Sirna for inhibiting APP gene expression and conjugate thereof, and use

Abstract

The present invention belongs to the field of biomedicine, and relates to an siRNA for inhibiting APP gene expression and a conjugate. The siRNA comprises a sense strand and an antisense strand. The sense strand comprises a nucleotide sequence I, and the antisense strand comprises a nucleotide sequence II. Each nucleotide in the nucleotide sequence I and the nucleotide sequence II is a modified or unmodified nucleotide. The nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region. The nucleotide sequence I is substantially identical to a first nucleotide sequence segment, and the first nucleotide sequence segment is a nucleotide sequence having a length of at least 15 nucleotides in an mRNA expressed by the APP gene. The siRNA of the present invention can specifically induce the degradation of APP mRNA, thereby inhibiting the synthesis of APP, inducing a persistent reduction in APP proteins, and reducing the pathological deposition of associated toxic proteins such as Aβ. The siRNA has a good prospects in becoming medicine.

Description

siRNAs and their conjugates that inhibit APP gene expression and their applications Technical Field

[0001] This invention belongs to the field of biomedicine and relates to an siRNA and conjugate that inhibits APP gene expression. Background Technology

[0002] Alzheimer's disease (AD) is the leading cause of dementia. It is defined as amyloid beta plaques and neurofibrillary tangles that manifest as forgetfulness or impairment of visual, language, executive, behavioral, or motor functions.

[0003] Cerebral amyloid angiopathy (CAA) is a common neurodegenerative disease in the brains of the elderly, characterized by the deposition of β-amyloid protein (Aβ) in intracranial microvessels (leptomeningeal arteries, cortical arterioles, and capillaries). Clinically, it can manifest in different subtypes, including lobar hemorrhage, cognitive impairment, rapidly progressive dementia, and cerebral amyloid attacks. It should be considered in middle-aged and elderly patients presenting with dementia, psychiatric symptoms, or recurrent or multiple lobar hemorrhages. Currently, there is no specific treatment.

[0004] The human amyloid precursor protein (APP) gene, located on chromosome 21q21.3, contains 20 exons and encodes amyloid precursor protein, the APP membrane protein. Studies have confirmed that β-amyloid protein (Aβ), produced from the breakdown of APP protein, is a significant pathological cause of both Alzheimer's disease (AD) and cerebrovascular accidental atrophy (CAA). Both APP and Aβ are associated with AD; the toxicity of Aβ depends on APP expression, and APP's effects extend beyond the production of toxic fragments. Aging accelerates Aβ accumulation by inducing APP processes, leading to upregulation of β-secretase. Transgenic mice overexpressing APP also exhibit significant alterations in vasoactive signaling, resulting in neurovascular dysfunction. Reduction in APP will lead to a simultaneous reduction in Aβ and all other toxic APP metabolites, mitigating the toxic environment associated with AD and slowing disease progression. Therefore, targeting APP to reduce Aβ formation is a key focus of drug development.

[0005] Currently, targeted therapies for APP include immunotherapy and APP secretase inhibitors. However, effective drug delivery to the brain is a key issue in immunotherapy, and other treatments have been terminated in clinical trials due to toxicity or side effects. Clinical evidence for antibody drugs targeting Aβ protein is concentrated in early-stage Alzheimer's disease (AD) patients; therefore, patients in the middle and late stages are not eligible for these drugs, and they also have toxic side effects such as cerebral edema. Other AD-specific drugs, such as acetylcholine inhibitors, can only alleviate mild to moderate AD symptoms and do not have significant therapeutic effects. Summary of the Invention

[0006] The purpose of this invention is to provide an siRNA molecule that can inhibit APP gene expression, in order to provide a new treatment for Alzheimer's disease and cerebral amyloid angiopathy.

[0007] In one aspect, the present invention provides an siRNA comprising a sense strand and an antisense strand, the sense strand comprising a nucleotide sequence I and the antisense strand comprising a nucleotide sequence II; each nucleotide in nucleotide sequence I and nucleotide sequence II is a modified or unmodified nucleotide; nucleotide sequence I and nucleotide sequence II are at least partially anticomplementary to form a double-stranded region; nucleotide sequence I is substantially identical to a first nucleotide sequence, which is a nucleotide sequence of at least 15 nucleotides in length in the mRNA expressed by the APP gene.

[0008] In a preferred embodiment, the first nucleotide sequence is a nucleotide sequence of 15 to 25 nucleotides in length from the mRNA expressed by the APP gene, for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides.

[0009] In a preferred embodiment, the first nucleotide sequence is a nucleotide sequence of at least 15 nucleotides in length from the highly active region of the mRNA expressed by the APP gene, such as a nucleotide sequence of 15-25 nucleotides. The highly active region is positions 334-804, 2305-2327, and 2813-2835 of the mRNA expressed by the APP gene, preferably positions 334-356, 490-512, 503-525, 635-656, 641-662, 782-804, 2305-2327, and 2813-2835. The mRNA expressed by the APP gene is shown in NCBI refseq ID NM_000484.4; specifically, the sequence of the mRNA expressed by the APP gene is shown in SEQ ID NO:1.

[0010] The high-activity region refers to the region in which the designed siRNA and siRNA conjugate can effectively reduce the APP mRNA level. In Figure 1, the regions were divided according to whether the observed maximum inhibition rate of siRNA and siRNA conjugate on APP mRNA fell into 40-60%, 60-80%, or greater than 80%.

[0011] In a preferred embodiment, the nucleotide sequence I has at least 70%, at least 80%, at least 85%, at least 90%, and at least 95% sequence identity with the first nucleotide segment.

[0012] In some implementations, the nucleotide sequence II in the above-described siRNA is substantially anticomplementary, substantially anticomplementary, or completely anticomplementary to the first nucleotide sequence.

[0013] The sense and antisense strands may be the same or different in length, with the sense strand being 16–23 nucleotides long and the antisense strand being 19–26 nucleotides long. In some embodiments, the length ratio of the siRNA sense and antisense strands is 16 / 21, 19 / 21, 21 / 23, or 19 / 24.

[0014] In a specific embodiment, sequence I comprises at least 15 consecutive nucleotides as shown in any of the sequences SEQ ID NO:2 to 111, such as at least 15, 16, 17, 18, 19, 20, or 21 nucleotides.

[0015] In a specific embodiment, the nucleic acid sequence of the positive strand is shown in Sequence I, which differs from any of the sequences in SEQ ID NO:2 to 111 by 1, 2 or 3 nucleotides.

[0016] In a specific implementation, the sequence I is as shown in any one of SEQ ID NO:2 to 111.

[0017] In a specific embodiment, sequence II comprises at least 15 consecutive nucleotides as shown in any of the sequences in SEQ ID NO:112 to 221, such as at least 15, 16, 17, 18, 19, 20, 21, 22 or 23 nucleotides.

[0018] In a specific embodiment, the nucleic acid sequence of the antisense strand is shown in Sequence II, which differs from any of the sequences in SEQ ID NO:112 to 221 by 1, 2 or 3 nucleotides.

[0019] In a specific embodiment, the sequence II is as shown in any one of SEQ ID NO:112 to 221.

[0020] In a specific embodiment, the siRNA is the siRNA molecules YGND22-1 to YGND22-110 shown in Table 1.

[0021] In one aspect, in the siRNA provided by the present invention, each nucleotide in nucleotide sequence I and nucleotide sequence II is a modified nucleotide.

[0022] In a specific embodiment, the modified nucleotide is a fluorinated nucleotide or a non-fluorinated nucleotide.

[0023] In specific embodiments, the fluorinated nucleotide refers to a nucleotide formed by replacing the hydroxyl group at the 2'-position of the ribosyl group with fluorine, having the structure shown in formula (1). The non-fluorinated nucleotide refers to a nucleotide or nucleotide analog formed by replacing the hydroxyl group at the 2'-position of the ribosyl group with a non-fluorinated group. In some embodiments, each non-fluorinated nucleotide is independently selected from one of the nucleotides or nucleotide analogs formed by replacing the hydroxyl group at the 2'-position of the ribosyl group with a non-fluorinated group. These nucleotides formed by replacing the hydroxyl group at the 2'-position of the ribosyl group with a non-fluorinated group are well known to those skilled in the art, and these nucleotides may be selected from one of 2'-alkoxy-modified nucleotides, 2'-substituted alkoxy-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-substituted alkyl-modified nucleotides, 2'-amino-modified nucleotides, 2'-substituted amino-modified nucleotides, and 2'-deoxynucleotides. In some embodiments, the 2'-alkoxy-modified nucleotide is a methoxy-modified nucleotide (2'-OMe), as shown in formula (2). In some embodiments, the 2'-substituted alkoxy-modified nucleotide, for example, may be a 2'-O-methoxyethyl-modified nucleotide (2'-MOE), as shown in formula (3). In some embodiments, the 2'-amino-modified nucleotide (2'-NH2) is shown in formula (4). In some embodiments, the 2'-deoxynucleotide (DNA) is shown in formula (5).

[0024] Nucleotide analogs are groups that can replace nucleotides in nucleic acids, but whose structure differs from that of adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine ribonucleotide. In some embodiments, nucleotide analogs can be isonucleotides, bridged nucleotides, or acyclic nucleotides.

[0025] A bridging nucleotide is a restricted or inaccessible nucleotide. Bridging nucleotides can contain a five-membered, six-membered, or seven-membered ring with a fixed C3-endoglucan condensation. In some embodiments, the bridging nucleotide can be LNA, ENA, cET BNA, etc.; wherein LNA is shown in formula (6), ENA is shown in formula (7), and cET BNA is shown in formula (8).

[0026] Acyclic nucleotides are a class of nucleotides formed by opening the sugar ring of a nucleotide. In some embodiments, acyclic nucleotides can be unlocked nucleic acids (UNA) or glycol nucleic acids (GNA), wherein UNA is shown in formula (9) and GNA is shown in formula (10).

[0027] In formulas (9) and (10) above, R is selected from H, OH, or alkoxy (O-alkyl).

[0028] Isonucleotides are compounds formed by altering the position of a base on the ribose ring in a nucleotide. In some embodiments, an isonucleotide can be a compound formed by moving a base from the 1'-position to the 2'-position or 3'-position on the ribose ring, as shown in formula (11) or formula (12).

[0029] In the compounds of formulas (11) and (12) above, R is selected from H, OH, F or non-fluorine groups as described above;

[0030] In the compounds of formulas (1) to (12) above, Base represents a base, such as A, U, G, C or T.

[0031] In a specific embodiment, one or more nucleotides at positions 7, 9, 10, and 11 of the nucleotide sequence I are fluorinated nucleotides, following the direction from the 5'-end to the 3'-end; and one or more nucleotides at positions 2, 6, 8, 9, 13, 14, 15, and 16 of the nucleotide sequence II are fluorinated nucleotides, following the direction from the 5'-end to the 3'-end.

[0032] In a specific embodiment, some nucleotides of the nucleotide sequence II may be modified with GNA; more specifically, the 7th position of the nucleotide sequence II is modified with GNA in the direction from the 5'-end to the 3'-end.

[0033] In a specific embodiment, in nucleotide sequences I and II, except for fluorinated and GNA modifications, all other nucleotides are methoxylated.

[0034] In specific embodiments, at least a portion of the phosphate ester groups in the phosphate-sugar backbone of at least one single strand of the sense and antisense strands of the siRNA provided by the present invention are phosphate ester groups with modifying groups. In some embodiments, the phosphate ester group with modifying groups is a thiophosphate ester group formed by replacing at least one oxygen atom in the phosphodiester bond of the phosphate ester group with a sulfur atom; in some embodiments, the phosphate ester group with modifying groups is a thiophosphate ester group having the structure shown in formula (13):

[0035] In some embodiments, the siRNA provided by the present invention preferably has the thiophosphate group linkage present at least one of the following positions: between the first and second nucleotides of the sense strand and / or antisense strand; between the second and third nucleotides of the sense strand and / or antisense strand; between the 19th and 20th nucleotides of the sense strand; between the 20th and 21st nucleotides of the sense strand; between the 21st and 22nd nucleotides of the antisense strand; between the 22nd and 23rd nucleotides of the antisense strand; or any combination thereof.

[0036] In some embodiments, the 5'-terminal nucleotide of the siRNA antisense strand is a 5'-phosphate nucleotide or a nucleotide modified with a 5'-phosphate analogue, as shown in formulas (14), (15), and (16):

[0037] In one aspect, the present invention provides an siRNA conjugate containing the aforementioned siRNA and a conjugate group conjugated to the siRNA.

[0038] In some embodiments, the pharmaceutically acceptable conjugation group in the siRNA conjugate can be galactose or N-acetylgalactosamine, wherein the galactose or N-acetylgalactosamine molecule can be monovalent, divalent, trivalent, or tetravalent.

[0039] In some embodiments, the conjugating group may be a n-chain alkyl group, such as n-hexadecyl (C16).

[0040] In some implementations, the conjugation site of siRNA and the conjugating group can be at the 3'-end or 5'-end of the sense strand of siRNA, at the 3'-end of the antisense strand, or within the internal sequence of siRNA.

[0041] In some specific embodiments, the siRNA conjugates involved in this invention have an L96 conjugation group, as shown in formula (I) below, or the conjugation group is C16, and the structure of the C16 conjugated nucleoside is shown in formula (II) below:

[0042] In specific embodiments, the siRNA conjugates are the siRNA conjugates (YGND22-1M~YGND22-110M) shown in Table 2, the siRNA conjugates (YGND22-1M'~YGND22-110M') shown in Table 3, and the siRNA conjugates (YGND22-9M'G, YGND22-16M'G, YGND22-92M'G) shown in Table 4.

[0043] In one aspect, the present invention provides a composition comprising the aforementioned siRNA or siRNA conjugate. In a preferred embodiment, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier or excipient. The pharmaceutically acceptable carriers or excipients involved in this invention include, but are not limited to, water for injection, sodium hydroxide, sodium dihydrogen phosphate monohydrate, sodium dihydrogen phosphate dihydrate, phosphoric acid, sodium chloride, potassium chloride, hydrochloric acid, anhydrous potassium dihydrogen phosphate, anhydrous disodium hydrogen phosphate, PEG2000, PEG6000, cholesterol, distearate, 1,2-dimyristate glyceryl, and dimethyl adipate.

[0044] In one aspect, the present invention provides the use of the aforementioned siRNA, siRNA conjugate, or pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of diseases related to excessive APP.

[0045] In one aspect, the present invention provides a method for preventing or treating diseases related to excessive APP, the method comprising administering to a subject a therapeutically effective amount or a preventatively effective amount of the siRNA, siRNA conjugate, or pharmaceutical composition of the present invention.

[0046] In a specific implementation, the diseases caused by excessive use of the APP are selected from cerebral amyloid angiopathy (CAA) and Alzheimer's disease (AD).

[0047] The pharmaceutical compositions involved in this invention can be used alone for the treatment of cerebral amyloid angiopathy (CAA) and Alzheimer's disease (AD), or in combination with standard oral medications, providing experimental support for diversified treatment options for clinical patients with the above diseases.

[0048] The appropriate dosage range for the siRNA that inhibits APP gene expression according to the present invention is generally from about 0.1 mg / kg to about 10.0 mg / kg, preferably from about 0.3 mg / kg to about 3.0 mg / kg.

[0049] The administration routes involved in this invention include intravenous administration, subcutaneous administration, intrathecal injection, intramuscular administration, subcutaneous administration, transdermal administration, airway administration (aerosol), ocular administration, nasal administration, rectal administration, pulmonary administration, and local administration (including oral administration and sublingual administration).

[0050] The siRNA of this invention can specifically induce the degradation of APP mRNA, thereby inhibiting APP synthesis, inducing a sustained reduction in APP protein, and reducing the pathological deposition of Aβ and other related toxic proteins, showing promising potential for drug development. Compared with traditional small molecule drugs and antibody drugs, small nucleic acid drugs can directly regulate upstream gene expression, making them relatively less prone to drug resistance; moreover, small nucleic acid drugs have a long half-life in vivo, thus requiring less frequent dosing (once every six months), resulting in better patient compliance. Therefore, this invention can effectively prevent or treat AD and CAA caused by the pathological deposition of toxic proteins such as Aβ resulting from APP protein degradation, providing these patients with more effective, safe, and convenient therapeutic drugs. Attached Figure Description

[0051] Figure 1 is a schematic diagram of the binding site of the siRNA of the present invention to the human APP gene;

[0052] Figure 2 shows the results of the siRNA conjugate of the present invention inhibiting APP expression in vivo;

[0053] Figure 3 shows the results of the siRNA conjugate of the present invention inhibiting APP expression in the brain;

[0054] Figure 4 shows the results of the siRNA conjugate of the present invention inhibiting APP expression in the brain;

[0055] Figure 5 shows the cell line toxicity results of the siRNA conjugate of the present invention. Detailed Implementation

[0056] definition

[0057] Unless otherwise specified, in the preceding and following text, uppercase letters C, G, U, A, and T represent cytosine, guanine, uracil, adenine, and thymine deoxynucleotides; lowercase letter m indicates that the nucleotide adjacent to the left of letter m is a methoxy-modified nucleotide; lowercase letter f indicates that the nucleotide adjacent to the left of letter f is a fluorinated nucleotide; lowercase letter s indicates that the two nucleotides adjacent to the left and right of letter s are linked by a thiophosphate subunit; the letter combination VP indicates that the nucleotide adjacent to the right of letter combination VP is a vinylphosphonate (5'-(E)-vinylphosphonate, E-VP) modified nucleotide; L96 has the structure of formula (I), which is linked to the 3'-terminus of the positive chain via a phosphate ester bond; C16 is linked to the 2'-O position of the nucleoside, and the resulting nucleoside structure is shown in formula (II).

[0058] In the preceding and following text, "fluorinated nucleotides" refers to nucleotides formed by replacing the 2'-hydroxyl group of the ribosome with fluorine, and "non-fluorinated nucleotides" refers to nucleotides or nucleotide analogs formed by replacing the 2'-hydroxyl group of the ribosome with a non-fluorinated group. "Nucleotide analogs" refer to groups that can replace nucleotides in nucleic acids but whose structure differs from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, or thymine deoxyribonucleotides. Examples include isonucleotides, bridged nucleic acids (BNAs), or acyclic nucleotides. "Methoxylated nucleotides" refers to nucleotides formed by replacing the 2'-hydroxyl group of the ribosome with a methoxy group.

[0059] In the context of this document, the terms "complementary" and "reverse complementary" are used interchangeably and have the meaning known to those skilled in the art: in a double-stranded nucleic acid molecule, the bases of one strand are paired complementaryly with the bases of the other strand. In DNA, the purine base adenine (A) always pairs with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (C) always pairs with the pyrimidine base cytosine (G). Each base pair consists of one purine and one pyrimidine. When adenine on one strand always pairs with thymine (or uracil) on the other strand, and guanine always pairs with cytosine, the two strands are considered complementary, and the sequence of the complementary strand can be inferred from its sequence. Correspondingly, "mismatch" in the art means, in a double-stranded nucleic acid, that the bases at corresponding positions are not paired complementaryly. Unless otherwise specified above and below, "substantially anticomplementary" means that there are no more than three base mismatches between the two nucleotide sequences involved; "substantially anticomplementary" means that there are no more than one base mismatch between the two nucleotide sequences; and "perfectly anticomplementary" means that there are no base mismatches between the two nucleotide sequences. For example, the siRNA molecule of the present invention comprises an oligonucleotide of 19 nucleotides in length and another oligonucleotide of 20 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 19 nucleotides that is perfectly complementary to the shorter oligonucleotide; for the purposes described herein, this situation may be referred to as "perfectly complementary." Therefore, as used herein, "perfectly complementary" means that all (100%) bases in the sequential sequence of the first polynucleotide hybridize with the same number of bases in the sequential sequence of the second polynucleotide. The sequential sequence may comprise all or part of the first or second nucleotide sequence.

[0060] The terms “complementary,” “substantially anticomplementary,” “truly anticomplementary,” or “completely anticomplementary” as used herein may refer to a base match between the sense and antisense strands of the APP siRNA, a base match between the antisense strand of the APP siRNA and the sequence of the target APP mRNA, or a base match between a single-stranded antisense oligonucleotide and the sequence of the target APP mRNA. It should be understood that the term “antisense strand of APP dsRNA” may refer to the same sequence as “APP antisense polynucleotide reagent.”

[0061] In the foregoing and hereinafter, particularly in the description of the preparation methods of the siRNA, pharmaceutical compositions, or siRNA conjugates of the present invention, unless otherwise specified, the nucleoside monomer refers to the modified or unmodified RNA phosphoramidites (sometimes also called nucleoside phosphoramidites) used in phosphoramidite solid-phase synthesis, depending on the type and sequence of nucleotides in the siRNA or siRNA conjugate to be prepared. Phosphoramidite solid-phase synthesis is a method known to those skilled in the art for the synthesis of siRNA. All nucleoside monomers used in the present invention are commercially available.

[0062] In Tables 1, 2, 3, and 4, if the left side of the 5'-terminal nucleotide of the positive strand or the modified positive strand with a connecting conjugation group is not marked with VP, it means that the 5'-terminal nucleotide is not connected to a 5'-phosphate group or a 5'-phosphate derivative group, and its structure is shown in Formula (III):

[0063] Wherein, Base represents a base, such as A, U, G, C or T; R is a hydroxyl group or is substituted by various groups known to those skilled in the art, for example, R can be 2'-fluoro(2'-F), 2'-alkoxy, 2'-substituted alkoxy, 2'-alkyl, 2'-substituted alkyl, 2'-amino, 2'-substituted amino, 2'-deoxynucleotide.

[0064] In Tables 1, 2, 3, and 4, if the left side of the 5'-terminal nucleotide of the antisense strand or modified antisense strand is not marked with VP, it means that the 5'-terminal nucleotide is not linked to a 5'-phosphate group or a 5'-phosphate derivative group, and its structure is as shown in Formula (III).

[0065] In Tables 1, 2, 3, and 4, the 3'-terminal nucleotide of the sense strand, the antisense strand, and the modified antisense strand has a hydroxyl group at the 3'-terminal position.

[0066] In this document, the term siRNA refers to small interfering RNA (RNAi) molecules. It is a class of double-stranded RNA molecules, also known in the art as short interfering RNA or silent RNA. siRNA typically comprises a sense strand (also called a guest strand) and an antisense strand (also called a leading strand), each strand being 15 to 30 nucleotides in length, typically 15 to 25 nucleotides. The antisense strand is complementary to the target nucleic acid (suitably a mature mRNA sequence) (e.g., at least 70% complementary, e.g., fully complementary), and the sense strand is complementary to the antisense strand, such that the sense and antisense strands form a double-stranded structure or a double-stranded region. The sense and antisense strands of siRNA can form a blunt-ended double-stranded structure (ends without unpaired nucleotides are called "blunt ends") or a double-stranded structure containing an overhang (ends with unpaired nucleotides or nucleotide analogues are called "overhangs"), which can be, for example, 1, 2, or 3 nucleotides long, similar to products produced by Dicer, which can form RISC substrates in vivo. The overhang can be a 5' overhang, a 3' overhang, or both. In some embodiments, one or more nucleotides in the overhang are replaced by a nucleoside phosphate thioester. In some embodiments, the first end of the siRNA is blunt-ended; in some embodiments, the second end of the siRNA is blunt-ended; and in some embodiments, both ends of the siRNA are blunt-ended. In some embodiments, both the sense and antisense strands have a 2-nucleotide-long 3' overhang. Therefore, the length of the double-stranded region can be, for example, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotide pairs, such as 19, 20, 21, 22, or 23 nucleotide pairs. One strand of the double-stranded region of the APP siRNA contains a sequence substantially complementary to the region of the target APP mRNA. The two strands forming the double-stranded structure can originate from a single RNA molecule having at least one self-complementary region, or can be formed by two or more separate RNA molecules. In some cases, when the double-stranded region is formed by a single molecule, the 5'-terminal portion of the sequence and the 3'-terminal portion of the sequence are anticomplementary, forming a double-stranded structure. The unpaired portion between the two portions forms a loop structure, i.e., the single molecule forms a "hairpin loop" structure. In some embodiments of the invention, the paired portion of the single molecule contains 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotide pairs, and the hairpin loop contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more unpaired nucleotides. When the essentially complementary two strands of the APP siRNA consist of separate RNA molecules, these molecules do not need to be covalently linked, but can be covalently linked.When two strands are covalently linked by means other than a hairpin loop, the connection structure is called a "connector". "siRNA" can also be referred to as dsRNA in this article.

[0067] In this document, "siRNA" sometimes also refers to "basic sequence." Specifically, "basic sequence" refers to a double-stranded siRNA in which each nucleotide is an unmodified nucleotide, as shown in Table 1. In this document, "siRNA," "basic sequence," "motif," and "siRNA motif" are used interchangeably, and their meanings also include the corresponding nucleotide sequence of the referred siRNA double strand. Those skilled in the art will clearly understand their precise technical meaning based on the context. Furthermore, the 5'-terminal nucleotide of the antisense strand of the motif may or may not be linked to a 5'-phosphate group or a 5'-phosphate-derived group.

[0068] In this document, "modified nucleotide" refers to the modification of one or more nucleotides in an siRNA molecule to improve its stability or efficacy. siRNA containing modified nucleotides can also be called "siRNA modifiers," referring to double-stranded ribonucleic acid containing at least one modified nucleotide; in some cases, it is also referred to as "double-stranded ribonucleic acid modifiers." In this document, different modification methods are used to modify the siRNA motif to prepare corresponding siRNA modifiers. For example, in some embodiments, the motif is modified using an alternating modification method to obtain alternatingly modified siRNA modifiers. In other embodiments, the motif is modified using a specific modification template to obtain siRNA modifiers modified with that specific template. In still other embodiments, the motif is modified using the off-target prevention modification method described herein to obtain off-target prevention modified siRNA modifiers. In some cases, multiple different modification methods can be used simultaneously to modify the same siRNA motif to obtain corresponding siRNA modifiers with multiple modification methods.

[0069] In this article, "siRNA conjugate" refers to a double-stranded ribonucleic acid conjugate or a conjugate of a double-stranded ribonucleic acid modifier obtained by attaching a conjugating group to a double-stranded ribonucleic acid or a double-stranded ribonucleic acid modifier.

[0070] In some instances herein, "siRNA" refers not only to the unmodified siRNA double strand (or siRNA motif) described above, but may also refer to its corresponding siRNA modifiers and / or siRNA conjugates. For example, in contexts involving, but not limited to, treatment methods and therapeutic agents, siRNA generally refers to at least one of siRNA motifs, siRNA modifiers, and / or siRNA conjugates. The specific technical meaning will be clearly understood by those skilled in the art in the context.

[0071] In this document, the term "antisense strand" refers to a strand of double-stranded ribonucleic acid (e.g., RNA double helix in this document) that includes a region substantially complementary to the target sequence. When used herein, the term "complementary region" refers to a region on the antisense strand that is substantially complementary to a sequence defined herein (e.g., the target sequence). When the complementary region is not perfectly complementary to the target sequence, mismatches can occur within the molecule or in terminal regions. Typically, the most permissible mismatches are in terminal regions, for example, within 5, 4, 3, 2, or 1 nucleotides at the 5'- and / or 3'-terminus.

[0072] In this document, the term "sense chain" as used herein refers to a chain of double-stranded RNA that includes regions substantially complementary to the regions of the antisense chain (as defined herein).

[0073] In this paper, the term "alternating modification" refers to the modification method in which nucleotides are modified with 2'-methoxy (2'-OMe), 2'-fluoro (2'-F), etc., according to the nucleotide sequence of the double-stranded ribonucleic acid.

[0074] In this document, "conjugation group" refers to a GalNAc derivative or a positive-chain alkyl group attached to an oligonucleotide. In some cases, the conjugation group includes a targeting group (also referred to as a ligand), and optionally also includes a linker, such as a GalNAc derivative attached to the oligonucleotide via a linker (e.g., a divalent, trivalent, or tetravalent branched linker arm), or a GalNAc derivative attached to the oligonucleotide via a monovalent linker arm. In most cases, "ligand" and "conjugation group" have meanings known in the art.

[0075] In this document, the term "inhibition" may be used interchangeably with "reduction," "silence," "downregulation," "suppression," and other similar terms, and includes any level of inhibition. In some instances, "regulation" refers to "inhibition," the specific meaning of which will be readily apparent to those skilled in the art from the context.

[0076] As used herein, the phrase “inhibit APP expression” includes inhibiting the expression of any APP gene (such as, for example, the mouse APP gene, rat APP gene, monkey APP gene, or human APP gene) and variants (e.g., naturally occurring variants) or mutants of the APP gene. Therefore, the APP gene can be a wild-type APP gene, a mutant APP gene, or, in the case of genetically manipulated cells, cell groups, or organisms, a transgenic APP gene.

[0077] "Inhibition of APP gene expression" includes inhibition of the APP gene at any level, such as at least partial inhibition of APP gene expression, such as inhibition of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0078] APP gene expression can be assessed based on the levels of any variable associated with APP gene expression, such as APP mRNA levels or APP protein levels. The inhibition can be assessed by a reduction in the absolute or relative level of one or more of these variables compared to a control level. A control level can be any type of control level utilized in the art, such as baseline levels before administration or levels determined from similar untreated or controlled (e.g., buffer-only control or inert agent control) subjects, cells, or samples.

[0079] In this document, "patient" or "subject" is intended to include humans or non-human animals, preferably mammals such as monkeys. More preferably, the subject or patient is a human.

[0080] In this article, "APP-related diseases" is intended to include any disease associated with the APP gene or protein. Such diseases can be caused, for example, by an overproduction of the APP protein. Exemplary APP-related diseases include cerebral amyloid angiopathy (CAA) and Alzheimer's disease (AD).

[0081] In this article, "therapeutic effective dose" is intended to include the amount of siRNA agent that is sufficient to achieve treatment of an APP-related disease (e.g., by attenuating, improving, or maintaining the existing disease or symptoms of one or more diseases) when administered to a patient. This "therapeutic effective dose" can vary depending on the siRNA agent, how it is administered, the disease and its severity, and medical history, age, weight, family history, genetic makeup, stage of the pathological process mediated by APP expression, type of prior or concomitant treatment (if any), and other individual characteristics of the patient to be treated.

[0082] In this article, "preventive effective dose" refers to the amount of siRNA agent sufficient to prevent or improve the disease or one or more symptoms of the disease when administered to subjects who have not yet experienced or exhibited symptoms of an APP-related disease but may be susceptible to it. Improving the disease includes slowing its progression or reducing the severity of subsequent disease development. This "preventive effective dose" can vary depending on the siRNA agent, how it is administered, the risk level for the disease, and medical history, age, weight, family history, genetic makeup, type of prior or concomitant treatment (if any), and other individual characteristics of the patient to be treated.

[0083] "Therapeutic effective amount" or "preventive effective amount" also includes the amount of siRNA agent that produces a desired local or systemic effect at a reasonable benefit / risk ratio applicable to any treatment. The siRNA agent used in the method of the present invention can be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0084] It is understood that the amount of APP siRNA or siRNA conjugate administered to the subject can be modified, at least in part, based on the determination of the subject's disease and / or condition and / or physiological characteristics. The therapeutic dose can be changed, for example, by altering the composition in which the APP siRNA or siRNA conjugate is administered, by altering the route of administration, by altering the timing of administration, etc., to increase or decrease the amount of APP siRNA or siRNA conjugate. The effective amount of APP siRNA or siRNA conjugate will vary depending on the specific condition being treated, the age and physical condition of the subject, the severity of the condition, the duration of treatment, the nature of any co-treatments, the specific route of administration, and other factors within the knowledge and expertise of the healthcare practitioner. For example, the effective amount may depend on the amount of APP peptide and / or the desired level of APP gene expression effective in treating APP-related diseases or conditions. Those skilled in the art can determine the effective amount of a particular APP siRNA or siRNA conjugate used in the methods of this invention empirically without excessive experimentation. Based on the teachings provided herein, effective preventative or therapeutic treatment regimens can be devised to effectively treat specific subjects by selecting from a variety of APP siRNAs or siRNA conjugates of the present invention and weighing factors such as potency, relative bioavailability, patient weight, severity of adverse side effects, and preferred route of administration. As used in embodiments of the present invention, the effective amount of the APP siRNA or siRNA conjugate of the present invention can be the amount that produces the desired biological effect in the cells upon contact with them.

[0085] It should be recognized that APP gene silencing can be performed constitutively or through genome engineering in any cell expressing APP, and can be determined by any suitable assay. In some embodiments of the invention, by administering the APP siRNA or siRNA conjugate of the invention, APP gene expression is reduced by at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments of the invention, by administering the APP siRNA or siRNA conjugate of the invention, APP gene expression is reduced by 5% to 10%, 5% to 25%, 10% to 50%, 10% to 75%, 25% to 75%, 25% to 100%, or 50% to 100%.

[0086] In this article, unless otherwise specified, when referring to any nucleotide position of any strand of siRNA motif, siRNA modifier, siRNA conjugate, siRNA duplex, etc., it means the 5' to 3' orientation.

[0087] In this document, the term "sequence identity" as used when referring to nucleic acid sequences means that a nucleic acid sequence contains at least about 70% or more sequence identity compared to a reference sequence, preferably at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity. The percentage of sequence identity is determined by comparing the best alignment of the two sequences in an alignment window. The percentage is calculated by determining the number of positions in both sequences where the same nucleic acid base occurs to generate the number of matching positions; dividing the number of matching positions by the total number of positions in the alignment window; and then multiplying the result by 100 to obtain the percentage of sequence identity. The inventions disclosed herein include those nucleotide sequences substantially the same as those disclosed herein (e.g., in Tables 1–4). In some embodiments, the nucleotide sequence is identical to, or has at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequences disclosed herein (e.g., in Tables 1-4).

[0088] Modification

[0089] In some embodiments of the present invention, the APP siRNA is chemically modified to obtain enhanced stability and / or one or more other beneficial properties. The nucleic acids in some embodiments of the present invention can be synthesized and / or modified using methods known in the art. Various methods exist in the prior art and can be applied to the present invention, and are included within the scope of protection of the present invention. Based on the structure of nucleic acids, modifications that can exist in certain embodiments of the APP siRNA of the present invention include, for example: 1) Base modification: mainly divided into three forms: purine modification, pyrimidine modification, and base substitution. Those skilled in the art will recognize that guanine, cytosine, adenine, and uracil can be replaced by other parts without significantly changing the base pairing characteristics of the oligonucleotide containing the nucleotide with such a substitution; 2) Ribose modification: this is one of the most important ways of modifying nucleic acids, which involves the modification and substitution of groups at specific positions on the ribose ring, including but not limited to 2'-position modification (such as 2'-OMe modification, 2'-F modification), 4'-position modification, 5'-position modification, isomerization modification, etc.; 3) Phosphate backbone modification: including but not limited to, modification of thiophosphate esters, replacing the phosphate ester group between nucleosides entirely with a group that does not contain phosphorus atoms, such as replacing P atoms with C atoms, S atoms, and N atoms, etc.; 4) Terminal modification: including but not limited to covalently linking specific groups at the 5'-end and / or 3'-end of the sense strand, phosphorylation modification at the 5'-end of the antisense strand, etc.

[0090] Those skilled in the art will understand that, depending on the siRNA molecule, there can be different numbers and types of modifications. In some embodiments of the present invention, the RNA molecule comprises the following numbers of modified ribonucleotides: at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or the full length of an APP siRNA molecule. For each of the multiple modified ribonucleotides in such an RNA molecule, the modification need not be the same.

[0091] RNA backbone modification

[0092] As is known in the art, RNA comprises a backbone structure formed by alternating links of phosphate and ribose. In RNA, the 3'-hydroxyl group of the ribose of one nucleotide is linked to the 5'-phosphate group of the next nucleotide, forming a 3'-5' phosphodiester bond. In some embodiments, the RNA analog has a 2'-5' linked phosphodiester bond, meaning that the 2'-hydroxyl group of the ribose of one nucleotide is linked to the 5'-phosphate group of the next nucleotide. In these cases, modifications to the RNA backbone include modifications to the phosphate and ribose groups. Modifications to the phosphate groups include, but are not limited to, the formation of: thiophosphates (such as thiophosphates, dithiophosphates, chiral thiophosphates, thioaminophosphates, thioalkylphosphonates, thioalkylphosphides), phosphate triesters, phosphinates, methyl and other alkylphosphonates (including 3'-alkylphosphonates and chiral phosphonates), aminophosphates (including aminoalkylphosphates, aminoalkylphosphides, 3'-aminophosphates, etc.), and other forms (such as borate phosphates).

[0093] In some embodiments, the RNA backbone is modified to be a non-phosphate-ribose backbone, typically a phosphorus-free backbone. For example, the RNA backbone has a backbone formed by short-chain alkyl or cycloalkyl nucleosides, mixed heteroatoms and alkyl or cycloalkyl nucleosides, or one or more short-chain heteroatoms or heterocyclic nucleosides. In some specific embodiments, the modified skeleton that does not contain phosphorus atoms is: 1) Morpholine bond skeleton: ribose is replaced by a morpholine ring (a six-membered nitrogen-containing heterocycle) to form morpholine oligonucleotide (PMO); 2) Siloxane skeleton: phosphodiester bond is replaced by a -Si-O- bond (-Si-O-Si-); 3) Sulfide skeleton: nucleosides are linked by thioether bonds (-S-), such as thiomethyl skeleton (-CH2-S-); 4) Amide skeleton: nucleosides are linked by a -CONH- bond, such as peptide nucleic acid (PNA); 5) Sulfonate / sulfonamide skeleton: containing -SO3- or -SO2NH- bonds; 6) Methylene imino skeleton: nucleosides are linked by a -CH2-NH- bond.

[0094] The above-mentioned RNA backbone modification methods are all conventional techniques in the field and can be used to prepare certain modified siRNAs or siRNA conjugates of the present invention.

[0095] In some embodiments of the invention, RNA mimics are included in APP siRNA or siRNA conjugates, siRNA sense strands, and siRNA antisense strands, for example, but not limited to replacing the sugar and nucleoside links (i.e., the backbone) of nucleotide units with novel groups. In such embodiments, the base units are preserved to hybridize with suitable APP nucleic acid target compounds. Methods for preparing RNA mimics are conventional practices in the art, and such methods can be used to prepare certain modified APP siRNAs or siRNA conjugates, siRNA sense strands, and siRNA antisense strands of the present invention.

[0096] In some embodiments, the APP siRNA or siRNA conjugate, the siRNA sense strand, and the siRNA antisense strand may include nucleobase modifications or substitutions (generally referred to in the art simply as "bases"). As used herein, "unmodified" or "native" nucleobases include purine bases adenine (A) and guanine (G), and pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include other synthetic and native nucleobases, such as 5-methylcytosine (5-me-C), pseudouracil (5-uracil), 2-thiouracil, 5-halouracil (e.g., 5-bromouracil), 8-hydroxyguanine, 7-methylguanine, 6-methyladenine, 2-aminoadenine, 8-azaguanine, 5-trifluoromethylcytosine, 7-azaadenine, and hypoxanthine. Additional nucleobases that may be included in certain embodiments of the APP siRNA or siRNA conjugate, siRNA sense strand, or siRNA antisense strand of the present invention are known in the art.

[0097] In some embodiments, the APP siRNA or siRNA conjugate of the present invention comprises at least one modified nucleotide, wherein the at least one modified nucleotide comprises: 2'-O-methyl nucleotide, 2'-fluoronucleotide, 2'-deoxynucleotide, 2'-methoxyethyl nucleotide, 2'-amino-modified nucleotide, 2'-alkyl-modified nucleotide, and 3'-OMe nucleotide, nucleotide comprising a 5'-thiophosphate group, or nucleotide comprising a non-natural base. In some embodiments, the 5'-terminus of the APP siRNA antisense strand comprises an E-vinylphosphonate nucleotide.

[0098] In some embodiments of the present invention, the APP siRNA or siRNA conjugate contains at least one modified nucleotide, wherein the at least one modified nucleotide comprises an open-ring nucleic acid (UNA, also known as an unlocked nucleic acid) and / or a glycol nucleic acid (GNA). These modifications disrupt the normal sugar-phosphate backbone linkages between nucleotides, causing UNA and GNA to exhibit lower stability under high-temperature conditions. This thermal instability not only endows them with unique physicochemical properties but also significantly improves the off-target profile of the siRNA or its conjugates, further optimizing their functional performance.

[0099] In some embodiments of the present invention, the APP siRNA or siRNA conjugate, the siRNA positive strand, and the siRNA negative strand may contain another modification comprising one or more ligands, partially or chemically linked conjugates to RNA that enhance one or more features of the APP dsRNA reagent, the siRNA positive strand, and the siRNA negative strand, respectively. Non-limiting examples of features that may be enhanced include: APP siRNA or siRNA conjugate, siRNA positive strand, siRNA negative strand activity, cellular distribution, delivery of the APP siRNA or siRNA conjugate, pharmacokinetic properties of the APP siRNA or siRNA conjugate, and cellular uptake of the APP siRNA or siRNA conjugate. In some embodiments of the present invention, the APP siRNA or siRNA conjugate comprises one or more targeting groups or linking groups, which are conjugated to the positive strand in some embodiments of the APP siRNA or siRNA conjugate of the present invention. Non-limiting examples of targeting groups are compounds containing N-acetyl-galactosamine (GalNAc) or positive-chain alkyl groups. The terms “targeting group,” “targeting agent,” “linker,” “targeting compound,” and “targeting ligand” are used interchangeably herein. In some embodiments of the invention, the APP siRNA or siRNA conjugate comprises a targeting compound conjugated to the 5'-terminus of the positive strand. In some embodiments of the invention, the APP siRNA or siRNA conjugate comprises a targeting compound conjugated to the 3'-terminus of the positive strand. In some embodiments of the invention, the APP siRNA or siRNA conjugate comprises a targeting compound conjugated to an intermediate nucleoside of the positive strand. In some embodiments of the invention, the APP siRNA or siRNA conjugate comprises a targeting group or a positive-chain alkyl group containing GalNAc. In some embodiments of the invention, the APP siRNA or siRNA conjugate does not contain a targeting compound conjugated to one or both of the 3'-terminus and 5'-terminus of the positive strand. In some embodiments of the invention, the APP siRNA or siRNA conjugate does not contain a GalNAc-containing targeting compound conjugated to one or both of the 5'-terminus and 3'-terminus of the positive strand. In some embodiments of the invention, the APP siRNA or siRNA conjugate comprises a positive-chain alkyl group conjugated to the sense strand. In some embodiments, the APP siRNA or siRNA conjugate comprises a hexadecyl (C16) group conjugated to the sixth nucleotide of the sense strand.

[0100] siRNA delivery

[0101] Some embodiments of the methods of the present invention include delivering APP siRNA or siRNA conjugates into cells. As used herein, the term "delivery" means promoting or influencing cellular uptake or absorption. Delivery methods suitable for the methods of the present invention include, but are not limited to, in vivo delivery.

[0102] Those skilled in the art will understand that by delivering (e.g., introducing) APP siRNA into cells, the level or amount of APP peptides in the cells can be reduced, and thus their activity decreased. Targeting agents and methods can be used to facilitate the delivery of APP siRNA to specific cell types, cell subtypes, organs, spatial regions, and / or subcellular regions within a target. Non-limiting examples of methods that can be used to deliver APP siRNA or siRNA conjugates to cells, tissues, and / or targets include: conjugation techniques (GalNAC conjugation, positive-chain alkyl conjugation, etc. as described above), LNP-based delivery methods, and naked RNA delivery, etc. These delivery methods have been successfully used in the art to deliver therapeutic RNAi agents for the treatment of various diseases and conditions. Those skilled in the art will understand and apply such delivery techniques, and such techniques, in conjunction with the content of this invention, are within the scope of protection of this invention.

[0103] It should be understood that, in addition to certain delivery methods described herein, other RNAi delivery methods may be used in conjunction with the implementation schemes of the APP siRNA and therapeutic methods described herein, such as, but not limited to, those described herein and those used in the art.

[0104] These and other therapeutic agents and behavioral modifiers are known in the art and can be used to treat APP-related diseases or conditions in subjects, and can also be combined with one or more of the APP siRNAs of the present invention for administration to subjects to treat APP-related diseases or conditions. The APP siRNAs of the present invention administered to cells or subjects to treat APP-related diseases or conditions can act synergistically with one or more other therapeutic agents or active ingredients, thereby increasing the effectiveness of one or more therapeutic agents or active ingredients and / or increasing the effectiveness of APP siRNAs in treating APP-related diseases or conditions.

[0105] Pharmaceutical compositions containing APP siRNA or siRNA conjugates

[0106] As used herein, a “pharmaceutical composition” comprises a pharmacologically effective amount of the APP siRNA or siRNA conjugate of the present invention and a pharmaceutically acceptable carrier or excipient. The term “pharmaceuticalally acceptable carrier or excipient” refers to a carrier or excipient used for the administration of a therapeutic agent. Such carriers or excipients include, but are not limited to, water for injection, sodium hydroxide, sodium dihydrogen phosphate monohydrate, sodium dihydrogen phosphate dihydrate, phosphoric acid, sodium chloride, potassium chloride, hydrochloric acid, anhydrous potassium dihydrogen phosphate, anhydrous disodium hydrogen phosphate, PEG2000, PEG6000, cholesterol, distearate, glyceryl 1,2-dimyristate, dimethyl adipic acid, and combinations thereof. Also included are nuclease inhibitors, such as bovine serum albumin (BSA), Poly I:C, nonspecific binding nucleases; pH buffers such as disodium citrate-hydrogen phosphate, histidine; and antioxidants such as vitamin E, BHT, etc.

[0107] The pharmaceutical compositions containing APP siRNA or siRNA conjugates of the present invention can be in any form known in the art, such as liquid formulations (infusions, injections), solid formulations (tablets, powders, pills, lyophilized powders), etc., and other forms can also be prepared according to actual needs. The preparation and use of pharmaceutical compositions are conventional techniques in the art. Those skilled in the art can know how to prepare the compositions and adjust the proportions to suit the user based on the disclosure herein. The preparation and use of pharmaceutical compositions also fall within the scope of protection of this invention.

[0108] Dosage or administration

[0109] The APP siRNA or its conjugates of the present invention can be administered to a subject in an amount and manner that effectively reduces the level and activity of APP peptides in cells and / or subjects, i.e., by drug administration. In some embodiments of the method of the present invention, the APP siRNA or its conjugates are administered to cells and / or subjects to treat diseases or conditions associated with APP expression and activity. In some embodiments, the method of the present invention includes administering the APP siRNA or its conjugates to a subject requiring such treatment to alleviate a disease or condition associated with APP expression in the subject. The APP siRNA or its conjugates of the present invention can be administered to reduce APP expression and / or activity in one or more types of cells in vitro, ex vivo, and in vivo.

[0110] In this invention, the APP siRNA or siRNA conjugate is delivered at a dose sufficient to inhibit APP gene expression. In some embodiments of this invention, the dose of APP siRNA or siRNA conjugate is 0.01 to 200.0 mg per kilogram of recipient body weight per day, such as 1–50 mg / kg body weight, 5–40 mg / kg body weight, 10–30 mg / kg body weight, 1–20 mg / kg body weight, 1–10 mg / kg body weight, 4–15 mg / kg body weight per day, including extreme values. For example, APP The dosage of siRNA or siRNA conjugates can be approximately 0.01 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3.0 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 21 mg / kg, 22 mg / kg daily. The dosage is 23 mg / kg, 24 mg / kg, 25 mg / kg, 26 mg / kg, 27 mg / kg, 28 mg / kg, 29 mg / kg, 30 mg / kg, 31 mg / kg, 32 mg / kg, 33 mg / kg, 34 mg / kg, 35 mg / kg, 36 mg / kg, 37 mg / kg, 38 mg / kg, 39 mg / kg, 40 mg / kg, 41 mg / kg, 42 mg / kg, 43 mg / kg, 44 mg / kg, 45 mg / kg, 46 mg / kg, 47 mg / kg, 48 mg / kg, 49 mg / kg, and 50 mg / kg body weight. In some other embodiments, the dosage is higher, such as 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg, and 100 mg / kg body weight per day.

[0111] The dosage and timing of administration of the APP siRNA or siRNA conjugate of the present invention can be determined based on a variety of factors, such as co-treatment, dose number, and individual subject parameters, including age, physical condition, and weight. These are factors well known to those skilled in the art and can be resolved through routine experiments. In some embodiments, a maximum dose may be used, i.e., the highest safe dose based on reasonable medical judgment.

[0112] In some embodiments, the APP siRNA or siRNA conjugates or compositions comprising them of the present invention may be administered to the subject at least daily, every other day, weekly, monthly, every three months, every six months, or annually. The frequency of administration may be once daily or more than once daily, for example, 2, 3, 4, 5, or more administrations at appropriate intervals within a 24-hour cycle. In some embodiments, the siRNA or siRNA conjugates of the present invention may be administered to the subject once or more daily, once or more weekly, once or more monthly, once or more every three months, every six months, or once or more annually.

[0113] In some embodiments, the APP siRNA or siRNA conjugate of the present invention may be administered before and / or while an APP-related disease or condition is present, including at all times before and after the early, middle, and late stages of the disease or condition. The physician may determine at which stage of the disease or condition to administer the siRNA or siRNA conjugate of the present invention based on the user's condition.

[0114] The APP siRNA or siRNA conjugates of the present invention can be administered or applied via a variety of medically acceptable routes of administration, resulting in effective treatment of APP-related diseases or conditions without causing clinically unacceptable side effects. In some embodiments of the present invention, the APP siRNA or siRNA conjugates can be administered orally, enterically, mucosatially, subcutaneously, and / or parenterally. The term "parentereal" includes subcutaneous, intravenous, intrathecal, intramuscular, intraperitoneal, and intrasternal injection or infusion techniques. Delivery routes of the present invention may include intrathecal, intraventricular, or intracranial. Various delivery methods, approaches, and reagents are known in the art.

[0115] treat

[0116] In some embodiments, the siRNA or siRNA conjugate of the present invention is applied alone. In other embodiments, it is applied in combination with one or more other treatment regimens for treating APP-related diseases or conditions. When applied in combination, other treatment regimens may be applied before, simultaneously with, and after the application of the APP siRNA of the present invention. As will be known to those skilled in the art, “simultaneously” as used herein means within 5 minutes, 10 minutes, 30 minutes, 45 minutes, and 60 minutes of zero time, where “zero time” is the time at which the APP siRNA reagent of the present invention is applied to the subject. Other treatment regimens may be other combinations of APP siRNA or siRNA conjugates or non-siRNA therapeutic agents. Non-siRNA therapeutics, without limitation, include: cholinesterase inhibitors; excitatory amino acid receptor antagonists such as memantine or memantine combined with donepezil, rivastigmine; medications for psychogenic symptoms such as risperidone, aripiprazole, olanzapine, quetiapine; monoclonal antibodies targeting Aβ, including lencanemab, adunatumab, and donepezil; antiepileptic and antimigraine medications such as levetiracetam, lamotrigine, lacosamide, clobazine, valproic acid, tiapride, verapamil, and other medications such as mannocycline, minocycline; immunosuppressive drugs such as cyclophosphamide, mycophenolate mofetil, azathioprine, intravenous immunoglobulin, rituximab, etc. Alternatively, they can be used in combination with dietary programs, cognitive therapy, and exercise therapy. Examples include the Mediterranean diet, the DASH diet, and the MIND diet; cognitive therapy includes activating brain regions through memory training and brain-training games (such as puzzles and Sudoku). Exercise therapy, such as moderate-intensity aerobic exercise (e.g., brisk walking, swimming).

[0117] As used herein, the terms “treatment,” “therapeutic,” or “cure” when applied to APP-related diseases or conditions can refer to preventative treatment, reducing the likelihood of a subject developing an APP-related disease or condition, or treatment undertaken after a subject has developed an APP-related disease or condition to eliminate or reduce the level of the APP-related disease or condition, prevent the APP-related disease or condition from becoming more severe, and / or slowing or improving the progression of one or more diseases or conditions associated with APP protein production in a subject compared to a subject without a therapy that reduces APP peptide activity. “Treatment” can also refer to prolonged survival compared to expected survival without treatment.

[0118] The term “prevention and / or treatment” includes not only the prevention and / or treatment of disease, but also generally includes preventing the onset of disease, slowing or reversing the progression of disease, preventing or slowing the onset of one or more symptoms associated with disease, and reducing and / or alleviating one or more symptoms associated with disease.

[0119] In this document, the treatment or prevention targets excessive APP production, thereby utilizing the siRNA or siRNA conjugate of the present invention to inhibit APP expression, thereby downregulating its expression level. As used herein, with respect to APP gene expression, the terms “inhibit,” “silence,” “reduce,” “downregulate,” and “knock down” refer to altering APP gene expression, for example, by one or more of the following: when cells, tissues, organs, or objects are contacted (e.g., treated or administered) with the APP siRNA or siRNA conjugate of the present invention, the level of RNA transcribed from the APP gene, the level of peptide and protein activity of the expressed APP are reduced, compared to a control level of RNA transcribed from the APP gene, a control level of the activity of the expressed APP, or a control level of APP translated from mRNA, respectively. In some embodiments, the control level is the level in cells, tissues, organs, or objects that have not been contacted with the APP siRNA or siRNA conjugate (e.g., treated or administered).

[0120] In some embodiments of the present invention, the APP content in serum samples before exposure to APP siRNA or siRNA conjugates is used as a baseline. After administration and treatment, the APP content is detected again in the obtained serum samples. When the APP peptide level decreases by at least 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, it indicates the efficacy level of the treatment given to the subject.

[0121] In some embodiments of the present invention, the physiological characteristics of APP-related diseases or conditions identified in the subject can serve as a control, and the results of identifying the physiological characteristics of the same subject at different times can be compared with the control results. For example, using the physiological characteristics or condition status of a subject who has not received APP treatment as a baseline, after one or more administrations of the APP siRNA or siRNA conjugate of the present invention, the onset, progression, or regression of APP-related diseases or conditions in the subject is assessed. When, for example, the condition regresses by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more, the efficacy level of the treatment given to the subject is indicated.

[0122] Cells, subjects, and controls

[0123] As used herein, the terms cell, tissue, organ, and / or object may be used in combination when referring to administration or delivery. In some aspects of the invention, the object is a human or vertebrate mammal; therefore, the invention can be used to treat APP-related diseases or conditions in both human and non-human subjects. In some embodiments of the invention, the object is a human and the methods of the invention can be used for human prevention and treatment programs.

[0124] Cells applicable to the method of this invention include in vivo and in vitro cells, which can be primary cultured tissue cells or immortalized cells of a specific lineage. Cells applicable to this invention can be brain tissue cells including neurons and glial cells (astrocytes, oligodendrocytes, microglia, ependymal cells); liver cells including hepatic parenchymal cells and non-parenchymal liver cells (hepatic stellate cells, hepatic sinusoidal endothelial cells, hepatic Kupffer cells, intrahepatic bile duct epithelial cells); kidney tissue cells including renal tubular cells, glomerular endothelial cells, renal interstitial fibroblasts, and renal vascular endothelial cells; thymic cells and thymic epithelial cells; testicular tissue cells; prostate tissue cells including prostate epithelial cells and interstitial cells; cardiomyocytes; and vascular endothelial cells.

[0125] Medicine box

[0126] In some embodiments, the APP siRNA or siRNA conjugate or composition of the present invention can be prepared as a pharmaceutical kit. The components of the kit, including the siRNA or siRNA conjugate of the present invention, and other necessary ingredients or devices, can be packaged in an aqueous medium or lyophilized form. In some embodiments, the kit of the present invention may comprise a carrier separated to enclose one or more container devices or a series of container devices (e.g., test tubes, vials, flasks, bottles, syringes, etc.). Different components are contained in different devices. Furthermore, the kit of the present invention may also include instructions. The instructions are typically in written form and describe the treatment method embodied in the kit and provide guidance for making decisions based on that treatment.

[0127] Example 1. siRNA Design and Synthesis

[0128] 1.1 siRNA Design

[0129]

[0130] 1.2 siRNA sequence synthesis

[0131] siRNA was synthesized according to a standard oligonucleotide solid-phase synthesis protocol, including a negative control siRNA (siCtrl).

[0132] Oligonucleotide solid-phase synthesis protocol: Commercially available 5'-DMT-2'-TBDMS-rU phosphorus amide monomers, 5'-DMT-2'-TBDMS-rA(Bz) phosphorus amide monomers, 5'-DMT-2'-TBDMS-rC(Ac) phosphorus amide monomers, and 5'-DMT-2'-TBDMS-rG(iBu) phosphorus amide monomers were used. RNA was synthesized on a 500 nmol scale. A phosphorus amide solution was prepared at a concentration of 50 mM, and 0.3 M benzylthiotetrazole (BTT) acetonitrile solution was used as an activator. During synthesis, a 0.1 M oxidizing agent (pyridine:THF:water = 20:78:2) was used to convert trivalent phosphorus to pentavalent phosphorus to stabilize the phosphate backbone. After synthesis, the sequence was ammonolyzed from the solid support and precipitated. The 2'-2'-O-tert-butyldimethylsilyl protecting group was removed with triethylamine trihydrofluoric acid.

[0133] For the synthesized RNA sequence, ammonolysis was performed at 55°C for 40 minutes using an ammonia:methylamine ratio of 1:1. After ammonolysis, the solid support CPG powder was removed, and the supernatant was dried. A protecting group remover was added, and the reaction was carried out at 60°C for 2 hours. Then, n-butanol was added at a 1:5 ratio, and the mixture was allowed to stand at -20°C for 30 minutes. The precipitate was collected by centrifugation. The precipitate was dissolved in RNase-free water and purified using reversed-phase chromatography (0.1M triethylamine acetic acid (TEAA) and acetonitrile). The purified sample was desalted by ultrafiltration with PBS and annealed to obtain siRNA. Verification of the obtained siRNA confirmed successful preparation of the target siRNA.

[0134] 1.3 siRNA sequence modification and conjugate synthesis

[0135] Modified siRNAs are synthesized according to oligonucleotide solid-phase synthesis schemes. The modified nucleotide groups can be introduced into the siRNAs disclosed herein using nucleoside monomers with corresponding modifications. The methods for preparing nucleoside monomers with corresponding modifications are also well known to those skilled in the art. L96 is conjugated to siRNA to synthesize siRNA conjugates according to the synthesis methods disclosed in WO2014025805A1 or WO2017015109A1. C16 (hexadecyl) is conjugated to siRNA to synthesize siRNA conjugates according to the synthesis method of Osborn MF, Khvorova A. Improving siRNA Delivery In Vivo Through Lipid Conjugation. Nucleic Acid Ther. 2018 Jun; 28(3):128-136. doi:10.1089 / nat.2018.0725. Epub 2018 May 10. PMID:29746209;PMCID:PMC5994667. The conjugating group L96 is shown in formula (I), and the nucleoside structure conjugated by C16 is shown in formula (II), with C16 attached to the 2'-O position of the nucleoside or nucleotide.

[0136] The structure of the conjugation group L96 is shown below:

[0137] The structure of the nucleoside conjugated with the C16 conjugation group is shown below:

[0138] Annealing of oligonucleotides to produce siRNA conjugates: The RNA oligomers to be annealed were prepared into a 200 μM solution using sterile RNase-free H2O (RNA hydrolase-free). The annealing reaction system was set up as follows: 100 μL of the above solution (10 nmol doublet concentration) was placed in a 95°C water bath for 10 minutes (≥100 nmol requires 20 minutes at high temperature) → immediately cooled in a 60°C water bath → the annealed solution was stored at 4°C. Equimolar amounts of RNA solution were combined to mix complementary strands. The siRNA molecule was confirmed to be correctly constructed. The siRNA solution was prepared into a dry powder for later use.

[0139] The synthesized siRNA molecule sequences are shown in Table 1 below, and the binding sites with the human APP gene are shown in Figure 1:

[0140] Table 1. siRNA sequence listing targeting APP

[0141] The sequences of the synthesized siRNA conjugates are shown in Table 2 below:

[0142] Table 2. siRNA conjugates targeting APP

[0143] Table 3. siRNA conjugates targeting APP

[0144] Table 4. siRNA conjugates targeting APP

[0145] In this context, uppercase letters C, G, U, A, and T represent cytosine, guanine, uracil, adenine nucleotide, and thymine deoxynucleotide, respectively; lowercase letter m indicates that the nucleotide adjacent to the left of m is methoxy-modified; lowercase letter f indicates that the nucleotide adjacent to the left of f is fluorinated; lowercase letter s indicates that the two nucleotides adjacent to s are linked by thiophosphate subunits; VP indicates that the nucleotide adjacent to the right of VP is 5'-(E)-vinylphosphonate (E-VP); GNA indicates that the nucleotide adjacent to the left of (GNA) is GNA-modified; L96 represents the L96 conjugate group linked to siRNA; hd represents the C16 conjugate group linked to siRNA; and (Nhd) indicates that the nucleotide N in siRNA is linked to C16, specifically, the 2'-O in the nucleotide N is linked to C16.

[0146] Example 2. In vitro activity screening of siRNA HCT116 cell line

[0147] 2.1 Experimental Procedure

[0148] 2.1.1 Cell Culture

[0149] HCT116 cells (BNCC, BNCC287750) were cultured in DMEM complete medium (Eallbio, with 10% FBS added) at 37°C and 5% CO2. When the confluence reached 80%–90%, the cells were digested with trypsin, counted, and transfected.

[0150] 2.1.2 Preparation of siRNA dilution buffer

[0151] (1) The dry powder of the siRNA to be tested was centrifuged at low temperature and high speed, and then dissolved in ultrapure distilled water to prepare a 100 μM siRNA stock solution.

[0152] (2) Prepare 200 nM siRNA dilution solution Y.

[0153] a) Take 50 μl of the 100 μM siRNA stock solution obtained in step (1) above, add 50 μl of ultrapure distilled water to obtain a siRNA dilution solution with a final concentration of 50 μM.

[0154] b) Take 2 μl of the 50 μM siRNA dilution solution obtained in step a) and add 18 μl of ultrapure distilled water to obtain siRNA stock solution X with a final concentration of 5 μM.

[0155] c) Take 2 μl of the prepared siRNA stock solution X and add 48 μl of Opti-medium (Gibco, 31985070) to obtain 200 nM siRNA dilution solution Y.

[0156] 2.1.3 HCT116 cell transfection

[0157] Pick 0.6 μl of transfection reagent was added to 10 μl of Opti-medium to obtain... Transfection reagent dilution solution; The transfection reagent diluent and the 200nM siRNA diluent Y prepared in step 2.1.2 were mixed at a volume ratio of 1:1 to prepare a transfection mixture. After standing for 5 minutes, 10 μl of the transfection mixture was added to a 96-well plate, along with 90 μl of HCT116 cells cultured in step 2.1.1 (final volume 100 μl / well, cell number 20,000 / well; taking siRNA diluent Y as 200nM as an example, the concentration of siRNA in this system is 10nM). The plate was then cultured for 24 hours after transfection.

[0158] 2.1.4 RNA Extraction

[0159] Total RNA was extracted from HCT116 cells obtained in step 2.1.3 according to the FlysisAmp Cells-to-CT 1-Step SYBR Green Kit product instructions.

[0160] 2.1.5 Quantitative Real-Time PCR

[0161] The extracted total RNA was analyzed by reverse transcription and real-time PCR using the FlysisAmp Cells-to-CT 1-Step SYBR Green Kit.

[0162] 2.1.6 Results Analysis

[0163] (1) Use the software of the 7500 real-time fluorescence quantitative PCR instrument (Thermo Fisher) to automatically calculate the Ct value;

[0164] (2) Calculate the relative expression level of the gene using the following formula:

[0165] ΔCt1=Ct(APP group)–Ct(APP group's ACTIN)

[0166] ΔCt2=Ct(siCtrl group)–Ct(siCtrl group's ACTIN)

[0167] ΔCt = ΔCt1 (APP group) - ΔCt2 (siCtrl group), where the siCtrl group is the negative control group;

[0168] mRNA expression relative to the siCtrl group = 2 -ΔΔCt

[0169] Inhibition rate (%) = (1 - mRNA expression relative to siCtrl group) × 100%.

[0170] 2.2 Experimental Results

[0171] The inhibitory effects of the siRNA of this invention are shown in Table 5 below:

[0172] Table 5. Results of in vitro screening of HCT116 cell lines using siRNA

[0173] As can be seen from Table 5, some of the siRNAs of the present invention can significantly inhibit the expression of the APP gene in HCT116 cells at 10 nM.

[0174] Example 3. In vitro activity screening of siRNA BE(2)-C cell line

[0175] 3.1 Experimental Procedure

[0176] 3.1.1 Cell Culture

[0177] BE(2)-C cells (BNCC, BNCC247047) were cultured in DMEM complete medium (Eallbio, with 10% FBS added) at 37°C and 5% CO2. When the confluence reached 80%–90%, the cells were digested with trypsin, counted, and transfected.

[0178] 3.1.2 Preparation of siRNA dilution buffer

[0179] (1) The dry powder of the siRNA to be tested was centrifuged at low temperature and high speed, and then dissolved in ultrapure distilled water to prepare a 100 μM siRNA stock solution.

[0180] (2) Prepare 200 nM siRNA dilution solution Y.

[0181] a) Take 50 μl of the 100 μM siRNA stock solution obtained in step (1) above, add 50 μl of ultrapure distilled water to obtain a siRNA dilution solution with a final concentration of 50 μM.

[0182] b) Take 2 μl of the 50 μM siRNA dilution solution obtained in step a) and add 18 μl of ultrapure distilled water to obtain siRNA stock solution X with a final concentration of 5 μM.

[0183] c) Take 2 μl of the prepared siRNA stock solution X and add 48 μl of Opti-medium to obtain a 200 nM siRNA dilution solution Y.

[0184] 3.1.3 BE(2)-C cell transfection

[0185] Pick 0.6 μl of transfection reagent was added to 10 μl of Opti-medium to obtain... Transfection reagent dilution solution; The transfection reagent diluent and the 200nM siRNA diluent Y prepared in step 3.1.2 were mixed at a volume ratio of 1:1 to prepare a transfection mixture. After standing for 5 minutes, 10 μl of the transfection mixture was added to a 96-well plate, along with 90 μl of BE(2)-C cells cultured in step 3.1.1 (final volume 100 μl / well, cell number 20,000 / well, siRNA concentration in this system 10 nM). The cells were then cultured for 24 hours after transfection.

[0186] 3.1.4 RNA Extraction

[0187] According to the instructions for the FlysisAmp Cells-to-CT 1-Step SYBR Green Kit, total RNA was extracted from BE(2)-C cells obtained in step 3.1.3.

[0188] 3.1.5 Quantitative Real-Time PCR

[0189] The extracted total RNA was analyzed by reverse transcription and real-time PCR using the FlysisAmp Cells-to-CT 1-Step SYBR Green Kit.

[0190] 3.1.6 Results Analysis

[0191] (1) Use the software of the 7500 real-time fluorescence quantitative PCR instrument (ThermoFisher) to automatically calculate the Ct value;

[0192] (2) Calculate the relative expression level of the gene using the following formula:

[0193] ΔCt1=Ct(APP group)–Ct(APP group's ACTIN)

[0194] ΔCt2=Ct(siCtrl group)–Ct(siCtrl group's ACTIN)

[0195] ΔCt = ΔCt1 (APP group) - ΔCt2 (siCtrl group), where the siCtrl group is the negative control group;

[0196] mRNA expression relative to the siCtrl group = 2 -ΔΔCt

[0197] Inhibition rate (%) = (1 - mRNA expression relative to siCtrl group) × 100%.

[0198] 3.2 Experimental Results

[0199] The inhibitory effect of the siRNA of the present invention is shown in Table 6 below:

[0200] Table 6. Results of in vitro screening of siRNA BE(2)-C cell lines

[0201] As shown in Table 6, some of the siRNAs of this invention can significantly inhibit the expression of the APP gene in BE(2)-C cells at 10 nM. Based on the in vitro screening results of Examples 2 and 3, the highly efficient activity range for inhibiting APP mRNA was determined to be positions 334-804, 2305-2327, and 2813-2835, calculated according to NCBI refseqID NM_000484.4.

[0202] Example 4. siRNA IC50 assay

[0203] This embodiment examines the dose-response relationship between drug dosage and biological effect by calculating the half-maximal inhibitory concentration (IC50) of each siRNA, thereby quantitatively reflecting the ability of the drug to cause changes in this indicator.

[0204] 4.1 The IC50 of siRNA inhibiting APP gene expression was determined using a method similar to that in Example 2, wherein the concentrations of transfected siRNA were 25 nM, 5 nM, 1 nM, 0.2 nM, 0.04 nM, 0.008 nM, and 0.0016 nM, respectively.

[0205] 4.2 Results Analysis

[0206] (1) Use the software of the 7500 real-time fluorescence quantitative PCR instrument (Thermo Fisher) to automatically calculate the Ct value;

[0207] (2) Calculate the relative expression level of the gene using the following formula:

[0208] ΔCt1=Ct(APP group)–Ct(APP group's ACTIN)

[0209] ΔCt2=Ct(siCtrl group)–Ct(siCtrl group's ACTIN)

[0210] ΔCt = ΔCt1 (APP group) - ΔCt2 (siCtrl group), where siCtrl group represents the negative control group;

[0211] mRNA expression relative to the siCtrl group = 2 -ΔΔCt

[0212] Inhibition rate (%) = (1 - mRNA expression relative to siCtrl group) × 100%.

[0213] Using the log value of siRNA concentration as the X-axis and the percentage inhibition rate as the Y-axis, dose-response curves were fitted using data analysis software to obtain the IC50 value of each siRNA.

[0214] The fitting formula is: Y = 100 / (1 + 10^((LogIC50 - X) × HillSlope))

[0215] Where HillSlope represents the slope of the percentage inhibition rate curve.

[0216] 4.3 Experimental Results

[0217] The IC50 assay results of the siRNA of this invention are shown in Table 7 below:

[0218] Table 7. IC50 assay results of siRNA in HCT116 cell line

[0219] As can be seen from Table 7, some of the siRNAs of the present invention can significantly inhibit APP gene expression.

[0220] Example 5. In vivo activity screening of siRNA conjugates

[0221] 5.1 Experimental Procedure

[0222] Six- to eight-week-old male mice (C57BL / 6) were purchased from Spiford (Beijing) Biotechnology Co., Ltd., weighing approximately 20g. Each mouse was intravenously injected with 1×10 11 A recombinant adeno-associated virus 8 (AAV8) vector (purchased from Yunzhou Biotechnology (Guangzhou) Co., Ltd.) containing one genome copy was used. The recombinant AAV8 vector consisted of an AAV8 capsid protein expression plasmid and a transfer plasmid carrying AAV8-hAPP. The transfer plasmid carried positions 151-3583 of the human APP sequence (NM_000484.4), controlled by the TBG promoter. The AAV8-hAPP transgenic mouse model was established 14 days after injection. Mice were then subcutaneously administered the conjugate at a dose of 3 mg / kg per mouse. Mice were sacrificed on days 7 (D7) and 14 (D14) after administration, with three mice from each group. Liver tissue was collected for mRNA expression detection. Total RNA was extracted using the Trizol method. mRNA was reverse transcribed using the HiScript III RT SuperMix for qPCR (+gDNA wiper) kit (catalog number: R323-01, Vazyme). Real-time quantitative PCR was performed using the ChamQ Universal SYBR qPCR Master Mix kit (catalog number: Q711-03, Vazyme). The in vivo activity results of different siRNA conjugates are shown in Table 8 and Figure 2.

[0223] Table 8. In vivo screening results of siRNA conjugates

[0224] 5.2 Explanation of Experimental Results

[0225] The chemically modified siRNA sequences exhibited good activity in mice. YGND22-9M, YGND22-16M, YGND22-17M, YGND22-23M, YGND22-24M, YGND22-28M, YGND22-71M, and YGND22-92M all significantly reduced APP expression levels in mice. At day 14, YGND22-28M showed an inhibition efficiency as high as 81.17%, while YGND22-16M achieved an inhibition efficiency of 71.21%.

[0226] Example 6. Screening of brain activity of siRNA conjugates

[0227] 6.1 Experimental Procedure

[0228] Six- to eight-week-old male mice (C57BL / 6) were purchased from Spiford (Beijing) Biotechnology Co., Ltd., weighing approximately 20g. Each mouse was intravenously injected with 1×10 11 A recombinant adeno-associated virus (AAV-PHP.eB) vector (purchased from Yunzhou Biotechnology (Guangzhou) Co., Ltd.) with 1 copy of the genome was used. The recombinant AAV-PHP.eB vector plasmid carried the human APP sequence (NM_000484.4). Fourteen days after injection, mouse cerebral cortex tissue was collected, and mRNA was detected by RT-qPCR. The results showed that human APP mRNA was overexpressed approximately 774-fold in the mouse cerebral cortex tissue, confirming the successful construction of the transgenic mouse model. The mice were then divided into 8 treatment groups and 1 control group, with 3 mice in each group. On day 0, 120 μg of siRNA conjugate was injected intracerebroventricularly (ICV) into each mouse. On day 14, central nervous system tissue and spinal cord tissue, including the cerebral cortex, hippocampus, and thoracic spinal cord, were collected, flash-frozen in liquid nitrogen, and mRNA was detected by RT-qPCR. The brain activity results of different siRNA conjugates are shown in Figure 3.

[0229] 6.2 Explanation of Experimental Results

[0230] The siRNA conjugates exhibited good activity in the mouse brain. YGND22-9M', YGND22-16M', YGND22-17M', YGND22-23M', YGND22-24M', YGND22-28M', YGND22-71M', and YGND22-92M' all significantly reduced APP expression levels in the mouse brain. On day 14, YGND22-92M' showed the highest inhibitory efficiency, with inhibition rates reaching 95.57% in the cerebral cortex, 95.59% in the hippocampus, and 95.51% in the thoracic spinal cord.

[0231] Example 7. Screening of brain activity of siRNA conjugates

[0232] 7.1 Experimental Procedure

[0233] Four-week-old male FAD3T transgenic mice (Alzheimer's disease model mice, purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.) were randomly divided into four treatment groups and one control group, with three mice in each group. On day 0, each mouse was injected with 120 μg of siRNA conjugate via ICV. On day 14, central nervous system tissues, including the cerebral cortex and hippocampus, were collected, flash-frozen in liquid nitrogen, and mRNA was detected by RT-qPCR. The brain activity results of different siRNA conjugates are shown in Figure 4.

[0234] 7.2 Explanation of Experimental Results

[0235] The siRNA conjugates showed good activity in the mouse brain, and YGND22-23M', YGND22-24M', YGND22-9M'G and YGND22-16M'G all significantly reduced the expression level of APP in the mouse brain.

[0236] Example 8. Cytotoxicity Results of siRNA Conjugates

[0237] 8.1 Experimental Procedure

[0238] Following experimental step 3.1 in Example 3, BE(2)-C cell culture and transfection were completed, with siRNA concentrations of 5 nM and 50 nM in the system. After 72 hours of culture, the cytotoxicity of each siRNA conjugate was measured by determining the cell viability / cytotoxicity ratio in each sample. Cell viability was measured by determining intracellular ATP content using a CellTiter-Glo (Promega, catalog number G7570) assay, according to the manufacturer's protocol. ToxiLight was used according to the manufacturer's protocol. TM (Lonza, catalog number LT07-217) Measures cytotoxicity in the supernatant.

[0239] 8.2 Experimental Results

[0240] The cytotoxicity results of the siRNA conjugates are shown in Figure 5.

[0241] The results showed that some of the siRNA conjugates of the present invention exhibited low cytotoxicity and good cell compatibility.

[0242] Example 9. Off-target analysis of siRNA conjugates

[0243] 9.1 Experimental Procedure

[0244] Following experimental step 3.1 in Example 3, BE(2)-C cell culture and transfection were completed. After 24 hours of culture, total RNA was extracted from BE(2)-C cells according to the instructions of the FlysisAmp Cells-to-CT 1-Step SYBR Green Kit. The RNA was sent to a third-party company for transcriptome analysis to identify differentially expressed genes. In the differentially expressed gene analysis, genes with padj≤0.05 and |log2FoldChange|>0.5145 were defined as differentially expressed genes. Here, padj≤0.05 indicates the significance level after multiple validation correction, ensuring that the identification results of differentially expressed genes have a confidence level of more than 95%; while |log2FoldChange|>0.5145 corresponds to a gene expression level that is at least upregulated by 1.43-fold (2^0.5145) or downregulated to 70% of the control group (2^-0.5145), thereby excluding minor fluctuations and focusing on genes with significant changes in expression levels. The specific results are shown in Table 9.

[0245] 9.2 Experimental Results

[0246] Transcriptome results showed that some of the siRNA conjugates of this invention caused less intracellular gene variation, had high safety, and could effectively prevent off-target effects.

[0247] Table 9. Transcriptome results of siRNA conjugates

Claims

1. A siRNA for inhibiting APP gene expression, the siRNA comprising a sense strand and an antisense strand, the sense strand comprising a nucleotide sequence I, and the antisense strand comprising a nucleotide sequence II; each nucleotide in nucleotide sequence I and nucleotide sequence II is a modified or unmodified nucleotide; nucleotide sequence I and nucleotide sequence II are at least partially anticomplementary to form a double-stranded region; nucleotide sequence I is substantially identical to a first nucleotide sequence, the first nucleotide sequence being a nucleotide sequence of at least 15 nucleotides in length in the mRNA expressing the APP gene, preferably, the first nucleotide sequence being a nucleotide sequence of 15 to 25 nucleotides in length in the mRNA expressing the APP gene, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides; Preferably, the first nucleotide sequence is a nucleotide sequence of at least 15 nucleotides in length from the highly active region of the mRNA expressed by the APP gene, preferably a nucleotide sequence of 15 to 25 nucleotides. The highly active region is positions 334 to 804, 2305 to 2327, and 2813 to 2835 of the mRNA expressed by the APP gene, preferably positions 334 to 356, 490 to 512, 503 to 525, 635 to 656, 641 to 662, 782 to 804, 2305 to 2327, and 2813 to 2835. The mRNA expressed by the APP gene is shown in NCBI refseqID NM_000484.4; specifically, the mRNA sequence is shown in SEQ ID NO:

1.

2. The siRNA according to claim 1, wherein, The nucleotide sequence I has at least 70%, at least 80%, at least 85%, at least 90%, and at least 95% sequence identity with the first nucleotide segment.

3. The siRNA according to claim 1 or 2, wherein, The sequence I comprises at least 15 consecutive nucleotides as shown in any of the sequences in SEQ ID NO:2 to 111, such as at least 15, 16, 17, 18, 19, 20, or 21 nucleotides; or, the nucleic acid sequence of the positive strand is as shown in sequence I, and sequence I differs from any of the sequences in SEQ ID NO:2 to 111 by 1, 2, or 3 nucleotides; preferably, sequence I is as shown in any of the sequences in SEQ ID NO:2 to 111. The sequence II comprises at least 15 consecutive nucleotides as shown in any of the sequences in SEQ ID NO:112 to 221, such as at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides; or, the nucleic acid sequence of the antisense strand is as shown in sequence II, and the sequence II differs from any of the sequences in SEQ ID NO:112 to 221 by 1, 2, or 3 nucleotides; preferably, the sequence II is as shown in any of the sequences in SEQ ID NO:112 to 221.

4. The siRNA according to claim 3, wherein the siRNA is selected from the siRNA molecules shown in Table 1.

5. The siRNA according to any one of claims 1 to 4, wherein, Each nucleotide in nucleotide sequence I and nucleotide sequence II is a modified nucleotide, wherein the modified nucleotide is a fluorinated nucleotide or a non-fluorinated nucleotide; preferably, the fluorinated nucleotide refers to a nucleotide formed by replacing the hydroxyl group at the 2' position of the ribosyl group with fluorine, having the structure shown in formula (1); the non-fluorinated nucleotide refers to a nucleotide or nucleotide analog formed by replacing the hydroxyl group at the 2' position of the ribosyl group with a non-fluorinated group, having the structure shown in any one of formulas (2) to (12): In equations (1) to (12), Base represents a base; In formulas (9) and (10), R is selected from H, OH or alkoxy (O-alkyl); In formulas (11) and (12), R is selected from H, OH, F or the non-fluorine group.

6. The siRNA according to claim 5, wherein, In the direction from the 5' end to the 3' end, one or more nucleotides at positions 7, 9, 10, and 11 of nucleotide sequence I are fluorinated nucleotides; and in the direction from the 5' end to the 3' end, one or more nucleotides at positions 2, 6, 8, 9, 13, 14, 15, and 16 of nucleotide sequence II are fluorinated nucleotides; or The nucleotide sequence II is partially modified with GNA; preferably, the 7th position of the nucleotide sequence II is modified with GNA in the direction from the 5' end to the 3' end.

7. The siRNA according to claim 5 or 6, wherein, At least a portion of the phosphate ester groups in the phosphate-sugar backbone of at least one single strand of the sense and antisense strands of the siRNA are phosphate ester groups with modifying groups, wherein the phosphate ester group with modifying groups is a thiophosphate ester group formed by replacing at least one oxygen atom in the phosphodiester bond of the phosphate ester group with a sulfur atom; preferably, the phosphate ester group with modifying groups is a thiophosphate ester group having the structure shown in formula (13): Preferably, the thiophosphate group linkage is present at least one of the following positions: between the first and second nucleotides of the sense and / or antisense strands; between the second and third nucleotides of the sense and / or antisense strands; between the 19th and 20th nucleotides of the sense strand; between the 20th and 21st nucleotides of the sense strand; between the 21st and 22nd nucleotides of the antisense strand; between the 22nd and 23rd nucleotides of the antisense strand; or any combination thereof. Most preferably, the 5'-terminal nucleotide of the siRNA antisense strand is a 5'-phosphate nucleotide or a nucleotide modified with a 5'-phosphate analogue, as shown in formulas (14), (15), and (16):

8. An siRNA conjugate comprising the siRNA according to any one of claims 5 to 7 and a conjugate group conjugated to the siRNA, wherein, The conjugating group is galactose, N-acetylgalactosamine, or a positive-chain alkyl group; Preferably, the galactose or N-acetylgalactosamine molecule can be monovalent, divalent, trivalent, or tetravalent; the positive-chain alkyl group is n-hexadecyl (C16); More preferably, the conjugating group is L96, with the structure shown in formula (I), or the conjugating group is C16, and the nucleoside structure conjugated by C16 is shown in formula (II). Most preferably, the siRNA conjugate is selected from the siRNA conjugates shown in Table 2, Table 3 or Table 4.

9. A composition comprising the siRNA of any one of claims 1 to 7 or the siRNA conjugate of claim 8; preferably, the composition is a pharmaceutical composition, further comprising a pharmaceutically acceptable carrier or excipient; more preferably, the carrier or excipient includes, but is not limited to, water for injection, sodium hydroxide, sodium dihydrogen phosphate monohydrate, sodium dihydrogen phosphate dihydrate, phosphoric acid, sodium chloride, potassium chloride, hydrochloric acid, anhydrous potassium dihydrogen phosphate, anhydrous disodium hydrogen phosphate, PEG2000, PEG6000, cholesterol, distearate, glyceryl 1,2-dimyristate, and dimethyl adipic acid.

10. Use of the siRNA according to any one of claims 1 to 7, or the siRNA conjugate according to claim 8, or the pharmaceutical composition according to claim 9 in the preparation of a medicament for the prevention and / or treatment of diseases related to excessive APP; preferably, the diseases related to excessive APP are selected from cerebral amyloid angiopathy (CAA) and Alzheimer's disease (AD).

11. A method for preventing or treating diseases related to excessive APP, comprising administering to a subject a therapeutically effective amount or a preventatively effective amount of the siRNA of any one of claims 1 to 7, or the siRNA conjugate of claim 8, or the pharmaceutical composition of claim 9; preferably, the diseases related to excessive APP are selected from cerebral amyloid angiopathy (CAA) and Alzheimer's disease (AD).

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