Sirna targeting APP gene and pharmaceutical composition thereof

Abstract

Provided in the present invention are a siRNA targeting an APP gene and a pharmaceutical composition thereof. The present invention also provides a conjugate of the siRNA, the conjugate comprising the siRNA that inhibits the expression of the APP gene and a pharmaceutically acceptable targeting molecule. The siRNA and the conjugate thereof provided by the present invention can effectively inhibit the expression of the APP gene, have good stability and negligible cytotoxicity and immunostimulatory activity, and thus can be used for treating diseases or conditions benefiting from reduced APP levels or inhibited expression.

Description

siRNA targeting the APP gene and its pharmaceutical composition

[0001] Cross-reference to related applications

[0002] This application claims priority to the following patent applications: patent application No. 202510198759.3, filed with the State Intellectual Property Office of China on February 21, 2025, entitled "siRNA targeting the APP gene and pharmaceutical composition thereof"; and priority to the earlier application No. 202610186468.7, filed with the State Intellectual Property Office of China on February 9, 2026, entitled "siRNA targeting the APP gene and pharmaceutical composition thereof"; the entire contents of the above patent applications are incorporated herein by reference. Technical Field

[0003] This invention provides an siRNA that inhibits the expression of amyloid precursor protein (APP) gene and a pharmaceutical composition thereof. The siRNA and pharmaceutical composition provided by this invention can treat diseases associated with amyloid precursor protein overexpression. Background Technology

[0004] Amyloid precursor protein (APP) is a single-pass transmembrane protein containing over 700 amino acids. β-amyloid protein (Aβ) is a product of abnormal APP cleavage. Under normal circumstances, APP is mainly cleaved by α and γ secretases in the body, while β secretase activity is low, resulting in the production of small amounts of Aβ. Under pathological conditions, β secretases synthesize and secrete large amounts, cleaving APP into Aβ. Aβ has neurotoxic effects; when its levels rise, cells cannot metabolize it, leading to its accumulation in large quantities outside the cell and the formation of Aβ fiber deposits. Aβ fiber deposits can damage nerve cells, including inducing apoptosis through the mitochondrial pathway, triggering inflammatory responses, and exacerbating the formation of neurofibrillary tangles, ultimately leading to cognitive impairment in patients. Therefore, inhibiting APP expression can reduce Aβ production, protect neurons, alleviate brain damage, and achieve the goal of preventing and treating the disease.

[0005] Alzheimer's disease (AD), cerebral amyloid angiopathy (CAA), and Down syndrome (DS) are all neurodegenerative diseases caused by abnormal Aβ deposition. AD is an insidious, progressive neurodegenerative disease affecting approximately 40 million people worldwide. Clinical manifestations include memory impairment, aphasia, apraxia, agnosia, visuospatial skill impairment, executive dysfunction, and personality and behavioral changes. Pathological features include Aβ deposition and Tau protein neurofibrillary tangles. CAA is a common neurodegenerative disease characterized by Aβ deposition in small blood vessels of the cerebral cortex and pia mater, leading to weakened vessel walls and increased susceptibility to rupture, resulting in cerebrovascular dysfunction. CAA is particularly common in the elderly; as age increases, the brain's ability to clear Aβ declines, causing the incidence of CAA to increase with age. Patients with Down syndrome have an extra chromosome 21, which contains the APP gene. Most patients with Down syndrome develop Aβ amyloid plaques before the age of 40, and more than 50% of patients will experience Alzheimer's disease before the age of 50.

[0006] Currently, approved drug treatments for APP-related diseases or conditions (including AD, CAA, and DS) are symptomatic treatments, not preventative or curative. Aβplague clearance antibody therapies, approved in recent years, have shown limited clinical benefit and cause significant treatment-related cerebral edema / hemorrhagic side effects. Therefore, there is an urgent clinical need for safe and effective treatment strategies for APP-related diseases. Summary of the Invention

[0007] This invention provides siRNA that can effectively inhibit APP gene expression, and thereby provides pharmaceutical compositions and methods for the prevention and / or treatment of APP-related diseases.

[0008] 1. siRNA

[0009] In one aspect, the present invention provides a small interfering RNA (siRNA) for inhibiting APP gene expression, the siRNA comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 consecutive nucleotides having a nucleotide sequence that is approximately 4 (e.g., 0, 1, 2, 3, or 4) nucleotides similar to the nucleotide sequence shown in any one of SEQ ID NO:1 to SEQ ID NO:200, SEQ ID NO:418 to SEQ ID NO:434, and the sense strand is at least partially complementary to the antisense strand.

[0010] The phrase "at least partially complementary" means that the two sequences can be completely complementary, or have no more than 6, 5, 4, 3, 2, or 1 mismatched base pairs in total, while retaining the ability to hybridize under relevant conditions. Those skilled in the art can determine the most suitable conditions for testing the complementarity of the two sequences based on the final application of the hybridized nucleotides. Such conditions can be, for example, stringent conditions, such as 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, at 50 or 70°C for 12-16 hours, followed by washing. Other conditions, such as physiologically relevant conditions that may be encountered in vivo, can also be applied.

[0011] In some embodiments, the antisense strand comprises at least 17 consecutive nucleotides that are approximately 4 (e.g., 0, 1, 2, 3, or 4) nucleotides similar to the nucleotide sequences shown in any one of SEQ ID NO:1 to SEQ ID NO:200, SEQ ID NO:418 to SEQ ID NO:434.

[0012] In some embodiments, the antisense strand comprises at least 17 consecutive nucleotides that differ from the nucleotide sequences shown in any one of SEQ ID NO:1 to SEQ ID NO:200, SEQ ID NO:418 to SEQ ID NO:434 by 0, 1, or 2 nucleotides.

[0013] In some embodiments, the antisense strand comprises at least 17 consecutive nucleotides that are approximately 4 (e.g., 0, 1, 2, 3, or 4) nucleotides similar to the nucleotide sequences shown in any one of SEQ ID NO:1 to SEQ ID NO:200, SEQ ID NO:418 to SEQ ID NO:434. In some embodiments, the antisense strand comprises at least 17 consecutive nucleotides that are approximately 3 (e.g., 0, 1, 2, or 3) nucleotides similar to the nucleotide sequences shown in any one of SEQ ID NO:1 to SEQ ID NO:200, SEQ ID NO:418 to SEQ ID NO:434. In some embodiments, the antisense strand comprises at least 17 consecutive nucleotides that are approximately 2 (e.g., 0, 1, or 2) nucleotides similar to the nucleotide sequences shown in any one of SEQ ID NO:1 to SEQ ID NO:200, SEQ ID NO:418 to SEQ ID NO:434. In some embodiments, the antisense strand comprises at least 17 consecutive nucleotides that are approximately equal to one (e.g., 0 or 1) nucleotide of the nucleotide sequence shown in any one of SEQ ID NO:1 to SEQ ID NO:200, SEQ ID NO:418 to SEQ ID NO:434. In some embodiments, the antisense strand comprises the nucleotide sequence shown in any one of SEQ ID NO:1 to SEQ ID NO:200, SEQ ID NO:418 to SEQ ID NO:434. In some embodiments, the antisense strand is the nucleotide sequence shown in any one of SEQ ID NO:1 to SEQ ID NO:200, SEQ ID NO:418 to SEQ ID NO:434.

[0014] In some embodiments, the sense strand and the antisense strand have a mismatch of no more than 6 nucleotides. In some embodiments, the sense strand and the antisense strand have a mismatch of no more than 5 nucleotides. In some embodiments, the sense strand and the antisense strand have a mismatch of no more than 4 nucleotides. In some embodiments, the sense strand and the antisense strand have a mismatch of no more than 3 nucleotides. In some embodiments, the sense strand and the antisense strand have a mismatch of no more than 2 nucleotides. In some embodiments, the sense strand and the antisense strand have a mismatch of no more than 1 nucleotide. In some embodiments, the sense strand and the antisense strand are completely complementary.

[0015] In some implementations, the sense strand and the antisense strand are complementary to each other by at least 15, 16, 17, 18, 19, 20 or 21 nucleotides.

[0016] In some embodiments, the sense strand and the antisense strand are at least 85% (e.g., at least 90%, at least 95%, at least 99%) complementary or completely complementary over at least 17 consecutive nucleotides.

[0017] In some embodiments, the sense strand and the antisense strand form a double-stranded region of 15 to 30 nucleotide pairs in length, such as 15 to 25 nucleotide pairs, 15 to 23 nucleotide pairs, 15 to 21 nucleotide pairs, such as 15, 16, 17, 18, 19, 20 or 21 nucleotide pairs.

[0018] In some embodiments, the antisense strand is 17 to 30 nucleotides long (e.g., 17 to 29, 17 to 28, 17 to 27, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 25, 19 to 23, 21 to 25, 21 to 23), and the sense strand is 17 to 30 nucleotides long (e.g., 17 to 29, 17 to 28, 17 to 27, 17 to 26, 17 to 25, 17 to 21, 19 to 21).

[0019] In some implementations, the antisense strand is 19 to 27 nucleotides long, and the sense strand is 17 to 25 nucleotides long.

[0020] In some implementations, the antisense strand is 21 to 23 nucleotides long, and the sense strand is 18 to 21 nucleotides long.

[0021] In some implementations, the antisense strand is 23 nucleotides long and the sense strand is 21 nucleotides long.

[0022] In some embodiments, the antisense strand is 23 nucleotides long and the sense strand is 18 nucleotides long.

[0023] In some implementations, the siRNA includes a blunt end and / or a protruding end.

[0024] In some embodiments, the siRNA comprises one or more single-stranded nucleotide overhangs, such as 1, 2, 3, 4, or 5 nucleotide overhangs. In some embodiments, the overhangs may be on the sense strand, the antisense strand, or any combination thereof. In some embodiments, 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.

[0025] In some embodiments, the 3' end of the antisense strand of the siRNA includes a two-nucleotide overhang. In some embodiments, the 3' end of the sense strand of the siRNA is blunt.

[0026] In some implementations, the 3' end of the antisense strand of the siRNA contains a two-nucleotide overhang.

[0027] In some implementations, the 3' end of the antisense strand of the siRNA contains a 5-nucleotide overhang.

[0028] In some embodiments, the positive chain comprises at least 15 consecutive nucleotides that are approximately 4 (e.g., 0, 1, 2, 3, or 4) nucleotides similar to the nucleotide sequences shown in any one of SEQ ID NO:201 to SEQ ID NO:400, SEQ ID NO:401 to SEQ ID NO:417.

[0029] In some embodiments, the sense strand of the siRNA comprises at least 17 consecutive nucleotides having a nucleotide sequence that is approximately equal to at least four nucleotides (e.g., 0, 1, 2, 3, or 4 nucleotides) of the nucleotide sequence shown in any one of SEQ ID NO:201 to SEQ ID NO:400, SEQ ID NO:401 to SEQ ID NO:417. The sense strand and the antisense strand of the siRNA comprise at least 17 consecutive nucleotides forming a double-stranded region. In some embodiments, the sense strand comprises at least 17 consecutive nucleotides having a nucleotide sequence that is approximately equal to at least three nucleotides (e.g., 0, 1, 2, or 3 nucleotides) of the nucleotide sequence shown in any one of SEQ ID NO:201 to SEQ ID NO:400, SEQ ID NO:401 to SEQ ID NO:417. In some embodiments, the positive strand comprises at least 17 consecutive nucleotides that are approximately two (e.g., 0, 1, or 2) nucleotides similar to the nucleotide sequence shown in any one of SEQ ID NO:201 to SEQ ID NO:400, SEQ ID NO:401 to SEQ ID NO:417. In some embodiments, the positive strand comprises at least 17 consecutive nucleotides that are approximately one (e.g., 0 or 1) nucleotide similar to the nucleotide sequence shown in any one of SEQ ID NO:201 to SEQ ID NO:400, SEQ ID NO:401 to SEQ ID NO:417.

[0030] In some embodiments, the positive chain comprises at least 17 consecutive nucleotides that differ from the nucleotide sequences shown in any one of SEQ ID NO:201 to SEQ ID NO:400, SEQ ID NO:401 to SEQ ID NO:417 by 0, 1, or 2 nucleotides.

[0031] In some embodiments, the sense strand has a region within the 17 consecutive nucleotides that is at least 85% (e.g., at least 90%, at least 95%, at least 99%) complementary or completely complementary to the antisense strand.

[0032] In some embodiments, the positive strand comprises at least 17 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NO:201 to SEQ ID NO:400, SEQ ID NO:401 to SEQ ID NO:417. In some embodiments, the positive strand is the nucleotide sequence shown in any one of SEQ ID NO:201 to SEQ ID NO:400, SEQ ID NO:401 to SEQ ID NO:417.

[0033] In some embodiments, the antisense strand of the siRNA comprises the antisense strand sequence or a portion thereof (e.g., at least 17 consecutive nucleotides thereof) of any duplex SJ-0001 to SJ-0200, SJ-0201 to SJ-0223 provided in Tables 1A and 1B, and the sense strand of the siRNA comprises the sense strand sequence or a portion thereof (e.g., at least 17 consecutive nucleotides thereof) of the duplex.

[0034] In some embodiments, the sequences of the sense and antisense strands of the siRNA comprise the sense and antisense strand sequences of any double-stranded form selected from SJ-0001 to SJ-0200 and SJ-0201 to SJ-0223 as described in Tables 1A and 1B.

[0035] In some embodiments, the antisense strand of the siRNA comprises an antisense strand sequence or a portion thereof (e.g., at least 17 consecutive nucleotides) as shown in any one of SEQ ID Nos: 165, 11, 15, 1, 173, 188, 177, 191, 182, 162, 7, 199, 170, 186, 13, 185, 74. Preferably, the sense strand of the siRNA comprises a sense strand sequence or a portion thereof (e.g., at least 17 consecutive nucleotides) as shown in any one of SEQ ID Nos: 365, 211, 215, 201, 373, 388, 377, 391, 382, ​​362, 207, 399, 370, 386, 213, 385, 274.

[0036] In some embodiments, the antisense strand of the siRNA comprises the antisense strand sequence or a portion thereof (e.g., at least 17 consecutive nucleotides) of SJ-0165, SJ-0011, SJ-0015, SJ-0001, SJ-0173, SJ-0188, SJ-0177, SJ-0191, SJ-0182, SJ-0162, SJ-0007, SJ-0199, SJ-0170, SJ-0186, SJ-0013, SJ-0185, and SJ-0074 provided in Table 1A. Preferably, the sense strand of the siRNA comprises the sense strand sequence or a portion thereof (e.g., at least 17 consecutive nucleotides) of the duplex.

[0037] 2. siRNA modification pattern

[0038] siRNAs can be modified in their nucleoside base structure or ribose-phosphate backbone to enhance their ability to inhibit target mRNA expression, reduce off-target effects, increase molecular biostability, increase the physical stability of the duplex formed between antisense and sense nucleic acids, and / or reduce immunogenicity. siRNA molecules containing ribonucleoside analogs or derivatives must retain the ability to form duplexes and allow or mediate specific degradation of target RNA via the RISC pathway.

[0039] In some embodiments of the invention, the siRNA contains at least one modifying nucleotide. The modifications need not be identical for each of the plurality of modified ribonucleotides in the siRNA.

[0040] 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.

[0041] In some embodiments of the present invention, the siRNA comprises 2'-modified nucleotides.

[0042] In some embodiments of the present invention, the modified nucleotide is selected from 2'-methoxynucleotides, 2'-fluoronucleotides, 2'-deoxynucleotides, 2',3'-cleaved nucleotide analogs, 2'-fluoroarabinonucleotides, 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 mimics, diol-modified nucleotides, deoxyxanthine nucleotides, 2'-O-methoxyethyl-modified nucleotides, 2'-methoxy and 2'-fluoro-modified nucleotides, debased nucleotides, morpholinonucleotides, locked nucleotides (LNA), unlocked nucleotides (UNA), or glycerol nucleotides (GNA), but the present invention is not limited thereto.

[0043] In some embodiments of the present invention, the nucleotides in the sense strand of the siRNA are selected from at least two of 2'-methoxynucleotides, 2'-fluoronucleotides, and 2'-deoxynucleotides; and / or the nucleotides in the antisense strand of the siRNA are selected from at least two of 2'-methoxynucleotides, 2'-fluoronucleotides, nucleotides modified with 2'-methoxy and 2'-fluoro, deoxyxanthine nucleotides, nucleotides modified with 2'-O-methoxyethyl, glycerol nucleotides, unlocking nucleotides, and nucleotides of 5'-phosphate mimics.

[0044] In some embodiments of the invention, the 5' and / or 3' ends of the positive strand of the siRNA optionally include capping residues (e.g., iab).

[0045] In some embodiments of the invention, the first nucleotide at the 5' end of the antisense strand of the siRNA optionally comprises (E)-vinylphosphonate modification.

[0046] In some embodiments of the present invention, the sense strand and / or antisense strand of the siRNA comprises modified internucleotide links; preferably, the 5' end and 3' end of the sense strand each independently comprise one or two thiophosphate groups; and / or the 5' end and 3' end of the antisense strand each independently comprise one or two thiophosphate groups.

[0047] In some embodiments of the present invention, the above-described antisense strand modification mode and / or sense strand modification mode are applied to small interfering RNA (siRNA) for suppressing APP gene expression.

[0048] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long and includes the following modification patterns:

[0049] (1)AS(5’-3’):Nms Nfs Nm Nm Nm Nf Nm Nm Nm Nm Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm;

[0050] (2)AS(5’-3’):Nms Nfs Nm Nm Nm Nf Nm Nf Nf Nm Nm Nf Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm;

[0051] (3)AS(5’-3’):Nms Nfs Nm Nm Nm Nf Nm Nf Nf Nm Nm Nf Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm;

[0052] (4)AS(5’-3’):Nms Nfs Nm Nm Nm Nm Nm Nm Nf Nm Nm Nf Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm;

[0053] (5)AS(5’-3’):Nms Nfs Nm Nf Nm Nm Nm Nm Nm Nm Nm Nf Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm;

[0054] (6)AS(5’-3’):Nms Nfs Nm Nm Nm Nf Nm Nf Nf Nf Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm;

[0055] (7)AS(5’-3’):Nms Nfs Nm Nm Nm Nf Nm Nf Nf Nf Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm;

[0056] (8)AS(5’-3’):Nms Nfs Nm Nf Nm Nf Nm Nf Nm Nf Nm Nm Nm Nf Nm Nf Nm Nf Nm Nf Nms Nms Nm;

[0057] (9)AS(5’-3’):Nms Nfs Nm Nm Nm Nf Nm Nm Nm Nm Nm Nm Nm Nf Nm Nf Nm Nf Nm Nm Nms Nms Nm;

[0058] (10)AS(5’-3’):Nms Nfs Nm Nm Nm Nm Nm Nf Nf Nm Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm;

[0059] (11)AS(5’-3’):Nms Nfs Nm Nm Nm Nm Nm Nf Nf Nm Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm;

[0060] (12)AS(5’-3’):Nms Nfs Nm Nm Nm Nm Nm Nf Nf Nm Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm;

[0061] (13)AS(5’-3’):Nms Nfs Nm Nm Nm Nm Nm Nf Nf Nm Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm;

[0062] (14)AS(5’-3’):Nms Nfs Nm Nm Nm Nm Nm Nf Nf Nm Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm;

[0063] (15)AS(5’-3’):Nms Nfs Nm Nm Nm Nm Nm Nf Nf Nm Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm;

[0064] (16)AS(5’-3’):Nms Nfs Nm Nm Nm Nm Nm Nf Nf Nm Nm Nm Nm Nf Nm Nf Nm Nf Nm Nm Nms Nms Nm;

[0065] (17)AS(5’-3’):Nms Nfs Nms Nm Nm Nm Nm Nf Nf Nm Nm Nm Nm Nf Nm Nm Nm Nf Nm Nm Nms Nms Nm;

[0066] (18)AS(5’-3’):Nms Nfs Nm Nm Nm Nm Nm Nf Nf Nm Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm;

[0067] (19)AS(5’-3’):Nms Nfs Nm Nm Nm Nm Nm Nf Nf Nm Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm;

[0068] (20)AS(5’-3’):Nms Nfs Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm;

[0069] (21)AS(5’-3’):Nms Nfs Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm;

[0070] (22)AS(5’-3’):Nms Nfs Nm Nm Nm Nf Nm Nm Nm Nm Nm Nf Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm;

[0071] (23)AS(5’-3’):Nms dNs Nms Nm dN Nm dN Nm Nm Nm Nm Nm Nm Nf Nm Nm Nm Nm Nm Nms Nms Nms Nm;

[0072] (24)AS(5’-3’):Nms Nfs Nms Nm dN Nm dN Nm Nm Nm Nm Nm Nm dN Nm Nm Nm Nm Nm Nms Nms Nms Nm;

[0073] (25)AS(5’-3’):(E)-VP Nms Nfs Nm Nm Nm Nm Nm Nm Nf Nm Nm Nf Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm;

[0074] (26)AS(5’-3’):(E)-VP Nms Nfs Nm Nm Nm Nf Nm Nf Nf Nf Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm;

[0075] (27)AS(5’-3’):(E)-VP Nms Nfs Nm Nm Nm Nf Nm Nf Nf Nf Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm;

[0076] (28)AS(5’-3’):(E)-VP Nms Nfs Nm Nf Nm Nf Nm Nf Nm Nf Nm Nm Nm Nf Nm Nf Nm Nf Nm Nf Nms Nms Nm;

[0077] (29)AS(5’-3’):(E)-VP Nms Nfs Nm Nm Nm Nf Nm Nm Nm Nm Nm Nm Nm Nf Nm Nf Nm Nf Nm Nm Nms Nms Nm;

[0078] (30)AS(5’-3’):(E)-VP Nms Nfs Nm Nm Nm Nm Nm Nf Nf Nm Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm;

[0079] (31)AS(5’-3’):(E)-VP Nms Nfs Nm Nm Nm Nm Nm Nf Nf Nm Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm;

[0080] (32)AS(5’-3’):(E)-VP Nms Nfs Nm Nm Nm Nm Nm Nf Nf Nm Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm;

[0081] (33)AS(5’-3’):(E)-VP Nms Nfs Nm Nm Nm Nm Nm Nf Nf Nm Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm;

[0082] (34)AS(5’-3’):(E)-VP Nms Nfs Nm Nm Nm Nm Nm Nf Nf Nm Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm;

[0083] (35)AS(5’-3’):(E)-VP Nms Nfs Nm Nm Nm Nm Nm Nf Nf Nm Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm;

[0084] (36)AS(5’-3’):(E)-VP Nms Nfs Nm Nm Nm Nm Nm Nf Nf Nm Nm Nm Nm Nf Nm Nf Nm Nf Nm Nm Nms Nms Nm;

[0085] (37)AS(5’-3’):(E)-VP Nms Nfs Nms Nm Nm Nm Nm Nf Nf Nm Nm Nm Nm Nf Nm Nm Nm Nf Nm Nm Nms Nms Nm;

[0086] (38)AS(5’-3’):(E)-VP Nms Nfs Nm Nm Nm Nm Nm Nf Nf Nm Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm;

[0087] (39)AS(5’-3’):(E)-VP Nms Nfs Nm Nm Nm Nm Nm Nf Nf Nm Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm;

[0088] (40)AS(5’-3’):(E)-VP Nms Nfs Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm;

[0089] (41)AS(5’-3’):(E)-VP Nms Nfs Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm;

[0090] (42)AS(5'-3'):(E)-VP Nms Nfs Nm Nm Nm Nf Nm Nm Nm Nm Nm Nf Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm;

[0091] (43)AS(5'-3'):(E)-VP Nms dNs Nms Nm dN Nm dN Nm Nm Nm Nm Nm Nm Nf Nm Nm Nm Nm Nm Nms Nms Nms Nm;

[0092] (44)AS(5'-3'):(E)-VP Nms Nfs Nms Nm dN Nm dN Nm Nm Nm Nm Nm Nm dN Nm Nm Nm Nm Nm Nms Nms Nms Nm;

[0093] Wherein, Nm is 2'-methoxynucleotide, Nf is 2'-fluoronucleotide, dN is 2'-deoxyribonucleotide, s is a thiophosphate group, and (E)-VP is 5'-(E)-vinyl phosphate (VP) modification.

[0094] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long and comprises:

[0095] (1) 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;

[0096] (2) The 2nd, 6th, 8th, 9th, 12th, 14th and 16th positions of the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides;

[0097] (3) The 2nd, 9th, 12th, 14th and 16th positions of the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides;

[0098] (4) The 2nd, 4th, 12th, 14th and 16th positions of the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides;

[0099] (5) The 2nd, 6th, 8th, 9th, 10th, 14th and 16th positions of the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides;

[0100] (6) The 2nd, 4th, 6th, 8th, 10th, 14th, 16th, 18th and 20th positions of the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides;

[0101] (7) The 2nd, 6th, 14th, 16th and 18th positions of the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides;

[0102] (8) The 2nd, 8th, 9th, 14th and 16th positions of the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides;

[0103] (9) The 2nd, 8th, 9th, 14th, 16th and 18th positions of the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides;

[0104] (10) The 2nd, 8th, 9th, 14th and 18th positions of the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides;

[0105] (11) The 2nd, 6th, 8th, 14th and 16th positions of the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides;

[0106] (12) The 2nd, 6th, 12th, 14th and 16th positions of the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides;

[0107] (13) The 14th position of the 5' end of the antisense strand is a 2'-fluoronucleotide, the 2nd, 5th and 7th positions are 2'-deoxyribonucleotides, and the remaining positions are 2'-methoxynucleotides;

[0108] (14) The second position of the 5' end of the antisense strand is 2'-fluoronucleotide, the 5th, 7th and 14th positions are 2'-deoxyribonucleotide, and the remaining positions are 2'-methoxynucleotide.

[0109] In some embodiments of the present invention, the siRNA has a positive strand length of 21 nucleotides and comprises the following modification patterns:

[0110] (1)SS(5'-3'):Nms Nms Nm Nm Nm Nm Nf Nm Nf Nf Nf Nm Nm Nm Nm Nm Nm Nm Nm Nm Nm;

[0111] (2)SS(5'-3'):Nms Nm Nm Nm Nm Nm Nm Nf Nm Nf Nm Nf Nm Nm Nm Nm Nm Nm Nm Nm Nms Nm;

[0112] (3)SS(5'-3'):Nms Nm Nm Nm Nm Nm Nm Nm Nm Nf Nm Nf Nm Nf Nm Nm Nm Nm Nm Nm Nms Nm;

[0113] (4)SS(5’-3’):Nms Nm Nm Nm Nm Nm Nm Nm Nf Nm Nf Nm Nf Nm Nm Nm Nm Nm Nm Nms Nm;

[0114] (5)SS(5’-3’):Nms Nm Nm Nm Nm Nm Nf Nm Nf Nm Nf Nm Nm Nm Nm Nm Nm Nm Nm Nms Nm;

[0115] (6)SS(5’-3’):Nms Nm Nm Nm Nm Nm Nf Nm Nf Nf Nf Nm Nm Nm Nf Nm Nm Nm Nm Nms Nm;

[0116] (7)SS(5’-3’):Nms Nm Nm Nm Nm Nm Nf Nm Nf Nf dN Nm Nm Nm Nm Nm Nm Nm Nm Nms Nm;

[0117] (8)SS(5’-3’):Nms Nm Nm Nm Nm Nm Nf Nm Nf Nf dN Nm Nm Nm Nm Nm Nm Nm Nm Nms Nm;

[0118] (9)SS(5’-3’):Nms Nm Nm Nm Nm Nm Nf Nm Nf Nf Nf Nm Nf Nm Nm Nm Nm Nm Nm Nms Nm;

[0119] (10)SS(5’-3’):Nms Nm Nm Nm Nm Nm Nf Nm Nf Nf Nf Nm Nm Nm Nm Nf Nm Nm Nm Nms Nm;

[0120] (11)SS(5’-3’):Nms Nm Nm Nm Nm Nm Nf Nm Nf Nf Nf Nm Nm Nm Nm Nm Nf Nm Nm Nms Nm;

[0121] (12)SS(5’-3’):Nms Nm Nm Nm Nm Nm Nf Nm Nf dN Nf Nm Nm Nm Nm Nm Nf Nm Nm Nms Nm;

[0122] (13)SS(5’-3’):Nms Nm Nm Nm Nm Nm Nf Nm dN Nf Nf Nm Nm Nm Nm Nm Nf Nm Nm Nms Nm;

[0123] (14)SS(5’-3’):Nms Nm Nm Nm Nm Nm Nf Nm Nf Nf dN Nm Nm Nm Nm Nm Nf Nm Nm Nms Nm;

[0124] (15)SS(5’-3’):Nms Nm Nm Nm Nm Nm Nm Nm Nf Nf Nf Nm Nm Nm Nm Nm Nf Nm Nm Nms Nm;

[0125] (16)SS(5’-3’):Nms Nm Nm Nm Nm Nm Nf Nm Nf Nf Nf Nm Nm Nm Nm Nm Nf Nm Nm Nms Nm;

[0126] (17)SS(5’-3’):Nms Nm Nm Nm Nm Nm Nf Nm Nf Nf Nf Nm Nm Nm Nm Nm Nf Nm Nm Nms Nm;

[0127] (18)SS(5’-3’):Nms Nm Nm Nm Nm Nm Nf Nm Nf Nf Nf Nm Nm Nm Nm Nf Nf Nm Nm Nms Nm;

[0128] (19)SS(5’-3’):Nms Nm Nm Nm Nm Nm Nf Nm Nf Nf Nf Nm Nm Nm Nf Nm Nf Nm Nm Nms Nm;

[0129] (20)SS(5’-3’):Nms Nm Nm Nm Nm Nm Nf Nm Nf Nf Nf Nm Nf Nm Nm Nm Nm Nm Nm Nms Nm;

[0130] (21)SS(5’-3’):Nms Nm Nm Nm Nm Nm Nf Nm Nf Nf Nf Nm Nm Nm Nf Nm Nm Nm Nm Nms Nm;

[0131] (22)SS(5'-3'):Nms Nms Nm Nm Nm Nm Nm Nm Nf dN Nf Nm Nm Nm Nm Nm Nf Nm Nms Nms Nm;

[0132] (23)SS(5'-3'):Nms Nms Nm Nm Nm Nm Nm Nf Nf Nf Nf Nm Nm Nm Nm Nm Nm Nm Nms Nms Nm;

[0133] (24)SS(5'-3'):Nms Nms Nm Nm Nf Nf Nf Nf Nf Nm Nm Nm Nm Nm Nf Nm Nms Nms Nm;

[0134] Wherein, Nm is a 2'-methoxy modified nucleotide, Nf is a 2'-fluoronucleotide, dN is a 2'-deoxyribonucleotide, and s is a thiophosphate ester linkage.

[0135] In some embodiments of the present invention, a positive strand comprising the following modification pattern (24)SS(5'-3'): Nms Nms Nm Nm Nf Nf Nf Nf Nm Nm Nm Nm Nm Nm Nm Nm Nms Nms Nm, having a length of 18 nucleotides, having a deletion of 3 nucleotides at the 5' end compared to the above modification patterns (1) to (23).

[0136] In some embodiments of the present invention, the siRNA has a positive strand length of 21 nucleotides and comprises:

[0137] (1) The 7th, 9th, 10th and 11th positions of the 5' end of the positive strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides;

[0138] (2) The 7th, 9th and 11th positions of the 5' end of the positive strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides;

[0139] (3) The 9th, 11th and 13th positions of the 5' end of the positive strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides;

[0140] (4) The 7th, 9th, 10th, 11th and 15th positions of the 5' end of the positive strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides;

[0141] (5) The 7th, 9th and 10th positions of the 5' end of the positive strand are 2'-fluoronucleotides, the 11th position is 2'-deoxyribonucleotides, and the remaining positions are 2'-methoxynucleotides;

[0142] (6) The 7th, 9th, 10th, 11th and 13th positions of the 5' end of the positive strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides;

[0143] (7) The 7th, 9th, 10th, 11th and 16th positions of the 5' end of the positive strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides;

[0144] (8) The 7th, 9th, 10th, 11th and 17th positions of the 5' end of the positive strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides;

[0145] (9) The 7th, 9th, 11th and 17th positions of the 5' end of the positive strand are 2'-fluoronucleotides, the 10th position is 2'-deoxyribonucleotides, and the remaining positions are 2'-methoxynucleotides;

[0146] (10) The 7th, 10th, 11th and 17th positions of the 5' end of the positive strand are 2'-fluoronucleotides, the 9th position is 2'-deoxyribonucleotides, and the remaining positions are 2'-methoxynucleotides;

[0147] (11) The 7th, 9th, 10th and 17th positions of the 5' end of the positive strand are 2'-fluoronucleotides, the 11th position is 2'-deoxyribonucleotides, and the remaining positions are 2'-methoxynucleotides;

[0148] (12) The 9th, 10th, 11th and 17th positions of the 5' end of the positive strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides;

[0149] (13) The 7th, 9th, 10th, 11th, 16th and 17th positions of the 5' end of the positive strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides;

[0150] (14) The 7th, 9th, 10th, 11th, 15th and 17th positions of the 5' end of the positive strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides;

[0151] (15) The 9th, 11th, and 17th positions at the 5' end of the positive strand are 2'-fluoronucleotides, the 10th position is a 2'-deoxyribonucleotide, and the remaining positions are 2'-methoxynucleotides; or

[0152] (16) The 8th, 9th, 10th and 11th positions of the 5' end of the positive strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0153] In some embodiments of the present invention, the siRNA has a positive strand length of 18 nucleotides and comprises:

[0154] (17) The 5th, 6th, 7th, 8th and 14th positions of the 5' end of the positive strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0155] 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.

[0156] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 6, 8, 9, 12, 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, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0157] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 6, 8, 9, 12, 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 9, 11, and 13 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0158] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 9, 12, 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 9, 11, and 13 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0159] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 4, 12, 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, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0160] 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.

[0161] 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, and 10 at the 5' end of the sense strand are 2'-fluoronucleotides, position 11 is 2'-deoxyribonucleotide, and the remaining positions are 2'-methoxynucleotides.

[0162] 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 7, 9, and 10 at the 5' end of the sense strand are 2'-fluoronucleotides, position 11 is 2'-deoxyribonucleotide, and the remaining positions are 2'-methoxynucleotides.

[0163] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 6, 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 21 nucleotides long, wherein positions 7, 9, 10, 11, and 13 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0164] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 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, 11, and 16 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0165] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 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, 11, and 17 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0166] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 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, 11, and 17 at the 5' end of the sense strand are 2'-fluoronucleotides, position 10 is a 2'-deoxyribonucleotide, and the remaining positions are 2'-methoxynucleotides.

[0167] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 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, 10, 11, and 17 at the 5' end of the sense strand are 2'-fluoronucleotides, position 9 is a 2'-deoxyribonucleotide, and the remaining positions are 2'-methoxynucleotides.

[0168] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 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 17 at the 5' end of the sense strand are 2'-fluoronucleotides, position 11 is 2'-deoxyribonucleotide, and the remaining positions are 2'-methoxynucleotides.

[0169] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 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 9, 10, 11, and 17 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0170] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 8, 9, 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 21 nucleotides long, wherein positions 7, 9, 10, 11, and 17 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0171] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 8, 9, 14, 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 21 nucleotides long, wherein positions 7, 9, 10, 11, and 17 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0172] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 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, 11, 16, and 17 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0173] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 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, 11, 15, and 17 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0174] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 6, 8, 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 13 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0175] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 6, 8, 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.

[0176] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 6, 12, 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 9, 11, and 17 at the 5' end of the sense strand are 2'-fluoronucleotides, position 10 is a 2'-deoxyribonucleotide, and the remaining positions are 2'-methoxynucleotides.

[0177] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein the 14th position of the 5' end of the antisense strand is a 2'-fluoronucleotide, the 2nd, 5th, and 7th positions are 2'-deoxyribonucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein the 8th, 9th, 10th, and 11th positions of the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0178] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein the second position of the 5' end of the antisense strand is a 2'-fluoronucleotide, the 5th, 7th, and 14th positions are 2'-deoxyribonucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 18 nucleotides long, wherein the 5th, 6th, 7th, 8th, and 14th positions of the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0179] In some embodiments of the present invention, the siRNA of the present invention comprises modified nucleoside links or a modified backbone. Modified nucleoside links or backbones include, but are not limited to: thiophosphates, 2'-O-methoxyethyl (MOE), 2'-fluorophosphates, alkyl phosphates, dithiophosphates, alkyl thiophosphates, aminophosphates, carbamates, carbonates, triphosphates, acetamipridates, carboxymethyl esters, and combinations thereof.

[0180] 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.

[0181] In some embodiments of the present invention, the 5' end and 3' end of the sense chain each independently contain one or two thiophosphate groups; and / or the 5' end and 3' end of the antisense chain each independently contain one, two, or three thiophosphate groups.

[0182] In some embodiments of the present invention, the nucleotides at positions 1 and 2 of the 5' end of the positive strand, the nucleotides at positions 2 and 3 of the 5' end of the positive strand, the nucleotides at positions 1 and 2 of the 3' end of the positive strand, the nucleotides at positions 2 and 3 of the 5' end of the positive strand, the nucleotides at positions 1 and 2 of the 5' end of the negative strand, the nucleotides at positions 2 and 3 of the 5' end of the negative strand, the nucleotides at positions 3 and 4 of the 5' end of the negative strand, the nucleotides at positions 1 and 2 of the 5' end of the negative strand, the nucleotides at positions 2 and 3 of the 5' end of the negative strand, and ... are all considered to be related to the 5' end of the negative strand. Between the nucleotides at positions 1 and 4, at least one is linked by a thiophosphate group; in some embodiments of the invention, at least two are linked by thiophosphate groups; in some embodiments of the invention, at least three are linked by thiophosphate groups; in some embodiments of the invention, at least four are linked by thiophosphate groups; in some embodiments of the invention, at least five are linked by thiophosphate groups; in some embodiments of the invention, at least six are linked by thiophosphate groups; in some embodiments of the invention, at least seven are linked by thiophosphate groups; in some embodiments of the invention, at least eight are linked by thiophosphate groups.

[0183] In some embodiments of the present invention, the nucleotides at positions 1 and 2 at the 5' end of the positive strand, and at positions 1 and 2 at the 3' end, are linked by thiophosphate groups.

[0184] In some embodiments of the present invention, the nucleotides at positions 1 and 2 at the 5' end of the positive strand are linked by thiophosphate groups, and the nucleotides at positions 1 and 2, and at positions 2 and 3 at the 3' end are linked by thiophosphate groups.

[0185] In some embodiments of the present invention, the nucleotides at positions 1 and 2 of the 5' end of the positive strand are linked by phosphate thioester groups.

[0186] 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.

[0187] 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.

[0188] 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.

[0189] 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.

[0190] In some embodiments of the present invention, the nucleotides at positions 1 and 2 of the 3' end of the antisense strand are linked by phosphate thioester groups.

[0191] 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 1 and 2, and at positions 2 and 3 at the 5' end are linked by thiophosphate groups.

[0192] 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 1 and 2 at the 5' end are also linked by thiophosphate groups.

[0193] In some embodiments of the present invention, the nucleotides at positions 1 and 2, and at positions 2 and 3, of the 3' end of the antisense strand are linked by phosphate thioester groups.

[0194] In some embodiments of the present invention, the nucleotides at positions 1 and 2, 2 and 3, 3 and 4 at the 3' end of the antisense strand, and at positions 1 and 2 at the 5' end are linked by thiophosphate groups.

[0195] In some embodiments of the present invention, the nucleotides at positions 1 and 2, 2 and 3 at the 3' end of the antisense strand, and at positions 1 and 2, 2 and 3, and 3 and 4 at the 5' end are linked by thiophosphate groups.

[0196] In some embodiments of the present invention, the nucleotides at positions 1 and 2, 2 and 3, and 3 and 4 at the 3' end of the antisense strand, and the nucleotides at positions 1 and 2, 2 and 3, and 3 and 4 at the 5' end are linked by thiophosphate groups.

[0197] In some embodiments of the present invention, the nucleotides at positions 1 and 2 at the 5' end of the sense strand, the nucleotides at positions 1 and 2 at the 3' end of the sense strand, the nucleotides at positions 1 and 2 at the 3' end of the antisense strand, the nucleotides at positions 2 and 3 at the 3' end of the antisense strand, the nucleotides at positions 1 and 2 at the 5' end of the antisense strand, and the nucleotides at positions 2 and 3 at the 5' end of the antisense strand are all linked by phosphate thioester groups.

[0198] 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 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 phosphate thioester groups.

[0199] In some embodiments of the present invention, the nucleotides at positions 1 and 2 at the 5' end of the sense strand, the nucleotides at positions 1 and 2 at the 3' end of the antisense strand, the nucleotides at positions 2 and 3 at the 3' end of the antisense strand, the nucleotides at positions 1 and 2 at the 5' end of the antisense strand, and the nucleotides at positions 2 and 3 at the 5' end of the antisense strand are all linked by phosphate thioester groups.

[0200] In some embodiments of the present invention, the nucleotides at positions 1 and 2 at the 5' end of the sense strand, the nucleotides at positions 1 and 2 at the 3' end of the antisense strand, the nucleotides at positions 2 and 3 at the 3' end of the antisense strand, and the nucleotides at positions 1 and 2 at the 5' end of the antisense strand are all linked by thiophosphate groups.

[0201] In some embodiments of the present invention, the nucleotides at positions 1 and 2 at the 5' end of the sense strand and the nucleotides at positions 1 and 2 at the 3' end of the antisense strand are linked by phosphate thioester groups.

[0202] In some embodiments of the present invention, the nucleotides at positions 1 and 2 at the 5' end of the sense strand, the nucleotides at positions 1 and 2 at the 3' end of the antisense strand, the nucleotides at positions 1 and 2 at the 5' end of the antisense strand, and the nucleotides at positions 2 and 3 at the 5' end of the antisense strand are all linked by thiophosphate groups.

[0203] 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 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 phosphate thioester groups.

[0204] In some embodiments of the present invention, the nucleotides at positions 1 and 2 at the 5' end of the sense strand, the nucleotides at positions 2 and 3 at the 5' end of the sense strand, and the nucleotides at positions 1 and 2 at the 3' end of the antisense strand are linked by thiophosphate groups.

[0205] 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 1 and 2 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, the nucleotides at positions 2 and 3 of the 5' end of the antisense strand, and the nucleotides at positions 3 and 4 of the 5' end of the antisense strand are linked by thiophosphate groups.

[0206] 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 linked by thiophosphate groups.

[0207] 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 3 and 4 of the 3' end of the antisense strand, the nucleotides at positions 1 and 2 of the 5' end of the antisense strand, the nucleotides at positions 2 and 3 of the 5' end of the antisense strand, and the nucleotides at positions 3 and 4 of the 5' end of the antisense strand are linked by thiophosphate groups.

[0208] In some embodiments of the present invention, the positive chain may include one or more end-capping residues or portions thereof, referred to as "end-capping residues". In some embodiments of the present invention, the end-capping residues are present at the 5' end, the 3' end, or both the 5' end and the 3' end of the positive chain.

[0209] In some embodiments of the present invention, a reverse debasing residue (iab) 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 reverse debasing deoxyribose moiety as a capping residue.

[0210] In some embodiments of the invention, one or more reverse abasing residues (iab) are added to the 3' end of the positive strand. In some embodiments of the invention, one or more reverse abasing residues (iab) are added to the 5' end of the positive strand. In some embodiments of the invention, one or more reverse abasing residues are included at or near one or more ends of the positive strand of the siRNA.

[0211] Reverse debasing residues can be linked via phosphate esters, thiophosphate esters, or other nucleosides.

[0212] In some embodiments of the invention, the first nucleotide at the 5' end of the antisense strand comprises a (E)-vinylphosphonate or analogue modification, such as (E)-VP modification. In some embodiments of the invention, the first nucleotide may optionally comprise other modifications, such as 2'-modification (e.g., 2'-methoxy modification).

[0213] In some embodiments, the modification patterns of the antisense and sense strands of the siRNA are selected from any one of the sense strand modification patterns and antisense strand modification patterns shown in Table 1D below: P0, P01-P20, 073, 078, 080, P03-E, P05-E-P20-E, 073-E, 078-E, 080-E.

[0214] Table 1D: Modification patterns of siRNA duplexes

[0215] In some embodiments of the present invention, the antisense strand modification mode and / or the sense strand modification mode described in the present invention are applied to the small interfering RNA (siRNA) for inhibiting APP gene expression described in the present invention.

[0216] In some embodiments of the present invention, the antisense strand of the small interfering RNA (siRNA) for inhibiting APP gene expression comprises the nucleotide sequence shown in any one of SEQ ID NO:1 to SEQ ID NO:200, SEQ ID NO:418 to SEQ ID NO:434.

[0217] In some embodiments of the present invention, the positive strand of the small interfering RNA (siRNA) for inhibiting APP gene expression comprises the nucleotide sequence shown in any one of SEQ ID NO:201 to SEQ ID NO:400, SEQ ID NO:401 to SEQ ID NO:417.

[0218] In some embodiments of the present invention, the sense and antisense sequences of the small interfering RNA (siRNA) used to suppress APP gene expression comprise the sense and antisense sequences of any siRNA selected from SJ-0001 to SJ-0200 and SJ-0201 to SJ-0223 as described in Tables 1A and 1B.

[0219] As those skilled in the art will understand, by applying the antisense strand modification patterns and / or the sense strand modification patterns in Table 1D to SJ-0001~SJ-0200 and SJ-0201~SJ-0223 described in Tables 1A and 1B, the modified siRNA of the present invention can be obtained.

[0220] In some embodiments of the present invention, the sequences of the sense and antisense strands of the siRNA are selected from at least 12 consecutive nucleotides of the sense and antisense strand sequences of any of the modified siRNA duplexes described in Tables 1E and 1F.

[0221] In some embodiments of the invention, the antisense strand comprises at least 15 consecutive modifying nucleotides that are approximately the same as the antisense strand sequence of any of the modified siRNA duplexes described in Tables 1E and 1F, consisting of no more than four (e.g., 0, 1, 2, 3, or 4) modifying nucleotides, and the sense strand is at least partially complementary to the antisense strand.

[0222] In some embodiments of the invention, the antisense strand comprises at least 17 consecutive nucleotides that differ from the antisense strand sequence of any of the modified siRNA duplexes described in Tables 1E and 1F by 0, 1, or 2 nucleotides.

[0223] In some embodiments of the invention, the positive strand comprises at least 15 consecutive modifying nucleotides that are approximately the same as the positive strand sequence of any of the modified siRNA duplexes described in Tables 1E and 1F, consisting of no more than four (e.g., 0, 1, 2, 3, or 4) modifying nucleotides.

[0224] In some embodiments of the invention, the sense strand has a region within the 15 consecutive modified nucleotides that is at least 85% complementary to the antisense strand.

[0225] In some embodiments of the invention, the positive strand comprises at least 17 consecutive modifying nucleotides that are approximately the same as the positive strand sequence of any of the modified siRNA duplexes described in Tables 1E and 1F by no more than two (e.g., 0, 1, or 2) modifying nucleotides.

[0226] In some embodiments of the present invention, the sequences of the sense and antisense strands of the siRNA are selected from the sense and antisense strand sequences of any of the modified siRNA duplexes described in Tables 1E and 1F.

[0227] In some embodiments of the present invention, the sequence information of the modified siRNA double strand is shown in Tables 1E and 1F, including the corresponding basic siRNA double strand sequence and modification pattern information.

[0228] Table 1E: Sequence information of modified siRNA duplexes for APP target design

[0229] Table 1F: Sequence information of modified siRNA duplexes designed for APP targets

[0230] As used herein, the sequence information of the modified siRNAs of the present invention is characterized by the information in Tables 1E and 1F. For example, SJ-0015-P01 comprises a base siRNA (SJ-0015) modified with the P01 modification pattern, wherein the sense strand of the base siRNA (SJ-0015) comprises the nucleotide sequence shown in SEQ ID NO: 215, and the antisense strand of the base siRNA (SJ-0015) comprises the nucleotide sequence shown in SEQ ID NO: 15. Therefore, SJ-0015-P01 can also be represented as follows: SJ-0015-P01 comprises: (i) a sense strand containing the following nucleotide sequence: Ums Am Cm Am Gm Am Af Um Cf Am Uf Um Gm Cm Um Um Am Um Gm Ams Um, and (ii) an antisense strand containing the following nucleotide sequence: Ams Ufs Cm Am Um Af Am Gf Cf Am Am ​​Uf Gm Af Um Uf Cm Um Gm Um AmsCms Am. Those skilled in the art can understand the sequence information of the modified siRNA of the present invention from the information in Tables 1E and 1F.

[0231] As used herein, compared to other modification patterns with a positive strand length of 21 nucleotides, the 5' end of the positive strand of modification patterns 080 and 080-E in Table 1D is deleted, resulting in a length of 18 nucleotides. When this modification pattern is applied to the base sequences (SJ-0001~SJ-0200, SJ-0201~SJ-0223) described in Tables 1A and 1B, the 5' end of the positive strand of these base sequences is deleted. For example, SJ-0074-080 contains: (i) a sense strand containing the following nucleotide sequence: Cms Ams Am Gm Uf Uf Cf Uf Um Um Gm Am Gm Cf Am Gms Ams Um, and (ii) an antisense strand containing the following nucleotide sequence: Ams Ufs Cms Um dG Um dU Cm Am Am ​​Am ​​Gm Am dA Cm Um Um Gm Um Ams Gms Gms Um.

[0232] In some exemplary embodiments of the present invention, the nucleic acid sequences of siRNA modified duplexes having modification modes 080 and 080-E are shown in the table below:

[0233] 3. Delivery

[0234] The siRNA of the present invention can be delivered or introduced by any means known in the art (e.g., in vitro delivery or introduction into cells, or in vivo delivery or introduction into a patient). For example, for in vivo delivery, the siRNA can be injected into a tissue site or administered systemically. In vivo delivery can also be performed using a β-glucan delivery system. In vitro introduction into cells includes methods known in the art, such as electroporation, lipid transfection, and receptor-mediated endocytosis.

[0235] In some embodiments, the delivery methods include, but are not limited to, viral delivery (retrovirus, adenovirus, lentivirus, baculovirus, AAV); liposomes (Lipofectamine, cationic DOTAP, neutral DOPC); nanoparticles (cationic polymers, PEI); bacterial delivery (tkRNAi); lipid nanoparticles (LNP); neutral liposomes (NL); polymer nanoparticles (low molecular weight polymers or high molecular weight polymers); double-stranded RNA binding motifs (dsRBMs); and other delivery systems known in the art that are suitable for nucleic acid or oligonucleotide delivery.

[0236] 4. Couplings

[0237] In one aspect, the present invention provides conjugates (also known as adjuvants) comprising at least one siRNA of the present invention and a pharmaceutically acceptable targeting molecule. The conjugates of the present invention are obtained by conjugating the siRNA of the present invention to a pharmaceutically acceptable targeting molecule, the conjugate comprising the pharmaceutically acceptable targeting molecule and optionally a linker. The siRNA may be non-covalently conjugated to the targeting molecule or covalently conjugated to the targeting molecule.

[0238] The pharmaceutically acceptable target molecule can be a target molecule commonly used in the field of siRNA drug delivery, which typically enhances the pharmacokinetic or biodistribution properties of the siRNA to which it is linked, and improves the cell-specific (or organ-specific) distribution and cell-specific (or organ-specific) uptake of the siRNA. Representative target molecules include, but are not limited to: compounds with affinity for cell surface molecules, cell receptor ligands, haptens, antibodies or antibody fragments, antibody mimics, etc. In some embodiments, the targeting molecule includes, but is not limited to, one or more of the following targeting molecules or their derivatives: integrins; 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); folate; 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.

[0239] The linker can be any linker commonly used in the field of siRNA drug delivery, including but not limited to one or more of the following linkers or their derivatives: amide linker, amino linker, carbonyl linker, carbamate linker, urea linker, ether linker, disulfide linker, succinylamino linker, etc.

[0240] In some embodiments, the targeting molecule may be directly or indirectly linked to the siRNA of the present invention via a linker / connector group. In some embodiments, the targeting molecule is linked to the siRNA via an unstable, cleavable, or reversible bond or linker. In some embodiments, the targeting molecule is linked to at least one end of the sense and / or antisense strand of the siRNA. In some embodiments, the targeting molecule is linked to the 5' and / or 3' end of the sense strand. In some embodiments, the targeting molecule is linked to the 5' and / or 3' end of the antisense strand.

[0241] In some embodiments, the coupling comprises the structure shown in formula (I-1-1).

[0242] in,

[0243] Z 1 Z 2 It does not exist, or each is independently selected from -O-, -S-, -N(R) a )-, -C(=O)-, -OC(=O)-, -C(=O)O-, -C(=O)NRa -、-NR a C(=O)-、-OC(=O)NR a -、-NR a C(=O)O-、-C 1-6 alkylene-, R a Each is independently selected from H and C. 1-6 alkyl;

[0244] R 1 C 12-32 Alkyl, the C 12-32 The alkyl group is optionally surrounded by one or more elements selected from H, halogens, OH, CN, NO2, COOH, and -OC. 1-6 Alkyl, -NHC 1-6 Alkyl and -N(C) 1-6 Alkyl group substitution.

[0245] In some implementations, the coupling agent, wherein, Selected from

[0246] In some embodiments, the conjugate comprises one or more nucleotide-lipid conjugate units or pharmaceutically acceptable salts, tautomers, enantiomers, or stereoisomers thereof, wherein the nucleotide-lipid conjugate unit comprises the structure shown in formula (II-1-1).

[0247] in,

[0248] Z 1 Z 2 R 1 As defined above;

[0249] X is O;

[0250] Y is O;

[0251] B can be a natural or non-natural base, or a modified or unmodified base.

[0252] In some embodiments, for the conjugate, B is uracil, cytosine, adenine, guanine, uracil with an amino protecting group, cytosine with an amino protecting group, adenine with an amino protecting group, or guanine with an amino protecting group.

[0253] In some implementations, for the aforementioned coupling, B is...

[0254] In some embodiments, for the aforementioned coupling, the structure of formula (II-1-1) is selected from...

[0255] In some embodiments, the coupling material, wherein the structure of formula (II-1-1) is selected from...

[0256] In some embodiments, the conjugate is selected from any of the following:

[0257] (1) LN15U-SJ-0165-P08-E, which contains an antisense strand sequence as shown in SEQ ID NO:446, and a positive strand sequence as shown in SEQ ID NO:435;

[0258] (2) LN17U-SJ-0165-P08-E, which contains the antisense strand sequence as shown in SEQ ID NO:447, and the positive strand sequence as shown in SEQ ID NO:436;

[0259] (3) LN14U-SJ-0165-P08-E, which contains the antisense strand sequence as shown in SEQ ID NO:448, and the positive strand sequence as shown in SEQ ID NO:437;

[0260] (4) LN15U-SJ-0165-073-E, which contains an antisense strand sequence as shown in SEQ ID NO:449, and a positive strand sequence as shown in SEQ ID NO:438;

[0261] (5) LN17U-SJ-0165-073-E, which contains an antisense strand sequence as shown in SEQ ID NO:450, and a positive strand sequence as shown in SEQ ID NO:439;

[0262] (6) LN14U-SJ-0165-073-E, which contains an antisense strand sequence as shown in SEQ ID NO:451, and a positive strand sequence as shown in SEQ ID NO:440;

[0263] (7) LN15C-SJ-0011-P09-E, which contains the antisense strand sequence as shown in SEQ ID NO:452, and the positive strand sequence as shown in SEQ ID NO:441;

[0264] (8) LN15C-SJ-0011-078-E, which contains an antisense strand sequence as shown in SEQ ID NO:453, and a positive strand sequence as shown in SEQ ID NO:442;

[0265] (9) LN15C-SJ-0011-080-E, which contains an antisense strand sequence as shown in SEQ ID NO:454, and a positive strand sequence as shown in SEQ ID NO:443;

[0266] (10)LN14C-SJ-0011-080E, which contains an antisense strand sequence as shown in SEQ ID NO:454, and a positive strand sequence as shown in SEQ ID NO:444;

[0267] (11)LN15C-SJ-0011-080E, which contains an antisense strand sequence as shown in SEQ ID NO:454, and a positive strand sequence as shown in SEQ ID NO:445.

[0268] In some embodiments, the structure of the nucleotide-lipid conjugate monomer is selected from the structures shown in Table A:

[0269] Table A

[0270] 5. Pharmaceutical Composition

[0271] In one aspect, the present invention provides a pharmaceutical composition comprising at least one siRNA of the present invention.

[0272] In some embodiments, the pharmaceutical composition contains one of the siRNAs described above.

[0273] In other embodiments, the pharmaceutical composition contains at least two of the siRNAs described above (e.g., but not limited to 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) as active ingredients. Preferably, each of the at least two siRNAs targets a different target sequence in the APP gene, thereby expecting a synergistic effect by acting simultaneously on different target sequences. Here, "different target sequences" means that there is no overlap between the target sequences, or the number of overlapping consecutive nucleotides between the 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 of the above-described siRNAs can be present in any different proportions. Preferably, the at least two of the above-described siRNAs can be present in a molar ratio of 1:100 to 100:1; more preferably, the at least two of the above-described siRNAs can be present in a molar ratio of 1:10 to 10:1, 1:5 to 5:1, or 1:2 to 2:1. In some implementations, the at least two of the above-described siRNAs are present in the same molar ratio.

[0274] In other embodiments, the pharmaceutical composition contains at least one siRNA of the present invention and also contains at least one siRNA targeting other targets (e.g., genes other than APP). In this case, the siRNA of the present invention and the siRNA targeting other targets can be present in any different proportions, for example, in a molar ratio of 1:100 to 100:1, for example, in a molar ratio of 1:10 to 10:1, 1:5 to 5:1 or 1:2 to 2:1, for example, in the same molar ratio.

[0275] In some embodiments, the pharmaceutical composition comprises an effective amount of siRNA. "Effective amount" refers to the amount of siRNA that is effective in producing the desired pharmacological, therapeutic, or preventative outcome. For example, if a given clinical treatment is considered effective when a measurable parameter associated with a disease or condition is reduced by at least 10%, then the therapeutically effective amount of a drug used to treat that disease or condition is the amount required to achieve that at least 10% reduction in the parameter. For example, a therapeutically effective amount of siRNA targeting APP can reduce APP mRNA levels by at least 10%.

[0276] In some embodiments, the siRNA in the pharmaceutical composition described in any of the above embodiments can be linked to a target molecule to form a conjugate. Therefore, in some embodiments, the pharmaceutical composition comprises the conjugate of the present invention.

[0277] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0278] In some embodiments, the pharmaceutically acceptable carrier and / or excipient is a delivery carrier. A delivery carrier is a substance that improves the delivery of nucleic acids or oligonucleotides to cells or tissues. Such substances can be any delivery carrier known in the art suitable for the delivery of nucleic acids or oligonucleotides, including but not limited to: viruses (retroviruses, adenoviruses, lentiviruses, baculoviruses, AAV); liposomes (Lipofectamine, cationic DOTAP, neutral DOPC); nanoparticles (cationic polymers, PEI); bacteria (tkRNAi); lipid nanoparticles (LNP); neutral liposomes (NL); polymer nanoparticles (low molecular weight polymers or high molecular weight polymers); double-stranded RNA binding motifs (dsRBMs), etc.

[0279] In some implementations, the siRNA may be encapsulated by the delivery vector.

[0280] In some embodiments, the siRNA may be linked to the delivery vector directly or indirectly via a linker / connector group. In some embodiments, the delivery vector is linked to the siRNA via an unstable, cleavable, or reversible bond or linker. In some embodiments, the delivery vector is linked to at least one end of the sense and / or antisense strand of the siRNA. In some embodiments, the delivery vector is linked to the 5' and / or 3' end of the sense strand. In some embodiments, the delivery vector is linked to the 5' and / or 3' end of the antisense strand.

[0281] In some embodiments, the pharmaceutical composition comprises one siRNA provided by the present invention, the siRNA being encapsulated by the delivery vector. The siRNA may be encapsulated in the same delivery vector or in separate delivery vectors.

[0282] In other embodiments, the pharmaceutical composition comprises at least two siRNAs provided by the present invention (e.g., but not limited to 2, 3, 4, 5, 6, 7, 8, 9, 10 or more), wherein the siRNAs are encapsulated by the delivery vector. The at least two siRNAs may be encapsulated in the same delivery vector or in separate delivery vectors. Preferably, the at least two siRNAs each target different target sequences in the APP gene.

[0283] In other embodiments, the pharmaceutical composition comprises at least one siRNA provided by the present invention and siRNAs targeting other targets (e.g., genes other than APP), wherein the siRNAs are encapsulated by the delivery vector. The siRNAs provided by the present invention and the siRNAs targeting other targets (e.g., genes other than APP) may be encapsulated in the same delivery vector or may be encapsulated in separate delivery vectors.

[0284] In some embodiments, the pharmaceutical compositions of the present invention are formulated into dosage forms compatible with their intended route of administration, such as local administration (e.g., direct injection or implantation), systemic administration, or subcutaneous, intravenous, intraperitoneal, or parenteral administration, including intracranial (e.g., intraventricular, intradural, or intrathecal), intramuscular, transdermal, airway (aerosol), nasal, oral, rectal, or topical (including buccal and sublingual) administration.

[0285] In some embodiments, the pharmaceutical composition is administered by inhalation, intranasal administration, intratracheal administration, or oropharyngeal inhalation. Formulations suitable for inhalation can be prepared by incorporating the desired amount of the active ingredient into a suitable solvent, followed by sterile filtration. Typically, formulations for inhalation are sterile solutions at physiological pH and have low viscosity. Salts may be added to the formulation to balance surface tension. In some cases, surfactants or co-solvents may be added to increase the solubility of the active ingredient and improve aerosol properties. In some cases, excipients may be added to control viscosity to ensure the size and distribution of atomized droplets.

[0286] In other embodiments, the pharmaceutical composition can be administered by injection, such as intravenously, intramuscularly, subcutaneously, intradermally, intra-articularly, intraocularly, intraperitoneally, or locally. Suitable pharmaceutical compositions for injectable use include sterile aqueous solutions or dispersions, as well as sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, antibacterial water, or phosphate-buffered saline (PBS). The pharmaceutical composition should remain stable under manufacturing and storage conditions and should be protected against contamination by microorganisms such as bacteria and fungi. For example, a sterile injectable solution can be prepared by incorporating the required dose of the active ingredient into a suitable solvent, and optionally, simultaneously incorporating other desired components (including, but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents for maintaining osmotic pressure, agents for delaying absorption, preservatives, or any combination thereof), followed by sterile filtration. Alternatively, the sterile injectable solution can be prepared as a sterile lyophilized powder (e.g., by vacuum drying or freeze-drying) for easy storage and use.

[0287] The siRNA of the present invention can be formulated in dosage units for easy administration. Dosage unit form refers to physically discrete units suitable for use as a single dose in the subject of treatment; each unit contains a predetermined amount of the active ingredient calculated to produce the desired therapeutic effect when combined with the desired drug carrier.

[0288] In this invention, the dosing regimen can be adjusted to obtain the optimal target response (e.g., treatment or prevention). For example, it can be administered as a single dose, multiple times over a period of time, or the dose can be reduced or increased proportionally according to the urgency of the treatment situation.

[0289] 6. Application

[0290] 6.1 Suppressing APP expression

[0291] The siRNA of this invention can be used to inhibit APP expression in vitro and / or in vivo.

[0292] In one aspect, the present invention provides a method for inhibiting APP expression in cells, the method comprising: introducing the siRNA, conjugate, or pharmaceutical composition of the present invention into the cells. In some embodiments, the method is performed in vitro. The siRNA of the present invention can be introduced by any nucleic acid delivery method known in the art, such as electroporation or lipid transfection.

[0293] The term "inhibition of APP expression" refers to at least partial repression of APP gene expression, which can be expressed as a decrease in the amount of detectable APP mRNA. For example, the degree of inhibition can be expressed as: ((mRNA in control cells) - (mRNA in treated cells)) / (mRNA in control cells) × 100%. Alternatively, the degree of inhibition can be given as a decrease in a parameter functionally associated with APP gene expression, such as the amount of protein encoded by the APP gene. In principle, APP gene silencing can be determined in any cell expressing APP (constitutive expression or expression via genetic engineering) and by any suitable assay. Measurements can be performed at multiple time points, before, during, and after siRNA administration, to determine the effect of siRNA. APP levels or expression can be measured by evaluating mRNA (e.g., by Northern blotting or PCR) or protein (e.g., by Western blotting or ELISA). For example, the effect of siRNA on APP expression can be determined by measuring the APP gene transcription rate (e.g., by RT-PCR). For example, the effect of siRNA on APP expression can be determined by measuring the expression level of a reporter gene (e.g., luciferase) fused to the APP gene.

[0294] In some embodiments, by applying the siRNA of the present invention, the expression of the APP gene is suppressed by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%. In some embodiments, by applying the siRNA of the present invention, the expression of the APP gene is suppressed by at least about 60%, 70%, or 80%. In some embodiments, by applying the siRNA of the present invention, the expression of the APP gene is suppressed by at least about 85%. In some embodiments, by applying the siRNA of the present invention, the expression of the APP gene is suppressed by at least about 90%. In some embodiments, by applying the siRNA of the present invention, the expression of the APP gene is suppressed by at least about 95%. In some embodiments, by applying the siRNA of the present invention, the expression of the APP gene is suppressed by at least about 96%, 97%, 98%, 99%, or 100%.

[0295] In some embodiments, the siRNA, the conjugate, or the pharmaceutical composition may be used alone or in combination with other pharmaceutically active agents, such as siRNAs targeting different target sequences in the APP gene or siRNAs targeting other targets.

[0296] In some embodiments, one siRNA provided by the present invention is used. The siRNA is optionally encapsulated in a delivery vector. In some embodiments, the one siRNA is encapsulated in the same delivery vector. In other embodiments, the one siRNA is encapsulated in different delivery vectors.

[0297] In other embodiments, at least two siRNAs provided by the present invention are used (e.g., but not limited to 2, 3, 4, 5, 6, 7, 8, 9, 10 or more), preferably each of the at least two siRNAs targets a different target sequence in the APP gene. The at least two siRNAs are optionally encapsulated in a delivery vector. In some embodiments, the at least two siRNAs are encapsulated in the same delivery vector. In other embodiments, the at least two siRNAs are encapsulated in separate delivery vectors.

[0298] In other embodiments, at least one siRNA provided by the present invention and siRNAs targeting other targets (e.g., genes other than APP) are used. The siRNA provided by the present invention and the siRNAs targeting other targets (e.g., genes other than APP) are optionally packaged in a delivery vector. In some embodiments, the siRNA provided by the present invention and the siRNAs targeting other targets (e.g., genes other than APP) are packaged in the same delivery vector. In other embodiments, the siRNA provided by the present invention and the siRNAs targeting other targets (e.g., genes other than APP) are packaged in separate delivery vectors.

[0299] 6.2 Treatment of APP-related diseases

[0300] The siRNA of this invention can be used to treat diseases or conditions that would benefit from reduced APP levels or suppressed APP expression.

[0301] In one aspect, the present invention provides a method for preventing and / or treating pathological conditions or diseases associated with APP in a subject, the method comprising administering an effective amount of the siRNA, conjugate, or pharmaceutical composition of the present invention to the subject in need. The present invention also relates to the use of the siRNA, conjugate, or pharmaceutical composition of the present invention in the preparation of a medicament for treating and / or preventing pathological conditions or diseases associated with APP.

[0302] In some implementations, the APP-related pathological condition or disease involves APP overexpression. APP overexpression refers to APP levels (e.g., APP levels present in the plasma or tissues of a subject, preferably in damaged tissues) that are higher than normal APP levels (e.g., the corresponding levels in healthy controls).

[0303] In some implementations, the APP-related pathological conditions or diseases will benefit from reduced APP levels or suppressed APP expression.

[0304] In some embodiments, the pathological condition or disease associated with the APP is Alzheimer's disease (AD), cerebral amyloid angiopathy (CAA), or Down syndrome (DS). In some embodiments, the Alzheimer's disease (AD) is early-onset familial Alzheimer's disease (EOFAD).

[0305] In some implementations, if APP expression is elevated for a specific disease, treatment with the siRNA of the present invention can reduce the elevated APP level or expression to a level considered normal for individuals without such disease.

[0306] In some implementations, the subject is a mammal, such as a human.

[0307] In some embodiments, the siRNA, the conjugate, or the pharmaceutical composition may be used alone or in combination with other pharmaceutically active agents (e.g., siRNAs targeting different target sequences in the APP gene or siRNAs targeting other targets), for example, administered simultaneously or sequentially.

[0308] In some embodiments, one siRNA provided by the present invention is used. The siRNA is optionally encapsulated in a delivery vector. In some embodiments, the one siRNA is encapsulated in the same delivery vector. In other embodiments, the one siRNA is encapsulated in different delivery vectors.

[0309] In other embodiments, at least two siRNAs provided by the present invention are used (e.g., but not limited to 2, 3, 4, 5, 6, 7, 8, 9, 10 or more), preferably each of the at least two siRNAs targets a different target sequence in the APP gene. The at least two siRNAs are optionally encapsulated in a delivery vector. In some embodiments, the at least two siRNAs are encapsulated in the same delivery vector. In other embodiments, the at least two siRNAs are encapsulated in separate delivery vectors.

[0310] In other embodiments, at least one siRNA provided by the present invention and siRNAs targeting other targets (e.g., genes other than APP) are used. The siRNA provided by the present invention and the siRNAs targeting other targets (e.g., genes other than APP) are optionally packaged in a delivery vector. In some embodiments, the siRNA provided by the present invention and the siRNAs targeting other targets (e.g., genes other than APP) are packaged in the same delivery vector. In other embodiments, the siRNA provided by the present invention and the siRNAs targeting other targets (e.g., genes other than APP) are packaged in separate delivery vectors.

[0311] 7. Terminology Definitions

[0312] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, to better understand this invention, definitions and explanations of relevant terms are provided below.

[0313] In this article, unless otherwise specified, the capital letters C, G, U, A, and T represent the base composition of nucleotides, including modified or unmodified nucleotides.

[0314] In this document, the term "modified nucleotide" refers to a nucleotide that independently has a modified ribose moiety, a modified nucleotide bond, or a modified base. Therefore, the term "modified nucleotide" encompasses substitution, addition, or removal (e.g., use of functional groups or atoms) of nucleotide bonds, ribose moieties, or bases. Modifications applicable to this invention include all types of modifications disclosed herein or known in the art. "Methoxy-modified nucleotide" refers to a nucleotide formed by replacing the 2' hydroxyl group of the ribose group with a methoxy group. "Fluoro-modified nucleotide" refers to a nucleotide formed by replacing the 2' hydroxyl group of the ribose group with a fluorine group. "2'-O-methoxyethyl (MOE) modified nucleotides" refer to nucleotides formed by replacing the 2'-hydroxyl group of the ribosome with 2'-O-methoxyethyl (MOE). "Deoxyxanthine nucleotides" refer to nucleotides formed by replacing the base of a nucleotide with deoxyxanthine (dI). "Glyceryl nucleotides (GNA)" refer to nucleotides formed by replacing the ribose of a nucleotide with an acyclic three-carbon propylene glycol (1,2-propanediol) backbone. "Unlocked nucleotides (UNA)" refer to nucleotides formed by replacing the ribose of a nucleotide with a ribose lacking the C2-C3 bond of RNA ribose rings. "Nucleotide analogs" refer to groups that can replace nucleotides in nucleic acids but have a different structure from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, or thymine deoxyribonucleotides. Examples include isonucleotides, bridged nucleic acids (BNA), or acyclic nucleotides.

[0315] The fluorinated nucleotide structures are shown below:

[0316] The structure of methoxylated nucleotides is shown below:

[0317] The nucleotide structure modified with 5'-(E)-vinylphosphonate is as follows:

[0318] Wherein, R represents H, OH, or any modifying group as defined above, such as those selected from H, OH, F, or methoxy; Base represents a natural or non-natural base, such as a base selected from A, U, C, G, or T: for example, the structure of VPmN is as follows:

[0319] s represents a thiophosphate group, and the structural diagram is as follows:

[0320] iab represents the reverse debasing residue, and its structural diagram is shown below:

[0321] dI is deoxyinosine nucleotide, and its structure is shown below:

[0322] MOE is modified with 2'-O-methoxyethyl, and its structural diagram is shown below:

[0323] GNA is a glycerol nucleotide, and its structural diagram is shown below;

[0324] UNA stands for unlocking nucleotide, and its structure is shown below:

[0325] In this document, the term "siRNA" refers to an RNA molecule capable of sequence-specifically inducing RNAi, consisting of a sense strand and an antisense strand, and having a partially or fully complementary double-stranded structure. In the siRNAs involved in this invention, the length of the complementary double-stranded structure can be 14 to 30 base pairs, for example 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 base pairs, for example 14 to 21 base pairs. In some embodiments of this invention, the siRNA may also contain modified nucleotides as needed, which do not cause a significant weakening or loss of the siRNA's function in inhibiting APP gene expression. Currently, there are various ways to modify siRNA in this field, including 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).

[0326] The term "antisense strand" includes a region substantially complementary to the target sequence. The term "sense strand," as used herein, refers to the strand of a region substantially complementary to the antisense strand. The term "complementary region" refers to a region on the antisense strand substantially complementary to the APP mRNA or a region on the sense strand substantially complementary to the antisense strand. When the complementary region is not perfectly complementary to the target sequence or the antisense strand, mismatches can occur within the molecule or at the terminal regions. Typically, the most permissible mismatches are at the terminal regions, for example, within 5, 4, 3, 2, or 1 nucleotides at the 5' and / or 3' ends.

[0327] In this document, unless otherwise specified, the term "complementary" refers to the ability of an oligonucleotide in a first sequence to hybridize with an oligonucleotide in 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 6, 5, 4, 3, 2, or 1 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 document, to satisfy the above hybridization ability requirements, a "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 article, 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.

[0328] Those skilled in the art can determine the most suitable conditions for testing the complementarity of two sequences based on the final application of the hybridized nucleotides. Such conditions can be, for example, stringent conditions, such as 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, at 50 or 70°C for 12–16 hours, followed by washing. Other conditions, such as physiologically relevant conditions that may be encountered in vivo, can also be applied.

[0329] In this article, unless otherwise specified, "nucleotide sequence difference" refers to a change in the type of bases of nucleotides at the same or corresponding positions 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 nucleotide sequence difference is considered to exist at that position. It should be noted that if the nucleotides at the same or corresponding positions differ only in the presence or type of modification compared to the original nucleotide sequence, then a nucleotide sequence difference at that position is not considered to exist.

[0330] Unless otherwise specified herein, the term "pharmaceutically acceptable carrier and / or excipient" means that the carrier, delivery carrier, diluent, excipient, and / or the salt / ester / hydrate formed therefrom are generally chemically or physically compatible with other components constituting a pharmaceutical dosage form (such as the siRNA of the present invention) and physiologically compatible with the subject. "Pharmaceutically acceptable carriers and / or excipients" do not exert or are not intended to exert a therapeutic effect at the intended dosage. Such components may serve to: a) assist in the processing of the drug delivery system during manufacturing; b) protect, support, or enhance the stability, bioavailability, or patient acceptability of the active ingredient; c) assist in product identification; and / or d) enhance the overall safety, efficacy, delivery, or any other property of the active ingredient during storage and use. For example, the siRNA of the present invention may be encapsulated by a delivery carrier. "Pharmaceutically acceptable carriers and / or excipients" include, but are not limited to: viruses, liposomes, nanoparticles, bacteria, lipid nanoparticles (LNP), neutral liposomes (NL), polymer nanoparticles, double-stranded RNA binding motifs (dsRBMs), pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents for maintaining osmotic pressure, agents for delaying absorption, and preservatives. For example, viruses include, but are not limited to, retroviruses, adenoviruses, lentiviruses, baculoviruses, and AAV. Liposomes include, but are not limited to, Lipofectamine, cationic DOTAP, and neutral DOPC. Nanoparticles include, but are not limited to, cationic polymers and PEI. Bacteria include, but are not limited to, tkRNAi. Polymer nanoparticles include, but are not limited to, low-molecular-weight polymers or high-molecular-weight polymers. pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Reagents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and their analogues. Reagents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol).

[0331] In this article, unless otherwise specified, the term "suppression" 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 levels of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 99%, or even 100%, compared to the absence of siRNA treatment. A 100% decrease in target gene expression levels means that there is no detectable level of target gene expression.

[0332] In this document, the term "protrusion" or "nucleotide protrusion" refers to at least one unpaired nucleotide protruding from the double-stranded structure of siRNA. For example, a protrusion exists when the 3' end of one strand of siRNA extends beyond the 5' end of the other strand (or vice versa). siRNA may contain at least one nucleotide protrusion, or the protrusion may contain at least 2 nt, at least 3 nt, at least 4 nt, at least 5 nt, or more. The protrusion may contain or be composed of nucleotide / nucleoside analogs, including deoxyribonucleotides / nucleosides. The protrusion may be on the sense strand, antisense strand, or any combination thereof. The nucleotide of the protrusion may be present at the 5' end, 3' end, or both ends of the antisense strand or sense strand of siRNA. Accordingly, the term "flat end" refers to the absence of a nucleotide protrusion.

[0333] In this document, the term "prevention" refers to a method implemented to prevent or delay the occurrence of a disease, condition, or symptom in a subject; the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical outcome. For the purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, alleviating symptoms, reducing the extent of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the progression of the disease, improving or alleviating the state of the disease, and relieving symptoms (whether partial or complete), whether detectable or undetectable. Furthermore, "treatment" can also refer to prolonged survival compared to the expected survival (if no treatment was received).

[0334] In this document, the term "effective amount" means an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective amount for disease prevention is an amount sufficient to prevent, stop, or delay the onset of a disease; an effective amount for disease treatment is an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic purposes will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the method of administration of the drug, and other concurrent treatments, etc.

[0335] 8. Beneficial effects of the invention

[0336] The siRNA of this invention can effectively inhibit APP gene expression in vitro and / or in vivo, and exhibits good stability, negligible cytotoxicity, and immunostimulatory activity. Therefore, the siRNA of this invention can be used to treat diseases or conditions that benefit from reduced or inhibited APP expression, and has significant clinical value for the treatment of APP-related diseases. Attached Figure Description

[0337] Figure 1 shows the results of the inhibitory effect of the APP siRNA of the present invention on APP expression in SK-N-MC cells. Detailed Implementation

[0338] Sequence information

[0339] Table 1A: siRNA double-stranded sequence information for APP target design

[0340] Table 1B: siRNA sequence information for APP target design

[0341] Table 1C: Sequence information of siRNA-lipid conjugates designed for APP targets

[0342] Table 1G: Other sequences

[0343] Detailed Implementation

[0344] The invention will now be described in the following non-limiting embodiments.

[0345] Those skilled in the art will understand that the embodiments are described by way of example only and are not intended to limit the scope of protection claimed in this application. Unless otherwise specified, the experimental methods in the embodiments are conventional methods. Where specific conditions are not specified in the embodiments, they are performed according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0346] 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.

[0347] Example 1: siRNA Synthesis

[0348] Sequence design was performed targeting the major transcript of human APP, NM_000484.4 (SEQ ID NO:457). Customized oligonucleotide sequences were synthesized using phosphoramide technology in solid-phase synthesis. All phosphoramide monomers were dissolved in anhydrous acetonitrile (50 mM) and dried using molecular sieves (3A).

[0349] Each coupling step in the sequence synthesis took 12 minutes. Phosphate bonds were constructed using an aqueous solution of iodine (50 mM). To introduce thiophosphate bonds, (E)-N,N-dimethyl-N'-(3-thio-3H-1,2,4-DITHIAZOL-5-yl)formamidin (DDTT) (50 mM pyridine solution) was used. Unreacted active groups were capped using a mixture of acetic anhydride, N-methylimidazolium, pyridine, and acetonitrile (N-methylimidazolium / acetonitrile ratio 1:4:acetic anhydride / pyridine / acetonitrile ratio 2:3:5, v / v).

[0350] After solid-phase synthesis, the dried synthetic support was deprotected by reacting at 55°C with ethanol / ammonium hydroxide (25% aqueous solution) = 1 / 3 (v / v) for 16 h. The support was filtered, the filtrate was concentrated, and the concentrate was reconstituted in water.

[0351] Crude siRNA was analyzed using Waters Acquity UPLC QDA. Buffer A was an aqueous solution of HFIP / DIEA / EDTA, and buffer B was HFIP / DIEA / EDTA in MeOH (80%) / water (20%). The gradient was: buffer B 25-45% for 6 min, 85% buffer B for 2 min. Samples were purified by RP-HPLC. Different fractions were collected by monitoring the UV absorbance at 260 nm. After purity analysis on the HPLC instrument, qualified fractions were collected and lyophilized to obtain the sample. Double-strand annealing: Complementary strands (SS and AS strands) of qualified purity were mixed in an equimolar ratio. The solution was placed in a 90°C metal bath and maintained at this temperature for 5 minutes, then slowly cooled to room temperature. Samples were taken for SEC testing to determine the double-strand annealing purity, which was above 90%. The obtained siRNA duplexes are shown in Tables 1A and 1B.

[0352] The aforementioned double strands were chemically modified according to methods known to those skilled in the art.

[0353] Example 2: Detection of APP siRNA Bioactivity

[0354] 2.1 APP siRNA biological assay method

[0355] For siRNA preparation, RNase-free H2O was used to dissolve the APP siRNA dry powder into a 20 μM siRNA stock solution. According to the experimental design, the siRNA was diluted 20-fold to the maximum working concentration, and then further diluted to the corresponding concentrations according to the experimental design. Initial single-concentration screening: first diluted to 20 nM, then mixed with RNAiMAX and cell suspension to a final concentration of 1 nM or 2 nM; IC50 screening: first diluted to 200 nM, then serially diluted 5-fold, resulting in final concentrations of 10 nM, 2 nM, 0.4 nM, 0.08 nM, 0.016 nM, 0.0032 nM, 0.00064 nM, 0.000128 nM, and 0.0000256 nM, for a total of 9 concentrations. The transfection reagent RNAiMAX (manufacturer: Invitrogen, catalog number: 13778075) was diluted 25 times with Opti-MEM medium and dispensed into 96-well dilution plates. An equal volume of the corresponding concentration of siRNA was added, and the mixture was mixed. Then, 10 μl of the solution was dispensed into each well of the 96-well cell culture plate and incubated at room temperature for 15 min to allow the siRNA / RNAiMAX complex to form. SK-N-MC cells (manufacturer: Nanjing Kebai Biotechnology Co., Ltd., catalog number: CBP60806) were cultured in EMEM medium (manufacturer: ATCC, catalog number: 30-2003) containing 10% FBS (manufacturer: Gibco, catalog number: A5669801). 90 μl of each cell was seeded into a cell culture plate containing siRNA / RNAiMAX. The culture plate was then transferred to a CO2 incubator and cultured at 37°C for 24 h. N2a cells (manufacturer: Wuhan Pronosai Life Science Co., Ltd., catalog number: CL-0168) were cultured in EMEM medium (manufacturer: Gibco, catalog number: 11140-050) containing 1% FBS and 10% FBS. In MEM medium (manufacturer: Gibco, catalog number: A5669801) containing FBS (manufacturer: Gibco, catalog number: 11095-098), 90 μl of cells containing 1×10^4 cells were seeded into a cell culture plate containing siRNA / RNAiMAX complex. The culture plate was then transferred to a CO2 incubator and cultured at 37°C for 24 h.

[0356] RNA acquisition (FlysisAmp Cells Lysis Kit, manufacturer: Vazyme, catalog number: CL101-02). Prepare the lysis working solution with the following ratio: FlysisAmp Cells Lysis Buffer:DNase I:Enhancer Solution = 23:1:1. After preparation, invert and mix thoroughly. Add 50 μl of the lysis working solution to each well of a 96-well cell culture plate after transfection and incubation for 5 min at room temperature to lyse the cells. Add 5 μl of FlysisAmp Cells Stop Buffer to each well and incubate for 2 min at room temperature to stop the reaction.

[0357] RT-PCR (manufacturer: Takara, catalog number: RR086A): Prepare the PCR reaction system according to the instructions and transfer it to a 384-well reaction chamber. Perform the RT-PCR reaction using the pre-set program of ABIQuantStudio 5System.

[0358] Data Processing: This assay uses the ΔΔCt method to calculate the relative expression level of the target gene. ΔCt is obtained by subtracting the ct value of the internal reference gene β-actin from the ct value of the target gene APP in the same reaction well. Then, ΔΔCt is obtained by subtracting the ΔCt of each reaction well from the ΔCt of the blank control group (normal cultured cells without any transfection sequence). Finally, 2^-ΔΔCt is calculated; this value represents the multiple of the expression level of the corresponding treatment well relative to the expression level of the blank control group. Inhibition level (KD efficiency) is expressed as: ((mRNA in control group cells) - (mRNA in treatment group cells)) ÷ (mRNA in control group cells), multiplied by 100%. Maximum inhibition rate (KD max) is expressed as the maximum knockdown efficiency.

[0359] 2.2 Single-point concentration test results

[0360] The single-point concentration test results of APP siRNA modified using the P0 modification mode are shown in Table 2. The results show that the tested APP siRNA can reduce the expression of target genes.

[0361] Table 2: Results of APP siRNA inhibition of APP expression in SK-N-MC cells

[0362] 2.3 IC50 Test Results

[0363] The IC50 results of APP siRNAs modified using the P0 modification mode are shown in Table 3 and Figure 1. The results showed that 17 APP siRNA activity sequences reduced the expression of target genes in a concentration-dependent manner.

[0364] Table 3: IC50 and KDmax results of APP siRNA inhibition of APP expression in SK-N-MC cells.

[0365] 2.4 Bare Sequence IC50 Test Results

[0366] IC50 tests were performed on the unmodified APP siRNAs in Table 3, and the results showed that 17 APP siRNA sequences reduced the expression of target genes in a concentration-dependent manner.

[0367] Table 4: IC50 results of unmodified APP siRNA inhibiting APP expression in SK-N-MC cells NA: Not Available

[0368] 2.5 Detection results of APP siRNA with different chemical modification modes

[0369] Multiple rounds of single-concentration screening and IC50 assays were performed on SK-N-MC and N2a cells using siRNAs with different modification patterns. The results are shown in Tables 5 to 8. The results showed that the tested APP siRNAs could reduce the expression of target genes.

[0370] Table 5: Initial screening of different modification patterns at single concentrations in SK-N-MC cells

[0371] Table 6: IC50 results of chemically modified APP siRNA inhibiting APP expression in SK-N-MC cells

[0372] Table 7: IC50 results of chemically modified APP siRNA inhibiting APP expression in cells

[0373] Table 8: IC50 results of chemically modified APP siRNA inhibiting APP expression in cells.

[0374] Example 3: Off-target analysis of APP siRNA

[0375] 3.1 APP siRNA Off-Target Analysis Methods

[0376] siRNA transfection of SK-N-MC cells: APP siRNA powder was dissolved in RNase-free H2O to prepare a 20 μM siRNA stock solution. According to the experimental design, the siRNA was diluted 20-fold to a working concentration of 0.2 μM. After mixing with RNAiMAX and cell suspension, the final concentration was 10 nM. The transfection reagent RNAiMAX (manufacturer: Invitrogen, catalog number: 13778075) was diluted 25-fold with Opti-MEM medium and aliquoted into 1.5 mL centrifuge tubes. An equal volume of the corresponding concentration of siRNA was added, and the mixture was thoroughly mixed. The resulting 250 μL samples were aliquoted into 6-well cell culture plates and incubated at room temperature for 15 min to allow the siRNA / RNAiMAX complex to form. Three replicates were prepared for each sequence. The MOCK group served as a blank control, without siRNA, and was otherwise consistent with the experimental groups. SK-N-MC cells (manufacturer: Nanjing Kebai Biotechnology Co., Ltd., catalog number: CBP60806) were cultured in EMEM medium (manufacturer: ATCC, catalog number: 30-2003) containing 10% FBS (manufacturer: Gibco, catalog number: A5669801). 2.25 ml of cells containing 6.25 × 10^5 cells were seeded into cell culture plates containing siRNA / RNAiMAX complex. The culture plates were then transferred to a CO2 incubator and cultured at 37°C for 24 h.

[0377] RNA extraction (TaKaRa MiniBEST Universal RNA Extraction Kit, manufacturer: TaKaRa, catalog number: 9767) and RNA-seq analysis: RNA was extracted from cell samples transfected 24 hours prior to presentation according to the instructions. Quality-tested RNA samples underwent transcriptome sequencing. Differentially expressed genes were screened using |logFold Change|≥1 and Padj≤0.05 as selection criteria. Differentially expressed genes from two transfections of the same sequence and the intersection of RNA-seq results were considered candidate off-target genes.

[0378] 3.2 Results of APP siRNA Off-Target Analysis

[0379] Differential gene analysis between the siRNA sequence group and the MOCK group used DESeq2 padj ≤ 0.05 and |log2FoldChange| ≥ 1.0 as thresholds. The statistical results are shown in Table 9. The results show that the off-target risk of the double-stranded siRNA conjugates in the table below is low. Among them, the SJ-0165-080-E, SJ-0169-073-E, SJ-0204-073-E, and SJ-0216-073-E sequences have no off-target genes (the number of downregulated differential genes in Table 9 is 0).

[0380] Table 9: Statistical Results of Differential Genes

[0381] Example 4: Stability analysis of APP siRNA

[0382] 4.1 APP siRNA Stability Analysis Method

[0383] Brain tissue from C57 mice after cardiac perfusion was isolated and homogenized using tissue:PBS buffer (pH 7.4) = 1:4. An appropriate amount of homogenate and siRNA were added, and the siRNA was diluted to a 2 μM incubation concentration with PBS buffer and incubated at 37°C for 0, 24, and 72 h. The siRNA after 0, 24, and 72 h of incubation was analyzed by liquid chromatography-high resolution mass spectrometry (LC-HRMS). The peak area (t-area) of the mass spectrometry response at 0, 24, and 72 h was recorded. The antisense (AS) and sense (SS) strands were calculated based on the relative peak area ratio with the internal standard (IS). Alnylam-Mivelsiran was used as the positive control molecule (PC-miv, sense strand sequence as shown in SEQ ID NO:455, and antisense strand sequence as shown in SEQ ID NO:456).

[0384] AS t(%)=(AS t area / IS t area) / (AS t0 area / IS t0 area)×100%;

[0385] SS t(%)=(SS t area / IS t area) / (SS t0 area / IS t0 area)×100%.

[0386] 4.2 Results of APP siRNA Stability Analysis

[0387] The stability analysis results of the double-stranded sequences in mouse brain tissue are shown in Table 10. With prolonged incubation time, a greater amount of remaining sequence indicates higher siRNA stability. The results showed that the siRNAs SJ-0202-073-E, SJ-0203-073-E, SJ-0204-073-E, SJ-0206-073-E, SJ-0207-073-E, SJ-0209-073-E, SJ-0214-073-E, SJ-0216-073-E, and SJ-0217-073-E were stable. The sequences SJ-0221-073-E, SJ-0223-073-E, SJ-0015-073-E, SJ-0001-073-E, SJ-0177-073-E, SJ-0173-073-E, SJ-0162-073-E, SJ-0185-073-E, SJ-0074-073-E, SJ-0169-073-E, SJ-0168-073-E, and SJ-0165-073-E exhibit excellent stability in mouse brain tissue.

[0388] Table 10: Stability of modified APP siRNA in C57 mouse perfused brain tissue homogenates NA: Not Available

[0389] Example 5: In vivo activity assay of C16 siRNA conjugates

[0390] 5.1 Preparation of C16 siRNA conjugates

[0391] The abbreviations used in this embodiment are shown in the table below.

[0392] 5.1.1 Synthesis of compound N15U

[0393] Step 1: Sodium azide (5.32 g, 82 mmol, 5 eq) was added to a solution of 1-bromohexadecane (1-1, 5 g, 16.4 mmol, 1 eq) in N,N-dimethylformamide (50 mL). After purging with nitrogen, the mixture was stirred at 70 °C for 1 h. TLC showed that the starting material reacted completely. The reaction solution was poured into ice water (500 mL), extracted with ethyl acetate (100 mL × 3), and the organic phase was washed with water and saturated sodium chloride solution (100 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give 1-azidohexadecane (compound 1-2, 4.2 g, yield: 96%).

[0394] 1H NMR (400MHz, CDCl3) δ3.25(t,J=7.0Hz,2H),1.64–1.55(m,2H),1.38–1.26(m,26H),0.88(t,J=6.6Hz,3H).

[0395] Step 2: Under nitrogen protection and in an ice bath, slowly add 1,3-dichloro-1,1,3,3-tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (compound 1-3,2 g, 8.2 mmol, 1 eq) to an ultra-dry pyridine (15 mL) solution. After the addition is complete, slowly raise the temperature to 25 °C and continue stirring for 17 hours. The reaction solution was concentrated under reduced pressure. The residue was diluted with ethyl acetate (20 mL) and poured into 2N dilute hydrochloric acid (50 mL). The residue was extracted with ethyl acetate (20 mL × 3). The organic phase was washed with 2N dilute hydrochloric acid (20 mL) and saturated sodium chloride solution (20 mL), respectively. The residue was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by normal column chromatography (silica gel, mobile phase A: petroleum ether, mobile phase B: ethyl acetate, elution gradient: 0% B to 50% B). The eluent was collected and concentrated under reduced pressure to give 1-((6aR,8R,9R,9aS)-9-hydroxy-2,2,4,4-tetraisopropyltetrahydro-6H-furano[3,2-f][1,3,5,2,4]trioxadisilicyclooctane-8-yl)pyrimidine-2,4(1H,3H)-dione (compound 1-4, 3.4 g, yield: 85.4%).

[0396] MS:m / z 487.22[M+H] + .

[0397] 1 H NMR (400MHz, DMSO-d6) δ11.37(s,1H),7.70(d,J=8.2Hz,1H),5.59(d,J=4.6Hz,1H) ,5.55(d,J=9.6Hz,2H),4.19–4.12(m,3H),4.03–3.91(m,2H),1.10–1.01(m,28H).

[0398] Step 3: Under ice bath conditions, add Na2CO3 (9.65g, 91mmol, 7.0eq) and tetrabutylammonium bromide (168mg, 0.52mmol, 0.04eq) to a mixed solution of compounds 1-4 (6.3g, 13mmol, 1.0eq) in dichloromethane (120mL) and water (240mL). Then, slowly add benzoyl chloride (2.36g, 16.8mmol, 1.3eq) dropwise to the reaction solution. After the addition is complete, raise the temperature to 25°C and continue stirring for 16 hours. TLC showed the reaction was complete. The reaction solution was allowed to stand and separate into layers. The aqueous phase was extracted with dichloromethane (150 mL). The organic phases were combined, washed with saturated sodium chloride aqueous solution (100 mL), dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by normal column chromatography (silica gel, mobile phase A: petroleum ether, mobile phase B: ethyl acetate, elution gradient: 0% B to 25% B). The eluent was collected and concentrated under reduced pressure to give 3-benzoyl-1-((6aR,8R,9S,9aS)-9-hydroxy-2,2,4,4-tetraisopropyltetrahydro-6H-furano[3,2-f][1,3,5,2,4]trioxadisilicyclooctane-8-yl)pyrimidine-2,4(1H,3H)-dione (compounds 1-5, 5.0 g, yield: 65%).

[0399] MS: m / z 613.26 [M+Na] + .

[0400] 1 H NMR(400MHz, DMSO-d6)δ8.04–7.96(m,2H),7.86(d,J=8.3Hz,1H),7.83–7.75(m,1H),7.61(t,J=7.8Hz,2H),5.82 (d,J=8.2Hz,1H),5.66(d,J=4.5Hz,1H),5.58(s,1H),4.24–4.13(m,3H),4.08–3.90(m,2H),1.14–0.93(m,28H).

[0401] Step 4: Under ice bath conditions, compounds 1-5 (2.0 g, 3.39 mmol, 1.0 eq) were dissolved in a mixed solvent of ethyl bromoacetate (10 mL) and toluene (10 mL). After purging with nitrogen, tetrabutylammonium iodide (626 mg, 1.69 mmol, 0.5 eq) was added, followed by slow dropwise addition of NaOH aqueous solution (10 mL, 60% w / v). The reaction was carried out at 0°C for 5 minutes. TLC showed that the reaction was essentially complete. The reaction solution was poured into water (60 mL), extracted with dichloromethane (120 mL), washed with saturated sodium chloride aqueous solution (60 mL), dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was then subjected to normal phase column chromatography (silica gel, mobile phase A: petroleum ether, mobile phase B: ethyl acetate, elution gradient: 0% B to 30%). B), the eluent was collected and concentrated under reduced pressure to give ethyl 2-(((6aR,8R,9S,9aR)-8-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furano[3,2-f][1,3,5,2,4]trioxadisilicyclooctane-9-yl)oxy)acetate (compound 1-6, 1.95 g, yield: 85%).

[0402] MS:m / z 677.30[M+H] + .

[0403] 1 HNMR (400MHz, DMSO-d6) δ8.03–7.95(m,2H),7.88–7.75(m,2H),7.65–7.56(m,2H),5.84(d,J=8.2Hz,1H),5.72(s,1H),4.40(d,J=16.5Hz ,1H),4.36–4.27(m,3H),4.22–4.13(m,1H),4.08–4.02(m,3H),3.94(dd,J=13.5,2.5Hz,1H),1.13(t,J=7.0Hz,3H),1.10–0.93(m,28H).

[0404] Step 5: Under ice bath conditions, add K2CO3 (815mg, 5.9mmol, 2.0eq) to a MeOH (25mL) solution of compounds 1-6 (2g, 2.95mmol, 1.0eq) and stir at room temperature for 2h. TLC showed that the reactants reacted completely. The reaction solution was diluted with dichloromethane (50 mL) and poured into water (50 mL). It was extracted with dichloromethane (30 mL). The organic phase was washed with dilute hydrochloric acid solution (0.1 N, 30 mL) and saturated sodium chloride solution (30 mL), respectively. It was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain methyl 2-(((6aR,8R,9S,9aR)-8-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furano[3,2-f][1,3,5,2,4]trioxadisili-9-yl)oxy)methyl acetate (compounds 1-7, 1.5 g, crude product), which was directly used in the next step.

[0405] MS:m / z 559.21[M+H] + .

[0406] Step 6: Under ice bath conditions, tetrabutylammonium fluoride (6.7 mL, 6.7 mmol, 2.5 eq, 1 M in THF) was added dropwise to a tetrahydrofuran (15 mL) solution of compounds 1-7 (1.5 g, 2.7 mmol, 1.0 eq). After the addition was complete, the temperature was slowly raised to 25 °C, and stirring was continued for 1 h. TLC showed that the reaction was complete. The reaction solution was concentrated and dissolved in ethyl acetate (30 mL), washed successively with water (20 mL) and saturated sodium chloride aqueous solution (20 mL), dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was subjected to normal-phase column chromatography (silica gel, mobile phase A: dichloromethane, mobile phase B: methanol, elution gradient: 0% B to 10% B). The eluent was collected and concentrated under reduced pressure to give methyl 2-(((2R,3S,5R)-2-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)acetate (compounds 1-8, 800 mg, crude product).

[0407] MS:m / z 317.02 [M+H] + .

[0408] 1HNMR(400MHz,DMSO-d6)δ11.35(s,1H),7.89–7.83(m,1H),5.90(d,J=5.4Hz,1H),5.63(dd,J=8.1,2.8Hz,1H), 5.22(s,2H),4.37–4.18(m,2H),4.14(t,J=4.5Hz,1H),4.06(t,J=5.2Hz,1H),3.62(s,3H),3.61–3.53(m,2H).

[0409] Step 7: Dissolve compounds 1-8 (2 g, 6.3 mmol, 1.0 eq) in MeOH (20 mL), and add propargylamine (3.5 g, 63 mmol, 10 eq). Heat to 65 °C and stir for 3 h. TLC showed that the reaction was complete. The reaction solution was concentrated to obtain the crude product, which was subjected to normal phase column chromatography (silica gel, mobile phase A: dichloromethane, mobile phase B: methanol, elution gradient: 0% B to 10% B). The eluent was collected and concentrated under reduced pressure to give 2-(((2R,3S,5R)-2-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)-N-(prop-2-yn-1-yl)acetamide (compounds 1-9, 900 mg, three-step yield: 45%).

[0410] 1 HNMR (400MHz, DMSO-d6) δ11.36(s,1H),8.35(t,J=5.7Hz,1H),7.94(d,J=8.1Hz,1H),5.83(d,J=3.4Hz,1H),5.62(d,J=8.1Hz,1H),5.40(d ,J=6.7Hz,1H),5.17(t,J=5.0Hz,1H),4.20–3.97(m,4H),3.97–3.86(m,3H),3.79–3.66(m,1H),3.66–3.52(m,1H),3.13(t,J=2.5Hz,1H).

[0411] Step 8: Pretreatment: Dissolve compounds 1-9 (750 mg, 2.21 mmol, 1 eq) in anhydrous pyridine (5 mL), concentrate under reduced pressure, and repeat three times.

[0412] Pretreated 2-(((2R,3S,5R)-2-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)-N-(prop-2-yn-1-yl)acetamide (compounds 1-9) was dissolved in anhydrous pyridine (10 mL), nitrogen gas was purged, the mixture was cooled to an ice bath, 4,4'-dimethoxytriphenylchloromethane (1.12 g, 3.32 mmol, 1.5 eq) was added, nitrogen gas was purged again, the mixture was heated to 23 °C, and stirred for 8 h. The reaction was monitored by TLC until complete. The reaction solution was diluted with dichloromethane (20 mL) and poured into ice-cold saturated NaHCO3 solution (60 mL). The solution was extracted with dichloromethane (20 × 3 mL), and the organic phase was washed with saturated sodium chloride solution (20 mL). The solution was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was subjected to normal phase column chromatography (silica gel, mobile phase A: dichloromethane, mobile phase B: methanol, elution gradient: 0% B to 6% B). The eluent was collected and concentrated under reduced pressure to give 2-(((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-2-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-hydroxytetrahydrofuran-3-yl)oxy)-N-(prop-2-yn-1-yl)acetamide (compounds 1-10, 930 mg, yield: 66%).

[0413] 1 H NMR (400MHz, DMSO-d6) δ11.40(s,1H),8.35(t,J=5.8Hz,1H),7.72(d,J=8.2Hz,1H ),7.40–7.30(m,4H),7.27–7.23(m,5H),6.91(d,J=8.6Hz,4H),5.82(d,J=2.1Hz,1 H),5.47(d,J=8.0Hz,1H),5.27(dd,J=8.2,1.8Hz,1H),4.26–4.19(m,2H),4.08–4 .02(m,3H),3.94–3.91(m,2H),3.74(s,6H),3.31–3.24(m,2H),3.13–3.12(m,1H).

[0414] Step 9: Dissolve compound 1-10 (930 mg, 1 eq, 1.45 mmol) and 1-azidohexadecane (compound 1-2, 582 mg, 1.5 eq, 2.17 mmol) in a mixed solution of tetrahydrofuran (15 mL) and H2O (3 mL). Under ice bath conditions, add CuSO4 (116 mg, 0.5 eq, 0.73 mmol) and sodium vitamin C (431 mg, 1.5 eq, 2.17 mmol). After purging with nitrogen, raise the temperature. The mixture was stirred at 28°C for 3 hours. The reaction was monitored by TLC until complete. The reaction solution was filtered through diatomaceous earth. The filter cake was washed with dichloromethane (40 mL). The filtrate was poured into a saturated sodium bicarbonate solution (40 mL), extracted with dichloromethane (20 mL × 3), and the organic phase was washed with a saturated sodium chloride solution (30 mL). The mixture was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was subjected to normal-phase column chromatography (silica gel, mobile phase A: petroleum ether, mobile phase B: methanol / ethyl acetate (1 / 1), elution gradient: 0%). B to 6% B), the eluent was collected and concentrated under reduced pressure to give 2-(((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-2-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-hydroxytetrahydrofuran-3-yl)oxy)-N-((1-hexadecyl-1H-1,2,3-triazol-4-yl)methyl)acetamide (compound 1-11,950 mg, yield: 72%).

[0415] 1 H NMR (400MHz, DMSO-d6) δ11.41(s,1H),8.44(t,J=6.0Hz,1H),7.91(s,1H),7.72(d,J=8.0Hz,1H),7.39– 7.30(m,4H),7.28–7.22(m,5H),6.90(d,J=8.6Hz,4H),5.81(d,J=2.0Hz,1H),5.47(d,J=7.8Hz,1H),5.2 6(dd,J=8.0,2.0Hz,1H),4.36(t,J=6.4Hz,2H),4.28(t,J=7.2Hz,2H),4.25–4.18(m,2H),4.08–3.99(m ,3H),3.74(s,6H),3.26–3.23(m,2H),1.77(q,J=7.2Hz,2H),1.22–1.21(m,26H),0.84(t,J=6.6Hz,3H).

[0416] Step 10: Pretreatment: Remove water from substrate compound 1-11 (380 mg, 0.42 mol, 1.0 eq) by azeotropic treatment with toluene (3.0 ml × 2).

[0417] Under nitrogen protection, 1H-tetrazole (58.6 mg, 0.84 mmol, 2.0 eq) was dissolved in anhydrous dichloromethane (1.2 mL). Under nitrogen protection, bis(diisopropylamino)(2-cyanoethoxy)phosphine (252 mg, 0.84 mol, 2.0 eq) was added, and the mixture was stirred at 30 °C for 1 hour. A dichloromethane solution (3 mL) of the pretreated compound 1-11 and N,N-diisopropylethylamine (108 mg, 0.84 mmol, 2.0 eq) was added to the reaction mixture. The reaction was continued at 25 °C under nitrogen protection for 2 hours. The reaction was monitored by TLC until it was complete. The mixture was then directly subjected to normal-phase column chromatography (silica gel, mobile phase A: dichloromethane (0.1% triethylamine), mobile phase B: methanol, elution gradient: 0% B to 4% B). The eluent was concentrated under reduced pressure to give (2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-(2-(((1-hexadecyl-1H-1,2,3-triazol-4-yl)methyl)amino)-2-oxoethoxy)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphonamide (compound N15U, 393 mg, yield: 85%).

[0418] MS:m / z 1109.66237[M+H] + .

[0419] 1 H NMR (400MHz, DMSO-d6) δ11.40(s,1H),8.11(t,J=6.0Hz,1H),7.86(d,J=8.8Hz,1H),7.75(dd,J=8.2,5.8Hz,1H),7.40–7.29(m, 4H),7.28–7.22(m,5H),6.90–6.86(m,4H),5.87(dd,J=7.6,2.8Hz,1H),5.27(dd,J=8.2,2.0Hz,1H),4.46–4.11(m,9H),3.79–3 .76(m,1H),3.73(d,J=2.2Hz,6H),3.70–3.58(m,1H),3.52–3.43(m,2H),3.39–3.35(m,1H),3.28–3.24(m,1H),2.75–2.71(m,1 H),2.61–2.58(m,1H),1.78–1.71(m,2H),1.22–1.21(m,26H),1.10–1.02(m,9H),0.91(d,J=6.8Hz,3H),0.84(t,J=6.6Hz,3H).

[0420] 31P NMR(162MHz,DMSO-d6)δ149.59,148.29.

[0421] 5.1.2 Synthesis of compound N15C

[0422] Step 1: Under ice bath conditions, add imidazole (509 mg, 7.5 mmol, 3 eq) to a solution of compound 1-10 (1.6 g, 2.5 mmol, 1 eq) in N,N-dimethylformamide (16 mL), stir for 10 min, then add tert-butyldimethylchlorosilane (752 mg, 5 mmol, 2 eq), replace with nitrogen, and then heat to 40 °C and stir for 14 hours. TLC monitoring showed that the reaction proceeded to completion. The reaction solution was poured into water (100 mL), extracted with ethyl acetate (30 mL × 3), washed with saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was directly subjected to normal phase column chromatography (silica gel, mobile phase A: dichloromethane, mobile phase B: methanol, elution gradient: 0% B to 5% B). The eluent was concentrated under reduced pressure to give 2-(((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsiloxy)-2-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-yl)oxy)-N-(prop-2-yn-1-yl)acetamide (compound 2-1, 1.6 g, yield: 85%).

[0423] 1 H NMR (400MHz, DMSO-d6) δ11.40(d,J=2.0Hz,1H),7.99(t,J=5.8Hz,1H),7.81(d,J=8.2Hz,1H), 7.40–7.29(m,4H),7.28–7.21(m,5H),6.92–6.86(m,4H),5.85(d,J=2.2Hz,1H),5.32(dd,J=8 .2,2.0Hz,1H),4.34–4.31(m,1H),4.14–3.99(m,4H),3.95–3.82(m,2H),3.74(s,6H),3.39–3 .36(m,1H),3.20–3.16(m,1H),3.08(t,J=2.6Hz,1H),0.73(s,9H),0.02(s,3H),-0.09(s,3H).

[0424] Step 2: Add 4-dimethylaminopyridine (518 mg, 4.24 mmol, 2 eq) and triethylamine (429 mg, 4.24 mmol, 2 eq) to a 16 mL acetonitrile solution of compound 2-1 (1.6 g, 2.12 mmol, 1 eq). After purging with nitrogen, cool to an ice bath. Then add 2,4,6-triisopropylbenzenesulfonyl chloride (962 mg, 3.18 mmol, 1.5 eq). After the addition is complete, purge with nitrogen again, raise the temperature to 20 °C, stir for 3 h, add ammonia (3 mL), and continue stirring at 20 °C for 17 h. TLC monitoring showed that the reaction proceeded to completion. The reaction solution was poured into ice water (100 mL), extracted with EA (50 mL × 2), and the organic phase was washed with saturated sodium chloride solution (50 mL). The solution was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 2-(((2R,3R,4R,5R)-2-(4-amino-2-oxopyrimidin-1(2H)-yl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsiloxy)tetrahydrofuran-3-yl)oxy)-N-(prop-2-yn-1-yl)acetamide (compound 2-2, 1.6 g, crude product), which was directly used in the next step of the reaction.

[0425] Step 3: Pretreatment: Dissolve compound 2-2 (1.6 g, crude product) in ultra-dry pyridine (10 mL), concentrate under reduced pressure, and repeat three times.

[0426] The pretreated compound 2-2 was dissolved in pyridine (16 mL), purged with nitrogen, and cooled to an ice bath. Benzoyl chloride (358 mg, 2.54 mmol, 1.2 eq) was then added. After the addition was complete, the temperature was raised to 20 °C and stirred for 2 h. The reaction mixture was monitored by TLC until the starting material was completely reacted. The reaction solution was diluted with dichloromethane (30 mL) and poured into a saturated sodium bicarbonate solution (100 mL). The solution was extracted with dichloromethane (50 mL × 3), and the organic phase was washed with a saturated sodium chloride solution (50 mL). The solution was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was directly subjected to normal phase column chromatography (silica gel, mobile phase A: dichloromethane, mobile phase B: methanol, elution gradient: 0% B to 4%). B), the eluent was concentrated under reduced pressure to give N-(1-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-3-(2-oxo-2-(prop-2-yn-1-ylamino)ethoxy)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide (compound 2-3, 1.6 g, yield: 88%).

[0427] 1H NMR (400MHz, DMSO-d6) δ11.34(s,1H),8.50(d,J=7.4Hz,1H),8.01–7.99(m,3H),,7.63(t,J=7.4Hz,1H),7.52(t ,J=7.6Hz,2H),7.42–7.33(m,4H),7.27(dd,J=8.7,1.8Hz,5H),7.18(d,J=7.4Hz,1H),6.92(d,J=8.6Hz,4H),5.9 3(s,1H),4.42–4.39(m,1H),4.33(d,J=15.2Hz,1H),4.25–4.22(m,1H),4.14–4.06(m,2H),4.00–3.84(m,2H),3. 76(s,6H),3.58–3.54(m,1H),3.26–3.23(m,1H),3.08(t,J=2.6Hz,1H),0.70(s,9H),0.01(s,3H),-0.12(s,3H).

[0428] Step 4: Dissolve compound 2-3 (1.6 g, 1.86 mol, 1 eq) in tetrahydrofuran (16 mL), add tetrabutylammonium fluoride (1 M in THF, 2.8 mL, 1.5 eq) under ice bath, and then raise the temperature to 20 °C and stir for 1 h. TLC monitoring showed that the reaction proceeded to completion. The reaction solution was concentrated under reduced pressure and directly subjected to normal phase column chromatography (silica gel, mobile phase A: dichloromethane, mobile phase B: methanol, elution gradient: 0% B to 4% B). The eluent was concentrated under reduced pressure to give N-(1-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxy-3-(2-oxo-2-(prop-2-yn-1-ylamino)ethoxy)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide (compound 2-4, 1.2 g, yield: 87%).

[0429] 1H NMR(400MHz, DMSO-d6)δ11.32(s,1H),8.40–8.35(m,2H),8.00(d,J=7.6Hz,2H),7.63(t, J=7.4Hz,1H),7.52(t,J=7.6Hz,2H),7.46–7.33(m,4H),7.29–7.25(m,5H),7.18(d,J=7.4 Hz,1H),6.93(d,J=8.2Hz,4H),5.88(s,1H),5.52(d,J=8.6Hz,1H),4.42–4.30(m,2H),4.2 1–4.13(m,2H),3.76(s,6H),3.96–3.93(m,3H),3.45–3.33(m,2H),3.13(d,J=2.4Hz,1H).

[0430] Step 5: Add 1-azidohexadecane (compound 1-2, 646 mg, 2.42 mmol, 1.5 eq) to a mixed solution of tetrahydrofuran (10 mL) and H2O (2 mL) of compound 2-4 (1.2 g, 1.61 mmol, 1 eq), cool to an ice bath, then add anhydrous CuSO4 (128.6 mg, 0.5 eq, 0.8 mmol) and sodium vitamin C (479 mg, 1.5 eq, 2.42 mmol), purge with nitrogen, raise the temperature to 30 °C, and continue stirring for 2 h. TLC monitoring showed the reaction proceeded to completion. The reaction solution was diluted with ethyl acetate (30 mL) and added to a saturated sodium bicarbonate solution (100 mL). Extraction was performed with ethyl acetate (30 mL x 3). The organic phase was washed successively with EDTA (0.5 M, pH = 7.4, 50 mL x 4) and saturated sodium chloride solution (30 mL). The solution was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was subjected to normal-phase column chromatography (silica gel, mobile phase A: dichloromethane, mobile phase B: methanol, elution gradient: 0% B to 5%). B), the eluent was concentrated under reduced pressure to give N-(1-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-(2-(((1-hexadecyl-1H-1,2,3-triazol-4-yl)methyl)amino)-2-oxoethoxy)-4-hydroxytetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide (compound 2-5, 1.4 g, yield: 86%).

[0431] 1H NMR (400MHz, DMSO-d6) δ11.32(s,1H),8.48(t,J=6.0Hz,1H),8.36(d,J=7.4Hz,1H),8.01(d,J=7.8Hz,2H),7.93(s, 1H),7.63(t,J=7.6Hz,1H),7.52(t,J=7.6Hz,2H),7.44–7.35(m,4H),7.31–7.26(m,5H),7.19(d,J=7.4Hz,1H),6.92 (dd,J=8.8,1.8Hz,4H),5.88(s,1H),5.52(d,J=8.6Hz,1H),4.44–4.37(m,3H),4.33–4.27(m,3H),,4.21–4.12(m,2H ),3.99(d,J=4.8Hz,1H),3.76(s,6H),3.44–3.37(m,2H),1.80–1.73(m,2H),1.22–1.21(m,26H),0.86–0.82(m,3H).

[0432] Step 6: Pretreatment: Remove water from substrate compound 2-5 (350 mg, 0.35 mmol, 1.0 eq) by azeotropic treatment with toluene (3.0 ml × 2).

[0433] Under nitrogen protection, 1H-tetrazole (49 mg, 0.7 mmol, 2.0 eq) was dissolved in anhydrous dichloromethane (1.4 mL). Under nitrogen protection, bis(diisopropylamino)(2-cyanoethoxy)phosphine (209 mg, 0.7 mmol, 2.0 eq) was added, and the mixture was stirred at 30 °C for 1 hour. A dichloromethane solution (2.5 mL) of the pretreated compound 2-5 and N,N-diisopropylethylamine (90 mg, 0.6 mmol, 2.0 eq) was added to the reaction mixture. The reaction was continued at 25 °C under nitrogen protection for 2 hours. The reaction was monitored by TLC until it was complete. The mixture was then directly subjected to normal-phase column chromatography (silica gel, mobile phase A: dichloromethane (0.1% triethylamine), mobile phase B: methanol, elution gradient: 0% B to 3% B). The eluent was concentrated under reduced pressure to give (2R,3R,4R,5R)-5-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(2-(((1-hexadecyl-1H-1,2,3-triazol-4-yl)methyl)amino)-2-oxoethoxy)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphonamide (compound N15C, 380 mg, yield: 91%).

[0434] MS:m / z 1212.67 [M+H] +

[0435] 1 H NMR (400MHz, DMSO-d6) δ11.31(s,1H),8.43–8.37(m,1H),8.16(t,J=6.4Hz,1H),8.01–7.99(m,2H),7.89(d,J=6.4Hz,1H), 7.63(t,J=7.6Hz,1H),7.51(t,J=7.6Hz,2H),7.45–7.32(m,4H),7.31–7.26(m,5H),7.17–7.12(m,1H),6.92–6.89(m,4H),5 .97(d,J=11.6Hz,1H),4.55–4.42(m,1H),4.40–4.16(m,8H),3.75(d,J=2.8Hz,6H),3.68–3.35(m,6H),2.73–2.69(m,1H), 2.61–2.57(m,1H),1.78–1.71(m,2H),1.21–1.19(m,26H),1.11–1.00(m,9H),0.90(d,J=6.8Hz,3H),0.84(t,J=6.6Hz,3H).

[0436] 31 P NMR(162MHz,DMSO-d6)δ150.08,148.16.

[0437] 5.1.3 Synthesis of compound N17U

[0438] Step 1: Under nitrogen protection at 21°C, dissolve lithium ethylenediamine complex (3.05 g, 29.8 mmol, 3.25 eq) in dimethyl sulfoxide (10 mL), add 1-bromohexadecane (compound 3-1, 2.8 g, 9.17 mmol, 1.0 eq) under nitrogen protection, react at 21°C for 2.5 h, then increase to 66°C and react for 0.5 h. The reaction was monitored by TLC until complete. The pH of the reaction solution was adjusted to 1 with 1M HCl aqueous solution. Saturated sodium chloride aqueous solution (10 mL) and ethyl acetate (10 mL × 3) were added to the reaction solution for extraction. The organic phase was washed twice with 1M HCl aqueous solution and twice with saturated sodium chloride aqueous solution. After drying with anhydrous sodium sulfate, the mixture was filtered and the residue was obtained by vacuum distillation. The residue was directly subjected to normal phase column chromatography (silica gel, mobile phase A: petroleum ether, mobile phase B: ethyl acetate, elution gradient: 0% B). The eluent was concentrated under reduced pressure to give octadecane-1-yne (compound 3-2, 2.3 g, yield: 98%).

[0439] 1H NMR (400MHz, CDCl3) δ2.18(td,J=7.1,2.6Hz,2H),1.93(t,J=2.6Hz,1H),1.52(p,J=7.0Hz,2H),1.44–1.34(m,2H),1.26(s,24H),0.88(t,J=6.8Hz,3H).

[0440] Step 2: Add ethyl 2-azidoacetate (compound 3-3, 1.0 g, 7.74 mmol, 1.0 eq) to a LiOH (464 mg, 19.4 mmol, 2.5 eq) H₂O (5 mL). After nitrogen purging, react at 21 °C for 2 hours. Monitor the reaction completion by TLC. Adjust the pH to 1-2 with 1N HCl, extract with ethyl acetate (20 mL × 5), combine the organic phases, dry to anhydrous magnesium sulfate, filter, and concentrate the filtrate under reduced pressure to obtain 2-azidoacetic acid (compound 3-4, 568 mg, crude product), which is directly used in the next step of the reaction.

[0441] 1 H NMR (400MHz, DMSO-d6) δ13.13(s,1H),3.98(s,2H).

[0442] Step 3: Under nitrogen protection, N-hydroxysuccinimide (645 mg, 5.60 mmol, 1.0 eq) in N,N-dimethylformamide (6 mL) was added dropwise to a solution of 2-azidoacetic acid (compound 3-4, 568 mg, 5.60 mmol, 1.0 eq) and N,N'-dicyclohexylcarbodiimide (1.16 g, 5.60 mmol, 1.0 eq) in N,N-dimethylformamide (10 mL). After the addition was complete, the mixture was incubated at 21°C. The reaction was monitored by TLC for 24 hours until complete. The reaction solution was filtered, and the filter cake was washed with ethyl acetate (30 mL). The filtrate was collected and washed with water (20 mL × 3), saturated NaHCO3 aqueous solution (20 mL × 2), and saturated NaCl aqueous solution (20 mL × 3), respectively. The filtrate was dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to obtain 2,5-dioxopyrrolidine-1-yl-2-azidoacetate (compound 3-5, 1.0 g, crude product), which was directly used in the next step of the reaction.

[0443] Step 4: At 0°C, 2,2'-dehydro-uridine (compounds 3-6, 5.00 g, 22.1 mmol, 1.0 eq) was dissolved in anhydrous pyridine (50 mL). 4,4'-bismethoxytriphenylmethyl chloride (4.12 g, 12.2 mmol, 0.55 eq) was added in portions, and the temperature was restored to 21°C, and the reaction continued for 1 hour. After 1 hour, the reaction temperature was lowered to 0°C, and 4,4'-bismethoxytriphenylmethyl chloride (4.12 g, 12.2 mmol, 0.55 eq) was added in portions to the reaction solution. The temperature was then restored to 21°C, and the reaction continued for 1 hour. The reaction was monitored by TLC until it was complete. Water (50 mL) was added to the reaction solution, and the mixture was extracted with dichloromethane (20 mL × 3). The combined organic phases were washed successively with saturated sodium chloride aqueous solution (20 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was subjected to normal phase column chromatography (silica gel, mobile phase A: dichloromethane, mobile phase B: methanol, elution gradient: 0% B to 15% B). The eluent was concentrated under reduced pressure to obtain (2R,3R,3aS,9aR)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-hydroxy-2,3,3a,9a-tetrahydro-6H-furano[2',3':4,5]oxazolo[3,2-a]pyrimidin-6-one (compound 3-7, 9.1 g, yield: 78%).

[0444] 1 H NMR(400MHz,DMSO-d6)δ7.95(d,J=7.4Hz,1H),7.35–7.19(m,5H),7.17–7.12(m,4H) ,6.87–6.81(m,4H),6.33(d,J=5.6Hz,1H),5.96(d,J=4.5Hz,1H),5.88(d,J=7.4Hz,1 H),5.21(dd,J=5.7,1.2Hz,1H),4.36–4.27(m,1H),4.22(ddd,J=7.3,4.4,2.8Hz,1H ), 3.73(d,J=1.6Hz,6H), 2.95(dd,J=10.3,4.4Hz,1H), 2.82(dd,J=10.3,7.3Hz,1H).

[0445] Step 5: At 21°C, under nitrogen protection, NaH (189.2 mg, 4.73 mmol, 0.5 eq, 60% in mineral oil) was added to trichloroacetonitrile (25 mL). After stirring the reaction solution for 5 minutes, compound 3-7 (5.0 g, 9.46 mmol, 1.0 eq) was added. The reaction temperature was then raised to 90°C and the reaction was continued for 16 hours. The reaction was monitored by TLC until it was complete. The reaction solution was concentrated under reduced pressure to obtain a crude product, which was then subjected to normal phase column chromatography (silica gel, mobile phase A: petroleum ether, mobile phase B: ethyl acetate, elution gradient: 0% B to 50% B). The eluent was concentrated under reduced pressure to give 1-((3aR,4R,6R,6aS)-6-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-2-(trichloromethyl)-3a,4,6,6a-tetrahydrofurano[3,4-d]oxazol-4-yl)pyrimidine-2,4(1H,3H)-dione (compound 3-8, 4.6 g, yield: 72%).

[0446] 1 H NMR (400MHz, DMSO-d6) δ11.42(d,J=2.2Hz,1H),7.83(d,J=8.0Hz,1H),7.42–7.27( m,4H),7.26–7.16(m,5H),6.86(dd,J=10.7,8.0Hz,4H),5.91(d,J=2.2Hz,1H),5.7 0–5.59(m,1H),5.50–5.41(m,1H),5.32–5.22(m,1H),4.14(dt,J=8.0,4.1Hz,1H), 3.73(d,J=3.1Hz,6H), 3.48(dd,J=10.2,7.8Hz,1H), 3.18(dd,J=10.2,3.9Hz,1H).

[0447] Step 6: Under nitrogen protection at 21°C, compound 3-8 (4.6 g, 6.84 mmol, 1.0 eq) was dissolved in anhydrous ethanol (18 mL), and then 6N NaOH aqueous solution (9 mL) was added. The reaction solution was heated to 70°C and the reaction was continued for 16 hours. The reaction was monitored by TLC until complete. The reaction solution was concentrated under reduced pressure, and saturated ammonium chloride aqueous solution (30 mL) was added. The solution was extracted with dichloromethane (40 mL × 3). The organic phases were combined and washed successively with water (20 mL) and saturated sodium chloride aqueous solution (20 mL × 2). After drying with anhydrous magnesium sulfate, the solution was filtered and concentrated under reduced pressure to obtain the crude product. The crude product was subjected to normal phase column chromatography (silica gel, mobile phase A: dichloromethane, mobile phase B: methanol, elution gradient: 0% B to 50% B). The eluent was concentrated under reduced pressure to obtain 1-((2R,3R,4S,5R)-3-amino-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)pyrimidin-2,4(1H,3H)-dione (compound 3-9, 2.62 g, yield: 72%).

[0448] 1 H NMR (400MHz, DMSO-d6) δ7.62(d,J=8.1Hz,1H),7.50–7.29(m,4H),7.28–7.18(m,5H),6.99–6.79(m,4H),5.66(d,J=7.1Hz,1 H),5.39(d,J=8.1Hz,1H),3.97(dq,J=7.2,3.3Hz,2H),3.74(s,6H),3.47–3.27(m,2H),3.20(ddd,J=28.4,10.4,3.9Hz,2H).

[0449] Step 7: Under nitrogen protection and in an ice bath, a solution of 2,5-dioxopyrrolidone-1-yl-2-azidoacetate (compound 3-5, 236.1 mg, crude product) in tetrahydrofuran (2 mL) was added dropwise to a solution of compound 3-9 (500 mg, 0.92 mmol, 1.0 eq) and N,N-diisopropylethylamine (296.1 mg, 2.29 mmol, 2.5 eq) in tetrahydrofuran (5 mL). After the addition was complete, the temperature was raised to 21 °C and the reaction was allowed to proceed for 16 hours. The reaction was monitored by TLC until complete. Water (20 mL) was added to the reaction mixture, and the mixture was extracted with dichloromethane (20 mL × 3). The organic phase was washed with saturated NaCl (10 mL × 2). The aqueous and saturated NaCl phases were combined, and the mixture was extracted again with ethyl acetate (10 mL × 2). The combined dichloromethane and ethyl acetate organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. Normal-phase column chromatography (silica gel, mobile phase A: dichloromethane, mobile phase B: methanol, elution gradient: 0% B to 4% B) was performed, and the eluent was concentrated under reduced pressure to give 2-azido-N-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-2-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-hydroxytetrahydrofuran-3-yl)acetamide (compound 3-10, 502 mg, yield: 87%).

[0450] 1 H NMR (400MHz, DMSO-d6) δ11.36(s,1H),8.25(d,J=8.5Hz,1H),7.65(d,J=8.1Hz,1H),7.37(dd, J=27.1,7.4Hz,4H),7.27(d,J=8.4Hz,5H),6.91(d,J=8.6Hz,4H),5.90(d,J=8.0Hz,1H),5.84 (d,J=4.7Hz,1H),5.43(d,J=8.0Hz,1H),4.67(td,J=8.2,5.7Hz,1H),4.23–4.15(m,1H),4.03 (d,J=3.7Hz,1H),3.91(s,2H),3.74(s,6H),3.32–3.26(m,1H),3.18(dd,J=10.6,3.2Hz,1H).

[0451] Step 8: Under nitrogen protection, dissolve compound 3-10 (300 mg, 0.48 mmol, 1.0 eq), octadecane-1-yne (compound 3-2, 179 mg, 0.72 mmol, 2.0 eq), CuSO4 (23 mg, 0.14 mmol, 0.3 eq), and sodium vitamin C (142 mg, 0.72 mmol, 1.5 eq) in a mixed solution of tetrahydrofuran / tert-butanol / water (3 / 1 / 1, 5 mL), and react at 21°C for 3 hours under nitrogen protection. The reaction was monitored by TLC until it was complete. NaHCO3 (10 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed successively with saturated sodium chloride aqueous solution (20 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was subjected to normal phase column chromatography (silica gel, mobile phase A: dichloromethane (0.1% triethylamine), mobile phase B: methanol, elution gradient: 0% B to 4% B). The eluent was concentrated under reduced pressure to obtain N-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-2-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-hydroxytetrahydrofuran-3-yl)-2-(4-hexadecyl-1H-1,2,3-triazol-1-yl)acetamide (compound 3-11, 400 mg, yield: 87%).

[0452] Step 9: Pretreatment: The substrate compound 3-11 (380 mg, 0.42 mol, 1.0 eq) was azeotropically dehydrated with toluene (3.0 ml × 2).

[0453] Under nitrogen protection, 1H-tetrazole (25 mg, 0.35 mmol, 1.8 eq) was dissolved in anhydrous dichloromethane (1.2 mL). Under nitrogen protection, bis(diisopropylamino)(2-cyanoethoxy)phosphine (108 mg, 0.35 mmol, 1.8 eq) was added, and the mixture was stirred at 30 °C for 1 hour. A dichloromethane solution (1 mL) of the pretreated compound 3-11 and N,N-diisopropylethylamine (50 mg, 0.39 mmol, 2.0 eq) was added to the reaction mixture. The reaction was continued at 25 °C under nitrogen protection for 2 hours. The reaction was monitored by TLC until it was complete. The mixture was then directly subjected to normal-phase column chromatography (silica gel, mobile phase A: dichloromethane (0.1% triethylamine), mobile phase B: methanol / acetone (1 / 1), elution gradient: 0% B to 3% B). The eluent was concentrated under reduced pressure to give (2R,3S,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-(2-(4-hexadecyl-1H-1,2,3-triazol-1-yl)acetamido)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphonamide (compound N17U, 188 mg, yield: 90%).

[0454] MS:m / z 1079.6188[M+H] + .

[0455] 1 H NMR (400MHz, DMSO-d6) δ11.47(s,1H),8.72(dd,J=15.0,8.5Hz,1H),7.76–7.64(m,2H),7.53–7.08(m,10H),6.94–6.81(m ,4H),6.01(d,J=7.9Hz,1H),5.43(d,J=8.1Hz,1H),5.23–5.00(m,2H),4.81(dq,J=40.7,7.8Hz,1H),4.23(d,J=41.1Hz,1H ),3.73(s,8H),3.53(dt,J=22.9,8.6Hz,2H),3.31–3.11(m,2H),2.82–2.75(m,1H),2.68(d,J=6.5Hz,1H),2.60(td,J=7.7 ,2.9Hz,2H),1.57(s,2H),1.23(s,26H),1.12(dd,J=6.8,3.5Hz,6H),1.00(dd,J=40.2,6.7Hz,6H),0.85(t,J=6.6Hz,3H).

[0456] 31P NMR(162MHz,DMSO-d6)δ149.55,147.07.

[0457] 5.1.4 Synthesis of compound N14U

[0458] Step 1: Under nitrogen protection at 21°C, dissolve lithium ethylenediamine complex (3.05 g, 29.8 mmol, 3.25 eq) in dimethyl sulfoxide (10 mL), add 1-bromohexadecane (compound 7-1, 2.8 g, 9.17 mmol, 1.0 eq) under nitrogen protection, react at 21°C for 2.5 h, then increase to 66°C and react for 0.5 h. The reaction was monitored by TLC until complete. The pH of the reaction solution was adjusted to 1 with 1M HCl aqueous solution. Saturated sodium chloride aqueous solution (10 mL) and ethyl acetate (10 mL × 3) were added to the reaction solution for extraction. The organic phase was washed twice with 1M HCl aqueous solution and twice with saturated sodium chloride aqueous solution. After drying with anhydrous sodium sulfate, the mixture was filtered and the residue was obtained by vacuum distillation. The residue was directly subjected to normal phase column chromatography (silica gel, mobile phase A: petroleum ether, mobile phase B: ethyl acetate, elution gradient: 0% B). The eluent was concentrated under reduced pressure to give octadecane-1-yne (compound 7-2, 2.3 g, yield: 98%).

[0459] 1 H NMR (400MHz, CDCl3) δ2.18(td,J=7.1,2.6Hz,2H),1.93(t,J=2.6Hz,1H),1.52(p,J=7.0Hz,2H),1.44–1.34(m,2H),1.26(s,24H),0.88(t,J=6.8Hz,3H).

[0460] Step 2: At 0°C, 2,2'-dehydro-uridine (compound 7-3, 5.00 g, 22.1 mmol, 1.0 eq) was dissolved in anhydrous pyridine (50 mL). 4,4'-bismethoxytriphenylmethylchloromethane (DMTrCl, 4.12 g, 12.2 mmol, 0.55 eq) was added in portions, and the temperature was restored to 21°C, and the reaction continued for 1 hour. After 1 hour, the reaction temperature was lowered to 0°C, and DMTrCl (4.12 g, 12.2 mmol, 0.55 eq) was added in portions to the reaction solution. The temperature was then restored to 21°C, and the reaction continued for 1 hour. The reaction was monitored by TLC until completion. Water (50 mL) was added to the reaction solution, and the mixture was extracted with DCM (20 mL × 3). The combined organic phases were washed successively with saturated sodium chloride aqueous solution (20 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. Purified by normal column chromatography (silica gel, mobile phase A: dichloromethane, mobile phase B: methanol, elution gradient: 7% B to 15% B), the eluent was collected, concentrated under reduced pressure, and dried under vacuum to give (2R,3R,3aS,9aR)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-hydroxy-2,3,3a,9a-tetrahydro-6H-furano[2',3':4,5]oxazolo[3,2-a]pyrimidin-6-one (compound 7-4, 9.1 g, yield: 78%).

[0461] 1 H NMR(400MHz,DMSO-d6)δ7.95(d,J=7.4Hz,1H),7.35–7.19(m,5H),7.17–7.12(m,4H) ,6.87–6.81(m,4H),6.33(d,J=5.6Hz,1H),5.96(d,J=4.5Hz,1H),5.88(d,J=7.4Hz,1 H),5.21(dd,J=5.7,1.2Hz,1H),4.36–4.27(m,1H),4.22(ddd,J=7.3,4.4,2.8Hz,1H ), 3.73 (d, J = 1.6Hz, 6H), 2.95 (dd, J = 10.3, 4.4Hz, 1H), 2.82 (dd, J = 10.3, 7.3Hz, 1H).

[0462] Step 3: At 21°C under nitrogen protection, lithium fluoride (353.3 mg, 13.62 mmol, 1.8 eq) was dissolved in N,N-dimethylformamide (27 mL). After heating to 110°C in an oil bath, N,N,N',N'-tetramethylethylenediamine (27 mL) and azidetrimethylsilane (1.57 g, 13.62 mmol, 1.8 eq) were added sequentially. After reacting for 1 hour under nitrogen protection at 110°C, a solution of N,N-dimethylformamide containing 4.0 g, 7.57 mmol, 1.0 eq of the starting compound 7-4 (7 mL) was added to the reaction solution. The reaction was carried out at 105°C under nitrogen protection for 16 hours. TLC monitoring showed that the reactants had largely reacted. After the reaction was brought back to room temperature, water (20 mL) was added to the reaction solution, and the mixture was extracted with EA (30 mL × 3). The organic phase was collected, washed with saturated sodium chloride aqueous solution (20 mL × 2), dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure to obtain the crude product. The crude product was purified by normal column chromatography (silica gel, mobile phase A: petroleum ether, mobile phase B: ethyl acetate, elution gradient: 55% B to 60% B). The eluent was collected, concentrated under reduced pressure, and dried under vacuum to give 1-((2R,3R,4S,5R)-3-azido-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)pyrimidin-2,4(1H,3H)-dione (compound 7-5, 1.42 g, yield: 33%).

[0463] 1 H NMR(400MHz,DMSO-d6)δ11.44(s,1H),7.69(d,J=8.1Hz,1H),7.40–7.29(m,4H ),7.24(dq,J=7.5,2.7Hz,5H),6.90(dd,J=8.8,1.3Hz,4H),5.99(d,J=5.9Hz,1 H),5.79–5.71(m,1H),5.36(d,J=8.1Hz,1H),4.42(q,J=6.1Hz,1H),4.26(dd,J =5.8,3.8Hz,1H),3.96(dt,J=7.4,3.8Hz,1H),3.74(s,6H),3.32–3.22(m,2H).

[0464] Step 4: Under nitrogen protection, compound 7-5 (500 mg, 0.88 mmol), octadecane-1-yne (compound 7-2, 438 mg, 1.75 mmol, 2.0 eq), CuSO4 (41.9 mg, 0.26 mmol, 0.3 eq) and sodium vitamin C (260.0 mg, 1.31 mmol, 1.5 eq) were dissolved in a tetrahydrofuran / tert-butanol / water (3 / 1 / 1, 5 mL) mixture and reacted at 21 °C for 5 hours under nitrogen protection. The reaction was monitored by TLC until it was complete. NaHCO3 (10 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed successively with saturated sodium chloride aqueous solution (20 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was subjected to normal phase column chromatography (silica gel, mobile phase A: dichloromethane, mobile phase B: methanol, elution gradient: 0% B to 2% B). The eluent was concentrated under reduced pressure to obtain 1-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-(4-hexadecyl-1H-1,2,3-triazol-1-yl)-4-hydroxytetrahydrofuran-2-yl)pyrimidin-2,4(1H,3H)-dione (compound 7-6, 582 mg, yield: 83%).

[0465] 1 H NMR(400MHz, DMSO-d6)δ11.41(d,J=2.1Hz,1H),7.89–7.78(m,2H),7.47–7.31(m,4H),7.30–7.12(m,5H) ),6.99–6.86(m,4H),6.40(d,J=4.8Hz,1H),5.75(d,J=5.6Hz,1H),5.43(ddd,J=11.5,7.4,3.4Hz,2H), 4.49(q,J=6.3Hz,1H),4.22(td,J=5.6,2.9Hz,1H),3.74(s,6H),3.35(dd,J=10.8,5.4Hz,1H),3.29(dd ,J=10.5,2.8Hz,1H),2.61(t,J=7.6Hz,2H),1.57(q,J=7.3Hz,2H),1.23(s,26H),0.85(t,J=6.6Hz,3H).

[0466] Step 5: Pretreatment: Substrate compound 7-6 (380 mg, 0.42 mol, 1.0 eq) was azeotropically dehydrated using toluene (3.0 mL × 2). Under nitrogen protection, 1H-tetrazole (47 mg, 0.66 mmol, 1.8 eq) was dissolved in anhydrous dichloromethane (1.2 mL). Under nitrogen protection, bis(diisopropylamino)(2-cyanoethoxy)phosphine (204 mg, 0.66 mmol, 1.8 eq) was added, and the mixture was stirred at 30 °C for 1 hour. A dichloromethane solution (3 mL) of the pretreated compound 7-6 and N,N-diisopropylethylamine (94 mg, 0.73 mmol, 2.0 eq) was added to the reaction mixture. The reaction was continued at 25 °C under nitrogen protection for 2 hours. The reaction was monitored by TLC until completion, and then directly subjected to normal-phase column chromatography (silica gel, mobile phase A: dichloromethane (0.1% triethylamine), mobile phase B: methanol / acetone (1 / 1), elution gradient: 0% B to 1% B). The eluent was concentrated under reduced pressure to give (2R,3S,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-(4-hexadecyl-1H-1,2,3-triazol-1-yl)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphonamide (compound N14U, 236 mg, yield: 82%).

[0467] MS: m / z 1022.59 [M+H] + .

[0468] 1 H NMR (400MHz, DMSO-d6) δ11.44(s,1H),7.87(dd,J=21.5,7.3Hz,2H),7.47–7.31(m,4H),7.29–7.20(m,5H),6. 89(ddd,J=8.4,5.2,2.7Hz,4H),6.38(t,J=4.4Hz,1H),5.70–5.49(m,1H),5.49–5.37(m,1H),4.41(d,J=18.7 Hz,1H),3.74(d,J=2.9Hz,6H),3.66–3.33(m,4H),3.16(s,2H),2.79(t,J=5.8Hz,1H),2.59(dt,J=14.9,7.6H z,3H),1.58(d,J=8.3Hz,2H),1.23(s,26H),0.99(d,J=7.7Hz,5H),0.87–0.81(m,7H),0.74(d,J=6.7Hz,3H). 31 P NMR(162MHz,DMSO-d6)δ150.04,148.28.

[0469] 5.1.5 Synthesis of compound N14C

[0470] Step 1: 1-((2R,3R,4S,5R)-3-azido-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)pyrimidin-2,4(1H,3H)-dione (compound 7-5, 1.3 g, 2.27 mmol, 1.0 eq) was dissolved in DMF (7 mL), and imidazole (465 mg, 6.82 mmol, 3.0 eq) and TBSCl (514 mg, 3.41 mmol, 1.5 eq) were added. The mixture was stirred at 30 °C for 16 hours. Water (20 mL) and EA (30 mL) were added to the reaction solution. The aqueous phase was extracted with EA (2 x 30 mL). The combined organic phases were washed with saturated NaCl (20 mL x 2), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by normal phase column chromatography (0–85% EA in PE). The eluent was concentrated under reduced pressure to obtain 1-((2R,3R,4S,5R)-3-azido-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-2-yl)pyrimidin-2,4(1H,3H)-dione (compound 8-1, 1.23 g, yield: 78%).

[0471] 1H NMR(400MHz,DMSO-d6)δ11.46(s,1H),7.82(d,J=8.1Hz,1H),7.39–7.19(m,9H),6.9 4–6.86(m,4H),5.77(s,1H),5.35(d,J=8.1Hz,1H),4.52(dd,J=7.1,5.6Hz,1H),4.3 5(dd,J=5.7,3.1Hz,1H),3.92(dt,J=7.2,3.8Hz,1H),3.75(s,6H),3.43(dd,J=11.1 ,2.7Hz,1H),3.16(dd,J=11.0,4.3Hz,1H),0.75(s,9H),0.06(s,3H),-0.07(s,3H).

[0472] Step 2: At 0°C, compound 8-1 (1.3 g, 1.90 mmol, 1 eq) was dissolved in ACN (10 mL), followed by the addition of DMAP (463 mg, 3.79 mmol, 2 eq), TEA (384 mg, 3.79 mmol, 2 eq), and TPSCl (464 mg, 3.79 mmol, 1.5 eq). After the addition was complete, the mixture was stirred continuously at 30°C for 3 hours. After the starting material was completely consumed by TLC monitoring, NH3·H2O (6 mL, 26–28% purity) was added, and the mixture was stirred continuously at room temperature for 17 hours. TLC showed the reaction was complete. Water (20 mL) and ethyl acetate (30 mL) were added to the reaction solution for extraction. The aqueous phase was extracted with ethyl acetate (2 x 30 mL). The combined organic phases were washed with saturated saline solution (2 x 20 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by normal phase column chromatography (0–10% MeOH in DCM). The eluent was concentrated under reduced pressure to obtain 4-amino-1-((2R,3R,4S,5R)-3-azido-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one (compound 8-2, 1.2 g).

[0473] 1 H NMR(400MHz,DMSO-d6)δ7.86(d,J=7.5Hz,1H),7.42–7.19(m,12H),6.95–6.84(m,4 H),5.82(d,J=3.0Hz,1H),5.55(d,J=7.5Hz,1H),4.51(dd,J=7.2,5.5Hz,1H),4.17( dd,J=5.5,3.0Hz,1H),3.92(dt,J=6.8,3.3Hz,1H),3.75(s,6H),3.45(dd,J=11.1, 2.7Hz,1H),3.13(dd,J=11.0,3.9Hz,1H),0.73(s,10H),0.05(s,3H),-0.09(s,3H).

[0474] Step 3: Dissolve compound 8-2 (1.2 g, 1.75 mmol, 1.0 eq) in 5 mL of anhydrous DCM, add DIPEA (566 mg, 4.38 mmol, 2.5 eq) and BzCl (283 mg, 2.01 mmol, 1.2 eq), and react for 16 hours under nitrogen protection. TLC showed the reaction was complete. Water (20 mL) and DCM (30 mL) were added to the reaction solution. The aqueous phase was extracted with DCM (2 x 30 mL). The combined organic phases were washed with saturated NaCl (20 mL x 2), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by normal-phase column chromatography (0–5% MeOH in DCM). The eluent was concentrated under reduced pressure to obtain N-(1-((2R,3R,4S,5R)-3-azido-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide (compound 8-3, 982 mg, yield: 75%). 1H NMR(400MHz, DMSO-d6)δ11.35(s,1H),8.49(d,J=7.5Hz,1H),8.05–7.95(m,2H),7.71–7.58(m,1H ),7.52(t,J=7.6Hz,2H),7.41–7.22(m,10H),6.92(d,J=8.5Hz,4H),5.80(d,J=1.3Hz,1H),4.63(d d,J=8.6,5.3Hz,1H),4.40(dd,J=5.3,1.4Hz,1H),4.02(dt,J=8.6,2.8Hz,1H),3.75(d,J=1.3Hz,6 H),3.61(d,J=10.7Hz,1H),3.20(dd,J=11.3,3.2Hz,1H),0.71(s,9H),0.06(s,3H),-0.10(s,3H).

[0475] Step 4: Dissolve compound 8-3 (980 mg, 1.24 mmol, 1.0 eq) in anhydrous THF (5 mL), add TBAF (1.86 mL, 1.86 mmol, 1 M in THF, 1.5 eq), and react for 3 h under nitrogen protection. Water (20 mL) and EA (30 mL) were added to the reaction solution. The aqueous phase was extracted with EA (2 x 30 mL). The combined organic phases were washed with saturated NaCl (20 mL x 2). The mixture was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by normal phase column chromatography (0-5% MeOH in DCM). The eluent was concentrated under reduced pressure to obtain N-(1-((2R,3R,4S,5R)-3-azido-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide (compound 8-4, 832 mg, yield: 95%).

[0476] 1 H NMR(400MHz,DMSO-d6)δ11.33(s,1H),8.30(d,J=7.5Hz,1H),8.07–7.95(m,2H),7 .68–7.60(m,1H),7.52(t,J=7.6Hz,2H),7.44–7.14(m,11H),6.92(d,J=8.8Hz,4H) ,5.99(d,J=6.0Hz,1H),5.77(d,J=1.8Hz,1H),4.54(dt,J=8.4,5.8Hz,1H),4.32(d d,J=5.5,1.9Hz,1H),4.05(dt,J=8.4,3.0Hz,1H),3.76(s,6H),3.41–3.34(m,2H).

[0477] Step 5: Dissolve compound 8-4 (400 mg, 0.60 mmol, 1.0 eq), octadecane-1-yne (compound 7-2, 223 mg, 0.89 mmol, 1.5 eq), CuSO4 (29 mg, 0.18 mmol, 0.3 eq), and sodium vitamin C (176 mg, 0.89 mmol, 1.5 eq) in 5 mL of mixed solution (THF:H2O = 3:1) and react for 16 hours under nitrogen protection. Water (20 mL) and EA (30 mL) were added to the reaction solution. The aqueous phase was extracted with EA (2 x 30 mL). The combined organic phases were washed with saturated NaCl (20 mL x 2), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by normal phase column chromatography (0–5% MeOH in DCM). The eluent was concentrated under reduced pressure to obtain N-(1-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-(4-hexadecyl-1H-1,2,3-triazol-1-yl)-4-hydroxytetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide (compound 8-5, 0.462 g, yield: 86%).

[0478] 1 H NMR (400MHz, DMSO-d6) δ11.33(s,1H),8.40(d,J=7.6Hz,1H),8.00(d,J=7.6Hz,2H),7.86(s,1H),7. 63(t,J=7.4Hz,1H),7.52(t,J=7.6Hz,2H),7.46–7.21(m,10H),6.92(d,J=8.7Hz,4H),6.41(d,J=2.6 Hz,1H),5.75(d,J=5.6Hz,1H),5.35(dd,J=6.7,2.6Hz,1H),4.63(q,J=6.9Hz,1H),4.29(dt,J=7.9, 3.4Hz,1H),3.76(s,6H),2.63(t,J=7.6Hz,2H),1.66–1.53(m,2H),1.23(s,27H),0.88–0.78(m,3H).

[0479] Step 6: Pretreatment: Substrate compound 8-5 (380 mg, 0.42 mol, 1.0 eq) was azeotropically dehydrated using toluene (1.0 mL × 2). Under nitrogen protection, 1H-tetrazole (56 mg, 0.10 mmol, 1.8 eq) was dissolved in anhydrous dichloromethane (1.6 mL). Under nitrogen protection, bis(diisopropylamino)(2-cyanoethoxy)phosphine (242 mg, 0.78 mmol, 2.0 eq) was added, and the mixture was stirred at 30 °C for 1 hour. A dichloromethane solution (2 mL) of the pretreated compound 8-5 and N,N-diisopropylethylamine (168 mg, 1.3 mmol, 3.0 eq) was added to the reaction mixture. The reaction was continued at 25 °C under nitrogen protection for 3 hours. The reaction was monitored by TLC until completion, and then directly subjected to normal-phase column chromatography (silica gel, mobile phase A: dichloromethane (0.1% triethylamine), mobile phase B: methanol / acetone (1 / 1), elution gradient: 0% B to 1% B). The eluent was concentrated under reduced pressure to give (2R,3S,4R,5R)-5-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(4-hexadecyl-1H-1,2,3-triazol-1-yl)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphonamide (compound N14C, 384 mg, yield: 72%).

[0480] MS: m / z 1125.63 [M+H] +

[0481] 1H NMR (400MHz, DMSO-d6) δ11.33(d,J=5.1Hz,1H),8.45(dd,J=28.6,7.6Hz,1H),8.00(d,J=7.6Hz,2H),7.88(d,J=13.9Hz,1H),7.63(t,J=7.9 Hz,1H),7.52(t,J=7.7Hz,2H),7.47–7.41(m,2H),7.39–7.16(m,8H),6.96–6.85(m,4H),6.39(dd,J=6.1,2.3Hz,1H),4.94–4.76(m,1H),4. 57–4.41(m,1H),3.75(d,J=1.8Hz,7H),3.64–3.34(m,4H),3.29(d,J=6.8Hz,1H),3.16(dt,J=10.1,6.5Hz,1H),2.86–2.80(m,1H),2.61(dt ,J=12.2,8.1Hz,2H),1.59(t,J=9.4Hz,2H),1.23(s,26H),1.01(dd,J=12.0,6.7Hz,6H),0.84(dt,J=7.0,3.4Hz,7H),0.73(d,J=6.7Hz,3H).

[0482] 31 P NMR(162MHz,DMSO-d6)δ149.46,148.82.

[0483] 5.1.6 Synthesis of siRNA-lipid conjugates

[0484] siRNA-lipid conjugates can be synthesized using methods commonly known in the art. This invention synthesizes the sense and antisense strands of siRNA-lipid conjugates using solid-phase phosphorous amide technology.

[0485] 1) Solid-phase synthesis and purification of single-stranded sequences

[0486] Depending on the scale, a solid support made of glass with controllable porosity (CPG.1000A) was used. All DNA monomers and 2'-modified RNA phosphoramide monomers were purchased from qualified suppliers of Chengdu Pioneer or Besun. Various special monomers: N15U, N15C, and N17U (Table A) were used on the positive strand and synthesized according to the following methods.

[0487] Specifically, the 2'-O-methylphosphoramide used below includes the following: (5'-O-dimethoxytriphenylmethyl-N 6 -(benzoyl)-2'-O-methyl-adenosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphamide, 5'-O-dimethoxy-triphenylmethyl-N4 -(acetyl)-2'-O-methyl-cytidine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphamide, (5'-O-dimethoxytriphenylmethyl-N 2 -(isobutyryl)-2'-O-methyl-guanosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphamide, 5'-O-dimethoxytriphenylmethyl-2'-O-methyl-uridine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphamide, 5'-dimethoxytriphenylmethyl-2'-O-methyl-inosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphamide. 2'-deoxy-2'-fluorophosphamide, 2'-O-propargylphosphamide, and 2'-O-methylphosphamide carry the same protecting group as 2'-O-methylphosphamide. Reverse debasing (3'-O-dimethoxytriphenylmethyl-2'-deoxyribose-5'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphamide, 5'-(4,4'-dimethoxytriphenylmethyl)-N 6 -(benzoyl)-2',3'-open-ring-adenosine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramide, 5'-(4,4'-dimethoxytriphenylmethyl)-N-acetyl-2',3'-open-ring-cytosine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramide, 5'-(4,4'- (dimethoxytriphenylmethyl)-N-isobutyryl-2',3'-open-ring-guanosine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramide, and 5'-(4,4'-dimethoxytriphenylmethyl)-2',3'-open-ring-uridine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramide, and deoxyphosphoramide. Special monomers used are compounds N15U, N15C, and N17U (Table A) phosphoramide monomers; the protecting groups on these monomers can be removed under alkaline conditions.

[0488] To introduce thiophosphate bonds, DDTT sulfidation reagent (50 mM pyridine solution) was used. For unreacted active groups, a mixture of acetic anhydride, N-methylimidazole, pyridine, and acetonitrile (N-methylimidazole / acetonitrile ratio 1:4:acetic anhydride / pyridine / acetonitrile ratio 2:3:5) was used as a capping agent.

[0489] Standard coupling and oxidation: All phosphoramide monomers or special monomers were dissolved in anhydrous dichloromethane, anhydrous acetonitrile, or anhydrous dichloromethane / acetonitrile mixture (50 mM), dried using molecular sieves (3A), and 5-benzylthio-1H-tetrazole (BTT, 250 mM in acetonitrile) or 5-ethylthio-1H-tetrazole (ETT, 250 mM in acetonitrile) was added as an activating agent solution. The coupling time for each step of sequence synthesis was 12 minutes.

[0490] Deprotection and pyrolysis: After solid-phase synthesis is completed, the dried synthesis support is deprotected by cutting with ammonium hydroxide (25% aqueous solution) at 55°C.

[0491] Purification: The carrier was filtered, concentrated, and then purified by RP-HPLC. The purity was monitored by LC-MS, and finally quantified by UV absorbance at 260 nm for further testing.

[0492] 2) Annealing

[0493] Complementary strands (SS strands (sense strands) and antisense strands) of acceptable purity were mixed in an equimolar ratio in 1×PBS (phosphate-buffered saline, 1×, Corning, Cellgro), and the solution was placed in a metal bath at 90°C for 5 minutes, then slowly cooled to room temperature. Samples were analyzed by SEC-HPLC, and the double-strand annealing purity was calculated using the integral value of the 260 nm UV absorption peak area.

[0494] Following the general procedure for oligonucleotide synthesis described in this article, Amyloid Precursor Protein (APP) siRNA agents (hereinafter also referred to as siRNA-lipid conjugates) were prepared using certain nucleotide-lipid conjugate monomers provided in this article (Table A) and commercially available 2'-modified RNA phosphorylamide monomers as raw materials.

[0495] Sequence information for exemplary siRNA-lipid conjugates is shown in Table 1C.

[0496] 5.2 IC50 test of siRNA-lipid conjugates in SK-N-MC and N2a cells

[0497] The experimental protocol for IC50 testing of siRNA-lipid conjugates coupled with different delivery systems on SK-N-MC cells and N2a cells is as described in Example 2.

[0498] IC50 data are shown in Table 11. The results show that the exemplary siRNA-lipid conjugates in the table below can all reduce the expression of target genes in a concentration-dependent manner.

[0499] Table 11: IC50 of siRNA-lipid conjugates in SK-N-MC and N2a cells

[0500] 5.3 Knockdown test of siRNA-lipid conjugates on hAPP and mAPP in 5XFAD 3'UTR mice

[0501] The efficacy of the selected RNAi agent was studied in B-Tg(5XFAD)mice(v5) mice (manufacturer: Biocytogen (Beijing) Pharmaceutical Technology Co., Ltd.). Six mice in each group received an intraventricular (ICV) injection of 120 μg of the RNAi agent or PBS (phosphate-buffered saline). On day 28 post-injection, the mice underwent cardiac perfusion, and brain tissue was harvested and the hippocampus isolated. The isolated hippocampus was weighed and, following the instructions of the RNA extraction kit (manufacturer: Takara, catalog number: 9767), an appropriate amount of lysis buffer was added for tissue homogenization and subsequent RNA extraction. The expression levels of human APP (hAPP) and mouse APP (mAPP) mRNA in the hippocampus were then measured and analyzed by quantitative PCR (qPCR, see Example 2).

[0502] Table 12 shows that the selected siRNA-lipid conjugates can effectively reduce the expression of human APP (hAPP) or mouse APP (mAPP) target genes in B-Tg(5XFAD)mice(v5) mice.

[0503] Table 12A: Knockdown results of siRNA-lipid conjugates on hAPP

[0504] Table 12B: Knockdown results of siRNA-lipid conjugates on mAPP

[0505] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The entire scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A small interfering RNA (siRNA) for inhibiting APP gene expression, said siRNA comprising a sense strand and an antisense strand, wherein, The antisense strand comprises at least 15 consecutive nucleotides that are approximately 4 (e.g., 0, 1, 2, 3, or 4) nucleotides similar to the nucleotide sequence shown in any one of SEQ ID NO:1 to SEQ ID NO:200, SEQ ID NO:418 to SEQ ID NO:434, and the sense strand is at least partially complementary to the antisense strand; Preferably, the antisense strand comprises at least 17 consecutive nucleotides having a nucleotide sequence that is approximately 4 (e.g., 0, 1, 2, 3, or 4) nucleotides similar to the nucleotide sequence shown in any one of SEQ ID NO:1 to SEQ ID NO:200, SEQ ID NO:418 to SEQ ID NO:434; Preferably, the antisense strand comprises at least 17 consecutive nucleotides that differ from the nucleotide sequences shown in any one of SEQ ID NO:1 to SEQ ID NO:200, SEQ ID NO:418 to SEQ ID NO:434 by 0, 1, or 2 nucleotides.

2. The siRNA according to claim 1, wherein, The positive strand comprises at least 15 consecutive nucleotides having a nucleotide sequence that is approximately 4 (e.g., 0, 1, 2, 3 or 4) nucleotides similar to the nucleotide sequence shown in any one of SEQ ID NO:201 to SEQ ID NO:400, SEQ ID NO:401 to SEQ ID NO:417; Preferably, the positive strand comprises at least 17 consecutive nucleotides having a nucleotide sequence that is approximately 4 (e.g., 0, 1, 2, 3, or 4) nucleotides similar to the nucleotide sequence shown in any one of SEQ ID NO:201 to SEQ ID NO:400, SEQ ID NO:401 to SEQ ID NO:417; Preferably, the positive strand comprises at least 17 consecutive nucleotides that differ from the nucleotide sequences shown in any one of SEQ ID NO:201 to SEQ ID NO:400, SEQ ID NO:401 to SEQ ID NO:417 by 0, 1, or 2 nucleotides; Preferably, the sense strand has a region within the 17 consecutive nucleotides that is at least 85% complementary to the antisense strand.

3. The siRNA according to claim 1 or 2, wherein, The siRNA contains a blunt end and / or a protruding end of 1 to 5 nucleotides; Preferably, the siRNA contains one or two nucleotide overhangs; Preferably, the protruding end is present at the 5' end and / or 3' end of the antisense chain and / or the justice chain; Preferably, the 3' end of the antisense strand of the siRNA contains a 2- or 5-nucleotide overhang.

4. The siRNA according to any one of claims 1-3, wherein, The antisense strand and the sense strand are each independently 17 to 30 nucleotides in length; preferably, the antisense strand is 19 to 27 nucleotides in length; preferably, the sense strand is 17 to 25 nucleotides in length.

5. The siRNA according to any one of claims 1-4, wherein, The antisense strand is 21-23 nucleotides long, and the sense strand is 18-21 nucleotides long.

6. The siRNA according to any one of claims 1-5, wherein, The sense strand and the antisense strand have a mismatch of no more than 6 nucleotides (e.g., 0, 1, 2, 3, 4, 5 or 6).

7. The siRNA according to any one of claims 1-6, wherein, The siRNA's sense and antisense strand sequences comprise the sense and antisense strand sequences of any double-stranded form selected from SJ-0001 to SJ-0200 and SJ-0201 to SJ-0223 as described in Tables 1A and 1B.

8. The siRNA according to any one of claims 1-7, wherein, The siRNA contains at least one modifying nucleotide.

9. The siRNA according to claim 8, wherein, All nucleotides in the sense and / or antisense strands of the siRNA are modified nucleotides or nucleotide analogs.

10. The siRNA according to claim 9, wherein, The modified nucleotide or nucleotide analogue is selected from 2'-methoxynucleotide, 2'-fluoronucleotide, 2'-deoxynucleotide, 2',3'-cleaved nucleotide analogue, 2'-fluoroarabinonucleotide, 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 group, nucleotide containing a 5'-phosphate mimic, diol-modified nucleotide, deoxyxanthine nucleotide, 2'-O-methoxyethyl-modified nucleotide, 2'-methoxy and 2'-fluoro-modified nucleotide, debased nucleotide, morpholinonucleotide, locked nucleotide, unlocked nucleotide, or glycerol nucleotide.

11. The siRNA according to any one of claims 8-10, wherein, The sense and / or antisense strands of the siRNA contain modified internucleotide links; Preferably, the 5' end and 3' end of the sense chain each independently contain one or two thiophosphate groups; and / or the 5' end and 3' end of the antisense chain each independently contain one or two or three thiophosphate groups.

12. The siRNA according to any one of claims 8-11, wherein, The nucleotides in the sense strand of the siRNA are selected from at least two of 2'-methoxynucleotides, 2'-fluoronucleotides, and 2'-deoxynucleotides; and / or, the nucleotides in the antisense strand of the siRNA are selected from at least two of 2'-methoxynucleotides, 2'-fluoronucleotides, and 2'-deoxynucleotides.

13. The siRNA according to any one of claims 8-12, wherein, The antisense strand of the siRNA contains a modification pattern selected from any of the following: (1)AS(5'-3'):Nms Nfs Nm Nm Nm Nf Nm Nm Nm Nm Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm; (2)AS(5'-3'):Nms Nfs Nm Nm Nm Nf Nm Nf Nf Nm Nm Nf Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm; (3)AS(5'-3'):Nms Nfs Nm Nm Nm Nf Nm Nf Nf Nm Nm Nf Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm; (4)AS(5’-3’):Nms Nfs Nm Nm Nm Nm Nm Nm Nf Nm Nm Nf Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm; (5)AS(5’-3’):Nms Nfs Nm Nf Nm Nm Nm Nm Nm Nm Nm Nf Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm; (6)AS(5’-3’):Nms Nfs Nm Nm Nm Nf Nm Nf Nf Nf Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm; (7)AS(5’-3’):Nms Nfs Nm Nm Nm Nf Nm Nf Nf Nf Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm; (8)AS(5’-3’):Nms Nfs Nm Nf Nm Nf Nm Nf Nm Nf Nm Nm Nm Nf Nm Nf Nm Nf Nm Nf Nms Nms Nm; (9)AS(5’-3’):Nms Nfs Nm Nm Nm Nf Nm Nm Nm Nm Nm Nm Nm Nf Nm Nf Nm Nf Nm Nm Nms Nms Nm; (10)AS(5’-3’):Nms Nfs Nm Nm Nm Nm Nm Nf Nf Nm Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm; (11)AS(5’-3’):Nms Nfs Nm Nm Nm Nm Nm Nf Nf Nm Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm; (12)AS(5’-3’):Nms Nfs Nm Nm Nm Nm Nm Nf Nf Nm Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm; (13)AS(5’-3’):Nms Nfs Nm Nm Nm Nm Nm Nf Nf Nm Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm; (14)AS(5’-3’):Nms Nfs Nm Nm Nm Nm Nm Nf Nf Nm Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm; (15)AS(5’-3’):Nms Nfs Nm Nm Nm Nm Nm Nf Nf Nm Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm; (16)AS(5’-3’):Nms Nfs Nm Nm Nm Nm Nm Nf Nf Nm Nm Nm Nm Nf Nm Nf Nm Nf Nm Nm Nms Nms Nm; (17)AS(5’-3’):Nms Nfs Nms Nm Nm Nm Nm Nf Nf Nm Nm Nm Nm Nf Nm Nm Nm Nf Nm Nm Nms Nms Nm; (18)AS(5’-3’):Nms Nfs Nm Nm Nm Nm Nm Nf Nf Nm Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm; (19)AS(5’-3’):Nms Nfs Nm Nm Nm Nm Nm Nf Nf Nm Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm; (20)AS(5’-3’):Nms Nfs Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm; (21)AS(5’-3’):Nms Nfs Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm; (22)AS(5’-3’):Nms Nfs Nm Nm Nm Nf Nm Nm Nm Nm Nm Nf Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm; (23)AS(5’-3’):Nms dNs Nms Nm dN Nm dN Nm Nm Nm Nm Nm Nm Nf Nm Nm Nm Nm Nm Nms Nms Nms Nm; (24)AS(5’-3’):Nms Nfs Nms Nm dN Nm dN Nm Nm Nm Nm Nm Nm dN Nm Nm Nm Nm Nm Nms Nms Nms Nm; (25)AS(5’-3’):(E)-VP Nms Nfs Nm Nm Nm Nm Nm Nm Nf Nm Nm Nf Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm; (26)AS(5’-3’):(E)-VP Nms Nfs Nm Nm Nm Nf Nm Nf Nf Nf Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm; (27)AS(5’-3’):(E)-VP Nms Nfs Nm Nm Nm Nf Nm Nf Nf Nf Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm; (28)AS(5’-3’):(E)-VP Nms Nfs Nm Nf Nm Nf Nm Nf Nm Nf Nm Nm Nm Nf Nm Nf Nm Nf Nm Nf Nms Nms Nm; (29)AS(5’-3’):(E)-VP Nms Nfs Nm Nm Nm Nf Nm Nm Nm Nm Nm Nm Nm Nf Nm Nf Nm Nf Nm Nm Nms Nms Nm; (30)AS(5’-3’):(E)-VP Nms Nfs Nm Nm Nm Nm Nm Nf Nf Nm Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm; (31)AS(5’-3’):(E)-VP Nms Nfs Nm Nm Nm Nm Nm Nf Nf Nm Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm; (32)AS(5’-3’):(E)-VP Nms Nfs Nm Nm Nm Nm Nm Nf Nf Nm Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm; (33)AS(5’-3’):(E)-VP Nms Nfs Nm Nm Nm Nm Nm Nf Nf Nm Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm; (34)AS(5’-3’):(E)-VP Nms Nfs Nm Nm Nm Nm Nm Nf Nf Nm Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm; (35)AS(5’-3’):(E)-VP Nms Nfs Nm Nm Nm Nm Nm Nf Nf Nm Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm; (36)AS(5'-3'):(E)-VP Nms Nfs Nm Nm Nm Nm Nm Nf Nf Nm Nm Nm Nm Nf Nm Nf Nm Nf Nm Nm Nms Nms Nm; (37)AS(5'-3'):(E)-VP Nms Nfs Nms Nm Nm Nm Nm Nf Nf Nm Nm Nm Nm Nf Nm Nm Nm Nf Nm Nm Nms Nms Nm; (38)AS(5'-3'):(E)-VP Nms Nfs Nm Nm Nm Nm Nm Nf Nf Nm Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm; (39)AS(5'-3'):(E)-VP Nms Nfs Nm Nm Nm Nm Nm Nf Nf Nm Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm; (40)AS(5'-3'):(E)-VP Nms Nfs Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm; (41)AS(5'-3'):(E)-VP Nms Nfs Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm; (42)AS(5'-3'):(E)-VP Nms Nfs Nm Nm Nm Nf Nm Nm Nm Nm Nm Nf Nm Nf Nm Nf Nm Nm Nm Nm Nms Nms Nm; (43)AS(5'-3'):(E)-VP Nms dNs Nms Nm dN Nm dN Nm Nm Nm Nm Nm Nm Nf Nm Nm Nm Nm Nm Nms Nms Nms Nm; (44)AS(5'-3'):(E)-VP Nms Nfs Nms Nm dN Nm dN Nm Nm Nm Nm Nm Nm dN Nm Nm Nm Nm Nm Nms Nms Nms Nm; Wherein, Nm is a 2'-methoxy modified nucleotide, Nf is a fluorinated modified nucleotide, dN is a deoxynucleotide, s is a thiophosphate ester linkage, and (E)-VP is a 5'-(E)-vinyl phosphate (VP) modification. And / or, The sense strand of the siRNA contains a modification pattern selected from any of the following: (1)SS(5’-3’):Nms Nms Nm Nm Nm Nm Nf Nm Nf Nf Nf Nm Nm Nm Nm Nm Nm Nm Nm Nm Nm; (2)SS(5’-3’):Nms Nm Nm Nm Nm Nm Nf Nm Nf Nm Nf Nm Nm Nm Nm Nm Nm Nm Nm Nms Nm; (3)SS(5’-3’):Nms Nm Nm Nm Nm Nm Nm Nm Nf Nm Nf Nm Nf Nm Nm Nm Nm Nm Nm Nms Nm; (4)SS(5’-3’):Nms Nm Nm Nm Nm Nm Nm Nm Nf Nm Nf Nm Nf Nm Nm Nm Nm Nm Nm Nms Nm; (5)SS(5’-3’):Nms Nm Nm Nm Nm Nm Nf Nm Nf Nm Nf Nm Nm Nm Nm Nm Nm Nm Nm Nms Nm; (6)SS(5’-3’):Nms Nm Nm Nm Nm Nm Nf Nm Nf Nf Nf Nm Nm Nm Nf Nm Nm Nm Nm Nms Nm; (7)SS(5’-3’):Nms Nm Nm Nm Nm Nm Nf Nm Nf Nf dN Nm Nm Nm Nm Nm Nm Nm Nm Nms Nm; (8)SS(5’-3’):Nms Nm Nm Nm Nm Nm Nf Nm Nf Nf dN Nm Nm Nm Nm Nm Nm Nm Nm Nms Nm; (9)SS(5’-3’):Nms Nm Nm Nm Nm Nm Nf Nm Nf Nf Nf Nm Nf Nm Nm Nm Nm Nm Nm Nms Nm; (10)SS(5’-3’):Nms Nm Nm Nm Nm Nm Nf Nm Nf Nf Nf Nm Nm Nm Nm Nf Nm Nm Nm Nms Nm; (11)SS(5’-3’):Nms Nm Nm Nm Nm Nm Nf Nm Nf Nf Nf Nm Nm Nm Nm Nm Nf Nm Nm Nms Nm; (12)SS(5’-3’):Nms Nm Nm Nm Nm Nm Nf Nm Nf dN Nf Nm Nm Nm Nm Nm Nf Nm Nm Nms Nm; (13) SS(5’-3’): Nms Nm Nm Nm Nm Nm Nf Nm dN Nf Nf Nm Nm Nm Nm Nm Nf Nm Nm Nms Nm; (14) SS(5’-3’): Nms Nm Nm Nm Nm Nm Nf Nm Nf Nf dN Nm Nm Nm Nm Nm Nf Nm Nm Nms Nm; (15) SS(5’-3’): Nms Nm Nm Nm Nm Nm Nm Nm Nf Nf Nf Nm Nm Nm Nm Nm Nf Nm Nm Nms Nm; (16) SS(5’-3’): Nms Nm Nm Nm Nm Nm Nf Nm Nf Nf Nf Nm Nm Nm Nm Nm Nf Nm Nm Nms Nm; (17) SS(5’-3’): Nms Nm Nm Nm Nm Nm Nf Nm Nf Nf Nf Nm Nm Nm Nm Nm Nf Nm Nm Nms Nm; (18) SS(5’-3’): Nms Nm Nm Nm Nm Nm Nf Nm Nf Nf Nf Nm Nm Nm Nm Nf Nf Nm Nm Nms Nm; (19) SS(5’-3’): Nms Nm Nm Nm Nm Nm Nf Nm Nf Nf Nf Nm Nm Nm Nf Nm Nf Nm Nm Nms Nm; (20) SS(5’-3’): Nms Nm Nm Nm Nm Nm Nf Nm Nf Nf Nf Nm Nf Nm Nm Nm Nm Nm Nm Nms Nm; (21) SS(5’-3’): Nms Nm Nm Nm Nm Nm Nf Nm Nf Nf Nf Nm Nm Nm Nf Nm Nm Nm Nm Nms Nm; (22) SS(5’-3’): Nms Nms Nm Nm Nm Nm Nm Nm Nf dN Nf Nm Nm Nm Nm Nm Nf Nm Nms Nms Nm; (23) SS(5’-3’): Nms Nms Nm Nm Nm Nm Nm Nf Nf Nf Nf Nm Nm Nm Nm Nm Nm Nm Nms Nms Nm; (24) SS(5’-3’): Nms Nms Nm Nm Nf Nf Nf Nf Nm Nm Nm Nm Nm Nf Nm Nms Nms Nm; Where, Nm is a 2’-methoxy-modified nucleotide, Nf is a fluoro-modified nucleotide, dN is a deoxynucleotide, and s is a phosphorothioate linkage.

14. The siRNA according to any one of claims 8-13, wherein, The first nucleotide at the 5' end of the antisense strand of the siRNA optionally includes (E)-vinylphosphonate modification.

15. The siRNA according to any one of claims 8-14, wherein, The modification patterns of the sense and antisense strands of the siRNA are selected from any one of the sense and antisense strand modification patterns shown in Table 1D: P0, P01-P20, 073, 078, 080, P03-E, P05-E-P20-E, 073-E, 078-E, 080-E.

16. The siRNA according to any one of claims 8-15, wherein, The sequences of the sense and antisense strands of the siRNA are selected from at least 12 consecutive nucleotides of the sense and antisense strand sequences of any of the modified siRNA duplexes described in Tables 1E and 1F. Preferably, the antisense strand comprises at least 15 consecutive modified nucleotides that are approximately the same as the antisense strand sequence of any of the modified siRNA duplexes described in Tables 1E and 1F by no more than 4 (e.g., 0, 1, 2, 3, or 4) modified nucleotides, and the sense strand is at least partially complementary to the antisense strand. Preferably, the antisense strand comprises at least 17 consecutive nucleotides that differ from the antisense strand sequence of any of the modified siRNA duplexes described in Tables 1E and 1F by 0, 1, or 2 nucleotides; Preferably, the positive strand comprises at least 15 consecutive modifying nucleotides that are approximately the same as the positive strand sequence of any of the modified siRNA duplexes described in Tables 1E and 1F, consisting of no more than 4 (e.g., 0, 1, 2, 3, or 4) modifying nucleotides. Preferably, the sense strand has a region within the 15 consecutive modified nucleotides that is at least 85% complementary to the antisense strand; Preferably, the positive strand comprises at least 17 consecutive modifying nucleotides that are approximately identical to the positive strand sequence of any of the modified siRNA duplexes described in Tables 1E and 1F by no more than two (e.g., 0, 1, or 2) modifying nucleotides. Preferably, the sequences of the sense and antisense strands of the siRNA are selected from the sense and antisense strand sequences of any of the modified siRNA duplexes described in Tables 1E and 1F.

17. A coupling, wherein, The conjugate comprises the siRNA as described in any one of claims 1-16 and a pharmaceutically acceptable targeting molecule.

18. The coupling of claim 17, comprising the structure shown in formula (I-1-1), in, Z 1 Z 2 It does not exist, or each is independently selected from -O-, -S-, -N(R) a )-, -C(=O)-, -OC(=O)-, -C(=O)O-, -C(=O)NR a -、-NR a C(=O)-、-OC(=O)NR a -、-NR a C(=O)O-、-C 1-6 alkylene-, R a Each is independently selected from H and C. 1-6 alkyl; R 1 C 12-32 Alkyl, the C 12-32 The alkyl group is optionally surrounded by one or more elements selected from H, halogens, OH, CN, NO2, COOH, -OC. 1-6 Alkyl, -NHC 1-6 Alkyl and -N(C) 1-6 Alkyl group substitution.

19. The coupling according to claim 17 or 18, wherein, Selected from 20. The coupling according to any one of claims 17-19, wherein, The conjugate comprises one or more nucleotide-lipid conjugate units or pharmaceutically acceptable salts, tautomers, enantiomers, or stereoisomers thereof, wherein the nucleotide-lipid conjugate unit comprises the structure shown in formula (II-1-1). in, Z 1 Z 2 R 1 As defined in claim 18; X is O; Y is O; B can be a natural or non-natural base, or a modified or unmodified base.

21. The coupling of claim 20, wherein, B represents uracil, cytosine, adenine, guanine, uracil with an amino protecting group, cytosine with an amino protecting group, adenine with an amino protecting group, or guanine with an amino protecting group.

22. The coupling according to claim 20 or 21, wherein, B is 23. The coupling compound according to any one of claims 20-22, wherein, The structure described in formula (II-1-1) is selected from 24. The coupling according to any one of claims 20-23, wherein, The structure described in formula (II-1-1) is selected from 25. The coupling according to any one of claims 17-24, wherein, The conjugate is selected from any of the following: (1) LN15U-SJ-0165-P08-E, which contains an antisense strand sequence as shown in SEQ ID NO:446, and a positive strand sequence as shown in SEQ ID NO:435; (2) LN17U-SJ-0165-P08-E, which contains the antisense strand sequence as shown in SEQ ID NO:447, and the positive strand sequence as shown in SEQ ID NO:436; (3) LN14U-SJ-0165-P08-E, which contains the antisense strand sequence as shown in SEQ ID NO:448, and the positive strand sequence as shown in SEQ ID NO:437; (4) LN15U-SJ-0165-073-E, which contains an antisense strand sequence as shown in SEQ ID NO:449, and a positive strand sequence as shown in SEQ ID NO:438; (5) LN17U-SJ-0165-073-E, which contains an antisense strand sequence as shown in SEQ ID NO:450, and a positive strand sequence as shown in SEQ ID NO:439; (6) LN14U-SJ-0165-073-E, which contains an antisense strand sequence as shown in SEQ ID NO:451, and a positive strand sequence as shown in SEQ ID NO:440; (7) LN15C-SJ-0011-P09-E, which contains the antisense strand sequence as shown in SEQ ID NO:452, and the positive strand sequence as shown in SEQ ID NO:441; (8) LN15C-SJ-0011-078-E, which contains an antisense strand sequence as shown in SEQ ID NO:453, and a positive strand sequence as shown in SEQ ID NO:442; (9) LN15C-SJ-0011-080-E, which contains an antisense strand sequence as shown in SEQ ID NO:454, and a positive strand sequence as shown in SEQ ID NO:443; (10)LN14C-SJ-0011-080E, which contains an antisense strand sequence as shown in SEQ ID NO:454, and a positive strand sequence as shown in SEQ ID NO:444; (11)LN15C-SJ-0011-080E, which contains an antisense strand sequence as shown in SEQ ID NO:454, and a positive strand sequence as shown in SEQ ID NO:

445.

26. A pharmaceutical composition, wherein, The pharmaceutical composition comprises the siRNA according to any one of claims 1-16 or the conjugate according to any one of claims 17-25, and a pharmaceutically acceptable carrier and / or excipient.

27. The pharmaceutical composition according to claim 26, wherein, The pharmaceutically acceptable carrier is a delivery carrier; preferably, the siRNA is encapsulated by the delivery carrier.

28. Use of the siRNA according to any one of claims 1-16, the conjugate according to any one of claims 17-25, or the pharmaceutical composition according to claim 26 or 27 in the preparation of a medicament for treating and / or preventing pathological conditions or diseases associated with APP; Preferably, the siRNA, the conjugate, or the pharmaceutical composition may be used alone or in combination with other pharmaceutically active agents (e.g., siRNAs targeting different target sequences in the APP gene or siRNAs targeting other targets).

29. The use according to claim 28, wherein, The pathological conditions or diseases associated with APP are Alzheimer's disease (AD), cerebral amyloid angiopathy (CAA), or Down syndrome (DS); Preferably, the Alzheimer's disease (AD) is early-onset familial Alzheimer's disease (EOFAD).

30. A method for preventing and / or treating pathological conditions or diseases associated with APP in a subject, the method comprising administering to a subject in need an effective amount of the siRNA of any one of claims 1-16, the conjugate of any one of claims 17-25, or the pharmaceutical composition of claim 26 or 27; Preferably, the siRNA, the conjugate, or the pharmaceutical composition may be used alone or in combination with other pharmaceutically active agents (e.g., siRNAs targeting different target sequences in the APP gene or siRNAs targeting other targets).

31. The method according to claim 30, wherein, The pathological conditions or diseases associated with APP are Alzheimer's disease (AD), cerebral amyloid angiopathy (CAA), or Down syndrome (DS); Preferably, the Alzheimer's disease (AD) is early-onset familial Alzheimer's disease (EOFAD).

32. The siRNA according to any one of claims 1-16, the conjugate according to any one of claims 17-25, or the pharmaceutical composition according to claim 26 or 27, used as a medicament; Preferably, the drug is used to prevent and / or treat pathological conditions or diseases related to APP; Preferably, the pathological condition or disease related to the APP is Alzheimer's disease (AD), cerebral amyloid angiopathy (CAA), or Down syndrome (DS); Preferably, the Alzheimer's disease (AD) is early-onset familial Alzheimer's disease (EOFAD); Preferably, the siRNA, the conjugate, or the pharmaceutical composition may be used alone or in combination with other pharmaceutically active agents (e.g., siRNAs targeting different target sequences in the APP gene or siRNAs targeting other targets).

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