siRNA for inhibiting APP gene expression and its conjugate and application

Abstract

The present invention belongs to the field of biomedicine and relates to an siRNA and conjugate for inhibiting APP gene expression. The siRNA comprises a sense strand and an antisense strand, wherein the sense strand comprises nucleotide sequence I and the antisense strand comprises 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 reverse-complementary to form a double-stranded region; nucleotide sequence I is substantially identical to a first nucleotide sequence segment, which is a nucleotide sequence of at least 19 nucleotides in length in the mRNA expressed by the APP gene. The siRNA of the present invention can specifically induce the degradation of APP mRNA, thereby inhibiting APP synthesis in the liver, inducing a sustained reduction in APP protein, and reducing the pathological deposition of related toxic proteins such as Aβ, and has good drug development prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to siRNA and a conjugate for inhibiting APP gene expression. Background Art

[0002] Alzheimer's disease (AD) is the leading cause of dementia and is defined by amyloid-β plaques and neurofibrillary tangles, manifesting as amnesia or impairment of vision, language, executive, behavioral, or motor function.

[0003] Cerebral amyloid angiopathy (CAA) is a common neurodegenerative disease in the elderly brain characterized by the deposition of amyloid β (Aβ) in intracranial microvasculature (pial arteries, cortical arterioles, and capillaries). Clinically, CAA can present with different subtypes, including lobar hemorrhage, cognitive impairment, rapidly progressive dementia, and cerebral amyloid attacks. CAA 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 APP (Amyloid Precursor Protein) gene, located on chromosome 21q21.3, contains 20 exons and encodes the amyloid precursor protein, the APP membrane protein. Studies have confirmed that β-amyloid (Aβ), produced by the breakdown of APP, is a key pathological factor in both AD and CAA. Both APP and Aβ are implicated in AD. The toxicity of Aβ depends on APP expression, and the effects of APP extend beyond the production of toxic fragments. Aging accelerates Aβ accumulation by inducing APP processing and upregulating β-secretase. Transgenic mice overexpressing APP also exhibit significant alterations in vasoactive signaling, leading to neurovascular dysfunction. Reducing APP expression leads 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, therapies targeting APP include immunotherapy or APP secretase inhibitors, but the key issue in immunotherapy is the effective delivery of drugs to the brain. Others may be terminated in clinical trials due to toxicity or side effects. Clinical evidence for antibody drugs targeting Aβ protein is concentrated in patients with early AD. Therefore, patients in the middle and late stages do not meet the indications for medication and have toxic side effects such as cerebral edema. Other specific drugs for AD, such as acetylcholine inhibitors, can only alleviate mild to moderate AD symptoms and have no significant therapeutic effect. Summary of the Invention

[0006] The purpose of the present invention is to provide a siRNA molecule capable of inhibiting 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, which comprises a sense chain and an antisense chain, the sense chain comprising a nucleotide sequence I, and the antisense chain comprising 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 basically consistent with the first nucleotide sequence, and the first nucleotide sequence is a nucleotide sequence with a length of at least 19 nucleotides in the mRNA expressed by the APP gene.

[0008] In a preferred embodiment, the first nucleotide sequence is a nucleotide sequence of 19 to 25 nucleotides in length in the mRNA expressed by the APP gene, for example, 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 19 nucleotides in length in the high activity interval of the mRNA expressed by the APP gene, such as a nucleotide sequence of 19 to 25 nucleotides. The high activity interval 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 range means that the siRNA and siRNA conjugate designed within this range can effectively reduce the level of APPmRNA. Figure 1 The results were divided into intervals based on whether the observed maximum inhibition rate of APP mRNA by siRNA and siRNA conjugates fell between 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%, or at least 95% sequence identity with the first stretch of nucleotides.

[0012] In some embodiments, in the above siRNA, the nucleotide sequence II is substantially reverse complementary, essentially reverse complementary, or completely reverse complementary to the first nucleotide sequence.

[0013] The sense strand and antisense strand are of the same or different lengths, the sense strand is 16-23 nucleotides long, and the antisense strand is 19-26 nucleotides long. In some embodiments, the length ratio of the siRNA sense strand to the antisense strand is 16 / 21, 19 / 21, 21 / 23, or 19 / 24.

[0014] In a specific embodiment, the sequence I comprises at least 15 consecutive nucleotides as shown in any one of SEQ ID NOs: 2-111, such as at least 15, 16, 17, 18, 19, 20 or 21 nucleotides. Preferably, the sequence I is as shown in any one of SEQ ID NOs: 2-111.

[0015] In a specific embodiment, the sequence II comprises at least 15 consecutive nucleotides as shown in any one of SEQ ID NOs: 112-221, such as at least 15, 16, 17, 18, 19, 20, 21, 22 or 23 nucleotides. Preferably, the sequence II is as shown in any one of SEQ ID NOs: 112-221.

[0016] In a specific embodiment, the siRNA includes a sense strand and an antisense strand, which are:

[0017] 1) The sequence of the sense strand is shown in SEQ ID NO: 2, and the sequence of the antisense strand is shown in SEQ ID NO: 112;

[0018] 2) the sequence of the sense strand is shown in SEQ ID NO: 3, and the sequence of the antisense strand is shown in SEQ ID NO: 113;

[0019] 3) the sequence of the sense strand is shown in SEQ ID NO: 4, and the sequence of the antisense strand is shown in SEQ ID NO: 114;

[0020] 4) the sequence of the sense strand is shown in SEQ ID NO: 5, and the sequence of the antisense strand is shown in SEQ ID NO: 115;

[0021] 5) the sequence of the sense strand is shown in SEQ ID NO: 6, and the sequence of the antisense strand is shown in SEQ ID NO: 116;

[0022] 6) the sequence of the sense strand is shown in SEQ ID NO: 7, and the sequence of the antisense strand is shown in SEQ ID NO: 117;

[0023] 7) the sequence of the sense strand is shown in SEQ ID NO: 8, and the sequence of the antisense strand is shown in SEQ ID NO: 118;

[0024] 8) the sequence of the sense strand is shown in SEQ ID NO: 9, and the sequence of the antisense strand is shown in SEQ ID NO: 119;

[0025] 9) the sequence of the sense strand is shown in SEQ ID NO: 10, and the sequence of the antisense strand is shown in SEQ ID NO: 120;

[0026] 10) The sequence of the sense strand is shown in SEQ ID NO: 11, and the sequence of the antisense strand is shown in SEQ ID NO: 121;

[0027] 11) The sequence of the sense strand is shown in SEQ ID NO: 12, and the sequence of the antisense strand is shown in SEQ ID NO: 122;

[0028] 12) the sequence of the sense strand is shown in SEQ ID NO: 13, and the sequence of the antisense strand is shown in SEQ ID NO: 123;

[0029] 13) the sequence of the sense strand is shown in SEQ ID NO: 14, and the sequence of the antisense strand is shown in SEQ ID NO: 124;

[0030] 14) the sequence of the sense strand is shown in SEQ ID NO: 15, and the sequence of the antisense strand is shown in SEQ ID NO: 125;

[0031] 15) the sequence of the sense strand is shown in SEQ ID NO: 16, and the sequence of the antisense strand is shown in SEQ ID NO: 126;

[0032] 16) The sequence of the sense strand is shown in SEQ ID NO: 17, and the sequence of the antisense strand is shown in SEQ ID NO: 127;

[0033] 17) The sequence of the sense strand is shown in SEQ ID NO: 18, and the sequence of the antisense strand is shown in SEQ ID NO: 128;

[0034] 18) The sequence of the sense strand is shown in SEQ ID NO: 19, and the sequence of the antisense strand is shown in SEQ ID NO: 129;

[0035] 19) The sequence of the sense strand is shown in SEQ ID NO: 20, and the sequence of the antisense strand is shown in SEQ ID NO: 130;

[0036] 20) The sequence of the sense strand is shown in SEQ ID NO: 21, and the sequence of the antisense strand is shown in SEQ ID NO: 131;

[0037] 21) the sequence of the sense strand is shown in SEQ ID NO: 22, and the sequence of the antisense strand is shown in SEQ ID NO: 132;

[0038] 22) the sequence of the sense strand is shown in SEQ ID NO: 23, and the sequence of the antisense strand is shown in SEQ ID NO: 133;

[0039] 23) the sequence of the sense strand is shown in SEQ ID NO: 24, and the sequence of the antisense strand is shown in SEQ ID NO: 134;

[0040] 24) the sequence of the sense strand is shown in SEQ ID NO: 25, and the sequence of the antisense strand is shown in SEQ ID NO: 135;

[0041] 25) the sequence of the sense strand is shown in SEQ ID NO: 26, and the sequence of the antisense strand is shown in SEQ ID NO: 136;

[0042] 26) the sequence of the sense strand is shown in SEQ ID NO: 27, and the sequence of the antisense strand is shown in SEQ ID NO: 137;

[0043] 27) The sequence of the sense strand is shown in SEQ ID NO: 28, and the sequence of the antisense strand is shown in SEQ ID NO: 138;

[0044] 28) The sequence of the sense strand is shown in SEQ ID NO: 29, and the sequence of the antisense strand is shown in SEQ ID NO: 139;

[0045] 29) The sequence of the sense strand is shown in SEQ ID NO: 30, and the sequence of the antisense strand is shown in SEQ ID NO: 140;

[0046] 30) The sequence of the sense strand is shown in SEQ ID NO: 31, and the sequence of the antisense strand is shown in SEQ ID NO: 141;

[0047] 31) The sequence of the sense strand is shown in SEQ ID NO: 32, and the sequence of the antisense strand is shown in SEQ ID NO: 142;

[0048] 32) the sequence of the sense strand is shown in SEQ ID NO: 33, and the sequence of the antisense strand is shown in SEQ ID NO: 143;

[0049] 33) the sequence of the sense strand is shown in SEQ ID NO: 34, and the sequence of the antisense strand is shown in SEQ ID NO: 144;

[0050] 34) the sequence of the sense strand is shown in SEQ ID NO: 35, and the sequence of the antisense strand is shown in SEQ ID NO: 145;

[0051] 35) the sequence of the sense strand is shown in SEQ ID NO: 36, and the sequence of the antisense strand is shown in SEQ ID NO: 146;

[0052] 36) the sequence of the sense strand is shown in SEQ ID NO: 37, and the sequence of the antisense strand is shown in SEQ ID NO: 147;

[0053] 37) The sequence of the sense strand is shown in SEQ ID NO: 38, and the sequence of the antisense strand is shown in SEQ ID NO: 148;

[0054] 38) The sequence of the sense strand is shown in SEQ ID NO: 39, and the sequence of the antisense strand is shown in SEQ ID NO: 149;

[0055] 39) The sequence of the sense strand is shown in SEQ ID NO: 40, and the sequence of the antisense strand is shown in SEQ ID NO: 150;

[0056] 40) The sequence of the sense strand is shown in SEQ ID NO:41, and the sequence of the antisense strand is shown in SEQ ID NO:151;

[0057] 41) The sequence of the sense strand is shown in SEQ ID NO: 42, and the sequence of the antisense strand is shown in SEQ ID NO: 152;

[0058] 42) the sequence of the sense strand is shown in SEQ ID NO: 43, and the sequence of the antisense strand is shown in SEQ ID NO: 153;

[0059] 43) the sequence of the sense strand is shown in SEQ ID NO: 44, and the sequence of the antisense strand is shown in SEQ ID NO: 154;

[0060] 44) the sequence of the sense strand is shown in SEQ ID NO: 45, and the sequence of the antisense strand is shown in SEQ ID NO: 155;

[0061] 45) the sequence of the sense strand is shown in SEQ ID NO: 46, and the sequence of the antisense strand is shown in SEQ ID NO: 156;

[0062] 46) the sequence of the sense strand is shown in SEQ ID NO: 47, and the sequence of the antisense strand is shown in SEQ ID NO: 157;

[0063] 47) The sequence of the sense strand is shown in SEQ ID NO: 48, and the sequence of the antisense strand is shown in SEQ ID NO: 158;

[0064] 48) The sequence of the sense strand is shown in SEQ ID NO: 49, and the sequence of the antisense strand is shown in SEQ ID NO: 159;

[0065] 49) The sequence of the sense strand is shown in SEQ ID NO: 50, and the sequence of the antisense strand is shown in SEQ ID NO: 160;

[0066] 50) The sequence of the sense strand is shown in SEQ ID NO: 51, and the sequence of the antisense strand is shown in SEQ ID NO: 161;

[0067] 51) The sequence of the sense strand is shown in SEQ ID NO: 52, and the sequence of the antisense strand is shown in SEQ ID NO: 162;

[0068] 52) the sequence of the sense strand is shown in SEQ ID NO: 53, and the sequence of the antisense strand is shown in SEQ ID NO: 163;

[0069] 53) the sequence of the sense strand is shown in SEQ ID NO: 54, and the sequence of the antisense strand is shown in SEQ ID NO: 164;

[0070] 54) the sequence of the sense strand is shown in SEQ ID NO: 55, and the sequence of the antisense strand is shown in SEQ ID NO: 165;

[0071] 55) the sequence of the sense strand is shown in SEQ ID NO: 56, and the sequence of the antisense strand is shown in SEQ ID NO: 166;

[0072] 56) The sequence of the sense strand is shown in SEQ ID NO: 57, and the sequence of the antisense strand is shown in SEQ ID NO: 167;

[0073] 57) The sequence of the sense strand is shown in SEQ ID NO: 58, and the sequence of the antisense strand is shown in SEQ ID NO: 168;

[0074] 58) The sequence of the sense strand is shown in SEQ ID NO: 59, and the sequence of the antisense strand is shown in SEQ ID NO: 169;

[0075] 59) The sequence of the sense strand is shown in SEQ ID NO: 60, and the sequence of the antisense strand is shown in SEQ ID NO: 170;

[0076] 60) The sequence of the sense strand is shown in SEQ ID NO: 61, and the sequence of the antisense strand is shown in SEQ ID NO: 171;

[0077] 61) The sequence of the sense strand is shown in SEQ ID NO: 62, and the sequence of the antisense strand is shown in SEQ ID NO: 172;

[0078] 62) The sequence of the sense strand is shown in SEQ ID NO: 63, and the sequence of the antisense strand is shown in SEQ ID NO: 173;

[0079] 63) The sequence of the sense strand is shown in SEQ ID NO: 64, and the sequence of the antisense strand is shown in SEQ ID NO: 174;

[0080] 64) The sequence of the sense strand is shown in SEQ ID NO: 65, and the sequence of the antisense strand is shown in SEQ ID NO: 175;

[0081] 65) The sequence of the sense strand is shown in SEQ ID NO: 66, and the sequence of the antisense strand is shown in SEQ ID NO: 176;

[0082] 66) The sequence of the sense strand is shown in SEQ ID NO: 67, and the sequence of the antisense strand is shown in SEQ ID NO: 177;

[0083] 67) The sequence of the sense strand is shown in SEQ ID NO: 68, and the sequence of the antisense strand is shown in SEQ ID NO: 178;

[0084] 68) The sequence of the sense strand is shown in SEQ ID NO: 69, and the sequence of the antisense strand is shown in SEQ ID NO: 179;

[0085] 69) The sequence of the sense strand is shown in SEQ ID NO: 70, and the sequence of the antisense strand is shown in SEQ ID NO: 180;

[0086] 70) The sequence of the sense strand is shown in SEQ ID NO: 71, and the sequence of the antisense strand is shown in SEQ ID NO: 181;

[0087] 71) The sequence of the sense strand is shown in SEQ ID NO: 72, and the sequence of the antisense strand is shown in SEQ ID NO: 182;

[0088] 72) The sequence of the sense strand is shown in SEQ ID NO: 73, and the sequence of the antisense strand is shown in SEQ ID NO: 183;

[0089] 73) The sequence of the sense strand is shown in SEQ ID NO: 74, and the sequence of the antisense strand is shown in SEQ ID NO: 184;

[0090] 74) The sequence of the sense strand is shown in SEQ ID NO: 75, and the sequence of the antisense strand is shown in SEQ ID NO: 185;

[0091] 75) The sequence of the sense strand is shown in SEQ ID NO: 76, and the sequence of the antisense strand is shown in SEQ ID NO: 186;

[0092] 76) The sequence of the sense strand is shown in SEQ ID NO: 77, and the sequence of the antisense strand is shown in SEQ ID NO: 187;

[0093] 77) The sequence of the sense strand is shown in SEQ ID NO: 78, and the sequence of the antisense strand is shown in SEQ ID NO: 188;

[0094] 78) The sequence of the sense strand is shown in SEQ ID NO: 79, and the sequence of the antisense strand is shown in SEQ ID NO: 189;

[0095] 79) The sequence of the sense strand is shown in SEQ ID NO: 80, and the sequence of the antisense strand is shown in SEQ ID NO: 190;

[0096] 80) The sequence of the sense strand is shown in SEQ ID NO: 81, and the sequence of the antisense strand is shown in SEQ ID NO: 191;

[0097] 81) The sequence of the sense strand is shown in SEQ ID NO: 82, and the sequence of the antisense strand is shown in SEQ ID NO: 192;

[0098] 82) the sequence of the sense strand is shown in SEQ ID NO: 83, and the sequence of the antisense strand is shown in SEQ ID NO: 193;

[0099] 83) The sequence of the sense strand is shown in SEQ ID NO: 84, and the sequence of the antisense strand is shown in SEQ ID NO: 194;

[0100] 84) the sequence of the sense strand is shown in SEQ ID NO: 85, and the sequence of the antisense strand is shown in SEQ ID NO: 195;

[0101] 85) the sequence of the sense strand is shown in SEQ ID NO: 86, and the sequence of the antisense strand is shown in SEQ ID NO: 196;

[0102] 86) The sequence of the sense strand is shown in SEQ ID NO: 87, and the sequence of the antisense strand is shown in SEQ ID NO: 197;

[0103] 87) The sequence of the sense strand is shown in SEQ ID NO: 88, and the sequence of the antisense strand is shown in SEQ ID NO: 198;

[0104] 88) The sequence of the sense strand is shown in SEQ ID NO: 89, and the sequence of the antisense strand is shown in SEQ ID NO: 199;

[0105] 89) The sequence of the sense strand is shown in SEQ ID NO: 90, and the sequence of the antisense strand is shown in SEQ ID NO: 200;

[0106] 90) The sequence of the sense strand is shown in SEQ ID NO: 91, and the sequence of the antisense strand is shown in SEQ ID NO: 201;

[0107] 91) The sequence of the sense strand is shown in SEQ ID NO: 92, and the sequence of the antisense strand is shown in SEQ ID NO: 202;

[0108] 92) The sequence of the sense strand is shown in SEQ ID NO: 93, and the sequence of the antisense strand is shown in SEQ ID NO: 203;

[0109] 93) The sequence of the sense strand is shown in SEQ ID NO: 94, and the sequence of the antisense strand is shown in SEQ ID NO: 204;

[0110] 94) The sequence of the sense strand is shown in SEQ ID NO: 95, and the sequence of the antisense strand is shown in SEQ ID NO: 205;

[0111] 95) The sequence of the sense strand is shown in SEQ ID NO: 96, and the sequence of the antisense strand is shown in SEQ ID NO: 206;

[0112] 96) The sequence of the sense strand is shown in SEQ ID NO: 97, and the sequence of the antisense strand is shown in SEQ ID NO: 207;

[0113] 97) The sequence of the sense strand is shown in SEQ ID NO: 98, and the sequence of the antisense strand is shown in SEQ ID NO: 208;

[0114] 98) The sequence of the sense strand is shown in SEQ ID NO: 99, and the sequence of the antisense strand is shown in SEQ ID NO: 209;

[0115] 99) The sequence of the sense strand is shown in SEQ ID NO: 100, and the sequence of the antisense strand is shown in SEQ ID NO: 210;

[0116] 100) The sequence of the sense strand is shown in SEQ ID NO: 101, and the sequence of the antisense strand is shown in SEQ ID NO: 211;

[0117] 101) The sequence of the sense strand is shown in SEQ ID NO: 102, and the sequence of the antisense strand is shown in SEQ ID NO: 212;

[0118] 102) the sequence of the sense strand is shown in SEQ ID NO: 103, and the sequence of the antisense strand is shown in SEQ ID NO: 213;

[0119] 103) The sequence of the sense strand is shown in SEQ ID NO: 104, and the sequence of the antisense strand is shown in SEQ ID NO: 214;

[0120] 104) The sequence of the sense strand is shown in SEQ ID NO: 105, and the sequence of the antisense strand is shown in SEQ ID NO: 215;

[0121] 105) The sequence of the sense strand is shown in SEQ ID NO: 106, and the sequence of the antisense strand is shown in SEQ ID NO: 216;

[0122] 106) The sequence of the sense strand is shown in SEQ ID NO: 107, and the sequence of the antisense strand is shown in SEQ ID NO: 217;

[0123] 107) The sequence of the sense strand is shown in SEQ ID NO: 108, and the sequence of the antisense strand is shown in SEQ ID NO: 218;

[0124] 108) The sequence of the sense strand is shown in SEQ ID NO: 109, and the sequence of the antisense strand is shown in SEQ ID NO: 219;

[0125] 109) The sequence of the sense strand is shown in SEQ ID NO: 110, and the sequence of the antisense strand is shown in SEQ ID NO: 220; or

[0126] 110) The sequence of the sense strand is shown in SEQ ID NO: 111, and the sequence of the antisense strand is shown in SEQ ID NO: 221.

[0127] In one aspect, in the siRNA provided by the present invention, each nucleotide in the nucleotide sequence I and the nucleotide sequence II is a modified nucleotide.

[0128] In a specific embodiment, the modified nucleotide is a fluorinated modified nucleotide or a non-fluorinated modified nucleotide.

[0129] In a specific embodiment, the fluoro-modified nucleotide refers to a nucleotide in which the hydroxyl group at the 2'-position of the ribose group of the nucleotide is replaced by fluorine, and has a structure shown in the following formula (1). The non-fluoro-modified nucleotide refers to a nucleotide or nucleotide analog in which the hydroxyl group at the 2'-position of the ribose group of the nucleotide is replaced by a non-fluoro group. In some embodiments, each non-fluoro-modified nucleotide is independently selected from one of the nucleotides or nucleotide analogs in which the hydroxyl group at the 2'-position of the ribose group of the nucleotide is replaced by a non-fluoro group. The nucleotides in which the hydroxyl group at the 2'-position of the ribose group is replaced by a non-fluoro group are well known to those skilled in the art, and these nucleotides can 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, can 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 as shown in formula (4). In some embodiments, the 2'-deoxynucleotide (DNA) is as shown in formula (5).

[0130]

[0131] Nucleotide analogs refer to groups that can replace nucleotides in nucleic acids but have structures different from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, or thymine ribonucleotides. In some embodiments, the nucleotide analogs can be isonucleotides, bridged nucleotides, or acyclic nucleotides.

[0132] A bridged nucleotide refers to a constrained or inaccessible nucleotide. The bridged nucleotide may contain a five-membered ring, a six-membered ring, or a seven-membered ring with a fixed C3-endo sugar condensation. In some embodiments, the bridged nucleotide may 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).

[0133]

[0134] Acyclic nucleotides are a type of nucleotide formed by opening the sugar ring of a nucleotide. In some embodiments, the acyclic nucleotide can be an unlocked nucleic acid (UNA) or a glycerol nucleic acid (GNA), wherein UNA is as shown in formula (9) and GNA is as shown in formula (10).

[0135]

[0136] In the above formulae (9) and (10), R is selected from H, OH, or alkoxy (O-alkyl).

[0137] An isonucleotide is a compound formed by a change in the position of a base on the ribose ring of a nucleotide. In some embodiments, an isonucleotide can be a compound formed by a base shifting from the 1' position to the 2' or 3' position of the ribose ring, as shown in Formula (11) or Formula (12).

[0138]

[0139] In the compounds of formula (11) and formula (12), R is selected from H, OH, F or the non-fluorine groups as described above;

[0140] In the compounds of formula (1) to formula (12), Base represents a base, such as A, U, G, C or T.

[0141] In a specific embodiment, from the 5' end to the 3' end, one or more nucleotides at positions 7, 9, 10, and 11 of the nucleotide sequence I are fluorinated modified nucleotides; and, from the 5' end to the 3' end, one or more nucleotides at positions 2, 6, 8, 9, 13, 14, 15, and 16 of the nucleotide sequence II are fluorinated modified nucleotides.

[0142] In a specific embodiment, at least a portion of the phosphate groups in the phosphate-sugar backbone of at least one single strand of the sense strand and the antisense strand of the siRNA provided by the present invention is a phosphate group having a modified group. In some implementation methods, the phosphate group having a modified group is a phosphorothioate group formed by replacing at least one oxygen atom in the phosphodiester bond of the phosphate group with a sulfur atom; in some embodiments, the phosphate group having a modified group is a phosphorothioate group having a structure as shown in formula (13):

[0143]

[0144] In some embodiments, in the siRNA provided by the present invention, preferably, the phosphorothioate linkage is present in at least one of the following positions: between the 1st and 2nd nucleotides of the sense strand and / or the antisense strand; between the 2nd and 3rd nucleotides of the sense strand and / or the antisense strand; between the 19th and 20th nucleotides of the antisense strand; between the 20th and 21st nucleotides of the antisense strand; between the 21st and 22nd nucleotides of the antisense strand; between the 22nd and 23rd nucleotides of the antisense strand; or any combination of the above.

[0145] In some embodiments, the 5'-terminal nucleotide of the antisense strand of the siRNA is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide, as shown in formula (14), formula (15) and formula (16):

[0146]

[0147] In one aspect, the present invention provides an siRNA conjugate comprising the above-described siRNA and a conjugated group conjugated to the siRNA. In some embodiments, the pharmaceutically acceptable conjugated 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. In some embodiments, the conjugation site of the siRNA to the conjugated group can be at the 3' or 5' end of the siRNA sense strand, at the 3' end of the antisense strand, or within the internal sequence of the siRNA.

[0148] In some specific embodiments, the siRNA conjugate of the present invention, the conjugated group is L96, and the structure is as follows:

[0149]

[0150] In a specific embodiment, the siRNA conjugate includes a sense strand and an antisense strand, which are:

[0151] 1) the sequence of the sense strand is shown in SEQ ID NO: 222, and the sequence of the antisense strand is shown in SEQ ID NO: 332;

[0152] 2) the sequence of the sense strand is shown in SEQ ID NO: 223, and the sequence of the antisense strand is shown in SEQ ID NO: 333;

[0153] 3) the sequence of the sense strand is shown in SEQ ID NO: 224, and the sequence of the antisense strand is shown in SEQ ID NO: 334;

[0154] 4) the sequence of the sense strand is shown in SEQ ID NO: 225, and the sequence of the antisense strand is shown in SEQ ID NO: 335;

[0155] 5) the sequence of the sense strand is shown in SEQ ID NO: 226, and the sequence of the antisense strand is shown in SEQ ID NO: 336;

[0156] 6) the sequence of the sense strand is shown in SEQ ID NO: 227, and the sequence of the antisense strand is shown in SEQ ID NO: 337;

[0157] 7) the sequence of the sense strand is shown in SEQ ID NO: 228, and the sequence of the antisense strand is shown in SEQ ID NO: 338;

[0158] 8) the sequence of the sense strand is shown in SEQ ID NO: 229, and the sequence of the antisense strand is shown in SEQ ID NO: 339;

[0159] 9) the sequence of the sense strand is shown in SEQ ID NO: 230, and the sequence of the antisense strand is shown in SEQ ID NO: 340;

[0160] 10) the sequence of the sense strand is shown in SEQ ID NO: 231, and the sequence of the antisense strand is shown in SEQ ID NO: 341;

[0161] 11) the sequence of the sense strand is shown in SEQ ID NO: 232, and the sequence of the antisense strand is shown in SEQ ID NO: 342;

[0162] 12) the sequence of the sense strand is shown in SEQ ID NO: 233, and the sequence of the antisense strand is shown in SEQ ID NO: 343;

[0163] 13) the sequence of the sense strand is shown in SEQ ID NO: 234, and the sequence of the antisense strand is shown in SEQ ID NO: 344;

[0164] 14) the sequence of the sense strand is shown in SEQ ID NO: 235, and the sequence of the antisense strand is shown in SEQ ID NO: 345;

[0165] 15) the sequence of the sense strand is shown in SEQ ID NO: 236, and the sequence of the antisense strand is shown in SEQ ID NO: 346;

[0166] 16) the sequence of the sense strand is shown in SEQ ID NO: 237, and the sequence of the antisense strand is shown in SEQ ID NO: 347;

[0167] 17) the sequence of the sense strand is shown in SEQ ID NO: 238, and the sequence of the antisense strand is shown in SEQ ID NO: 348;

[0168] 18) The sequence of the sense strand is shown in SEQ ID NO: 239, and the sequence of the antisense strand is shown in SEQ ID NO: 349;

[0169] 19) The sequence of the sense strand is shown in SEQ ID NO: 240, and the sequence of the antisense strand is shown in SEQ ID NO: 350;

[0170] 20) the sequence of the sense strand is shown in SEQ ID NO: 241, and the sequence of the antisense strand is shown in SEQ ID NO: 351;

[0171] 21) the sequence of the sense strand is shown in SEQ ID NO: 242, and the sequence of the antisense strand is shown in SEQ ID NO: 352;

[0172] 22) the sequence of the sense strand is shown in SEQ ID NO: 243, and the sequence of the antisense strand is shown in SEQ ID NO: 353;

[0173] 23) the sequence of the sense strand is shown in SEQ ID NO: 244, and the sequence of the antisense strand is shown in SEQ ID NO: 354;

[0174] 24) the sequence of the sense strand is shown in SEQ ID NO: 245, and the sequence of the antisense strand is shown in SEQ ID NO: 355;

[0175] 25) the sequence of the sense strand is shown in SEQ ID NO: 246, and the sequence of the antisense strand is shown in SEQ ID NO: 356;

[0176] 26) the sequence of the sense strand is shown in SEQ ID NO: 247, and the sequence of the antisense strand is shown in SEQ ID NO: 357;

[0177] 27) the sequence of the sense strand is shown in SEQ ID NO: 248, and the sequence of the antisense strand is shown in SEQ ID NO: 358;

[0178] 28) the sequence of the sense strand is shown in SEQ ID NO: 249, and the sequence of the antisense strand is shown in SEQ ID NO: 359;

[0179] 29) The sequence of the sense strand is shown in SEQ ID NO: 250, and the sequence of the antisense strand is shown in SEQ ID NO: 360;

[0180] 30) the sequence of the sense strand is shown in SEQ ID NO: 251, and the sequence of the antisense strand is shown in SEQ ID NO: 361;

[0181] 31) The sequence of the sense strand is shown in SEQ ID NO: 252, and the sequence of the antisense strand is shown in SEQ ID NO: 362;

[0182] 32) the sequence of the sense strand is shown in SEQ ID NO: 253, and the sequence of the antisense strand is shown in SEQ ID NO: 363;

[0183] 33) the sequence of the sense strand is shown in SEQ ID NO: 254, and the sequence of the antisense strand is shown in SEQ ID NO: 364;

[0184] 34) the sequence of the sense strand is shown in SEQ ID NO: 255, and the sequence of the antisense strand is shown in SEQ ID NO: 365;

[0185] 35) the sequence of the sense strand is shown in SEQ ID NO: 256, and the sequence of the antisense strand is shown in SEQ ID NO: 366;

[0186] 36) the sequence of the sense strand is shown in SEQ ID NO: 257, and the sequence of the antisense strand is shown in SEQ ID NO: 367;

[0187] 37) the sequence of the sense strand is shown in SEQ ID NO: 258, and the sequence of the antisense strand is shown in SEQ ID NO: 368;

[0188] 38) the sequence of the sense strand is shown in SEQ ID NO: 259, and the sequence of the antisense strand is shown in SEQ ID NO: 369;

[0189] 39) The sequence of the sense strand is shown in SEQ ID NO: 260, and the sequence of the antisense strand is shown in SEQ ID NO: 370;

[0190] 40) the sequence of the sense strand is shown in SEQ ID NO: 261, and the sequence of the antisense strand is shown in SEQ ID NO: 371;

[0191] 41) The sequence of the sense strand is shown in SEQ ID NO: 262, and the sequence of the antisense strand is shown in SEQ ID NO: 372;

[0192] 42) the sequence of the sense strand is shown in SEQ ID NO: 263, and the sequence of the antisense strand is shown in SEQ ID NO: 373;

[0193] 43) the sequence of the sense strand is shown in SEQ ID NO: 264, and the sequence of the antisense strand is shown in SEQ ID NO: 374;

[0194] 44) the sequence of the sense strand is shown in SEQ ID NO: 265, and the sequence of the antisense strand is shown in SEQ ID NO: 375;

[0195] 45) the sequence of the sense strand is shown in SEQ ID NO: 266, and the sequence of the antisense strand is shown in SEQ ID NO: 376;

[0196] 46) the sequence of the sense strand is shown in SEQ ID NO: 267, and the sequence of the antisense strand is shown in SEQ ID NO: 377;

[0197] 47) The sequence of the sense strand is shown in SEQ ID NO: 268, and the sequence of the antisense strand is shown in SEQ ID NO: 378;

[0198] 48) The sequence of the sense strand is shown in SEQ ID NO: 269, and the sequence of the antisense strand is shown in SEQ ID NO: 379;

[0199] 49) The sequence of the sense strand is shown in SEQ ID NO: 270, and the sequence of the antisense strand is shown in SEQ ID NO: 380;

[0200] 50) The sequence of the sense strand is shown in SEQ ID NO: 271, and the sequence of the antisense strand is shown in SEQ ID NO: 381;

[0201] 51) The sequence of the sense strand is shown in SEQ ID NO: 272, and the sequence of the antisense strand is shown in SEQ ID NO: 382;

[0202] 52) the sequence of the sense strand is shown in SEQ ID NO: 273, and the sequence of the antisense strand is shown in SEQ ID NO: 383;

[0203] 53) The sequence of the sense strand is shown in SEQ ID NO: 274, and the sequence of the antisense strand is shown in SEQ ID NO: 384;

[0204] 54) the sequence of the sense strand is shown in SEQ ID NO: 275, and the sequence of the antisense strand is shown in SEQ ID NO: 385;

[0205] 55) The sequence of the sense strand is shown in SEQ ID NO: 276, and the sequence of the antisense strand is shown in SEQ ID NO: 386;

[0206] 56) The sequence of the sense strand is shown in SEQ ID NO: 277, and the sequence of the antisense strand is shown in SEQ ID NO: 387;

[0207] 57) The sequence of the sense strand is shown in SEQ ID NO: 278, and the sequence of the antisense strand is shown in SEQ ID NO: 388;

[0208] 58) The sequence of the sense strand is shown in SEQ ID NO: 279, and the sequence of the antisense strand is shown in SEQ ID NO: 389;

[0209] 59) The sequence of the sense strand is shown in SEQ ID NO: 280, and the sequence of the antisense strand is shown in SEQ ID NO: 390;

[0210] 60) The sequence of the sense strand is shown in SEQ ID NO: 281, and the sequence of the antisense strand is shown in SEQ ID NO: 391;

[0211] 61) The sequence of the sense strand is shown in SEQ ID NO: 282, and the sequence of the antisense strand is shown in SEQ ID NO: 392;

[0212] 62) The sequence of the sense strand is shown in SEQ ID NO: 283, and the sequence of the antisense strand is shown in SEQ ID NO: 393;

[0213] 63) The sequence of the sense strand is shown in SEQ ID NO: 284, and the sequence of the antisense strand is shown in SEQ ID NO: 394;

[0214] 64) The sequence of the sense strand is shown in SEQ ID NO: 285, and the sequence of the antisense strand is shown in SEQ ID NO: 395;

[0215] 65) The sequence of the sense strand is shown in SEQ ID NO: 286, and the sequence of the antisense strand is shown in SEQ ID NO: 396;

[0216] 66) The sequence of the sense strand is shown in SEQ ID NO: 287, and the sequence of the antisense strand is shown in SEQ ID NO: 397;

[0217] 67) The sequence of the sense strand is shown in SEQ ID NO: 288, and the sequence of the antisense strand is shown in SEQ ID NO: 398;

[0218] 68) The sequence of the sense strand is shown in SEQ ID NO: 289, and the sequence of the antisense strand is shown in SEQ ID NO: 399;

[0219] 69) The sequence of the sense strand is shown in SEQ ID NO: 290, and the sequence of the antisense strand is shown in SEQ ID NO: 400;

[0220] 70) The sequence of the sense strand is shown in SEQ ID NO: 291, and the sequence of the antisense strand is shown in SEQ ID NO: 401;

[0221] 71) The sequence of the sense strand is shown in SEQ ID NO: 292, and the sequence of the antisense strand is shown in SEQ ID NO: 402;

[0222] 72) The sequence of the sense strand is shown in SEQ ID NO: 293, and the sequence of the antisense strand is shown in SEQ ID NO: 403;

[0223] 73) The sequence of the sense strand is shown in SEQ ID NO: 294, and the sequence of the antisense strand is shown in SEQ ID NO: 404;

[0224] 74) The sequence of the sense strand is shown in SEQ ID NO: 295, and the sequence of the antisense strand is shown in SEQ ID NO: 405;

[0225] 75) The sequence of the sense strand is shown in SEQ ID NO: 296, and the sequence of the antisense strand is shown in SEQ ID NO: 406;

[0226] 76) The sequence of the sense strand is shown in SEQ ID NO: 297, and the sequence of the antisense strand is shown in SEQ ID NO: 407;

[0227] 77) The sequence of the sense strand is shown in SEQ ID NO: 298, and the sequence of the antisense strand is shown in SEQ ID NO: 408;

[0228] 78) The sequence of the sense strand is shown in SEQ ID NO: 299, and the sequence of the antisense strand is shown in SEQ ID NO: 409;

[0229] 79) The sequence of the sense strand is shown in SEQ ID NO: 300, and the sequence of the antisense strand is shown in SEQ ID NO: 410;

[0230] 80) The sequence of the sense strand is shown in SEQ ID NO: 301, and the sequence of the antisense strand is shown in SEQ ID NO: 411;

[0231] 81) The sequence of the sense strand is shown in SEQ ID NO: 302, and the sequence of the antisense strand is shown in SEQ ID NO: 412;

[0232] 82) The sequence of the sense strand is shown in SEQ ID NO: 303, and the sequence of the antisense strand is shown in SEQ ID NO: 413;

[0233] 83) The sequence of the sense strand is shown in SEQ ID NO: 304, and the sequence of the antisense strand is shown in SEQ ID NO: 414;

[0234] 84) The sequence of the sense strand is shown in SEQ ID NO: 305, and the sequence of the antisense strand is shown in SEQ ID NO: 415;

[0235] 85) The sequence of the sense strand is shown in SEQ ID NO: 306, and the sequence of the antisense strand is shown in SEQ ID NO: 416;

[0236] 86) The sequence of the sense strand is shown in SEQ ID NO: 307, and the sequence of the antisense strand is shown in SEQ ID NO: 417;

[0237] 87) The sequence of the sense strand is shown in SEQ ID NO: 308, and the sequence of the antisense strand is shown in SEQ ID NO: 418;

[0238] 88) The sequence of the sense strand is shown in SEQ ID NO: 309, and the sequence of the antisense strand is shown in SEQ ID NO: 419;

[0239] 89) The sequence of the sense strand is shown in SEQ ID NO: 310, and the sequence of the antisense strand is shown in SEQ ID NO: 420;

[0240] 90) The sequence of the sense strand is shown in SEQ ID NO: 311, and the sequence of the antisense strand is shown in SEQ ID NO: 421;

[0241] 91) The sequence of the sense strand is shown in SEQ ID NO: 312, and the sequence of the antisense strand is shown in SEQ ID NO: 422;

[0242] 92) The sequence of the sense strand is shown in SEQ ID NO: 313, and the sequence of the antisense strand is shown in SEQ ID NO: 423;

[0243] 93) The sequence of the sense strand is shown in SEQ ID NO: 314, and the sequence of the antisense strand is shown in SEQ ID NO: 424;

[0244] 94) The sequence of the sense strand is shown in SEQ ID NO: 315, and the sequence of the antisense strand is shown in SEQ ID NO: 425;

[0245] 95) The sequence of the sense strand is shown in SEQ ID NO: 316, and the sequence of the antisense strand is shown in SEQ ID NO: 426;

[0246] 96) The sequence of the sense strand is shown in SEQ ID NO: 317, and the sequence of the antisense strand is shown in SEQ ID NO: 427;

[0247] 97) The sequence of the sense strand is shown in SEQ ID NO: 318, and the sequence of the antisense strand is shown in SEQ ID NO: 428;

[0248] 98) The sequence of the sense strand is shown in SEQ ID NO: 319, and the sequence of the antisense strand is shown in SEQ ID NO: 429;

[0249] 99) The sequence of the sense strand is shown in SEQ ID NO: 320, and the sequence of the antisense strand is shown in SEQ ID NO: 430;

[0250] 100) The sequence of the sense strand is shown in SEQ ID NO: 321, and the sequence of the antisense strand is shown in SEQ ID NO: 431;

[0251] 101) The sequence of the sense strand is shown in SEQ ID NO: 322, and the sequence of the antisense strand is shown in SEQ ID NO: 432;

[0252] 102) The sequence of the sense strand is shown in SEQ ID NO: 323, and the sequence of the antisense strand is shown in SEQ ID NO: 433;

[0253] 103) The sequence of the sense strand is shown in SEQ ID NO: 324, and the sequence of the antisense strand is shown in SEQ ID NO: 434;

[0254] 104) The sequence of the sense strand is shown in SEQ ID NO: 325, and the sequence of the antisense strand is shown in SEQ ID NO: 435;

[0255] 105) The sequence of the sense strand is shown in SEQ ID NO: 326, and the sequence of the antisense strand is shown in SEQ ID NO: 436;

[0256] 106) The sequence of the sense strand is shown in SEQ ID NO: 327, and the sequence of the antisense strand is shown in SEQ ID NO: 437;

[0257] 107) The sequence of the sense strand is shown in SEQ ID NO: 328, and the sequence of the antisense strand is shown in SEQ ID NO: 438;

[0258] 108) The sequence of the sense strand is shown in SEQ ID NO: 329, and the sequence of the antisense strand is shown in SEQ ID NO: 439;

[0259] 109) The sequence of the sense strand is shown in SEQ ID NO: 330, and the sequence of the antisense strand is shown in SEQ ID NO: 440; or

[0260] 110) The sequence of the sense strand is shown in SEQ ID NO: 331, and the sequence of the antisense strand is shown in SEQ ID NO: 441.

[0261] 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. Pharmaceutically acceptable carriers or excipients of the present 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, distearoylphosphatidylcholine, 1,2-dimyristyl glyceride, and dimethyl adipate.

[0262] In one aspect, the present invention provides use of the aforementioned siRNA, siRNA conjugate or pharmaceutical composition in the preparation of a medicament for preventing and / or treating diseases related to excessive APP.

[0263] In one aspect, the present invention provides a method for preventing or treating diseases related to excessive APP, comprising administering the siRNA, siRNA conjugate or pharmaceutical composition of the present invention to a subject.

[0264] In a specific embodiment, the disease caused by excessive APP is selected from cerebral amyloid angiopathy (CAA) and Alzheimer's disease (AD).

[0265] The pharmaceutical composition of the present invention can be used alone to treat cerebral amyloid angiopathy (CAA) and Alzheimer's disease (AD), or in combination with standard oral medications, providing experimental support for the implementation of diversified treatment options for clinical patients with the above diseases.

[0266] The suitable dosage of the siRNA for inhibiting APP gene expression of the present invention is generally in the range of about 0.1 mg / kg to about 10.0 mg / kg, preferably about 0.3 mg / kg to about 3.0 mg / kg.

[0267] The administration routes involved in the present 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 buccal administration and sublingual administration).

[0268] The siRNA of the present invention can specifically induce the degradation of APP mRNA, thereby inhibiting the synthesis of APP in the liver, inducing a lasting reduction in APP protein, and reducing the pathological deposition of related toxic proteins such as Aβ, and has good drug development prospects. Compared with traditional small molecule drugs and antibody drugs, small nucleic acid drugs can directly regulate upstream gene expression and are relatively less likely to develop drug resistance; and small nucleic acid drugs have a long half-life in the body, so the frequency of administration is low (the drug can be given once every six months), and patient compliance is good. Therefore, the present invention can effectively prevent or treat two diseases, AD and CAA, which are caused by the pathological deposition of toxic proteins such as Aβ produced by the decomposition of APP protein, and provide more effective, safe and convenient therapeutic drugs for the above patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0269] Figure 1 Schematic diagram of the site where the siRNA of the present invention binds to the human APP gene;

[0270] Figure 2 This is the result of the siRNA conjugate of the present invention inhibiting APP expression in vivo. DETAILED DESCRIPTION

[0271] definition

[0272] In the above and below texts, unless otherwise specified, capital letters C, G, U, and A represent cytosine, guanine, uracil, and adenine nucleotides; lowercase letter m indicates that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide; lowercase letter f indicates that the nucleotide adjacent to the left of the letter f is a fluorine-modified nucleotide; lowercase letter s indicates that the two nucleotides adjacent to the left and right of the letter s are connected by thiophosphate subunits; the letter combination VP indicates that the nucleotide adjacent to the right of the letter combination VP is a vinyl phosphate (5'-(E)-vinylphosphonate, E-VP)-modified nucleotide; L96 has the structure of formula (I) and is connected to the 3' end of the sense chain through a phosphate bond.

[0273] In the above and below, the term "fluoro-modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2'-position of the ribose group of a nucleotide is replaced by fluorine, and a "non-fluorinated modified nucleotide" refers to a nucleotide or nucleotide analog in which the hydroxyl group at the 2'-position of the ribose group of a nucleotide is replaced by a non-fluorinated group. A "nucleotide analog" refers to a group that can replace a nucleotide in a nucleic acid but has a structure different from that of adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine deoxyribonucleotide. Examples include isonucleotides, bridged nucleic acids (BNAs), or acyclic nucleotides. The term "methoxy-modified nucleotide" refers to a nucleotide in which the 2'-hydroxyl group of the ribose group is replaced by a methoxy group. In the context of this article, the expressions "complementary" and "reverse complementary" are used interchangeably and have the meanings known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one chain are paired with the bases on the other chain in a complementary manner. In DNA, the purine base adenine (A) consistently pairs with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (C) consistently pairs with the pyrimidine base cytosine (G). Each base pair consists of a purine and a pyrimidine. When adenine on one strand consistently pairs with thymine (or uracil) on the other strand, and guanine consistently pairs with cytosine, the two strands are considered complementary to each other, and the sequence of the complementary strand can be inferred from the sequence of the complementary strand. Accordingly, the term "mismatch" in the art means that the bases at corresponding positions in a double-stranded nucleic acid are not paired in a complementary manner. In the above and below text, unless otherwise specified, "substantially reverse complementary" means that there are no more than three base mismatches between the two nucleotide sequences involved; "substantially reverse complementary" means that there are no more than one base mismatch between the two nucleotide sequences; and "perfectly reverse complementary" means that there are no base mismatches between the two nucleotide sequences. In the above and below, particularly when describing the preparation method of siRNA, pharmaceutical composition or siRNA conjugate of the present disclosure, unless otherwise specified, the nucleoside monomer (nucleoside monomer) refers to, according to the kind and order of nucleotides in the siRNA or siRNA conjugate to be prepared, the modification or unmodified nucleoside phosphoramidite monomer (unmodified or modified RNAphosphoramidites, sometimes RNAphosphoramidites is also referred to as Nucleoside phosphora-midites) used in phosphoramidite solid phase synthesis. Phosphoramidite solid phase synthesis is the method used in siRNA synthesis well known to those skilled in the art. The nucleoside monomer used in the present invention all can be commercially obtained.

[0274] In Tables 1 and 2, if there is no VP on the left side of the 5'-terminal nucleotide of the sense strand or the modified sense strand connected to the conjugated group, 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 (II):

[0275]

[0276] 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, or 2'-deoxynucleotide.

[0277] In Tables 1 and 2, if there is no VP on the left side of the 5' terminal nucleotide of the antisense strand or modified antisense strand, 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 also shown in Formula (II).

[0278] In Tables 1 and 2, the 3'-terminal nucleotide of the sense strand, the 3'-terminal nucleotide of the antisense strand, and the modified antisense strand has a hydroxyl group at its 3'-terminal position.

[0279] Example 1. siRNA design and synthesis

[0280] 1.1 siRNA design

[0281]

[0282] 1.2 siRNA sequence synthesis

[0283] siRNAs, including negative control siRNA (siCtrl), were synthesized according to standard oligonucleotide solid-phase synthesis protocols.

[0284] Oligonucleotide solid-phase synthesis protocol: Commercially available 5'-DMT-2'-TBDMS-rU phosphoramidite monomers, 5'-DMT-2'-TBDMS-rA(Bz) phosphoramidite monomers, 5'-DMT-2'-TBDMS-rC(Ac) phosphoramidite monomers, and 5'-DMT-2'-TBDMS-rG(iBu) phosphoramidite monomers were used. RNA was synthesized at a 500 nmol scale. The phosphoramidite solution was prepared at a concentration of 50 mM, and a 0.3 M benzylthiotetrazolium (BTT) solution in acetonitrile was used as an activator. During the synthesis process, a 0.1 M oxidizing agent (pyridine:THF:water = 20:78:2) was used to convert trivalent phosphorus to a pentavalent phosphorus-stabilized phosphate backbone. After completion of the synthesis, the sequence was aminolyzed from the solid support and precipitated. The 2'-O-tert-butyldimethylsilyl protecting group at the 2' end was removed using triethylamine trihydrofluoride.

[0285] For the RNA sequence that has been synthesized, it is subjected to aminolysis at 55°C for 40 minutes using an aminolysis solution of ammonia water: methylamine = 1:1. After the aminolysis is completed, the solid phase carrier CPG powder is removed and the supernatant is drained. A protecting group removal agent is added, and the reaction is carried out at 60°C for 2 hours. N-butanol is added in a ratio of 1:5, and the mixture is allowed to stand for 30 minutes at -20°C. The precipitate is then centrifuged. RNase-free water is added for dissolution, and the mixture is purified by reverse phase chromatography (0.1M triethylamine acetic acid (TEAA) and acetonitrile). The purified sample is desalted by ultrafiltration using PBS. Annealing is performed to obtain siRNA, which is then verified. The result shows that the target siRNA was successfully prepared.

[0286] 1.3 siRNA sequence modification and conjugate synthesis

[0287] Modified siRNAs can be synthesized according to oligonucleotide solid-phase synthesis protocols. Modified nucleotide groups can be introduced into the siRNAs disclosed herein by using correspondingly modified nucleoside monomers. Methods for preparing correspondingly modified nucleoside monomers are also well known to those skilled in the art. SiRNA conjugates can be synthesized by conjugating L96 to siRNA using the synthesis methods disclosed in WO2014025805A1 or WO2017015109A1.

[0288] The structure of the conjugated group L96 is shown below:

[0289]

[0290] Annealing of oligoribonucleotides to produce siRNA conjugates: Prepare a 200 μM solution of the RNA oligomers to be annealed in sterile RNase-free HO (RNA hydrolase-free). Set up the annealing reaction as follows: Place 100 μL of the above solution (duplex concentration 10 nmol) in a 95°C water bath for 10 minutes (amounts ≥ 100 nmol require 20 minutes at high temperature). Immediately cool the solution to a 60°C water bath. Store the annealed solution at 4°C. Combine equimolar amounts of RNA solution to mix the complementary strands. Verify that the siRNA molecules are correctly constructed. Prepare the siRNA solution into a dry powder for later use.

[0291] The sequences of the synthesized siRNA molecules are shown in Table 1 below. The sites that bind to the human APP gene are as follows: Figure 1 As shown:

[0292] Table 1. Sequence list of siRNA targeting APP

[0293]

[0294]

[0295]

[0296]

[0297]

[0298] Wherein, A, U, G, and C represent adenine, uracil, guanine, and cytosine nucleotides. The sequences of the synthesized siRNA conjugates are shown in Table 2 below:

[0299] Table 2. Sequence list of siRNA conjugates targeting APP

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307] Among them, the lowercase letter m indicates that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide; the lowercase letter f indicates that the nucleotide adjacent to the left of the letter f is a fluorine-modified nucleotide; the lowercase letter s indicates that the two nucleotides adjacent to the left and right of the letter s are connected by thiophosphate subunits; VP indicates that the nucleotide adjacent to the right of the letter combination VP is a vinyl phosphate (5'-(E)-vinylphosphonate, E-VP)-modified nucleotide; L96 represents the L96 conjugate group connected to the siRNA.

[0308] Example 2. siRNA activity screening in HCT116 cell line in vitro

[0309] 2.1 Experimental steps

[0310] 2.1.1 Cell culture

[0311] HCT116 cells (BNCC, BNCC287750) were cultured in a 37°C, 5% CO2 environment using DMEM complete medium (Eallbio, supplemented with 10% FBS). When the confluency reached 80%-90%, the cells were trypsinized, counted, and transfected.

[0312] 2.1.2 Preparation of siRNA dilution solution

[0313] (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.

[0314] (2) Prepare 200 nM siRNA diluent Y.

[0315] a) taking 50 μl of the 100 μM siRNA stock solution prepared in step (1) above, adding 50 μl of ultrapure distilled water to obtain a siRNA dilution solution with a final concentration of 50 μM;

[0316] b) taking 2 μl of the 50 μM siRNA dilution prepared in step a) and adding 18 μl of ultrapure distilled water to obtain siRNA stock solution X with a final concentration of 5 μM;

[0317] 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.

[0318] 2.1.3 HCT116 cell transfection

[0319] Pick 0.6 μl of RNAiMAX transfection reagent was added with 10 μl of Opti-medium to obtain RNAiMAX transfection reagent diluent; RNAiMAX transfection reagent diluent and 200 nM siRNA diluent Y prepared in step 2.1.2 were mixed in a 1:1 volume ratio to prepare a transfection mixture. The mixture was allowed to stand for 5 minutes. 10 μl of the transfection mixture was added to a 96-well plate, and 90 μl of HCT116 cells cultured in step 2.1.1 were added (final volume 100 μl / well, 20,000 cells / well, assuming 200 nM siRNA diluent Y, the siRNA concentration in this system is 10 nM); the cells were cultured for 24 hours after the above transfection.

[0320] 2.1.4 RNA extraction

[0321] According to the FlysisAmp Cells-to-CT 1-Step SYBR Green Kit product instructions, extract total RNA from HCT116 cells obtained in step 2.1.3.

[0322] 2.1.5 Fluorescence quantitative PCR

[0323] The extracted total RNA was reverse transcribed and analyzed by real-time PCR using the FlysisAmp Cells-to-CT 1-Step SYBR Green Kit.

[0324] 2.1.6 Result Analysis

[0325] (1) The Ct value was automatically calculated using the software of the 7500 real-time fluorescence quantitative PCR instrument (Thermo Fisher);

[0326] (2) The relative expression of genes was calculated using the following formula:

[0327] ΔCt1=Ct(APP group)–Ct(ACTIN in APP group)

[0328] ΔCt2=Ct(siCtrl group)–Ct(ACTIN in siCtrl group)

[0329] ΔΔCt=ΔCt1 (APP group)-ΔCt2 (siCtrl group), where siCtrl group represents nonspecific siRNA sequence and serves as negative control group;

[0330] mRNA expression relative to siCtrl group = 2 -ΔΔCt

[0331] Inhibition rate (%) = (1 - mRNA expression relative to the siCtrl group) × 100%.

[0332] 2.2 Experimental Results

[0333] The inhibitory effects of the siRNA of the present invention are shown in Table 3 below:

[0334] Table 3. siRNA HCT116 cell line in vitro screening results

[0335]

[0336]

[0337]

[0338] As can be seen from Table 3, some of the siRNAs of the present invention can significantly inhibit the expression of APP gene in HCT116 cells at 10 nM.

[0339] Example 3. In vitro activity screening of siRNA BE(2)-C cell line

[0340] 3.1 Experimental steps

[0341] 3.1.1 Cell culture

[0342] BE(2)-C cells (BNCC, BNCC247047) were cultured in DMEM complete medium (Eallbio, supplemented with 10% FBS) at 37°C in a 5% CO2 environment. When the confluence rate reached 80%-90%, the cells were trypsinized, counted, and transfected.

[0343] 3.1.2 Preparation of siRNA dilution solution

[0344] (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.

[0345] (2) Prepare 200 nM siRNA diluent Y.

[0346] a) taking 50 μl of the 100 μM siRNA stock solution prepared in step (1) above, adding 50 μl of ultrapure distilled water to obtain a siRNA dilution solution with a final concentration of 50 μM;

[0347] b) taking 2 μl of the 50 μM siRNA dilution prepared in step a) and adding 18 μl of ultrapure distilled water to obtain siRNA stock solution X with a final concentration of 5 μM;

[0348] c) Take 2 μl of the prepared siRNA stock solution X and add 48 μl of Opti-medium to obtain 200 nM siRNA dilution solution Y.

[0349] 3.1.3BE(2)-C cell transfection

[0350] Pick 0.6 μl of RNAiMAX transfection reagent was added with 10 μl of Opti-medium to obtain RNAiMAX transfection reagent diluent; RNAiMAX transfection reagent diluent and 200nM siRNA diluent Y prepared in step 3.1.2 were mixed in a 1:1 volume ratio to prepare a transfection mixture. The mixture was allowed to stand for 5 minutes. 10μl of the transfection mixture was added to a 96-well plate, and 90μl of the BE(2)-C cells cultured in step 3.1.1 were added (final volume 100μl / well, the number of cells was 20,000 / well, and the concentration of siRNA in this system was 10nM); the cells were cultured for 24 hours after the above transfection.

[0351] 3.1.4 RNA extraction

[0352] According to the FlysisAmp Cells-to-CT 1-Step SYBR Green Kit product instructions, total RNA was extracted from the BE(2)-C cells obtained in step 3.1.3.

[0353] 3.1.5 Fluorescence quantitative PCR

[0354] The extracted total RNA was reverse transcribed and analyzed by real-time PCR using the FlysisAmp Cells-to-CT 1-Step SYBR Green Kit.

[0355] 3.1.6 Result Analysis

[0356] (1) The Ct value was automatically calculated using the software of the 7500 real-time fluorescence quantitative PCR instrument (ThermoFisher);

[0357] (2) The relative expression of genes was calculated using the following formula:

[0358] ΔCt1=Ct(APP group)–Ct(ACTIN in APP group)

[0359] ΔCt2=Ct(siCtrl group)–Ct(ACTIN in siCtrl group)

[0360] ΔΔCt=ΔCt1 (APP group)-ΔCt2 (siCtrl group), where siCtrl group represents nonspecific siRNA sequence and serves as negative control group;

[0361] mRNA expression relative to siCtrl group = 2 -ΔΔCt

[0362] Inhibition rate (%) = (1 - mRNA expression relative to the siCtrl group) × 100%.

[0363] 3.2 Experimental Results

[0364] The inhibitory effects of the siRNA of the present invention are shown in Table 4 below:

[0365] Table 4. Results of in vitro screening of siRNABE(2)-C cell lines

[0366]

[0367]

[0368]

[0369]

[0370] As can be seen from Table 4, some of the siRNAs of the present 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 effective activity ranges for inhibiting APP mRNA were determined to be positions 334-804, 2305-2327, and 2813-2835 calculated according to NCBI refseqID NM_000484.4.

[0371] Example 4. siRNA IC50 determination

[0372] This example investigates the dose-effect 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 the indicator.

[0373] 4.1 The IC50 of siRNA inhibiting APP gene expression was determined in a manner similar to that of Example 2, wherein the concentrations of the transfected siRNA were 25 nM, 5 nM, 1 nM, 0.2 nM, 0.04 nM, 0.008 nM, and 0.0016 nM, respectively.

[0374] 4.2 Results Analysis

[0375] (1) The Ct value was automatically calculated using the software of the 7500 real-time fluorescence quantitative PCR instrument (Thermo Fisher);

[0376] (2) The relative expression of genes was calculated using the following formula:

[0377] ΔCt1=Ct(APP group)–Ct(ACTIN in APP group)

[0378] ΔCt2=Ct(siCtrl group)–Ct(ACTIN in siCtrl group)

[0379] ΔΔCt=ΔCt1 (APP group)-ΔCt2 (siCtrl group), where siCtrl group represents nonspecific siRNA sequence and serves as negative control group;

[0380] mRNA expression relative to siCtrl group = 2 -ΔΔCt

[0381] Inhibition rate (%) = (1 - mRNA expression relative to the siCtrl group) × 100%.

[0382] The log value of siRNA concentration was used as the X-axis and the percentage inhibition rate was used as the Y-axis. The analysis software GraphPad

[0383] Prism 8's "log(inhibitor) vs. normalized response--Variable slope" function module was used to fit the dose-effect curve to obtain the IC50 value of each siRNA.

[0384] The fitting formula is: Y = 100 / (1 + 10^((LogIC50-X)×HillSlope))

[0385] Where: HillSlope represents the slope of the percentage inhibition curve.

[0386] 4.3 Experimental Results

[0387] The IC50 determination results of the siRNA of the present invention are shown in Table 5 below:

[0388] Table 5. siRNA HCT116 cell line IC50 determination results

[0389]

[0390]

[0391] As can be seen from Table 5, some of the siRNAs of the present invention can significantly inhibit APP gene expression.

[0392] Example 5. In vivo activity screening of siRNA conjugates

[0393] Based on the in vitro screening results of Examples 2, 3, and 4 above, some siRNA sequences with good in vitro activity were selected, and their conjugates were used to verify their in vivo activity.

[0394] 5.1 Experimental steps

[0395] Male mice (C57BL / 6) aged 6-8 weeks were purchased from Spefox (Beijing) Biotechnology Co., Ltd., weighing about 20 g. Each mouse was intravenously injected with 1×10 11 The recombinant adeno-associated virus 8 (AAV8) vector contains 100 genome copies. The recombinant AAV8 vector is packaged by an AAV8 capsid protein expression plasmid and a transfer plasmid carrying AAV8-hAPP, wherein the transfer plasmid carries positions 151-3583 of the human APP sequence (NM_000484.4) and is controlled by the TBG promoter. The AAV8-hAPP transgenic mouse model was constructed 14 days after injection. The conjugate was then administered subcutaneously to each mouse at a dose of 3 mg / kg. Mice were killed on the 7th day (D7) and the 14th day (D14) after administration, with 6 mice in each group, and liver tissue was taken for mRNA expression detection. Total RNA was extracted by Trizol, mRNA was reverse transcribed using the HiScript III RT SuperMix for qPCR (+ gDNA wiper) kit (Cat. No. R323-01, Vazyme), and real-time fluorescence quantitative PCR was performed using the ChamQ Universal SYBR qPCR Master Mix kit (Cat. No. Q711-03, Vazyme). The results of in vivo activity screening of different siRNA conjugates are shown in Tables 6 and Figure 2 shown.

[0396] Table 6. In vivo screening results of siRNA conjugates

[0397]

[0398]

[0399] 5.2 Experimental Results

[0400] The chemically modified siRNA sequences showed excellent 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 in mice. On day 14, the inhibition efficiency of YGND22-28M reached 81.17%, and that of YGND22-16M reached 71.21%.

Claims

1. An siRNA for inhibiting APP gene expression, the siRNA comprising a sense strand and an antisense strand, the sense strand comprising a nucleotide sequence I, the antisense strand comprising 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, the first nucleotide sequence being a nucleotide sequence of at least 19 nucleotides in length in the mRNA expressed by the APP gene, wherein: The sequence of the sense strand is shown in SEQ ID NO: 29, and the sequence of the antisense strand is shown in SEQ ID NO:

139.

2. An siRNA conjugate, wherein the sequence of the sense strand is shown in SEQ ID NO: 249, and the sequence of the antisense strand is shown in SEQ ID NO:

359.

3. A composition comprising the siRNA according to claim 1 or the siRNA conjugate according to claim 2.

4. The composition according to claim 3, wherein The composition is a pharmaceutical composition and further comprises a pharmaceutically acceptable carrier or excipient, wherein 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, distearoylphosphatidylcholine, 1,2-dimyristyl glyceride, and dimethyl adipate.

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