Rnai agent targeting SARM1 and pharmaceutical use thereof

Abstract

The present disclosure relates to an RNAi agent targeting SARM1 and a pharmaceutical use thereof. Specifically, the present disclosure further relates to a pharmaceutical composition, cell or kit comprising the RNAi agent, and a method of using the RNAi agent for treating and / or preventing related disorders suffered by a subject.

Description

RNAi agents targeting SARM1 and their pharmaceutical applications

[0001] This disclosure claims priority to Chinese Patent Application No. 202410789680.3, filed June 19, 2024; Chinese Patent Application No. 202411004439.1, filed July 25, 2024; and Chinese Patent Application No. 202510007682.7, filed January 3, 2025, the full text of which is incorporated herein by reference. Technical Field

[0002] This disclosure pertains to the field of biomedicine and specifically relates to RNAi agents, pharmaceutical compositions, and their pharmaceutical uses that target sterile alpha and TIR motif 1 (SARM1). Background Technology

[0003] Axonal degeneration occurs after nerve injury and is a hallmark of various neurological diseases, including peripheral nerve disorders, traumatic brain injury, and neurodegenerative diseases. Due to the disruption of neural circuit integrity, it leads to progressive loss of neurological function in patients. Wallerian degeneration, as a form of programmed subcellular death, promotes axonal breakdown in disease and injury and is an intrinsic axonal damage-activating molecular pathway. SARM1 (sterile alpha and TIR motif containing 1) is one of the main regulators of active axonal destruction in this pathway.

[0004] SARM1, a Toll-like receptor adaptor protein, is a complex multi-domain protein containing an ARM (Armadillo / HEAT repeat) domain, a SAM (Sterile alpha motif) domain, and a TIR (Toll / interleukin-receptor) domain. SARM1 can regulate the NF-κB post-regulation signaling pathway after activating the Toll-like receptor. Its intrinsic NADase activity within its TIR domain can also catalyze the generation of ADPR (Adenosine 5,-diphosphate ribose) and cyclic cADPR (Cyclic adenosine 5,-diphosphate ribose) from NAD+. SARM1 activation is strictly regulated. In healthy neurons, SARM1 is inactive. When responding to various pathological triggers, the decrease in the level of the axon maintenance factor NMNAT2 (Nicotinamide mononucleotide adenylyl transferase 2) leads to SARM1 activation. Damaged axons actively drive their own destruction through the SAM domain and TIR motif, resulting in Ca2+ entry, calpain activation and cytoskeleton degradation. It plays an important pro-degenerative role in programmed axon death (Wallerian degeneration).

[0005] Axonal degeneration is a common feature of many acute, chronic, sporadic, and familial neurological diseases. SARM1, as a regulator of active axonal destruction, can serve as a target for axon-specific therapeutic interventions. This disclosure aims to provide a novel nucleotide drug that inhibits SARM1, which can effectively block the pathway by inhibiting the expression of SARM1 protein to maintain the integrity of the patient's axon, providing a potential treatment for such diseases for which there is currently a lack of effective clinical interventions. Summary of the Invention

[0006] This disclosure provides an RNAi agent that targets SARM1.

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

[0008] The sense strand comprises at least 15 consecutive nucleotide sequences that differ from any nucleotide sequence in SEQ ID NO:1 to SEQ ID NO:17, SEQ ID NO:18 to SEQ ID NO:87, and SEQ ID NO:385 to SEQ ID NO:391 by no more than 3 nucleotides; the antisense strand comprises at least 15 consecutive nucleotide sequences that differ from any nucleotide sequence in SEQ ID NO:88 to SEQ ID NO:104, SEQ ID NO:105 to SEQ ID NO:174, and SEQ ID NO:392 to SEQ ID NO:398 by no more than 3 nucleotides.

[0009] In some schemes, "difference of no more than 3 nucleotides" means that the difference can be 0, 1, 2, or 3 nucleotides; in some schemes, "at least 15 consecutive" means at least 15, 16, 17, 18, 19, 20, or 21 consecutive; preferably, at least 17, 18, 19, 20, or 21 consecutive.

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

[0011] In some implementations, the justice chain and antisense chain are at least partially anticomplementary to form a dual-chain region. In some implementations, there are no more than 5, 4, 3, 2, or 1 mismatches between the justice chain and antisense chain. In some implementations, the justice chain and antisense chain are completely anticomplementary.

[0012] In some embodiments, the RNAi agent disclosed herein comprises one or two blunt ends.

[0013] In some embodiments, the sense and / or antisense strands of the RNAi agent disclosed herein each independently contain one or two unpaired nucleotides. In some embodiments, the 3' end of the antisense strand includes a protrusion formed by unpaired nucleotides.

[0014] In some embodiments, the sense strand and antisense strand are each independently composed of 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. In some embodiments, the sense strand and antisense strand are each independently composed of 18, 19, 20, 21, 22, or 23 nucleotides.

[0015] In some implementations, the lengths of the sense strand and the antisense strand may be the same or different, with the sense strand being 19-23 nucleotides long and the antisense strand being 19-26 nucleotides long. Therefore, the length ratio of the sense strand to the antisense strand of the RNAi agent provided in this 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 / 19, 19 / 21, 21 / 21, 21 / 23, 23 / 23, or 23 / 25. In some embodiments, the length ratio of the sense strand to the antisense strand is 19 / 21 or 21 / 23.

[0016] In some embodiments, the sense strand comprises or is selected from any one of the nucleotide sequences shown in SEQ ID NO:1 to SEQ ID NO:17, SEQ ID NO:18 to SEQ ID NO:87, and SEQ ID NO:385 to SEQ ID NO:391; the antisense strand comprises or is selected from any one of the nucleotide sequences shown in SEQ ID NO:88 to SEQ ID NO:104, SEQ ID NO:105 to SEQ ID NO:174, and SEQ ID NO:392 to SEQ ID NO:398.

[0017] In some embodiments, the RNAi agent comprises or is selected from any of the following groups of sense and antisense strands:

[0018] Group 1), such as the sense chain shown in SEQ ID NO: 1 and the antisense chain shown in SEQ ID NO: 88;

[0019] Group 2), such as the sense chain shown in SEQ ID NO: 2 and the antisense chain shown in SEQ ID NO: 89, or, such as the sense chain shown in SEQ ID NO: 3 and the antisense chain shown in SEQ ID NO: 90;

[0020] Group 3), such as the sense chain shown in SEQ ID NO: 4 and the antisense chain shown in SEQ ID NO: 91, or, such as the sense chain shown in SEQ ID NO: 5 and the antisense chain shown in SEQ ID NO: 92;

[0021] Group 4), such as the sense chain shown in SEQ ID NO: 6 and the antisense chain shown in SEQ ID NO: 93, or, such as the sense chain shown in SEQ ID NO: 7 and the antisense chain shown in SEQ ID NO: 94;

[0022] Group 5), such as the sense chain shown in SEQ ID NO: 8 and the antisense chain shown in SEQ ID NO: 95;

[0023] Group 6), such as the sense chain shown in SEQ ID NO: 9 and the antisense chain shown in SEQ ID NO: 96;

[0024] Group 7), such as the sense chain shown in SEQ ID NO: 10 and the antisense chain shown in SEQ ID NO: 97;

[0025] Group 8), such as the sense chain shown in SEQ ID NO: 11 and the antisense chain shown in SEQ ID NO: 98;

[0026] Group 9), such as the sense chain shown in SEQ ID NO: 12 and the antisense chain shown in SEQ ID NO: 99, or, such as the sense chain shown in SEQ ID NO: 13 and the antisense chain shown in SEQ ID NO: 100;

[0027] Group 10), such as the sense chain shown in SEQ ID NO: 14 and the antisense chain shown in SEQ ID NO: 101, or, such as the sense chain shown in SEQ ID NO: 15 and the antisense chain shown in SEQ ID NO: 102;

[0028] Group 11), such as the sense chain shown in SEQ ID NO: 16 and the antisense chain shown in SEQ ID NO: 103;

[0029] Group 12), such as the sense chain shown in SEQ ID NO: 17 and the antisense chain shown in SEQ ID NO: 104;

[0030] For the sense chains shown in SEQ ID NO:18 to SEQ ID NO:87, SEQ ID NO:385 to SEQ ID NO:391, and the antisense chains shown in SEQ ID NO:105 to SEQ ID NO:174, SEQ ID NO:392 to SEQ ID NO:398, the pairwise correspondences are shown in Table 1B, and are considered to have been fully described with reference to the above content and format.

[0031] In some embodiments, at least one nucleotide in the sense strand and / or antisense strand is a modified nucleotide.

[0032] In some implementations, all nucleotides in the sense strand and / or antisense strand are modified nucleotides.

[0033] In some embodiments, the positive strand contains three consecutive 2'-fluorinated nucleotides. In some embodiments, the three consecutive nucleotides at positions 7-9 of the 5' end of the positive strand are 2'-fluorinated nucleotides. In some embodiments, the three consecutive nucleotides at positions 7-9 of the 5' end of the positive strand are 2'-fluorinated nucleotides, and the remaining nucleotides are non-fluorinated nucleotides; preferably, the non-fluorinated nucleotides are 2'-methoxylated nucleotides.

[0034] In some embodiments, the antisense strand contains at least five 2'-fluoro-modified nucleotides, for example, it may contain five, six, seven, eight, nine or ten 2'-fluoro-modified nucleotides.

[0035] In some embodiments, the nucleotides at positions 2, 6, 12, 14, and 16 are 2'-fluorinated nucleotides, arranged from the 5' end to the 3' end. In some embodiments, the antisense strand contains five 2'-fluorinated nucleotides, with the nucleotides at positions 2, 6, 12, 14, and 16 being 2'-fluorinated nucleotides, and the remaining nucleotides being non-fluorinated nucleotides. In some embodiments, the antisense strand contains eight 2'-fluorinated nucleotides, with the nucleotides at positions 2, 4, 6, 10, 12, 14, 16, and 18 being 2'-fluorinated nucleotides, and the remaining nucleotides being non-fluorinated nucleotides. Preferably, the non-fluorinated nucleotides are 2'-methoxylated nucleotides.

[0036] In some embodiments, at least one phosphodiester group in the sense strand and / or antisense strand is a phosphodiester group with a modifying group. The phosphodiester group with the modifying group increases the stability of the RNAi agent in a biological sample or environment. In some embodiments, the sense strand and / or antisense strand includes multiple phosphodiester groups with modifying groups. In some embodiments, both the sense strand and the antisense strand contain multiple phosphodiester groups with modifying groups, for example, two, three, four, or five phosphodiester groups with modifying groups.

[0037] In some embodiments, the phosphodiester group with the modifying group is present at one or more locations selected from: between any two adjacent nucleotides from the first to the fourth nucleotide from the 5' end and / or the 3' end of the sense strand, and / or between any two adjacent nucleotides from the first to the fourth nucleotide from the 5' end and / or the 3' end of the antisense strand. For example, it may be located at at least one of the following locations:

[0038] Between the first and second nucleotides at the 5' end of the positive strand;

[0039] Between the second and third nucleotides at the 5' end of the positive strand;

[0040] Between the first and second nucleotides at the 3' end of the positive strand;

[0041] Between the second and third nucleotides at the 3' end of the positive strand;

[0042] Between the first and second nucleotides at the 5' end of the antisense strand;

[0043] Between the second and third nucleotides at the 5' end of the antisense strand;

[0044] Between the first and second nucleotides at the 3' end of the antisense strand;

[0045] Between the second and third nucleotides at the 3' end of the antisense strand.

[0046] In some embodiments, the sense and / or antisense chains include a plurality of phosphothioester groups, the phosphothioester groups being present in:

[0047] Between the first and second nucleotides at the 5' end of the positive strand; and

[0048] Between the second and third nucleotides at the 5' end of the positive strand; and

[0049] Between the first and second nucleotides at the 3' end of the positive strand; and

[0050] Between the second and third nucleotides at the 3' end of the positive strand; and

[0051] Between the first and second nucleotides at the 5' end of the antisense strand; and

[0052] Between the second and third nucleotides at the 5' end of the antisense strand; and

[0053] Between the first and second nucleotides at the 3' end of the antisense strand; and

[0054] Between the second and third nucleotides at the 3' end of the antisense strand.

[0055] In some embodiments, the phosphodiester group with the modifying group is a thiophosphate diester group. In some embodiments, the phosphodiester group with the modifying group is a 5'-vinylphosphate diester group. In some embodiments, the first nucleotide at the 5' end of the sense or antisense strand is a nucleotide containing a 5' phosphate modification; preferably, the 5' phosphate modified nucleotide is a nucleotide with a 5' end modified with vinyl phosphate (5'-VP).

[0056] In some embodiments, the positive chain is selected from or includes the nucleotide sequences shown in any one of SEQ ID NO: 175 to SEQ ID NO: 208, SEQ ID NO: 209 to SEQ ID NO: 279.

[0057] In some embodiments, the antisense strand is selected from or includes the nucleotide sequence shown in any one of SEQ ID NO: 280 to SEQ ID NO: 313, SEQ ID NO: 314 to SEQ ID NO: 384.

[0058] In some embodiments, the RNAi agent further comprises one or more delivery groups linked to the sense and / or antisense strands. In the context of this disclosure, "linked" includes both covalent and non-covalent linkages. The delivery group enables the delivery of the RNAi agent of this disclosure to a target site where SARM1 expression is present.

[0059] In some embodiments, the delivery group is a lipophilic group, and the one or more lipophilic groups are attached to any one or more nucleotides in the sense or antisense strand of the RNAi agent. In some embodiments, all of the one or more lipophilic groups are attached to the sense strand of the RNAi agent. In some embodiments, all of the one or more lipophilic groups are attached to the antisense strand of the RNAi agent. In some embodiments, at least one of the lipophilic groups is attached to the sense strand of the RNAi agent, and at least one of the lipophilic groups is attached to the antisense strand of the RNAi agent.

[0060] In some embodiments, the lipophilic group is linked to a base of the nucleotide; in some embodiments, the lipophilic group is linked to a sugar ring of the nucleotide. In some embodiments, the lipophilic group is linked to an internucleotide linker between two adjacent nucleotides. In some embodiments, the lipophilic group comprises a saturated or unsaturated C group. 4-30 A hydrocarbon chain, and optionally a functional group selected from halogens, alkoxy groups, hydroxyl groups, amines, carboxylic acids, sulfonates, phosphates, thiols, azides, and alkynes.

[0061] In some embodiments, the lipophilic group comprises saturated or unsaturated C. 6-18 Hydrocarbon chain. In some embodiments, the lipophilic group comprises saturated or unsaturated C atoms. 16 Hydrocarbon chain.

[0062] In some embodiments, the delivery group comprises a targeting ligand that targets the liver. In some embodiments, the targeting ligand binds to the asialic acid glycoprotein receptor (ASGPR). In some embodiments, the targeting ligand comprises a galactose cluster or a cluster of galactose derivatives selected from N-acetyl-galactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine, or N-isobutyrylgalactosamine.

[0063] In some embodiments, the delivery group is attached to the 3' end of the positive strand of the RNAi agent.

[0064] In some embodiments, the delivery group is attached to the end of the RNAi agent via a phosphodiester group, a thiophosphate diester group, or a phosphonate group. In some embodiments, the delivery group is attached to the end of the RNAi agent via a phosphodiester group.

[0065] In some embodiments, the delivery group is indirectly linked to the end of the RNAi agent via a phosphodiester group, a thiophosphate diester group, or a phosphonic acid group. In some embodiments, the delivery group is indirectly linked to the end of the RNAi agent via a phosphodiester group.

[0066] In some embodiments, the delivery group is directly attached to the end of the RNAi agent via a phosphodiester group, a thiophosphate diester group, or a phosphonic acid group. In some embodiments, the delivery group is directly attached to the end of the RNAi agent via a phosphodiester group.

[0067] In some embodiments, the delivery group is directly attached to the 3' end of the positive strand of the RNAi agent via a phosphodiester group or a thiophosphate diester group.

[0068] On the other hand, this disclosure provides a pharmaceutical composition comprising the RNAi agent described herein and one or more pharmaceutically acceptable excipients, such as vehicles, carriers, diluents, and / or drug delivery systems (such as delivery polymers). Various drug delivery systems are known and can be used with the RNAi agent of this disclosure, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the RNAi agent, receptor-mediated endocytosis, and constructing nucleic acids as part of retroviruses or other vectors.

[0069] In some embodiments, the pharmaceutical composition may further comprise pharmaceutically acceptable excipients and / or adjuvants, which may be one or more formulations or compounds conventionally used in the art. For example, the pharmaceutically acceptable excipients may include at least one of pH buffers, protectants, and osmotic pressure regulators.

[0070] In some embodiments, the unit dose of the pharmaceutical composition is 0.001 mg to 1000 mg.

[0071] In some embodiments, the pharmaceutical composition contains 0.01-99.99% of the aforementioned RNAi agent based on the total weight of the composition. In some embodiments, the pharmaceutical composition contains 0.1-99.9% of the aforementioned RNAi agent. In some embodiments, the pharmaceutical composition contains 0.5%-99.5% of the aforementioned RNAi agent. In some embodiments, the pharmaceutical composition contains 1%-99% of the aforementioned RNAi agent. In some embodiments, the pharmaceutical composition contains 2%-98% of the aforementioned RNAi agent.

[0072] In some embodiments, the pharmaceutical composition contains 0.01% to 99.99% pharmaceutically acceptable excipients based on the total weight of the composition. In some embodiments, the pharmaceutical composition contains 0.1% to 99.9% pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical composition contains 0.5% to 99.5% pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical composition contains 1% to 99% pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical composition contains 2% to 98% pharmaceutically acceptable excipients.

[0073] In some embodiments, when the RNAi agent or pharmaceutical composition of this disclosure comes into contact with cells expressing a target gene, it is 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 assays, such as Western blotting or flow cytometry, that the RNAi agent or pharmaceutical composition of this 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%.

[0074] In some embodiments, when the RNAi agent or pharmaceutical composition of this disclosure comes into contact with cells expressing the target gene, the percentage of residual expression of the target gene mRNA induced by the RNAi agent or pharmaceutical composition of this disclosure is 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 blotting or flow cytometry, to be no higher than 99%, no higher than 95%, no higher than 90%, no higher than 85%, no higher than 80%, no higher than 75%, no higher than 70%, no higher than 65%, no higher than 60%, no higher than 55%, no higher than 50%, no higher than 45%, no higher than 40%, no higher than 35%, no higher than 30%, no higher than 25%, no higher than 20%, no higher than 15%, or no higher than 10%.

[0075] In some embodiments, when the RNAi agent or pharmaceutical composition described herein comes into contact with cells expressing a target gene, 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 assays, such as Western blotting, or flow cytometry, 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.

[0076] In some embodiments, when the RNAi agent or pharmaceutical composition described herein comes into contact with cells expressing a target gene, the RNAi agent, 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 assays, such as Western blotting, or flow cytometry, 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 reducing target activity by at least 20%, at least 19%, at least 15%, at most 10%, at most 5%, or more than 1%.

[0077] In some embodiments, when the RNAi agent or pharmaceutical composition described herein comes into contact with cells expressing a target gene, the RNAi agent, 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 assays, such as Western blotting, or flow cytometry, 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%, at least 75%, or at least 80%, while increasing 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%, or at least 75%.

[0078] On the other hand, this disclosure provides a method for reducing SARM1 expression, comprising administering to a subject an effective amount or effective dose of the RNAi agent and / or pharmaceutical composition of this disclosure.

[0079] On the other hand, this disclosure provides a method for treating and / or preventing diseases associated with SARM1 gene expression in a subject, comprising administering to the subject an effective amount or effective dose of the RNAi agent and / or pharmaceutical composition of this disclosure.

[0080] On the other hand, this disclosure provides the use of the RNAi agent and / or pharmaceutical composition described herein in the preparation of a medicament for treating and / or preventing diseases related to SARM1 gene expression.

[0081] On the other hand, this disclosure provides the use of the RNAi agent and / or pharmaceutical composition described herein in the preparation of a medicament for reducing the expression of SARM1.

[0082] On the other hand, this disclosure provides an RNAi agent and / or pharmaceutical composition described herein that can be used to reduce SARM1 expression.

[0083] On the other hand, this disclosure provides an RNAi agent and / or pharmaceutical composition described herein that can be used to treat and / or prevent diseases associated with SARM1 gene expression.

[0084] In some embodiments, the disease is a central nervous system degenerative disease. In some embodiments, the central nervous system degenerative disease is a central nervous system degenerative disease related to axonal degeneration, such as one or more of multiple sclerosis (MS), spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), Alzheimer's disease (AD), neuralgia (e.g., post-chemotherapy neuralgia), axonal peroneal muscular atrophy, and / or peripheral neuropathy.

[0085] On the other hand, this disclosure provides a method for in vivo delivery of an RNAi agent that inhibits SARM1 expression and / or replication, the method comprising administering to a subject the RNAi agent and / or pharmaceutical composition of this disclosure.

[0086] The RNAi agents or pharmaceutical compositions and methods disclosed herein can reduce the level of target mRNA in cells, cell populations, tissues, or subjects, including administering a therapeutically effective amount of the RNAi agent or pharmaceutical composition of this disclosure to a subject, wherein the RNAi agent is linked to the delivery group thereby inhibiting the expression of target mRNA in the subject.

[0087] In some embodiments, the subject has been identified as having pathological upregulation of the target gene in the targeted cells or tissues prior to administration of the RNAi agent and / or pharmaceutical composition disclosed herein.

[0088] The subjects mentioned in this disclosure refer to subjects who are diagnosed with (or suspected of having, or are susceptible to) a disease or condition that would benefit from a reduction or inhibition of target mRNA expression.

[0089] The RNAi agents and / or pharmaceutical compositions disclosed herein may be delivered via any suitable mode of administration common in the art, such as local administration (e.g., direct injection, implantation), systemic administration, or subcutaneous, intravenous, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal, and intrasheathal), intramuscular, transdermal, airway (aerosol), nasal, oral, rectal, buccal, or sublingual administration.

[0090] In an optional embodiment, the pharmaceutical composition provided in this disclosure can be administered by injection, for example, intravenous, intramuscular, intradermal, subcutaneous, duodenal, or intraperitoneal injection.

[0091] On the other hand, this disclosure provides a cell containing the RNAi agent disclosed herein. This cell cannot develop into a complete plant or animal individual.

[0092] On the other hand, this disclosure provides a kit comprising the RNAi agent and / or pharmaceutical composition of this disclosure.

[0093] This disclosure also provides a method for silencing mRNA of a target gene in cells, the method comprising the step of introducing an RNAi agent and / or pharmaceutical composition of the present disclosure into the cell.

[0094] This disclosure also provides a method for silencing a target gene or its mRNA in cells in vivo or in vitro, the method comprising the step of introducing an RNAi agent and / or pharmaceutical composition according to this disclosure into the cell.

[0095] This disclosure also provides a method for inhibiting the expression of a target gene or its mRNA, the method comprising administering to a subject in need an effective amount or effective dose of an RNAi agent and / or pharmaceutical composition according to this disclosure.

[0096] In some embodiments, the effective amount or 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.

[0097] In some implementations, the target gene is the SARM1 gene, and the target mRNA is the mRNA expressed by the target gene.

[0098] This disclosure also provides a method for preparing an RNAi agent, comprising: synthesizing the RNAi agent described in this disclosure.

[0099] The pharmaceutically acceptable salts of the compounds described in this disclosure are selected from inorganic or organic salts, and the compounds described in this disclosure can react with acidic or basic substances to form the corresponding salts.

[0100] In the context of this disclosure, the compounds comprise the RNAi agents of this disclosure.

[0101] On the other hand, without specifying the configuration, the compounds disclosed herein may exist in specific geometric or stereoisomeric forms, including cis and trans isomers, (-)- and (+)- enantiomers, (R)- and (S)- enantiomers, diastereomers, (D)- isomers, (L)- isomers, and racemic mixtures thereof, as well as other mixtures, such as mixtures enriched with enantiomers or diastereomers, all of which are within the scope of this disclosure. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and mixtures thereof are included within the scope of this disclosure.

[0102] Furthermore, without specifying the configuration, the compounds and intermediates of this disclosure may also exist in different tautomer forms, and all such forms are included within the scope of this disclosure. The terms "tautomer" or "tautomer form" refer to structural isomers with different energies that can interconvert via low energy barriers.

[0103] The compounds disclosed herein may be asymmetric, for example, having one or more stereoisomers. Unless otherwise stated, all stereoisomers include, for example, enantiomers and diastereomers. The compounds containing asymmetric carbon atoms of this disclosure can be isolated in optically active pure form or in racemic form. The optically active pure form can be resolved from racemic mixtures or synthesized using chiral starting materials or chiral reagents.

[0104] Optically active (R)- and (S)- isomers, as well as D- and L- isomers, can be prepared by chiral synthesis, chiral reagents, or other conventional techniques. If an enantiomer of a compound of this disclosure comprising the RNAi agent of this 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 a 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 salt of the diastereomeric isomer is formed with a suitable optically active acid or base, followed by diastereomeric resolution by conventional methods known in the art, and then recovery of the pure enantiomer. Furthermore, the separation of enantiomers and diastereomeric isomers is typically accomplished by using chromatography employing a chiral stationary phase and optionally combined with chemical derivatization (e.g., from amines to carbamates).

[0105] This disclosure also includes compounds identical to those described herein, but in which one or more atoms are labeled with isotopes whose atomic weights or mass numbers differ from those commonly found in nature. Examples of isotopes that can be incorporated into compounds of this 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, etc.

[0106] Without specifying the configuration, in the chemical structure of this disclosure, the bonds are... This indicates that the configuration is not specified; that is, if chiral isomers exist in the chemical structure, the bond... It can be Or simultaneously include and Two configurations. Although all structural formulas described herein are represented in certain isomer forms for simplicity, this disclosure can include all isomers, such as tautomers, rotatimers, geometric isomers, diastereomers, racemates, and enantiomers. In the chemical structure of this disclosure, bonds... No configuration was specified, i.e., key The configuration can be E-type or Z-type, or it can contain both E-type and Z-type configurations.

[0107] This publication incorporates the full text of WO2022028462A1, WO2023274395A1, and WO2023208023A1.

[0108] Terminology Explanation

[0109] To facilitate understanding of this disclosure, some technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0110] As used herein, “RNAi agent” (also known as “RNAi trigger”) refers to an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule capable of degrading or inhibiting (e.g., under appropriate conditions, degrading or inhibiting) the translation of a messenger RNA (mRNA) transcript of a target gene in a sequence-specific manner. RNAi agents as used herein may act via RNA interference mechanisms (i.e., by interacting with RNA interference pathway mechanisms in mammalian cells, such as RNA-induced silencing complexes or RISC) or through any alternative mechanism or pathway. Although the term RNAi agent as used herein is considered to act primarily through RNA interference mechanisms, the disclosed RNAi agents are not bound to or limited to 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 agent described herein is at least partially complementary to the targeted mRNA. RNAi agents may contain one or more modified nucleotides and / or one or more non-phosphodiester bonds.

[0111] Unless otherwise specified, in the context of this disclosure, the terms "containing Stellile Alpha and TIR motif 1" and "SARM1" are used interchangeably. SARM1 includes, but is not limited to, human SARM1, cynomolgus monkey SARM1, mouse SARM1, and rat SARM1, whose amino acid and complete coding sequences and mRNA sequences are readily available from publicly available databases, such as GenBank, UniProt, OMIM, and the Macaca Genome Project website.

[0112] The term "SARM1" also refers to naturally occurring DNA sequence variations in the SARM1 gene, such as single nucleotide polymorphisms (SNPs) in the SARM1 gene. Exemplary SNPs can be found in the dbSNP database.

[0113] The term "target sequence" refers to a continuous portion of the nucleotide sequence of the mRNA molecule formed during SARM1 transcription, including mRNA processed from RNA as the primary transcription product. In one embodiment, the target sequence is located within the protein-coding region of SARM1.

[0114] As used herein, in the context of RNA-mediated gene silencing, the sense strand (also known as SS, SS chain, or sense strand) refers to a strand containing a sequence that is identical or substantially identical to the target mRNA sequence. The antisense strand (also known as AS, AS chain) refers to a strand containing a sequence that is complementary or substantially complementary to the target mRNA sequence.

[0115] In the context of describing the sense strand of the RNAi agent described herein, the term "at least 15 consecutive nucleotide sequences differing by no more than 3 nucleotides from any of the nucleotide sequences in SEQ ID NO: 1 to SEQ ID NO: 17" is intended to mean that the sense strand of the RNAi agent described herein comprises at least 15 consecutive nucleotides as in any of the sense strands in SEQ ID NO: 1 to SEQ ID NO: 17, or differs from at least 15 consecutive nucleotides in any of the sense strands in SEQ ID NO: 1 to SEQ ID NO: 17 by no more than 3 nucleotide sequences (optionally, no more than 2 nucleotide sequences; optionally, 1 nucleotide sequence). Other similar descriptions in the context of this disclosure should also be understood similarly, and the term "difference" in this disclosure does not include nucleotides containing different modifications; that is, nucleotides containing the same bases but different modifications are not considered differing nucleotides in the context of this disclosure. Other similar descriptions of the sense strand and / or antisense strand in the context of this disclosure should also be understood similarly.

[0116] In this disclosure, the "5' region," also known as the "5' end" or "5' terminus," of the sense or antisense strand can be used interchangeably. For example, the nucleotides at positions 2 to 8 of the 5' region of the antisense strand can be replaced with the nucleotides at positions 2 to 8 of the 5' terminus of the antisense strand. Similarly, the "3' region," "3' terminus," and "3' terminus" of the sense or antisense strand can also be used interchangeably.

[0117] Unless otherwise specified, in the context of this disclosure, "G", "C", "A", "T" and "U" represent nucleotides, which respectively contain the bases of guanine, cytosine, adenine, thymidine, and uracil. It is well known to those skilled in the art that substitutions of bases T and U do not significantly affect the properties of the RNAi agent sequence, and U in the sequences of this disclosure can be arbitrarily replaced with T; the resulting sequences are also within the scope of protection of this disclosure. In the sequences disclosed herein, for the same nucleic acid chain, the direction from the 5' end to the 3' end is defined as left to right. 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; the lowercase letter s indicates that the two nucleosides adjacent to the letter s are linked by a thiophosphate diester group. Unless otherwise specified, the two nucleosides are linked by a phosphate diester group; VP indicates that the 5' end of the nucleoside adjacent to the left / right of the letter is a 5'-vinyl phosphate diester group. The 5'-vinyl phosphate diester group can be cis or trans, that is, it can be the 5'-E-VP isomer (i.e., trans-vinyl phosphate diester group), the 5'-Z-VP isomer (i.e., cis-vinyl phosphate diester group), or a mixture thereof; IB indicates a reverse abase-free nucleoside. Unless otherwise specified, the terms "RNAi agent," "nucleotide," "compound," "chemical modification," "oligonucleotide," "double-stranded RNAi inhibitor molecule," "siRNA," "dsRNA," "nucleic acid," and "RNAi" in this disclosure can exist independently as salts, mixed salts, or non-salts (e.g., free acids or free bases). When present as salts or mixed salts, they are pharmaceutically acceptable salts. The term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts. When present as salts, some groups may ionize to form anions / cations; for example, phosphodiester groups and thiophosphonate diester groups can exist in anionic form. The salt forms of the following structures are also within the scope of protection of this disclosure.

[0118] The above-mentioned modifications and linking groups have the structures shown in Table 1A below, where Base represents a base:

[0119] Table 1A

[0120] As used in this disclosure, the term "2'-fluorinated modification" refers to the substitution of a 2'-hydroxyl group at the ribosome position of a nucleoside / nucleotide with a fluorine group, and "non-2'-fluorinated modification" refers to the substitution of a 2'-hydroxyl group at the ribosome position of a nucleoside / nucleotide with a non-fluorinated group. For example, as used in this disclosure, the term "2'-methoxylated modification" refers to the substitution of a methoxy group for the 2'-hydroxyl group of the ribosome.

[0121] The terms "lipophilic group" or "lipophilic moiety" broadly refer to any compound or chemical part that has an affinity for lipids. One way to characterize the lipophilicity of a lipophilic moiety is through the octanol-water partition coefficient logK. ow K ow This represents the ratio of the concentration of a chemical substance in the octanol phase to its concentration in the aqueous phase at equilibrium in a two-phase system. In principle, logK... ow When the value exceeds 0, the chemical substance exhibits lipophilicity. Typically, the logK of the lipophilic portion... ow Values ​​exceeding 1, 1.5, 2, 3, 4, 5, or 10, such as the logK of 6-aminohexanol. ow The logK of cholesterol-based N-(hexyl-6-ol)carbamate is approximately 0.7. ow It is 10.7.

[0122] The lipophilicity of a molecule can be altered relative to the functional groups it carries. For example, adding a hydroxyl or amino group to the end of the lipophilic moiety can increase or decrease the partition coefficient (e.g., logK) of the lipophilic moiety. ow The lipophilic moiety can be aliphatic, cyclic (e.g., alicyclic), or polycyclic (e.g., polycyclic alicyclic compounds), such as steroids (e.g., sterols) or straight-chain or branched aliphatic hydrocarbons. The lipophilic moiety may generally comprise a hydrocarbon chain, which may be cyclic or acyclic. The hydrocarbon chain may contain various substituents and / or one or more heteroatoms, such as oxygen or sulfur atoms. Such lipophilic aliphatic moieties include, but are not limited to, saturated or unsaturated C4-C... 30 Hydrocarbons (e.g., C) 10 -C 30 Hydrocarbons), saturated or unsaturated fatty acids, waxes (e.g., monohydric esters of fatty acids and fatty diamides), terpenes (e.g., C464 ... 10 Terpenes, C 15 Sesquiterpenes, C 20 Diterpenes, C 30 Triterpenes and C 40 Tetraterpenes and other polycyclic hydrocarbons; for example, the lipophilic moiety may be optionally substituted C 10-30 Straight-chain alkyl; for example, the lipophilic moiety may be an optionally substituted C. 14-24 Straight-chain alkyl groups.

[0123] As used herein, the terms “complementary” or “reverse complementary” are used interchangeably and have the meaning known to those skilled in the art: in a double-stranded nucleic acid molecule, the bases of one strand are paired in a complementary manner with the bases of the other strand. In DNA, the purine base adenine always pairs with the pyrimidine base thymine (or uracil in RNA); the purine base guanine always pairs with the pyrimidine base cytosine. Each base pair consists of one purine and one pyrimidine. When adenine on one strand always pairs with thymine (or uracil) on the other strand, and guanine always pairs with cytosine, the two strands are considered complementary, and the sequence of the complementary strand can be inferred from its sequence. Correspondingly, “mismatch” in the art means, in the case of a double-stranded nucleic acid, that the bases at corresponding positions are not paired in a complementary manner.

[0124] As used herein, the term “inhibition” may be used interchangeably with “reduction,” “silencing,” “downregulation,” “blocking,” and other similar terms, and includes any level of inhibition. Inhibition can be assessed by a reduction in one or more of these variables at an absolute or relative level compared to a control level. This 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 a subject, cell, or sample that has been treated untreated or with a control (e.g., a buffer-only control or an inert control). For example, the degree of inhibition of target gene expression by an RNAi agent can be characterized by residual mRNA expression levels such as not exceeding 99%, not exceeding 95%, not exceeding 90%, not exceeding 85%, not exceeding 80%, not exceeding 75%, not exceeding 70%, not exceeding 65%, not exceeding 60%, not exceeding 55%, not exceeding 50%, not exceeding 45%, not exceeding 40%, not exceeding 35%, not exceeding 30%, not exceeding 25%, not exceeding 20%, not exceeding 15%, or not exceeding 10%. The inhibition rate of target gene expression can be measured using... The Luciferase Assay System was used to detect the chemiluminescence values ​​of fireflies (Fir) and sea urchins (Ren), and the relative value Ratio = Ren / Fir was calculated. In this disclosure, the proportion of remaining mRNA expression (or remaining activity %) = Ratio (RNAi agent treatment group) / Ratio (no RNAi agent control group), and the inhibition rate (%) = 100% - remaining mRNA expression (%).

[0125] Unless otherwise specified, the terms "compound," "ligand," "nucleic acid ligand conjugate," "nucleic acid," "conjugate," "chemical modification," "targeting ligand," "dsRNA," "siRNA," and "RNAi agent" in this disclosure can exist independently as a salt, a mixture of salts, or a non-salt (e.g., a free acid or a free base). When present as a salt or a mixture of salts, it may be a pharmaceutically acceptable salt. In the context of this disclosure, "compound" also includes RNAi agents.

[0126] The term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0127] "Pharmaceutically acceptable acid addition salts" refer to salts formed with inorganic or organic acids that retain the bioavailability of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochlorides, hydrobroms, sulfates, nitrates, and phosphates; organic acid salts include, but are not limited to, formates, acetates, 2,2-dichloroacetate, trifluoroacetate, propionates, hexanoates, octanoates, decanoates, undecenoates, glycolates, gluconates, lactates, sebates, adipates, glutarate, malonates, oxalates, maleates, succinates, fumarates, tartrates, citrates, palmitates, stearates, oleates, cinnamates, laurates, malates, glutamates, pyroglutamates, aspartate, benzoates, methanesulfonates, benzenesulfonates, p-toluenesulfonates, alginates, ascorbic acid salts, salicylates, 4-aminosalicylic acid salts, and naphthalene disulfonates. These salts can be prepared by methods known in the art.

[0128] "Pharmaceutically acceptable base addition salts" refer to salts formed with inorganic or organic bases that retain the bioavailability 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, and aluminum salts. In some implementations, the inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, the following: primary amines, secondary amines, and tertiary amines; substituted amines, including naturally occurring 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, methylglucosamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Some embodiments include organic bases such as isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. These salts can be prepared by methods known in the art.

[0129] "Effective amount" or "effective dose" refers to the amount of RNAi agent, compound, or pharmaceutical composition necessary to achieve any one or more beneficial or desired therapeutic outcomes. For prophylactic use, beneficial or desired outcomes include eliminating or reducing risk, mitigating severity, or delaying the onset of symptoms, including the symptoms, their complications, and the biochemical, histological, and / or behavioral symptoms of intermediate pathological phenotypes presented during the development of the symptoms. For therapeutic applications, beneficial or desired outcomes include clinical outcomes such as reducing the incidence of various symptoms associated with the target genes, target mRNAs, or target proteins of this disclosure, or improving one or more symptoms of said symptoms, reducing the dosage of other agents required to treat the symptoms, enhancing the efficacy of another agent, and / or delaying the progression of the symptoms associated with the target genes, target mRNAs, or target proteins of this disclosure in patients.

[0130] As used herein, the terms “patient,” “subject,” or “individual” are used interchangeably and include human or non-human animals, such as mammals, such as humans or monkeys.

[0131] The RNAi agents provided in this disclosure can be obtained using conventional preparation methods in the art (e.g., solid-phase synthesis and liquid-phase synthesis). Solid-phase synthesis is already available as a commercially available custom service. Modified nucleotide groups can be introduced into the RNAi agents described in this disclosure using appropriately modified nucleoside monomers. Methods for preparing appropriately modified nucleoside monomers and for introducing modified nucleotide groups into RNAi agents are also well known to those skilled in the art.

[0132] The term “chemical modification” or “modification” includes all alterations to nucleotides by chemical means, such as the addition or removal of a chemical moiety, or the substitution of one chemical moiety for another.

[0133] The term "base" includes any known DNA and RNA base, base analogues such as purines or pyrimidines, and also includes natural compounds such as adenine, thymine, guanine, cytosine, uracil, hypoxanthoside, and natural analogues.

[0134] The terms "blunt-ended" or "knuckle-ended" are used interchangeably and refer to the absence of unpaired nucleotides or nucleotide analogues at a given end of an RNAi agent; that is, no nucleotide protrusions. In most cases, RNAi agents with blunt ends will be double-stranded throughout their entire length.

[0135] The terms “about” and “approximately” mean that a numerical value is within an acceptable margin of error for a specific value as determined by a person skilled in the art, the numerical value depending in part on how it is measured or measured (i.e., the limits of the measurement system). For example, “about” may mean within or above 1 standard deviation. Alternatively, “about” or “substantially includes” may 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 of a number or range of values ​​preceded by the term “about” also includes embodiments of a given number. Unless otherwise stated, when a specific value appears in this application and claims, the meaning of “about” or “substantially includes” should be assumed to be within an acceptable margin of error for that specific value.

[0136] Unless otherwise stated, "optionally," "optionally," "optional," or "optional" means that the event or situation described below may but does not have to occur, and this description includes the possibility that the event or situation may or may not occur. For example, "optionally, R1 and R2 are directly connected to form a loop" means that R1 and R2 being directly connected to form a loop may occur but is not required to exist, and this description includes both the case where R1 and R2 are directly connected to form a loop and the case where R1 and R2 are not connected to form a loop.

[0137] In the chemical structural formula disclosed herein, It can be connected with one or more groups according to the scope of the invention described herein.

[0138] The term "connection" refers to the link between two molecules, either through a covalent bond or through a non-covalent bond (e.g., hydrogen bond or ionic bond), including direct and indirect connections.

[0139] The term "direct link" refers to the connection between a first compound or group and a second compound or group without any intercalating atoms or atomic groups.

[0140] The term "indirect link" refers to the connection between a first compound or group and a second compound or group through an intermediate group, compound, or molecule (e.g., a linking group).

[0141] The term "substituted" means that any one or more hydrogen atoms on a specified atom (typically carbon, oxygen, or nitrogen atoms) are replaced by any group defined herein, provided that the substitution does not exceed the normal valence of the specified atom and yields a stable compound. Non-limiting examples of substituents include C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, cyano, hydroxyl, oxo, carboxyl, cycloalkyl, cycloalkenyl, heterocyclic, heteroaryl, aryl, ketone, alkoxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, or halogens (e.g., F, Cl, Br, I). When the substituent is a ketone or oxo (i.e., =O), two (2) hydrogen atoms on the atom are substituted.

[0142] "Being replaced by one or more..." means that it can be replaced by a single or multiple substituents. When replaced by multiple substituents, it can be a plurality of identical substituents or a combination of one or a plurality of different substituents. Detailed Implementation

[0143] The present disclosure is further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the disclosure. Experimental methods in the embodiments of this disclosure that do not specify specific conditions are generally performed under conventional conditions, such as those described in Cold Spring Harbor's Antibody Technology Manual or Molecular Cloning Manual; or under conditions recommended by the raw material or commercial manufacturer. Reagents that do not specify a specific source are commercially available, conventional reagents.

[0144] Example 1. Design of SARM1 RNAi Agent

[0145] Human SARM1 gene (NM_015077.4) was used as the target gene to design a 19 / 21nt RNAi agent in accordance with the general rules for active RNAi agents. The unmodified sense and antisense strand sequences are shown in Table 1B, and the modified sense and antisense strand sequences are detailed in Table 2.

[0146] Table 1B. Unmodified RNAi agents

[0147] Table 2. Modified RNAi agents

[0148] In Tables 1B and 2 above, within the same nucleic acid chain, the direction from the 5' end to the 3' end is considered as left to right. The lowercase letter 'm' indicates that the nucleoside adjacent to the left of 'm' is 2'-methoxy modified; the lowercase letter 'f' indicates that the nucleoside adjacent to the left of 'f' is 2'-fluoro modified; the uppercase letter 'VP' indicates that the 5' end of the sugar ring of the nucleoside adjacent to its right is modified with an E-vinyl phosphate group; the lowercase letter 's' indicates that the two nucleosides adjacent to 's' are connected by a thiophosphate diester group. Unless otherwise specified, the first nucleotide at the 3' end of each chain has a hydroxyl group at its 3' position; the first nucleotide at the 5' end of each chain has a hydroxyl group at its 5' position. The structures of the 2'-methoxy modified nucleosides, 2'-fluoro modified nucleosides, thiophosphate diester groups, phosphate diester groups, and E-vinyl phosphate modified nucleosides are shown in Table 1A. The scope of protection of this disclosure also includes the ionic forms of the listed structures when they form salts.

[0149] Example 2. Synthesis of RNAi Agents

[0150] The synthesis of the RNAi agent disclosed herein is no different from the conventional solid-phase synthesis of phosphorus amides. The synthesis process is briefly described below: Using a Dr. Oligo48 synthesizer (Biolytic), starting with a universal CPG carrier, unmodified nucleoside phosphorus amide monomers or nucleoside phosphorus amide monomers modified at corresponding positions in the sequence are sequentially linked according to the synthesis program. The nucleoside phosphorus amide monomers used in the embodiments of this disclosure were purchased from Shanghai Zhaowei Co., Ltd. and Suzhou Jima Co., Ltd. 5-Ethylthio-1H-tetrazole (ETT) was used as the activator (0.6M acetonitrile solution), 0.22M PADS dissolved in a 1:1 volume ratio of acetonitrile and 3-methylpyridine (Shanghai Lingjiang) solution was used as the sulfiding agent, and iodopyridine / aqueous solution (Shanghai Lingjiang) was used as the oxidizing agent.

[0151] After solid-phase synthesis, the oligonucleotides 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 concentration and evaporation to dryness, purification was performed using C18 reverse-phase chromatography with 0.1M TEAA and acetonitrile as the mobile phase. The target oligonucleotides were collected, lyophilized, identified as the target product by LC-MS, and then quantified by UV (260 nm).

[0152] The obtained single-stranded oligonucleotides were annealed according to complementary pairing in equimolar ratios. The resulting double-stranded RNAi agent was then dissolved in 1×PBS and adjusted to the required concentration for the experiment.

[0153] Example 3. Inhibition of human SARM1 in cells by RNAi agents - Inhibitory activity at 3 concentration points

[0154] The RNAi agent disclosed herein was screened for in vitro molecular-level activity at three concentration gradients (10 nM, 1 nM, 0.1 nM) in A172 cells.

[0155] A172 cells were cultured in Dulbecco's modified eagle medium containing 10% fetal bovine serum at 37°C and 5% CO2. 24 hours before transfection, A172 cells were seeded into 96-well plates at a density of 1 × 10⁶ cells per well. 4 100 μL of culture medium per well for each cell.

[0156] Following the product manual, Lipofectamine RNAi MAX (ThermoFisher, 13778150) was used to transfect RNAi agents at final concentrations of 10 nM, 1 nM, and 0.1 nM, with double replicates for each concentration. After 48 hours of treatment, total RNA was extracted from cells using a high-throughput cell RNA extraction kit FG0417-L / FG0418-XL (ZhiFan Medical, magnetic bead method). RNA reverse transcription experiments (Takara, RR037A) and quantitative real-time PCR (Thermo, 4444557) were performed to determine the SARM1 mRNA level in A172 cells. The human SARM1 mRNA level was corrected for the level of the GAPDH internal reference gene.

[0157] The instruments involved in this experiment are shown in Table 3.

[0158] Table 3. Experimental Instruments

[0159] In the real-time quantitative PCR detection, probe Q-PCR detection experiment was used, and its primer information is shown in Table 4.

[0160] Table 4. Taqman Primer Information Table

[0161] After the Q-PCR assay is completed, the corresponding Ct value is obtained according to the threshold automatically set by the system. The expression of a specific gene can be relatively quantified by comparing Ct values: comparing Ct refers to calculating the difference in gene expression by comparing the Ct value with that of the internal reference gene, also known as 2-1. -△△Ct △△Ct=[(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%.

[0162] Results are expressed as the percentage of remaining human SARM1 mRNA expression relative to cells treated with the control RNAi agent.

[0163] Table 5. Remaining percentage of RNAi agent expression at 3 concentration points in A172 cells

[0164] Example 4. Inhibitory activity of RNAi agents on human SARM1 in cells - multi-concentration site inhibition

[0165] The RNAi agent disclosed herein was screened for in vitro molecular-level activity in A172 cells using nine concentration gradients (10 nM, 2 nM, 0.4 nM, 0.08 nM, 0.016 nM, 0.0032 nM, 0.00064 nM, 0.000128 nM, 0.000026 nM). The experimental procedures, reagents, instruments, and data processing methods were all as described in Example 3.

[0166] Results are expressed as the percentage of remaining human SARM1 mRNA expression relative to cells treated with the control RNAi agent.

[0167] Table 6. Residual percentage of RNAi agent expression after multiple doses in A172 cells

[0168] Example 5. Inhibition of human SARM1 in cells by RNAi agents - 3 concentration point inhibition activity

[0169] The RNAi agent disclosed herein was screened for in vitro molecular-level activity in A172 cells using three concentration gradients (10 nM, 1 nM, 0.1 nM). The experimental procedures, reagents, instruments, and data processing methods were all as described in Example 3.

[0170] Results are expressed as the percentage of remaining human SARM1 mRNA expression relative to cells treated with the control RNAi agent.

[0171] Table 7. Remaining percentage of RNAi agent expression at 3 concentration points in A172 cells

[0172] Example 6. Inhibitory activity of RNAi agents on SARM1 expression in glioblastoma cells (A172)

[0173] A172 cells were cultured in DMEM medium (Gibco) (containing 10% FBS) at 37°C and 5% CO2.

[0174] 24 hours before transfection, A172 cells were inoculated at 1.0 × 10⁶ cells per cell line. 4Cells were seeded at a density of 100 μL / well in 96-well plates. Following the manufacturer's instructions, cells were transfected with RNAi MAX transfection reagent (ThermoFisher, 13778150) to obtain the RNAi agent. Eight concentration points were set for the RNAi agent, with the highest concentration at 20 nM (5-fold dilution) and the lowest concentration at 0.000256 nM. Forty-eight hours after transfection, total RNA was extracted using a high-throughput cell RNA extraction kit (ThermoFisher, A27828), RNA reverse transcription was performed (Takara, RR037B), and quantitative real-time PCR was conducted (ThermoFisher, 4444557) to determine the mRNA level of human SARM1. The mRNA level of human SARM1 was corrected for the level of the GAPDH internal reference gene.

[0175] In the real-time quantitative PCR detection, probe-based Q-PCR was used, and its primer information is shown in Table 8.

[0176] Table 8. Taqman Primer Information

[0177] Results Analysis Methods

[0178] After the Q-PCR assay is completed, the corresponding Ct value is obtained according to the threshold automatically set by the system. The expression of a specific gene can be relatively quantified by comparing Ct values: comparing Ct refers to calculating the difference in gene expression by comparing the Ct value with that of the internal reference gene, also known as 2-1. -△△Ct △△Ct=[(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%.

[0179] The results were expressed as IC50 of the inhibition rate of human SARM1 mRNA relative to cells treated with the control RNAi agent. 50 The results are shown in Table 9.

[0180] The data in Table 9 indicate that all RNAi agents disclosed herein exhibit high levels of inhibitory activity against the SARM1 gene in A172 cells.

[0181] Table 9. Inhibition of SARM1 IC50 by the RNAi agent of this disclosure in A172 cells 50 active

[0182] Example 7. Inhibitory activity of RNAi agents on human SARM1 expression in glioblastoma cells (A172)

[0183] A172 cells were cultured in DMEM medium (Gibco) (containing 10% FBS) at 37°C and 5% CO2.

[0184] During transfection, A172 cells were loaded at a concentration of 1.0 × 10⁻⁶. 4 Cells were seeded at a density of 1 cell / well in 96-well plates, with each well containing 90 μL of culture medium. Following the manufacturer's instructions, RNAi MAX transfection reagent (ThermoFisher, 13778150) was used to transfect cells with RNAi. Seven concentration points were set for the RNAi reagent, with the highest concentration at 20 nM (5-fold dilution) and the lowest concentration at 0.00128 nM. 24 h after transfection, total RNA was extracted from the cells using a high-throughput cell RNA extraction kit (Zhiang Biotechnology, MNTR / FX96 human samples (Lr)), RNA reverse transcription assays (Takara, RR037B), and quantitative real-time PCR detection (ThermoFisher, 4444557). The mRNA level of human SARM1 was measured, and the mRNA level was corrected based on the level of the GAPDH internal reference gene.

[0185] In the real-time quantitative PCR detection, probe Q-PCR detection experiment was used, and its primer information is shown in Table 8.

[0186] The results were expressed as IC50 of the inhibition rate of human SARM1 mRNA relative to cells treated with the control RNAi agent. 50 The results are shown in Tables 10-12.

[0187] Table 10. Inhibition of SARM1 IC50 by the RNAi agent of this disclosure in A172 cells 50 active

[0188] Table 11. Inhibition of SARM1 IC50 activity by the RNAi agent of this disclosure in A172 cells.

[0189] Table 12. Inhibition of SARM1 IC50 by the RNAi agent of this disclosure in A172 cells 50 active

[0190] Example 8. Single-concentration site-specific inhibitory activity of RNAi agents against human SARM1 in glioblastoma cells (A172).

[0191] A172 cells were cultured in DMEM medium (Gibco) (containing 10% FBS) at 37°C and 5% CO2.

[0192] During transfection, A172 cells were loaded at a concentration of 1.0 × 10⁻⁶.4 Cells were seeded at a density of 1 cell / well in 96-well plates, with 90 μL of culture medium per well. Following the manufacturer's instructions, RNAi MAX transfection reagent (ThermoFisher, 13778150) was used to transfect cells with RNAi agent at a final concentration of 20 nM, with double replicates per concentration. 24 h after transfection, total RNA was extracted using a high-throughput cell RNA extraction kit (Zhiang Biotechnology, MNTR / FX96 human samples (Lr)), RNA reverse transcription assays (Takara, RR037B), and quantitative real-time PCR detection (ThermoFisher, 4444557). Human SARM1 mRNA levels were measured and corrected for GAPDH internal reference gene levels.

[0193] In the real-time quantitative PCR detection, probe Q-PCR detection experiment was used, and its primer information is shown in Table 8.

[0194] Results are expressed as the percentage of remaining human SARM1 mRNA expression relative to cells treated with the control RNAi agent, as shown in Table 13.

[0195] Table 13. Percentage of remaining SARM1 mRNA expression at single concentration points of RNAi agent in A172 cells

Claims

1. An RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, wherein: The positive strand comprises at least 15 consecutive nucleotide sequences that differ from any nucleotide sequence in SEQ ID NO:1 to SEQ ID NO:17 by no more than 3 nucleotides; The antisense strand comprises at least 15 consecutive nucleotide sequences that differ from any of the nucleotide sequences in SEQ ID NO:88 to SEQ ID NO:104 by no more than 3 nucleotides.

2. The RNAi agent according to claim 1, wherein: The positive strand comprises at least 17 consecutive nucleotide sequences that differ from any nucleotide sequence in SEQ ID NO:1 to SEQ ID NO:17 by no more than 3 nucleotides; The antisense strand comprises at least 17 consecutive nucleotide sequences that differ from any nucleotide sequence in SEQ ID NO:88 to SEQ ID NO:104 by no more than 3 nucleotides; Preferably, the positive strand comprises at least 19 consecutive nucleotide sequences that differ from any nucleotide sequence in SEQ ID NO:1 to SEQ ID NO:17 by no more than 3 nucleotides; Preferably, the antisense strand comprises at least 21 consecutive nucleotide sequences that differ from any nucleotide sequence in SEQ ID NO:88 to SEQ ID NO:104 by no more than 3 nucleotides.

3. The RNAi agent according to any one of claims 1 or 2, comprising the sense and antisense strands shown in any one of the following groups: Group 1), such as the sense chain shown in SEQ ID NO: 1 and the antisense chain shown in SEQ ID NO: 88; Group 2), such as the sense chain shown in SEQ ID NO: 2 and the antisense chain shown in SEQ ID NO: 89, or, such as the sense chain shown in SEQ ID NO: 3 and the antisense chain shown in SEQ ID NO: 90; Group 3), such as the sense chain shown in SEQ ID NO: 4 and the antisense chain shown in SEQ ID NO: 91, or, such as the sense chain shown in SEQ ID NO: 5 and the antisense chain shown in SEQ ID NO: 92; Group 4), such as the sense chain shown in SEQ ID NO: 6 and the antisense chain shown in SEQ ID NO: 93, or, such as the sense chain shown in SEQ ID NO: 7 and the antisense chain shown in SEQ ID NO: 94; Group 5), such as the sense chain shown in SEQ ID NO: 8 and the antisense chain shown in SEQ ID NO: 95; Group 6), such as the sense chain shown in SEQ ID NO: 9 and the antisense chain shown in SEQ ID NO: 96; Group 7), such as the sense chain shown in SEQ ID NO: 10 and the antisense chain shown in SEQ ID NO: 97; Group 8), such as the sense chain shown in SEQ ID NO: 11 and the antisense chain shown in SEQ ID NO: 98; Group 9), such as the sense chain shown in SEQ ID NO: 12 and the antisense chain shown in SEQ ID NO: 99, or, such as the sense chain shown in SEQ ID NO: 13 and the antisense chain shown in SEQ ID NO: 100; Group 10), such as the sense chain shown in SEQ ID NO: 14 and the antisense chain shown in SEQ ID NO: 101, or, such as the sense chain shown in SEQ ID NO: 15 and the antisense chain shown in SEQ ID NO: 102; Group 11), such as the sense chain shown in SEQ ID NO: 16 and the antisense chain shown in SEQ ID NO: 103; Group 12), such as the sense chain shown in SEQ ID NO: 17 and the antisense chain shown in SEQ ID NO:

104.

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

5. The RNAi agent according to claim 4, wherein: The positive strand contains three consecutive 2'-fluorinated nucleotides; and / or The antisense strand contains at least five 2'-fluorinated nucleotides; Preferably, the nucleotides at positions 7, 8, and 9 of the positive strand are 2'-fluorinated nucleotides, following the direction from the 5' end to the 3' end; and / or, Preferably, the nucleotides at positions 2, 6, 12, 14, and 16 of the antisense strand are 2'-fluorinated nucleotides, in the direction from the 5' end to the 3' end; or, the nucleotides at positions 2, 4, 6, 10, 12, 14, 16, and 18 of the antisense strand are 2'-fluorinated nucleotides. The nucleotides at the remaining positions in the sense strand and the antisense strand are nucleotides modified with 2'-methoxy groups.

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

7. The RNAi agent according to claim 6, wherein the phosphodiester group having the modifying group is present at at least one of the following positions: Between any two adjacent nucleotides from the first to the fourth nucleotide at the 5' end and / or the 3' end of the positive strand, and / or Between any two adjacent nucleotides from the first to the fourth nucleotide at the 5' end and / or the 3' end of the antisense strand; Preferably, The sense and / or antisense chains contain multiple phosphodiester groups with modifying groups, wherein the phosphodiester groups with modifying groups are present in: Between the first and second nucleotides, and between the second and third nucleotides at the 5' end of the positive strand; Between the first and second nucleotides, and between the second and third nucleotides at the 3' end of the positive strand; Between the first and second nucleotides, and between the second and third nucleotides at the 5' end of the antisense strand; and Between the first and second nucleotides, and between the second and third nucleotides, at the 3' end of the antisense strand.

8. The RNAi agent according to any one of claims 1-7, wherein, The positive strand comprises the nucleotide sequence shown in any one of SEQ ID NO:175 to SEQ ID NO:208; and / or, The antisense strand comprises the nucleotide sequence shown in any one of SEQ ID NO:280 to SEQ ID NO:

313.

9. The RNAi agent according to any one of claims 1-8, wherein the RNAi agent further comprises one or more delivery groups, the delivery groups being linked to the sense strand and / or antisense strand; Preferably, the delivery group comprises a lipophilic group; More preferably, the delivery group comprises saturated or unsaturated C 16 Hydrocarbon chain.

10. A pharmaceutical composition comprising: The RNAi agent according to any one of claims 1-9, and Pharmaceutically acceptable excipients.

11. A cell comprising the RNAi agent according to any one of claims 1-9.

12. A reagent kit comprising: The RNAi agent according to any one of claims 1-9; and / or The pharmaceutical composition of claim 10.

13. A method for reducing the expression of genes containing Stellile Alpha and TIR motif 1 (SARM1) in vitro or in vivo, comprising: The RNAi agent of any one of claims 1-9 and / or the pharmaceutical composition of claim 10 are administered to the subject in an effective amount or dose.

14. A method for treating and / or preventing a disease in a subject, comprising: The subject is given an effective amount or effective dose of the RNAi agent of any one of claims 1-9 and / or the pharmaceutical composition of claim 10; Preferably, the disease is a degenerative disease of the central nervous system; More preferably, the central nervous system degenerative disease is selected from multiple sclerosis (MS), spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), Alzheimer's disease (AD), neuralgia, axonal peroneal muscular atrophy, and peripheral neuropathy.

15. A method for in vivo delivery of an RNAi agent, the method comprising administering to a subject the RNAi agent of any one of claims 1-9 and / or the pharmaceutical composition of claim 10.

16. A method for preparing an RNAi agent, comprising: Synthesize the RNAi agent according to any one of claims 1-9.

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