DSARN TARGETING MAPT

Abstract

The invention relates to a dsRNA targeting MAPT, a dsRNA complex. The invention also relates to a pharmaceutical, cell or kit containing dsRNA.

Description

dsRNA targeting MAPT and its medical use Technical Field

[0001] The present disclosure belongs to the field of biomedicine, and specifically relates to dsRNA targeting the microtubule-associated protein tau gene (MAPT), a composition, and medical uses thereof. Background Art

[0002] The microtubule-associated protein tau gene (MAPT) encodes the microtubule-associated protein tau and is located on chromosome 17q21.31. Tau protein is a microtubule-associated protein that, under normal physiological conditions, is primarily located in the axons of adult neurons, regulating the stability of microtubule assembly. Exons 9, 10, 11, and 12 of the MAPT gene encode the microtubule-binding repeat sequences of tau protein. Due to the alternative splicing of exon 10, tau protein isoforms are classified as 3R-tau and 4R-tau, where "R" refers to the number of repeats of the microtubule-binding domain. In the adult brain, the ratio of 3R-tau protein to 4R-tau protein isoforms is approximately 1. The 3R-tau / 4R-tau ratio is skewed in disease states, and deviations from the normal 3R / 4R tau ratio are characteristic of neurodegenerative tauopathies.

[0003] Tau protein has a very high content of proline and lysine residues and over 80 potential phosphorylation sites, making it easier for tau to cross-link and aggregate with other tau molecules through condensation reactions due to hyperphosphorylation under pathological conditions. Under pathological conditions, tau phosphorylation levels are 2-3 times higher than normal. This hyperphosphorylation causes tau protein to detach from microtubules, increasing tau content in the cytoplasm and causing aggregation to form insoluble fibrillar bundles, ultimately leading to neurofibrillary entanglements, a key pathological feature of Alzheimer's disease (AD). Tau pathology is also seen in other neurodegenerative dementias, such as frontotemporal dementia and Parkinson's disease linked to chromosome 17 (FTDP-17), Pick disease (PiD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), argyrophilic grain disease (AGD), tau dementia (TOD), and chronic traumatic encephalopathy (CTE). In particular, patients with FTDP-17 exhibit mutations in numerous exons and introns of the tau gene, leading to tau accumulation. These findings suggest that abnormalities in tau protein lead to tau protein accumulation and neuronal degeneration.

[0004] Lack the method for acceptable treatment this class neurodegenerative disease at present.Therefore, need to have selectivity and effectively suppress or regulate the medicine of MAPT gene expression, so that effective treatment has the experimenter of MAPT associated disease (for example Alzheimer's disease, frontotemporal lobe dementia FTD, PSP or other tau diseases).The purpose of this paper is to provide dsRNA and the method for the treatment of this class disease.

[0005] Summary of the Invention

[0006] The present disclosure provides a dsRNA targeting MAPT.

[0007] In some embodiments, the present disclosure provides a dsRNA comprising a sense strand and an antisense strand forming a double-stranded region;

[0008] The positive strand comprises a sequence of at least 15 (e.g., 16, 17, 18, 19, 20, 21) consecutive nucleotides that differs from the nucleotide sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 10, SEQ ID NO: 21 to SEQ ID NO: 78 by no more than 3 (e.g., 0, 1, 2, 3) nucleotides.

[0009] The antisense strand comprises a sequence of at least 15 (e.g., 16, 17, 18, 19, 20, 21) consecutive nucleotides that differs from the nucleotide sequence of any one of SEQ ID NO: 11 to SEQ ID NO: 20, SEQ ID NO: 79 to SEQ ID NO: 136 by no more than 3 (e.g., 0, 1, 2, 3) nucleotides.

[0010] In some embodiments, the sense strand comprises at least 17 consecutive nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 10, SEQ ID NO: 21 to SEQ ID NO: 78; and the antisense strand comprises at least 17 consecutive nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of any one of SEQ ID NO: 11 to SEQ ID NO: 20, SEQ ID NO: 79 to SEQ ID NO: 136.

[0011] In some embodiments, the sense strand comprises at least 19 consecutive nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 10, SEQ ID NO: 21 to SEQ ID NO: 78; and / or the antisense strand comprises at least 21 consecutive nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of any one of SEQ ID NO: 11 to SEQ ID NO: 20, SEQ ID NO: 79 to SEQ ID NO: 136.

[0012] In some embodiments, the antisense strand is at least partially reverse complementary to the target sequence to mediate RNA interference. In some embodiments, there are no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 mismatch between the antisense strand and the target sequence. In some embodiments, the antisense strand is completely reverse complementary to the target sequence.

[0013] In some embodiments, the sense strand and the antisense strand are at least partially reverse complementary to form a double-stranded region. In some embodiments, there are no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 mismatch between the sense strand and the antisense strand. In some embodiments, the sense strand and the antisense strand are completely reverse complementary.

[0014] In some embodiments, the dsRNA of the present disclosure comprises one or two blunt ends. In some specific embodiments, each strand of the dsRNA independently comprises 1 to 2 unpaired nucleotides. In some embodiments, the dsRNA of the present disclosure comprises an overhang at the 3' end of the antisense strand of the dsRNA.

[0015] In some embodiments, the sense and antisense strands each independently have 16 to 35, 16 to 34, 17 to 34, 17 to 33, 18 to 33, 18 to 32, 18 to 31, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 19 to 25, 19 to 24, or 19 to 23 nucleotides (e.g., 19, 20, 21, 22, 23 nucleotides).

[0016] In some embodiments, the sense strand and antisense strand are the same or different in length, with the sense strand being 19-23 nucleotides in length and the antisense strand being 19-26 nucleotides in length. The ratio of the length of the sense and antisense strands of the dsRNA provided by the present disclosure can be 19 / 19, 19 / 20, 19 / 21, 19 / 22, 19 / 23, 19 / 24, 19 / 25, 19 / 26, 20 / 19, 20 / 20, 20 / 21, 20 / 22, 20 / 23, 20 / 24, 20 / 25, 20 / 26, 21 / 20, 21 / 21, 21 / 22, 21 / 23, 21 / 24, 21 / 25, 21 / 26, 22 / 20, 22 / 21, 22 / 22, 22 / 23, 22 / 24, 22 / 25, 22 / 26, 23 / 20, 23 / 21, 23 / 22, 23 / 23, 23 / 24, 23 / 25 or 23 / 26. In some embodiments, the length ratio of the sense strand to the antisense strand of the dsRNA is 19 / 21, 21 / 23, or 23 / 25. In some embodiments, the length ratio of the sense strand to the antisense strand of the dsRNA is 19 / 21.

[0017] In some embodiments, the sense strand comprises or is selected from the group consisting of any one of the following nucleotide sequences: SEQ ID NO: 1 to SEQ ID NO: 10, SEQ ID NO: 21 to SEQ ID NO: 78.

[0018] In some embodiments, the antisense strand comprises or is selected from the group consisting of any one of the following nucleotide sequences: SEQ ID NO: 11 to SEQ ID NO: 20, SEQ ID NO: 79 to SEQ ID NO: 136.

[0019] In some embodiments, the dsRNA of the present disclosure comprises a sense strand and an antisense strand, or is selected from any one of the following groups:

[0020] Group 1), the sense strand as shown in SEQ ID NO: 1 and the antisense strand as shown in SEQ ID NO: 11;

[0021] Group 2), the sense strand as shown in SEQ ID NO: 2 and the antisense strand as shown in SEQ ID NO: 12;

[0022] Group 3), the sense strand as shown in SEQ ID NO: 3 and the antisense strand as shown in SEQ ID NO: 13;

[0023] Group 4), the sense strand as set forth in SEQ ID NO: 4 and the antisense strand as set forth in SEQ ID NO: 14;

[0024] Group 5), the sense strand as set forth in SEQ ID NO: 5 and the antisense strand as set forth in SEQ ID NO: 15;

[0025] Group 6), the sense strand as set forth in SEQ ID NO: 6 and the antisense strand as set forth in SEQ ID NO: 16;

[0026] Group 7), the sense strand as set forth in SEQ ID NO: 7 and the antisense strand as set forth in SEQ ID NO: 17;

[0027] Group 8), the sense strand as set forth in SEQ ID NO: 8 and the antisense strand as set forth in SEQ ID NO: 18;

[0028] Group 9), the sense strand as shown in SEQ ID NO: 9 and the antisense strand as shown in SEQ ID NO: 19; and

[0029] Group 10), the sense strand as shown in SEQ ID NO: 10 and the antisense strand as shown in SEQ ID NO: 20.

[0030] In some embodiments, at least one nucleotide in the sense strand and / or antisense strand is a modified nucleotide. In some embodiments, all nucleotides in the sense strand and / or antisense strand are modified nucleotides.

[0031] In some embodiments, all nucleotides are modified nucleotides.

[0032] In some embodiments, the modified nucleotide is selected from the group consisting of: a 2'-fluoro modified nucleotide, a 2-methoxy modified nucleotide, and a nucleotide in which the 5' end of the nucleoside is modified with a trans-vinyl phosphodiester group.

[0033] In some embodiments, three consecutive nucleotides in the sense strand of the dsRNA are 2'-fluoro-modified nucleotides. In some embodiments, in the sense strand of the dsRNA, from the 5' end to the 3' end, three consecutive nucleotides at positions 7-9 in the sense strand are 2'-fluoro-modified nucleotides. In some embodiments, three consecutive nucleotides at positions 7-9 in the sense strand of the dsRNA are 2'-fluoro-modified nucleotides, and the remaining positions in the sense strand are 2'-methoxy-modified nucleotides.

[0034] In some embodiments, the nucleotides at positions 2, 6, 12, 14, and 16 of the antisense strand, from the 5' end to the 3' end, are each independently a 2'-fluoro-modified nucleotide. In some embodiments, the nucleotides at positions 2, 4, 6, 10, 12, 14, 16, or 18 of the antisense strand, from the 5' end to the 3' end, are each independently a 2'-fluoro-modified nucleotide. In some embodiments, the remaining positions in the antisense strand are all 2'-methoxy-modified nucleotides.

[0035] In some embodiments, the 5' end of the antisense strand comprises a phosphorus-containing group, and the phosphorus-containing group is a 5'-vinyl phosphodiester group (5'-VP). When the 5'-terminal phosphorus-containing group is a 5'-vinyl phosphodiester group (5'-Vp), the 5'-Vp can be a 5'-E-VP isomer (i.e., a trans-vinyl phosphodiester group), a 5'-Z-VP isomer (i.e., a cis-vinyl phosphodiester group), or a mixture thereof. In some embodiments, the 5' end of the antisense strand comprises a phosphorus-containing group, and the phosphorus-containing group is a 5'-E-VP isomer (i.e., a trans-vinyl phosphodiester group).

[0036] In some embodiments, at least one phosphodiester group in the sense strand and / or antisense strand is a phosphodiester group with a modifying group. The modifying group allows the dsRNA to have increased stability in a biological sample or environment. In some embodiments, the phosphodiester group with a modifying group is a thiophosphate diester group. In some embodiments, the phosphodiester group with a modifying group is a 5'-vinyl phosphodiester group.

[0037] In some embodiments, the phosphodiester group having a modifying group is present in at least one position selected from the group consisting of:

[0038] between the first and second nucleotides at the 5' end of the sense strand;

[0039] between the second and third nucleotides at the 5' end of the sense strand;

[0040] between the first and second nucleotides at the 3' end of the sense strand;

[0041] between the second and third nucleotides at the 3' end of the sense strand;

[0042] between the first and second nucleotides at the 5' end of the antisense strand;

[0043] between the second and third nucleotides at the 5' end of the antisense strand;

[0044] between the first nucleotide and the second nucleotide at the 3' end of the antisense strand; and

[0045] between the second and third nucleotides at the 3' end of the antisense strand.

[0046] In some embodiments, the sense strand and / or the antisense strand comprises a plurality of phosphodiester groups with modified groups, and the phosphodiester groups with modified groups are present at a plurality of the aforementioned positions.

[0047] In some embodiments, the phosphodiester group having a modifying group is present in:

[0048] between the first and second nucleotides at the 5' end of the sense strand;

[0049] between the second and third nucleotides at the 5' end of the sense strand;

[0050] between the first and second nucleotides at the 3' end of the sense strand;

[0051] between the second and third nucleotides at the 3' end of the sense strand;

[0052] between the first and second nucleotides at the 5' end of the antisense strand;

[0053] between the second and third nucleotides at the 5' end of the antisense strand;

[0054] between the first nucleotide and the second nucleotide at the 3' end of the antisense strand; and

[0055] between the second and third nucleotides at the 3' end of the antisense strand.

[0056] In some embodiments, in the direction from the 5' end to the 3' end, at least one nucleotide at positions 2 to 8 (e.g., positions 2, 3, 4, 5, 6, 7, 8) of the antisense strand comprises a chemical modification represented by formula (I) or a pharmaceutically acceptable salt thereof, wherein the chemical modification represented by formula (I) is selected from:

[0057] Wherein: B represents the bases corresponding to positions 2-8 at the 5' end of the antisense strand.

[0058] In some embodiments, the sense strand is selected from or comprises the nucleotide sequence shown in any one of SEQ ID NO: 137 to SEQ ID NO: 201.

[0059] In some embodiments, the antisense strand is selected from or comprises the nucleotide sequence shown in any one of SEQ ID NO: 202 to SEQ ID NO: 266.

[0060] In some more specific embodiments, the dsRNA comprises or is selected from the group consisting of a sense strand and an antisense sequence:

[0061] The sense strand set forth in SEQ ID NO: 137 and the antisense strand set forth in SEQ ID NO: 202;

[0062] The sense strand set forth in SEQ ID NO: 138 and the antisense strand set forth in SEQ ID NO: 203;

[0063] The sense strand as set forth in SEQ ID NO: 139 and the antisense strand as set forth in SEQ ID NO: 204;

[0064] The sense strand set forth in SEQ ID NO: 140 and the antisense strand set forth in SEQ ID NO: 205;

[0065] The sense strand set forth in SEQ ID NO: 141 and the antisense strand set forth in SEQ ID NO: 206;

[0066] The sense strand set forth in SEQ ID NO: 142 and the antisense strand set forth in SEQ ID NO: 207;

[0067] The sense strand set forth in SEQ ID NO: 143 and the antisense strand set forth in SEQ ID NO: 208;

[0068] The sense strand set forth in SEQ ID NO: 144 and the antisense strand set forth in SEQ ID NO: 209;

[0069] The sense strand set forth in SEQ ID NO: 145 and the antisense strand set forth in SEQ ID NO: 210;

[0070] The sense strand is shown as SEQ ID NO: 146 and the antisense strand is shown as SEQ ID NO: 211.

[0071] In some embodiments, the dsRNA is linked to one or more lipophilic groups.In some embodiments, the sense strand of the dsRNA is linked to one or more lipophilic groups.

[0072] In some embodiments, the one or more lipophilic groups are attached to one or more nucleotides on the sense strand of the dsRNA; in some embodiments, the one or more lipophilic groups are attached to one or more nucleotides on the antisense strand of the dsRNA; in some embodiments, the one or more lipophilic groups are attached to multiple nucleotides on both the sense and antisense strands of the dsRNA.

[0073] In some embodiments, the one or more lipophilic groups are attached to one or more bases; in some embodiments, the one or more lipophilic groups are attached to one or more sugar rings; in some embodiments, the one or more lipophilic groups are attached to one or more internucleoside linking groups.

[0074] In some embodiments, the one or more lipophilic groups are attached to at least one of the following positions:

[0075] The first nucleotide at the 5' end or 3' end of the sense strand; the first nucleotide at the 5' end or 3' end of the antisense strand; the first nucleotide at the 3' end of the antisense strand; a nucleotide in the middle of the sense strand; a nucleotide in the middle of the antisense strand.

[0076] In some embodiments, the one or more lipophilic groups are attached to nucleotides 2-8 of the sense strand, in a 5' to 3' direction; in some embodiments, the one or more lipophilic groups are attached to nucleotides 2-8 of the antisense strand; in some embodiments, the one or more lipophilic groups are attached to nucleotides 6 of the sense strand.

[0077] In some embodiments, the lipophilic group comprises a saturated or unsaturated C 4-30 a hydrocarbon chain, and optionally a functional group selected from halogen, alkoxy, hydroxy, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.

[0078] In some embodiments, the lipophilic group comprises a saturated or unsaturated C 6-18 In some embodiments, the lipophilic group comprises a saturated or unsaturated C 16 Hydrocarbon chain.

[0079] In some embodiments, the saturated or unsaturated C 16 The hydrocarbon chain is attached to the first nucleotide at the 5' end of the sense strand.

[0080] In some embodiments, the sense strand comprises or is selected from the sequence shown in any one of SEQ ID NO: 267 to SEQ ID NO: 276, and / or the antisense strand comprises or is selected from the sequence shown in any one of SEQ ID NO: 277 to SEQ ID NO: 286.

[0081] In some embodiments, the dsRNA comprises a sense strand and an antisense strand, or is selected from any one of the following groups:

[0082] The sense strand set forth in SEQ ID NO: 267 and the antisense strand set forth in SEQ ID NO: 277;

[0083] The sense strand as set forth in SEQ ID NO: 268 and the antisense strand as set forth in SEQ ID NO: 278;

[0084] The sense strand set forth in SEQ ID NO: 269 and the antisense strand set forth in SEQ ID NO: 279;

[0085] The sense strand as set forth in SEQ ID NO: 270 and the antisense strand as set forth in SEQ ID NO: 280;

[0086] The sense strand set forth in SEQ ID NO: 271 and the antisense strand set forth in SEQ ID NO: 281;

[0087] The sense strand set forth in SEQ ID NO: 272 and the antisense strand set forth in SEQ ID NO: 282;

[0088] The sense strand as set forth in SEQ ID NO: 273 and the antisense strand as set forth in SEQ ID NO: 283;

[0089] The sense strand set forth in SEQ ID NO: 274 and the antisense strand set forth in SEQ ID NO: 284;

[0090] The sense strand set forth in SEQ ID NO: 275 and the antisense strand set forth in SEQ ID NO: 285;

[0091] The sense strand is shown as SEQ ID NO:276 and the antisense strand is shown as SEQ ID NO:286.

[0092] The present disclosure also provides a dsRNA comprising any one of the above dsRNAs and a targeting ligand attached to the dsRNA. In some embodiments, the dsRNA and the targeting ligand are covalently or non-covalently attached.

[0093] In some embodiments, the targeting ligand targets the liver; in some embodiments, the targeting ligand binds to the asialoglycoprotein receptor (ASGPR); in some embodiments, the targeting ligand comprises a galactose cluster or a galactose derivative cluster, wherein the galactose derivative is selected from N-acetyl-galactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine or N-isobutyrylgalactosamine.

[0094] In some embodiments, the targeting ligand is linked to the 3' end of the sense strand of the dsRNA.

[0095] In some embodiments, the targeting ligand is linked to the terminus of the dsRNA through a phosphodiester group, a phosphorothioate diester group, or a phosphonic acid group; in some embodiments, the targeting ligand is linked to the terminus of the dsRNA through a phosphodiester group.

[0096] In some embodiments, the targeting ligand is indirectly linked to the dsRNA terminus via a phosphodiester group, a phosphorothioate diester group, or a phosphonic acid group; in some embodiments, the targeting ligand is indirectly linked to the dsRNA terminus via a phosphodiester group.

[0097] In some embodiments, the targeting ligand is directly linked to the terminus of the dsRNA through a phosphodiester group, a phosphorothioate diester group, or a phosphonic acid group; in some embodiments, the targeting ligand is directly linked to the terminus of the dsRNA through a phosphodiester group.

[0098] In some embodiments, the targeting ligand is directly linked to the 3' end of the sense strand of the dsRNA via a phosphodiester group or a phosphorothioate diester group; in some embodiments, the targeting ligand is directly linked to the 3' end of the sense strand of the dsRNA via a phosphodiester group.

[0099] In some embodiments, the targeting portion of a targeting ligand is composed of one or more targeting groups or targeting moieties that assist in directing the delivery of the therapeutic agent to which it is attached to the desired target location. In some cases, the targeting moiety can bind to cells or cell receptors and initiate endocytosis to promote the entry of the therapeutic agent into the cell. The targeting moiety can include a compound with affinity for a cell receptor or a cell surface molecule or antibody. Various targeting ligands containing a targeting moiety can be linked to therapeutic agents and other compounds to target the agent to cells and specific cell receptors.

[0100] In some embodiments, the types of targeting moieties include carbohydrates, cholesterol and cholesterol groups or steroids. Targeting moieties that can bind to cell receptors include sugars such as galactose, galactose derivatives (e.g., N-acetyl-galactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine, N-isobutyrylgalactosamine), mannose and mannose derivatives).

[0101] Targeting moieties that are known to bind to the asialoglycoprotein receptor (ASGPR) are particularly useful for directing the delivery of oligomeric compounds to the liver. The asialoglycoprotein receptor is abundantly expressed on liver cells (hepatocytes). Cell receptor targeting moieties that target the ASCPR include galactose and galactose derivatives. Specifically, clusters of galactose derivatives, including clusters consisting of 2, 3, 4, or more than 4 N-acetyl-galactosamine (GalNAc or NAG) can promote the uptake of certain compounds in hepatocytes. The GalNAc clusters conjugated to the oligomeric compound are used to direct the composition to the liver, where the N-acetyl-galactosamine sugar can bind to the asialoglycoprotein receptor on the surface of liver cells. Binding of the asialoglycoprotein receptor is believed to initiate receptor-mediated endocytosis, thereby promoting the entry of the compound into the cell interior.

[0102] In some embodiments, the targeting ligands may include 2, 3, 4 or more than 4 targeting moieties. In some embodiments, the targeting ligands disclosed herein may include 1, 2, 3, 4 or more than 4 targeting moieties connected to the branching group via L2.

[0103] In some embodiments, each targeting moiety comprises a galactosamine derivative, which is N-acetyl-galactosamine. Other sugars that can be used as targeting moieties and have affinity for asialoglycoprotein receptors can be selected from galactose, galactosamine, N-formyl-galactosamine, N-acetyl-galactosamine, N-propionyl-galactosamine, N-n-butyryl-galactosamine, and N-isobutyryl-galactosamine.

[0104] In some embodiments, the targeting ligands of the present disclosure include N-acetylgalactosamine as a targeting moiety.

[0105] In some embodiments, the targeting ligand comprises three terminal galactosamines or galactosamine derivatives (such as N-acetyl-galactosamine), each of which has affinity for sialoglycoprotein receptors. In some embodiments, the targeting ligand comprises three terminal N-acetyl-galactosamines (GalNAc or NAG) as targeting moieties.

[0106] In some embodiments, the targeting ligand comprises four terminal galactosamines or galactosamine derivatives (such as N-acetyl-galactosamine), each of which has affinity for the asialoglycoprotein receptor. In some embodiments, the targeting ligand comprises four terminal N-acetyl-galactosamines (GalNAc or NAG) as targeting moieties.

[0107] When referring to three terminal N-acetyl-galactosamines, terms commonly used in the art include tri-antennary, tri-valent, and trimer. When referring to four terminal N-acetyl-galactosamines, terms commonly used in the art include tetra-antennary, tetra-valent, and tetramer.

[0108] In some embodiments, the targeting ligand provided by the present disclosure is a compound shown below or a pharmaceutically acceptable salt thereof,

[0109] In some embodiments, the N-acetyl-galactosamine moiety in the above targeting ligands can be replaced with N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine, or N-isobutyrylgalactosamine.

[0110] On the other hand, the present disclosure provides a composition comprising the dsRNA described herein and one or more pharmaceutically acceptable excipients, such as a vehicle, a carrier, a diluent, and / or a delivery polymer. The term "pharmaceutical composition" refers to a mixture containing one or more compounds described herein or their physiologically pharmaceutically acceptable salts or prodrugs with other chemical components, as well as other components such as physiologically pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitate the absorption of the active ingredient, and thus exert biological activity.

[0111] Various delivery systems are known and can be used for the disclosed dsRNAs, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the dsRNA, receptor-mediated endocytosis, constructing the nucleic acid as part of a retroviral or other vector.

[0112] "Pharmaceutically acceptable excipients" include, but are not limited to, any adjuvant, carrier, glidant, sweetener, diluent, preservative, dye / colorant, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier approved by the U.S. Food and Drug Administration (FDA) for use by humans or domestic animals.

[0113] In some embodiments, the pharmaceutical composition may further comprise a pharmaceutically acceptable excipient and / or adjuvant, which may be one or more of various preparations or compounds conventionally used in the art. For example, the pharmaceutically acceptable excipient may include at least one of a pH buffer, a protective agent, and an osmotic pressure regulator.

[0114] In some embodiments, the unit dose of the pharmaceutical composition is 0.001 mg-1000 mg.

[0115] In certain embodiments, the pharmaceutical composition contains 0.01-99.99% of the aforementioned compound or its pharmaceutically acceptable salt or its isotopic substitution, based on the total weight of the composition. In certain embodiments, the pharmaceutical composition contains 0.1-99.9% of the aforementioned compound or its pharmaceutically acceptable salt or its isotopic substitution. In certain embodiments, the pharmaceutical composition contains 0.5%-99.5% of the aforementioned compound or its pharmaceutically acceptable salt or its isotopic substitution. In certain embodiments, the pharmaceutical composition contains 1%-99% of the aforementioned compound or its pharmaceutically acceptable salt or its isotopic substitution. In certain embodiments, the pharmaceutical composition contains 2%-98% of the aforementioned compound or its pharmaceutically acceptable salt or its isotopic substitution.

[0116] The pharmaceutical composition can be conveniently presented in unit dosage form. Generally, the pharmaceutical composition can be formulated into any suitable dosage form, such as, but not limited to, injection, tablet, capsule, gel, etc. Pharmaceutical compositions include, but are not limited to, solutions, emulsions, and liposome-containing formulations.

[0117] In certain embodiments, the pharmaceutical composition comprises 0.01% to 99.99% of a pharmaceutically acceptable excipient, based on the total weight of the composition. In certain embodiments, the pharmaceutical composition comprises 0.1% to 99.9% of a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition comprises 0.5% to 99.5% of a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition comprises 1% to 99% of a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition comprises 2% to 98% of a pharmaceutically acceptable excipient.

[0118] On the other hand, the present disclosure provides a dsRNA or composition of the present disclosure for use in the preparation of a medicament for treating a disease in a subject, which in some embodiments is selected from a liver-derived disease, and in some embodiments is selected from a central nervous system degenerative disease. In some embodiments, the disease is a disease associated with MAPT gene expression. The present disclosure provides a dsRNA and / or pharmaceutical composition of the present disclosure for use in the preparation of a medicament for treating and / or preventing a disease associated with MAPT gene expression; in some embodiments, the disease is selected from a neurodegenerative disease associated with the MAPT gene; in some embodiments, the disease is selected from tauopathy, Alzheimer's disease (AD), frontotemporal dementia and Parkinson's disease associated with chromosome 17 (FTDP-17), frontotemporal dementia (FTD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), chronic traumatic encephalopathy (CTE), epilepsy, Dravet syndrome (DS), Pick disease (PiD), argyrophilic grain disease (AGD), glial cell globular inclusion tauopathy (GGT).

[0119] The present disclosure provides use of the aforementioned dsRNA and / or pharmaceutical composition in preparing a medicament for inhibiting the expression of MAPT.

[0120] On the other hand, the present disclosure provides a kind of method for treating and / or preventing subject's disease, including giving dsRNA and / or compositions of the present disclosure to subject.In some embodiments, described disease is the method for the disease relevant to MAPT gene expression.In some embodiments, described disease is selected from tau disease, Alzheimer's disease (AD), frontotemporal dementia and Parkinson's disease (FTDP-17) related to chromosome 17, frontotemporal dementia (FTD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), chronic traumatic encephalopathy (CTE), epilepsy, Dravet syndrome (DS), Pick disease (PiD), argyrophilic grain disease (AGD), glial cell spherical inclusion tau disease (GGT).

[0121] In another aspect, the present disclosure provides a method for reducing MAPT gene expression, comprising administering to a subject an effective amount or effective dose of the dsRNA and / or pharmaceutical composition of the present disclosure.

[0122] In another aspect, the present disclosure provides a method for inhibiting MAPT mRNA expression in a subject, the method comprising administering the dsRNA and / or pharmaceutical composition of the present disclosure to the subject.

[0123] In another aspect, the present disclosure provides a method for delivering an expression-inhibiting oligomeric compound to the liver in vivo, the method comprising administering to a subject a dsRNA and / or a pharmaceutical composition of the present disclosure.

[0124] In another aspect, the present disclosure provides a method for in vivo delivery of a dsRNA that inhibits MAPT expression and / or replication, the method comprising administering to a subject a dsRNA and / or pharmaceutical composition of the present disclosure.

[0125] In another aspect, the present disclosure provides a method for delivering an expression-inhibiting oligomeric compound to the central nervous system in vivo, comprising administering the above-mentioned dsRNA or composition to a subject.

[0126] In another aspect, the disclosure provides a kit comprising the dsRNA and / or pharmaceutical composition of the disclosure.

[0127] In another aspect, the present disclosure provides a cell comprising the dsRNA of the present disclosure.

[0128] The dsRNA or compositions and methods disclosed herein can reduce the level of a target mRNA in a cell, a cell population, a cell population, a tissue or a subject, comprising administering to the subject a therapeutically effective amount of a dsRNA or composition of the present disclosure, the dsRNA being linked to a targeting ligand, thereby inhibiting the expression of the target mRNA in the subject. In some embodiments, the subject has been previously identified as having pathological upregulation of the target gene in the targeted cell or tissue. The subject described in the present disclosure is a subject diagnosed with (or suspected of having, or susceptible to) a disease or condition that would benefit from reduction or inhibition of target mRNA expression.

[0129] The delivery can be by local administration (e.g., direct injection, implantation, or topical administration), systemic administration, or subcutaneous, intravenous, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intramuscular, transdermal, airway (aerosol), nasal, oral, rectal, or topical (including buccal and sublingual) administration.

[0130] In alternative embodiments, the pharmaceutical compositions provided herein can be administered by injection, for example, intravenously, intramuscularly, intradermally, subcutaneously, intraduodenally, or intraperitoneally.

[0131] In an alternative embodiment, after the lipophilic group and / or targeting ligand described above is linked to the dsRNA to form a conjugate, the conjugate can be packaged in a kit.

[0132] In some embodiments, the dsRNA or pharmaceutical composition, when contacted with a cell expressing the target gene, inhibits the expression of the target gene by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, as measured by, for example, psiCHECK activity screening and luciferase reporter gene assays, other methods such as PCR or branched DNA (bDNA)-based methods, or protein-based methods such as immunofluorescence analysis, e.g., Western Blot or flow cytometry.

[0133] In some embodiments, when the dsRNA or pharmaceutical composition is contacted with a cell expressing the target gene, the percentage of target gene mRNA remaining expression caused by the dsRNA or pharmaceutical composition is no greater than 99%, no greater than 95%, no greater than 90%, no greater than 85%, no greater than 80%, no greater than 75%, no greater than 70%, no greater than 65%, no greater than 60%, no greater than 55%, no greater than 50%, no greater than 45%, no greater than 40%, no greater than 35%, no greater than 30%, no greater than 25%, no greater than 20%, no greater than 15%, or no greater than 10%, as measured by, for example, psiCHECK activity screening and luciferase reporter gene assays, other methods such as PCR or branched DNA (bDNA)-based methods, or protein-based methods such as immunofluorescence analysis, for example, Western Blot or flow cytometry.

[0134] In some embodiments, when the above-mentioned dsRNA or pharmaceutical composition is contacted with a cell expressing the target gene, the dsRNA reduces off-target activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70% or at least 75% while maintaining on-target activity as measured by, for example, psiCHECK activity screening and luciferase reporter gene assays, other methods such as PCR or branched DNA (bDNA)-based methods, or protein-based methods such as immunofluorescence analysis, e.g., Western Blot, or flow cytometry.

[0135] In some embodiments, the dsRNA or pharmaceutical composition described above, when contacted with a cell expressing a target gene, reduces off-target activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% while reducing target activity by at most 20%, at most 19%, at most 15%, at most 10%, at most 5%, or more than 1%, as measured by, for example, psiCHECK activity screening and luciferase reporter gene assays, other methods such as PCR or branched DNA (bDNA)-based methods, or protein-based methods such as immunofluorescence analysis, e.g., Western Blot, or flow cytometry.

[0136] In some embodiments, the dsRNA or pharmaceutical composition described above, when contacted with a cell expressing the target gene, increases target activity by at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%, while reducing off-target activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%, as measured by, for example, psiCHECK activity screening and luciferase reporter assays, other methods such as PCR or branched DNA (bDNA)-based methods, or protein-based methods such as immunofluorescence analysis, e.g., Western Blot, or flow cytometry.

[0137] The present disclosure also provides a method for preparing the dsRNA and / or pharmaceutical composition of the present disclosure, comprising: synthesizing the dsRNA and / or pharmaceutical composition of the present disclosure.

[0138] The present disclosure also provides a dsRNA, characterized in that one or more bases U of any dsRNA of the present disclosure are replaced with bases T, for example, 1, 2, 3, 3, 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments, all bases U are replaced with bases T.

[0139] The pharmaceutically acceptable salts of the compounds described in the present disclosure are selected from inorganic salts or organic salts. The compounds described in the present disclosure can react with acidic or basic substances to form corresponding salts.

[0140] On the other hand, without specifying a configuration, the compounds of the present disclosure may exist in specific geometric or stereoisomeric forms. The present disclosure contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the present disclosure. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All of these isomers and their mixtures are included within the scope of the present disclosure.

[0141] In addition, without specifying the configuration, the compounds and intermediates of the present disclosure may also exist in different tautomeric forms, and all such forms are included within the scope of the present disclosure. The term "tautomer" or "tautomeric form" refers to structural isomers of different energies that are interconvertible via a low energy barrier.

[0142] The compounds of the present disclosure may be asymmetric, for example, having one or more stereoisomers. Unless otherwise indicated, all stereoisomers are included, such as enantiomers and diastereomers. The compounds of the present disclosure containing asymmetric carbon atoms can be isolated in optically pure forms or racemic forms. Optically pure forms can be resolved from racemic mixtures or synthesized by using chiral starting materials or chiral reagents.

[0143] Optically active (R)- and (S)-isomers, as well as D and L isomers, can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the present disclosure is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide the pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), a diastereomeric salt is formed with an appropriate optically active acid or base, and then the diastereoisomers are separated by conventional methods known in the art, and then the pure enantiomer is recovered. In addition, the separation of enantiomers and diastereomers is typically accomplished using chromatography, which employs a chiral stationary phase and is optionally combined with a chemical derivatization method (e.g., carbamate formation from an amine).

[0144] The present disclosure also includes isotopically labeled compounds of the present disclosure that are identical to those described herein, but where one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into the compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as 2 H.3 H. 11 C. 13 C. 14 C. 13 N. 15 N. 15 O. 17 O. 18 O. 31 P. 32 P. 35 S. 18 F. 123 I. 125 I and 36 Cl et al.

[0145] Unless otherwise stated, when a position is specifically designated as deuterium (D), the position is understood to have at least 1000 times the abundance of deuterium greater than the natural abundance of deuterium (which is 0.015%) (i.e., at least 10% deuterium incorporation). In the example, the natural abundance of the compound greater than deuterium can be at least 1000 times the abundance of deuterium, at least 2000 times the abundance of deuterium, at least 3000 times the abundance of deuterium, at least 4000 times the abundance of deuterium, at least 5000 times the abundance of deuterium, at least 6000 times the abundance of deuterium or more abundant deuterium. The disclosure also includes various deuterated forms of formula I and formula II compounds. Each available hydrogen atom connected to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can synthesize deuterated forms of formula I and formula II compounds with reference to relevant literature. Commercially available deuterated starting materials may be used to prepare deuterated forms of compounds of Formula I and Formula II, or they may be synthesized using conventional techniques using deuterated reagents, including but not limited to deuterated borane, trideuterated borane in tetrahydrofuran, deuterated lithium aluminum hydride, deuterated iodoethane, deuterated iodomethane, and the like.

[0146] In the chemical structures of the compounds disclosed herein, the bonds Indicates that the configuration is not specified, that is, if chiral isomers exist in the chemical structure, the bond Can be or include both Although all the above structural formulas are drawn as certain isomers for the sake of simplicity, the present disclosure may include all isomers, such as tautomers, rotational isomers, geometric isomers, diastereomers, racemates and enantiomers. In the chemical structures of the compounds described in the present disclosure, the bonds No configuration is specified, i.e. the bond The configuration can be E-type or Z-type, or include both E and Z configurations.

[0147] The present disclosure incorporates the full text of WO2022028462A1 and WO2023274395A1.

[0148] Explanation of terms

[0149] In order to make the present disclosure more easily understood, some technical and scientific terms are specifically defined below. Unless otherwise clearly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present disclosure belongs.

[0150] Unless otherwise specified, in the context of this disclosure, the terms "microtubule-associated protein tau gene," "MAPT," and "Microtubule associated protein tau" are used interchangeably. MAPT includes, but is not limited to, human MAPT, cynomolgus macaque MAPT, mouse MAPT, and rat MAPT. Their amino acid sequences, complete coding sequences, and mRNA sequences are readily available using publicly available databases, such as GenBank, UniProt, OMIM, and the Macaca mulatta genome project website.

[0151] The term "MAPT" also refers to naturally occurring DNA sequence variations of the MAPT gene, such as single nucleotide polymorphisms (SNPs) in the MAPT gene. Exemplary SNPs can be found in the dbSNP database.

[0152] The term "target sequence" refers to a continuous portion of the nucleotide sequence of the mRNA molecule formed during MAPT transcription, including the mRNA of the RNA processing product of the primary transcription product. The targeted portion in the target sequence should be long enough to serve as a substrate for the cutting (iRNA-directed cleavage) guided by iRNA. In one embodiment, the target sequence is in the protein coding region of MAPT.

[0153] The term "neurodegenerative diseases associated with the MAPT gene" includes any disease or condition associated with reduced MAPT expression and / or activity. Symptoms or signs typically include decreased cognitive ability, memory impairment, loss of ability to understand or express language, abnormal behavior, impaired motor function, and an increase in the number or volume of neurofibrillary inclusions. Typical "neurodegenerative diseases associated with the MAPT gene" include tauopathy, Alzheimer's disease (AD), frontotemporal dementia and Parkinson's disease associated with chromosome 17 (FTDP-17), frontotemporal dementia (FTD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), chronic traumatic encephalopathy (CTE), epilepsy, Dravet syndrome (DS), Pick disease (PiD), argyrophilic grain disease (AGD), and glial cell spherical inclusion tauopathy (GGT).

[0154] As used herein, in the context of RNA-mediated gene silencing, the positive strand (also known as SS, SS strand or sense strand) refers to the strand comprising a sequence identical or substantially identical to the target mRNA sequence; the antisense strand (also known as AS or AS strand) refers to the strand having a sequence complementary to the target mRNA sequence.

[0155] In the context of describing the sense strand of the dsRNA described herein, the term "the sense strand comprises a sequence of at least 15 consecutive nucleotides that differs from the nucleotide sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 10, SEQ ID NO: 21 to SEQ ID NO: 78 by no more than 3 nucleotides." is intended to mean that the sense strand of the dsRNA described herein comprises at least 15 consecutive nucleotides as the sense strand of any one of SEQ ID NO: 1 to SEQ ID NO: 10, SEQ ID NO: 21 to SEQ ID NO: 78, or differs from the at least 15 consecutive nucleotides of the sense strand of any one of SEQ ID NO: 1 to SEQ ID NO: 10, SEQ ID NO: 21 to SEQ ID NO: 78 by no more than 3 nucleotide sequences (optionally, differs by no more than 2 nucleotide sequences; optionally, differs by 1 nucleotide sequence; optionally, differs by 0 nucleotides). Other similar descriptions in the context of the present disclosure should also be understood in the same way.

[0156] In this disclosure, the "5' region," also known as the "5' end," or "5' terminus" of the sense or antisense strand may be used interchangeably. For example, nucleotides 2 through 8 in the 5' region of the antisense strand may be replaced with nucleotides 2 through 8 at the 5' terminus of the antisense strand. Similarly, the "3' region," "3' terminus," and "3' terminus" of the sense or antisense strand may be used interchangeably.

[0157] Unless otherwise specified, in the context of this disclosure, "G," "C," "A," "T," and "U" represent nucleosides, including the bases guanine, cytosine, adenine, thymidine, and uracil, respectively. It is well known to those skilled in the art that substitution of T and U does not significantly affect the properties of the dsRNA sequence. U in the sequences disclosed herein can be arbitrarily substituted with T, and the resulting sequences are also within the scope of protection of this disclosure. In the sequence disclosed herein, for the same nucleic acid chain, from the 5' end to the 3' end, the lowercase letter m indicates that the nucleoside adjacent to the left of the letter m is a 2'-methoxy-modified nucleoside; the lowercase letter f indicates that the nucleoside adjacent to the left of the letter f is a 2'-fluoro-modified nucleoside; VP indicates that the 5' end of the nucleoside adjacent to the left / right of the letter is a trans-vinyl phosphodiester group (5'-E-VP); the lowercase letter s indicates that the two nucleosides adjacent to the letter s are connected by a thiophosphate diester group. Unless otherwise specified, the two adjacent nucleosides are connected by a phosphodiester group. Unless otherwise specified, the "RNAi agent", "nucleotide", "compound", "chemical modification", "oligonucleotide", "double-stranded RNAi inhibitor molecule", "siRNA", "siRNA conjugate", "dsRNA", "nucleic acid" and "RNAi", "nucleoside" of the present disclosure can independently exist in the form of a salt, a mixed salt or a non-salt (e.g., a free acid or a free base). When present in the form of a salt or a mixed salt, it can be a pharmaceutically acceptable salt. The term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts. When present in salt form, some groups may ionize to form anions or cations. For example, phosphodiester and thiophosphonate diester groups can exist as anions. The salt forms corresponding to the following structures are also within the scope of protection of this disclosure. Unless otherwise specified, the 3' position of the first nucleotide at the 3' end of each chain is a hydroxyl group; the 5' position of the first nucleotide at the 5' end of each chain is a hydroxyl group.

[0158] The above-mentioned modification and linking groups have the structures shown in the following table, where Base represents a base:

[0159] Table 1

[0160] The term "lipophilic group" or "lipophilic moiety" refers broadly to any compound or chemical moiety that has an affinity for lipids. One way to characterize the lipophilicity of a lipophilic moiety is by the octanol-water partition coefficient log K ow , where K ow is the ratio of the concentration of a chemical substance in the octanol phase to its concentration in the water phase at equilibrium in a two-phase system. In principle, logK ow When it exceeds 0, the chemical is lipophilic. Typically, the logK of the lipophilic moiety isow More than 1, more than 1.5, more than 2, more than 3, more than 4, more than 5, or more than 10, for example, the log K of 6-aminohexanol ow The logK of cholesteryl N-(hexan-6-ol) carbamate is about 0.7. ow It is 10.7.

[0161] The lipophilicity of a molecule can be altered relative to the functional groups it carries. For example, the addition of a hydroxyl or amine group to the terminus of a lipophilic moiety can increase or decrease the partition coefficient (e.g., log K) of the lipophilic moiety. ow ) value. For example, the lipophilic portion can be aliphatic, cyclic such as alicyclic, or polycyclic such as polyalicyclic compounds, such as steroids (e.g., sterols) or straight or branched aliphatic hydrocarbons. The lipophilic portion can generally comprise a hydrocarbon chain, which can be cyclic or acyclic. The hydrocarbon chain can contain various substituents and / or one or more heteroatoms, such as oxygen or sulfur atoms. Such lipophilic aliphatic portions include, but are not limited to, saturated or unsaturated C4-C 30 Hydrocarbons (such as C 10 -C 30 hydrocarbons), saturated or unsaturated fatty acids, waxes (e.g. monoalcohol esters of fatty acids and fatty diamides), terpenes (e.g., C 10 Terpenes, C 15 Sesquiterpenes, C 20 Diterpenes, C 30 Triterpenes and C 40 tetraterpenes) and other polyalicyclic hydrocarbons; for example, the lipophilic portion may be optionally substituted C 10-30 For example, the lipophilic portion can be an optionally substituted C 14-24 of a straight chain alkyl group.

[0162] As used herein, the terms "complementary" and "reverse complement" are used interchangeably and have the meanings known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one strand are paired with bases on the other strand in a complementary manner. In DNA, the purine base adenine is always paired with the pyrimidine base thymine (or uracil in RNA); the purine base guanine is always paired with the pyrimidine base cytosine. Each base pair consists of a purine and a pyrimidine. When adenine on one strand is always paired with thymine (or uracil) on the other strand, and guanine is always paired with cytosine, the two strands are considered to be complementary to each other, and the sequence of the strand can be inferred from the sequence of its complementary strand. Accordingly, "mismatch" is used in the art to mean that in a double-stranded nucleic acid, the bases at corresponding positions are not paired in a complementary manner.

[0163] As used herein, the term "inhibition" can be used interchangeably with "reduce", "silence", "downregulate", "suppression" and other similar terms, and includes the inhibition of any level. Inhibition can be assessed by the reduction of the absolute or relative level of one or more of these variables compared to the control level. The control level can be any type of control level used in the art, such as baseline level before administration or the level determined from an untreated or control (such as buffer control or inert agent control) treated subject, cell, or sample. For example, mRNA remaining expression can be used to characterize the degree of inhibition of dsRNA to target gene expression, such as mRNA remaining expression being not higher than 99%, not higher than 95%, not higher than 90%, not higher than 85%, not higher than 80%, not higher than 75%, not higher than 70%, not higher than 65%, not higher than 60%, not higher than 55%, not higher than 50%, not higher than 45%, not higher than 40%, not higher than 35%, not higher than 30%, not higher than 25%, not higher than 20%, not higher than 15%, or not higher than 10%. The inhibition rate of target gene expression can be measured using The Luciferase Assay System was used to measure the firefly chemiluminescence value (Fir) and the Renilla chemiluminescence value (Ren), and the relative value (Ratio) was calculated as Ren / Fir. In the present disclosure, the ratio of remaining mRNA expression (or remaining activity %) = Ratio (dsRNA-treated group) / Ratio (no-dsRNA control group), and the inhibition rate (%) = 100% - remaining mRNA expression (%).

[0164] Unless otherwise specified, the "compound," "ligand," "nucleic acid-ligand conjugate," "nucleic acid," "conjugate," "chemical modification," "targeting ligand," "dsRNA," "siRNA," and "RNAi" of the present disclosure may independently exist in the form of a salt, a mixed salt, or a non-salt form (e.g., a free acid or a free base). When present in the form of a salt or a mixed salt, it may be a pharmaceutically acceptable salt.

[0165] The term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0166] "Pharmaceutically acceptable acid addition salts" refer to salts formed with inorganic or organic acids that retain the biological effectiveness of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochlorides, hydrobromides, sulfates, nitrates, and phosphates; organic acid salts include, but are not limited to, formates, acetates, 2,2-dichloroacetates, trifluoroacetates, propionates, caproates, octanoates, decanoates, undecylenates, glycolates, gluconates, lactates, sebacates, adipates, glutarates, malonates, oxalates, maleates, succinates, fumarates, tartrates, citrates, palmitates, stearates, oleates, cinnamates, laurates, malates, glutamate, pyroglutamate, aspartate, benzoates, methanesulfonates, benzenesulfonates, p-toluenesulfonates, alginate, ascorbate, salicylates, 4-aminosalicylates, and naphthalene disulfonates. These salts can be prepared by methods known in the art.

[0167] "Pharmaceutically acceptable base addition salts" refer to salts formed with inorganic or organic bases that retain the biological effectiveness of the free acid without other side effects. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts, and the like. Preferred inorganic salts are ammonium salts, sodium salts, potassium salts, calcium salts, and magnesium salts, with sodium salts being preferred. Salts derived from organic bases include, but are not limited to, following salts: primary amines, secondary amines and tertiary amines, substituted amines, including natural substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine. These salts can be prepared by methods known in the art.

[0168] "Effective amount" or "effective dose" refers to the amount of a drug, compound, or pharmaceutical composition necessary to achieve any one or more beneficial or desired therapeutic results. For prophylactic uses, beneficial or desired results include eliminating or reducing the risk, lessening the severity, or delaying the onset of a condition, including the biochemical, histological, and / or behavioral symptoms of the condition, its complications, and intermediate pathological phenotypes that present during the progression of the condition. For therapeutic applications, beneficial or desired results include clinical results, such as reducing the incidence of various conditions associated with the disclosed target genes, target mRNAs, or target proteins, or ameliorating one or more symptoms of the condition, reducing the dose of another agent required to treat the condition, enhancing the efficacy of another agent, and / or delaying the progression of a condition associated with the disclosed target genes, target mRNAs, or target proteins in a patient.

[0169] As used herein, "patient," "subject," or "individual" are used interchangeably and include humans or non-human animals, such as mammals, eg, humans or monkeys.

[0170] The dsRNA provided herein can be obtained by conventional preparation methods in the art (e.g., solid-phase synthesis and liquid-phase synthesis methods). Solid-phase synthesis is commercially available on a custom basis. Modified nucleotide groups can be introduced into the dsRNA disclosed herein by using correspondingly modified nucleoside monomers. Methods for preparing correspondingly modified nucleoside monomers and methods for introducing modified nucleotide groups into dsRNA are also well known to those skilled in the art.

[0171] The term "chemically modified" or "modification" includes all changes in a nucleotide by chemical means, such as the addition or removal of a chemical moiety, or the substitution of one chemical moiety for another.

[0172] The term "base" encompasses any known DNA and RNA base, base analogues such as purines or pyrimidines, and also includes the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogues.

[0173] The terms "blunt end" or "blunt terminus" are used interchangeably to refer to a dsRNA having no unpaired nucleotides or nucleotide analogs at a given terminus, i.e., no nucleotide overhangs. In most cases, a dsRNA having both termini blunt-ended will be double-stranded throughout its entire length.

[0174] The terms "about" and "approximately" refer to values ​​that are within an acceptable error range for a specific value as determined by one of ordinary skill in the art, which depends in part on how it is measured or determined (i.e., the limits of the measurement system). For example, "about" can mean a standard deviation within or exceeding 1. Alternatively, "about" or "substantially comprising" can mean a range of up to 20%, such as between 1% and 15%, between 1% and 10%, between 1% and 5%, between 0.5% and 5%, or between 0.5% and 1%. In this disclosure, each instance in which a number or numerical range is preceded by the term "about" also includes embodiments of the given number. Unless otherwise stated, when a specific value appears in the application and claims, the meaning of "about" or "substantially comprising" should be assumed to be within an acceptable error range for that specific value.

[0175] Unless otherwise indicated, "optionally," "optional," "optional," or "optional" means that the subsequently described event or circumstance may but need not occur, and the description includes situations in which the event or circumstance occurs or does not occur. For example, "optionally, R1 and R2 are directly linked to form a ring" means that R1 and R2 may but need not be directly linked to form a ring, and the description includes situations in which R1 and R2 are directly linked to form a ring and situations in which R1 and R2 do not form a ring.

[0176] In the chemical structural formula disclosed herein, It may be linked to one or more of any groups in accordance with the scope of the invention described herein.

[0177] The term "linked" when referring to a connection between two molecules means that the two molecules are connected by a covalent bond or the two molecules are associated via a non-covalent bond (eg, hydrogen bond or ionic bond), including direct connection and indirect connection.

[0178] The term "directly linked" refers to a first compound or group being linked to a second compound or group without any intervening atoms or groups of atoms.

[0179] The term "indirectly linked" means that a first compound or group is linked to a second compound or group through an intermediate group, compound or molecule (eg, a linking group).

[0180] The term "substituted" means that any one or more hydrogen atoms on a designated atom (usually a carbon, oxygen, or nitrogen atom) are replaced with any group as defined herein, provided that the normal valence of the designated atom is not exceeded and that the substitution results in a stable compound. Non-limiting examples of substituents include C 1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, cyano, hydroxy, oxo, carboxyl, cycloalkyl, cycloalkenyl, heterocyclyl, heteroaryl, aryl, keto, alkoxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, or halogen (e.g., F, Cl, Br, I). When the substituent is keto or oxo (i.e., =0), then two (2) hydrogens on the atom are replaced.

[0181] "Substituted by one or more..." means that the compound may be substituted by a single or multiple substituents. When substituted by multiple substituents, the substituents may be multiple identical substituents or a combination of one or more different substituents. DETAILED DESCRIPTION

[0182] The present disclosure is further described below with reference to the following examples, but these examples are not intended to limit the scope of this disclosure. Experimental methods in the examples herein where specific conditions are not specified generally follow conventional conditions, such as those in the Cold Spring Harbor Laboratory Manual of Antibody Techniques and the Molecular Cloning Manual, or the conditions recommended by the raw material or product manufacturer. Reagents where the specific source is not specified are commercially available.

[0183] Example 1. Design of human MAPT dsRNA

[0184] The human MAPT gene (NM_016841.4) was used as the target gene, and 19 / 21 nt dsRNA was designed to meet the general rules for active dsRNA. The unmodified sense and antisense strand sequences are shown in Tables 2A and 2B, and the modified sense and antisense strand sequences are shown in Table 3.

[0185] Table 2A. Unmodified sense and antisense strands of dsRNAs disclosed herein

[0186] Table 2B. Sense and antisense strands of dsRNAs disclosed herein

[0187] Table 3. Sense and antisense strands of dsRNAs of the present disclosure

[0188] In Tables 2A, 2B, and 3 above, the sequence represents the direction from the 5' end to the 3' end from left to right, and "G," "C," "A," "T," and "U" represent nucleosides, including guanine, cytosine, adenine, thymidine, and uracil, respectively. A lowercase letter m indicates that the nucleoside adjacent to the left of the letter m is a 2'-methoxy-modified nucleoside; a lowercase letter f indicates that the nucleoside adjacent to the left of the letter f is a 2'-fluoro-modified nucleoside; VP indicates that the 5' end of the nucleoside adjacent to the left / right of the letter is a trans-vinyl phosphodiester group (5'-E-VP); a lowercase letter hd indicates that the nucleoside adjacent to the left of the letter hd is a nucleoside in which the 2' position of the sugar ring is connected to an -O-n-hexadecyl group; a lowercase letter s indicates that the two nucleosides adjacent to the letter s are connected by a thiophosphate diester group. Unless otherwise specified, the two adjacent nucleosides are connected by a phosphodiester group. Unless otherwise specified, the 3' position of the first nucleotide at the 3' end of each chain is a hydroxyl group; the 5' position of the first nucleotide at the 5' end of each chain is a hydroxyl group.

[0189] The structures of the 2'-methoxy modified nucleosides, 2'-fluoro modified nucleosides, nucleosides in which the 2' position of the sugar ring is connected to -O-n-hexadecyl, trans-vinyl phosphodiester group (5'-E-VP), phosphorothioate diester group, and phosphodiester group are shown in the following table. When the dsRNA of the present disclosure exists in salt form, for example, in sodium salt form, the salt form corresponding to the structure in Table 4 below is also within the scope of protection of the present disclosure. In Table 4, Base represents the base at the corresponding position:

[0190] Table 4

[0191] Example 2. Synthesis of dsRNA

[0192] The synthesis of dsRNA is similar to conventional phosphoramidite solid-phase synthesis. The synthetic process is briefly described as follows: Nucleoside phosphoramidite monomers were linked one by one according to the synthetic protocol, starting with a universal CPG carrier on a Dr. Oligo 48 synthesizer (Biolytic). Nucleoside phosphoramidite monomers, including those with 2'-methoxy modifications, 2'-fluoro modifications, E-vinyl phosphate groups, and -O-n-hexadecyl linkages at the 2' position of the sugar ring, were purchased from Shanghai Zhaowei, Suzhou Jima, or Jiangsu Shenji. 5-Ethylthio-1H-tetrazole (ETT) was used as the activating agent (0.6 M in acetonitrile), a 0.22 M PADS solution in a 1:1 volume ratio of acetonitrile and collidine (Suzhou Kelema) was used as the sulfurizing agent, and an iodine pyridine / water solution (Kelema) was used as the oxidizing agent.

[0193] After solid-phase synthesis, the oligoribonucleotides were cleaved from the solid support and soaked in a 3:1 solution of 28% ammonia and ethanol at 50°C for 16 hours. The mixture was then centrifuged, and the supernatant was transferred to another centrifuge tube. After evaporation, the supernatant was purified using C18 reverse-phase chromatography with a mobile phase of 0.1 M TEAA and acetonitrile. DMTr was removed using a 3% trifluoroacetic acid solution. The target oligonucleotide was collected, lyophilized, identified as the target product by LC-MS, and quantified by UV (260 nm).

[0194] The obtained single-stranded oligonucleotides were annealed according to complementary pairing in an equal molar ratio, and the resulting dsRNA was dissolved in 1× PBS and adjusted to the required concentration for the experiment.

[0195] Example 3. Inhibition of human MAPT in MCF7 cells by dsRNA - Inhibitory activity at three concentrations

[0196] In vitro molecular activity screening of dsRNA was performed in MCF7 cells using three concentration gradients (10 nM, 1 nM, 0.1 nM).

[0197] MCF7 cells were cultured in Dulbecco's modified eagle medium supplemented with 10% fetal bovine serum at 37°C and 5% CO2. 24 h before transfection, MCF7 cells were seeded in 96-well plates at a density of 1 × 10 cells per well. 4 cells, 100 μL culture medium per well.

[0198] Lipofectamine RNAi MAX (ThermoFisher, 13778150) was used for dsRNA transfection according to the product manual. The final concentration of dsRNA transfection was 10 nM, 1 nM, and 0.1 nM, with duplicate wells for each concentration. After 48 hours of treatment, total RNA was extracted from cells using the high-throughput cell RNA extraction kit FG0417-L / FG0418-XL (Zhifan Medical, magnetic bead method). RNA reverse transcription was performed (Takara, RR037A) and quantitative real-time PCR (Thermo, 4444557) to measure human MAPT mRNA levels. Human MAPT mRNA levels were corrected based on the level of the GAPDH internal reference gene.

[0199] The instruments involved in this experiment are shown in Table 5.

[0200] Table 5. Experimental instruments

[0201] Among them, in the real-time quantitative PCR detection, a probe Q-PCR detection experiment was used, and its primer information is shown in Table 6.

[0202] Table 6 Taqman primer information

[0203] Result analysis method:

[0204] After the Q-PCR test is completed, the corresponding Ct value is obtained according to the threshold value automatically set by the system. The expression of a gene can be relatively quantified by comparing the Ct value: Comparative Ct refers to calculating the gene expression difference by the difference between the Ct value and the internal reference gene, also known as 2 -△△Ct , ΔΔCt=[(Ct experimental group target gene-Ct experimental group internal reference)-(Ct control group target gene-Ct control group internal reference)]. Inhibition rate (%)=(1-remaining amount of target gene expression)*100%.

[0205] The results are expressed as the remaining percentage of human MAPT mRNA expression relative to that of cells treated with dsRNA. The results in Table 7 indicate that the dsRNAs disclosed herein all have good MAPT mRNA inhibitory activity.

[0206] Table 7 Single-dose inhibitory activity of dsRNA in MCF7 cells

[0207] Example 4. Inhibition of human MAPT mRNA in MCF7 cells by the dsRNA disclosed herein

[0208] The dsRNA disclosed herein was screened for in vitro molecular activity in MCF7 cells. The concentration of each dsRNA sample was 10 nM from the beginning of transfection, with 5-fold serial dilutions and 7-9 concentration points.

[0209] MCF7 cells were cultured in Dulbecco's modified eagle medium supplemented with 10% fetal bovine serum at 37°C and 5% CO2. 24 h before transfection, MCF7 cells were seeded in 96-well plates at a density of 1 × 10 cells per well. 4 cells, 100 μL culture medium per well.

[0210] Following the product instructions, dsRNA was transfected using Lipofectamine RNAi MAX (ThermoFisher, 13778150). Seven or nine dsRNA concentrations were set, starting at 10 nM, and duplicate wells were used for each concentration. After 48 hours of treatment, total RNA was extracted from cells using the high-throughput cell RNA extraction kit FG0417-L / FG0418-XL (Zhifan Medical, magnetic bead method). RNA reverse transcription was performed (Takara, RR037A), and quantitative real-time PCR (Thermo, 4444557) was performed to measure human MAPT mRNA levels. Human MAPT mRNA levels were corrected for GAPDH levels.

[0211] Among them, in the real-time quantitative PCR detection, a probe Q-PCR detection experiment was used, and its primer information is shown in Table 8 above.

[0212] Table 8 Taqman primer information

[0213] Result analysis method:

[0214] After the Q-PCR test is completed, the corresponding Ct value is obtained according to the threshold value automatically set by the system. The expression of a gene can be relatively quantified by comparing the Ct value: Comparative Ct refers to calculating the gene expression difference by the difference between the Ct value and the internal reference gene, also known as 2 -△△Ct , ΔΔCt=[(Ct experimental group target gene-Ct experimental group internal reference)-(Ct control group target gene-Ct control group internal reference)]. Inhibition rate (%)=(1-remaining amount of target gene expression)*100%.

[0215] The results are expressed as the percentage of human MAPT mRNA expression remaining in cells treated with dsRNA. 50 The results are shown in Tables 9 and 10. The results in Tables 9 and 10 indicate that the dsRNAs disclosed herein all have good MAPT inhibitory activity.

[0216] Table 9. Activity results of dsRNA at 9 concentrations in MCF-7 cells (IC 50 )

[0217] Table 10. Activity results of dsRNA at 7 concentrations in MCF-7 cells (IC 50 )

[0218] Example 5. Inhibitory activity of the dsRNA disclosed herein on human MAPT in A172 cells

[0219] The disclosed dsRNA was screened for in vitro molecular activity in A172 cells. The concentration of each dsRNA sample was 10 nM from the beginning of transfection, with 5-fold serial dilutions and 7-9 concentration points.

[0220] A172 cells were cultured in Dulbecco's modified eagle medium supplemented with 10% fetal bovine serum at 37°C and 5% CO2. 24 h before transfection, A172 cells were seeded into 96-well plates at a density of 1 × 10 cells per well. 4 cells, 100 μL culture medium per well.

[0221] The experimental operation and result analysis refer to Example 4. In the real-time quantitative PCR detection, a probe Q-PCR detection experiment was used, and the primer information is shown in Table 8.

[0222] In vitro molecular activity screening of dsRNA sequences was performed in A172 cells using a 7-9 concentration gradient. Results were expressed as the percentage of human MAPT mRNA expression remaining relative to cells treated with control dsRNA. IC50 inhibition 50 The results are shown in Tables 11 and 12.

[0223] Table 11. Activity results of dsRNA of the present disclosure at 9 concentration points in A172 cells (IC 50 )

[0224] Table 12. Activity results of dsRNA of the present disclosure at 7 concentrations in A172 cells (IC 50 )

[0225] Example 6. dsRNA psiCHECK on-target activity

[0226] The dsRNA of the present disclosure was screened for on-target activity at the molecular level in vitro using 9 concentration gradients in HEK293A cells.

[0227] The on-target plasmid GSCM, containing a dsRNA target sequence, was constructed and inserted into the psiCHECK-2 plasmid, which contains both the Renilla luciferase and firefly luciferase genes. As a dual-reporter system, the dsRNA target sequence was inserted into the 3' UTR region of the Renilla luciferase gene. The activity of the dsRNA against the target sequence was measured by measuring Renilla luciferase expression after calibration with firefly luciferase.

[0228] HEK293A cells were cultured in DMEM high glucose medium containing 10% fetal bovine serum at 37°C and 5% CO2. 24 h before transfection, HEK293A cells were seeded in 96-well plates at a seeding density of 8 × 10 cells per well. 3 cells, 100 μL culture medium per well.

[0229] Cells were co-transfected with dsRNA and the corresponding GSCM plasmid using Lipofectamine 2000 (ThermoFisher, 11668019) according to the manufacturer's instructions. 0.2 μL of Lipofectamine 2000 was used per well. A total of 20 ng of plasmid was transfected per well. For the target sequence plasmid, 9 dsRNA concentrations were set, starting at 20 nM, and diluted fourfold in duplicate. 24 hours after transfection, target levels were detected using the Dual-Luciferase Reporter Assay System (Promega, E2940).

[0230] Table 13. Activity results of dsRNA of the present disclosure at 9 concentration points in the psi-Check system (IC 50 )

[0231] Table 14. Activity results of dsRNA of the present disclosure at 9 concentration points in the psi-Check system (IC 50 )

[0232] Example 7. Inhibitory activity of dsRNA on human MAPT in A172 cells

[0233] The dsRNA disclosed herein was screened for in vitro molecular activity in A172 cells. The concentration of each dsRNA sample was 10 nM from the beginning of transfection, with 5-fold serial dilution and 7 concentration points.

[0234] A172 cells were cultured in Dulbecco's modified eagle medium supplemented with 10% fetal bovine serum at 37°C and 5% CO2. 24 h before transfection, A172 cells were seeded into 96-well plates at a density of 1 × 10 cells per well. 4 cells, 100 μL culture medium per well.

[0235] The experimental operation and result analysis refer to Example 4. In the real-time quantitative PCR detection, a probe Q-PCR detection experiment was used, and the primer information is shown in Table 6.

[0236] In vitro molecular activity screening of dsRNA sequences was performed in A172 cells using a seven-point concentration gradient. Results were expressed as the percentage of human MAPT mRNA expression remaining relative to that in cells treated with control dsRNA. IC 50 The results are shown in Table 15. The results in Table 15 indicate that TJR103374 of the present disclosure exhibits significantly superior MAPT mRNA inhibitory activity compared to TJR103374, TJR103375, and TJR103376.

[0237] Table 15-1. Activity results (IC50) of dsRNA at 7 concentrations in A172 cells

[0238] Table 15-2. Activity results (IC50) of dsRNA at 7 concentrations in A172 cells

[0239] Example 8. dsRNA psiCHECK on-target activity

[0240] The dsRNA of the present disclosure was screened for on-target activity at the molecular level in vitro using 9 concentration gradients in HEK293A cells.

[0241] The on-target plasmid GSCM, containing a dsRNA target sequence, was constructed and inserted into the psiCHECK-2 plasmid, which contains both the Renilla luciferase and firefly luciferase genes. As a dual-reporter system, the dsRNA target sequence was inserted into the 3' UTR region of the Renilla luciferase gene. The activity of the dsRNA against the target sequence was measured by measuring Renilla luciferase expression after calibration with firefly luciferase.

[0242] HEK293A cells were cultured in DMEM high glucose medium containing 10% fetal bovine serum at 37°C and 5% CO2. 24 h before transfection, HEK293A cells were seeded in 96-well plates at a seeding density of 8 × 10 cells per well. 3 cells, 100 μL culture medium per well.

[0243] Cells were co-transfected with dsRNA and the corresponding GSCM plasmid using Lipofectamine 2000 (ThermoFisher, 11668019) according to the manufacturer's instructions. 0.2 μL of Lipofectamine 2000 was used per well. A total of 20 ng of plasmid was transfected per well. For the target sequence plasmid, 9 dsRNA concentrations were set, starting at 20 nM, and serially diluted threefold, with duplicate wells for each concentration. 24 hours after transfection, on-target levels were detected using the Dual-Luciferase Reporter Assay System (Promega, E2940).

[0244] Table 16. Activity results of dsRNA of the present disclosure at 9 concentration points in the psi-Check system (IC 50 )

[0245] Example 9. Inhibitory activity of dsRNA on human MAPT in A172 cells

[0246] The dsRNA disclosed herein was screened for in vitro molecular activity in A172 cells. The concentration of each dsRNA sample was 10 nM from the beginning of transfection, with 5-fold serial dilution and 7 concentration points.

[0247] A172 cells were cultured in Dulbecco's modified eagle medium supplemented with 10% fetal bovine serum at 37°C and 5% CO2. 24 h before transfection, A172 cells were seeded into 96-well plates at a density of 1 × 10 cells per well. 4 cells, 100 μL culture medium per well.

[0248] The experimental operation and result analysis refer to Example 4. In the real-time quantitative PCR detection, a probe Q-PCR detection experiment was used, and the primer information is shown in Table 6.

[0249] In vitro molecular activity screening of dsRNA sequences was performed in A172 cells using a seven-point concentration gradient. Results were expressed as the percentage of human MAPT mRNA expression remaining relative to that in cells treated with control dsRNA. IC 50 The results are shown in Table 17. The results in Table 17 indicate that the dsRNA of the present disclosure exhibits significantly excellent MAPT mRNA inhibitory activity.

[0250] Table 17. Activity results (IC50) of dsRNA at 7 concentrations in A172 cells

Claims

1. A dsRNA comprising a sense strand and an antisense strand forming a double-stranded region; The sense strand comprises at least 15 consecutive nucleotides and differs from any nucleotide sequence in SEQ ID NO: 1 to SEQ ID NO: 10 by no more than 3 nucleotides; and The antisense strand comprises at least 15 consecutive nucleotides and differs from any nucleotide sequence of SEQ ID NO: 11 to SEQ ID NO: 20 by no more than 3 nucleotides.

2. The dsRNA according to claim 1, wherein the sense strand comprises at least 17 consecutive nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 10; and / or the antisense strand comprises at least 17 consecutive nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of any one of SEQ ID NO: 11 to SEQ ID NO: 20; Preferably, the sense strand comprises at least 19 consecutive nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of any one of SEQ ID NO:1 to SEQ ID NO:10; and / or, the antisense strand comprises at least 21 consecutive nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of any one of SEQ ID NO:11 to SEQ ID NO:

20.

3. The dsRNA according to claim 1 or 2, comprising or selected from the sense strand and the antisense strand of any one of the following groups: Group 1), the sense strand as shown in SEQ ID NO: 1 and the antisense strand as shown in SEQ ID NO: 11; Group 2), the sense strand as shown in SEQ ID NO: 2 and the antisense strand as shown in SEQ ID NO: 12; Group 3), the sense strand as shown in SEQ ID NO: 3 and the antisense strand as shown in SEQ ID NO: 13; Group 4), the sense strand as shown in SEQ ID NO:4 and the antisense strand as shown in SEQ ID NO:14; Group 5), the sense strand as shown in SEQ ID NO:5 and the antisense strand as shown in SEQ ID NO:15; Group 6), the sense strand as shown in SEQ ID NO: 6 and the antisense strand as shown in SEQ ID NO: 16; Group 7), the sense strand as shown in SEQ ID NO:7 and the antisense strand as shown in SEQ ID NO:17; Group 8), the sense strand as shown in SEQ ID NO: 8 and the antisense strand as shown in SEQ ID NO: 18; Group 9), a sense strand as shown in SEQ ID NO: 9 and an antisense strand as shown in SEQ ID NO: 19; and Group 10), the sense strand as shown in SEQ ID NO: 10 and the antisense strand as shown in SEQ ID NO:

20.

4. The dsRNA according to any one of claims 1 to 3, wherein at least one nucleotide in the sense strand and / or the antisense strand is a modified nucleotide; Preferably, all nucleotides in the sense strand and / or antisense strand are modified nucleotides.

5. The dsRNA according to claim 4, wherein three consecutive nucleotides in the sense strand are 2'-fluoro-modified nucleotides; and / or In the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 12, 14 and 16 of the antisense strand are each independently a 2'-fluoro-modified nucleotide; Preferably, in the direction from the 5' end to the 3' end, the nucleotides at positions 7, 8 and 9 of the sense strand are 2'-fluoro-modified nucleotides; the nucleotides at positions 2, 6, 12, 14 and 16 of the antisense strand are each independently 2'-fluoro-modified nucleotides or The nucleotides at positions 2, 4, 6, 10, 12, 14, 16 or 18 of the antisense strand are each independently a 2'-fluoro-modified nucleotide, and optionally, the 5' end of the antisense strand comprises a 5'-vinyl phosphodiester group; The nucleotides at the remaining positions in the sense strand and the antisense strand are 2-methoxy-modified nucleotides.

6. The dsRNA according to any one of claims 1 to 5, wherein at least one phosphodiester group in the sense strand and / or the antisense strand is a phosphodiester group having a modified group, preferably a thiophosphate diester group.

7. The dsRNA according to claim 6, wherein the phosphodiester group having a modification group is present in at least one position selected from the following positions: Between the first nucleotide and the second nucleotide at the 5' end of the sense strand; Between the second nucleotide and the third nucleotide at the 5' end of the sense strand; Between the first nucleotide and the second nucleotide at the 3' end of the sense strand; Between the second nucleotide and the third nucleotide at the 3' end of the sense strand; Between the first nucleotide and the second nucleotide at the 5' end of the antisense strand; Between the second nucleotide and the third nucleotide at the 5' end of the antisense strand; between the first nucleotide and the second nucleotide at the 3' end of the antisense strand; and Between the second nucleotide and the third nucleotide at the 3' end of the antisense strand; Preferably, The sense strand comprises 1, 2, 3 or 4 phosphodiester groups having a modifying group, preferably a thiophosphodiester group; and / or The antisense strand comprises 1, 2, 3 or 4 phosphodiester groups having a modifying group, preferably a phosphorothioate diester group.

8. The dsRNA according to any one of claims 4 to 7, wherein The sense strand comprises the nucleotide sequence shown in any one of SEQ ID NO: 137 to SEQ ID NO: 146; And / or, the antisense strand comprises the nucleotide sequence shown in any one of SEQ ID NO:202 to SEQ ID NO:

211.

9. The dsRNA according to any one of claims 1 to 8, wherein The sense strand of the dsRNA is linked to one or more lipophilic groups.

10. The dsRNA according to claim 9, wherein The sense strand comprises a sequence as shown in any one of SEQ ID NO: 267 to SEQ ID NO: 276, and the antisense strand comprises a sequence as shown in any one of SEQ ID NO: 277 to SEQ ID NO:

286.

11. A pharmaceutical composition comprising the dsRNA according to any one of claims 1 to 10, and a pharmaceutically acceptable carrier.

12. A cell comprising the dsRNA according to any one of claims 1-10.

13. A kit comprising the dsRNA according to any one of claims 1 to 10 or the pharmaceutical composition according to claim 11.

14. A method for reducing the expression of the microtubule-associated protein tau gene (MAPT), the method comprising administering to a subject an effective amount or an effective dose of the dsRNA according to any one of claims 1-10 or a pharmaceutical composition according to claim 11.

15. A method for treating and / or preventing a disease associated with MAPT gene expression in a subject, the method comprising administering to the subject an effective amount or an effective dose of a dsRNA according to any one of claims 1-10, or a pharmaceutical composition according to claim 11; preferably, the disease associated with MAPT gene expression is selected from tauopathy, Alzheimer's disease (AD), frontotemporal dementia and Parkinson's disease associated with chromosome 17 (FTDP-17), frontotemporal dementia (FTD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), chronic traumatic encephalopathy (CTE), epilepsy, Dravet syndrome (DS), Pick disease (PiD), argyrophilic grain disease (AGD), glial globular inclusion tauopathy (GGT).

16. A method for treating and / or preventing a disease, comprising administering to a subject an effective amount or an effective dose of the dsRNA according to any one of claims 1 to 10, or the pharmaceutical composition according to claim 11; preferably, the disease is selected from tauopathy, Alzheimer's disease (AD), frontotemporal dementia and Parkinson's disease associated with chromosome 17 (FTDP-17), frontotemporal dementia (FTD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), chronic traumatic encephalopathy (CTE), epilepsy, Dravet syndrome (DS), Pick disease (PiD), argyrophilic grain disease (AGD), glial globular inclusion tauopathy (GGT). 17 . A method for in vivo delivery of a dsRNA that inhibits MAPT expression and / or replication, the method comprising administering the dsRNA according to any one of claims 1 to 10 or the pharmaceutical composition according to claim 11 to a subject.

18. A method for preparing a dsRNA or a pharmaceutical composition, comprising: Synthesize the dsRNA according to any one of claims 1 to 10, or the pharmaceutical composition according to claim 11.