RNAi Agents with Modified Nucleotides

Abstract

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

Description

[Technical Field]

[0001] (Sequence Listing) This application has been submitted with a Sequence Listing in ST.26 XML format. The Sequence Listing is provided as a file entitled "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 technology]

[0002] RNA interference (RNAi) is a highly conserved regulatory mechanism by which sequence-specific gene silencing is achieved by double-stranded RNA (dsRNA) molecules (Fire et al., Nature 391:806-811, 1998). Physiologically, RNAi is initiated by the Dicer enzyme, which cleaves long dsRNA molecules into short double-stranded fragments of approximately 21-23 nucleotides called siRNAs. After the siRNAs are unwound, the antisense strand is loaded into the RNA-induced silencing complex (RISC) and hybridizes to complementary sequences in the target mRNA, while the sense strand is degraded (Nakanishi, Wiley Interdiscip. Rev. RNA, Vol. 7:637-660, 2016). Silencing of target mRNAs is then mediated by Ago2, the catalytic component of RISC (Bobbin and Rossi, Annu. Rev. Pharmacol. Toxicol., Vol. 56:103-122, 2016).

[0003] RNAi agents are susceptible to nuclease degradation. One of the challenges of RNAi-based therapy is the ability to deliver intact RNAi agents to target tissues and cells. Chemical modification and / or ligand conjugation can be used to improve the stability and delivery of RNAi agents to target tissues and cells. However, some chemical modification and / or ligand conjugation are not well tolerated and cause safety concerns in human patients (Chi, et al. Today. 2017 May; 22(5): 823-833).

[0004] There remains a need for safe and effective RNAi agents suitable for therapeutic use, eg, for the treatment of human disease. Summary of the Invention

[0005] 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.

[0006] In one aspect, provided herein is a compound having the following formula:

[0007] [ka] (wherein n is an integer of 1 to 4),

[0008] [ka] (wherein n is an integer of 0 to 2),

[0009] [ka] and wherein B is a nucleobase selected from adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), or derivatives 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.

[0010] In some embodiments, the compound comprising any one of Formulas Ia, Ib, Ic, II-IV, or XXI is a nucleoside, nucleotide, or analog thereof.

[0011] In another aspect, provided herein is an RNAi agent comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a double-stranded region, and wherein the sense strand or the antisense strand has the following formula:

[0012] [ka] (wherein n is an integer of 1 to 4),

[0013] [ka] (wherein n is an integer of 0 to 2),

[0014] [ka] and wherein B is a nucleobase selected from adenine, cytosine, guanine, thymine, uracil, or derivatives 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.

[0015] In some embodiments, the sense strand is 15 to 50 nucleotides in length, hi some embodiments, the antisense strand is 15 to 30 nucleotides in length.

[0016] In some embodiments, the sense strand comprises a modified nucleotide of any one of Formulas Ia, Ib, Ic, II-IV, or XXI, e.g., 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 one of Formulas Ia, Ib, Ic, II-IV, or XXI, e.g., at any one of positions 6-10 or 15-18 from the 5' end.

[0017] In some embodiments, the sense and antisense strands further comprise one or more 2'-fluoro- and 2'-O-methyl-modified nucleotides. In some embodiments, the sense and antisense strands comprise one or more modified internucleotide linkages, such as phosphorothioate linkages.

[0018] In some embodiments, the antisense strand comprises a phosphate analog (e.g., 5'-vinylphosphonate) at the 5'-terminus. In some embodiments, the sense strand comprises an abasic portion or an inverted abasic portion.

[0019] 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.

[0020] In another aspect, provided herein is a pharmaceutical composition comprising a compound or RNAi agent described herein and a pharmaceutically acceptable carrier.

[0021] In a further aspect, provided herein are methods of treating a neurodegenerative disease (e.g., a synucleinopathy or a tauopathy) in a patient in need thereof, such methods comprising administering to the patient an effective amount of a compound, RNAi agent, or pharmaceutical composition described herein. In some embodiments, the compound, RNAi agent, or pharmaceutical composition is administered to the patient via intrathecal, intracerebroventricular, or intracisternal injection.

[0022] Also provided herein are methods for inhibiting or reducing a target mRNA in a cell, the method comprising contacting a cell containing the target mRNA with a compound, RNAi agent, or pharmaceutical composition described herein.

[0023] In another aspect, provided herein are compounds, RNAi agents, or pharmaceutical compositions for use in therapy. Also provided herein are compounds, RNAi agents, or pharmaceutical compositions for use in treating neurodegenerative diseases, such as synucleinopathy or tauopathy. Also provided herein is the use of a compound or RNAi agent in the manufacture of a medicament for treating a neurodegenerative disease, such as a synucleinopathy or tauopathy. DETAILED DESCRIPTION OF THE INVENTION

[0024] 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.

[0025] In one aspect, provided herein is a compound having the following formula:

[0026] [ka] (wherein n is an integer of 1 to 4),

[0027] [ka] (wherein n is an integer of 0 to 2),

[0028] [ka] and 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, or 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-carboxy-cytidine, 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.

[0029] In some embodiments, the compound comprising any one of Formulas Ia, Ib, Ic, II-IV, or XXI is a nucleoside, nucleotide, or analog thereof.

[0030] In some embodiments, provided herein are compounds of formula Ia

[0031] [ka] Including, 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, or 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-carboxy-cytidine, phenoxazine, N6-alkyl-A, or O6-alkyl-G.

[0032] In some embodiments, provided herein are compounds of formula Ib

[0033] [ka] Including, 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, or 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-carboxy-cytidine, phenoxazine, N6-alkyl-A, or O6-alkyl-G.

[0034] In some embodiments, provided herein are compounds of formula Ic

[0035] [ka] (wherein n is an integer of 1 to 4), 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, or 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-carboxy-cytidine, 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.

[0036] In some embodiments, provided herein are compounds of formula II

[0037] [ka] Including, 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, or 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-carboxy-cytidine, phenoxazine, N6-alkyl-A, or O6-alkyl-G.

[0038] In some embodiments, provided herein are compounds of formula III

[0039] [ka] Including, 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, or 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-carboxy-cytidine, phenoxazine, N6-alkyl-A, or O6-alkyl-G.

[0040] In some embodiments, provided herein are compounds of formula IV

[0041] [ka] (wherein n is an integer of 0 to 2), 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, or 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-carboxy-cytidine, 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.

[0042] In some embodiments, provided herein are compounds of formula XXI

[0043] [ka] Including, 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, or 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-carboxy-cytidine, phenoxazine, N6-alkyl-A, or O6-alkyl-G.

[0044] In some embodiments, provided herein are compounds comprising any one of Formulas Va-VIIIa:

[0045] [ka]

[0046] In some embodiments, the compound comprising any one of Formulas Va-VIIIa is a nucleoside, nucleotide, or analog thereof.

[0047] In some embodiments, provided herein are compounds comprising any one of Formulas Vb-VIIIb:

[0048] [ka]

[0049] In some embodiments, the compound comprising any one of Formulas Vb-VIIIb is a nucleoside, nucleotide, or analog thereof.

[0050] In some embodiments, provided herein are compounds comprising any one of Formulas Vc-VIIIc:

[0051] [ka] (wherein n is an integer of 1 to 4),

[0052] [ka] (wherein n is an integer of 1 to 4),

[0053] [ka] (wherein n is an integer of 1 to 4),

[0054] [ka] (wherein n is an integer of 1 to 4)

[0055] In some embodiments, n in Formulas Vc-VIIIc is 1. In some embodiments, n in Formulas Vc-VIIIc is 2. In some embodiments, n in Formulas Vc-VIIIc is 3. In some embodiments, n in Formulas Vc-VIIIc is 4.

[0056] In some embodiments, the compound comprising any one of Formulas Vc-VIIIc is a nucleoside, nucleotide, or analog thereof.

[0057] In some embodiments, provided herein are compounds comprising any one of Formulas IX-XII:

[0058] [ka]

[0059] In some embodiments, the compound comprising any one of Formulas IX-XII is a nucleoside, nucleotide, or analog thereof.

[0060] In some embodiments, provided herein are compounds comprising any one of Formulas XIII-XVI:

[0061] [ka]

[0062] In some embodiments, the compound comprising any one of Formulas XIII-XVI is a nucleoside, nucleotide, or analog thereof.

[0063] In some embodiments, provided herein are compounds comprising any one of Formulas XVII-XX:

[0064] [ka] (wherein n is an integer of 0 to 2),

[0065] [ka] (wherein n is an integer of 0 to 2),

[0066] [ka] (wherein n is an integer of 0 to 2),

[0067] [ka] (wherein n is an integer of 0 to 2).

[0068] In some embodiments, n in Formulas XVII-XX is 0. In some embodiments, n in Formulas XVII-XX is 1. In some embodiments, n in Formulas XVII-XX is 2. In some embodiments, a compound comprising any one of Formulas XVII-XX is a nucleoside, nucleotide, or analog thereof.

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

[0070] [ka]

[0071] In some embodiments, the compound comprising any one of Formulas XXII-XXV is a nucleoside, nucleotide, or analog thereof.

[0072] In another aspect, provided herein is an RNAi agent comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a double-stranded region, and wherein the sense strand or the antisense strand is represented by Formula Ia, Ib, Ic, II-IV, or XXI:

[0073] [ka] (wherein n is an integer of 1 to 4),

[0074] [ka] (wherein n is an integer of 0 to 2),

[0075] [ka] and 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, or 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-carboxy-cytidine, 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.

[0076] 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 double-stranded region, and wherein the sense strand or the antisense strand is a RNAi agent having a structure represented by Formula Ia

[0077] [ka] and a modified nucleotide of 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-carboxy-cytidine, phenoxazine, N6-alkyl-A, or O6-alkyl-G.

[0078] 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 double-stranded region, and wherein the sense strand or the antisense strand is represented by Formula Ib

[0079] [ka] and a modified nucleotide of 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, or 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-carboxy-cytidine, phenoxazine, N6-alkyl-A, or O6-alkyl-G.

[0080] 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 double-stranded region, and wherein the sense strand or the antisense strand is represented by Formula Ic

[0081] [ka] (wherein n is an integer of 1 to 4), and a modified nucleotide of 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, or 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-carboxy-cytidine, phenoxazine, N6-alkyl-A, or O6-alkyl-G. 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.

[0082] 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 double-stranded region, and wherein the sense strand or the antisense strand has a structure represented by Formula II

[0083] [ka] and a modified nucleotide of 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-carboxy-cytidine, phenoxazine, N6-alkyl-A, or O6-alkyl-G.

[0084] 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 double-stranded region, and wherein the sense strand or the antisense strand has a structure represented by Formula III

[0085] [ka] and a modified nucleotide of 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-carboxy-cytidine, phenoxazine, N6-alkyl-A, or O6-alkyl-G.

[0086] 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 double-stranded region, and wherein the sense strand or the antisense strand has a structure represented by Formula IV

[0087] [ka] (wherein n is an integer of 0 to 2), and a modified nucleotide of 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, or 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-carboxy-cytidine, 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.

[0088] 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 double-stranded region, and wherein the sense strand or the antisense strand has a structure represented by Formula XXI

[0089] [ka] and a modified nucleotide of 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, or 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-carboxy-cytidine, phenoxazine, N6-alkyl-A, or O6-alkyl-G.

[0090] In some embodiments, provided herein is an RNAi agent comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a double-stranded region, and wherein the sense strand or the antisense strand comprises a modified nucleotide of any one of Formulas Va-VIIIa:

[0091] [ka]

[0092] In some embodiments, provided herein is an RNAi agent comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a double-stranded region, and wherein the sense strand or the antisense strand comprises a modified nucleotide of any one of Formulas Vb-VIIIb:

[0093] [ka]

[0094] In some embodiments, provided herein is an RNAi agent comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a double-stranded region, and wherein the sense strand or the antisense strand comprises a modified nucleotide of any one of Formulas Vc-VIIIc:

[0095] [ka] (wherein n is an integer of 1 to 4),

[0096] [ka] (wherein n is an integer of 1 to 4),

[0097] [ka] (wherein n is an integer of 1 to 4),

[0098] [ka] (wherein n is an integer of 1 to 4).

[0099] In some embodiments, n in Formulas Vc-VIIIc is 1. In some embodiments, n in Formulas Vc-VIIIc is 2. In some embodiments, n in Formulas Vc-VIIIc is 3. In some embodiments, n in Formulas Vc-VIIIc is 4.

[0100] In some embodiments, provided herein is an RNAi agent comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a double-stranded region, and wherein the sense strand or the antisense strand comprises a modified nucleotide of any one of Formulas IX-XII:

[0101] [ka]

[0102] In some embodiments, provided herein is an RNAi agent comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a double-stranded region, and wherein the sense strand or the antisense strand comprises a modified nucleotide of any one of Formulas XIII-XVI:

[0103] [ka]

[0104] In some embodiments, provided herein is an RNAi agent comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a double-stranded region, and wherein the sense strand or the antisense strand comprises a modified nucleotide of any one of Formulas XVII-XX:

[0105] [ka] (wherein n is an integer of 0 to 2),

[0106] [ka] (wherein n is an integer of 0 to 2),

[0107] [ka] (wherein n is an integer of 0 to 2),

[0108] [ka] (wherein n is an integer of 0 to 2).

[0109] In some embodiments, n in Formulas XVII-XX is 0. In some embodiments, n in Formulas XVII-XX is 1. In some embodiments, n in Formulas XVII-XX is 2.

[0110] In some embodiments, provided herein is an RNAi agent comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a double-stranded region, and wherein the sense strand or the antisense strand comprises a modified nucleotide of any one of Formulas XXII-XXV:

[0111] [ka]

[0112] 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. In some embodiments, both the sense strand and the antisense strand are 15 to 30 nucleotides in length, e.g., 20 to 25 nucleotides in length. In some embodiments, the sense strand is 21 nucleotides in length. In some embodiments, the antisense strand is 23 nucleotides in length. In some embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length. In some embodiments, the double-stranded region is 15 to 21 nucleotides in length. In some embodiments, the double-stranded region is 21 nucleotides in length. In some embodiments, the sense strand and the antisense strand may have an overhang (i.e., a 5' overhang or a 3' overhang) at either the 5' end or the 3' end. For example, the sense strand and the antisense strand may have a 5' overhang or a 3' overhang of 1 to 5 nucleotides or 1 to 3 nucleotides. In some embodiments, the antisense strand includes a 3' overhang of two nucleotides.

[0113] In some embodiments, the sense strand comprises a modified nucleotide of any one of Formulae 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 Formulae 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 at position 13 from the 5' end 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.

[0114] In some embodiments, the antisense strand comprises a modified nucleotide of any one of Formulae 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 Formulae 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.

[0115] 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 in the sense strand are 2'-O-methyl-modified nucleotides.

[0116] 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, the nucleotides at other positions in the antisense strand are 2'-O-methyl modified nucleotides.

[0117] 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 in the sense strand are 2'-O-methyl modified nucleotides.

[0118] 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, the nucleotides at other positions in the antisense strand are 2'-O-methyl modified nucleotides.

[0119] 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, the nucleotides at other positions in the antisense strand are 2'-O-methyl modified nucleotides.

[0120] 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, the nucleotides at other positions in the antisense strand are 2'-O-methyl modified nucleotides.

[0121] 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.

[0122] In some embodiments, the antisense strand comprises a phosphate analog at the 5'-terminus. In some embodiments, the antisense strand comprises a 5'-vinyl phosphonate at the 5'-terminus.

[0123] In some embodiments, the sense strand comprises an abasic portion or an inverted abasic portion, eg, an abasic portion or an inverted abasic portion from Table 3.

[0124] 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.

[0125] Exemplary sense and antisense strand sequences of RNAi agents targeting human SNCA mRNA (SNCA RNAi agents) are provided in Table 1.

[0126] Table 1. Nucleic acid sequences of exemplary SNCA RNAi agents [Table 1-1]

[0127] (Continued from Table 1) [Table 1-2]

[0128] (Continued from Table 1) [Table 1-3]

[0129] (Continued from Table 1) [Table 1-4]

[0130] (Continued from Table 1) [Table 1-5] Abbreviations - "m" indicates 2'-OMe, "f" indicates 2'-fluoro, *" indicates a phosphorothioate linkage, "VP" indicates 5'-vinylphosphonate, "ads" indicates Formula I(a), "ss" indicates Formula II, "L1" indicates Formula III, "L2" indicates Formula IV where n is 0, "L3" indicates Formula XXI, "adsII" indicates Formula I(b), and "iAb" indicates the inverted abasic position of Table 3.

[0131] In some embodiments, provided herein are: (a) the sense strand comprises a first nucleic acid sequence having 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 having 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; (b) the sense strand comprises a first nucleic acid sequence having 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 having 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; (c) the sense strand comprises a first nucleic acid sequence having 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 having 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; (d) the sense strand comprises a first nucleic acid sequence having 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 having 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; (e) the sense strand comprises a first nucleic acid sequence having 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 having 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; (f) the sense strand comprises a first nucleic acid sequence having 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 having 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; (g) the sense strand comprises a first nucleic acid sequence having 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 having 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; (h) the sense strand comprises a first nucleic acid sequence having 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 having 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 (i) an SNCA RNAi agent comprising a sense strand and an antisense strand comprising a pair of nucleic acid sequences selected from the group consisting of: (i) the sense strand comprises a first nucleic acid sequence having 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 (ii) the antisense strand comprises a second nucleic acid sequence having 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.

[0132] In some embodiments, provided herein are: (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 NOs: 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 to 19, 37, 38, or 67 to 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; (l) 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) an SNCA RNAi agent comprising a sense strand and an antisense strand comprising a pair of nucleic acid sequences selected from the group consisting of: the sense strand comprising SEQ ID NO: 123, and the antisense strand comprising SEQ ID NO: 124.

[0133] In some embodiments, provided herein is an SNCA RNAi agent comprising a sense strand comprising SEQ ID NO: 3 and an antisense strand comprising SEQ ID NO: 4. In some embodiments, provided herein is an SNCA RNAi agent comprising a sense strand comprising SEQ ID NO: 19 and an antisense strand comprising SEQ ID NO: 7. In some embodiments, provided herein is an SNCA RNAi agent comprising a sense strand comprising SEQ ID NO: 87 and an antisense strand comprising SEQ ID NO: 88. In some embodiments, provided herein is an SNCA RNAi agent comprising a sense strand comprising SEQ ID NO: 89 and an antisense strand comprising SEQ ID NO: 90.

[0134] In some embodiments, provided herein are: (a) the sense strand consists of any one of SEQ ID NOs: 3, 5, or 20, and the antisense strand consists of SEQ ID NO: 4; (b) the sense strand consists of any one of SEQ ID NOs: 6, 8 to 19, 37, 38, or 67 to 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; (l) 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) An SNCA RNAi agent comprising a sense strand and an antisense strand consisting of a pair of nucleic acid sequences selected from the group consisting of: the sense strand consisting of SEQ ID NO: 123 and the antisense strand consisting of SEQ ID NO: 124.

[0135] In some embodiments, provided herein is an SNCA RNAi agent 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 is an SNCA RNAi agent 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 is an SNCA RNAi agent 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 is an SNCA RNAi agent comprising a sense strand consisting of SEQ ID NO: 89 and an antisense strand consisting of SEQ ID NO: 90.

[0136] Exemplary sense and antisense strand sequences of RNAi agents targeting human MAPT mRNA (MAPT RNAi agents) are provided in Table 2.

[0137] Table 2. Nucleic acid sequences of exemplary MAPT RNAi agents [Table 2-1]

[0138] (Continued from Table 2) [Table 2-2]

[0139] (Continued from Table 2) [Table 2-3]

[0140] (Continued from Table 2) [Table 2-4]

[0141] (Continued from Table 2) [Table 2-5] * The last nucleotide does not match the transcript. Abbreviations - "m" indicates 2'-OMe, "f" indicates 2'-fluoro, * " indicates a 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).

[0142] Table 3. Abasic or inverted abasic (iAb) moieties [Table 3] "5'" and "3'" indicate the 5' to 3' direction of the sequence.

[0143] In some embodiments, provided herein are: (a) the sense strand comprises a first nucleic acid sequence having 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 having 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; (b) the sense strand comprises a first nucleic acid sequence having 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 having 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; (c) the sense strand comprises a first nucleic acid sequence having 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 having 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; (d) the sense strand comprises a first nucleic acid sequence having 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 having 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; (e) the sense strand comprises a first nucleic acid sequence having 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 having 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; (f) the sense strand comprises a first nucleic acid sequence having 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 having 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; (g) the sense strand comprises a first nucleic acid sequence having 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 having 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; (h) the sense strand comprises a first nucleic acid sequence having 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 having 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; (i) the sense strand comprises a first nucleic acid sequence having 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 having 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 (j) A MAPT RNAi agent comprising a sense strand and an antisense strand comprising a pair of nucleic acid sequences selected from the group consisting of: wherein the sense strand comprises a first nucleic acid sequence having 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 having 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.

[0144] In some embodiments, provided herein are: (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 to 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 to 51, and 53, and the antisense strand comprises SEQ ID NO: 30; (f) the sense strand comprises SEQ ID NOs: 31, 36, 43, 52, 154, 155, 161 to 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; (l) 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 to 159, 164, and 165; (y) the sense strand comprises SEQ ID NO: 160 and the antisense strand comprises SEQ ID NO: 152; and (z) A MAPT RNAi agent comprising a sense strand and an antisense strand comprising a pair of nucleic acid sequences selected from the group consisting of: the sense strand comprising SEQ ID NO: 43 or 166, and the antisense strand comprising SEQ ID NO: 156.

[0145] In some embodiments, provided herein are: (a) the sense strand consists of any one of SEQ ID NOs: 27, 33, 39, 40, and 47 to 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 to 51, and 53, and the antisense strand consists of SEQ ID NO: 30; (c) the sense strand consists of SEQ ID NOs: 31, 36, 43, 52, 154, 155, 161 to 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; (l) 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 to 159, 164, and 165; (o) the sense strand consists of SEQ ID NO: 160 and the antisense strand consists of SEQ ID NO: 152; and (p) A MAPT RNAi agent comprising a sense strand and an antisense strand consisting of a pair of nucleic acid sequences selected from the group consisting of: the sense strand consisting of SEQ ID NO: 43 or 166, and the antisense strand consisting of SEQ ID NO: 156.

[0146] The sense and antisense strands of RNAi agents can be synthesized using any nucleic acid polymerization method known in the art, such as solid-phase synthesis using phosphoramidite chemistry (e.g., Current Protocols in Nucleic Acid Chemistry, Beaucage, SLet et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA), H-phosphonate, phosphotriester chemistry, or enzymatic synthesis. Automated commercially available synthesizers, such as the MerMade™ 12 from LGC Biosearch Technologies, or other synthesizers from BioAutomation or Applied Biosystems, can be used. Phosphorothioate linkages can be introduced using sulfurizing reagents such as phenylacetyl disulfide or DDTT (((dimethylaminomethylidene)amino)-3H-1,2,4-dithiazoline-3-thione). The use of similar techniques and commercially available modified amidites and Controlled-Pore Glass (CPG) products to synthesize modified and conjugated oligonucleotides is well known.

[0147] Purification methods can be used to remove unwanted impurities from the final oligonucleotide product.The purification techniques commonly used for single-stranded oligonucleotides include reverse-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, oligonucleotides can be analyzed by mass spectrometry and quantified by spectrophotometry at a wavelength of 260 nm.Then, sense strand and antisense strand can be annealed to form a double strand.

[0148] In another aspect, the present disclosure provides a pharmaceutical composition comprising a compound or RNAi agent described herein and a pharmaceutically acceptable carrier. Such pharmaceutical compositions may also comprise 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).

[0149] In a further aspect, provided herein is a method of treating a neurodegenerative disease in a patient in need thereof, such method comprising administering to the patient an effective amount of a compound, RNAi agent, or pharmaceutical composition described herein.

[0150] 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.

[0151] In some embodiments, the neurodegenerative disease is 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-logopenic (PPA-L), multiple system tauopathy with presenile dementia (MSD). Tauopathy with presenile dementia (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 amyloid angiopathy, cerebral amyloid angiopathy, chronic traumatic encephalopathy (CTE), corticobasal degeneration (CBD), Creutzfeldt-Jakob diseaseCJD, dementia pugilistica, diffuse neurofibrillary tangles with calcification, Down syndrome, epilepsy, Gerstmann-Straussler-Scheinker disease, Hallervorden-Spatz disease, Huntington's disease, inclusion body myositis, lead encephalopathy, Litiko-Bodig disease, meningioangiomatosis, multiple system atrophy, myotonic dystrophy, Niemann-Pick disease type C (NP-C), non-Guamanian motor neuron disease with neurofibrillary tangles, postencephalitic parkinsonism, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, neurofibrillary tangle-type senile dementia, neurofibrillary tangle-predominant dementia, ganglioglioma, gangliocytoma, subacute sclerosing panencephalitis, tuberous sclerosis, lipofuscinosis, primary age-related tauopathy The tauopathy is selected from the group consisting of glioma, glial tauopathy (PART), and globular glial tauopathies (GGT).

[0152] In some embodiments, the compound, RNAi agent, or pharmaceutical composition is administered to the patient via intrathecal, intracerebroventricular, or intracisternal injection.

[0153] Also provided herein are methods of inhibiting or reducing a target mRNA in a cell, the method comprising contacting a cell containing the target mRNA with a compound, RNAi agent, or pharmaceutical composition 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 within a subject. In some embodiments, the subject is a human subject.

[0154] Dosage regimens may be adjusted to provide the optimum 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.

[0155] Dosage values ​​may vary depending on the type and severity of the condition to be alleviated. It will 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.

[0156] In another aspect, provided herein are compounds, RNAi agents, or pharmaceutical compositions for use in therapy. Also provided herein are compounds, RNAi agents, or pharmaceutical compositions for use in treating neurodegenerative diseases, such as synucleinopathy or tauopathy. Also provided herein is the use of a compound or RNAi agent in the manufacture of a medicament for treating a neurodegenerative disease, such as a synucleinopathy or tauopathy.

[0157] As used herein, the terms "a," "an," "the," and similar terms as used in the context of this disclosure (particularly in the context of the claims) should be construed to cover both the singular and the plural, unless otherwise specified herein or clearly contradicted by context.

[0158] As used herein, the term "alkyl" means a saturated straight- or branched-chain monovalent hydrocarbon radical containing the indicated number of carbon atoms. For example, "C1-C 20 Alkyl (C1-C 20 "Alkyl" means a radical having from 1 to 20 carbon atoms in a linear or branched arrangement.

[0159] As used herein, "antisense strand" refers to an oligonucleotide that is complementary to a region of a target sequence. Similarly, as used herein, "sense strand" refers to an oligonucleotide that is complementary to a region of the antisense strand.

[0160] As used herein, "complementary" refers to a structural relationship between two nucleotides (e.g., on two opposing nucleic acids or on opposing regions of a single nucleic acid strand) that allows the two nucleotides to base pair with each other. For example, purine nucleotides of one nucleic acid that are complementary to pyrimidine nucleotides of an opposing nucleic acid may base pair together by forming hydrogen bonds with each other. Complementary nucleotides may 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 regions of multiple nucleotides that are complementary to each other and form a region of complementarity, as described herein.

[0161] As used herein, a "delivery moiety" refers to a chemical moiety that facilitates entry of an oligonucleotide or RNAi agent into a cell. A delivery moiety can be a lipid, cholesterol, vitamin E, carbohydrate, amino sugar, polypeptide, or protein.

[0162] As used herein, "duplex," with respect to a nucleic acid or oligonucleotide, means the structure formed through complementary base pairing of two antiparallel sequences of nucleotides, whether formed by two separate nucleic acid strands or by a single folded strand (e.g., via a hairpin).

[0163] "Effective amount" refers to the amount (duration and means of administration) necessary to achieve the desired therapeutic result. The effective amount of an RNAi agent may vary depending on factors such as the individual's condition, age, sex, and weight, as well as the ability of the RNAi agent to induce a desired response in the individual. An effective amount is also one in which the therapeutically beneficial effects outweigh any toxic or harmful effects of the RNAi agent.

[0164] The term "knockdown" or "expression knockdown" refers to reduced mRNA or protein expression of a gene following treatment with a reagent, eg, an RNAi agent.

[0165] As used herein, "modified internucleotide linkage" refers to an internucleotide linkage that has one or more chemical modifications compared to a reference internucleotide linkage having a phosphodiester bond. Typically, the modified internucleotide linkage confers one or more desirable 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.

[0166] As used herein, a "modified nucleotide" refers to a nucleotide having one or more chemical modifications compared to a corresponding reference nucleotide selected from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, adenine deoxyribonucleotides, guanine deoxyribonucleotides, cytosine deoxyribonucleotides, and thymidine deoxyribonucleotides. The modified nucleotide may have one or more chemical modifications, for example, in its sugar, nucleobase, and / or phosphate group. Additionally or alternatively, the modified nucleotide may have one or more chemical moieties conjugated to the corresponding reference nucleotide. In some embodiments, the modified nucleotide is a 2'-fluoro-modified nucleotide, a 2'-O-methyl-modified nucleotide, or a 2'-O-alkyl-modified nucleotide, e.g., a 2'-O-C16 alkyl-modified nucleotide. In some embodiments, the modified nucleotide has a phosphate analog, e.g., a 5'-vinyl phosphonate. In some embodiments, the modified nucleotide is an abasic moiety or an inverted abasic moiety.

[0167] As used herein, the term "synucleinopathy" refers to diseases characterized by fibrillar aggregates of alpha-synuclein protein in the cytoplasm of selective populations of neurons and glia in the central and / or peripheral nervous system.

[0168] As used herein, the term "tauopathy" refers to a disease associated with abnormal tau protein expression, secretion, phosphorylation, cleavage, and / or aggregation.

[0169] As used herein, "nucleotide" means an organic compound having a nucleoside (a nucleic acid base (e.g., adenine, cytosine, guanine, thymine, or uracil) and a pentose sugar (e.g., ribose or 2'-deoxyribose)) linked to a phosphate group, which can serve as a monomer unit of nucleic acid polymers (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)).

[0170] As used herein, "oligonucleotide" means a polymer of linked nucleotides, each of which may be modified or unmodified. Oligonucleotides are typically less than about 100 nucleotides in length.

[0171] As used herein, "overhang" refers to an unpaired nucleotide(s) that protrudes from the double-stranded structure of a double-stranded oligonucleotide. An overhang may include one or more unpaired nucleotides extending from the double-stranded region at the 5'-end or 3'-end of a double-stranded oligonucleotide. An overhang may be a 3'-overhang or a 5'-overhang on the antisense strand or the sense strand of a double-stranded oligonucleotide.

[0172] As used herein, the term "patient" refers to a human patient.

[0173] As used herein, "phosphate analog" refers to a chemical moiety that mimics the electrostatic and / or steric properties of a phosphate group. In some embodiments, the phosphate analog is located at the 5'-terminal nucleotide of an oligonucleotide in place of the 5'-phosphate, which is often susceptible to enzymatic removal. The 5' phosphate analog may include a phosphatase-resistant linkage. Examples of phosphate analogs include 5' methylene phosphonate (5'-MP) and 5'-(E)-vinyl phosphonate (5'-VP). In some embodiments, the phosphate analog is 5'-VP.

[0174] 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 in a candidate sequence that are identical to the nucleotides, nucleosides, or nucleobases in the reference nucleic acid sequence, after optimally aligning the sequences and introducing gaps or overhangs, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid sequence identity can be accomplished in a variety of ways within the skill of the art, for example, using publicly available computer software programs, such as those described in Current Protocols in Molecular Biology (Ausubel et al., eds., 1987, Supp. 30, section 7.7.18, Table 7.7.1), and computer software programs including BLAST, BLAST-2, ALIGN, Clustal W2.0, Clustal X2.0, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment across the entire length of the sequences being compared. The percentage of "sequence identity" can be determined by comparing two optimally aligned sequences over a comparison window, although the nucleic acid sequence fragment within the comparison window may contain additions or deletions (e.g., gaps or overhangs) compared to the reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. The percentage can be calculated by determining the number of positions where the same nucleotide, nucleoside, or nucleic acid base is present in both sequences to determine the number of matched positions, dividing the number of matched positions by the total number of positions within the comparison window, and multiplying the result by 100 to determine the percentage of sequence identity. The output is the percent identity of the subject sequence to the query sequence.

[0175] As used herein, "RNAi," "RNAi agent," "iRNA," "iRNA agent," and "RNA interference agent" refer to an agent that mediates sequence-specific degradation of 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, where 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 a nucleotide of the sense strand.

[0176] As used herein, a "strand" refers to a single, contiguous sequence of nucleotides linked together by internucleotide linkages (e.g., phosphodiester or phosphorothioate linkages). A strand may have two free ends (e.g., a 5' end and a 3' end).

[0177] As used herein, "SNCA" refers to the alpha-synuclein (SNCA) mRNA transcript. The nucleic acid sequence of the human SNCA mRNA transcript can be found in NM_000345.4:

[0178] [Table 4]

[0179] The amino acid sequence of the human SNCA protein can be found in NP_000336.1:

[0180] [Table 5]

[0181] 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.

[0182] As used herein, "MAPT" refers to the human MAPT mRNA transcript, which encodes 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 below.

[0183] i. MAPT transcript variant 1 → tau protein isoform 1: NM_016835.5 (nucleotide sequence) → NP_058519.3 (amino acid sequence), ii. MAPT transcript variant 2 → tau protein isoform 2: NM_005910.6 (nucleotide sequence) → NP_005901.2 (amino acid sequence), iii. MAPT transcript variant 3 → tau protein isoform 3: NM_016834.5 (nucleotide sequence) → NP_058518.1 (amino acid sequence), iv. MAPT transcript variant 4 → tau protein isoform 4: NM_016841.5 (nucleotide sequence) → NP_058525.1 (amino acid sequence), v.MAPT transcript variant 5 → tau protein isoform 5: NM_001123067.4 (nucleotide sequence) → NP_001116539.1 (amino acid sequence), vi. MAPT transcript variant 6 → Tau protein isoform 6: NM_001123066.4 (nucleotide sequence) → NP_001116538.2 (amino acid sequence), vii. MAPT transcript variant 7 → Tau protein isoform 7: NM_001203251.2 (nucleotide sequence) → NP_001190180.1 (amino acid sequence), viii. MAPT transcript variant 8 → tau protein isoform 8: NM_001203252.2 (nucleotide sequence) → NP_001190181.1 (amino acid sequence), ix. MAPT transcript variant 9 → tau protein isoform 9: NM_001377265.1 (nucleotide sequence) → NP_001364194.1 (amino acid sequence), x.MAPT transcript variant 10 → tau protein isoform 10: NM_001377266.1 (nucleotide sequence) → NP_001364195.1 (amino acid sequence), xi. MAPT transcript variant 11 → Tau protein isoform 11: NM_001377267.1 (nucleotide sequence) → NP_001364196.1 (amino acid sequence), xii. MAPT transcript variant 12 → tau protein isoform 4: NM_001377268.1 (nucleotide sequence) → NP_001364197.1 (amino acid sequence).

[0184] The nucleotide sequence of human MAPT transcript variant 6 (encoding 2N4R tau) can be found in NM_001123066.4:

[0185] [Table 6-1]

[0186] [Table 6-2]

[0187] [Table 6-3]

[0188] The corresponding amino acid sequence of human tau protein isoform 6 can be found in NP_001116538.2:

[0189] [Table 7]

[0190] The nucleotide sequence of human MAPT transcript variant 5 (encoding 1N4R tau) can be found in NM_001123067.4:

[0191] [Table 8-1]

[0192] [Table 8-2]

[0193] [Table 8-3]

[0194] The corresponding amino acid sequence of human tau protein isoform 5 can be found in NP_001116539.1:

[0195] [Table 9]

[0196] The nucleotide sequence of human MAPT transcript variant 4 (encoding 0N3R tau) can be found in NM_016841.5:

[0197] [Table 10-1]

[0198] [Table 10-2]

[0199] [Table 10-3]

[0200] The corresponding amino acid sequence of human tau protein isoform 4 can be found in NP_058525.1:

[0201] [Table 11]

[0202] As used herein, "subject" means a mammal (including cats, dogs, mice, rats, chimpanzees, apes, monkeys, and humans). Preferably, the subject is a human.

[0203] As used herein, "treatment" or "treating" refers to any process that may slow, control, retard, or stop the progression of a disorder or disease disclosed herein, or ameliorate the symptoms of the disorder or disease, but does not necessarily indicate the complete elimination of all disorder 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]

[0204] Example 1. Synthesis of Compounds and RNAi Agents 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 cerebral spinal 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, and "DMSO" refers to dimethyl sulfoxide. sulfoxide, "DMT" refers to 4,4'-dimethoxytrityl, "EDCI" refers to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, "ES / MS" refers to electrospray mass spectrometry, "EtOAc" refers to ethyl acetate, "EtOH" refers to ethanol and ethyl alcohol, "IP-RP" refers to ion-pair reverse phase, "LC / MS" refers to liquid chromatography-mass spectrometry, "MeOH" refers to methanol and methyl alcohol, and "MPA" refers to mobile phase A.A: "MPB" refers to mobile phase B; "MWCO" refers to molecular weight cut-off; "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; and "TFA" refers to trifluoracetic acid. "THF" refers to tetrahydrofuran, "UPLC" refers to ultra-performance liquid chromatography, and "UV" refers to ultraviolet light.

[0205] Scheme 1

[0206] [ka]

[0207] Scheme 1, Step A, shows the reaction of compound 1 with 2,2'-dipyridyl disulfide 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 reagent 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.

[0208] Scheme 2

[0209] [ka]

[0210] Scheme 2, Step A, depicts the ring-opening addition of an appropriately substituted (disulfanyl)ethanol reagent to compound 6 using boron trifluoride diethyl etherate in a solvent such as DMA to give compound 7. Step B depicts 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.

[0211] Scheme 3

[0212] [ka]

[0213] Scheme 3, Steps AC, illustrates the conversion of compound (6) to compound (12), which is essentially similar to the process found in Scheme 2, Steps AC.

[0214] Scheme 4

[0215] [ka]

[0216] Scheme 4, Step A shows 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).

[0217] Scheme 5

[0218] [ka]

[0219] Scheme 5, Step A, depicts the alkylation of compound 15 with (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 depicts the coupling of compound 14 with compound 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 through the use of TFA and triethylsilane in a solvent such as DCM to give compound 18. Step D depicts the coupling of compound 18 to an appropriately modified sense strand partner in the presence of TCEP to give compound 19.

[0220] Scheme 6

[0221] [ka]

[0222] Scheme 6, Step A shows the reaction of compound 20 with a suitable thiol, such as 2-((3r,5r,7r)-adamantan-1-yl)ethane-1-thiol or dodecane-1-thiol, in the presence of borate buffer to give compound 21. Step B shows the addition of compound 21 to a suitable modified sense strand partner in the presence of AMA solution to give compound 22.

[0223] Scheme 7

[0224] [ka]

[0225] Scheme 7, Step A shows the conversion of compound 8 to compound 23 by first adding chlorotrimethylsilane in a solvent such as pyridine, followed by treatment with 1,2,4-triazole, TEA, and phosphoryl chloride, and finally adding 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, which is essentially similar to the process found in Scheme 2, Step C.

[0226] Preparation 1 2-Dodecyldisulfaneylpyridine

[0227] [ka]

[0228] 1-Dodecanethiol (12.7 g, 61.4 mmol) was added to a solution of 2,2'-dipyridyl disulfide (20.5 g, 92.1 mmol) in MeOH (90 mL) and THF (5 mL). The mixture was stirred at ambient temperature for 16 h and then concentrated in vacuo. The resulting residue was purified via silica gel flash chromatography 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).

[0229] The compounds in Table 15 were prepared in an essentially similar manner to those found in Preparation 1.

[0230] Table 15 [Table 12]

[0231] Preparation 2 3-(Dodecyldisulfanyl)propanoic acid

[0232] [ka]

[0233] 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 h and then concentrated in vacuo. The resulting residue was purified via silica gel flash chromatography eluting with 5-30% EtOAc in hexanes to afford the title compound (14 g, 76%) as a colorless oil. 1 H NMR (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).

[0234] The compounds in Table 16 were prepared in an essentially similar manner to that found in Preparation 2.

[0235] Table 16 [Table 13]

[0236] Preparation 3 2,5-Dioxopyrrolidin-1-yl 3-(dodecyldisulfanyl)propanoate

[0237] [ka]

[0238] 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 h and then concentrated in vacuo. The resulting residue was purified via silica gel flash chromatography eluting with 0 to 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).

[0239] The compounds in Table 17 were prepared in an essentially similar manner to that found in Preparation 3.

[0240] Table 17 [Table 14]

[0241] Preparation 4 1-((2R,3R,4R,5R)-3-(2-tert-butyldisulfanyl)ethoxy)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione

[0242] [ka]

[0243] To a suspension of 2,2'-anhydro-1-(beta-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 diethyl etherate (4.0 mL, 31.2 mmol). The mixture was heated to 130 °C for 24 h, then cooled to ambient temperature and diluted with EtOAc (150 mL). The solution was washed with saturated aqueous sodium chloride (4 × 50 mL). Silica gel (10 g) was added to the organics, which were then concentrated in vacuo to a dry powder and purified via silica gel flash chromatography, eluting with 50–100% (5% MeOH / EtOAc) in hexanes, to give the title compound (2.10 g, 25%) as a viscous, 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).

[0244] Preparation 5 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

[0245] [ka]

[0246] 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'-dimethoxytrityl 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 silica gel flash chromatography eluting with 20-70% EtOAc in hexanes to afford 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).

[0247] Preparation 6 (2R,3R,4R,5R)-5-(4-acetamido-2-oxopyrimidin-1(2H)-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(2-tert-butyldisulfanyl)ethoxy)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite

[0248] [ka]

[0249] The title compound was synthesized using a method similar to that described in WO 2019 / 217459 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.

[0250] 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 chlorotrimethylsilane (2.14 mL, 16.85 mmol) was stirred at ambient temperature for 30 minutes. After this time, 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 before being cooled to 0° C. Phosphoryl chloride (0.98 mL, 10.53 mmol) was added, and the reaction mixture was stirred at 0° C. for 2 hours. Ammonia (10.53 mL, 465 mmol) was then added, and the mixture was stirred at ambient temperature for 4.5 hours. The reaction mixture was quenched with 50 / 50 water / saturated aqueous sodium chloride, extracted with EtOAc (3 times), dried over sodium sulfate, and concentrated in vacuo. The resulting residue was purified via silica gel flash chromatography, eluting with 0-100% MeOH in EtOAc, to give 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 as a tan foam (2.26 g, 77%). ES / MS (m / z): 692 (MH).

[0251] 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 (3 times). The combined organics were washed with water and saturated aqueous sodium chloride, dried over sodium sulfate, filtered, and concentrated in vacuo. The resulting residue was purified via silica gel flash chromatography 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).

[0252] 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 1 h, additional 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.11 mL, 0.455 mmol) was added to the mixture. After 1 h at ambient temperature, DCM (25 mL) was added. The mixture was washed with saturated aqueous sodium bicarbonate (3 times), dried over sodium sulfate, filtered, and concentrated in vacuo. The resulting residue was purified via silica gel flash chromatography 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.

[0253] Preparation 7 (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

[0254] [ka]

[0255] 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 h, additional 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (0.36 mL, 1.6 mmol) was added. After 1 h, the crude reaction was poured into a slurry of silica gel (10 g) in 20 mL of 1% TEA / DCM, concentrated in vacuo to a dry powder, and purified via silica gel flash chromatography eluting with 20-70% EtOAc in hexanes containing 1% TEA to afford the title compound (2.60 g, 75%) as a white foam. 1 H NMR(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),4.56 -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.

[0256] Preparation 8 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

[0257] [ka]

[0258] To a suspension of 2,2'-anhydro-1-(beta-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 diethyl etherate (1.4 mL, 11.0 mmol). The mixture was heated to 130 °C for 12 h and then cooled to ambient temperature. The mixture was diluted with EtOAc (50 mL) and washed with saturated aqueous sodium chloride (4 × 20 mL). Silica gel (10 g) was added to the organics, concentrated in vacuo to a dry powder, and purified via silica gel flash chromatography, eluting with 50–100% (5% MeOH / EtOAc) in hexanes, to give the title compound (0.76 g, 22%) as a 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 (brs,1H),3.21(brs,1H),2.91(t,2H),2.11-2.05(m,3H),1.90-1.85(m,6H),1.78-1.67(m,6H).

[0259] Preparation 9 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

[0260] [ka]

[0261] A solution of 1-((2R,3R,4R,5R)-3-(2-(((3S,5S,7S)-adamantan-1-yl)disulfaninyl)ethoxy)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (0.76 g, 1.6 mmol), 4,4′-dimethoxytrityl 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 in vacuo to a dry powder, and purified via silica gel flash chromatography eluting with 20-70% EtOAc in hexanes to afford the title compound (0.80 g, 64%) as a white foam. 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).

[0262] Preparation 10 (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

[0263] [ka]

[0264] 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 h, additional 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (0.12 mL, 0.53 mmol) was added. After 1 h, the crude reaction was poured into a slurry of silica gel (3 g) in 10 mL of 1% TEA / DCM, concentrated in vacuo to a dry powder, and purified via silica gel flash chromatography eluting with 20-70% EtOAc in hexanes containing 1% TEA to afford 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.

[0265] Preparation 11 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-dimethylpropanethioate

[0266] [ka]

[0267] To a suspension of 2,2'-anhydro-1-(beta-D-arabinofuranosyl)uracil (1.5 g, 6.6 mmol), S-(2-hydroxyethyl) 2,2-dimethylpropanethioate (4.3 g, 26.53 mmol), and DMA (7.37 mL) was added boron trifluoride diethyl etherate (4.38 mL, 16.6 mmol). The mixture was heated to 100 °C for 6 h, then cooled to ambient temperature and concentrated in vacuo to remove excess ether. The resulting residue was purified via silica gel flash chromatography, eluting with 0 to 100% (0.1% formic acid / water) in ACN to give the title compound (0.5 g, 19.4%) as a white foam. 1 H NMR(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).

[0268] Preparation 12 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-dimethylpropanethioate

[0269] [ka]

[0270] 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-dimethylpropanethioate (2.0 g, 5.1 mmol), 4,4′-dimethoxytrityl 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 silica gel flash chromatography eluting with 0 to 100% EtOAc containing 1% TEA in hexane to afford the title compound (2.92 g, 82.1%) as a white foam. 1 H 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).

[0271] Preparation 13 S-(2-(((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(((2-cyanoethoxy)(diisopropylamino)phosphanyl)oxy)-2-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-yl)oxy)ethyl) 2,2-dimethylpropanethioate

[0272] [ka]

[0273] 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-dimethylpropanethioate (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 h, additional 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (0.94 mL, 4.23 mmol) was added. After 1 h, additional 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (0.19 mL, 0.85 mmol) was added. After 10 min, the crude reaction was loaded onto silica gel and purified via silica gel flash chromatography eluting with 0 to 100% EtOAc in hexanes containing 1% TEA to give the title compound (2.32 g, 61.5%) as a white foam. 1 H 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.

[0274] Preparation 14 2-(Tritylthio)ethyl 4-methylbenzenesulfonate

[0275] [ka]

[0276] A solution of 2-(tritylthio)ethanol (1.00 g, 3.03 mmol), DCM (9 mL), p-toluenesulfonyl chloride (0.8665 g, 4.545 mmol), and pyridine (0.50 mL, 6.06 mmol) was stirred at ambient temperature for 16 h. The mixture was diluted with water (50 mL) and then extracted with EtOAc (3 × 75 mL). The combined organic layers were washed with saturated aqueous sodium chloride (2 × 150 mL), dried over NaSO, and concentrated in vacuo. The crude reaction was diluted with DCM, loaded onto silica gel, and purified via silica gel flash chromatography, eluting with 5–40% EtOAc in hexanes, to afford the title compound (330 mg, 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).

[0277] Preparation 15 2-((4R,8R)-4,8,12-trimethyltridecyl)-1,2,3,4-tetrahydroisoquinolin-6-ol

[0278] [ka]

[0279] Potassium bicarbonate (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 via silica gel flash chromatography, eluting with a gradient of 0 to 100% EtOAc in hexanes, to afford the title compound (0.81 g, 65%) as a white solid. 1H 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).

[0280] Preparation 16 2-((4R,8R)-4,8,12-trimethyltridecyl)-6-(2-(tritylthio)ethoxy)-1,2,3,4-tetrahydroisoquinoline

[0281] [ka]

[0282] 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 h. The reaction was concentrated in vacuo, then diluted with DCM, loaded onto silica gel, and purified via silica gel flash chromatography eluting with 0-40% EtOAc in hexanes to give the title compound (0.143 g, 39%) as a viscous 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).

[0283] Preparation 17 2-((2-((4R,8R)-4,8,12-trimethyltridecyl)-1,2,3,4-tetrahydroisoquinolin-6-yl)oxy)ethane-1-thiol

[0284] [ka]

[0285] 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 h. The mixture was concentrated in vacuo and then diluted with EtOAc (75 mL). The organic layer was washed with saturated aqueous NaHCO3 (1 × 50 mL), and the aqueous was back-extracted with EtOAc (1 × 75 mL). The organic layers were combined, dried over NaSO4, 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 afford 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).

[0286] Preparation 18 6-((2-((3r,5r,7r)-adamantan-1-yl)ethyl)disulfanyl)nicotinic acid

[0287] [ka]

[0288] 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 h. The reaction was then concentrated to a total volume of approximately 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).

[0289] Preparation 19 6-(dodecyldisulfanyl)nicotinic acid

[0290] [ka]

[0291] Prepare the title compound from dodecane-1-thiol in a manner essentially similar 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).

[0292] C12 ADS-linked siRNA

[0293] [ka]

[0294] The sense strand (3.1 g, 0.44 mmol), synthesized using the conditions found in the following protocol, in 4x borate buffer water (113 mL) was treated with a solution of 2,5-dioxopyrrolidin-1-yl 3-(dodecyldisulfanyl)propanoate (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 obtain the crude oligonucleotide.

[0295] Crude oligonucleotides were purified via an AKTA™ Pure purification system using reverse phase on a Source 15RPC column (MPA: 50 mM NaOAc with 10% ACN and MPB: 80% ACN / water). In all cases, fractions with mass purity greater than 85% and no impurities greater than 5% were combined.

[0296] The purified oligonucleotides were desalted using a 15 mL 3K MWCO centrifuge spin tube at 3500 x g for approximately 30 minutes. The oligonucleotides were rinsed with RNAse-free water until the eluate reached a conductivity of less than 100 μm / cm. After desalting was complete, 2-3 mL of RNAse-free water was added, followed by 10 cycles of aspiration, and the retentate was transferred to a 50 mL Falcon tube. This was repeated until complete transfer of the oligos was confirmed by measuring the compound concentration on the filter via nanodrop. The final oligonucleotides were then nanofiltered twice at 3500 x g for 2 minutes through a 15 mL 100K MWCO centrifuge spin tube. The final desalted oligonucleotides were analyzed for concentration (nanodrop at A260) and characterized by IP-RP LCMS for mass purity and UPLC for UV purity. ES / MS (m / z): 7324.6 (M+H).

[0297] The following compounds were prepared in a manner essentially similar to that found for the C12 ADS-linked siRNA.

[0298] Table 18 [Table 15]

[0299] SS-C12 linked siRNA

[0300] [ka]

[0301] After oligo synthesis (sense strand synthesized using the conditions found in the protocol below), the oligo-loaded CPG 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 shaking at ambient temperature. After 1 hour, over 80% of the desired product mass was observed. The solution was then concentrated in a Genevac to remove organic matter and obtain the crude oligonucleotide. The crude oligonucleotide was filtered using a 0.2 micron syringe filter and then purified via an AKTA™ Pure purification system using an anion exchange (AEX) Source 15Q column. For AEX, a Source™ 15Q column was used containing MPA: 20 mM NaH2PO4, pH 7.4 with 15% ACN, and MPB: 1 M NaBr, 20 mM NaH2PO4, pH 7.4 with 15% ACN. In all cases, fractions with >85% mass purity and >5% impurity content were combined.

[0302] The purified oligonucleotides were desalted using a 15 mL 3K MWCO centrifuge spin tube at 3500 x g for approximately 30 minutes. The oligonucleotides were rinsed with RNAse-free water until the eluate reached a conductivity of less than 100 μS / cm. After desalting was complete, 2-3 mL of RNAse-free water was added, followed by 10 cycles of aspiration and transfer of the retention to a 50 mL Falcon tube. This was repeated until complete transfer of the oligos by measuring the concentration of the compound on the filter via nanodrop. The final desalted oligonucleotides were analyzed for concentration (nanodrop at A260) and characterized by IP-RP LCMS for mass purity and UPLC for UV purity. ES / MS (m / e): 7239.6.

[0303] The compounds in Table 4 were prepared in a manner essentially similar to that found in the USS-C12 linked siRNA preparations.

[0304] Table 4. Exemplary modified nucleotides [Table 16]

[0305] SS-adamantyl-linked siRNA

[0306] [ka]

[0307] 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)propanoate (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 achieve an organic solvent concentration of 10% or less. Excess 2,5-dioxopyrrolidin-1-yl 3-(pyridin-2-yldisulfanyl)propanoate was removed using a 15 mL 3K MWCO centrifuge spin tube at 3500 x g for approximately 30 minutes. The oligonucleotides were rinsed three times with RNAse-free water. After removing the 2,5-dioxopyrrolidin-1-yl 3-(pyridin-2-yldisulfanyl)propanoate, 1 mL of RNAse-free water was added, followed by 10 aspiration cycles, and the retentate was transferred to a 5 mL Falcon tube. This was repeated until complete transfer of the oligos by measuring the compound concentration on the filter via nanodrop. The final oligonucleotides were analyzed for concentration (nanodrop at A260) and characterized by IP-RP, LCMS, and UPLC for mass purity and UV purity. ES / MS (m / z): 7196.02 (M+H).

[0308] 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 yield the crude oligonucleotide. The crude oligonucleotide was purified via an AKTA™ Pure purification system using a Source 15RPC 10 x 200 mm column (MPA: 10 mM NaOAc with 2% ACN and MPB: 80% ACN in water) using a reverse-phase gradient from 2 to 50% over 8 column volumes. The desired product eluted at 10%. In all cases, fractions with a mass purity of >85% and no impurities of >5% were combined. The solution was then concentrated via Genevac to remove the organic solvent and yield the purified oligonucleotide. The purified oligonucleotides were desalted using a 15 mL 3K MWCO centrifuge spin tube at 3500 x g for approximately 30 minutes. The oligonucleotides were rinsed with RNAse-free water until the eluate reached a conductivity of less than 100 μg / cm. After desalting was complete, 1 mL of RNAse-free water was added, followed by 10 cycles of aspiration, and the retentate was transferred to a 5 mL Falcon tube. This was repeated until complete transfer of the oligos by measuring the concentration of the compound on the filter via nanodrop. The final desalted oligonucleotides were analyzed for concentration (nanodrop at A260) and characterized by IP-RP, LCMS for mass purity, and UPLC for UV purity. ES / MS (m / z): 7253.15 (M+H).

[0309] SS-C2-tetraisoquinoline-linked siRNA

[0310] [ka]

[0311] The sense strand (1 mM solution in water), synthesized using the conditions found in the protocol below, was treated with 10 equivalents of TCEP. The reaction was shaken at 45°C for 18 hours. The solution was then transferred to a 15 mL 3K MWCO centrifugal spin filter and spun at 3500 x g for approximately 30 minutes. After adding 15 mL of water, the process was repeated. An aqueous solution of 0.5 mM siRNA was treated with a solution of dipyridyl disulfide in ACN (20 equivalents). The final ACN concentration was 20%. After 1 hour, the reaction was diluted with water to bring the ACN content to 10%. The solution was then transferred to a 15 mL 3K MWCO centrifugal spin filter and spun at 3500 x g for approximately 30 minutes. After adding 15 mL of water, the process was 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 (Source 15 RPC column; MPA: 50 mM NaOAc containing 10% ACN and MPB: 50 mM NaOAc containing 80% ACN). The purified oligonucleotide was desalted at 3500 x g for approximately 30 minutes using a 15 mL 3K MWCO centrifuge spin tube. The oligonucleotide was rinsed with RNAse-free water until the conductivity of the eluate reached less than 100 μg / cm. The final oligonucleotide was then nanofiltered twice through a 15 mL 100K MWCO centrifuge spin tube at 3500 x g for 2 minutes. 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).

[0312] Synthesis of dsRNA The single strands (sense and antisense) of the RNA duplex were synthesized on a solid support via MerMade™ 12. The sequences of the sense and antisense strands are shown in Tables 1 and 2. Oligonucleotides were synthesized via phosphoromidite chemistry on either a 5, 10, 25, or 50 μmol scale.

[0313] All single strands were synthesized from commercially available standard support mA. Standard reagents were used for oligo synthesis (Table 5), where 0.1 M xanthan gum in pyridine was used as the sulfurizing reagent and 20% DEA in ACN was used as a post-synthesis auxiliary wash. All monomers (Table 6) were made up at 0.1 M in ACN and contained molecular sieve trap bags.

[0314] The oligonucleotides were cleaved and deprotected (C / D) at 45°C for 20 hours. The sense strand was C / Ded from the CPG using ammonium hydroxide (28-30%, cold), while 3% DEA in ammonium hydroxide (28-30%, cold) was used for the antisense strand. C / D was determined to be complete by IP-RP LCMS when the resulting mass data confirmed sequence identity. Depending on the scale, the CPG was filtered through a 0.45 μm PVDF syringeless filter, a 0.22 μm PVDF Steriflip® vacuum filter, or a 0.22 μm PVDF Stericup® Quick Release filter. The CPG was backwashed / rinsed with either 30% ACN / RNAse-free water or 30% EtOH / RNAse-free water, then filtered through the same filtration device and combined with the first filtrate. This was repeated twice. The material was then divided equally between 50 mL Falcon tubes and passed through a Genevac™ to remove organics. After concentration, the crude oligonucleotide was returned to synthesis scale by dilution with RNAse-free water and filtered through either a 0.45 μm PVDF syringeless filter, a 0.22 μm PVDF Steriflip® vacuum filtration, or a 0.22 μm PVDF Stericup® Quick release.

[0315] Crude oligonucleotides were purified via an AKTA™ Pure purification system using either ion exchange (AEX) or reverse phase (RP) Source 15Q-RP columns. For AEX, an ES Industry Source™ 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, with the column temperature maintained at 65°C. For RP, a Source™ 15Q-RP column with MPA: 50 mM NaOAc with 10% ACN, and MPB: 50 mM NaOAc with 80% ACN. In all cases, fractions with mass purity greater than 85% and containing less than 5% impurities were combined.

[0316] The purified oligonucleotides were desalted using a 15 mL 3K MWCO centrifuge spin tube at 3500 x g for approximately 30 minutes. The oligonucleotides were rinsed with RNAse-free water until the conductivity of the eluate reached less than 100 mSemi / cm. After desalting was complete, 2-3 mL of RNAse-free water was added, followed by 10 cycles of aspiration. The retentate was transferred to a 50 mL Falcon tube, and this process was repeated until complete transfer of the oligos by measuring the concentration of the compound on the filter via nanodrop. The final oligonucleotides were then nanofiltered twice at 3500 x g for 2 minutes through a 15 mL 100K MWCO centrifuge spin tube. The final desalted oligonucleotides were analyzed for concentration (nanodrop at A260) and characterized by IP-RP LCMS for mass purity and UPLC for UV purity.

[0317] To prepare the duplex, equimolar amounts of the sense and antisense strands were combined and heated to 65°C for 10 minutes, then slowly cooled to ambient temperature over 40 minutes. The integrity of the duplex was confirmed by UPLC analysis and characterized by LCMS using IP-RP. All duplexes were nanofiltered, and endotoxin levels were then measured using a Charles River Endosafe® Cartridge Device to obtain the final RNAi conjugate compound. For in vivo analysis, an appropriate amount of duplex was lyophilized and then reconstituted in 1x PBS for rodent studies or CSF for non-human primate studies.

[0318] The molecular weights of exemplary SNCA and MAPT RNAi agents are shown in Tables 7 and 8.

[0319] Table 5 - Oligonucleotide synthesis reagents [Table 17]

[0320] Table 6 - Phosphoramidites [Table 18-1]

[0321] (Continued from Table 6) [Table 18-2]

[0322] Table 7 - Molecular weights of exemplary SNCA RNAi agents [Table 19-1]

[0323] (Continued from Table 7) [Table 19-2] "S" refers to the sense strand and "AS" refers to the antisense strand.

[0324] Table 8 - Molecular weights of exemplary MAPT RNAi agents [Table 20-1]

[0325] (Continued from Table 8) [Table 20-2] "S" refers to the sense strand and "AS" refers to the antisense strand.

[0326] Example 2. In vitro characterization of RNAi agents Selected RNAi agents were tested in vitro for target mRNA inhibition in cultured cells, including 293T cells, mouse cortical neurons (MCN), and / or human induced pluripotent stem cells (hiPSC).

[0327] Materials and Methods 293T luciferase transfection, RNAi treatment, and analysis: 293T cells transfected with a pMIR-luciferase construct (Invitrogen, Waltham, MA) containing the target sequence were plated overnight at 37°C, 5% CO2. On day 2, cells were transfected with siRNA using RNAiMAX (Invitrogen, Waltham, MA) according to the protocol provided by the manufacturer. Cells were incubated for 48 hours at 37°C, 5% CO2. Plates were cooled to room temperature, and then an equal volume of Bio-Glo (Promega, Madison, WI) was added to each well. Plates were incubated in the dark at room temperature and read on a BioTek Neos2 plate reader (Agilent, Santa Clara, CA).

[0328] Mouse primary cortical neuron (MCN) culture and RNAi treatment and analysis: Mouse primary cortical neurons were isolated from wild-type C57BL6 mouse embryos at E18 or from hTau C57BL6 transgenic mouse embryos expressing a human tau transgene at E18. Cells were plated at a density of 40k cells / well in poly-D-lysine-coated 96-well plates and cultured for 7 days at 37°C in NbActiv1 (BrainBits, LLC) containing 1% antibiotic / antimycotic (Corning) in a tissue culture incubator in a humidified chamber at 5% CO2. On day 7, half of the medium was removed from each well, and 2x the concentration of RNAi in culture medium with 2% FBS was added for treatment as CRCs. The cells were incubated for an additional 7, 14, or 21 days. Half of the medium was replaced with fresh culture medium every 7 days. At the end of the RNAi treatment, RT-qPCR was performed to quantify SNCA or MAPT mRNA levels using the TaqMan Fast Advanced Cell-to-CT kit. Specifically, cells were lysed, cDNA was generated using a Mastercycler X50a (Eppendorf), and qPCR was performed using a QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems). Alpha-synuclein (ThermoFisher, Mm00447333_m1) and human MAPT (ThermoFisher, Hs00902194_m1) gene expression levels were normalized to β-actin (ThermoFisher, Mm02619580_g1) using the respective probes.

[0329] Human induced pluripotent stem cell-derived neuron (hiPSC) 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 3 days (DIV3) to initiate neuronal differentiation. They were plated at 30 kJ / well onto 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% CO). Cells were half-fed every 7 days and treated with RNAi on DIV21 by serially diluting the RNAi agent in NDM, aspirating 75 mL according to the dilution, and adding 75 mL of 2x the RNAi concentration for a final 1x RNAi. After treatment, cells were half-fed every 7 days by removing half the medium and adding back fresh NDM. Cell lysates were collected on DIV35 (14 days later) or DIV42 (21 days later) and analyzed using TaqMan Fast Advanced Cells-to-C. T RT-qPCR was performed using a kit (ThermoFisher, A35377) to determine mRNA knockdown using an SNCA probe (ThermoFisher, Hs00240907_m1) or MAPT probe (ThermoFisher, Hs00902194_m1) as the gene of interest and an ACTb probe (ThermoFisher, Hs99999903_m1) as the housekeeping gene.

[0330] result Tables 9A-9C summarize the in vitro activity of selected SNCA RNAi agents. As shown in Tables 9A-9C, the RNAi agents tested knock down SNCA expression in several different cell lines.

[0331] Table 9A. In vitro activity of selected SNCA RNAi agents in mouse primary cortical neurons [Table 21]

[0332] Table 9B. In vitro activity of selected SNCA RNAi agents in the T293 luciferase assay [Table 22]

[0333] Table 9C. In vitro activity of selected SNCA RNAi agents in hiPSC neurons [Table 23]

[0334] Table 10 summarizes the in vitro activity of selected MAPT RNAi agents. As shown in Table 10, the tested RNAi agents knock down MAPT expression in mouse cortical neurons.

[0335] Table 10. In vitro activity of MAPT RNAi agents in mouse primary cortical neurons [Table 24]

[0336] Example 3. In vivo characterization of selected RNAi agents The efficacy of selected RNAi agents was also studied in Sprague Dawley rats. Six rats were given 300 μg or 100 μg of SNCA RNAi agent or PBS (phosphate-buffered saline) intrathecally and sacrificed 7 days after injection. Rat SNCA mRNA expression in the spinal cord and brain was measured and analyzed by qPCR. The results are shown in Table 11A.

[0337] Similar studies were carried out using 0.4mg, 1.2mg, or 2.4mg of SNCA RNAi agent, and rats were sacrificed two months after administration of SNCA RNAi agent. Rat SNCA mRNA expression was measured in the spinal cord and brain and analyzed by qPCR. The results are shown in Table 11B.

[0338] Table 11A. Percentage of SNCA mRNA knockdown (KD) in rats [Table 25] ND means not determined.

[0339] Table 11B. Percentage of knockdown (KD) of SNCA mRNA in rats [Table 26]

[0340] The efficacy of selected SNCA RNAi agents was studied in wild-type C56BL / 6N mice. 59 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.

[0341] Table 11C. Percentage of knockdown (KD) of SNCA mRNA in mice [Table 27] ND means not detected.

[0342] We also studied the efficacy of selected MAPT RNAi agents in hTau transgenic mice expressing human MAPT RNA but lacking mouse MAPT RNA (Andorfer et al., J Neurochem 2003, 86, 582-590). Six mice received intracerebroventricular (ICV) injections of 100 μg or 250 μg of MAPT RNAi agent or PBS (phosphate-buffered saline) and were sacrificed 14, 35, or 59 days after injection. MAPT mRNA expression in the brain was measured and analyzed by quantitative PCR (qPCR). The results are shown in Tables 11D-11F.

[0343] Table 11D. MAPT mRNA knockdown (KD) percentage in hTau mice after 14 days of 100 μg MAPT RNAi agent treatment [Table 28]

[0344] Table 11E. MAPT mRNA knockdown (KD) percentage in hTau mice after 35 days of 100 μg MAPT RNAi agent treatment [Table 29]

[0345] Table 11F. MAPT mRNA knockdown (KD) percentage in hTau mice after 59 days of 250 μg MAPT RNAi agent treatment [Table 30]

[0346] RNAi agent tissue distribution and microgliosis analysis Fixed rat right hemispheric brains and spinal cords (fourth cervical segment [C4 or SC2], fourth thoracic segment [T4 or SC5], eleventh thoracic segment [T11 or SC8], and first lumbar segment [L1 or SC10]) were stored in cold (4°C) 1x PBS (phosphate-buffered saline, CAS number: 7732-18-5) until tissue processing. Samples were processed using a Leica ASP6025S Tissue Processor and embedded using a Leica HistoCore Arcadia H-Heated Paraffin Embedding Station and a HistoCore Arcadia C-Cold Plate. Brains were embedded sagitally, and spinal cords were embedded transversely. Blocks were stored at room temperature until sectioning.

[0347] The blocks were sectioned using a HistoCore AUTOCUT-Automated Rotary Microtome (Leica Biosystems, 149AUTO00C1). Briefly, the blocks were trimmed to fully expose the tissue, and 5-um-thick sections were taken and placed on Fisherbrand™ Superfrost™ Plus Microscope Slides (Fisherbrand, 12-550-15). The brains were sectioned in stages from the midline (0 um, 500 um, and 1000 um from the midline), and the spinal cords were serially sectioned. The slides were dried overnight at room temperature before staining.

[0348] Slides were stained on a Leica BOND RX (Leica Biosystems, 21.2821). One slide from each brain was stained from each stage level, and one serial section from the spinal cord was stained. All slides were stained using the Advanced Cell Diagnostics (ACD) miRNAscope™ LS Reagent Kit-RED (Advanced Cell Diagnostics, 324600). For detection of the antisense siRNA strand, a probe was used (Advanced Cell Diagnostics, Eli Lilly & Co., 1063228-S1). Other reagents used included the miRNAscope™ LS Negative Control Probe-SR-Scramble-S1 (Advanced Cell Diagnostics, 727888-S1) and BOND Polymer Refine Red Detection (Leica Biosystems, DS9390). All slides were stained according to the manufacturer's protocol for miRNAscope™ with minor modifications. The washes in steps 75, 85, and 92 were modified to open washes. After staining, the slides were washed in DI water for 2 minutes, dried at 60°C for 30 minutes, and cover slipped.

[0349] Slides were scanned using a Leica Aperio GT450 Slide Scanner and uploaded to Aperio eSide Manager for analysis. Using Aperio ImageScope, the frontal cortex, brainstem, C4, T4, T11, and L1 regions were manually delineated, and an image analysis algorithm was run on each delineated region to calculate the "pixel positivity percentage." Briefly, the algorithm was adapted from the Aperio ImageScope "Positive Pixel Count 2002-08-11" algorithm. The output of the algorithm included pixel positivity, where positive pixels were equal to the antisense strand of the siRNA molecule, and all other pixels were negative pixels. The "pixel positivity percentage" was calculated by dividing the number of positive pixels in the image by the total number of pixels in the image, including negative pixels, and multiplying by 100. The results are shown in Table 12 and demonstrate that the tested RNAi agents have good distribution profiles throughout the brain and spinal cord.

[0350] Table 12. RNAi agent tissue distribution as measured by miRNAscope™ pixel positivity percent. [Table 31]

[0351] 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 (anti-rabbit poly-HRP-IgG (<25 μg / mL) containing 10% (v / v) animal serum in Tris-buffered saline / 0.1% ProClin™ 950) was applied, followed by DAB Part 1 (66 mM 3,3'-diaminobenzidine tetrahydrochloride hydrate in stabilizer solution), Part B (<0.1% (v / v) hydrogen peroxide in stabilizer solution), and hematoxylin (<0.1% hematoxylin) counterstain. After staining, slides were dehydrated using a Leica ST5010 Autostainer XL and coverslipped with Surgipath Micromount mounting medium (Leica, 3801731). Slides were scanned using a Leica Aperio GT450 Slide Scanner and uploaded to Aperio eSlide Manager for analysis. Images were opened using Aperio ImageScope and assessed for microgliosis using the scoring parameters shown in Table 13.

[0352] Table 13. Microgliosis Scoring [Table 32]

[0353] The results of the microgliosis assessment are shown in Table 14.

[0354] Table 14. Microgliosis Assessment [Table 33-1]

[0355] (Continued from Table 14) [Table 33-2]

[0356] (Continued from Table 14) [Table 33-3]

[0357] Sequence Listing

[0358] [Table 34-1]

[0359] (Continuation of the above table) [Table 34-2]

[0360] (Continuation of the above table) [Table 34-3]

[0361] (Continuation of the above table) [Table 34-4]

[0362] (Continuation of the above table) [Table 34-5]

[0363] (Continuation of the above table) [Table 34-6]

[0364] (Continuation of the above table) [Table 34-7]

[0365] (Continuation of the above table) [Table 34-8]

[0366] (Continuation of the above table) [Table 34-9]

[0367] (Continuation of the above table) [Table 34-10]

[0368] (Continuation of the above table) [Table 34-11]

[0369] (Continuation of the above table) [Table 34-12]

[0370] (Continuation of the above table) [Table 34-13]

[0371] (Continuation of the above table) [Table 34-14]

Claims

1. Formula Ia, Ib, Ic, II-IV, or XXI: 【Chemistry 1】 (wherein n is an integer from 1 to 4). 【Chemistry 2】 (wherein n is an integer from 0 to 2). 【Transformation 3】 and A compound of the formula: wherein B is a nucleobase selected from adenine, cytosine, guanine, thymine, uracil, or derivatives thereof.

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

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

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

5. 10. The compound of claim 1, wherein the compound comprises Formula IV:

6. The compound of claim 5 , wherein n is 0.

7. 6. The compound of claim 5, wherein n is 2.

8. 2. 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, a 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 double-stranded region, and the sense strand or the antisense strand has a structure represented by Formula Ia, Ib, Ic, II-IV, or XXI: 【Chemistry 4】 (wherein n is an integer from 1 to 4). 【Transformation 5】 (wherein n is an integer from 0 to 2). 【Transformation 6】 and wherein B is a nucleobase selected from adenine, cytosine, guanine, thymine, uracil, or derivatives thereof.

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

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

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

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

15. The RNAi agent of claim 14, wherein n is 0.

16. 15. The RNAi agent of claim 14, wherein n is 2.

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

18. The RNAi agent according to any one of claims 10 to 17, wherein the sense strand has a length of 15 to 50 nucleotides.

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

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

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

22. The RNAi agent of any one of claims 10 to 21, wherein the length of the double-stranded region is 21 nucleotides.

23. 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 to 6 or 12 to 21 from the 5' end.

24. 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. 25. The RNAi agent of any one of claims 10 to 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 to 10 or 15 to 18 from the 5' end.

26. The RNAi agent of any one of claims 10 to 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. 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 nucleotides at other positions in the sense strand are 2'-O-methyl modified nucleotides.

29. The RNAi agent of any one of claims 26 to 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 in the antisense strand are 2'-O-methyl modified nucleotides.

31. 27. 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 nucleotides at other positions in the sense strand are 2'-O-methyl modified nucleotides.

33. The RNAi agent of any one of claims 26 to 28, 31, and 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 in the antisense strand are 2'-O-methyl modified nucleotides.

35. The RNAi agent of any one of claims 26 to 28, 31, and 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 in the antisense strand are 2'-O-methyl modified nucleotides.

37. The RNAi agent of any one of claims 26 to 28, 31, and 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 in the antisense strand are 2'-O-methyl modified nucleotides.

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

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

41. 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 to 41, wherein the antisense strand comprises four or five phosphorothioate linkages.

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

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

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

46. 46. ​​The RNAi agent of any one of claims 10 to 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. 47. The RNAi agent of claim 46, wherein the antisense strand is complementary to SNCA mRNA.

48. The sense strand and the antisense strand are: (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 NOs: 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; (l) 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; 48. The RNAi agent of claim 47, comprising a pair of nucleic acid sequences selected from the group consisting of:

49. The sense strand and the antisense strand are: (a) the sense strand consists of any one of SEQ ID NOs: 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 to 19, 37, 38, or 67 to 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; (l) 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; 49. The RNAi agent of claim 47 or 48, comprising a pair of nucleic acid sequences selected from the group consisting of:

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

51. The sense strand and the antisense strand are: (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, and 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; (l) 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, and 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; 51. The RNAi agent of claim 50, comprising a pair of nucleic acid sequences selected from the group consisting of:

52. The sense strand and the antisense strand are: (a) the sense strand consists of any one of SEQ ID NOs: 27, 33, 39, 40, 47 to 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, and 53, and the antisense strand consists of SEQ ID NO: 30; (c) the sense strand consists of any one of SEQ ID NOs: 31, 36, 43, 52, 154, 155, 161 to 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; (l) 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 to 159, 164, and 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; 52. The RNAi agent of claim 50 or 51, consisting of a pair of nucleic acid sequences selected from the group consisting of:

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

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

55. 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 neurodegenerative disease is 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), non-fluent variant primary progressive aphasia (nfvPPA), Parkinson's disease, Pick's disease (PiD), primary semantic progressive aphasia (PPA-S), primary logopenic progressive aphasia (P PA-L), Multisystem Tauopathy with Presenile Dementia (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), Amyloid Angiopathy of the British Type, Cerebral Amyloid Angiopathy, Chronic Traumatic Encephalopathy (CTE), Corticobasal Degeneration (CBD) , Creutzfeldt-Jakob disease (CJD), dementia pugilistica, diffuse neurofibrillary tangles with calcification, Down syndrome, epilepsy, Gerstmann-Straussler-Scheinker disease, Hallervorden-Spatz disease, Huntington's disease, inclusion body myositis, lead encephalopathy, Litiko-Bodig disease, meningioangiomatosis, multiple system atrophy, myotonic dystrophy, Niemann-Pick disease type C (NP-C), non-Guamanian encephalopathy with neurofibrillary tangles 55. The method of claim 54, wherein the tauopathy is selected from motor neuron disease, post-encephalitic parkinsonism, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, neurofibrillary tangle senile dementia, neurofibrillary tangle-predominant dementia, ganglioglioma, gangliocytoma, subacute sclerosing panencephalitis, tuberous sclerosis, lipofuscinosis, primary age-related tauopathy (PART), or globular glial tauopathy (GGT).

57. 57. The method of any one of claims 54-56, wherein the compound or RNAi agent is administered to the patient via intrathecal, intracerebroventricular, or intracisternal injection.

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

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

60. 54. A compound according to any one of claims 1 to 9, an RNAi agent according to any one of claims 10 to 52, or a pharmaceutical composition according to claim 53 for use in the treatment of a neurodegenerative disease.

61. 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 neurodegenerative disease is Alzheimer's disease, frontotemporal dementia (FTD), frontotemporal dementia linked to chromosome 17 with parkinsonism (FTDP-17), frontotemporal lobar degeneration (FTLD), behavioral variant frontotemporal dementia (bvFTD), non-fluent variant primary progressive aphasia (nfvPPA), Parkinson's disease, Pick's disease (PiD), primary semantic progressive aphasia (PPA-S), primary logopenic progressive aphasia (PPA-L). , multisystem tauopathy with presenile dementia (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), amyloid angiopathy of the British type, cerebral amyloid angiopathy, chronic traumatic encephalopathy (CTE), corticobasal degeneration (CBD), Creutzfeldt-Jakob disease Cobb's disease (CJD), dementia pugilistica, diffuse neurofibrillary tangles with calcification, Down's syndrome, epilepsy, Gerstmann-Straussler-Scheinker disease, Hallervorden-Spatz disease, Huntington's disease, inclusion body myositis, lead encephalopathy, Litiko-Bodig disease, meningioangiomatosis, multiple system atrophy, myotonic dystrophy, Niemann-Pick disease type C (NP-C), non-Guamanian motor neuron disease with neurofibrillary tangles, post-encephalitic paroxysmal nodular syndrome (PNS).

61. The compound, RNAi agent, or pharmaceutical composition for use according to claim 60, wherein the tauopathy is selected from kinsonism, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, neurofibrillary tangle senile dementia, neurofibrillary tangle-predominant dementia, ganglioglioma, gangliocytoma, subacute sclerosing panencephalitis, tuberous sclerosis, lipofuscinosis, primary age-related tauopathy (PART), or globular glial tauopathy (GGT).

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

64. 64. The use of 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 neurodegenerative disease is 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), non-fluent variant primary progressive aphasia (nfvPPA), Parkinson's disease, Pick's disease (PiD), primary semantic progressive aphasia (PPA-S), primary logopenic progressive aphasia (P PA-L), Multisystem Tauopathy with Presenile Dementia (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), Amyloid Angiopathy of the British Type, Cerebral Amyloid Angiopathy, Chronic Traumatic Encephalopathy (CTE), Corticobasal Degeneration (CBD) , Creutzfeldt-Jakob disease (CJD), dementia pugilistica, diffuse neurofibrillary tangles with calcification, Down syndrome, epilepsy, Gerstmann-Straussler-Scheinker disease, Hallervorden-Spatz disease, Huntington's disease, inclusion body myositis, lead encephalopathy, Litiko-Bodig disease, meningioangiomatosis, multiple system atrophy, myotonic dystrophy, Niemann-Pick disease type C (NP-C), non-Guamanian encephalopathy with neurofibrillary tangles 64. The use of claim 63, wherein the tauopathy is selected from motor neuron disease, post-encephalitic parkinsonism, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, neurofibrillary tangle senile dementia, neurofibrillary tangle-predominant dementia, ganglioglioma, gangliocytoma, subacute sclerosing panencephalitis, tuberous sclerosis, lipofuscinosis, primary age-related tauopathy (PART), or globular glial tauopathy (GGT).

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