a white foam (0.80 g, 64%). 1 H NMR(CD3CN)δ7.75(d,1H),7.46(d,2H),7.40-7.25(m,7H),6.92(d,4H),5.86(d,1H),5.28(d,1H),4.36(q,1H),4.05-3.87( m,4H),3.80(s,6H),3.46-3.34(m,2H),3.24(d,1H),2.93(t,2H),2.11-2.05(m,3H),1.90-1.85(m,6H),1.78-1.67(m,6H).

[0429] Preparation 10

[0430] (2R,3R,4R,5R)-4-(2-(((3S,5S,7S)-adamantan-1-yl)disulfanyl)ethoxy)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite

[0431]

[0432] A solution of 1-((2R,3R,4R,5R)-3-(2-(((3S,5S,7S)-adamantan-1-yl)disulfanyl)ethoxy)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (0.69 g, 0.89 mmol), 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (0.35 mL, 1.5 mmol), DIPEA (0.47 mL, 2.7 mmol) and DCM (5 mL) was stirred at ambient temperature. After 1 hour, additional 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (0.12 mL, 0.53 mmol) was added. After 1 h, the crude reaction was poured into 10 mL of a 1% TEA / DCM slurry of silica gel (3 g), concentrated to dryness in vacuo, and purified via flash chromatography on silica gel eluting with 20-70% EtOAc in hexanes containing 1% TEA to give the title compound (0.63 g, 73%) as a white foam. 1 H NMR(CD3CN)δ7.84(d,0.5H),7.75(d,0.5H),7.52-7.25(m,9H),6.96-6.8 6(m,4H),5.91-5.85(m,1H),5.29-5.21(m,1H),4.56-4.41(m,1H),4.21-3 .35(m,17H),2.96-2.85(m,2H),2.73-2.67(m,1H),2.58-2.52(m,1H),2.1 1-2.05(m,3H),1.90-1.85(m,6H),1.78-1.67(m,6H),1.26-0.97(m,12H). 31 P NMR (CD3CN) δ 149.7, 149.1.

[0433] Preparation 11

[0434] S-(2-(((2R,3R,4R,5R)-2-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)ethyl) 2,2-dimethylpropylthioate

[0435]

[0436] To a suspension of 2,2'-anhydro-1-(β-D-arabinofuranosyl)uracil (1.5 g, 6.6 mmol), S-(2-hydroxyethyl) 2,2-dimethylpropylthioate (4.3 g, 26.53 mmol), and DMA (7.37 mL) was added boron trifluoride etherate (4.38 mL, 16.6 mmol). The mixture was heated to 100°C for 6 hours, then cooled to ambient temperature and concentrated in vacuo to remove excess ether. The resulting residue was purified via flash chromatography on silica gel eluting with 0-100% (0.1% formic acid / water) in ACN to give the title compound (0.5 g, 19.4%) as a white foam. 1 HNMR(CDCl3)δ7.72(d,1H),5.73(m,2H),4.31(t,1H),4.17(dd,1H),4.07-3.93(m,4H),3.70(dt,1H),3.10(m,2H),1.24(s,9H).

[0437] Preparation 12

[0438] S-(2-(((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-2-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-hydroxytetrahydrofuran-3-yl)oxy)ethyl) 2,2-dimethylpropylthioate

[0439]

[0440] A solution of S-(2-(((2R,3R,4R,5R)-2-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)ethyl)2,2-dimethylpropylthioate (2.0 g, 5.1 mmol), 4,4'-bismethoxytrityl chloride (1.92 g, 5.7 mmol), DMAP (6.3 mg, 51.5 μmol) and pyridine (14.3 mL) was stirred at ambient temperature for 14.5 hours. The reaction was then concentrated in vacuo. The residue was loaded onto silica gel and purified via flash chromatography on silica gel eluting with 0-100% EtOAc containing 1% TEA in hexanes containing 1% TEA to give the title compound (2.92 g, 82.1%) as a white foam. 1H NMR(DMSO-d6)δ11.38(s,1H),8.57(m,1H),7.78(tt,1H),7.70(d,1H),7.40-7.23(m,10H),6.90(d,4H),5.79(d,1H), 5.29(d,1H),5.19(d,1H),4.18(q,1H),3.97m,2H),3.74(s,6H),3.61(m,1H),3.26(m,2H),3.02(m,2H),1.16(s,9H).

[0441] Preparation 13

[0442] S-(2-(((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(((2-cyanoethoxy)(diisopropylamino)phosphaneyl)oxy)-2-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-yl)oxy)ethyl) 2,2-dimethylpropylthioate

[0443]

[0444] A solution of S-(2-(((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-2-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-hydroxytetrahydrofuran-3-yl)oxy)ethyl)2,2-dimethylpropylthioate (2.9 g, 4.2 mmol), 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (1.13 mL, 5.07 mmol), DIPEA (1.84 mL, 10.57 mmol) and DCM (42.3 mL) was stirred at ambient temperature. After 1 hour, additional 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (0.94 mL, 4.23 mmol) was added. After 1 hour, additional 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (0.19 mL, 0.85 mmol) was added. After 10 minutes, the crude reaction was loaded onto silica gel and purified via flash chromatography on silica gel eluting with 0-100% EtOAc in hexanes containing 1% TEA to give the title compound as a white foam (2.32 g, 61.5%). 1H NMR(DMSO-d6)δ11.38(s,1H),7.77(q,1H),7.41-7.22(m,9H),6.9(m,4H),5.8(t,1H),5.27(dd,1H) ,4.39(m,1H),4.18-4.07(m,1H),3.84-3.50(m,12H),3.01(m,2H),2.79(t,1H),1.25-1.10(m,21H). 31 P NMR(DMSO-d6)δ149.3,148.5.

[0445] Preparation 14

[0446] 2-(Triphenylmethylthio)ethyl 4-methylbenzenesulfonate

[0447]

[0448] The product of 2-(tritylthio)ethanol (1.00g, 3.03mmol), DCM (9mL), p-toluenesulfonyl chloride (0.8665g, 4.545mmol) and pyridine (0.50mL, 6.06mmol) was stirred at ambient temperature for 16 hours.Mixture was diluted with water (50mL), then extracted with EtOAc (3x75mL).The organic layer merged was washed with saturated sodium chloride aqueous solution (2x150mL), dried with NaSO4, and concentrated in a vacuum.The crude reaction was diluted with DCM, loaded onto silica gel, and purified via flash chromatography on silica gel eluting with the hexane solution of 5-40% EtOAc to give the title compound (330mg, 23%) as a brown oil. 1 H NMR(CDCl3)7.75-7.67(m,2H),7.38-7.17(m,17H),3.62(t,2H),2.52(t,2H),2.47(s,3H).

[0449] Preparation 15

[0450] 2-((4R,8R)-4,8,12-Trimethyltridecyl)-1,2,3,4-tetrahydroisoquinolin-6-ol

[0451]

[0452] Potassium carbonate (0.51 g, 3.7 mmol) was added to a solution of 1,2,3,4-tetrahydroisoquinolin-6-ol (0.50 g, 3.4 mmol) in DMF (25 mL). (4R, 8R)-1-iodo-4,8,12-trimethyltridecane (1.3 g, 3.7 mmol) was then added to the reaction. The mixture was stirred at 65 ° C for 4 hours, then cooled to ambient temperature and concentrated in vacuo. The resulting crude material was purified by flash chromatography on silica gel eluted with a gradient of 0-100% EtOAc in hexane to give the title compound (0.81 g, 65%) as a white solid. 1 H NMR(CDCl3)δ6.89(d,1H),6.61(dd,1H),6.53(d,1H),3.61(s,2H),2.91-2.68(m,4H),2.53(t,2H),1.77-1.00(m,19H),0.94-0.81(m,12H).

[0453] Preparation 16

[0454] 2-((4R,8R)-4,8,12-Trimethyltridecyl)-6-(2-(tritylthio)ethoxy)-1,2,3,4-tetrahydroisoquinoline

[0455]

[0456] A solution of 2-((4R,8R)-4,8,12-trimethyltridecyl)-1,2,3,4-tetrahydroisoquinolin-6-ol (0.20 g, 0.54 mmol), DMF (2.1 mL), cesium carbonate (0.35 g, 1.10 mmol) and 2-(tritylthio)ethyl 4-methylbenzenesulfonate (0.33 g, 0.70 mmol) was stirred at 45° C. for 3 hours. The reaction was concentrated in vacuo, then diluted with DCM, loaded onto silica gel, and purified via flash chromatography on silica gel eluting with 0-40% EtOAc in hexanes to give the title compound (0.143 g, 39%) as a viscous, light yellow oil. 1 H NMR(CDCl3)7.48-7.43(m,8H),7.32-7.21(m,7H),6.89(d,1H),6.53-6.49(m,2H),3.71(t,2H),3.5 5(s,2H),2.88-2.82(m,2H),2.72-2.66(m,2H),2.63(t,2H),2.50-2.44(m,2H),1.69-0.78(m,31H).

[0457] Preparation 17

[0458] 2-((2-((4R,8R)-4,8,12-Trimethyltridecyl)-1,2,3,4-tetrahydroisoquinolin-6-yl)oxy)ethane-1-thiol

[0459]

[0460] A solution of 2-((4R,8R)-4,8,12-trimethyltridecyl)-6-(2-(tritylthio)ethoxy)-1,2,3,4-tetrahydroisoquinoline (0.1426 g, 0.21 mmol), DCM (0.7 mL), TFA (0.41 mL, 5.3 mmol), and triethylsilane (0.07 mL, 0.4 mmol) was stirred at ambient temperature for 1 hour. The reaction was concentrated in vacuo and then diluted with EtOAc (75 mL). The organic layer was washed with saturated aqueous NaHCO (1 x 50 mL), and the aqueous layer was back-extracted with EtOAc (1 x 75 mL). The organic layers were combined, dried over NaSO, and concentrated in vacuo. The resulting material was diluted with DCM, then loaded onto silica gel and purified via silica gel flash chromatography eluting with 0-100% EtOAc in hexanes containing 0.5% TEA to give the title compound as a clear oil (0.065 g, 71%). 1 H NMR(CDCl3)7.04(d,1H),6.83(dd,1H),6.73(d,1H),4.62(d,1H),4.11(t,2 H),3.99(d,1H),3.80-3.72(m,1H),3.38-2.87(m,7H),2.26-0.70(m,31H).

[0461] Preparation 18

[0462] 6-((2-((3r,5r,7r)-adamantan-1-yl)ethyl)disulfanyl)nicotinic acid

[0463]

[0464] 6-[(5-Carboxy-2-pyridyl)disulfanyl]pyridine-3-carboxylic acid (617 mg, 2 mmol) was stirred in THF (10 mL) and 20X borate buffer (10 mL) until all solids dissolved. 2-((3r,5r,7r)-adamantan-1-yl)ethane-1-thiol (196 mg, 1 mmol) was added in one portion and the reaction was stirred at ambient temperature for 3 hours. The reaction was then concentrated to a total volume of ~5 mL, and the residue was purified by reverse phase flash chromatography (C18 column) eluting with a gradient of 0-70% acetonitrile / 10 mM ammonium bicarbonate to give the title compound (180 mg, 52%) as a white solid. 1 H NMR(DMSO-d6)8.82(d,1H),8.18(dd,1H),7.74(d,1H),2.86-2.77(m,2H),1.94-1.84(m,3H),1.68-1.53(m,6H),1.48-1.37(m,8H).

[0465] Preparation 19

[0466] 6-(Dodecyldisulfanyl)nicotinic acid

[0467]

[0468] The title compound was prepared from dodecan-1-thiol in a manner essentially analogous to the procedure found in Preparation 18. 1 H NMR(DMSO-d6)8.91(d,1H),8.27(dd,1H),7.91(d,1H),2.87(t,2H),1.67-1.55(m,2H),1.40-1.14(m,18H),0.86(t,3H).

[0469] C12 ADS-linked siRNA

[0470]

[0471] The sense strand (3.1 g, 0.44 mmol) synthesized using the conditions found in the protocol below in 4X borate buffer (113 mL) was treated with a solution of 2,5-dioxopyrrolidin-1-yl 3-(dodecyldisulfanyl)propionate (5.3 g, 4.4 mmol) in ACN (113 mL). The solution was shaken at 30° C. for 1.5 hours. The reaction was quenched by diluting with water and adjusting the pH to 7 with 1.2 M aqueous HCl. The solution was then concentrated via Genevac to remove the organic solvent and provide the crude oligonucleotide.

[0472] The AKTA HPLC was performed using reverse phase on a source 15RPC column (MPA: 50 mM NaOAc with 10% ACN and MPB: 80% acetonitrile / water). TM Crude oligonucleotides were purified using the ELISA Pure purification system. In all cases, fractions containing >85% purity by mass and >5% impurities were pooled.

[0473] The oligonucleotide of purification uses 15mL 3K MWCO centrifugal spin tube with 3500xg desalination~30 minutes.Rinse oligonucleotide with RNase-free water, until eluate conductivity reaches<100usemi / cm.After desalination is complete, add 2-3mL RNase-free water, then suction 10x, and retaining material (retainment) is transferred in 50mL falcon tube.By measuring the compound concentration on the filter via nanodrop, this repeats until the complete transfer of oligonucleotide.Then final oligonucleotide carries out nanofiltration 2x with 3500xg for 2 minutes via 15mL 100K MWCO centrifugal spin tube.The final desalted oligonucleotide is analyzed for concentration (nano drop at A260 place), characterized by IP-RP, the LCMS for mass purity and the UPLC for UV purity.ES / MS(m / z):7324.6(M+H).

[0474] The compounds below were prepared in a manner substantially similar to that found for the preparation of C12 ADS-linked siRNA.

[0475] Table 18

[0476]

[0477] SS-C12-linked siRNA

[0478]

[0479] After oligonucleotide synthesis (sense strand was synthesized using the conditions found in the scheme below), the CPG loaded with the oligonucleotide was washed with diethylamine and then dried under vacuum. 50 μmol of loaded CPG was added to a 50 mL falcon tube and 50 mg of 6-(dodecyldisulfanyl)nicotinic acid was added to the same tube, followed by 15 mL of AMA solution (29% ammonium hydroxide in water: 40% methylamine in water, 1: 1) and shaken at ambient temperature. After 1 hour, >80% of the desired product mass was observed. The solution was then concentrated on a Genevac to remove organic matter and provide the crude oligonucleotide. The crude oligonucleotide was filtered using a 0.2 micron needle filter and then purified by AKTA TMPure purification system uses anion exchange (AEX) source 15Q column for purification. For AEX, use Source with the following TM 15Q column: MPA: 20 mM NaH2PO4 with 15% ACN, pH 7.4 and MPB: 20 ​​mM NaH2PO4 with 1 M NaBr, 15% ACN, pH 7.4. In all cases, fractions containing greater than 85% mass purity and no impurities >5% were pooled.

[0480] The oligonucleotide of purification uses 15mL 3K MWCO centrifugal spin tube with 3500xg desalination~30 minutes.Rinse oligonucleotide with RNA-free water, until eluate conductivity reaches<100 μ S / cm.After desalination is completed, add the RNA-free water of 2-3mL, then suction 10x, retainer is transferred in 50mL falcon pipe, by measuring the compound concentration on the filter via nanodrop, this repeats until the complete transfer of oligonucleotide.Finally desalted oligonucleotide is analyzed (nano drop at A260 place) for concentration, characterized by the IP-RP LCMS for mass purity and the UPLC for UV purity.ES / MS(m / e):7239.6.

[0481] The compounds in Table 4 were prepared in a manner substantially similar to that found in the preparation of USS-C12 linked siRNAs.

[0482] Table 4. Exemplary modified nucleotides

[0483]

[0484] SS-adamantyl-linked siRNA

[0485]

[0486] The sense strand (0.0077 mmol in 15 mL of water), synthesized using the conditions found in the protocol below, was added to 20X borate buffer (2.25 mL) and then treated with a solution of 2,5-dioxopyrrolidin-1-yl 3-(pyridin-2-yldisulfanyl)propionate (0.0241 g, 0.0772 mmol) (CAS No. 68181-17-9) in MeCN (3.75 mL). The solution was shaken at ambient temperature for 30 minutes. The solution was then diluted to 40 mL with RNase-free water to bring the organic solvent concentration to ≤10%. Excess 2,5-dioxopyrrolidin-1-yl 3-(pyridin-2-yldisulfanyl)propionate was removed using a 15 mL 3K MWCO centrifuge spin tube at 3500 x g for ~30 minutes. The oligonucleotide was rinsed three times with RNase-free water. After removing 2,5-dioxopyrrolidine-1-base 3-(pyridin-2-yl disulfanyl) propionate, add the RNase-free water of 1mL, then suction 10x, and retentate is transferred in 5mL falcon pipe.By measuring the compound concentration on the filter via nanodrop, this repeats until the complete transfer of oligonucleotide.Final oligonucleotide is analyzed (nano drop at A260 place) for concentration, characterized by IP-RP, LCMS for mass purity and UPLC for UV purity.ES / MS (m / z): 7196.02 (M+H).

[0487] The sense strand synthesized above (0.0035 mmol in 1.4 mL of water) was treated with a solution of 1-adamantanethiol (0.0119 g, 0.0705 mmol) (CAS No. 34301-54-7) in THF (1.40 mL). The solution was shaken at 50° C. for 16 hours. The solution was then concentrated via Genevac to remove the organic solvent and provide the crude oligonucleotide. The oligonucleotide was purified by reverse phase chromatography on a source 15RPC 10×200 mm column (MPA: 10 mM NaOAc with 2% ACN and MPB: 80% ACN in water) via AKTA chromatography. TMThe crude oligonucleotides were purified using the Pure purification system. The desired product eluted at 10% using a 2-50% gradient over 8 column volumes. In all cases, fractions containing a mass purity greater than 85% and no impurities >5% were merged. The solution was then concentrated via Genevac to remove the organic solvent, and purified oligonucleotides were provided. Purified oligonucleotides were desalted using a 15mL 3K MWCO centrifugal spin tube at 3500xg for ~30 minutes. The oligonucleotides were rinsed with RNase-free water until the eluate conductivity reached <100usemi / cm. After desalination was complete, 1mL of RNase-free water was added, followed by aspiration 10x, and the retentate was transferred to a 5mL falcon tube. The concentration of the compound on the filter was measured via nanodrop, which was repeated until the complete transfer of the oligonucleotide. The final desalted oligonucleotides were analyzed (nano drop at A260) for concentration and characterized by IP-RP, LCMS for mass purity, and UPLC for UV purity. ES / MS (m / z): 7253.15 (M+H).

[0488] SS-C2-tetraisoquinoline-linked siRNA

[0489]

[0490] The sense strand (1mM aqueous solution) synthesized using the conditions found in the scheme below is treated with 10 equivalents of TCEP. The reaction is allowed to vibrate at 45°C for 18 hours. The solution is then transferred to a 15mL 3K MWCO centrifugal spin filter and rotated at 3500xg for 30 minutes. After adding 15mL of water, this process is repeated. The aqueous solution of siRNA (0.5mM) is treated with an ACN solution of disulfide dipyridine (20 equivalents). The final ACN content is 20%. After 1 hour, the reaction is diluted with water so that the ACN content reaches 10%. The solution is then transferred to a 15mL 3K MWCO centrifugal spin filter and rotated at 3500xg for 30 minutes. After adding 15mL of water, this process is repeated. An aqueous solution of siRNA (1 mM) was treated with 2-((2-((4R,8R)-4,8,12-trimethyltridecyl)-1,2,3,4-tetrahydroisoquinolin-6-yl)oxy)ethane-1-thiol (10 equivalents) dissolved in THF. The concentration of the thiol solution in THF was calculated to give a final THF content of 60%. The solution was shaken at 45°C for 48 hours. The THF was then removed via vacuum centrifugation, and the conjugated siRNA was purified via reverse phase chromatography (Source15 RPC column; MPA: 50 mM NaOAc with 10% ACN and MPB: 50 mM NaOAc with 80% ACN). The purified oligonucleotides were desalted using a 15 mL 3K MWCO centrifugal spin tube at 3500 x g for ~30 minutes. The oligonucleotides were rinsed with RNase-free water until the eluate conductivity reached <100 usemi / cm. The final oligonucleotide was then nanofiltered 2x at 3500 x g for 2 minutes using a 15 mL 100K MWCO centrifugal spin tube. The final desalted oligonucleotide was analyzed for concentration (nanodrop at A260) and characterized by IP-RP LCMS for mass purity and UPLC for UV purity. ES / MS (m / z): 7423.6 (M+H).

[0491] Synthesis of dsRNA

[0492] The single strands of the RNA duplex (sense and antisense strands) are synthesized by MerMade TM 12 was synthesized on a solid support. The sequences of the sense and antisense strands are shown in Tables 1 and 2. Oligonucleotides were synthesized via phosphoramidite chemistry at a 5, 10, 25, or 50 μmol scale.

[0493] All single strands were synthesized using commercially available standard support mA. Standard reagents were used for oligonucleotide synthesis (Table 5), with 0.1 M hydroxanthin in pyridine as the sulfurizing agent and 20% DEA in ACN as the auxiliary detergent after synthesis. All monomers (Table 6) were prepared at 0.1 M in ACN and contained molecular sieve capture bags.

[0494] Oligonucleotides were cleaved and deprotected (C / D) at 45°C for 20 hours. The sense strand was C / Ded from CPG using ammonium hydroxide (28-30%, cold), while 3% DEA in ammonium hydroxide (28-30%, cold) was used for the antisense strand. When the quality data obtained confirmed the identity of the sequence, the C / D was completed by IP-RPLCMS. Depending on the scale, CPG was filtered through a 0.45um PVDF non-pinhole filter, a 0.22um PVDF filter, and a 100ml PBS filter. Vacuum filtration or 0.22um PVDF Quickrelease filtration was performed. The CPG was backwashed / rinsed with 30% ACN / RNase-free water or 30% EtOH / RNase-free water, then filtered through the same filtration apparatus and combined with the first filtrate. This was repeated twice. The material was then evenly distributed into 50 mL falcon tubes for filtration via Genevac. TM After concentration, crude oligonucleotides were diluted back to the synthesis scale with RNase-free water and passed through a 0.45 μm PVDF syringe filter, a 0.22 μm PVDF Vacuum filtration or 0.22 μm PVDF Quick release for filtration.

[0495] Using ion exchange (AEX) or reverse phase (RP) source 15Q-RP column, by AKTA TM Pure purification system to purify crude oligonucleotides. For AEX, the column temperature was maintained at 65°C. TM 15Q column with MPA: 20 mM NaH2PO4, 15% ACN, pH 7.4 and MPB: 20 ​​mM NaH2PO4, 1 M NaBr, 15% ACN, pH 7.4. For RP, Source TM 15Q-RP column with MPA: 50 mM NaOAc containing 10% ACN and MPB: 50 mM NaOAc containing 80% ACN. In all cases, fractions containing a mass purity greater than 85% and no impurities >5% were pooled.

[0496] The oligonucleotide of purification uses 15mL 3K MWCO centrifugal spin tube with 3500xg desalination~30 minutes.With RNAse-free water, rinse oligonucleotide, until eluate conductivity reaches<100msemi / cm.After desalination is completed, add the RNAse-free water of 2-3mL, then suction 10x, retentate is transferred in 50mL falcon pipe, by measuring the compound concentration on the filter via nanodrop, this repeats until the complete transfer of oligonucleotide.Final oligonucleotide then carries out nanofiltration 2x with 3500xg totally in 2 minutes via 15mL 100KMWCO centrifugal spin tube.The oligonucleotide of final desalination is analyzed (nano drop at A260 place) for concentration, characterizes by the IP-RP LCMS for mass purity and the UPLC for UV purity.

[0497] For duplex preparation, equimolar amounts of sense and antisense strands were combined and heated at 65°C for 10 minutes, then slowly cooled to ambient temperature over 40 minutes. The integrity of the duplexes was confirmed by UPLC analysis and characterized by LCMS using IP-RP. All duplexes were nanofiltered and then purified by Charles River Endotoxin levels were measured by the Cartridge Device to give the final compound of the RNAi conjugate.For in vivo analysis, appropriate amounts of duplexes were lyophilized and then reconstituted in 1X PBS for rodent studies and in aCSF for non-human primate studies.

[0498] The molecular weights of exemplary SNCA and MAPT RNAi agents are shown in Tables 7 and 8.

[0499] Table 5 - Oligonucleotide synthesis reagents

[0500]

[0501]

[0502] Table 6 - Phosphoramidites

[0503]

[0504]

[0505] Table 7 - Molecular weights of exemplary SNCA RNAi agents

[0506]

[0507]

[0508]

[0509]

[0510] "S" refers to the sense strand; "AS" refers to the antisense strand.

[0511] Table 8 - Molecular weights of exemplary MAPT RNAi agents

[0512]

[0513]

[0514]

[0515] "S" refers to the sense strand; "AS" refers to the antisense strand.

[0516] Example 2. In vitro characterization of RNAi reagents

[0517] Selected RNAi agents were tested in vitro for target mRNA inhibition in cultured cells, including 293T cells, mouse cortical neurons (MCNs), and / or human induced pluripotent stem cells (hiPSCs).

[0518] Materials and Methods

[0519] 293T luciferase transfection, RNAi treatment and analysis: 293T cells transfected with pMIR-luciferase constructs (Invitrogen, Waltham, MA) containing the target sequence were plated overnight at 37°C; 5% CO2. On the second day, cells were transfected with siRNA using RNAiMAX (Invitrogen, Waltham, MA) using the protocol provided by the manufacturer. The cells were incubated for 48 hours at 37°C; 5% CO2. The plates were cooled to room temperature, and then an equal volume of Bio-Glo (Promega, Madison, WI) was added to each well. The plates were incubated at room temperature in the dark and read on a BioTek Neos2 plate reader (Agilent, Santa Clara, CA).

[0520] Mouse primary cortical neurons (MCN) culture and RNAi treatment and analysis: Mouse primary cortical neurons were isolated from wild-type C57BL6 mouse embryos at E18 or hTau C57BL6 transgenic mouse embryos expressing human tau transgenes at E18. Cells were plated in poly-D-lysine-coated 96-well plates at a density of 40k cells / well and cultured in NbActiv1 (BrainBits, LLC) containing 1% antibiotic / antimycotic (Corning) at 37°C in a humidified chamber with 5% CO2 in a tissue culture incubator for 7 days. On the 7th day, half the culture medium was removed from each well, and a 2x concentration of RNAi in a culture medium with 2% FBS was added for CRC treatment and incubated with the cells for another 7, 14, or 21 days. Half of the culture medium was replaced with fresh culture medium every 7 days. At the end of RNAi treatment, RT-qPCR was performed using the TaqMan Fast Advanced Cell-to-CT kit to quantify SNCA or MAPT mRNA levels. Specifically, cells were lysed, cDNA was generated on a Mastercycler X50a (Eppendorf), and qPCR was performed on a QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems). α-synuclein (ThermoFisher, Mm00447333_m1) and human MAPT (ThermoFisher, Hs00902194_m1) gene expression levels were normalized using respective probes for β-actin (ThermoFisher, Mm02619580_g1).

[0521] Human induced pluripotent stem cell-derived neurons (hiPSC neurons) culture and RNAi treatment and analysis: Doxycycline-inducible Neurogenin 2 (NGN2) human induced pluripotent stem cells (hiPSCs) were developed by Bioneer for Eli Lilly. hiPSCs were induced with doxycycline for three days (DIV3) to initiate neuronal differentiation and plated at 30k / well on 96-well PDL and laminin-coated plates and grown in Neuronal Differentiation Media (NDM) consisting of DMEM / F12 (Life Technologies 11330-057), Neurobasal medium (Gibco 15240062), antibiotics, supplements, growth factors, and doxycycline in an incubator (37°C / 5% CO2). Cells were half-fed every seven days, and at DIV21, RNAi reagents were serially diluted in NDM, and cells were treated with RNAi by aspirating 75 mL and adding 75 mL of 2x RNAi concentration for the final concentration of 1x RNAi according to the dilution. Cells were half-fed every seven days after treatment by removing half of the culture medium and re-adding fresh NDM. Cell lysates were harvested at DIV35 (after 14 days) or DIV42 (after 21 days) and analyzed using TaqMan Fast Advanced Cells-to-C T RT-qPCR was performed using a PCR amplification kit (ThermoFisher, A35377), and mRNA knockdown was determined using the SNCA probe (ThermoFisher, Hs00240907_m1) or MAPT probe (ThermoFisher, Hs00902194_m1) as the gene of interest, and the ACTb probe (ThermoFisher, Hs99999903_m1) as the housekeeping gene.

[0522] result

[0523] Tables 9A-9C summarize the in vitro activities of selected SNCA RNAi agents. As shown in Tables 9A-9C, the tested RNAi agents knocked down SNCA expression in several different cell lines.

[0524] Table 9A. In vitro activity of selected SNCA RNAi agents in primary mouse cortical neurons

[0525]

[0526]

[0527] Table 9B. In vitro activity of selected SNCA RNAi agents in the T293 luciferase assay

[0528]

[0529] Table 9C. In vitro activity of selected SNCA RNAi reagents in hiPSC neurons

[0530]

[0531] The in vitro activities of selected MAPT RNAi agents are summarized in Table 10. As shown in Table 10, the tested RNAi agents knocked down MAPT expression in mouse cortical neurons.

[0532] Table 10. In vitro activity of MAPT RNAi reagents in primary mouse cortical neurons

[0533]

[0534]

[0535] Example 3. In vivo characterization of selected RNAi agents

[0536] The efficacy of selected RNAi agents was also studied in Sprague Dawley rats. Six rats received intrathecal delivery of 300 μg or 100 μg of SNCA RNAi agent or PBS (phosphate-buffered saline) and were sacrificed 7 days after infusion. Rat SNCA mRNA expression in the spinal cord and brain was measured and analyzed by qPCR. The results are shown in Table 11A.

[0537] Similar studies were performed using 0.4 mg, 1.2 mg, or 2.4 mg of SNCA RNAi reagent, and rats were sacrificed 2 months after administration of the SNCA RNAi reagent. Rat SNCA mRNA expression in the spinal cord and brain was measured and analyzed by qPCR. The results are shown in Table 11B.

[0538] Table 11A. Percent knockdown (KD) of SNCA mRNA in rats

[0539]

[0540] ND means not determined.

[0541] Table 11B. Percent knockdown (KD) of SNCA mRNA in rats

[0542]

[0543] The efficacy of selected SNCA RNAi agents was studied in wild-type C56BL / 6N mice. Fifty-nine mice received an intracerebroventricular (ICV) injection of 30 μg of RNAi agent or PBS (phosphate-buffered saline) and were sacrificed 21 days after injection. Mouse SNCA mRNA expression in the spinal cord and brain was measured and analyzed by quantitative PCR (qPCR). The results are shown in Table 11C.

[0544] Table 11C. Percent knockdown (KD) of SNCA mRNA in mice

[0545]

[0546] ND means not detected.

[0547] The efficacy of the selected MAPT RNAi reagent was also studied in hTau transgenic mice (Andorfer et al., J Neurochem 2003, 86, 582–590) that express human MAPT RNA and lack mouse MAPT RNA. Six mice received intracerebroventricular (ICV) injections of 100 μg or 250 μg of MAPT RNAi reagent or PBS (phosphate-buffered saline) and were killed on the 14th, 35th, or 59th day after injection. The MAPT mRNA expression in the brain was measured and analyzed by quantitative PCR (qPCR). The results are shown in Tables 11D-11F.

[0548] Table 11D. Percent knockdown (KD) of MAPT mRNA in hTau mice 14 days after treatment with 100 μg of MAPT RNAi reagent

[0549]

[0550] Table 11E. Percent knockdown (KD) of MAPT mRNA in hTau mice 35 days after treatment with 100 μg of MAPT RNAi reagent

[0551]

[0552] Table 11F. Percent knockdown (KD) of MAPT mRNA in hTau mice 59 days after treatment with 250 μg of MAPT RNAi reagent

[0553]

[0554] RNAi reagent tissue distribution and microgliosis analysis

[0555] Fixed rat right hemisphere brain and spinal cord (fourth cervical vertebral segment [C4 or SC2], fourth thoracic vertebral segment [T4 or SC5], eleventh thoracic vertebral segment [T11 or SC8], and first lumbar vertebral segment [L1 or SC10]) were stored in ice-cold (4°C) 1x PBS (phosphate-buffered saline, CAS number: 7732-18-5) until tissue processing. Samples were processed on a Leica ASP6025S Tissue Processor and embedded using a Leica HistoCore Arcadia H-Heated Paraffin Embedding Station and a HistoCore Arcadia C-Cold Plate. Brain tissue was embedded along the sagittal plane, and spinal cord was embedded along the transverse plane. Blocks were stored at room temperature until sectioning.

[0556] Blocks were sectioned using a HistoCore AUTOCUT-Automated Rotary Microtome (Leica Biosystems, 149AUTO00C1). Briefly, blocks were trimmed to fully expose the tissue, and 5 μm thick sections were obtained and mounted on Fisherbrand TM Superfrost TM The brain was sectioned from the midline at 0 μm, 500 μm, and 1000 μm, and the spinal cord was sectioned serially. The sections were dried overnight at room temperature before staining.

[0557] Slides were stained on a LeicaBOND RX (Leica Biosystems, 21.2821). For each brain, one slide from each step level was stained, and for the spinal cord, one serial section was stained. All slides were stained using the Advanced Cell Diagnostics (ACD) miRNAscope TM LS Reagent Kit-RED (Advanced Cell Diagnostics, 324600) was used for staining. Antisense siRNA strands were detected using a probe (Advanced Cell Diagnostics, 1063228-S1, for Eli Lilly & Co.). Other reagents used included miRNAscope TMLS Negative Control Probe-SR-Scramble-S1 (Advanced Cell Diagnostics, 727888-S1) and BOND Polymer Refine Red Detection (Leica Biosystems, DS9390). All slides were prepared according to the manufacturer's instructions for miRNAscope. TM The staining was performed according to the protocol of with slight modifications. The washes in steps 75, 85, and 92 were modified to open washes. Once stained, the slides were washed in DI water for 2 minutes, dried at 60° C. for 30 minutes, and coverslipped.

[0558] Slides are scanned on a Leica Aperio GT450 Slide Scanner and uploaded to Aperio eSlide Manager for analysis. Use Aperio ImageScope, manually outline frontal cortex, brainstem, C4, T4, T11 and L1, and run image analysis algorithms for each outlined area to calculate "percent pixel positive rate". In brief, the algorithm is adapted from Aperio ImageScope "Positive Pixel Count 2002-08-11" algorithm. The output of the algorithm comprises a pixel positive rate, in which positive pixels equal the antisense strand of the siRNA molecule, and all other pixels are negative pixels. "Percent pixel positive rate" is the positive pixel number in the image divided by the total pixel number that comprises negative pixels in the image, then multiplied by 100. The results are shown in Table 12, which shows that the RNAi reagent tested has a good distribution overview across brain and spinal cord.

[0559] Table 12. miRNA scope TM Tissue distribution of RNAi reagents measured by percent pixel positivity.

[0560]

[0561] Additional slides were stained with anti-Iba1 antibody (FUJIFILM Wako, 013-27691, 1:2000) diluted in BOND Primary Antibody Diluent (Leica, AR9352) using IHC Protocol F (Leica) and the BOND Polymer Refine Detection Kit (Leica, DS9800). Briefly, after blocking with H2O2 (3–4% (v / v)), the primary antibody was applied. Polymer (in tris-buffered saline / 0.1% ProClin TM950 containing 10% (v / v) animal serum anti-rabbit poly HRP-IgG (<25μg / mL), followed by DAB part 1 (66mM 3,3'-diaminobenzidine tetrahydrochloride hydrate in stabilizer solution) part B (≤0.1% (v / v) hydrogen peroxide in stabilizer solution) and hematoxylin (<0.1% hematoxylin) counterstaining. After staining, the slides were dehydrated using a Leica ST5010 Autostainer XL and coverslipped with Surgipath Micromount mounting medium (Leica, 3801731). The slides were scanned on a Leica Aperio GT450 Slide Scanner and uploaded to Aperio eSlide Manager for analysis. Using Aperio Image Scope, images were opened and microgliosis was evaluated using the scoring parameters shown in Table 13.

[0562] Table 13. Microgliosis score

[0563]

[0564] The results of microgliosis evaluation are shown in Table 14.

[0565] Table 14. Evaluation of microgliosis

[0566]

[0567]

[0568]

[0569]

[0570]

[0571] Sequence Listing

[0572]

[0573]

[0574]

[0575]

[0576]

[0577]

[0578]

[0579]

[0580]

[0581]

[0582]

[0583]

Claims

1. A compound comprising any one of Formulas Ia, Ib, Ic, II-IV, or XXI: Where n is an integer from 1 to 4, Where n is an integer from 0 to 2, wherein B is a nucleobase selected from adenine, cytosine, guanine, thymine, uracil or a derivative thereof.

2. The compound of claim 1, wherein the compound comprises Formula Ia, Ib, or Ic.

3. The compound of claim 1, wherein the compound comprises Formula II.

4. The compound of claim 1, wherein the compound comprises Formula III.

5. The compound of claim 1, wherein the compound comprises Formula IV. The compound according to claim 5 , wherein n is 0. The compound according to claim 5 , wherein n is 2.

8. The compound of claim 1, wherein the compound comprises Formula XXI.

9. The compound according to any one of claims 1 to 8, wherein the compound is a nucleoside, nucleotide or an analog thereof.

10. An RNAi agent comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a duplex region, wherein the sense strand or the antisense strand comprises a modified nucleotide of any one of Formulas Ia, Ib, Ic, II-IV, or XXI: Where n is an integer from 1 to 4, Where n is an integer from 0 to 2, and wherein B is a nucleobase selected from adenine, cytosine, guanine, thymine, uracil or a derivative thereof.

11. The RNAi agent of claim 10, wherein the sense strand or antisense strand comprises modified nucleotides of Formula Ia, Ib, or Ic.

12. The RNAi agent of claim 10, wherein the sense strand or antisense strand comprises a modified nucleotide of Formula II.

13. The RNAi agent of claim 10, wherein the sense strand or antisense strand comprises a modified nucleotide of Formula III.

14. The RNAi agent of claim 10, wherein the sense strand or antisense strand comprises a modified nucleotide of Formula IV. The RNAi agent of claim 14 , wherein n is 0. The RNAi agent of claim 14 , wherein n is 2.

17. The RNAi agent of claim 10, wherein the sense strand or antisense strand comprises modified nucleotides of Formula XXI.

18. The RNAi agent of any one of claims 10-17, wherein the sense strand is 15 to 50 nucleotides in length.

19. The RNAi agent of any one of claims 10-18, wherein the antisense strand is 15 to 30 nucleotides in length.

20. The RNAi agent of any one of claims 10-19, wherein the sense strand is 21 nucleotides in length.

21. The RNAi agent of any one of claims 10-20, wherein the antisense strand is 23 nucleotides in length.

22. The RNAi agent of any one of claims 10-21, wherein the duplex region is 21 nucleotides in length.

23. The RNAi agent of any one of claims 10-22, wherein the sense strand comprises a modified nucleotide of any one of Formulas Ia, Ib, Ic, II-IV, or XXI at any one of positions 1-6 or 12-21 from the 5' end.

24. The RNAi agent of claim 23, wherein the sense strand comprises a modified nucleotide of any one of Formulas Ia, Ib, Ic, II-IV, or XXI at position 13 from the 5' end.

25. The RNAi agent of any one of claims 10-24, wherein the antisense strand comprises a modified nucleotide of any one of Formulas Ia, Ib, Ic, II-IV, or XXI at any one of positions 6-10 or 15-18 from the 5' end.

26. The RNAi agent of any one of claims 10-25, wherein the sense strand and the antisense strand further comprise one or more 2'-fluoro modified nucleotides and 2'-O-methyl modified nucleotides.

27. The RNAi agent of claim 26, wherein the sense strand comprises four 2'-fluoro modified nucleotides at positions 7, 9, 10, and 11 from the 5' end of the sense strand.

28. The RNAi agent of claim 27, wherein the nucleotides at other positions of the sense strand are 2'-O-methyl modified nucleotides.

29. The RNAi agent of any one of claims 26-28, wherein the antisense strand comprises four 2'-fluoro modified nucleotides at positions 2, 6, 14, and 16 from the 5' end of the antisense strand.

30. The RNAi agent of claim 29, wherein nucleotides at other positions of the antisense strand are 2'-O-methyl modified nucleotides.

31. The RNAi agent of claim 26, wherein the sense strand comprises three 2'-fluoro modified nucleotides at positions 9, 10, and 11 from the 5' end of the sense strand.

32. The RNAi agent of claim 31, wherein the nucleotides at other positions of the sense strand are 2'-O-methyl modified nucleotides.

33. The RNAi agent of any one of claims 26-28, 31, 32, wherein the antisense strand comprises five 2'-fluoro modified nucleotides at positions 2, 5, 7, 14, and 16 from the 5' end of the antisense strand.

34. The RNAi agent of claim 33, wherein nucleotides at other positions of the antisense strand are 2'-O-methyl modified nucleotides.

35. The RNAi agent of any one of claims 26-28, 31, 32, wherein the antisense strand comprises five 2'-fluoro modified nucleotides at positions 2, 5, 8, 14, and 16 from the 5' end of the antisense strand.

36. The RNAi agent of claim 35, wherein nucleotides at other positions of the antisense strand are 2'-O-methyl modified nucleotides.

37. The RNAi agent of any one of claims 26-28, 31, 32, wherein the antisense strand comprises five 2'-fluoro modified nucleotides at positions 2, 3, 7, 14, and 16 from the 5' end of the antisense strand.

38. The RNAi agent of claim 37, wherein nucleotides at other positions of the antisense strand are 2'-O-methyl modified nucleotides.

39. The RNAi agent of any one of claims 10-38, wherein the sense and antisense strands comprise one or more modified internucleotide linkages.

40. The RNAi agent of claim 39, wherein the one or more modified internucleotide linkages are phosphorothioate linkages.

41. The RNAi agent of claim 39 or 40, wherein the sense strand comprises four or five phosphorothioate linkages.

42. The RNAi agent of any one of claims 39-41, wherein the antisense strand comprises four or five phosphorothioate linkages.

43. The RNAi agent of any one of claims 10-42, wherein the antisense strand comprises a phosphate analog at the 5' end.

44. The RNAi agent of claim 43, wherein the phosphate analog is 5'-vinylphosphonate.

45. The RNAi agent of any one of claims 10-44, wherein the sense strand comprises an abasic portion or an inverted abasic portion.

46. ​​The RNAi agent of any one of claims 10-45, wherein the antisense strand is complementary to a target mRNA selected from SNCA, MAPT, APP, ATXN2, ATXN3, SARM1, APOE, BACE1, FMR1, LRRK2, HTT, SOD1, SCN10A, SCN9A, or CACNA1B mRNA.

47. The RNAi agent of claim 46, wherein the antisense strand is complementary to SNCA mRNA.

48. The RNAi agent of claim 47, wherein the sense strand and antisense strand comprise a pair of nucleic acid sequences selected from the group consisting of: (a) the sense strand comprises SEQ ID NO: 1, and the antisense strand comprises SEQ ID NO: 2; (b) the sense strand comprises any one of SEQ ID NO: 3, 5 or 20, and the antisense strand comprises SEQ ID NO: 4; (c) the sense strand comprises any one of SEQ ID NOs: 6, 8-19, 37, 38, or 67-81, and the antisense strand comprises SEQ ID NO: 7; (d) the sense strand comprises SEQ ID NO: 19, and the antisense strand comprises SEQ ID NO: 66; (e) the sense strand comprises SEQ ID NO: 9 or 16, and the antisense strand comprises SEQ ID NO: 82; (f) the sense strand comprises SEQ ID NO: 83, and the antisense strand comprises SEQ ID NO: 84; (g) the sense strand comprises SEQ ID NO: 85, and the antisense strand comprises SEQ ID NO: 86; (h) the sense strand comprises SEQ ID NO: 87, and the antisense strand comprises SEQ ID NO: 88; (i) the sense strand comprises SEQ ID NO: 89, and the antisense strand comprises SEQ ID NO: 90; (j) the sense strand comprises SEQ ID NO: 91, and the antisense strand comprises SEQ ID NO: 92 or 93; (k) the sense strand comprises SEQ ID NO: 94, and the antisense strand comprises SEQ ID NO: 95; (1) the sense strand comprises SEQ ID NO: 96, and the antisense strand comprises SEQ ID NO: 97; (m) the sense strand comprises SEQ ID NO: 98, and the antisense strand comprises SEQ ID NO: 99; (n) the sense strand comprises SEQ ID NO: 100, and the antisense strand comprises SEQ ID NO: 101; (o) the sense strand comprises SEQ ID NO: 102, and the antisense strand comprises SEQ ID NO: 103; (p) the sense strand comprises SEQ ID NO: 104, and the antisense strand comprises SEQ ID NO: 105; (q) the sense strand comprises SEQ ID NO: 106, and the antisense strand comprises SEQ ID NO: 107; (r) the sense strand comprises SEQ ID NO: 108, and the antisense strand comprises SEQ ID NO: 109 or 122; (s) the sense strand comprises SEQ ID NO: 110, and the antisense strand comprises SEQ ID NO: 111; (t) the sense strand comprises SEQ ID NO: 112, and the antisense strand comprises SEQ ID NO: 113; (u) the sense strand comprises SEQ ID NO: 114, and the antisense strand comprises SEQ ID NO: 115; (v) the sense strand comprises SEQ ID NO: 116, and the antisense strand comprises SEQ ID NO: 117; (w) the sense strand comprises SEQ ID NO: 118, and the antisense strand comprises SEQ ID NO: 119; (x) the sense strand comprises SEQ ID NO: 120, and the antisense strand comprises SEQ ID NO: 121; and (y) the sense strand comprises SEQ ID NO: 123, and the antisense strand comprises SEQ ID NO:

124.

49. The RNAi agent of claim 47 or 48, wherein the sense strand and the antisense strand consist of a pair of nucleic acid sequences selected from the group consisting of: (a) the sense strand consists of any one of SEQ ID NO: 3, 5 or 20, and the antisense strand consists of SEQ ID NO: 4; and (b) the sense strand consists of any one of SEQ ID NOs: 6, 8-19, 37, 38, or 67-81, and the antisense strand consists of SEQ ID NO: 7; (c) the sense strand consists of SEQ ID NO: 19, and the antisense strand consists of SEQ ID NO: 66; (d) the sense strand consists of SEQ ID NO: 9 or 16, and the antisense strand consists of SEQ ID NO: 82; (e) the sense strand consists of SEQ ID NO: 87, and the antisense strand consists of SEQ ID NO: 88; (f) the sense strand consists of SEQ ID NO: 89, and the antisense strand consists of SEQ ID NO: 90; (g) the sense strand consists of SEQ ID NO: 91, and the antisense strand consists of SEQ ID NO: 92 or 93; (h) the sense strand consists of SEQ ID NO: 106, and the antisense strand consists of SEQ ID NO: 107; (i) the sense strand consists of SEQ ID NO: 108, and the antisense strand consists of SEQ ID NO: 109 or 122; (j) the sense strand consists of SEQ ID NO: 110, and the antisense strand consists of SEQ ID NO: 111; (k) the sense strand consists of SEQ ID NO: 112, and the antisense strand consists of SEQ ID NO: 113; (1) the sense strand consists of SEQ ID NO: 114, and the antisense strand consists of SEQ ID NO: 115; (m) the sense strand consists of SEQ ID NO: 116, and the antisense strand consists of SEQ ID NO: 117; (n) the sense strand consists of SEQ ID NO: 118, and the antisense strand consists of SEQ ID NO: 119; (o) the sense strand consists of SEQ ID NO: 120, and the antisense strand consists of SEQ ID NO: 121; and (p) the sense strand consists of SEQ ID NO: 123, and the antisense strand consists of SEQ ID NO:

124.

50. The RNAi agent of claim 46, wherein the antisense strand is complementary to MAPT mRNA.

51. The RNAi agent of claim 50, wherein the sense strand and antisense strand comprise a pair of nucleic acid sequences selected from the group consisting of: (a) the sense strand comprises SEQ ID NO: 21, and the antisense strand comprises SEQ ID NO: 22; (b) the sense strand comprises SEQ ID NO: 23, and the antisense strand comprises SEQ ID NO: 24; (c) the sense strand comprises SEQ ID NO: 25, and the antisense strand comprises SEQ ID NO: 26; (d) the sense strand comprises any one of SEQ ID NOs: 27, 33, 39, 40, 47-49, and the antisense strand comprises SEQ ID NO: 28; (e) the sense strand comprises any one of SEQ ID NOs: 29, 34, 35, 42, 50-51, 53, and the antisense strand comprises SEQ ID NO: 30; (f) the sense strand comprises any one of SEQ ID NOs: 31, 36, 43, 52, 154, 155, 161-163, and the antisense strand comprises SEQ ID NO: 32; (g) the sense strand comprises SEQ ID NO: 39 or 40, and the antisense strand comprises SEQ ID NO: 41; (h) the sense strand comprises SEQ ID NO: 44 or 46, and the antisense strand comprises SEQ ID NO: 45; (i) the sense strand comprises SEQ ID NO: 53, and the antisense strand comprises SEQ ID NO: 54 or 55; (j) the sense strand comprises SEQ ID NO: 56, and the antisense strand comprises SEQ ID NO: 57; (k) the sense strand comprises SEQ ID NO: 125, and the antisense strand comprises SEQ ID NO: 126; (1) the sense strand comprises SEQ ID NO: 127, and the antisense strand comprises SEQ ID NO: 128; (m) the sense strand comprises SEQ ID NO: 129, and the antisense strand comprises SEQ ID NO: 130; (n) the sense strand comprises SEQ ID NO: 131, and the antisense strand comprises SEQ ID NO: 132; (o) the sense strand comprises SEQ ID NO: 133, and the antisense strand comprises SEQ ID NO: 134; (p) the sense strand comprises SEQ ID NO: 135, and the antisense strand comprises SEQ ID NO: 136; (q) the sense strand comprises SEQ ID NO: 137, and the antisense strand comprises SEQ ID NO: 138; (r) the sense strand comprises SEQ ID NO: 139, and the antisense strand comprises SEQ ID NO: 140; (s) the sense strand comprises SEQ ID NO: 141, and the antisense strand comprises SEQ ID NO: 142; (t) the sense strand comprises SEQ ID NO: 143, and the antisense strand comprises SEQ ID NO: 144; (u) the sense strand comprises SEQ ID NO: 145, and the antisense strand comprises SEQ ID NO: 146; (v) the sense strand comprises SEQ ID NO: 147, and the antisense strand comprises SEQ ID NO: 148; (w) the sense strand comprises SEQ ID NO: 34, and the antisense strand comprises any one of SEQ ID NOs: 149, 150, and 151; (x) the sense strand comprises SEQ ID NO: 31, and the antisense strand comprises any one of SEQ ID NOs: 152, 153, 156-159, 164, 165; (y) the sense strand comprises SEQ ID NO: 160, and the antisense strand comprises SEQ ID NO: 152; and (z) the sense strand comprises SEQ ID NO: 43 or 166, and the antisense strand comprises SEQ ID NO:

156.

52. The RNAi agent of claim 50 or 51, wherein the sense strand and the antisense strand consist of a pair of nucleic acid sequences selected from the group consisting of: (a) the sense strand consists of any one of SEQ ID NOs: 27, 33, 39, 40, 47-49, and the antisense strand consists of SEQ ID NO: 28; (b) the sense strand consists of any one of SEQ ID NOs: 29, 34, 35, 42, 50-51, 53, and the antisense strand consists of SEQ ID NO: 30; and (c) the sense strand consists of any one of SEQ ID NOs: 31, 36, 43, 52, 154, 155, 161-163, and the antisense strand consists of SEQ ID NO: 32; (d) the sense strand consists of SEQ ID NO: 39 or 40, and the antisense strand consists of SEQ ID NO: 41; (e) the sense strand consists of SEQ ID NO: 44 or 46, and the antisense strand consists of SEQ ID NO: 45; (f) the sense strand consists of SEQ ID NO: 53, and the antisense strand consists of SEQ ID NO: 54 or 55; (g) the sense strand consists of SEQ ID NO: 137, and the antisense strand consists of SEQ ID NO: 138; (h) the sense strand consists of SEQ ID NO: 139, and the antisense strand consists of SEQ ID NO: 140; (i) the sense strand consists of SEQ ID NO: 141, and the antisense strand consists of SEQ ID NO: 142; (j) the sense strand consists of SEQ ID NO: 143, and the antisense strand consists of SEQ ID NO: 144; (k) the sense strand consists of SEQ ID NO: 145, and the antisense strand consists of SEQ ID NO: 146; (1) the sense strand consists of SEQ ID NO: 147, and the antisense strand consists of SEQ ID NO: 148; (m) the sense strand consists of SEQ ID NO: 34, and the antisense strand consists of any one of SEQ ID NOs: 149, 150, and 151; (n) the sense strand consists of SEQ ID NO: 31, and the antisense strand consists of any one of SEQ ID NOs: 152, 153, 156-159, 164, 165; (o) the sense strand consists of SEQ ID NO: 160, and the antisense strand consists of SEQ ID NO: 152; and (p) the sense strand consists of SEQ ID NO: 43 or 166, and the antisense strand consists of SEQ ID NO:

156.

53. A pharmaceutical composition comprising the compound according to any one of claims 1-9, or the RNAi agent according to any one of claims 10-52, and a pharmaceutically acceptable carrier.

54. A method of treating a neurodegenerative disease in a patient in need thereof, the method comprising administering to the patient an effective amount of a compound according to any one of claims 1-9, an RNAi agent according to any one of claims 10-52, or a pharmaceutical composition according to claim 53.

55. The method of claim 54, wherein the neurodegenerative disease is a synucleinopathy selected from Parkinson's disease, Alzheimer's disease, multiple system atrophy, or dementia with Lewy bodies.

56. The method of claim 54, wherein the neurodegenerative disease is a tauopathy selected from the group consisting of Alzheimer's disease, frontotemporal dementia (FTD), frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), frontotemporal lobar degeneration (FTLD), behavioral variant frontotemporal dementia (bvFTD), nonfluent variant primary progressive aphasia (nfvPPA), Parkinson's disease, Pick's disease (PiD), primary progressive aphasia - semantic (PPA-S), primary progressive aphasia - pauci-lexic (PPA-L), multisystem tauopathy with Alzheimer's disease (MSTD), neurofibrillary tangle (NFT) dementia, FTD with motor neuron disease, progressive supranuclear palsy (PSP), amyotrophic lateral sclerosis / parkinsonism-dementia complex (ALS-PDC), argyrophilic grain dementia (AGD), British amyloidosis (BAM) and tauopathies. Powdery mildew, cerebral amyloid angiopathy, chronic traumatic encephalopathy (CTE), corticobasal degeneration (CBD), Creutzfeldt-Jakob disease (CJD), dementia pugilistica, diffuse neurofibrillary tangles with calcifications, Down syndrome, epilepsy, Gerstmann-Straussler-Schenck disease, Hallervorden-Spatz disease, Huntington's disease, inclusion body myositis, lead encephalopathy, Lytico-Bodig disease, meningioangiomatosis, multiple system atrophy, myotonic dystrophy , Niemann-Pick disease type C (NP-C), non-Guam motor neuron disease with neurofibrillary tangles, postencephalitic Parkinsonism, prion amyloid angiopathy, progressive subcortical gliosis, dementia with tangles, dementia with predominant tangles, ganglioglioma, gangliocytoma, subacute sclerosing encephalitis, tuberous sclerosis, lipofuscinosis, primary age-related tauopathy (PART), or globular glial tauopathy (GGT).

57. The method of any one of claims 54-56, wherein the compound or RNAi agent is administered to the patient intrathecally, intracerebroventricularly, or via intracisterna magna injection.

58. A method of inhibiting or reducing a target mRNA in a cell, the method comprising contacting a cell comprising the target mRNA with a compound according to any one of claims 1-9, an RNAi agent according to any one of claims 10-52, or a pharmaceutical composition according to claim 53.

59. A compound according to any one of claims 1-9, an RNAi agent according to any one of claims 10-52, or a pharmaceutical composition according to claim 53 for use in therapy.

60. Use of the compound of any one of claims 1-9, the RNAi agent of any one of claims 10-52, or the pharmaceutical composition of claim 53 for treating a neurodegenerative disease.

61. The compound, RNAi agent or pharmaceutical composition for use according to claim 60, wherein the neurodegenerative disease is a synucleinopathy selected from Parkinson's disease, Alzheimer's disease, multiple system atrophy or dementia with Lewy bodies.

62. The compound, RNAi agent or pharmaceutical composition for use according to claim 60, wherein the neurodegenerative disease is a tauopathy selected from the group consisting of Alzheimer's disease, frontotemporal dementia (FTD), frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), frontotemporal lobar degeneration (FTLD), behavioral variant frontotemporal dementia (bvFTD), nonfluent variant primary progressive aphasia (nfvPPA), Parkinson's disease, Pick's disease (PiD), primary progressive aphasia - semantic (PPA-S), primary progressive aphasia - pauci-verbal (PPA-L), multisystem tauopathy with Alzheimer's disease (MSTD), neurofibrillary tangles (NFT) dementia, FTD with motor neuron disease, progressive supranuclear palsy (PSP), amyotrophic lateral sclerosis / parkinsonism-dementia complex (ALS-PDC), argyrophilic grain dementia (AGD), and tauopathies. GD), British amyloid angiopathy, cerebral amyloid angiopathy, chronic traumatic encephalopathy (CTE), corticobasal degeneration (CBD), Creutzfeldt-Jakob disease (CJD), dementia pugilistica, diffuse neurofibrillary tangles with calcifications, Down syndrome, epilepsy, Gerstmann-Straussler-Schenck disease, Hallervorden-Spatz disease, Huntington's disease, inclusion body myositis, lead encephalopathy, Lytico-Bodig disease, meningioangiomatosis, multiple system atrophy, myotonic leukocytosis Malnutrition, Niemann-Pick disease type C (NP-C), non-Guam motor neuron disease with neurofibrillary tangles, postencephalitic Parkinson's disease, prion amyloid angiopathy, progressive subcortical gliosis, dementia with tangles, dementia with predominant tangles, ganglioglioma, gangliocytoma, subacute sclerosing encephalitis, tuberous sclerosis, lipofuscinosis, primary age-related tauopathy (PART), or globular glial tauopathy (GGT).

63. Use of a compound according to any one of claims 1-9 or an RNAi agent according to any one of claims 10-52 in the preparation of a medicament for treating a neurodegenerative disease.

64. The use according to claim 63, wherein the neurodegenerative disease is a synucleinopathy selected from Parkinson's disease, Alzheimer's disease, multiple system atrophy or dementia with Lewy bodies.

65. The method of claim 63, wherein the neurodegenerative disease is a tauopathy selected from the group consisting of Alzheimer's disease, frontotemporal dementia (FTD), frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), frontotemporal lobar degeneration (FTLD), behavioral variant frontotemporal dementia (bvFTD), nonfluent variant primary progressive aphasia (nfvPPA), Parkinson's disease, Pick's disease (PiD), primary progressive aphasia - semantic (PPA-S), primary progressive aphasia - pauci-lexic (PPA-L), multisystem tauopathy with Alzheimer's disease (MSTD), neurofibrillary tangle (NFT) dementia, FTD with motor neuron disease, progressive supranuclear palsy (PSP), amyotrophic lateral sclerosis / parkinsonism-dementia complex (ALS-PDC), argyrophilic grain dementia (AGD), British amyloidosis Powdery mildew, cerebral amyloid angiopathy, chronic traumatic encephalopathy (CTE), corticobasal degeneration (CBD), Creutzfeldt-Jakob disease (CJD), dementia pugilistica, diffuse neurofibrillary tangles with calcifications, Down syndrome, epilepsy, Gerstmann-Straussler-Schenck disease, Hallervorden-Spatz disease, Huntington's disease, inclusion body myositis, lead encephalopathy, Lytico-Bodig disease, meningioangiomatosis, multiple system atrophy, myotonic dystrophy , Niemann-Pick disease type C (NP-C), non-Guam motor neuron disease with neurofibrillary tangles, postencephalitic Parkinsonism, prion amyloid angiopathy, progressive subcortical gliosis, dementia with tangles, dementia with predominant tangles, ganglioglioma, gangliocytoma, subacute sclerosing encephalitis, tuberous sclerosis, lipofuscinosis, primary age-related tauopathy (PART), or globular glial tauopathy (GGT).

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