Rnai agent targeting APP and pharmaceutical use thereof

Abstract

The present invention relates to an RNAi agent targeting APP and the pharmaceutical use thereof. Specifically, the present invention relates to an RNAi agent targeting APP, a pharmaceutical composition, and the pharmaceutical use thereof. The present invention further relates to a pharmaceutical composition, cell or kit containing the RNAi agent, and a method involving using the RNAi agent for treating and / or preventing a related disorder in a subject.

Description

RNAi agents targeting APP and their medical uses Technical Field

[0001] The present disclosure belongs to the field of biomedicine, and particularly relates to an RNAi agent targeting amyloid precursor protein (APP), a pharmaceutical composition containing the same, and medical uses thereof. Background Art

[0002] The amyloid precursor protein (APP) gene encodes a membrane protein expressed in neurons and glia. APP is cleaved by α-secretase to produce soluble, non-neurotoxic sAPPα; APP is cleaved by β- and γ-secretases to produce hydrophobic, aggregation-prone Aβ peptides, which are secreted extracellularly. Aβ40 and Aβ42 are two common isoforms and, as the main components of amyloid plaques, are believed to be involved in the development and progression of Alzheimer's disease (AD). Furthermore, both Alzheimer's disease and cerebral amyloid angiopathy in patients with Down syndrome are characterized by the abnormal accumulation of amyloid plaques.

[0003] Inhibiting APP expression and / or activity with a drug that selectively and effectively inhibits APP, thereby blocking or inhibiting the production and / or levels of Aβ, will help prevent or treat various APP-related diseases. Such drugs, methods, and pharmaceutical compositions can be used to improve at least one symptom or hallmark of a neurodegenerative disease, including cognitive impairment, such as decreased memory and language skills; behavioral and psychological symptoms, such as apathy and lack of motivation, gait disturbances, and seizures; progressive dementia; and abnormal amyloid deposition. Neurodegenerative diseases include sporadic Alzheimer's disease, hereditary / familial Alzheimer's disease, Down syndrome, frontotemporal dementia, and cerebral amyloid angiopathy.

[0004] Currently lacking acceptable methods for treating such neurodegenerative diseases. Therefore, there is a need for drugs that selectively and effectively inhibit or regulate APP gene expression in order to effectively treat subjects with APP-related diseases. The purpose of this article is to provide methods for treating such diseases.

[0005] Summary of the Invention

[0006] The present disclosure provides an RNAi agent targeting APP.

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

[0008] The sense strand comprises a nucleotide sequence that differs from the nucleotide sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 11, SEQ ID NO: 23 to SEQ ID NO: 65 by no more than 3 nucleotides (e.g., 0, 1, 2, 3), and comprises at least 15 (e.g., 16, 17, 18, 19, 20, 21) consecutive nucleotides;

[0009] The antisense strand comprises a nucleotide sequence that differs from the nucleotide sequence of any one of SEQ ID NO: 11 or SEQ ID NO: 22, SEQ ID NO: 66 to SEQ ID NO: 108 by no more than 3 (e.g., 0, 1, 2, 3) nucleotides, and comprises at least 15 (e.g., 16, 17, 18, 19, 20, 21) consecutive nucleotides.

[0010] In some embodiments, the antisense strand is at least partially reverse complementary to the target sequence to mediate RNA interference. In some embodiments, there are no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 mismatch between the antisense strand and the target sequence. In some embodiments, the antisense strand is completely reverse complementary to the target sequence.

[0011] In some embodiments, the sense strand and the antisense strand are at least partially reverse complementary to form a double-stranded region. In some embodiments, there are no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 mismatch between the sense strand and the antisense strand. In some embodiments, the sense strand and the antisense strand are completely reverse complementary.

[0012] In some embodiments, the RNAi agents of the present disclosure comprise one or two blunt ends.

[0013] In some specific embodiments, each strand of the RNAi agent independently comprises 1 to 2 unpaired nucleotides.

[0014] In some embodiments, the RNAi agents of the present disclosure comprise an overhang located at the 3' end of the antisense strand of the RNAi agent.

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

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

[0017] In some embodiments, the sense strand comprises at least 15 consecutive nucleotides and differs from the nucleotide sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 11, and SEQ ID NO: 23 to SEQ ID NO: 65 by no more than 2 nucleotides, in some embodiments, by no more than 1 nucleotide, and in some embodiments, by 1 nucleotide.

[0018] In some embodiments, the antisense strand comprises at least 15 consecutive nucleotide sequences and differs from the nucleotide sequence shown in any one of SEQ ID NO: 12 to SEQ ID NO: 22, and SEQ ID NO: 66 to SEQ ID NO: 108 by no more than 2 nucleotides; in some embodiments, the nucleotide sequence differs by no more than 1 nucleotide; in some embodiments, by 1 nucleotide.

[0019] In some embodiments, the sense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence set forth in any one of SEQ ID NO: 1 to SEQ ID NO: 11, and SEQ ID NO: 23 to SEQ ID NO: 65. In some embodiments, the sense strand comprises at least 16 consecutive nucleotides of the nucleotide sequence set forth in any one of SEQ ID NO: 1 to SEQ ID NO: 11, and SEQ ID NO: 23 to SEQ ID NO: 65. In some embodiments, the sense strand comprises at least 17 consecutive nucleotides of the nucleotide sequence set forth in any one of SEQ ID NO: 1 to SEQ ID NO: 11, and SEQ ID NO: 23 to SEQ ID NO: 65. In some embodiments, the sense strand comprises at least 19 consecutive nucleotides of the nucleotide sequence set forth in any one of SEQ ID NO: 1 to SEQ ID NO: 11, and SEQ ID NO: 23 to SEQ ID NO: 65.

[0020] In some embodiments, the antisense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence set forth in any one of SEQ ID NOs: 12 to SEQ ID NOs: 22, and SEQ ID NOs: 66 to SEQ ID NOs: 108. In some embodiments, the antisense strand comprises at least 17 consecutive nucleotides of the nucleotide sequence set forth in any one of SEQ ID NOs: 12 to SEQ ID NOs: 22, and SEQ ID NOs: 66 to SEQ ID NOs: 108. In some embodiments, the antisense strand comprises at least 19 consecutive nucleotides of the nucleotide sequence set forth in any one of SEQ ID NOs: 12 to SEQ ID NOs: 22, and SEQ ID NOs: 66 to SEQ ID NOs: 108. In some embodiments, the antisense strand comprises at least 20 consecutive nucleotides of the nucleotide sequence set forth in any one of SEQ ID NOs: 12 to SEQ ID NOs: 22, and SEQ ID NOs: 66 to SEQ ID NOs: 108. In some embodiments, at least 21 consecutive nucleotides.

[0021] In some embodiments, the sense strand comprises or is selected from any one of the following nucleotide sequences: SEQ ID NO: 1 to SEQ ID NO: 11, and SEQ ID NO: 23 to SEQ ID NO: 65.

[0022] In some embodiments, the antisense strand comprises or is selected from any one of the following nucleotide sequences: SEQ ID NO: 12 to SEQ ID NO: 22, and SEQ ID NO: 66 to SEQ ID NO: 108.

[0023] In some embodiments, the RNAi agent comprises or consists of a sense strand and an antisense strand of any one of the following:

[0024] Group 1), the sense strand as shown in SEQ ID NO: 1 and the antisense strand as shown in SEQ ID NO: 12;

[0025] or the sense strand as shown in SEQ ID NO: 2 and the antisense strand as shown in SEQ ID NO: 13;

[0026] Group 2), the sense strand as set forth in SEQ ID NO: 3 and the antisense strand as set forth in SEQ ID NO: 14;

[0027] or the sense strand as shown in SEQ ID NO:4 and the antisense strand as shown in SEQ ID NO:15;

[0028] Group 3), the sense strand as set forth in SEQ ID NO: 5 and the antisense strand as set forth in SEQ ID NO: 16;

[0029] Group 4), the sense strand as set forth in SEQ ID NO: 6 and the antisense strand as set forth in SEQ ID NO: 17;

[0030] or the sense strand as shown in SEQ ID NO: 7 and the antisense strand as shown in SEQ ID NO: 18;

[0031] Group 6), the sense strand as set forth in SEQ ID NO: 8 and the antisense strand as set forth in SEQ ID NO: 19;

[0032] or the sense strand as shown in SEQ ID NO: 9 and the antisense strand as shown in SEQ ID NO: 20; and

[0033] Group 7), the sense strand as shown in SEQ ID NO: 10 and the antisense strand as shown in SEQ ID NO: 21;

[0034] Or the sense strand as shown in SEQ ID NO:11 and the antisense strand as shown in SEQ ID NO:22.

[0035] In some embodiments, at least one nucleotide in the sense strand and / or the antisense strand is a modified nucleotide. In some embodiments, all nucleotides are modified nucleotides.

[0036] In some embodiments, three consecutive nucleotides in the sense strand of the RNAi agent are 2'-fluoro-modified nucleotides. In some embodiments, in the sense strand of the RNAi agent, in the direction from the 5' end to the 3' end, the three consecutive nucleotides at positions 7-9 of the 5' end are 2'-fluoro-modified nucleotides. In some embodiments, the three consecutive nucleotides at positions 7-9 of the 5' end of the sense strand of the RNAi agent are 2'-fluoro-modified nucleotides, and the remaining positions of the sense strand are 2'-methoxy-modified nucleotides.

[0037] In some embodiments, the nucleotides at positions 2, 6, 12, 14, and 16 of the antisense strand, from the 5' end to the 3' end, are each independently a 2'-fluoro-modified nucleotide. In some embodiments, the nucleotides at positions 2, 6, 12, 14, and 16 of the antisense strand, from the 5' end to the 3' end, are each independently a 2'-fluoro-modified nucleotide; or the nucleotides at positions 2, 4, 6, 10, 12, 14, 16, or 18 of the antisense strand are each independently a 2'-fluoro-modified nucleotide, and the remaining nucleotides in the antisense strand are all 2'-methoxy-modified nucleotides.

[0038] In some embodiments, the 5' end of the antisense strand comprises a phosphorus-containing group, and the phosphorus-containing group is a 5'-vinyl phosphodiester group (5'-VP). When the 5'-terminal phosphorus-containing group is a 5'-vinyl phosphodiester group (5'-VP), the 5'-VP can be a 5'-E-VP isomer (i.e., a trans-vinyl phosphodiester group), a 5'-Z-VP isomer (i.e., a cis-vinyl phosphodiester group), or a mixture thereof. In some embodiments, the 5' end of the antisense strand comprises a phosphorus-containing group, and the phosphorus-containing group is a 5'-E-VP isomer (i.e., a trans-vinyl phosphodiester group).

[0039] In some embodiments, at least one phosphodiester group in the sense strand and / or antisense strand of the RNAi agent of the present disclosure is a phosphodiester group with a modifying group. The modifying group enables the RNAi agent to have increased stability in a biological sample or environment. In some embodiments, the phosphodiester group with a modifying group is a thiophosphate diester group.

[0040] In some embodiments, the phosphorothioate diester group is present in at least one position selected from:

[0041] between the first and second nucleotides at the 5' end of the sense strand;

[0042] between the second and third nucleotides at the 5' end of the sense strand;

[0043] between the first and second nucleotides at the 3' end of the sense strand;

[0044] between the second and third nucleotides at the 3' end of the sense strand;

[0045] between the first and second nucleotides at the 5' end of the antisense strand;

[0046] between the second and third nucleotides at the 5' end of the antisense strand;

[0047] between the first nucleotide and the second nucleotide at the 3' end of the antisense strand; and

[0048] between the second and third nucleotides at the 3' end of the antisense strand.

[0049] In some embodiments, the sense strand and / or antisense strand includes multiple phosphodiester groups with modifying groups, such as 1, 2, 3, 4, 5, or 6 phosphorothioate diester groups.

[0050] In some embodiments, the sense strand of the RNAi agents of the present disclosure includes two phosphorothioate diester groups, present at the following positions:

[0051] between the first nucleotide and the second nucleotide at the 5' end of the sense strand; and

[0052] between the second and third nucleotides at the 5' end of the sense strand; and

[0053] The antisense strand of the RNAi agents of the present disclosure includes four phosphorothioate diester groups, present at the following positions:

[0054] between the first and second nucleotides at the 5' end of the antisense strand;

[0055] between the second and third nucleotides at the 5' end of the antisense strand;

[0056] between the first nucleotide and the second nucleotide at the 3' end of the antisense strand; and

[0057] between the second and third nucleotides at the 3' end of the antisense strand.

[0058] In some embodiments, the sense and antisense strands each include four phosphorothioate diester groups, present at the following positions:

[0059] between the first and second nucleotides at the 5' end of the sense strand;

[0060] between the second and third nucleotides at the 5' end of the sense strand;

[0061] between the first and second nucleotides at the 3' end of the sense strand;

[0062] between the second and third nucleotides at the 3' end of the sense strand;

[0063] between the first and second nucleotides at the 5' end of the antisense strand;

[0064] between the second and third nucleotides at the 5' end of the antisense strand;

[0065] between the first nucleotide and the second nucleotide at the 3' end of the antisense strand; and

[0066] between the second and third nucleotides at the 3' end of the antisense strand.

[0067] In some embodiments, the RNAi agent is linked to one or more lipophilic groups.

[0068] In some embodiments, the one or more lipophilic groups are attached to one or more nucleotides on the sense strand of the RNAi agent; in some embodiments, the one or more lipophilic groups are attached to one or more nucleotides on the antisense strand of the RNAi agent; in some embodiments, the one or more lipophilic groups are attached to multiple nucleotides on both the sense and antisense strands of the RNAi agent.

[0069] In some embodiments, the one or more lipophilic groups are attached to one or more bases; in some embodiments, the one or more lipophilic groups are attached to one or more sugar rings; in some embodiments, the one or more lipophilic groups are attached to one or more internucleoside linking groups.

[0070] In some embodiments, the one or more lipophilic groups are attached to nucleotides 2-8 in the middle of the sense strand; in some embodiments, the one or more lipophilic groups are attached to nucleotides 2-8 in the middle of the antisense strand.

[0071] In some embodiments, the one or more lipophilic groups are attached to at least one of the following positions:

[0072] The first nucleotide from the 5' end of the sense strand; the first nucleotide from the 5' end of the antisense strand; the first nucleotide from the 3' end of the sense strand; the first nucleotide from the 3' end of the antisense strand; and the sixth nucleotide from the 5' end of the sense strand. In some embodiments, the one or more lipophilic groups are attached to: the sixth nucleotide from the 5' end of the sense strand.

[0073] In some embodiments, the lipophilic group is attached to the sixth nucleotide in the 5' to 3' direction of the sense strand. In some embodiments, the lipophilic group is attached to the first nucleotide at the 5' end of the sense strand.

[0074] In some embodiments, the lipophilic group comprises a saturated or unsaturated C 4-30 A hydrocarbon chain, and optionally a functional group selected from halogen, alkoxy, hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide and alkyne. In some embodiments, the lipophilic group comprises a saturated or unsaturated C 6-18 In some embodiments, the lipophilic group comprises a saturated or unsaturated C 16 In some embodiments, the saturated or unsaturated C 16 The hydrocarbon chain is attached to the first nucleotide from the 5' end of the sense strand.

[0075] In some embodiments, the lipophilic group is connected to the 2' position of the sugar ring of the first nucleoside or the sixth nucleoside at the 5' end of the sense strand. In some embodiments, the nucleoside to which the lipophilic group is connected is a nucleoside in which the 2' position of the sugar ring is connected to -O-n-hexadecyl or a uridine nucleoside in which the 2' position of the sugar ring is connected to -CH2O-n-hexadecyl, and has the structures shown in the following formula (2) and formula (3), respectively:

[0076] In formula (2) and formula (3), Base represents a base.

[0077] The present disclosure also provides an RNAi agent, comprising any one of the RNAi agents described in the present disclosure and a targeting ligand linked to the RNAi agent.

[0078] In some embodiments, the RNAi agent and the targeting ligand are covalently or non-covalently linked.

[0079] In some embodiments, the targeting ligand targets the liver; in some embodiments, the targeting ligand binds to the asialoglycoprotein receptor (ASGPR); in some embodiments, the targeting ligand comprises a galactose cluster or a galactose derivative cluster, wherein the galactose derivative is selected from N-acetyl-galactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine or N-isobutyrylgalactosamine.

[0080] In some embodiments, the targeting ligand is linked to the 3' end of the sense strand of the RNAi agent.

[0081] In some embodiments, the targeting ligand is linked to the end of the RNAi agent through a phosphodiester group, a phosphorothioate diester group, or a phosphonic acid group; in some embodiments, the targeting ligand is linked to the end of the RNAi agent through a phosphodiester group.

[0082] In some embodiments, the targeting ligand is indirectly linked to the terminus of the RNAi agent through a phosphodiester group, a phosphorothioate diester group, or a phosphonic acid group; in some embodiments, the targeting ligand is indirectly linked to the terminus of the RNAi agent through a phosphodiester group.

[0083] In some embodiments, the targeting ligand is directly linked to the end of the RNAi agent through a phosphodiester group, a phosphorothioate diester group, or a phosphonic acid group; in some embodiments, the targeting ligand is directly linked to the end of the RNAi agent through a phosphodiester group.

[0084] In some embodiments, the targeting ligand is directly linked to the 3' end of the sense strand of the RNAi agent via a phosphodiester group or a phosphorothioate diester group; in some embodiments, the targeting ligand is directly linked to the 3' end of the sense strand of the RNAi agent via a phosphodiester group.

[0085] In some embodiments, the targeting portion of a targeting ligand is composed of one or more targeting groups or targeting moieties that assist in directing the delivery of the therapeutic agent to which it is attached to the desired target location. In some cases, the targeting moiety can bind to cells or cell receptors and initiate endocytosis to promote the entry of the therapeutic agent into the cell. The targeting moiety can include a compound with affinity for a cell receptor or a cell surface molecule or antibody. Various targeting ligands containing a targeting moiety can be linked to therapeutic agents and other compounds to target the agent to cells and specific cell receptors.

[0086] In some embodiments, the types of targeting moieties include carbohydrates, cholesterol and cholesterol groups or steroids. Targeting moieties that can bind to cell receptors include sugars such as galactose, galactose derivatives (e.g., N-acetyl-galactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine, N-isobutyrylgalactosamine), mannose and mannose derivatives).

[0087] Targeting moieties that are known to bind to the asialoglycoprotein receptor (ASGPR) are particularly useful for directing the delivery of oligomeric compounds to the liver. The asialoglycoprotein receptor is abundantly expressed on liver cells (hepatocytes). Cell receptor targeting moieties that target the ASCPR include galactose and galactose derivatives. Specifically, clusters of galactose derivatives, including clusters consisting of 2, 3, 4, or more than 4 N-acetyl-galactosamine (GalNAc or NAG) can promote the uptake of certain compounds in hepatocytes. The GalNAc clusters conjugated to the oligomeric compound are used to direct the composition to the liver, where the N-acetyl-galactosamine sugar can bind to the asialoglycoprotein receptor on the surface of liver cells. Binding of the asialoglycoprotein receptor is believed to initiate receptor-mediated endocytosis, thereby promoting the entry of the compound into the cell interior.

[0088] In some embodiments, the targeting ligands may include 2, 3, 4 or more than 4 targeting moieties. In some embodiments, the targeting ligands disclosed herein may include 1, 2, 3, 4 or more than 4 targeting moieties connected to the branching group via L2.

[0089] In some embodiments, each targeting moiety comprises a galactosamine derivative, which is N-acetyl-galactosamine. Other sugars that can be used as targeting moieties and have affinity for asialoglycoprotein receptors can be selected from galactose, galactosamine, N-formyl-galactosamine, N-acetyl-galactosamine, N-propionyl-galactosamine, N-n-butyryl-galactosamine, and N-isobutyryl-galactosamine.

[0090] In some embodiments, the targeting ligands of the present disclosure include N-acetylgalactosamine as a targeting moiety.

[0091] In some embodiments, the targeting ligand comprises three terminal galactosamines or galactosamine derivatives (such as N-acetyl-galactosamine), each of which has affinity for sialoglycoprotein receptors. In some embodiments, the targeting ligand comprises three terminal N-acetyl-galactosamines (GalNAc or NAG) as targeting moieties.

[0092] In some embodiments, the targeting ligand comprises four terminal galactosamines or galactosamine derivatives (such as N-acetyl-galactosamine), each of which has affinity for the asialoglycoprotein receptor. In some embodiments, the targeting ligand comprises four terminal N-acetyl-galactosamines (GalNAc or NAG) as targeting moieties.

[0093] When referring to three terminal N-acetyl-galactosamines, terms commonly used in the art include tri-antennary

[0094] antennary), trivalent and trimer.

[0095] When referring to the four terminal N-acetyl-galactosamine groups, terms commonly used in the art include tetra-antennary

[0096] antennary), tetravalents, and tetramers.

[0097] In some embodiments, the targeting ligand provided by the present disclosure is a compound shown below or a pharmaceutically acceptable salt thereof,

[0098] In some embodiments, the N-acetyl-galactosamine moiety in the above targeting ligands can be replaced with N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine, or N-isobutyrylgalactosamine.

[0099] In some embodiments, the sense and antisense strands of the RNAi agents of the present disclosure, wherein the sense strand comprises the nucleotide sequence shown in any one of SEQ ID NO: 109 to SEQ ID NO: 125, and SEQ ID NO: 143 to SEQ ID NO: 185;

[0100] And / or, the antisense strand comprises the nucleotide sequence shown in any one of SEQ ID NO: 126 to SEQ ID NO: 142, and SEQ ID NO: 186 to SEQ ID NO: 228.

[0101] In some more specific embodiments, the RNAi agent comprises or consists of a sense strand and an antisense strand of any one of the following:

[0102] The sense strand set forth in SEQ ID NO: 109 and the antisense strand set forth in SEQ ID NO: 126;

[0103] The sense strand as set forth in SEQ ID NO: 110 and the antisense strand as set forth in SEQ ID NO: 127;

[0104] The sense strand set forth in SEQ ID NO: 111 and the sense strand set forth in SEQ ID NO: 128;

[0105] The sense strand set forth in SEQ ID NO: 112 and the antisense strand set forth in SEQ ID NO: 129;

[0106] The sense strand as set forth in SEQ ID NO: 113 and the antisense strand as set forth in SEQ ID NO: 130;

[0107] The sense strand as set forth in SEQ ID NO: 114 and the antisense strand as set forth in SEQ ID NO: 131;

[0108] The sense strand set forth in SEQ ID NO: 115 and the antisense strand set forth in SEQ ID NO: 132;

[0109] The sense strand set forth in SEQ ID NO: 116 and the antisense strand set forth in SEQ ID NO: 133;

[0110] The sense strand set forth in SEQ ID NO: 117 and the antisense strand set forth in SEQ ID NO: 134;

[0111] The sense strand set forth in SEQ ID NO: 118 and the antisense strand set forth in SEQ ID NO: 135;

[0112] The sense strand set forth in SEQ ID NO: 119 and the antisense strand set forth in SEQ ID NO: 136;

[0113] The sense strand set forth in SEQ ID NO: 120 and the antisense strand set forth in SEQ ID NO: 137;

[0114] The sense strand set forth in SEQ ID NO: 121 and the antisense strand set forth in SEQ ID NO: 138;

[0115] The sense strand set forth in SEQ ID NO: 122 and the antisense strand set forth in SEQ ID NO: 139;

[0116] The sense strand as set forth in SEQ ID NO: 123 and the antisense strand as set forth in SEQ ID NO: 140;

[0117] The sense strand as set forth in SEQ ID NO: 124 and the antisense strand as set forth in SEQ ID NO: 141; and

[0118] The sense strand is shown as SEQ ID NO:125 and the antisense strand is shown as SEQ ID NO:142.

[0119] On the other hand, the present disclosure provides a pharmaceutical composition comprising the RNAi agent described herein, and one or more pharmaceutically acceptable excipients, such as a vehicle, a carrier, a diluent, and / or a delivery polymer. The term "pharmaceutical composition" refers to a mixture containing one or more compounds described herein or their physiologically pharmaceutically acceptable salts or prodrugs with other chemical components, as well as other components such as physiologically pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote administration to an organism, facilitate the absorption of the active ingredient, and thus exert biological activity.

[0120] Various delivery systems are known and can be used for the RNAi agents of the present disclosure, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the RNAi agent, receptor-mediated endocytosis, constructing the nucleic acid as part of a retroviral or other vector.

[0121] In some embodiments, the pharmaceutical composition may further comprise a pharmaceutically acceptable excipient and / or adjuvant, which may be one or more of various preparations or compounds conventionally used in the art. For example, the pharmaceutically acceptable excipient may include at least one of a pH buffer, a protective agent, and an osmotic pressure regulator.

[0122] In some embodiments, the unit dose of the pharmaceutical composition is 0.001 mg-1000 mg.

[0123] In some embodiments, the effective amount or effective dose of the RNAi agent and / or pharmaceutical composition is about 0.001 mg / kg body weight to about 200 mg / kg body weight, about 0.01 mg / kg body weight to about 100 mg / kg body weight, or about 0.5 mg / kg body weight to about 50 mg / kg body weight.

[0124] In certain embodiments, the pharmaceutical composition contains 0.01-99.99% of the aforementioned compound or its pharmaceutically acceptable salt or its isotopic substitution, based on the total weight of the composition. In certain embodiments, the pharmaceutical composition contains 0.1-99.9% of the aforementioned compound or its pharmaceutically acceptable salt or its isotopic substitution. In certain embodiments, the pharmaceutical composition contains 0.5%-99.5% of the aforementioned compound or its pharmaceutically acceptable salt or its isotopic substitution. In certain embodiments, the pharmaceutical composition contains 1%-99% of the aforementioned compound or its pharmaceutically acceptable salt or its isotopic substitution. In certain embodiments, the pharmaceutical composition contains 2%-98% of the aforementioned compound or its pharmaceutically acceptable salt or its isotopic substitution.

[0125] The pharmaceutical composition can be conveniently presented in unit dosage form. Generally, the pharmaceutical composition can be formulated into any suitable dosage form, such as, but not limited to, injection, tablet, capsule, gel, etc. Pharmaceutical compositions include, but are not limited to, solutions, emulsions, and liposome-containing formulations.

[0126] In certain embodiments, the pharmaceutical composition comprises 0.01% to 99.99% of a pharmaceutically acceptable excipient, based on the total weight of the composition. In certain embodiments, the pharmaceutical composition comprises 0.1% to 99.9% of a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition comprises 0.5% to 99.5% of a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition comprises 1% to 99% of a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition comprises 2% to 98% of a pharmaceutically acceptable excipient.

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

[0128] In another aspect, the present disclosure further provides a kit comprising the RNAi agent or pharmaceutical composition of the present disclosure.

[0129] On the other hand, the present disclosure also provides a method for silencing the APP gene or the mRNA expressed by the APP gene in a cell, the method comprising the step of introducing the RNAi agent and / or the pharmaceutical composition according to the present disclosure into the cell. The present disclosure also provides a method for silencing the APP gene or the mRNA expressed by the APP gene in a cell in vivo or in vitro, the method comprising the step of introducing the RNAi agent and / or the pharmaceutical composition according to the present disclosure into the cell. The present disclosure also provides a method for reducing APP gene expression, the method comprising administering the RNAi agent and / or the pharmaceutical composition of the present disclosure to a subject.

[0130] On the other hand, the present disclosure provides a kind of RNAi agent or pharmaceutical composition described in the present disclosure for the preparation of a medicine for treating and / or preventing a subject's disease. In some embodiments, the disease is a disease associated with the APP gene. In some embodiments, the disease is a hepatocellular disease or a central nervous system degenerative disease. In some embodiments, the disease is selected from sporadic Alzheimer's disease, hereditary / familial Alzheimer's disease, Down syndrome, frontotemporal dementia and cerebral amyloid angiopathy, Alzheimer's disease and cerebral amyloid angiopathy in patients with Down syndrome.

[0131] On the other hand, the present disclosure also provides a method for treating and / or preventing a disease, comprising administering to a subject an RNAi agent and / or pharmaceutical composition of the present disclosure. In some embodiments, the disease is a disease associated with the APP gene. In some embodiments, the disease is a hepatocellular disease or a central nervous system degenerative disease. In some embodiments, the disease is selected from sporadic Alzheimer's disease, hereditary / familial Alzheimer's disease, Down syndrome, frontotemporal dementia and cerebral amyloid angiopathy, Alzheimer's disease and cerebral amyloid angiopathy in patients with Down syndrome.

[0132] The present disclosure provides a method for treating and / or preventing diseases related to APP gene expression in a subject, comprising administering to the subject an effective amount or effective dose of the RNAi agent or pharmaceutical composition of the present disclosure, which can inhibit APP gene expression and / or replication.

[0133] The present disclosure provides use of the RNAi agent and / or pharmaceutical composition of the present disclosure in preparing a drug for treating and / or preventing diseases associated with APP gene expression.

[0134] The present disclosure provides use of the RNAi agent and / or pharmaceutical composition of the present disclosure in preparing a medicament for inhibiting the expression of APP.

[0135] The present disclosure provides a method for inhibiting APP expression, comprising administering an effective amount or effective dose of the RNAi agent and / or pharmaceutical composition of the present disclosure to a subject.

[0136] The present disclosure provides a method for in vivo delivery of an RNAi agent that inhibits APP expression and / or replication, comprising administering the RNAi agent and / or pharmaceutical composition of the present disclosure to a subject.

[0137] In another aspect, the present disclosure provides a method for in vivo delivery of a RNAi agent that inhibits APP expression and / or replication, the method comprising administering the RNAi agent and / or pharmaceutical composition of the present disclosure to a subject.

[0138] In some embodiments, the subject has been previously identified as having pathological upregulation of the target gene in the targeted cells or tissues.

[0139] The subject described in the present disclosure refers to a subject diagnosed with (or suspected of having, or susceptible to) a disease or disorder that would benefit from reduction or inhibition of target mRNA expression.

[0140] Delivery can be by local administration (e.g., direct injection, implantation, or topical administration), systemic administration, or subcutaneous, intravenous, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intramuscular, transdermal, airway (aerosol), nasal, oral, rectal, or topical (including buccal and sublingual) administration.

[0141] In alternative embodiments, the pharmaceutical compositions provided herein can be administered by injection, for example, intravenously, intramuscularly, intradermally, subcutaneously, intraduodenally, or intraperitoneally.

[0142] In an alternative embodiment, after the targeting ligand and the RNAi agent are linked to form a conjugate, the conjugate can be packaged in a kit.

[0143] In some embodiments, when the RNAi agent or pharmaceutical composition of the present disclosure is contacted with a cell expressing the target gene, the RNAi agent or pharmaceutical composition of the present disclosure inhibits the expression of the target gene by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, as measured by, for example, psiCHECK activity screening and luciferase reporter gene assays, other methods such as PCR or branched DNA (bDNA)-based methods, or protein-based methods such as immunofluorescence analysis, e.g., Western Blot or flow cytometry.

[0144] In some embodiments, when the RNAi agent or pharmaceutical composition of the present disclosure is contacted with a cell expressing the target gene, the percentage of remaining target gene mRNA expression caused by the RNAi agent or pharmaceutical composition of the present disclosure is no more than 99%, no more than 95%, no more than 90%, no more than 85%, no more than 80%, no more than 75%, no more than 70%, no more than 65%, no more than 60%, no more than 55%, no more than 50%, no more than 45%, no more than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, or no more than 10%, as determined by, for example, psiCHECK activity screening and luciferase reporter gene assay, other methods such as PCR or branched DNA (bDNA)-based methods, or protein-based methods such as immunofluorescence analysis, such as Western Blot or flow cytometry.

[0145] In some embodiments, when the RNAi agent or pharmaceutical composition of the present disclosure is contacted with a cell expressing a target gene, the RNAi agent reduces off-target activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70% or at least 75% while maintaining target activity, as determined by, for example, psiCHECK activity screening and luciferase reporter gene assays, other methods such as PCR or branched DNA (bDNA)-based methods, or protein-based methods such as immunofluorescence analysis, e.g., Western Blot, or flow cytometry.

[0146] In some embodiments, the RNAi agent or pharmaceutical composition of the present disclosure, when contacted with cells expressing the target gene, reduces the target activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%, while reducing the off-target activity by at least 20%, at most 19%, at most 15%, at most 10%, at most 5%, or more than 1%, as measured by, for example, psiCHECK activity screening and luciferase reporter gene assays, other methods such as PCR or branched DNA (bDNA)-based methods, or protein-based methods such as immunofluorescence analysis, e.g., Western Blot, or flow cytometry.

[0147] In some embodiments, the RNAi agent or pharmaceutical composition of the present disclosure, when contacted with a cell expressing a target gene, increases target activity by at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%, while reducing off-target activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%, as measured by, for example, psiCHECK activity screening and luciferase reporter gene assays, other methods such as PCR or branched DNA (bDNA)-based methods, or protein-based methods such as immunofluorescence analysis, e.g., Western Blot, or flow cytometry.

[0148] In some embodiments, administration is by a route of administration that includes intramuscular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic, intravenous, subcutaneous, cerebrospinal, or a combination thereof.

[0149] The present disclosure also provides a method for preparing an RNAi agent or pharmaceutical composition, which comprises: synthesizing the RNAi agent or pharmaceutical composition described in the present disclosure.

[0150] The present disclosure also provides an RNAi agent, characterized in that one or more bases U of any RNAi agent of the present disclosure are replaced by base T, for example, 1, 2, 3, 3, 5, 6, 7, 8, 9, or 10.

[0151] The pharmaceutically acceptable salts of the compounds described in the present disclosure are selected from inorganic salts or organic salts. The compounds described in the present disclosure can react with acidic or basic substances to form corresponding salts.

[0152] On the other hand, without specifying a configuration, the compounds of the present disclosure may exist in specific geometric or stereoisomeric forms. The present disclosure contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the present disclosure. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All of these isomers and their mixtures are included within the scope of the present disclosure.

[0153] In addition, without specifying the configuration, the compounds and intermediates of the present disclosure may also exist in different tautomeric forms, and all such forms are included within the scope of the present disclosure. The term "tautomer" or "tautomeric form" refers to structural isomers of different energies that can interconvert via a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include interconversions via proton migration, such as keto-enol and imine-enamine, lactam-lactim isomerizations. All tautomeric forms are within the scope of the present disclosure. The naming of the compounds does not exclude any tautomers.

[0154] The compounds of the present disclosure may be asymmetric, for example, having one or more stereoisomers. Unless otherwise indicated, all stereoisomers are included, such as enantiomers and diastereomers. The compounds of the present disclosure containing asymmetric carbon atoms can be isolated in optically pure forms or racemic forms. Optically pure forms can be resolved from racemic mixtures or synthesized by using chiral starting materials or chiral reagents.

[0155] Optically active (R)- and (S)-isomers, as well as D and L isomers, can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the present disclosure is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide the pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), a diastereomeric salt is formed with an appropriate optically active acid or base, and then the diastereoisomers are separated by conventional methods known in the art, and then the pure enantiomer is recovered. In addition, the separation of enantiomers and diastereomers is typically accomplished using chromatography, which employs a chiral stationary phase and is optionally combined with a chemical derivatization method (e.g., carbamate formation from an amine).

[0156] The present disclosure also includes isotopically labeled compounds of the present disclosure that are identical to those described herein, but where one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into the compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as 2 H. 3 H. 11 C. 13 C. 14 C. 13 N. 15 N. 15 O. 17 O. 18 O. 31 P. 32 P. 35 S. 18 F. 123 I. 125 I and 36 Cl et al.

[0157] Unless otherwise stated, when a position is specifically designated as deuterium (D), the position is understood to have at least 1000 times the abundance of deuterium greater than the natural abundance of deuterium (which is 0.015%) (i.e., at least 10% deuterium incorporation). In the example, the natural abundance of the compound greater than deuterium can be at least 1000 times the abundance of deuterium, at least 2000 times the abundance of deuterium, at least 3000 times the abundance of deuterium, at least 4000 times the abundance of deuterium, at least 5000 times the abundance of deuterium, at least 6000 times the abundance of deuterium or more abundant deuterium. The disclosure also includes various deuterated forms of formula I and formula II compounds. Each available hydrogen atom connected to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can synthesize deuterated forms of formula I and formula II compounds with reference to relevant literature. Commercially available deuterated starting materials may be used to prepare deuterated forms of compounds of Formula I and Formula II, or they may be synthesized using conventional techniques using deuterated reagents, including but not limited to deuterated borane, trideuterated borane in tetrahydrofuran, deuterated lithium aluminum hydride, deuterated iodoethane, deuterated iodomethane, and the like.

[0158] In the chemical structures of the compounds disclosed herein, the bonds Indicates that the configuration is not specified, that is, if chiral isomers exist in the chemical structure, the bond Can be or include both Although all the structural formulas described in this disclosure are drawn as certain isomers for the sake of simplicity, the disclosure may include all isomers, such as tautomers, rotational isomers, geometric isomers, diastereomers, racemates and enantiomers. In the chemical structures of the compounds described in this disclosure, the bond No configuration is specified, i.e. the bond The configuration can be E-type or Z-type, or include both E and Z configurations.

[0159] The present disclosure incorporates the full texts of WO2022028462A1, WO2023274395A1, and WO2023208023A1.

[0160] Explanation of terms

[0161] In order to make the present disclosure more easily understood, some technical and scientific terms are specifically defined below. Unless otherwise clearly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present disclosure belongs.

[0162] As used herein, "RNAi agents" (also referred to as "RNAi triggers") refer to pharmaceutical compositions containing RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecules that are capable of degrading or inhibiting (e.g., degrading or inhibiting under appropriate conditions) the translation of messenger RNA (mRNA) transcripts of target mRNA in a sequence-specific manner. The RNAi agents used herein can act through an RNA interference mechanism (i.e., by inducing RNA interference through interaction with the RNA interference pathway machinery of mammalian cells (RNA-induced silencing complex or RISC)) or through any alternative mechanism or pathway. Although it is believed that the term RNAi agents used herein act primarily through an RNA interference mechanism, the disclosed RNAi agents are not bound or limited by any particular pathway or mechanism of action. The RNAi agents disclosed herein consist of a sense strand and an antisense strand, and include, but are not limited to, short (or small) interfering RNAs (siRNAs). The antisense strand of the RNAi agents described herein is at least partially complementary to the targeted mRNA. The RNAi agent may comprise one or more modified nucleotides and / or one or more non-phosphodiester linkages.

[0163] Unless otherwise specified, in the context of this disclosure, the terms "amyloid precursor protein," "amyloid β precursor protein," "APP," "ABPP," "amyloid precursor protein," "amyloid beta precursor protein," and "β-amyloid precursor protein" are used interchangeably. APP includes, but is not limited to, human APP, cynomolgus monkey APP, mouse APP, and rat APP, and their amino acid sequences, complete coding sequences, and mRNA sequences are readily available from publicly available databases, such as GenBank, UniProt, OMIM, and the Macaca mulatta genome project website.

[0164] The term "APP" also refers to naturally occurring DNA sequence variations of the APP gene, such as single nucleotide polymorphisms (SNPs) in the APP gene. Exemplary SNPs can be found in the dbSNP database.

[0165] The term "target sequence" refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during transcription of APP, including mRNA that is an RNA processing product of the primary transcription product. In one embodiment, the target sequence is within the protein coding region of APP.

[0166] The term "neurodegenerative disease associated with the APP gene" includes any disease or condition associated with decreased APP expression and / or activity. Symptoms or signs typically include cognitive impairment, such as decreased memory and language skills; behavioral and psychological symptoms, such as apathy and lack of motivation, gait disorders, and seizures; progressive dementia; and abnormal amyloid deposition. Typical "neurodegenerative diseases associated with the APP gene" include sporadic Alzheimer's disease, hereditary / familial Alzheimer's disease, Alzheimer's disease in patients with Down syndrome, and cerebral amyloid angiopathy.

[0167] As used herein, in the context of RNA-mediated gene silencing, the positive strand (also known as SS, SS strand or sense strand) refers to the strand comprising a sequence identical or substantially identical to the target mRNA sequence; the antisense strand (also known as AS or AS strand) refers to the strand having a sequence complementary to the target mRNA sequence.

[0168] In the context of describing the sense strand of the RNAi agent described herein, the term "differs from any one of SEQ ID NO: 1 to SEQ ID NO: 11 by no more than 3 nucleotide sequences and comprises at least 15 consecutive nucleotides" is intended to mean that the sense strand of the RNAi agent described herein comprises at least 15 consecutive nucleotides of the sense strand of any one of SEQ ID NO: 1 to SEQ ID NO: 11, or differs from at least 15 consecutive nucleotides of the sense strand of any one of SEQ ID NO: 1 to SEQ ID NO: 11 by no more than 3 nucleotide sequences (optionally, differs by no more than 2 nucleotide sequences; optionally, differs by 1 nucleotide sequence).

[0169] In this disclosure, the "5' region," also known as the "5' end," or "5' terminus" of the sense or antisense strand may be used interchangeably. For example, nucleotides 2 through 8 in the 5' region of the antisense strand may be replaced with nucleotides 2 through 8 at the 5' terminus of the antisense strand. Similarly, the "3' region," "3' terminus," and "3' terminus" of the sense or antisense strand may be used interchangeably.

[0170] Unless otherwise specified, in the context of this disclosure, "G," "C," "A," "T," and "U" represent nucleosides, including guanine, cytosine, adenine, thymidine, and uracil, respectively. It is well known to those skilled in the art that substitution of T and U does not significantly affect the properties of the RNAi agent sequence. U in the sequences disclosed herein can be arbitrarily substituted with T, and the resulting sequences are also within the scope of protection of this disclosure. In the sequence disclosed herein, for the same nucleic acid chain, from the 5' end to the 3' end, the lowercase letter m indicates that the nucleoside adjacent to the left of the letter m is a 2'-methoxy-modified nucleoside; the lowercase letter f indicates that the nucleoside adjacent to the left of the letter f is a 2'-fluoro-modified nucleoside; VP indicates that the 5' end of the nucleoside adjacent to the left / right of the letter is a trans-vinyl phosphodiester group (5'-E-VP); the lowercase letter s indicates that the two nucleosides adjacent to the letter s are connected by a thiophosphate diester group. Unless otherwise specified, the two adjacent nucleosides are connected by a phosphodiester group. Unless otherwise specified, the "RNAi agent", "nucleotide", "compound", "chemical modification", "oligonucleotide", "double-stranded RNAi inhibitor molecule", "siRNA", "siRNA conjugate", "dsRNA", "nucleic acid" and "RNAi", "nucleoside" of the present disclosure can independently exist in the form of a salt, a mixed salt or a non-salt (e.g., a free acid or a free base). When present in the form of a salt or a mixed salt, it can be a pharmaceutically acceptable salt. The term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts. When present in salt form, some groups may ionize to form anions or cations. For example, phosphodiester and thiophosphonate diester groups can exist as anions. The salt forms corresponding to the following structures are also within the scope of protection of this disclosure. Unless otherwise specified, the 3' position of the first nucleotide at the 3' end of each chain is a hydroxyl group; the 5' position of the first nucleotide at the 5' end of each chain is a hydroxyl group.

[0171] The above-mentioned modification and linking groups have the structures shown in the following table, where Base represents a base:

[0172] Table 1

[0173] The term "lipophilic group" or "lipophilic moiety" refers broadly to any compound or chemical moiety that has an affinity for lipids. One way to characterize the lipophilicity of a lipophilic moiety is by the octanol-water partition coefficient log K ow , where K ow is the ratio of the concentration of a chemical substance in the octanol phase to its concentration in the water phase at equilibrium in a two-phase system. In principle, logK owWhen it exceeds 0, the chemical is lipophilic. Typically, the logK of the lipophilic moiety is ow More than 1, more than 1.5, more than 2, more than 3, more than 4, more than 5, or more than 10, for example, the log K of 6-aminohexanol ow The logK of cholesteryl N-(hexan-6-ol) carbamate is about 0.7. ow It is 10.7.

[0174] The lipophilicity of a molecule can be altered relative to the functional groups it carries. For example, the addition of a hydroxyl or amine group to the terminus of a lipophilic moiety can increase or decrease the partition coefficient (e.g., log K) of the lipophilic moiety. ow ) value. For example, the lipophilic portion can be aliphatic, cyclic such as alicyclic, or polycyclic such as polyalicyclic compounds, such as steroids (e.g., sterols) or straight or branched aliphatic hydrocarbons. The lipophilic portion can generally comprise a hydrocarbon chain, which can be cyclic or acyclic. The hydrocarbon chain can contain various substituents and / or one or more heteroatoms, such as oxygen or sulfur atoms. Such lipophilic aliphatic portions include, but are not limited to, saturated or unsaturated C4-C 30 Hydrocarbons (such as C 10 -C 30 hydrocarbons), saturated or unsaturated fatty acids, waxes (e.g. monoalcohol esters of fatty acids and fatty diamides), terpenes (e.g., C 10 Terpenes, C 15 Sesquiterpenes, C 20 Diterpenes, C 30 Triterpenes and C 40 tetraterpenes) and other polyalicyclic hydrocarbons; for example, the lipophilic portion may be optionally substituted C 10-30 For example, the lipophilic portion can be an optionally substituted C 14-24 of a straight chain alkyl group.

[0175] As used herein, the terms "complementary" and "reverse complement" are used interchangeably and have the meanings known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one strand are paired with bases on the other strand in a complementary manner. In DNA, the purine base adenine is always paired with the pyrimidine base thymine (or uracil in RNA); the purine base guanine is always paired with the pyrimidine base cytosine. Each base pair consists of a purine and a pyrimidine. When adenine on one strand is always paired with thymine (or uracil) on the other strand, and guanine is always paired with cytosine, the two strands are considered to be complementary to each other, and the sequence of the strand can be inferred from the sequence of its complementary strand. Accordingly, "mismatch" is used in the art to mean that in a double-stranded nucleic acid, the bases at corresponding positions are not paired in a complementary manner.

[0176] As used herein, the term "inhibit" can be used interchangeably with "reduce", "silence", "downregulate", "suppress" and other similar terms, and includes any level of inhibition. Inhibition can be assessed by the reduction of the absolute or relative level of one or more of these variables compared to the control level. The control level can be any type of control level used in the art, such as a baseline level before administration or a level determined from an untreated or control-treated subject, cell, or sample (e.g., only a buffer control or an inert agent control). For example, the remaining expression of mRNA can be used to characterize the degree of inhibition of RNAi agent expression of the target gene, such as the remaining expression of mRNA being no more than 99%, no more than 95%, no more than 90%, no more than 85%, no more than 80%, no more than 75%, no more than 70%, no more than 65%, no more than 60%, no more than 55%, no more than 50%, no more than 45%, no more than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, or no more than 10%. The inhibition rate of target gene expression can be measured using The Luciferase Assay System was used to measure the firefly chemiluminescence value (Fir) and the Renilla chemiluminescence value (Ren), and the relative value Ratio = Ren / Fir was calculated. In the present disclosure, the ratio of remaining mRNA expression (or remaining activity %) = Ratio (RNAi-treated group) / Ratio (no-RNAi control group), and the inhibition rate (%) = 100% - remaining mRNA expression (%).

[0177] Unless otherwise specified, the "compound", "ligand", "nucleic acid ligand conjugate", "nucleic acid", "conjugate", "chemical modification", "targeting ligand", "dsRNA", "siRNA", and "RNAi agent" of the present disclosure can independently exist in the form of salt, mixed salt or non-salt (e.g., free acid or free base). When present in the form of a salt or a mixed salt, it can be a pharmaceutically acceptable salt. In the context of the present disclosure, "dsRNA" and "RNAi agent" can be interchangeable in some embodiments.

[0178] The term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0179] "Pharmaceutically acceptable acid addition salts" refer to salts formed with inorganic or organic acids that retain the biological effectiveness of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochlorides, hydrobromides, sulfates, nitrates, and phosphates; organic acid salts include, but are not limited to, formates, acetates, 2,2-dichloroacetates, trifluoroacetates, propionates, caproates, octanoates, decanoates, undecylenates, glycolates, gluconates, lactates, sebacates, adipates, glutarates, malonates, oxalates, maleates, succinates, fumarates, tartrates, citrates, palmitates, stearates, oleates, cinnamates, laurates, malates, glutamate, pyroglutamate, aspartate, benzoates, methanesulfonates, benzenesulfonates, p-toluenesulfonates, alginate, ascorbate, salicylates, 4-aminosalicylates, and naphthalene disulfonates. These salts can be prepared by methods known in the art.

[0180] "Pharmaceutically acceptable base addition salts" refer to salts formed with inorganic or organic bases that retain the biological effectiveness of the free acid without other side effects. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like. In some embodiments, the inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, primary amines, secondary amines, and tertiary amines, substituted amines, including natural substituted amines, cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like. Some embodiments of the organic base include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. These salts can be prepared by methods known in the art.

[0181] "Effective amount" or "effective dose" refers to the amount of a drug, compound, or pharmaceutical composition necessary to achieve any one or more beneficial or desired therapeutic results. For prophylactic uses, beneficial or desired results include eliminating or reducing the risk, lessening the severity, or delaying the onset of a condition, including the biochemical, histological, and / or behavioral symptoms of the condition, its complications, and intermediate pathological phenotypes that present during the progression of the condition. For therapeutic applications, beneficial or desired results include clinical results, such as reducing the incidence of various conditions associated with the disclosed target genes, target mRNAs, or target proteins, or ameliorating one or more symptoms of the condition, reducing the dose of another agent required to treat the condition, enhancing the efficacy of another agent, and / or delaying the progression of a condition associated with the disclosed target genes, target mRNAs, or target proteins in a patient.

[0182] As used herein, "patient," "subject," or "individual" are used interchangeably and include humans or non-human animals, such as mammals, eg, humans or monkeys.

[0183] The RNAi agents provided herein can be obtained by conventional preparation methods in the art (e.g., solid phase synthesis and liquid phase synthesis methods). Among them, solid phase synthesis already has commercial customization services. Modified nucleotide groups can be introduced into the RNAi agents described in the present disclosure by using nucleoside monomers with corresponding modifications. Methods for preparing nucleoside monomers with corresponding modifications and methods for introducing modified nucleotide groups into RNAi agents are also well known to those skilled in the art.

[0184] The term "chemically modified" or "modification" includes all changes in a nucleotide by chemical means, such as the addition or removal of a chemical moiety, or the substitution of one chemical moiety for another.

[0185] The term "base" encompasses any known DNA and RNA base, base analogues such as purines or pyrimidines, and also includes the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogues.

[0186] The terms "blunt end" or "blunt end" are used interchangeably and refer to the absence of unpaired nucleotides or nucleotide analogs at a given end of an RNAi agent, i.e., no nucleotide overhangs. In most cases, an RNAi agent having both ends blunt-ended will be double-stranded throughout its entire length.

[0187] The terms "about" and "approximately" refer to values ​​that are within an acceptable error range for a specific value as determined by one of ordinary skill in the art, which depends in part on how it is measured or determined (i.e., the limits of the measurement system). For example, "about" can mean a standard deviation within or exceeding 1. Alternatively, "about" or "substantially comprising" can mean a range of up to 20%, such as between 1% and 15%, between 1% and 10%, between 1% and 5%, between 0.5% and 5%, or between 0.5% and 1%. In this disclosure, each instance in which a number or numerical range is preceded by the term "about" also includes embodiments of the given number. Unless otherwise stated, when a specific value appears in the application and claims, the meaning of "about" or "substantially comprising" should be assumed to be within an acceptable error range for that specific value.

[0188] Unless otherwise indicated, "optionally," "optional," "optional," or "optional" means that the subsequently described event or circumstance may but need not occur, and the description includes situations in which the event or circumstance occurs or does not occur. For example, "optionally, R1 and R2 are directly linked to form a ring" means that R1 and R2 may but need not be directly linked to form a ring, and the description includes situations in which R1 and R2 are directly linked to form a ring and situations in which R1 and R2 do not form a ring.

[0189] In the chemical structural formula disclosed herein, It may be linked to one or more of any groups in accordance with the scope of the invention described herein.

[0190] The term "linked" when referring to a connection between two molecules means that the two molecules are connected by a covalent bond or the two molecules are associated via a non-covalent bond (eg, hydrogen bond or ionic bond), including direct connection and indirect connection.

[0191] The term "directly linked" refers to a first compound or group being linked to a second compound or group without any intervening atoms or groups of atoms.

[0192] The term "indirectly linked" means that a first compound or group is linked to a second compound or group through an intermediate group, compound or molecule (eg, a linking group).

[0193] The term "substituted" means that any one or more hydrogen atoms on a designated atom (typically a carbon, oxygen, and nitrogen atom) are replaced by any group as defined herein, provided that the normal valence of the designated atom is not exceeded and the substitution produces a stable compound. Non-limiting examples of substituents include C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, cyano, hydroxyl, oxo, carboxyl, cycloalkyl, cycloalkenyl, heterocyclyl, heteroaryl, aryl, ketone, alkoxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, or halogen (e.g., F, Cl, Br, I). When a substituent is ketone or oxo (i.e., =O), then two (2) hydrogen atoms on the atom are replaced.

[0194] "Substituted by one or more..." means that the compound may be substituted by a single or multiple substituents. When substituted by multiple substituents, the substituents may be multiple identical substituents or a combination of one or more different substituents. DETAILED DESCRIPTION

[0195] The present disclosure is further described below with reference to the following examples, but these examples are not intended to limit the scope of this disclosure. Experimental methods in the examples herein where specific conditions are not specified generally follow conventional conditions, such as those in the Cold Spring Harbor Laboratory Manual of Antibody Techniques and the Molecular Cloning Manual, or the conditions recommended by the raw material or product manufacturer. Reagents where the specific source is not specified are commercially available.

[0196] Example 1. Design of human APP RNAi agent

[0197] Using the human APP gene (NM_000484.3) as the target gene, a 19 / 21 nt RNAi agent was designed to meet the general criteria for active RNAi agents. The unmodified sense and antisense strand sequences are shown in Tables 2a and 2b, and the modified sense and antisense strand sequences are shown in Table 3.

[0198] Table 2a. Sense and antisense strands of the RNAi agents of the present disclosure

[0199] Table 2b. Sense and antisense strands of the RNAi agents of the present disclosure

[0200] Table 3. Sense and antisense strands of the RNAi agents of the present disclosure

[0201] In Tables 2a, 2b, and 3 above, the sequences are from left to right in the direction from 5' to 3', and "G", "C", "A", "T", and "U" represent nucleosides, which include guanine, cytosine, adenine, thymidine, and uracil, respectively. The lowercase letter m indicates that the nucleoside adjacent to the left of the letter m is a 2'-methoxy-modified nucleoside; the lowercase letter f indicates that the nucleoside adjacent to the left of the letter f is a 2'-fluoro-modified nucleoside; VP indicates that the 5' end of the nucleoside adjacent to the left / right of the letter is a trans-vinyl phosphodiester group (5'-E-VP); the lowercase letter hd indicates that the nucleoside adjacent to the left of the letter hd is a nucleoside with a -O-n-hexadecyl group attached to the 2' position of the sugar ring; NA0133' indicates that the nucleoside adjacent to the right of NA0133' is a nucleoside with a -CH2O-n-hexadecyl group attached to the 2' position of the sugar ring, i.e., it has the structure shown in formula (3) in the specification; the lowercase letter s indicates that the two nucleosides adjacent to the letter s are connected by a thiophosphate diester group. Unless otherwise specified, the two adjacent nucleosides are connected by a phosphodiester group. Unless otherwise specified, the 3' position of the first nucleotide at the 3' end of each chain is a hydroxyl group; the 5' position of the first nucleotide at the 5' end of each chain is a hydroxyl group.

[0202] The structures of the 2'-methoxy modified nucleoside, 2'-fluoro modified nucleoside, nucleoside in which the 2' position of the sugar ring is connected to -O-n-hexadecyl, trans-vinyl phosphodiester group (5'-E-VP), phosphorothioate diester group, glycoside in which the 2' position of the sugar ring is connected to -CH2O-n-hexadecyl, and phosphodiester group are shown in the following table. When the RNAi agent of the present disclosure exists in the form of a salt, for example, in the form of a sodium salt, the structure of the salt form corresponding to the structure in Table 4 below is also within the scope of protection of the present disclosure. In Table 4, Base represents the base at the corresponding position:

[0203] Table 4

[0204] Example 2. Synthesis of RNAi Agents

[0205] The synthesis of the RNAi agent disclosed herein is no different from the conventional phosphoramidite solid phase synthesis method. The synthesis process is briefly described as follows: On a Dr.Oligo48 synthesizer (Biolytic), starting with a universal CPG carrier, nucleoside phosphoramidite monomers are connected one by one according to the synthesis procedure. Nucleoside monomer raw materials include nucleoside phosphoramidite monomers with E-VP at the 5' position, 2'-methoxy modification, 2'-fluoro modification, E-vinyl phosphate group, and -O-n-hexadecyl group at the 2' position of the sugar ring. Nucleoside phosphoramidite monomers were purchased from Shanghai Zhaowei, Suzhou Jima or Jiangsu Shenji. 5-Ethylthio-1H-tetrazole (ETT) was used as an activator (0.6M acetonitrile solution), 0.22M PADS dissolved in acetonitrile and trimethylpyridine (Suzhou Kelema) solution in a volume ratio of 1:1 was used as a sulfurizing reagent, and iodine pyridine / water solution (Kelema) was used as an oxidant.

[0206] After solid-phase synthesis, the oligoribonucleotides were cleaved from the solid support and soaked in a 3:1 solution of 28% ammonia and ethanol at 50°C for 16 hours. The mixture was then centrifuged, and the supernatant was transferred to another centrifuge tube. After evaporation, the supernatant was purified using C18 reverse-phase chromatography with a mobile phase of 0.1 M TEAA and acetonitrile. DMTr was removed using a 3% trifluoroacetic acid solution. The target oligonucleotide was collected, lyophilized, identified as the target product by LC-MS, and quantified by UV (260 nm).

[0207] The obtained single-stranded oligonucleotides were annealed according to the complementary pairing in an equal molar ratio, and the resulting RNAi agent was dissolved in 1× PBS and adjusted to the required concentration for the experiment.

[0208] The RNAi agent containing NA0133' can be prepared according to the method described in PCT application WO2024125556A1.

[0209] Example 3. Inhibition of human APP in MCF-7 cells by RNAi agents - Inhibitory activity at three concentrations

[0210] In vitro molecular activity screening of RNAi agents was performed in MCF-7 cells using three concentration gradients (10 nM, 1 nM, 0.1 nM).

[0211] MCF-7 cells were cultured in Dulbecco's modified eagle medium supplemented with 10% fetal bovine serum at 37°C and 5% CO2. 24 h before transfection, MCF-7 cells were seeded in 96-well plates at a density of 1 × 10 cells per well. 4 cells, 100 μL culture medium per well.

[0212] Following the product instructions, cells were transfected with the RNAi agent using Lipofectamine RNAi MAX (ThermoFisher, 13778150) at a gradient of 10 nM, 1 nM, and 0.1 nM concentrations, with duplicate wells for each concentration. After 24 hours of treatment, total RNA was extracted using the High-Throughput Cell RNA Extraction Kit FG0417-L / FG0418-XL (Zhifan Medical, magnetic bead method). RNA reverse transcription (Takara, RR037A) and quantitative real-time PCR (Thermo, 4444557) were performed to measure human APP mRNA levels. These levels were corrected for GAPDH, an internal reference gene.

[0213] The instruments involved in this experiment are shown in Table 5.

[0214] Table 5 Experimental instruments

[0215] Among them, in the real-time quantitative PCR detection, a probe Q-PCR detection experiment was used, and its primer information is shown in Table 6.

[0216] Table 6 Taqman primer information

[0217] Result analysis method

[0218] After the Q-PCR test is completed, the corresponding Ct value is obtained according to the threshold value automatically set by the system. The expression of a gene can be relatively quantified by comparing the Ct value: Comparative Ct refers to calculating the gene expression difference by the difference between the Ct value and the internal reference gene, also known as 2 -△△Ct , ΔΔCt=[(Ct experimental group target gene-Ct experimental group internal reference)-(Ct control group target gene-Ct control group internal reference)]. Inhibition rate (%)=(1-remaining amount of target gene expression)*100%.

[0219] The results are shown in Table 7. The results of this example are expressed as the remaining percentage of human APP mRNA expression relative to cells treated with a control RNAi agent.

[0220] Table 7 Single-dose inhibitory activity of RNAi agents in MCF-7 cells

[0221] Example 4. Inhibition of human APP by RNAi agents in MCF-7 cells

[0222] The RNAi agent sequences were screened for in vitro molecular activity in MCF-7 cells using 9 concentration gradients in the same manner as in Example 3. The results of this example are shown in Table 8, and the results are expressed as the remaining percentage of human APP mRNA expression relative to cells treated with the control RNAi agent. The IC 50 The results are shown in Table 8.

[0223] Table 8 Activity results of RNAi agents at 9 concentrations in MCF-7 cells (IC 50 )

[0224] Example 5. Inhibition of human APP in A172 cells by RNAi agents - Inhibitory activity at 7 concentration points

[0225] In vitro molecular activity screening of the RNAi agent of the present invention was performed in A172 cells using 7 concentration gradients. Each RNAi agent sample had a concentration of 10 nM from the beginning of transfection, 7-fold gradient dilution, and 7 concentration points.

[0226] A172 cells were cultured in Dulbecco's modified eagle medium supplemented with 10% fetal bovine serum at 37°C and 5% CO2. 24 h before transfection, A172 cells were seeded into 96-well plates at a density of 1 × 10 cells per well. 4 cells, 100 μL culture medium per well.

[0227] Following the product instructions, cells were transfected with the RNAi agent using Lipofectamine RNAi MAX (ThermoFisher, 13778150). Seven concentrations of RNAi agent were set, starting at 10 nM, and replicates were performed for each concentration. After 24 hours of treatment, total RNA was extracted using the High-Throughput Cell RNA Extraction Kit FG0417-L / FG0418-XL (Zhifan Medical, magnetic bead method). RNA reverse transcription was performed (Takara, RR037A), and quantitative real-time PCR (Thermo, 4444557) was performed to measure human APP mRNA levels. Human APP mRNA levels were corrected for GAPDH levels.

[0228] The instruments involved in this experiment are shown in Table 5. In the real-time quantitative PCR detection, a probe Q-PCR detection experiment was used, and its primer information is shown in Table 6.

[0229] Result analysis method

[0230] After the Q-PCR test is completed, the corresponding Ct value is obtained according to the threshold value automatically set by the system. The expression of a gene can be relatively quantified by comparing the Ct value: Comparative Ct refers to calculating the gene expression difference by the difference between the Ct value and the internal reference gene, also known as 2 -△△Ct , ΔΔCt=[(Ct experimental group target gene-Ct experimental group internal reference)-(Ct control group target gene-Ct control group internal reference)]. Inhibition rate (%)=(1-remaining amount of target gene expression)*100%.

[0231] The results are expressed as the percentage of human APP mRNA expression remaining relative to that in cells treated with a control RNAi agent. 50 The results are shown in Table 9.

[0232] Table 9 Activity results of RNAi agents at 7 concentrations in A172 cells (IC 50 )

[0233] Example 6. Inhibitory activity of RNAi agents on human APP in primary human hepatocytes (PHH)

[0234] The RNAi agents in Table 10 were screened for activity in human primary hepatocytes (PHH) using 7 concentration gradients. The transfection concentration of each RNAi agent sample was 10 nM, with 5-fold gradient dilution and 7 concentration points.

[0235] PHH were frozen in liquid nitrogen. 24 h before transfection, primary human hepatocytes (PHH) were revived and seeded into 96-well plates at a seeding density of 3 × 10 cells per well. 4 cells, 80 μL of culture medium per well.

[0236] According to the product manual, RNAi agents were transfected using Lipofectamine RNAi MAX (ThermoFisher, 13778150) at a gradient final concentration of 10 nM, 2 nM, 0.4 nM, 0.08 nM, 0.016 nM, 0.0032 nM, and 0.00064 nM. After 24 hours of treatment, total RNA was extracted from cells using a high-throughput cell RNA extraction kit, followed by RNA reverse transcription and quantitative real-time PCR. Human APP mRNA levels were measured and corrected for GAPDH mRNA levels.

[0237] In the real-time quantitative PCR detection, a probe Q-PCR detection experiment was used, and its primer information is shown in Table 10. The results of this example are shown in Table 10. The results in Table 10 show that the RNAi agent disclosed herein has a high level of on-target inhibitory activity against the APP gene in PHH cells.

[0238] Table 10 Activity results of RNAi agents at 7 concentrations in PHH cells (IC 50 )

[0239] Example 7. Inhibitory activity of RNAi agents on monkey APP in monkey primary hepatocytes (PCH)

[0240] Seven concentration gradients were used in monkey primary hepatocytes (PCH) to screen the activity of the RNAi agents listed in Table 10. The concentration of each RNAi agent sample was 10 nM from the beginning of transfection, with 5-fold gradient dilution and 7 concentration points.

[0241] PCH was frozen in liquid nitrogen. 24 h before transfection, monkey primary hepatocytes (PCH) were revived and seeded into 96-well plates at a seeding density of 3 × 10 4 cells, 80 μL of culture medium per well.

[0242] According to the product manual, RNAi agents were transfected using Lipofectamine RNAi MAX (ThermoFisher, 13778150) at a gradient of 10 nM, 2 nM, 0.4 nM, 0.08 nM, 0.016 nM, 0.0032 nM, and 0.00064 nM. After 24 hours of treatment, total RNA was extracted from cells using a high-throughput cell RNA extraction kit, followed by RNA reverse transcription and quantitative real-time PCR. Monkey APP mRNA levels were measured and corrected for GAPDH levels.

[0243] In the real-time quantitative PCR detection, a probe Q-PCR detection experiment was used, and its primer information is shown in Table 11. The results of this example are shown in Table 12, which shows that the RNAi agent disclosed herein has a high level of on-target inhibitory activity against the APP gene in PCH cells.

[0244] Table 11 Taqman primer information

[0245] Table 12 Activity results of RNAi agents at 7 concentrations in PCH cells (IC 50 )

[0246] Example 8. Inhibitory activity of RNAi agents on human APP in A172 cells

[0247] The RNAi agent of the present invention was tested for in vitro molecular activity in A172 cells using 7 concentration gradients. Each RNAi agent sample had a concentration of 10 nM from the beginning of transfection, 7-fold gradient dilution, and 7 concentration points.

[0248] A172 cells were cultured in Dulbecco's modified eagle medium supplemented with 10% fetal bovine serum at 37°C and 5% CO2. 24 h before transfection, A172 cells were seeded in 96-well plates at a density of 1 × 10 cells per well. 4 cells, 100 μL culture medium per well.

[0249] Following the product instructions, cells were transfected with the RNAi agent using Lipofectamine RNAi MAX (ThermoFisher, 13778150). Seven concentrations of RNAi agent were set, starting at 10 nM, and duplicate wells were used for each concentration. After 48 hours of treatment, total RNA was extracted using the MNTR / FX96 high-throughput cell RNA extraction kit (Zhiang Bio, magnetic bead method). RNA reverse transcription was performed (Takara, RR037A) and quantitative real-time PCR (Thermo, 4444557) to measure human APP mRNA levels. Human APP mRNA levels were corrected for GAPDH levels.

[0250] Result analysis method

[0251] After the Q-PCR test is completed, the corresponding Ct value is obtained according to the threshold value automatically set by the system. The expression of a gene can be relatively quantified by comparing the Ct value: Comparative Ct refers to calculating the gene expression difference by the difference between the Ct value and the internal reference gene, also known as 2 -△△Ct , ΔΔCt=[(Ct experimental group target gene-Ct experimental group internal reference)-(Ct control group target gene-Ct control group internal reference)]. Inhibition rate (%)=(1-remaining amount of target gene expression)*100%.

[0252] The results are expressed as the percentage of human APP mRNA expression remaining relative to that in cells treated with a control RNAi agent. 50 The results are shown in Table 13. As shown in the results in Table 13, the RNAi agent of the present disclosure has good APP mRNA inhibitory activity.

[0253] Table 13 Activity results of RNAi agents at 7 concentrations in A172 cells (IC 50 )

[0254] Example 9. On-target activity of the RNAi agent psiCHECK

[0255] The RNAi agents of the present invention were screened for on-target activity at the molecular level in vitro using 9 concentration gradients in HEK293A cells.

[0256] The target plasmid GSCM, containing the target sequence of the RNAi agent disclosed herein, was inserted into the psiCHECK-2 plasmid, which contains both the Renilla luciferase gene and the firefly luciferase gene. As a dual-reporter gene system, the RNAi agent's target sequence was inserted into the 3' UTR region of the Renilla luciferase gene. The activity of the RNAi agent against the target sequence was detected by measuring Renilla luciferase expression after calibration with firefly luciferase.

[0257] HEK293A cells were cultured in DMEM high glucose medium containing 10% fetal bovine serum at 37°C and 5% CO2. 24 h before transfection, HEK293A cells were seeded in 96-well plates at a seeding density of 8 × 10 cells per well. 3 cells, 100 μL culture medium per well.

[0258] According to the instructions, Lipofectamine 2000 (ThermoFisher, 11668019) was used to co-transfect the cells with the RNAi agent and the corresponding GSCM plasmid, using 0.2 μL Lipofectamine 2000 per well. The plasmid transfection amount was 20 ng per well. For the target sequence plasmid, a total of 9 concentration points were set, with a starting concentration of 20 nM, 3-fold gradient dilution, and double replicates for each concentration. 24 h after transfection, the target level was detected using the Dual-Luciferase Reporter Assay System (Promega, E2940). The results are shown in Table 14. As shown in the results of Table 14, the RNAi agent disclosed herein exhibits excellent APP mRNA inhibitory activity.

[0259] Table 14 Activity results of RNAi agents at 9 concentration points in the psi-Check system (IC 50 )

[0260] Example 10. On-target activity of the RNAi agent psiCHECK

[0261] The sequences of RNAi agents were screened for on-target activity at the in vitro molecular level using two concentrations in HEK293A cells.

[0262] The on-target plasmid GSCM, which contains the RNAi agent's target sequence, is inserted into the psiCHECK-2 plasmid, which contains both the Renilla luciferase and firefly luciferase genes. As a dual-reporter system, the RNAi agent's target sequence is inserted into the 3'UTR region of the Renilla luciferase gene. RNAi agent activity against the target sequence is measured by measuring Renilla luciferase expression in a calibrated assay using firefly luciferase.

[0263] HEK293A cells were cultured in DMEM high glucose medium containing 10% fetal bovine serum at 37°C and 5% CO2. 24 h before transfection, HEK293A cells were seeded in 96-well plates at a seeding density of 8 × 10 cells per well. 3 cells, 100 μL culture medium per well.

[0264] Cells were co-transfected with the RNAi agent and the corresponding GSCM plasmid using Lipofectamine 2000 (ThermoFisher, 11668019) according to the manufacturer's instructions. 0.2 μL of Lipofectamine 2000 was used per well. A total of 20 ng of plasmid was transfected per well. Two concentrations of the target sequence plasmid were set: 0.03 nM and 0.01 nM, with duplicate wells for each concentration. 24 hours after transfection, on-target levels were measured using the Dual-Luciferase Reporter Assay System (Promega, E2940).

[0265] The results are shown in Table 15. The results in Table 15 indicate that the RNAi agent of the present disclosure exhibits excellent inhibitory activity against APP mRNA in the psi-Check system even at low concentrations.

[0266] Table 15 Activity results of RNAi agents at two concentration points in the psi-Check system

[0267] Example 11. Inhibitory activity of RNAi agents on human APP gene expression in A172 cells

[0268] The compounds were tested for in vitro molecular activity in A172 cells using eight concentration gradients, following the same method as in Example 8. Each RNAi agent sample was transfected at a concentration of 10 nM, with a 7-fold gradient dilution and eight concentration points. The results were expressed as the percentage of human APP mRNA expression remaining relative to cells treated with the control RNAi agent. The IC of inhibition was 50 The results are shown in Table 16. The results in Table 16 indicate that the RNAi agent of the present disclosure has excellent inhibitory activity against APP mRNA.

[0269] Table 16 Activity results of RNAi agents at 8 concentrations in A172 cells (IC 50 )

Claims

1. A RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region; The sense strand comprises at least 15 consecutive nucleotides and differs from any nucleotide sequence in SEQ ID NO: 1 to SEQ ID NO: 11 by no more than 3 nucleotides; and The antisense strand comprises at least 15 consecutive nucleotides and differs from any nucleotide sequence of SEQ ID NO: 12 to SEQ ID NO: 22 by no more than 3 nucleotides.

2. The RNAi agent according to claim 1, wherein the sense strand comprises at least 17 consecutive nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 11; the antisense strand comprises at least 17 consecutive nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of any one of SEQ ID NO: 12 to SEQ ID NO: 22; Preferably, the sense strand comprises at least 19 consecutive nucleotides that differ from any one of SEQ ID NO: 1 to SEQ ID NO: 11 by no more than 3 nucleotides; preferably the nucleotide sequence differs by no more than 1 nucleotide, and / or, The antisense strand comprises at least 21 consecutive nucleotides that differ from the nucleotide sequence of any one of SEQ ID NO: 12 to SEQ ID NO: 22 by no more than 3 nucleotides; preferably, the nucleotide sequence differs by no more than 1 nucleotide.

3. The RNAi agent according to claim 1 or claim 2, wherein the RNAi agent comprises or consists of any combination of the following sense strands and antisense strands: Group 1), the sense strand as shown in SEQ ID NO: 1 and the antisense strand as shown in SEQ ID NO: 12; or the sense strand as shown in SEQ ID NO:2 and the antisense strand as shown in SEQ ID NO:13; Group 2), the sense strand as shown in SEQ ID NO:3 and the antisense strand as shown in SEQ ID NO:14; or the sense strand as shown in SEQ ID NO:4 and the antisense strand as shown in SEQ ID NO:15; Group 3), the sense strand as shown in SEQ ID NO:5 and the antisense strand as shown in SEQ ID NO:16; Group 4), the sense strand as shown in SEQ ID NO:6 and the antisense strand as shown in SEQ ID NO:17; or the sense strand as shown in SEQ ID NO:7 and the antisense strand as shown in SEQ ID NO:18; Group 6), the sense strand as shown in SEQ ID NO:8 and the antisense strand as shown in SEQ ID NO:19; or the sense strand as shown in SEQ ID NO:9 and the antisense strand as shown in SEQ ID NO:20; and Group 7), the sense strand as shown in SEQ ID NO: 10 and the antisense strand as shown in SEQ ID NO: 21; Or the sense strand as shown in SEQ ID NO:11 and the antisense strand as shown in SEQ ID NO:

22.

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

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

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

7. The RNAi agent according to claim 6, wherein the phosphodiester group having a modifying group is present in at least one of the positions selected from the group consisting of: Between the first nucleotide and the second nucleotide at the 5' end of the sense strand; Between the second nucleotide and the third nucleotide at the 5' end of the sense strand; Between the first nucleotide and the second nucleotide at the 3' end of the sense strand; Between the second nucleotide and the third nucleotide at the 3' end of the sense strand; Between the first nucleotide and the second nucleotide at the 5' end of the antisense strand; Between the second nucleotide and the third nucleotide at the 5' end of the antisense strand; between the first nucleotide and the second nucleotide at the 3' end of the antisense strand; and The 3' end of the antisense strand is between the second nucleotide and the third nucleotide.

8. The RNAi agent of any one of claims 1-7, wherein the 5' end of the antisense strand comprises a phosphorus-containing group, and the phosphorus-containing group is a 5'-vinyl phosphodiester group.

9. The RNAi agent according to any one of claims 1 to 8, wherein The RNAi agent is linked to one or more lipophilic groups; Preferably, the lipophilic group is linked to the first or sixth nucleotide at the 5' end of the sense strand.

10. The RNAi agent according to any one of claims 1 to 9, wherein The sense strand comprises the nucleotide sequence shown in any one of SEQ ID NO: 109 to SEQ ID NO: 125; and / or, the antisense strand comprises the nucleotide sequence shown in any one of SEQ ID NO: 126 to SEQ ID NO: 142; Preferably, the RNAi agent comprises or consists of any combination of the following sense strands and antisense strands: The sense strand as set forth in SEQ ID NO: 109 and the antisense strand as set forth in SEQ ID NO: 126; The sense strand as shown in SEQ ID NO: 110 and the antisense strand as shown in SEQ ID NO: 127; The sense strand as shown in SEQ ID NO:111 and the antisense strand as shown in SEQ ID NO:128; The sense strand as set forth in SEQ ID NO: 112 and the antisense strand as set forth in SEQ ID NO: 129; The sense strand as shown in SEQ ID NO: 113 and the antisense strand as shown in SEQ ID NO: 130; The sense strand as set forth in SEQ ID NO: 114 and the antisense strand as set forth in SEQ ID NO: 131; The sense strand as set forth in SEQ ID NO: 115 and the antisense strand as set forth in SEQ ID NO: 132; The sense strand as set forth in SEQ ID NO: 116 and the antisense strand as set forth in SEQ ID NO: 133; The sense strand as set forth in SEQ ID NO: 117 and the antisense strand as set forth in SEQ ID NO: 134; The sense strand as set forth in SEQ ID NO: 118 and the antisense strand as set forth in SEQ ID NO: 135; The sense strand as set forth in SEQ ID NO: 119 and the antisense strand as set forth in SEQ ID NO: 136; The sense strand as set forth in SEQ ID NO: 120 and the antisense strand as set forth in SEQ ID NO: 137; The sense strand as shown in SEQ ID NO: 121 and the antisense strand as shown in SEQ ID NO: 138; The sense strand as set forth in SEQ ID NO: 122 and the antisense strand as set forth in SEQ ID NO: 139; The sense strand as shown in SEQ ID NO: 123 and the antisense strand as shown in SEQ ID NO: 140; The sense strand as set forth in SEQ ID NO: 124 and the antisense strand as set forth in SEQ ID NO: 141; and The sense strand is shown as SEQ ID NO:125 and the antisense strand is shown as SEQ ID NO:

142.

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

12. A cell comprising the RNAi agent of any one of claims 1-10.

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

14. A method for reducing the expression of amyloid precursor protein (APP), comprising administering to a subject the RNAi agent according to any one of claims 1-10 or the pharmaceutical composition according to claim 11.

15. A method for treating and / or preventing a disease, comprising administering to a subject the RNAi agent according to any one of claims 1-10, or the pharmaceutical composition according to claim 11; preferably, the disease is a neurodegenerative disease associated with the APP gene; more preferably, the disease is selected from Alzheimer's disease, frontotemporal dementia and cerebral amyloid angiopathy.

16. A method for in vivo delivery of a RNAi agent that inhibits APP expression and / or replication, comprising administering the RNAi agent according to any one of claims 1 to 10, or the pharmaceutical composition according to claim 11, to a subject.

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

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