Sirna targeting MAPT gene expression, and conjugate thereof and use thereof

Abstract

Provided are an siRNA targeting microtubule-associated protein tau (MAPT) gene expression, and a conjugate thereof and the use thereof, wherein the siRNA contains a sense strand and an antisense strand. The provided siRNA, siRNA conjugate and pharmaceutical composition have good stability and good MAPT gene inhibitory activity.

Description

A siRNA targeting MAPT gene expression, its conjugates, and its uses Technical Field

[0001] This invention relates to siRNA and its conjugates for inhibiting MAPT (Microtubule-associated protein tau) gene expression, pharmaceutical compositions, and their use in reducing MAPT gene expression in individuals and preventing pathological conditions or diseases associated with MAPT gene overexpression. Specifically, pathological conditions or diseases associated with MAPT gene overexpression include Alzheimer's disease, supranuclear palsy, cortical-basal degeneration, Argyll Robertson pupillary syndrome, Pick's disease, and tau proteinopathy. Background Technology

[0002] The microtubule-associated protein tau (MAPT) gene, which encodes TAU protein, is located at locus 17q21 on the long arm of human chromosome 17. It contains 16 exons that are spliced ​​into different transcripts. In the human brain, tau protein is primarily encoded by 11 of these exons, which combine to form different TAU ​​proteins with 352 to 441 amino acids. In the CNS, it is mainly distributed in nerve cell axons / dendries, cytoplasm, and cell membranes, and is also secreted into the extracellular cerebrospinal fluid. Its main function is to stabilize the microtubules in nerve cells; these microtubules are part of the cytoskeleton and are crucial for maintaining cell morphology and axonal transport.

[0003] Alzheimer's disease (AD) is an insidious, progressive neurodegenerative disease. Patients typically experience multiple types of symptoms, which become increasingly severe as the disease progresses. While medications can help control the condition and sometimes even slow its progression, they cannot stop it. The main characteristics of the disease include progressive cognitive impairment and behavioral disturbances, making it the most common type of dementia in old age. The risk of developing the disease increases with age; approximately 10% of people over 65 and about 32% of people over 85 are considered to have AD. In 2023, there were approximately 50 million people with AD worldwide, and with an aging population, this number is projected to grow to 150 million by 2050. It seriously threatens people's health and poses a significant socioeconomic burden.

[0004] The pathogenesis of Alzheimer's disease (AD) is complex and not yet fully understood, but several main hypotheses exist to explain its pathological process, including the Aβ cascade hypothesis, the Tau protein aberrant phosphorylation hypothesis, the cholinergic hypothesis, the neuroinflammation hypothesis, and the metal ion disorder hypothesis. Among these, the Tau protein aberrant phosphorylation hypothesis is one of the more thoroughly studied hypotheses, with numerous animal and clinical studies demonstrating that Tau protein plays a crucial role in the pathogenesis of Alzheimer's disease (AD).

[0005] In Alzheimer's disease (AD), Tau protein undergoes abnormal phosphorylation, specifically the addition of phosphate groups at multiple sites. This phosphorylation causes Tau protein to detach from microtubules, disrupting microtubule stability and impairing axonal transport. Abnormally phosphorylated Tau protein readily aggregates to form double-helix filaments, which further aggregate to form neurofibrillary tangles (NFTs). This abnormal aggregation of Tau protein and the formation of NFTs damage the intraneuronal transport system, affecting the transport of nutrients and signaling molecules, ultimately leading to neuronal dysfunction and death. Pathological changes in Tau protein primarily affect the hippocampus and cortical regions of the brain, areas closely related to learning and memory functions. Therefore, abnormal Tau protein aggregation is associated with cognitive decline in AD patients. Diseases caused by abnormal Tau protein include, but are not limited to, Alzheimer's disease, as well as progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Argyll Robertson's pupillary syndrome (AGD), and Pick's disease (PiD). Currently, there are no effective treatments for these diseases, making the development of related therapies of great clinical value.

[0006] This application provides a small interfering RNA (siRNA) formulation targeting MAPT, which can specifically bind to MAPT mRNA, disrupt the normal translation template function of MAPT mRNA, thereby preventing its translation of Tau protein, inhibiting the production of abnormal Tau protein from the source, and can be used to treat / prevent diseases caused by abnormal Tau protein, such as AD, PSP, etc.

[0007] Compared to traditional drugs, siRNAs exhibit poor stability and are easily degraded by nucleases when administered systemically. Furthermore, it is necessary to explore ways to further enhance activity while avoiding off-target effects, immune stimulation, cytotoxicity, and other side effects. Therefore, developing more candidate siRNAs that are stable in the blood, possess good biological activity, exhibit low cytotoxicity, and can effectively inhibit MAPT gene expression over a long period is an urgent problem to be solved. Simultaneously, developing drugs using these candidate siRNAs that inhibit MAPT gene expression to effectively prevent and / or treat diseases caused by Tau protein abnormalities is both necessary for clinical research and a realistic possibility for commercialization. Summary of the Invention

[0008] This invention provides an siRNA for inhibiting MAPT gene expression, the siRNA comprising a sense strand and an antisense strand; wherein the antisense strand comprises at least 17 consecutive nucleotides differing from the nucleotide sequences shown in any of SEQ ID NO:2 to SEQ ID NO:86 by no more than 4 nucleotides, the antisense strand being 17 to 30 nucleotides in length; the sense strand being 17 to 30 nucleotides in length and at least partially complementary to the antisense strand.

[0009] The phrase "at least partially complementary" means that the two sequences can be completely complementary, or have no more than 5, 4, 3, or 2 mismatched base pairs in total, while retaining the ability to hybridize under relevant conditions.

[0010] In some embodiments of the present invention, the antisense strand differs from any nucleotide sequence shown in SEQ ID NO:1 to SEQ ID NO:86 by no more than 4 nucleotides; in some embodiments of the present invention, the antisense strand differs from any nucleotide sequence shown in SEQ ID NO:1 to SEQ ID NO:86 by no more than 3 nucleotides; in some embodiments of the present invention, the antisense strand differs from any nucleotide sequence shown in SEQ ID NO:1 to SEQ ID NO:86 by no more than 2 nucleotides; in some embodiments of the present invention, the antisense strand differs from any nucleotide sequence shown in SEQ ID NO:1 to SEQ ID NO:86 by no more than 1 nucleotide; in some embodiments of the present invention, the antisense strand is any nucleotide sequence shown in SEQ ID NO:1 to SEQ ID NO:86.

[0011] In some embodiments of the present invention, the sense strand and the antisense strand have a mismatch of no more than 3 nucleotides; in some embodiments of the present invention, the sense strand and the antisense strand have a mismatch of no more than 2 nucleotides; in some embodiments of the present invention, the sense strand and the antisense strand have a mismatch of no more than 1 nucleotide; in some embodiments of the present invention, the sense strand and the antisense strand are completely complementary.

[0012] Preferably, the sense strand and the antisense strand are complementary to each other by at least 15, 16, 17, 18, 19, 20 or 21 nucleotides.

[0013] In some embodiments of the present invention, the antisense strand is 19 to 27 nucleotides in length; the sense strand is 19 to 25 nucleotides in length.

[0014] In some embodiments of the present invention, the antisense strand is 19 to 23 nucleotides in length; the sense strand is 19 to 21 nucleotides in length.

[0015] In some embodiments of the present invention, the antisense strand is 23 nucleotides long and the sense strand is 21 nucleotides long. In some embodiments of the present invention, the antisense strand is 22 nucleotides long and the sense strand is 20 nucleotides long. In some embodiments of the present invention, the antisense strand is 21 nucleotides long and the sense strand is 21 nucleotides long. In some embodiments of the present invention, the antisense strand is 21 nucleotides long and the sense strand is 19 nucleotides long. In some embodiments of the present invention, the antisense strand is 19 nucleotides long and the sense strand is 19 nucleotides long.

[0016] In some embodiments of the invention, the siRNA comprises one or more single-stranded nucleotide overhangs, such as 1, 2, 3, or 4 nucleotide overhangs. In some embodiments of the invention, the overhangs may be on the sense strand, the antisense strand, or any combination thereof. In some embodiments of the invention, the overhangs are located at the 5' end, the 3' end, or both ends of the antisense strand or the sense strand of the siRNA.

[0017] In some embodiments of the present invention, the siRNA has a two-nucleotide overhang at the 3' end of the antisense strand.

[0018] In some embodiments of the present invention, the siRNA has a blunt end. In some embodiments of the present invention, the siRNA has at least one blunt end located at the 5' end of the antisense strand (or the 3' end of the sense strand).

[0019] In some embodiments of the present invention, the siRNA has two blunt ends.

[0020] In some embodiments of the present invention, the nucleotide sequence (5'→3') of the siRNA is selected from duplex 1 to duplex 86:

[0021] Table 1. Sense and antisense strand sequences of unmodified siRNA duplexes

[0022] In some embodiments of the present invention, the siRNA contains at least one modifying nucleotide.

[0023] In some embodiments of the present invention, all nucleotides in the sense strand and / or antisense strand of the siRNA are modified nucleotides or nucleotide analogs.

[0024] In some embodiments of the present invention, the modified nucleotide is selected from 2'-methoxynucleotide, 2'-fluoronucleotide, 2'-deoxynucleotide, 2',3'-cleaved nucleotide analog, 2'-fluoro-arabinonucleotide, 2'-methoxyethylnucleotide, 2'-amino-modified nucleotide, 2'-alkyl-modified nucleotide, 3'-methoxynucleotide, 2'-allyl-modified nucleotide, nucleotide containing a thiophosphate group, nucleotide containing a methylphosphonate group, nucleotide containing a 5'-phosphate, nucleotide containing a 5'-phosphate mimic, diol-modified nucleotide, debaseted nucleotide, morpholinonucleotide, locked nucleotide (LNA), unlocked nucleotide (UNA), threononucleotide (TNA), or glycerol nucleotide (GNA), but the present invention is not limited thereto.

[0025] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 7, 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0026] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 6, 8, 9, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 7, 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0027] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein the 2nd, 6th, 14th, and 16th positions of the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein the 9th, 10th, and 11th positions of the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0028] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 6, 8, 9, 10, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 7, 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0029] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 6, 8, 9, 10, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 7, 9, 10, 11, and 15 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0030] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 6, 10, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 3, 7, 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0031] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 7, 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0032] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 3, 4, 6, 8, 10, 14, 16, 18, 20, and 22 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein position 2 at the 5' end of the sense strand is a 2'-fluoronucleotide, and the remaining positions are 2'-methoxynucleotides.

[0033] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 4, 5, 6, 8, 10, 14, 16, 18, 20, and 22 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein position 2 at the 5' end of the sense strand is a 2'-fluoronucleotide, and the remaining positions are 2'-methoxynucleotides.

[0034] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 4, 6, 7, 8, 10, 14, 16, 18, 20, and 22 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein position 2 at the 5' end of the sense strand is a 2'-fluoronucleotide, and the remaining positions are 2'-methoxynucleotides.

[0035] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 4, 6, 8, 10, 14, 16, 18, and 20 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 1, 3, 5, 7, 9, 10, 11, 13, 15, 17, 19, and 21 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0036] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 4, 6, 8, 10, 14, 16, 18, 20, and 22 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 1, 3, 5, 7, 9, 10, 11, 13, 15, 17, 19, and 21 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0037] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 6, 8, 9, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, position 7 is glycerol nucleotide (GNA), and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 7, 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0038] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 8, 9, 10, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, position 6 is glycerol nucleotide (GNA), and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 7, 9, 10, 11, and 15 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0039] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 8, 9, 10, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, position 4 is glycerol nucleotide (GNA), and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 7, 9, 10, 11, and 15 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0040] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 8, 9, 10, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, position 5 is glycerol nucleotide (GNA), and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 7, 9, 10, 11, and 15 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0041] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 8, 9, 10, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, position 7 is glycerol nucleotide (GNA), and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 7, 9, 10, 11, and 15 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0042] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 8, 9, 10, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, position 6 is a glycerol nucleotide (GNA), and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0043] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 5, 7, and 12 at the 5' end of the antisense strand are 2'-deoxynucleotides, position 14 is a 2'-fluoronucleotide, and the remaining positions are 2'-methoxynucleotides. In some embodiments of the present invention, the sense strand of the siRNA is 21 nucleotides long, wherein positions 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0044] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 5, 7, and 12 at the 5' end of the antisense strand are 2'-deoxynucleotides, positions 6, 8, 9, 10, 14, and 16 are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides. In some embodiments of the present invention, the sense strand of the siRNA is 21 nucleotides long, wherein positions 7, 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0045] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides long, wherein the 2nd, 6th, 14th, and 16th positions of the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein the 5th, 7th, and 9th positions of the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0046] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides long, wherein the 2nd, 6th, 14th, and 16th positions of the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein the 7th, 9th, 10th, and 11th positions of the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0047] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides long, wherein positions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0048] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides long, wherein positions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0049] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides long, wherein the 2nd, 6th, 14th, and 16th positions of the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 19 nucleotides long, wherein the 7th, 8th, and 9th positions of the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0050] In some embodiments of the present invention, the antisense strand of the siRNA is 19 nucleotides long, wherein positions 2, 4, 6, 8, 10, 12, 14, 16, and 18 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 19 nucleotides long, wherein positions 7, 8, and 9 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0051] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein the 14th position at the 5' end of the antisense strand is a 2'-fluoronucleotide, the 2nd, 5th, and 7th positions are 2'-deoxynucleotides, the 12th position is a threonucleotide, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein the 9th, 10th, and 11th positions at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0052] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein the 2nd, 6th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein the 9th, 10th, and 11th positions at the 5' end of the sense strand are 2'-fluoronucleotides, the 1st position is a threonucleotide, and the remaining positions are 2'-methoxynucleotides.

[0053] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, position 22 is a threonucleotide, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0054] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, position 23 is a threonucleotide, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0055] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides long, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 20 nucleotides long, wherein positions 8, 9, and 10 at the 5' end of the sense strand are 2'-fluoronucleotides, position 1 is a threonucleotide, and the remaining positions are 2'-methoxynucleotides.

[0056] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides long, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, position 21 is a threonucleotide, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 20 nucleotides long, wherein positions 8, 9, and 10 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0057] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides long, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, position 22 is a threonucleotide, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 20 nucleotides long, wherein positions 8, 9, and 10 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0058] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides long, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 20 nucleotides long, wherein positions 8, 9, and 10 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0059] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides long, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 20 nucleotides long, wherein positions 6, 8, 9, and 10 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0060] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides long, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 20 nucleotides long, wherein positions 8, 9, and 10 at the 5' end of the sense strand are 2'-fluoronucleotides, position 6 is 2'-deoxynucleotide, and the remaining positions are 2'-methoxynucleotides.

[0061] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides long, wherein positions 2, 6, 8, 9, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 20 nucleotides long, wherein positions 6, 8, 9, and 10 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0062] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides long, wherein the 2nd, 6th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 19 nucleotides long, wherein the 7th, 8th, and 9th positions at the 5' end of the sense strand are 2'-fluoronucleotides, the 1st position is a threonucleotide, and the remaining positions are 2'-methoxynucleotides.

[0063] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides long, wherein the 2nd, 6th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoronucleotides, the 20th position is a threonucleotide, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 19 nucleotides long, wherein the 7th, 8th, and 9th positions at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0064] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides long, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, position 21 is a threonucleotide, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 19 nucleotides long, wherein positions 7, 8, and 9 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0065] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 4, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, positions 5 and 7 are 2'-deoxynucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0066] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein the 2nd, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoronucleotides, the 5th position is a 2'-deoxynucleotide, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein the 9th, 10th, 11th, and 12th positions at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0067] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein the 2nd, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoronucleotides, the 5th position is a 2'-deoxynucleotide, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein the 9th, 10th, and 11th positions at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0068] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein the 2nd, 12th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoronucleotides, the 7th position is a 2'-deoxynucleotide, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein the 9th, 10th, and 11th positions at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0069] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein the 2nd, 12th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoronucleotides, the 5th position is a 2'-deoxynucleotide, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein the 9th, 10th, and 11th positions at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0070] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides long, wherein positions 2, 12, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, position 7 is a 2'-deoxynucleotide, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 20 nucleotides long, wherein positions 8, 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0071] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides long, wherein the 2nd, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoronucleotides, the 5th position is a 2'-deoxynucleotide, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 20 nucleotides long, wherein the 8th, 9th, 10th, and 11th positions at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0072] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides long, wherein the 2nd, 12th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoronucleotides, the 7th position is a 2'-deoxynucleotide, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 19 nucleotides long, wherein the 7th, 8th, and 9th positions at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0073] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides long, wherein the 2nd, 12th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoronucleotides, the 7th position is a 2'-deoxynucleotide, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 19 nucleotides long, wherein the 7th, 9th, and 11th positions at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0074] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides long, wherein positions 2, 5, 12, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, position 7 is a 2'-deoxynucleotide, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 19 nucleotides long, wherein positions 7, 8, and 9 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0075] In some embodiments of the present invention, the modified nucleotide is a nucleotide in which the phosphate group is modified by a thiophosphate group. That is, a sulfur atom replaces the non-bridging oxygen atom in the phosphodiester bond, thereby replacing the phosphodiester bond with a thiophosphate diester bond.

[0076] In some embodiments of the present invention, the 5' end and 3' end of the sense chain each independently contain 0, 1, or 2 thiophosphate groups; and / or the 5' end and 3' end of the antisense chain each independently contain 1 or 2 thiophosphate groups.

[0077] In some embodiments of the present invention, at least one of the nucleotides at positions 1 and 2 of the 5' end of the sense strand, the nucleotides at positions 2 and 3 of the 5' end of the sense strand, the nucleotides at positions 1 and 2 of the 3' end of the sense strand, the nucleotides at positions 2 and 3 of the 3' end of the sense strand, the nucleotides at positions 1 and 2 of the 3' end of the antisense strand, the nucleotides at positions 2 and 3 of the 3' end of the antisense strand, the nucleotides at positions 1 and 2 of the 5' end of the antisense strand, and the nucleotides at positions 2 and 3 of the 5' end of the antisense strand are linked by a thiophosphate group; preferably, at least four are linked by thiophosphate groups; in some embodiments of the present invention, at least six are linked by thiophosphate groups; in some embodiments of the present invention, all eight are linked by thiophosphate groups.

[0078] In some embodiments of the present invention, the nucleotides at positions 1 and 2, and at positions 2 and 3, of the 5' end of the positive strand are linked by phosphate thioester groups.

[0079] In some embodiments of the present invention, the nucleotides at positions 1 and 2, and positions 2 and 3 at the 5' end of the positive strand are linked by thiophosphate groups, and the nucleotides at positions 1 and 2, and positions 2 and 3 at the 3' end are linked by thiophosphate groups.

[0080] In some embodiments of the present invention, the nucleotides at positions 1 and 2 at the 3' end of the antisense strand are linked by thiophosphate groups, and the nucleotides at positions 2 and 3 at the 5' end are linked by thiophosphate groups.

[0081] In some embodiments of the present invention, the nucleotides at positions 1 and 2 of the 5' end of the sense strand, the nucleotides at positions 2 and 3 of the 5' end of the sense strand, the nucleotides at positions 1 and 2 of the 3' end of the sense strand, the nucleotides at positions 2 and 3 of the 3' end of the sense strand, the nucleotides at positions 1 and 2 of the 3' end of the antisense strand, the nucleotides at positions 2 and 3 of the 3' end of the antisense strand, the nucleotides at positions 1 and 2 of the 5' end of the antisense strand, and the nucleotides at positions 2 and 3 of the 5' end of the antisense strand are all linked by thiophosphate groups.

[0082] In some embodiments of the present invention, the positive strand may include one or more capping residues or portions, referred to as “capping residues”. A “capping residue” is a nonnucleotide compound or other portion that can be incorporated into one or more ends of a nucleotide sequence of siRNA. In some embodiments of the present invention, the capping residues are present at the 5' end, the 3' end, or both the 5' end and the 3' end of the positive strand.

[0083] In some embodiments of the present invention, an inverse debasing residue (invAb) is added as a capping residue. See F. Czauderna, Nucleic Acids Res., 2003, 31(11), 2705-16. In some embodiments of the present invention, the 5' end and / or 3' end of the positive strand may contain more than one inverse debasing deoxyribose moiety as a capping residue.

[0084] In some embodiments of the invention, one or more inverse debase residues (invAb) are added to the 3' end of the positive strand. In some embodiments of the invention, one or more inverse debase residues (invAb) are added to the 5' end of the positive strand. In some embodiments of the invention, one or more inverse debase residues may be inserted between the linker-target ligand portion and the nucleotide sequence of the siRNA positive strand. In some embodiments of the invention, one or more inverse debase residues are included at or near one or more ends of the siRNA positive strand.

[0085] In some embodiments of the invention, one or more inverse abase residues (invAb) are added to the 5' end of the positive strand. In some embodiments of the invention, one or more inverse abase residues may be inserted between the linker-target ligand portion and the nucleotide sequence of the siRNA positive strand.

[0086] Reverse debasing residues can be linked to nucleic acids via phosphodiester bonds, thiophosphate diester bonds, etc.

[0087] In some embodiments of the present invention, the first nucleotide at the 5' end of the antisense strand is selected from the following structures:

[0088] Wherein, Base is a base A, U, G, C, T or other modified bases.

[0089] In some embodiments of the present invention, the first nucleotide at the 5' end of the antisense strand is a (E)-vinyl phosphate modified nucleotide.

[0090] In some embodiments of the present invention, the siRNA contains at least one base-modified nucleotide.

[0091] In some embodiments of the present invention, the bases of the base-modified nucleotide are selected from the following structures:

[0092] In some embodiments of the present invention, the base-modified nucleotide may be located at positions 5, 6, 7, and 8 of the siRNA antisense strand.

[0093] In some embodiments of the present invention, the base-modified nucleotide may be located at the single-stranded nucleotide overhang in the siRNA.

[0094] Preferably, the siRNA antisense strand contains two nucleotide overhangs, and the base-modified nucleotide is the first nucleotide of the siRNA antisense strand overhang.

[0095] Preferably, the siRNA antisense strand contains two nucleotide overhangs, and the base-modified nucleotide is the second nucleotide of the siRNA antisense strand overhang.

[0096] In some embodiments of the present invention, the nucleotide sequence (5'→3') of the modified siRNA is selected from duplex 87 to duplex 172:

[0097] Table 2. Sensitive and antisense strand sequences of the modified siRNA duplex

[0098] In some embodiments of the present invention, the nucleotide sequence (5'→3') of the modified siRNA is selected from duplex 173 to duplex 237:

[0099] Table 3. Sensitive and antisense strand sequences of the modified siRNA duplex

[0100] The present invention also provides siRNA conjugates obtained by conjugating the above-mentioned siRNA with conjugating molecules.

[0101] In this invention, unless otherwise specified, "conjugation" refers to the covalent connection between two or more chemical parts; "conjugated compound" refers to a compound formed by the covalent connection between various chemical parts; and "siRNA conjugated compound" refers to a compound formed by the covalent attachment of one or more chemical parts to siRNA. It should be noted that the chemical parts can be directly attached to the siRNA or attached to the siRNA via a linker.

[0102] In this invention, unless otherwise specified, the "-" in "connector-targeting ligand" refers to the covalent connection between the connector and the targeting ligand.

[0103] In some embodiments of the present invention, the targeting ligand is linked to siRNA via a linker to form a conjugate molecule.

[0104] In some embodiments of the present invention, the targeting ligand forms a conjugated molecule via a linker, which is independently or simultaneously attached to the 3' or 5' end of the siRNA's positive strand.

[0105] In some embodiments of the present invention, the siRNA of the present invention can be conjugated with a pharmaceutically acceptable conjugating molecule to obtain an siRNA conjugate. In some embodiments of the present invention, the siRNA is covalently conjugated to the conjugating molecule. To reduce the potential impact of conjugation on siRNA activity, the conjugation site between the siRNA and the conjugating molecule can be at the 3' or 5' end of the siRNA's sense strand, or at the 5' end of the antisense strand. In some embodiments, the conjugation site between the siRNA and the conjugating molecule can also be within the internal sequence of the siRNA.

[0106] The pharmaceutically acceptable targeting ligand may be a targeting ligand commonly used in the field of siRNA drug delivery, such as, but not limited to, one or more of the following targeting ligands or their derivatives: lipophilic molecules, such as cholesterol, bile acids, vitamins (e.g., vitamin E), lipid molecules of different chain lengths; polymers, such as polyethylene glycol; polypeptides, such as transmembrane peptides; aptamers; antibodies; quantum dots; carbohydrates, such as lactose, polylactose, mannose, galactose, N-acetylgalactosamine (GalNAc); folic acid; or receptor ligands expressed by hepatocytes, such as desialyl glycoprotein, desialyl sugar residues, lipoproteins (e.g., high-density lipoprotein, low-density lipoprotein, etc.), glucagon, neurotransmitters (e.g., adrenaline), growth factors, transferrin, etc.

[0107] In some embodiments of the present invention, the targeting ligand is N-acetylgalactosamine.

[0108] In some embodiments of the present invention, the targeting ligand is directly attached to the 3' end of the siRNA positive strand. In some embodiments of the present invention, the targeting ligand is directly attached to the 5' end of the siRNA positive strand.

[0109] In some embodiments of the present invention, the targeting ligand is attached to the 3' end of the siRNA positive strand via a adapter.

[0110] In some embodiments of the present invention, the targeting ligand is attached to the 5' end of the siRNA positive strand via a adapter.

[0111] In some embodiments of the present invention, the targeting ligand is N-acetylgalactosamine, which is attached to the 3' end of the siRNA sense strand via a linker.

[0112] In some embodiments of the present invention, the targeting ligand linker portion is GalNAc(L96) and has the following structure:

[0113] In some embodiments of the present invention, GalNAc(L96) is linked to the 3' end of the siRNA positive strand.

[0114] In some embodiments of the present invention, GalNAc(L96) is linked to the 5' end of the siRNA positive strand.

[0115] In some embodiments of the present invention, GalNAc(L96) is linked to the inverse ablation residue (invAb) at the 3' end of the siRNA positive strand.

[0116] In some embodiments of the present invention, GalNAc(L96) is linked to the inverse ablation residue (invAb) at the 5' end of the siRNA's positive strand.

[0117] In some embodiments of the present invention, the targeting ligand connector portion is Ser(GN) and has the following structure:

[0118] In some embodiments of the present invention, Ser(GN) is attached to the 3' end of the siRNA positive strand. In some embodiments of the present invention, Ser(GN) is attached to the 5' end of the siRNA positive strand.

[0119] In some embodiments of the present invention, Ser(GN) is simultaneously linked to both the 3' and 5' ends of the siRNA positive strand.

[0120] In some embodiments of the present invention, the targeting ligand connector portion is LP-GalNAc (attached to the 5' end of the positive chain) and has the following structure:

[0121] In some embodiments of the present invention, the targeting ligand connector portion is XY-GalNAc (attached to the 3' end of the positive chain) and has the following structure:

[0122] In some embodiments of the present invention, the siRNA conjugate is selected from conjugate 1 to conjugate 10.

[0123] Table 4. Sequences of modified siRNA conjugates

[0124] In some embodiments of the present invention, the siRNA conjugates of the present invention are formed by covalently linking siRNA with a ligand, and the ligand is linked at any position on the sense or antisense strand of the siRNA.

[0125] In some embodiments of the present invention, the ligand is a lipophilic molecule.

[0126] In some embodiments of the present invention, the ligand is directly attached to the 3' end of the siRNA positive strand.

[0127] In some embodiments of the present invention, the ligand is directly attached to the 5' end of the siRNA positive strand.

[0128] In some embodiments of the present invention, the ligand is attached to the 3' end of the siRNA positive strand via a adapter.

[0129] In some embodiments of the present invention, the ligand is attached to the 5' end of the siRNA positive strand via a adapter.

[0130] In some embodiments of the present invention, the lipid ligand is covalently linked to any position of the siRNA nucleotide glycosyl group via a hydroxyl group; wherein the covalent bond is an ether bond, ester bond, amino carbonate bond, phosphodiester bond, or thiophosphate diester bond; preferably, it is linked to the 2', 3', or 5' position of the nucleotide glycosyl group. In some embodiments of the present invention, the lipophilic moiety comprises a saturated or unsaturated C4-C30 hydrocarbon chain (e.g., C4-C30 alkyl or alkenyl), the hydrocarbon chain being linked to the siRNA via a lipophilic moiety containing a hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, or alkyne functional group.

[0131] In some embodiments of the present invention, the ligand portion is a C16 alkyl group, which, upon coupling with the 2'-position of at least one nucleotide nucleotide glycosyl group of the siRNA, forms the following structure:

[0132] Among them The siRNA is linked to the rest of the siRNA via phosphodiester bonds or thiophosphate bonds.

[0133] In some embodiments of the present invention, the lipid ligand is covalently linked to any position of the siRNA nucleoside glycosyl group via a hydroxyl group; wherein the covalent bond is an ether bond, ester bond, amino carbonate bond, phosphodiester bond or thiophosphate diester bond; preferably, it is linked to the 2'-position, 3'-position or 5'-position of the nucleoside glycosyl group.

[0134] In some embodiments of the present invention, the lipid ligand coupled with siRNA forms the following structure:

[0135] in,

[0136] exist The part is connected to the rest of the siRNA;

[0137] X is selected from O or S;

[0138] Base is selected from A, T, G, C, I or unconventional bases;

[0139] R is a C4-C30 alkyl chain, a deuterated or fluoroalkyl chain, or an optional functional group including hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.

[0140] R' can be OH, F, OMe, or H.

[0141] In some embodiments of the present invention, the lipid ligand is coupled to the 2'-position of at least one nucleotide nucleotide glycosyl group of the siRNA to form the following structure:

[0142] in,

[0143] exist The portion of the siRNA is linked to the rest of the siRNA via a phosphodiester bond or a phosphothiodiester bond.

[0144] R is a C4-C30 alkyl chain, a deuterated or fluoroalkyl chain, or an optional functional group including hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, or alkyne, or Oleyl, Arachidonic acid, Vitamin A.

[0145] In some embodiments of the present invention, the lipid ligand is coupled to the 2'-position of at least one nucleotide nucleotide glycosyl group of the siRNA to form the following structure:

[0146] B can be a conventional base or a modified base.

[0147] In some embodiments of the present invention, the lipid ligand portion has the following structure:

[0148] Where m is selected from integers 1-12; p is selected from integers 1-20.

[0149] In some embodiments of the present invention, the lipid ligand portion has the following structure:

[0150] In some embodiments of the present invention, the lipid ligand portion has the following structure:

[0151] The present invention also provides a pharmaceutical composition comprising any of the above-described siRNAs and / or any of the above-described siRNA conjugates and a pharmaceutically acceptable carrier.

[0152] In some embodiments of the invention, the pharmaceutical composition contains one siRNA as described in the first aspect. In other embodiments of this disclosure, the pharmaceutical composition contains at least two siRNAs as described in the first aspect (e.g., but not limited to 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) as active ingredients. Preferably, the at least two siRNAs as described in the first aspect each target different target sequences in the MAPT gene, thereby expecting to exert synergistic effects by acting simultaneously on different target sequences. Here, "different target sequences" means that there is no overlap between target sequences, or the number of overlapping consecutive nucleotides between target sequences is less than 5 (e.g., the number of overlapping consecutive nucleotides is 4, 3, 2, 1, or 0). In this case, the at least two siRNAs as described in the first aspect can be present in any different proportions. Preferably, the at least two siRNAs as described in the first aspect may exist in a molar ratio of 1:100 to 100:1; more preferably, the at least two siRNAs as described in the first aspect may exist in a molar ratio of 1:10 to 10:1, 1:5 to 5:1, or 1:2 to 2:1. In some embodiments of the present invention, the at least two siRNAs as described in the first aspect exist in the same molar ratio.

[0153] The present invention also provides the use of any of the above-described siRNAs and / or any of the above-described siRNA conjugates and / or the above-described pharmaceutical compositions in the preparation of medicaments for treating and / or preventing pathological conditions or diseases associated with Tau protein abnormalities.

[0154] Further, the pathological condition or disease is a CNS disease. More preferably, the CNS disease is a disease caused by abnormal Tau protein, including but not limited to Alzheimer's disease, as well as Progressive Supranuclear Palsy (PSP), Corticobasal Degeneration (CBD), Argyll Robertson Pupil Syndrome (AGD), and Pick's Disease (PiD).

[0155] The siRNA, siRNA conjugates, and pharmaceutical compositions provided by this invention exhibit good stability, excellent MAPT gene inhibitory activity, satisfactory cytotoxicity and immunostimulatory effects, and can improve diseases caused by abnormal Tau protein aggregation.

[0156] The sequence of the MAPT gene targeted by siRNA in this invention is shown in SEQ ID NO:197:

[0157] SEQ ID NO:197 (MAPT gene)

[0158] In this invention, unless otherwise specified, uppercase letters C, G, U, A, and T represent the base composition of nucleotides, including modified and unmodified nucleotides; lowercase letter m indicates that the nucleotide adjacent to the right of the identifier m is a 2'-methoxynucleotide; letter i2F indicates that the nucleotide adjacent to the right of the identifier f is a 2'-fluoronucleotide; lowercase letter d indicates that the nucleotide adjacent to the right of the identifier d is a 2'-deoxynucleotide; cet indicates 2'-O-ethyl-bridged nucleic acid; and gn indicates that the nucleotide adjacent to the right of the identifier gn is glycerol. Nucleotide (GNA); tn indicates that the nucleotide adjacent to the right of the tn identifier is a threonucleotide (TNA); the asterisk (*) indicates that the two nucleotides adjacent to the asterisk (or the nucleotide and the linker-targeting ligand) are linked by a thiophosphate group; eVP indicates that the nucleotide adjacent to its right is a (E)-vinyl phosphate modified nucleotide; invAb indicates a reverse debasing residue; hd is a 2'-O-C16 nucleotide; L96 indicates that the linker-targeting ligand is conjugated at this location (GalNAc(L96)). Ser(GN) indicates that the linker-targeting ligand is conjugated at this location (Ser(GN)).

[0159] In this invention, unless otherwise specified, the capital letter I indicates the base composition of the base-modified nucleotide, and the base is... mI represents Inosine with a methoxy-substituted ribose 2'-position; m6A indicates the base composition of the modified nucleotide, with the bases being... The uppercase letter X indicates the base composition of a base-modified nucleotide, where the base is... The uppercase letter B indicates the base composition of a base-modified nucleotide, where the base is... The number 15 indicates the base of the base-modified nucleotide, and the base is... Unless otherwise specified, all the nucleotides containing special bases have a methoxy group substituted at the 2'-position of the ribose.

[0160] In this invention, unless otherwise specified, the term "complementary" refers to the ability of an oligonucleotide of a first sequence to hybridize with an oligonucleotide of a second sequence under certain conditions and form a double-stranded structure. "At least partially complementary" means that the two sequences can be completely complementary, or have no more than 5, 4, 3, or 2 mismatched base pairs in total, while retaining the ability to hybridize under the relevant conditions. Furthermore, where the two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs should not be considered mismatches for determining complementarity. In this invention, to satisfy the above hybridization ability requirements, the "complementary" sequence may also include or consist entirely of base pairs formed from non-Watson-Crick base pairs and / or from non-natural and modified nucleotides. Such non-Watson-Crick base pairs include, but are not limited to, G:U swing base pairs or Hoogstein base pairs. Correspondingly, in this invention, unless otherwise specified, "mismatch" refers to a situation in the siRNA double-stranded molecule where the bases at corresponding positions are not paired in a complementary manner. In this invention, unless otherwise specified, "difference in nucleotide sequence" refers to a change in the type of bases at the same or corresponding positions of the nucleotides compared to the original nucleotide sequence. For example, if a nucleotide base in the original nucleotide sequence is A, and the nucleotide base at the same or corresponding position is changed to U, C, G, or dT, dC, dG, etc., then a difference in nucleotide sequence is considered to exist at that position. It should be noted that if, compared to the original nucleotide sequence, the nucleotides at the same or corresponding positions differ only in the presence or type of modification, then a difference in nucleotide sequence is not considered to exist at that position.

[0161] In this invention, unless otherwise specified, the term "pharmaceutical acceptable" means that the carrier, transporter, diluent, excipient and / or the salt / ester / hydrate formed therefrom is generally chemically or physically compatible with other components constituting a drug dosage form and physiologically compatible with the receptor.

[0162] In this invention, unless otherwise specified, the term "inhibition" refers to the down-regulation of target gene expression due to siRNA-mediated mRNA degradation. "Down-regulation" refers to a decrease in target gene expression level of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more, or even 100%, compared to the absence of siRNA treatment. A 100% decrease in target gene expression level means that there is no detectable level of target gene expression.

[0163] In this invention, the siRNA may also contain modified nucleotides as needed, and the modified nucleotides will not cause a significant weakening or loss of the siRNA's function in inhibiting MAPT gene expression. Currently, there are various ways to modify siRNA in the art, including, for example, backbone modification (such as phosphate group modification), ribose group modification, and base modification (Watts, JK, G.F. Deleavey, and M.J. Damha, Chemically Modified siRNA: Tools and Applications. Drug Discov Today, 2008, 13(19-20): p. 842-55).

[0164] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0165] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Detailed Implementation

[0166] Those skilled in the art will recognize that the siRNA described in this invention can be obtained using conventional siRNA preparation methods (e.g., solid-phase synthesis and liquid-phase synthesis), both of which are commercially available custom-made services. Those skilled in the art will also understand that modified nucleotide groups can be introduced into the siRNA described in this invention using appropriately modified nucleotide monomers. Methods for preparing appropriately modified nucleotide monomers are well known to those skilled in the art, and commercially available monomers are also available.

[0167] Example 1: siRNA Synthesis

[0168] For the sense and antisense strands of the nucleic acid double-stranded conjugate sequences disclosed herein, as well as the sense and antisense strands of the modified double strands, CPG was used as a solid-phase support; CPG was used as the starting cycle for the synthesis of the sense and antisense strands.

[0169] Using a YB-192S synthesizer, a solid-phase synthesis method of phosphoramidite was employed. Starting with a solid support, nucleoside monomers were sequentially linked in the 3'-5' direction to achieve a synthesis scale of 0.2 μmol.

[0170] The phosphorus amide monomer is linked through a continuous cycle of four chemical reactions: deprotection, coupling, oxidation / sulfidation, and capping. The phosphorus amide monomer is prepared as a 0.05 M acetonitrile solution, with 0.3 M BTT in acetonitrile as the activator, a 3% trichloroacetic acid / dichloromethane solution as the deprotecting agent, a 0.05 M iodine / pyridine / tetrahydrofuran / aqueous solution (v / v / v = 2 / 1 / 7) as the oxidizing agent, acetic anhydride / acetonitrile solution as capping agent A (v / v = 2 / 8), pyridine / N-methylimidazolium / tetrahydrofuran solution as capping agent B (v / v / v = 10 / 16 / 74) as the capping agent, and a 0.05 M DDTT solution of pyridine / acetonitrile as the thiochemical agent (v / v = 4 / 6).

[0171] After solid-phase synthesis, the support was transferred to a 2 mL centrifuge tube, and 0.8 mL of concentrated ammonia was added. The mixture was then sealed and reacted at 55 °C for 16 h. After cooling to room temperature, the solution was transferred to a 2 mL centrifuge tube and concentrated to dryness. 0.2 mL of anhydrous DMSO was added to dissolve the solution, followed by 0.25 mL of triethylamine trihydrofluoride. The reaction was carried out at 65 °C for 2 h. After the reaction was complete, the mixture was cooled to room temperature, and the crude sequence was obtained by ethanol precipitation.

[0172] The crude product was purified by reversed-phase HPLC, and the collected fraction was lyophilized. Ethanol precipitation was performed by adding 0.3 mL of 1M sodium acetate solution and 0.9 mL of ethanol to replace the sequence with sodium salt. Then, desalting was carried out using a 3KD ultrafiltration tube to remove excess free salt.

[0173] The sense and antisense chains were prepared into an aqueous solution of a certain concentration. The sense and antisense chains were mixed at a molar ratio of 1:1.05, incubated at 95°C for 5 minutes, and then naturally cooled to room temperature. The solution was then freeze-dried to obtain the target product.

[0174] Representative LC-MS test method: When the test sample is subjected to denaturing IP·RP-LC detection, the complementary double strands are untied into single strands (sense and antisense strands). Then, the parent ions of the sense and antisense strands are fragmented by tandem mass spectrometry. All detected fragment ions are analyzed and resolved using the software CONFIRM Sequence. The sequence of the test sample is consistent with the theoretical sequence, that is, the deviation between the actual molecular weight (MW) and the theoretical molecular weight (MW) is less than 0.05%. The results are shown in Tables 5 and 6.

[0175] Table 5. Molecular weight (MW) of nucleic acid double-stranded conjugates

[0176] Table 6. Molecular weight (MW) of lipid-delivered nucleic acid double-stranded conjugates

[0177] Example 2: In vitro activity detection

[0178] T98G cell activity assay

[0179] Cell culture: T98G cells (ATCC) were cultured at 37°C and 5% CO2 in MEM complete medium (Gibco, supplemented with 10% FBS and 1% P / S) until near confluence. Cells were then trypsinized and seeded into 96-well plates, with 2.0 × 10⁶ cells added to each well. 4 T98G cells were cultured in 0.1 mL of MEM complete medium (Gibco, with 10% FBS and 1% P / S) at 37°C and 5% CO2 for 16-24 h.

[0180] Cell transfection: Add 0.15 μL of lipofectamine RNAiMax (Invitrogen) to each well of opti-MEM, then add 5.0 μL of siRNA and mix. Add the mixture to a PCR tube and incubate at room temperature for 5 minutes. Finally, add the siRNA mixture to the cells and continue culturing for 24 hours before RNA extraction. Single-dose experiments were performed at concentrations of 10 nM and 1 nM or 1 nM and 0.1 nM siRNA duplexes.

[0181] RNA extraction

[0182] Using the Total RNA Isolation Kit (Omega, CAT: R6834-02): Discard the original culture medium, collect the cells, wash with PBS, then add 200 μL of lysis buffer (containing 1% β-mercaptoethanol) to lyse the cells. Follow the instructions for the RNA isolation kit. Finally, add 30 μL of RNase-free water, let stand for 2 minutes, and then centrifuge at 14000g for 2 minutes to collect the RNA.

[0183] cDNA synthesis

[0184] cDNA synthesis was performed using the TransGold gDNA Removal and cDNA Synthesis Kit (TransGold Biotechnology Co., Ltd., Beijing, China, Cat#AE311-03). 1 μg of total RNA was added to each sample, and cDNA synthesis was performed using a gradient thermal cycler (LongGene, A600) following the manufacturer's instructions.

[0185] Real-time quantitative PCR

[0186] Add the synthesized cDNA and the mixed stock solution (containing primers, qPCR premix and ultrapure water) to a 384-well plate (Bokcom Biosystems Cat#PC-0040-9U) to make the final real-time quantitative PCR system contain 0.25 μM each of upstream and downstream primers of the target gene (MAPT) or internal reference gene (GADPH) and 1×SYBR Green premix (Applied Biosystems Cat#A25742).

[0187] The ΔΔCt measurement method was used in ABI QuantStudio. TM 6. Perform real-time fluorescence PCR in a real-time fluorescence PCR system. Perform 3-4 independent transfection tests for each double-stranded strain, with 3-4 assays per transfection.

[0188] psiCHECK in vitro activity assay

[0189] Bioinformatics analysis was used to screen for cross-reactive MAPT siRNA candidate sequences in humans and non-human primates, and potential MAPT inhibitors of this invention were synthesized. For screening of target siRNAs, human MAPT cDNA sequences were cloned from commercially available mammalian expression vectors (oriene, rockville, md) into a commercially available, report-based screening plasmid (psicheck2(promega, madison, wi)), which produces Renilla luciferase.

[0190] / MAPT fusion mRNA. siRNA activity was detected in Hep3B cells using psiCHECK screening. Hep3B cells were seeded at 20,000 cells / well in 96-well plates. The MAPT siRNA of this invention was transfected at two concentrations: 50 ng MAPT-pSiCHECK2 plasmid and 0.3 μL lipofectamine 2000 per well. After culturing at 37°C and 5% CO2 for 24 h, MAPT gene knockout activity was determined using a Dual-Glo luciferase assay system (Promega E2920). Each duplex was subjected to 3-4 independent transfection assays. Gene knockout was determined by measuring Renila luciferase levels normalized to constitutively expressed firefly luciferase levels.

[0191] Table 7. In vitro activity of modified sequences, i.e., residual mRNA expression levels.

[0192] Table 8-1 In vitro activity of modified sequences, i.e., residual mRNA expression levels

[0193] Table 8-2 In vitro activity of modified sequences, i.e., residual mRNA expression levels

[0194] Table 9. In vitro activity assay of conjugates, i.e., residual mRNA expression level.

[0195] Example 3: Effects of intraventricular administration of siRNA on target gene expression in the brain of hMAPT mice

[0196] After 1 week of acclimatization, male hMAPT mice were randomly divided into 13 groups of 3 mice each on Day 0 according to their body weight. The mice were then administered the drugs according to Table 10. The first day of administration was Day 0.

[0197] Lateral ventricle administration: Experimental mice were anesthetized by inhalation of isoflurane. The mouse head was prepared and disinfected with povidone-iodine solution. The anesthetized mice were then fixed in a stereotaxic apparatus. Specifically, the upper incisors were inserted into the horizontal bar, and the knob was adjusted to press the nose bar firmly. The ear bars were inserted into the mouse's ear canal, and the left and right ear bars were adjusted to ensure that the line connecting the two ears was aligned with the ear bars. After ensuring that the scale positions of the left and right ear bars were the same, the knob was adjusted to lock the ear bars. The criteria for proper mouse fixation were: nose aligned with the midline, head still, tail not falling off when lifted, and skull level. Isoflurane was continuously inhaled throughout the entire procedure.

[0198] ASO814907 is a positive compound with the following structure:

[0199] mCesmCeoGesTesTesTdsTdsmCdsTdsTdsAdsmCdsmCdsAesmCeomCesmCesTe

[0200] “e” represents 2'-MOE nucleotide, “d” is 2'-deoxynucleotide, “o” is phosphate ester bond, “s” is thiophosphate ester bond, and “mC” is m5C.

[0201] The number of animals in each group and the detailed route of administration, dosage, and regimen are shown in Table 10 below:

[0202] Table 10. Administration routes, dosages, and regimens in animal models.

[0203] Note: aCSF is artificial cerebrospinal fluid.

[0204] After group administration, the effects of the compound on the animals' normal behavior were routinely monitored, including the animals' activity level, feeding and drinking, and any abnormalities in the eyes, coat, or other areas. On Day 7, mice were anesthetized with isoflurane, their abdominal cavities were opened, the abdominal aorta was severed, and the mice were euthanized. After opening the skull, the brain tissue was separated, and the hippocampus, frontal cortex, hypothalamus, and other remaining brain tissue from the right side of the brain were collected and preserved in RNAlater for PCR detection.

[0205] Table 11: Residual expression levels of hMAPT mRNA in various tissues 7 days after drug administration

[0206] Example 4: Study on PD activity of a single intrathecal injection of the test substance in hMAPT mice

[0207] After maintaining the depth of anesthesia with 2.5% isoflurane inhalation, the animal is placed in a prone position, and the hair on its waist is shaved. The puncture site is wiped with alcohol. The L5-6 interspinous space is used as the puncture point, and the vertical distance between the puncture point and the highest point of the mouse's back is maintained >3cm. The experimenter locates the hip tubercle with their left hand; its horizontal position is the L5-6 interspinous space of the mouse. The left thumb and middle finger are placed on both sides of the L5-6 interspinous space of the mouse, and the skin is stretched outward. With the index finger in position, the right hand holds a microsyringe and slowly inserts the needle vertically into the interspinous space. The mouse's tail is observed during needle insertion. When a tail-flicking motion occurs, the syringe plunger is gently pulled back. When cerebrospinal fluid reflux is observed, the injection site is determined, and intrathecal injection is performed. The injection time and needle retention time are 30-40 seconds after needle insertion. The drug volume is 10μL, and the day of drug administration is defined as Day 0.

[0208] Table 12. Administration routes, dosages, and regimens in animal models.

[0209] Seven days after administration (Day 7), mice were anesthetized with isoflurane and euthanized by exsanguination from the abdominal aorta. Dissection was performed to collect tissues from the lumbar spinal cord (administration site), cervical spinal cord, frontal cortex, midbrain, hippocampus, striatum, hypothalamus, and remaining brain tissue. The remaining brain tissue was flash-frozen in liquid nitrogen and then stored at -80°C for later analysis. qPCR was used to detect changes in hMAPT mRNA expression in each tissue, and the results are shown in Table 13 below.

[0210] Table 13 Residual expression levels of hMAPT mRNA in various tissues 7 days after drug administration.

[0211] Conclusion: In vivo testing in transgenic mice showed that conjugates 2, 5, and 8 achieved good gene silencing effects on target genes in CNS tissues, especially in the hippocampus, frontal cortex, and temporal cortex tissues associated with Alzheimer's disease, where the gene silencing effect was superior to that of the clinical compound ASO814907.

Claims

1. A siRNA for inhibiting MAPT gene expression, characterized in that: The siRNA comprises a sense strand and an antisense strand; wherein the antisense strand comprises at least 17 consecutive nucleotides that differ from the nucleotide sequences shown in any of SEQ ID NO:1 to SEQ ID NO:86 by no more than 4 nucleotides, and the antisense strand is 17 to 30 nucleotides in length; the sense strand is 17 to 30 nucleotides in length and is at least partially complementary to the antisense strand.

2. The siRNA according to claim 1, characterized in that: The antisense strand is 19–27 nucleotides long; the sense strand is 19–25 nucleotides long.

3. The siRNA according to claim 1, characterized in that: The antisense strand differs from any of the nucleotide sequences shown in SEQ ID NO:1 to SEQ ID NO:86 by no more than 4 nucleotides.

4. The siRNA according to claim 1, characterized in that: The sense strand and antisense strand have a mismatch of no more than 3 nucleotides.

5. The siRNA according to claims 1-4, characterized in that: The siRNA sequence is selected from duplex 1 to duplex 86.

6. The siRNA according to any one of claims 1-5, characterized in that: The siRNA contains at least one modifying nucleotide.

7. The siRNA according to claim 6, characterized in that: All nucleotides in the sense and / or antisense strands of the siRNA are modified nucleotides or nucleotide analogs.

8. The siRNA according to claim 7, characterized in that: The modified nucleotide is selected from 2'-methoxynucleotides, 2'-fluoronucleotides, 2'-deoxynucleotides, 2',3'-cleaved nucleotide analogs, 2'-fluoro-arabinonucleotides, 2'-methoxyethylnucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, 3'-methoxynucleotides, 2'-allyl-modified nucleotides, nucleotides containing thiophosphate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphate groups, nucleotides containing 5'-phosphate analogs, diol-modified nucleotides, debased nucleotides, morpholinonucleotides, locked nucleotides, unlocked nucleotides, threonucleotides, or glycerol nucleotides.

9. The siRNA according to any one of claims 1-8, characterized in that: The 5' end and 3' end of the sense chain each independently contain 0, 1, or 2 thiophosphate groups; and / or the 5' end and 3' end of the antisense chain each independently contain 1 or 2 thiophosphate groups.

10. The siRNA-modified duplex according to any one of claims 6-9, characterized in that: The siRNA-modified double strands are selected from the modified double strands shown in Tables 2 and 3: double strand 87 to double strand 237.

11. The siRNA according to any one of claims 1-10, characterized in that: The first nucleotide at the 5' end of the antisense strand is a (E)-vinyl phosphate modified nucleotide, and / or the 5' and / or 3' ends of the sense strand optionally contain capping residues (e.g., invAb).

12. The siRNA according to any one of claims 1-11, characterized in that: The siRNA contains at least one base-modified nucleotide.

13. The siRNA conjugate obtained by conjugating the siRNA according to any one of claims 1-12 with a conjugating molecule.

14. The siRNA conjugate according to claim 13, characterized in that: The targeting ligand forms a conjugated molecule via a linker, attaching independently or simultaneously to the 3' or 5' end of the siRNA's positive strand; or, The siRNA conjugate is formed by covalently linking siRNA with a ligand, wherein the ligand is a lipophilic molecule and is attached to any position on either the sense or antisense strand of the siRNA. Preferably, the ligand portion has the following structure:

15. The siRNA conjugate according to claims 13-14, characterized in that: The siRNA conjugates are selected from conjugate 1 to conjugate 10.

16. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises the siRNA of any one of claims 1-12 and / or the siRNA conjugate of any one of claims 13-15 and a pharmaceutically acceptable carrier.

17. Use of the siRNA of any one of claims 1-12 and / or the siRNA conjugate of any one of claims 13-15 and / or the pharmaceutical composition of claim 16 in the preparation of a medicament for reducing MAPT gene expression in an individual and for treating and / or preventing pathological conditions or diseases associated with MAPT gene overexpression.

18. The use according to claim 17, characterized in that: The pathological conditions or diseases mentioned include Alzheimer's disease, supranuclear palsy, cortical-basal degeneration, Argyll Robertson pupillary syndrome, Pick's disease, and tau proteinosis.

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