Rnai agent targeting LRRK2 and medical use thereof

Abstract

An RNAi agent targeting LRRK2 and medical use thereof, including a pharmaceutical composition, cell, or kit comprising the RNAi agent, and a method for using the RNAi agent to treat and / or prevent a subject suffering from a related disorder.

Description

RNAi agents targeting LRRK2 and their medical uses

[0001] The present disclosure claims the priority of Chinese patent application 202410292322.1 with an application date of March 14, 2024 and the priority of Chinese patent application 202411444130.4 with an application date of October 16, 2024, and the present disclosure cites the full text of the above-mentioned Chinese patent applications. Technical Field

[0002] The present disclosure belongs to the field of biomedicine, and specifically relates to an RNAi agent targeting the leucine-rich repeat kinase 2 (LRRK2) gene, a composition, and medical uses thereof. Background Art

[0003] The LRRK2 gene is a member of the leucine-rich repeat kinase family, consisting of 51 exons. The protein it encodes has an ankyrin repeat region, a leucine-rich repeat (LRR) domain, a kinase domain, a DFG-like motif, a RAS domain, a GTPase domain, an MLK-like domain, and a WD40 domain. The LRRK2 gene is primarily expressed in the cytoplasm, with high expression in the lungs of healthy individuals and low expression in the brain.

[0004] Genome-wide association studies (GWAS) have shown that mutations in the LRRK2 gene are a strong risk locus for Parkinson's disease (PD). Accumulating evidence suggests that LRRK2 kinase function is enhanced in PD. Overactivation of the LRRK2 protein kinase is also found in neurons of patients with idiopathic Parkinson's disease who do not have mutations. Overactive LRRK2 protein kinase in neurons impairs vesicle trafficking and autophagy-lysosomal function, leading to abnormal accumulation of α-synuclein and an increased risk of PD.

[0005] LRRK2 mutant knock-in mice show early pathological changes of PD, including increased susceptibility to nigrostriatal neurotransmission, development of motor and non-motor symptoms, mitochondrial and autophagy-lysosomal defects, and synucleinopathy. In a mouse model of Parkinson's disease, ASO-mediated LRRK2 inhibition can prevent the formation of pathological synuclein α-syn inclusions in dopamine neurons. In some preclinical studies and early clinical trials, LRRK2 inhibitors have shown neuroprotective effects. Therefore, there is a need for drugs that selectively and effectively inhibit or regulate LRRK2 gene expression in order to effectively treat subjects with LRRK2-related diseases. The purpose of the present disclosure is to provide methods for treating such diseases. Summary of the Invention

[0006] The present disclosure provides an RNAi agent targeting LRRK2.

[0007] In some embodiments, the present disclosure provides an RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region; the sense strand comprises at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence shown in any one of SEQ ID NO:4, SEQ ID NO:1 to SEQ ID NO:3, SEQ ID NO:5 to SEQ ID NO:12, SEQ ID NO:20, SEQ ID NO:26, SEQ ID NO:35, and SEQ ID NO:64; the antisense strand comprises at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence shown in any one of SEQ ID NO:74, SEQ ID NO:71 to SEQ ID NO:73, SEQ ID NO:75 to SEQ ID NO:82, SEQ ID NO:90, SEQ ID NO:96, SEQ ID NO:105, and SEQ ID NO:134.

[0008] In some embodiments, the "difference is no more than 3 nucleotides" means that it can differ by 0, 1, 2, or 3 nucleotides; in some embodiments, the "at least 15 consecutive nucleotides" means that at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides are consistent; preferably, at least 17, 18, 19, 20, or 21 consecutive nucleotides are consistent.

[0009] In some embodiments, the antisense strand is at least partially complementary to the target sequence to mediate RNA interference. In some embodiments, there are no more than 5, 4, 3, 2, or 1 nucleotide mismatches between the antisense strand and the target sequence. In some embodiments, the antisense strand is completely reverse complementary to the target sequence.

[0010] 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, 4, 3, 2, or 1 nucleotide mismatches between the sense strand and the antisense strand. In some embodiments, the sense strand and the antisense strand are completely reverse complementary.

[0011] In some embodiments, the RNAi agents of the present disclosure comprise one or two blunt ends.

[0012] In some embodiments, the sense strand and / or antisense strand of the RNAi agent of the present disclosure each independently comprises 1 or 2 unpaired nucleotides. In some embodiments, the 3' end of the antisense strand comprises an overhang formed by unpaired nucleotides.

[0013] In some embodiments, the sense and antisense strands each independently consist 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 and antisense strands each independently consist of 18, 19, 20, 21, 22, or 23 nucleotides.

[0014] In some embodiments, the sense strand and antisense strand are the same or different in length, with the sense strand being 19-23 nucleotides in length and the antisense strand being 19-26 nucleotides in length. Thus, the ratio of the length of the sense and antisense strands of the RNAi agents provided herein 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.

[0015] In some embodiments, the sense strand comprises at least 15 consecutive nucleotides and differs by no more than 2 nucleotides from any one of SEQ ID NO:4, SEQ ID NO:1 to SEQ ID NO:3, SEQ ID NO:5 to SEQ ID NO:12, SEQ ID NO:20, SEQ ID NO:26, SEQ ID NO:35, and SEQ ID NO:64. In some embodiments, the nucleotide sequence differs by no more than 1 nucleotide. In some embodiments, the difference is 1 nucleotide.

[0016] In some embodiments, the antisense strand comprises at least 15 contiguous nucleotides and differs by no more than 2 nucleotides from the nucleotide sequence of SEQ ID NO: 74, SEQ ID NO: 71 to SEQ ID NO: 73, SEQ ID NO: 75 to SEQ ID NO: 82, SEQ ID NO: 90, SEQ ID NO: 96, SEQ ID NO: 105, or SEQ ID NO: 134. In some embodiments, the nucleotide sequence differs by no more than 1 nucleotide. In some embodiments, the difference is 1 nucleotide.

[0017] In some embodiments, the sense strand comprises at least 15 consecutive nucleotides of any one of SEQ ID NO:4, SEQ ID NO:1 to SEQ ID NO:3, SEQ ID NO:5 to SEQ ID NO:12, SEQ ID NO:20, SEQ ID NO:26, SEQ ID NO:35, and SEQ ID NO:64. In some embodiments, at least 16 consecutive nucleotides. In some embodiments, at least 17 consecutive nucleotides. In some embodiments, at least 19 consecutive nucleotides. In some embodiments, at least 18 consecutive nucleotides.

[0018] In some embodiments, the antisense strand comprises at least 15 consecutive nucleotides of any one of SEQ ID NO:74, SEQ ID NO:71 to SEQ ID NO:73, SEQ ID NO:75 to SEQ ID NO:82, SEQ ID NO:90, SEQ ID NO:96, SEQ ID NO:105, and SEQ ID NO:134. In some embodiments, at least 16 consecutive nucleotides. In some embodiments, at least 17 consecutive nucleotides. In some embodiments, at least 18 consecutive nucleotides. In some embodiments, at least 19 consecutive nucleotides. In some embodiments, at least 20 consecutive nucleotides. In some embodiments, at least 21 consecutive nucleotides.

[0019] In some embodiments, the sense strand comprises or is selected from any one of the following nucleotide sequences: SEQ ID NO:4, SEQ ID NO:1 to SEQ ID NO:3, SEQ ID NO:5 to SEQ ID NO:12, SEQ ID NO:20, SEQ ID NO:26, SEQ ID NO:35, SEQ ID NO:64.

[0020] In some embodiments, the antisense strand comprises or is selected from any one of the following nucleotide sequences: SEQ ID NO:74, SEQ ID NO:71 to SEQ ID NO:73, SEQ ID NO:75 to SEQ ID NO:82, SEQ ID NO:90, SEQ ID NO:96, SEQ ID NO:105, SEQ ID NO:134.

[0021] In some embodiments, the RNAi agent comprises a sense strand and an antisense strand, or is selected from any one of the following groups:

[0022] Group 1), the sense strand set forth in SEQ ID NO: 4 and the antisense strand set forth in SEQ ID NO: 74;

[0023] Group 2), the sense strand set forth in SEQ ID NO: 2 and the antisense strand set forth in SEQ ID NO: 72;

[0024] Group 3), the sense strand set forth in SEQ ID NO: 3 and the antisense strand set forth in SEQ ID NO: 73, or the sense strand set forth in SEQ ID NO: 64 and the antisense strand set forth in SEQ ID NO: 134;

[0025] Group 4), the sense strand set forth in SEQ ID NO: 1 and the antisense strand set forth in SEQ ID NO: 71;

[0026] Group 5), the sense strand set forth in SEQ ID NO: 5 and the antisense strand set forth in SEQ ID NO: 75;

[0027] Group 6), the sense strand set forth in SEQ ID NO: 6 and the antisense strand set forth in SEQ ID NO: 76, or the sense strand set forth in SEQ ID NO: 26 and the antisense strand set forth in SEQ ID NO: 96;

[0028] Group 7), the sense strand set forth in SEQ ID NO: 7 and the antisense strand set forth in SEQ ID NO: 77;

[0029] Group 8), the sense strand set forth in SEQ ID NO: 8 and the antisense strand set forth in SEQ ID NO: 78;

[0030] Group 9), the sense strand set forth in SEQ ID NO: 9 and the antisense strand set forth in SEQ ID NO: 79;

[0031] Group 10), the sense strand set forth in SEQ ID NO: 10 and the antisense strand set forth in SEQ ID NO: 80, or the sense strand set forth in SEQ ID NO: 35 and the antisense strand set forth in SEQ ID NO: 105;

[0032] Group 11), the sense strand set forth in SEQ ID NO: 11 and the antisense strand set forth in SEQ ID NO: 81;

[0033] Group 12), the sense strand as shown in SEQ ID NO: 12 and the antisense strand as shown in SEQ ID NO: 82, or the sense strand as shown in SEQ ID NO: 20 and the antisense strand as shown in SEQ ID NO: 90.

[0034] In some embodiments, at least one nucleotide in the sense strand and / or antisense strand is a modified nucleotide. In some embodiments, all nucleotides in the sense strand and / or antisense strand are modified nucleotides.

[0035] In some embodiments, the sense strand contains three consecutive 2'-fluoro-modified nucleotides. In some embodiments, from the 5' end to the 3' end, the nucleotides at positions 7, 8, and 9 in the sense strand are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are non-fluoro-modified nucleotides, preferably, the non-fluoro-modified nucleotides are 2'-methoxy-modified nucleotides.

[0036] In some embodiments, the antisense strand contains at least five 2'-fluoro-modified nucleotides. In some embodiments, 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.

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

[0038] In some embodiments, in the 5' to 3' direction, the nucleotides at positions 2, 6, 12, 14, and 16 of the antisense strand are each independently 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are non-2'-fluoro-modified nucleotides. Preferably, the non-fluoro-modified nucleotides are 2'-methoxy-modified nucleotides.

[0039] 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 modifying group enables the RNAi agent to have increased stability in a biological sample or environment. In some embodiments, the sense strand and / or antisense strand include a plurality of phosphodiester groups with a modifying group. In some embodiments, the phosphodiester group with a modifying group is present in one or more of the following positions: between any two adjacent nucleotides from the 1st nucleotide to the 3rd nucleotide of the 5' end end and / or the 3' end end of the antisense strand, and / or between any two adjacent nucleotides from the 1st nucleotide to the 3rd nucleotide of the 5' end end and / or the 3' end end of the antisense strand. 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 of the following positions:

[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 phosphorothioate diester groups present in:

[0050] between the first nucleotide and the second nucleotide at the 5' end of the sense strand; and

[0051] between the second and third nucleotides at the 5' end of the sense strand; and

[0052] between the first nucleotide and the second nucleotide at the 3' end of the sense strand; and

[0053] between the second and third nucleotides at the 3' end of the sense strand; and

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

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

[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 strand is selected from or comprises the nucleotide sequence shown in any one of SEQ ID NO: 141 to SEQ ID NO: 164, and SEQ ID NO: 287 to SEQ ID NO: 290.

[0059] In some embodiments, the antisense strand is selected from or comprises the nucleotide sequence shown in any one of SEQ ID NO: 214 to SEQ ID NO: 237, and SEQ ID NO: 291 to SEQ ID NO: 294.

[0060] In some embodiments, the RNAi agent further comprises one or more delivery groups, which are connected to the sense strand and / or antisense strand. In the context of the present disclosure, "connected" includes covalent and non-covalent connections. The delivery group is capable of delivering the RNAi agent of the present disclosure to a location where LRRK2 gene expression is present.

[0061] In some embodiments, the delivery group comprises a lipophilic group, and the one or more lipophilic groups are connected to any one or more nucleotides in the sense strand or antisense strand of the RNAi agent. In some embodiments, the one or more lipophilic groups are all connected to the sense strand in the RNAi agent. In some embodiments, the one or more lipophilic groups are all connected to the antisense strand in the RNAi agent. In some embodiments, at least one of the lipophilic groups is connected to the sense strand in the RNAi agent, and at least one of the lipophilic groups is connected to the antisense strand in the RNAi agent.

[0062] In some embodiments, the lipophilic group is connected to the base of the nucleotide. In some embodiments, the lipophilic group is connected to the sugar ring of the nucleotide. In some embodiments, the lipophilic group is connected to the internucleoside linking group between two adjacent nucleotides. 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, hydroxy, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.

[0063] 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 Hydrocarbon chain.

[0064] In some embodiments, the RNAi agent contains a lipophilic group, and the lipophilic group is n-hexadecyl.

[0065] In some embodiments, the lipophilic group is covalently linked to the 2' position of the sugar ring of the nucleoside in the nucleotide. In this case, the nucleoside to which the lipophilic group is linked has a structure as shown in Formula (I):

[0066] in,

[0067] X1 is selected from O, S, N, C atoms;

[0068] X2 is selected from O, S atoms;

[0069] R1 is selected from C 10 -C 30 A straight-chain alkyl group, or a C alkyl group interrupted by one or more O or S atoms 10 -C 30 A straight chain alkyl group; optionally, the C 10 -C 30 The straight chain alkyl group may be replaced by one or more R a Replace, or optionally, the C 10 -C 30 The adjacent two carbon atoms of the straight chain alkyl form a C 3-6 Cycloalkyl;

[0070] R a Each is independently selected from hydrogen, deuterium, halogen, hydroxy, cyano, alkyl, haloalkyl, alkoxy, cycloalkyl, heterocycloalkyl;

[0071] n is 1;

[0072] B represents a base.

[0073] In some embodiments, the nucleoside of formula (I) has the structure of the nucleoside portion of the compound of formula (I) or a pharmaceutically acceptable salt thereof as described in PCT application WO2024125556A1, which is incorporated herein by reference in its entirety.

[0074] In some embodiments, X1 in formula (I) is selected from an O or S atom. In some embodiments, X1 is an O atom.

[0075] In some embodiments, X2 in Formula (I) is selected from an O or S atom. In some embodiments, it is an O atom.

[0076] In some embodiments, R1 in formula (I) is selected from C 14 -C 24 Straight chain alkyl (such as C 14 、C 15 、C 16 、C 17 、C 18 、C 19 、C 20 、C 21 、C 22 、C 23 、C 24 A straight-chain alkyl group) or a C alkyl group interrupted by one or more O or S atoms 14 -C 24 Straight chain alkyl (such as C 14 、C 15 、C 16 、C 17 、C 18 、C 19 、C 20 、C 21 、C 22 、C 23 、C 24 straight-chain alkyl).

[0077] In some embodiments, R a are each independently selected from hydrogen, deuterium, halogen (such as fluorine, chlorine, bromine), C 1-6 Alkyl (e.g., C1, C2, C3, C4, C5, C6 alkyl, including but not limited to methyl, ethyl, isopropyl), C 1-6 Alkoxy (e.g., C1 alkoxy, C2 alkoxy, C3 alkoxy, C4 alkoxy, C5 alkoxy, C6 alkoxy, including but not limited to methoxy, ethoxy, propoxy, isopropoxy).

[0078] In some embodiments, R a Each is independently selected from hydrogen, deuterium, fluorine, methyl, and methoxy.

[0079] In some embodiments, R1 in formula (I) is selected from: The a end is connected to X2.

[0080] In some embodiments, B is selected from adenine, guanine, cytosine, uracil, and thymine.

[0081] In some embodiments, the nucleoside of formula (I) is selected from:

[0082] In some embodiments, the nucleoside of formula (I) is selected from:

[0083] In some embodiments, the nucleoside of formula (I) is:

[0084] Here, B represents a base.

[0085] In some embodiments, the nucleoside as shown in formula (I) can be located at any position in the sense strand and / or antisense strand of the RNAi agent. In some embodiments, the nucleoside as shown in formula (I) is located in the sense strand. In some embodiments, the nucleoside as shown in formula (I) is located at one or more positions from the 1st to the 8th position from the 5' end or the 3' end of the sense strand, for example, can be located at the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th or 8th position. In some embodiments, the nucleoside as shown in formula (I) is located at one or more positions from the 1st, 2nd, 6th and 7th, 19th, 20th or 21st position from the 5' end of the sense strand.

[0086] In some embodiments, the nucleoside of formula (I) is located at one or more of position 2, position 7, or position 20 from the 5' end of the sense strand.

[0087] In some embodiments, the nucleoside linked to the lipophilic group has a structure as shown in formula (II):

[0088] Here, Base represents a base.

[0089] In some embodiments, the delivery group comprises a targeting ligand that targets the liver. In some embodiments, the targeting ligand binds to asialoglycoprotein receptor (ASGPR). In some embodiments, the targeting ligand comprises a galactose cluster or a galactose derivative cluster, the galactose derivative being selected from N-acetyl-galactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine, or N-isobutyrylgalactosamine.

[0090] In some embodiments, the delivery group is attached to the 3' end of the sense strand of the RNAi agent.

[0091] In some embodiments, the delivery group is linked to the end of the RNAi agent via a phosphodiester group, a phosphorothioate diester group, or a phosphonic acid group. In some embodiments, the delivery group is linked to the end of the RNAi agent via a phosphodiester group.

[0092] In some embodiments, the delivery group is indirectly 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 delivery group is indirectly linked to the end of the RNAi agent through a phosphodiester group.

[0093] In some embodiments, the delivery group 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 delivery group is directly linked to the end of the RNAi agent through a phosphodiester group.

[0094] In some embodiments, the delivery group 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 delivery group is directly linked to the 3' end of the sense strand of the RNAi agent via a phosphodiester group.

[0095] On the other hand, the present disclosure provides a pharmaceutical composition comprising the RNAi agent described herein and one or more pharmaceutically acceptable carriers thereof, such as, but not limited to, excipients, such as vehicles, diluents, and / or drug delivery systems (such as delivery polymers). Various drug 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 RNAi agents, receptor-mediated endocytosis, constructing nucleic acids as part of retroviruses or other vectors.

[0096] 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.

[0097] In some embodiments, the unit dose of the pharmaceutical composition is 0.001 mg-1000 mg.

[0098] In certain embodiments, the pharmaceutical composition comprises 0.01-99.99% of the aforementioned RNAi agent based on the total weight of the composition. In certain embodiments, the pharmaceutical composition comprises 0.1-99.9% of the aforementioned RNAi agent. In certain embodiments, the pharmaceutical composition comprises 0.5%-99.5% of the aforementioned RNAi agent. In certain embodiments, the pharmaceutical composition comprises 1%-99% of the aforementioned RNAi agent. In certain embodiments, the pharmaceutical composition comprises 2%-98% of the aforementioned RNAi agent.

[0099] In certain embodiments, the pharmaceutical composition comprises 0.01% to 99.99% of a pharmaceutically acceptable carrier, based on the total weight of the composition. In certain embodiments, the pharmaceutical composition comprises 0.1% to 99.9% of a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition comprises 0.5% to 99.5% of a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition comprises 1% to 99% of a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition comprises 2% to 98% of a pharmaceutically acceptable carrier.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] In another aspect, the present disclosure provides a method for reducing LRRK2 expression, comprising administering to a subject an effective amount or effective dose of the RNAi agent and / or pharmaceutical composition of the present disclosure.

[0106] In another aspect, the present disclosure provides a method for treating and / or preventing a disease associated with LRRK2 gene expression in a subject, comprising administering an effective amount or effective dose of the RNAi agent and / or pharmaceutical composition of the present disclosure to the subject.

[0107] In another aspect, the present disclosure provides use of the RNAi agent and / or pharmaceutical composition of the present disclosure in preparing a medicament for treating and / or preventing a disease associated with LRRK2 gene expression.

[0108] In another aspect, the present disclosure provides use of the RNAi agent and / or pharmaceutical composition of the present disclosure in the preparation of a medicament for inhibiting the expression of LRRK2.

[0109] In some embodiments, the disease is a disease associated with LRRK2. In some embodiments, the disease is a neurodegenerative disease. In some embodiments, the neurodegenerative disease can be a familial disease or a sporadic disease, including Parkinson's disease, amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Huntington's disease, schizophrenia, progressive myoclonic epilepsy (Unver-Richt-Lundberg Lafora disease), Hallervorden-Spatz disease, etc. In some embodiments, the disease is Parkinson's disease.

[0110] In another aspect, the present disclosure discloses a method for in vivo delivery of an RNAi agent that inhibits LRRK2 expression and / or replication, comprising administering to a subject an effective amount or effective dose of the RNAi agent and / or pharmaceutical composition of the present disclosure.

[0111] The RNAi agents or pharmaceutical compositions and methods disclosed herein can reduce the level of target mRNA in a cell, a cell population, a cell population, a tissue or a subject, comprising: administering to the subject a therapeutically effective amount of the RNAi agent or pharmaceutical composition of the present disclosure, wherein the RNAi agent is linked to the delivery group, thereby inhibiting the expression of the target mRNA in the subject.

[0112] In some embodiments, prior to administration of the RNAi agents and / or pharmaceutical compositions of the present disclosure, the subject has been identified as having pathological upregulation of a target gene in the targeted cells or tissues.

[0113] 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.

[0114] Delivery of the RNAi agents and / or pharmaceutical compositions disclosed herein can be by local administration (e.g., direct injection, implantation, or local 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, and any other suitable mode of administration commonly used in the art.

[0115] In alternative embodiments, the pharmaceutical compositions provided herein can be administered by injection, for example, intravenously, intramuscularly, intradermally, subcutaneously, intraduodenally, or intraperitoneally.

[0116] In another aspect, the present disclosure provides a cell comprising the RNAi agent of the present disclosure, wherein the cell is incapable of developing into an animal or plant individual.

[0117] In another aspect, the present disclosure provides a kit comprising the RNAi agent and / or pharmaceutical composition of the present disclosure. In an optional embodiment, after the delivery group is linked to the RNAi agent, the RNAi agent can be packaged in the kit in the form of a conjugate.

[0118] The present disclosure also provides a method for silencing mRNA of a target gene in a cell, the method comprising the step of introducing the RNAi agent and / or pharmaceutical composition of the present disclosure into the cell.

[0119] The present disclosure also provides a method for silencing a target gene or mRNA of a target gene in a cell in vivo or in vitro, the method comprising the step of introducing the RNAi agent and / or pharmaceutical composition according to the present disclosure into the cell.

[0120] The present disclosure also provides a method for inhibiting the expression of a target gene or target gene mRNA, comprising administering an effective amount or effective dose of the RNAi agent and / or pharmaceutical composition according to the present disclosure to a subject in need thereof.

[0121] 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.

[0122] In some embodiments, the target gene is the LRRK2 gene, and the target mRNA is the mRNA expressed by the target gene.

[0123] The present disclosure also provides a method for preparing an RNAi agent, which comprises: synthesizing the RNAi agent described in the present disclosure.

[0124] The pharmaceutically acceptable salts of the compounds described in the present disclosure are selected from inorganic salts or organic salts, and the compounds described in the present disclosure can react with acidic or basic substances to form corresponding salts. In the context of the present disclosure, the compounds include the RNAi agents disclosed in the present disclosure.

[0125] 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.

[0126] 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 are interconvertible via a low energy barrier.

[0127] 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.

[0128] 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).

[0129] 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.

[0130] 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.

[0131] The present disclosure incorporates the entire text of WO2023274395A1.

[0132] Explanation of terms

[0133] 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.

[0134] As used herein, "RNAi agent" means an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule containing a messenger RNA (mRNA) transcript that can degrade or inhibit (e.g., degrade or inhibit under appropriate conditions) a target mRNA in a sequence-specific manner. The RNAi agent used herein can act by an RNA interference mechanism (i.e., by inducing RNA interference by interacting with the RNA interference pathway machinery (RNA-induced silencing complex or RISC) of mammalian cells) or by any alternative mechanism or pathway. Although it is believed that the term RNAi agent used herein acts primarily by 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 RNA (siRNA). The antisense strand of the RNAi agent 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.

[0135] Unless otherwise specified, in the context of the present disclosure, the terms "leucine-rich repeat kinase 2", "LRRK2", etc. are used interchangeably in the present disclosure. LRRK2 includes but is not limited to human LRRK2, cynomolgus monkey LRRK2, mouse LRRK2, and rat LRRK2, and their amino acid and complete coding sequences and mRNA sequences are easily obtained using publicly available databases, for example, GenBank, UniProt, OMIM, and the Macaca genome project website.

[0136] The term "LRRK2" also refers to naturally occurring DNA sequence variations of the LRRK2 gene, such as single nucleotide polymorphisms (SNPs) in the LRRK2 gene. Exemplary SNPs can be found in the dbSNP database.

[0137] The term "target sequence" refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during transcription of LRRK2, including mRNAs that are RNA processing products of the primary transcription product. The targeted portion of the target sequence should be long enough to serve as a substrate for iRNA-directed cleavage. In one embodiment, the target sequence is within the protein coding region of LRRK2. 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 a strand containing a sequence that is identical or substantially identical to the target mRNA sequence; the antisense strand (also known as AS or AS strand) refers to a strand having a sequence that is complementary to the target mRNA sequence.

[0138] In the context of describing the sense strand of the RNAi agent described herein, the term "at least 15 consecutive nucleotide sequences that differ by no more than 3 nucleotides from the nucleotide sequence set forth in SEQ ID NO: 1" is intended to indicate that the sense strand of the RNAi agent described herein comprises at least 15 consecutive nucleotides of the nucleotide sequence set forth in SEQ ID NO: 1, or differs by no more than 3 nucleotides (optionally, differs by no more than 2 nucleotides; optionally, differs by 1 nucleotide) from at least 15 consecutive nucleotide sequences of the nucleotide sequence set forth in SEQ ID NO: 1. Other similar descriptions in the context of this disclosure should also be understood similarly. The "difference" in this disclosure does not include nucleotides containing different modifications, that is, nucleotides containing the same base but different modifications do not belong to the nucleotides that differ in this disclosure. Other similar descriptions of the sense strand and / or antisense strand in the context of this disclosure should also be understood similarly.

[0139] 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.

[0140] Unless otherwise specified, in the context of the present disclosure, "G", "C", "A", "T" and "U" represent nucleotides, respectively, which include the bases of guanine, cytosine, adenine, thymidine and uracil, respectively. It is well known to those skilled in the art that the replacement of bases T and U will not significantly affect the properties of the RNAi agent sequence. In the sequence of the present disclosure, U can be arbitrarily replaced by T, and the sequence obtained after the replacement is also within the scope of protection of the present disclosure. In the sequence of the present disclosure, for the same nucleic acid chain, with the direction from the 5' end to the 3' end as the left to right direction, 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 connected by a thiophosphate diester group. Unless otherwise specified, the two nucleosides are connected by a phosphodiester group. Unless otherwise specified, the "RNAi agent", "nucleotide", "compound", "chemical modification", "oligonucleotide", "double-stranded RNAi inhibitor molecule", "siRNA", "dsRNA", "nucleic acid" and "RNAi" of the present disclosure can independently exist in the form of salt, mixed salt or non-salt (such as free acid or free base). When present in the form of a salt or a mixed salt, it may be a pharmaceutically acceptable salt. The term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts. When it exists in the form of a salt, some groups may ionize to form anions / cations, for example, phosphodiester groups and thiophosphonic acid diester groups can exist in the form of anions. Unless otherwise specified, the salt forms corresponding to the following structures are also within the scope of protection of the present 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. The above-mentioned modifications and linking groups have the structures shown in the following table, respectively, where Base represents the base at the corresponding position:

[0141] Table 1

[0142] 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 ow When 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 owThe logK of cholesteryl N-(hexan-6-ol) carbamate is about 0.7. ow It is 10.7.

[0143] 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.

[0144] 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.

[0145] 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 by Dual- 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 (%).

[0146] The term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts. As an example, the pharmaceutically acceptable salt of the RNAi agent of the present disclosure is a sodium salt.

[0147] "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.

[0148] "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.

[0149] "Effective amount" or "effective dose" refers to the amount of a drug, RNAi agent, 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, reducing 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.

[0150] As used herein, "patient," "subject," or "individual" are used interchangeably and include humans or non-human animals, such as mammals, eg, humans or monkeys.

[0151] 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.

[0152] 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.

[0153] 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.

[0154] 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.

[0155] 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.

[0156] 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.

[0157] 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.

[0158] 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.

[0159] 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.

[0160] 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).

[0161] 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.

[0162] "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

[0163] The present disclosure is further described below with reference to the examples, but these examples are not intended to limit the scope of the present disclosure. Experimental methods in the examples of the present disclosure that do not specify specific conditions are generally performed under conventional conditions, such as the Cold Spring Harbor Laboratory Manual of Antibody Technology and the Molecular Cloning Manual; or according to the conditions recommended by the raw material or product manufacturer. Reagents whose specific sources are not specified can be obtained from any supplier of molecular biology reagents with a quality / purity suitable for molecular biology applications. Unless otherwise specified, the reagents used in the following examples are all commercially available products.

[0164] Example 1. Design of LRRK2 RNAi Agents

[0165] The sequences of the sense and antisense strands in the RNAi agent disclosed herein are shown in Table 2 below. The human LRRK2 gene (NM_198578.4) is used as the target gene, and the 19 / 21 nt unmodified sense / antisense strands are designed to meet the general rules of active double-stranded oligonucleotides. The modified sequences of the RNAi agent disclosed herein are shown in Table 2.

[0166] Table 2. Sequences of unmodified RNAi agents targeting the LRRK2 gene

[0167] Table 3. Modified RNAi agent sequences targeting the LRRK2 gene

[0168] In Tables 2 and 3, within the same nucleic acid sequence, from left to right, in the 5' to 3' direction, G, C, A, and U represent nucleosides containing guanine, cytosine, adenine, and uracil, respectively. The sequence is oriented from left to right in the 5' to 3' direction. A lowercase letter m indicates that the nucleoside adjacent to the left of the letter m is a 2'-methoxy-modified nucleoside; a lowercase letter f indicates that the nucleoside adjacent to the left of the letter f is a 2'-fluoro-modified nucleoside; and a lowercase letter s indicates that the two nucleosides adjacent to the letter s are linked by a phosphorothioate diester linkage. Unless otherwise specified, two adjacent nucleosides are linked by a phosphodiester linkage. 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.

[0169] The structures of the 2'-methoxy modified nucleosides, 2'-fluoro modified nucleosides, phosphorothioate diester groups, and phosphodiester groups are shown in Table 1. When the RNAi agent of the present disclosure exists in salt form, for example, in the form of a sodium salt, the structure of the salt form corresponding to the structure in Table 1 is also within the scope of protection of the present disclosure.

[0170] Example 2. Synthesis of RNAi Agents of the Present Disclosure

[0171] The synthesis of the RNAi agent disclosed in the present invention 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, unmodified nucleoside phosphoramidite monomers or nucleoside phosphoramidite monomers with modifications at corresponding positions in the sequence are connected one by one according to the synthesis procedure. The nucleoside phosphoramidite monomers used in the embodiments of the present invention were purchased from Shanghai Zhaowei Company and Suzhou Jima Company. 5-Ethylthio-1H-tetrazole (ETT) was used as an 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 a sulfurizing reagent, and iodine pyridine / water solution (Shanghai Lingjiang) was used as an oxidant.

[0172] 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 reversed-phase chromatography with a mobile phase of 0.1 M TEAA and acetonitrile. The target oligonucleotides were collected, lyophilized, identified as the desired product by LC-MS, and quantified by UV light (260 nm).

[0173] The obtained single-stranded oligonucleotides were annealed according to the complementary pairing in an equimolar ratio, and the resulting RNAi agent of the present disclosure was dissolved in 1×PBS and adjusted to the concentration required for the experiment.

[0174] Example 3. Single concentration point activity of RNAi agents in endogenous cells

[0175] RNAi agents were screened at a single concentration for molecular mimicry of endogenous cellular activities.

[0176] A549 cells were cultured in RPMI1640 medium (Gibco) (containing 10% FBS) at 37°C and 5% CO2. 24 h before transfection, A549 cells were seeded in 24-well plates at a seeding density of 5 × 10 cells per well. 4 cells, 500 μL culture medium per well.

[0177] According to the instructions, RNAi MAX transfection reagent (ThermoFisher, 13778150) was used to transfect the cells with RNAi agents. A single concentration point of 20 nM was set for the RNAi agent. After 48 h of transfection, total RNA was extracted from the cells using a high-throughput cell RNA extraction kit (ThermoFisher, A27828), RNA reverse transcription experiments (Takara, RR037B) and quantitative real-time PCR detection (ThermoFisher, 4444557). The mRNA level of human LRRK2 was measured and corrected for the mRNA level of human LRRK2 according to the GAPDH internal reference gene level.

[0178] Result analysis method:

[0179] 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%.

[0180] The results are expressed as the percentage of human LRRK2 expression remaining in cells treated with RNAi agents. The results of the inhibition rate are shown in Table 4.

[0181] Table 4. Single-site activity results of the RNAi agents disclosed herein in A549 cells

[0182] Example 4. psiCHECK on-target activity of RNAi agents in human embryonic kidney cells (HEK293A)

[0183] RNAi agents were screened for on-target activity at the in vitro molecular level using nine concentration gradients in HEK293A cells.

[0184] 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 1 × 10 cells per well. 4 cells, 100 μL culture medium per well.

[0185] According to the instructions, Lipofectamine 2000 (ThermoFisher, 11668019) was used to co-transfect the cells with RNAi agents and corresponding plasmids, using 0.3 μL Lipofectamine 2000 per well. The amount of plasmid transfection was 20 ng per well. For the target sequence plasmid, a total of 9 concentration points were set for the RNAi agent, with the highest concentration point having a final concentration of 10 nM, 5-fold gradient dilution, and the lowest concentration point having a final concentration of 0.0000256 nM. 24 h after transfection, the target level was detected using the Dual-Luciferase Reporter Assay System (Promega, E2940). The results are shown in Table 5. The results show that the RNAi agent disclosed herein has good target inhibitory activity.

[0186] Table 5. psiCHECK on-target activity of the RNAi agents of the present disclosure in HEK293A cells

[0187] Example 5. Endogenous Cellular Activity of RNAi Agents

[0188] RNAi agents were screened for in vitro molecular mimicry of endogenous cellular activity in A549 cells using a seven-point concentration gradient. dsRNA was diluted fivefold using RNAi MAX transfection reagent (ThermoFisher, 13778150) at an initial concentration of 20 nM to form seven concentration points (20 nM, 4 nM, 0.8 nM, 0.16 nM, 0.032 nM, 0.0064 nM, and 0.00128 nM). A549 cells were cultured in RPMI1640 medium (Gibco) containing 10% FBS at 37°C and 5% CO2. After adding RNAi agents to 96-well plates, the seeding density was 1.4 × 10 4A549 cells were transfected at 400 μL / well (90 μL culture medium per well). Twenty-four hours after transfection, total RNA was extracted using a high-throughput cell RNA extraction kit (Zhiang Biotechnology, MNTR / FX96). Human LRRK2 mRNA levels were measured by RNA reverse transcription (Takara, RR037B) and quantitative real-time PCR (ThermoFisher, 4444557). GAPDH was used as an internal reference gene for expression level correction.

[0189] Results were analyzed with reference to Example 3. dsRNA sequences were screened for in vitro molecular activity in A549 cells using a seven-point concentration gradient. Results are expressed as the percentage of human LRRK2 expression remaining relative to that in cells treated with dsRNA. Table 6 shows the inhibition rates.

[0190] Table 6. Endogenous activity of RNAi agents of the present disclosure in A549 cells

Claims

1. An RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, The sense strand comprises at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence shown in any one of SEQ ID NO:4, SEQ ID NO:1 to SEQ ID NO:3, SEQ ID NO:5 to SEQ ID NO:12, SEQ ID NO:20, SEQ ID NO:26, SEQ ID NO:35, and SEQ ID NO:64; The antisense strand comprises at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence shown in any one of SEQ ID NO:74, SEQ ID NO:71 to SEQ ID NO:73, SEQ ID NO:75 to SEQ ID NO:82, SEQ ID NO:90, SEQ ID NO:96, SEQ ID NO:105, and SEQ ID NO:

134.

2. The RNAi agent of claim 1, wherein: The sense strand comprises at least 17 consecutive nucleotides that differ by no more than 3 nucleotides from any one of SEQ ID NO:4, SEQ ID NO:1 to SEQ ID NO:3, SEQ ID NO:5 to SEQ ID NO:12, SEQ ID NO:20, SEQ ID NO:26, SEQ ID NO:35, and SEQ ID NO:64; The antisense strand comprises at least 17 consecutive nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence shown in any one of SEQ ID NO:74, SEQ ID NO:71 to SEQ ID NO:73, SEQ ID NO:75 to SEQ ID NO:82, SEQ ID NO:90, SEQ ID NO:96, SEQ ID NO:105, and SEQ ID NO:134; Preferably, the sense strand comprises at least 18 consecutive nucleotides that differ from any one of the nucleotide sequences of SEQ ID NO:4, SEQ ID NO:1 to SEQ ID NO:3, SEQ ID NO:5 to SEQ ID NO:12, SEQ ID NO:20, SEQ ID NO:26, SEQ ID NO:35, and SEQ ID NO:64 by no more than 3 nucleotides; preferably, differs by no more than 1 nucleotide; and / or, Preferably, the antisense strand comprises at least 20 consecutive nucleotides that differ from any one of the nucleotide sequences of SEQ ID NO:74, SEQ ID NO:71 to SEQ ID NO:73, SEQ ID NO:75 to SEQ ID NO:82, SEQ ID NO:90, SEQ ID NO:96, SEQ ID NO:105, and SEQ ID NO:134 by no more than 3 nucleotides; preferably, differs by no more than 1 nucleotide; More preferably, the sense strand comprises at least 19 consecutive nucleotides that differ from any one of SEQ ID NO:4, SEQ ID NO:1 to SEQ ID NO:3, SEQ ID NO:5 to SEQ ID NO:12, SEQ ID NO:20, SEQ ID NO:26, SEQ ID NO:35, and SEQ ID NO:64 by no more than 3 nucleotides; preferably, differs by no more than 1 nucleotide; and / or, More preferably, the antisense strand comprises at least 21 consecutive nucleotides that differ by no more than 3 nucleotides from any one of the nucleotide sequences of SEQ ID NO:74, SEQ ID NO:71 to SEQ ID NO:73, SEQ ID NO:75 to SEQ ID NO:82, SEQ ID NO:90, SEQ ID NO:96, SEQ ID NO:105, and SEQ ID NO:134; preferably, the difference is no more than 1 nucleotide.

3. The RNAi agent according to claim 1 or 2, comprising a sense strand and an antisense strand as shown in any one of the following groups: Group 1), the sense strand set forth in SEQ ID NO: 4 and the antisense strand set forth in SEQ ID NO: 74; Group 2), the sense strand set forth in SEQ ID NO: 2 and the antisense strand set forth in SEQ ID NO: 72; Group 3), the sense strand set forth in SEQ ID NO: 3 and the antisense strand set forth in SEQ ID NO: 73, or the sense strand set forth in SEQ ID NO: 64 and the antisense strand set forth in SEQ ID NO: 134; Group 4), the sense strand set forth in SEQ ID NO: 1 and the antisense strand set forth in SEQ ID NO: 71; Group 5), the sense strand set forth in SEQ ID NO: 5 and the antisense strand set forth in SEQ ID NO: 75; Group 6), the sense strand set forth in SEQ ID NO: 6 and the antisense strand set forth in SEQ ID NO: 76, or the sense strand set forth in SEQ ID NO: 26 and the antisense strand set forth in SEQ ID NO: 96; Group 7), the sense strand set forth in SEQ ID NO: 7 and the antisense strand set forth in SEQ ID NO: 77; Group 8), the sense strand set forth in SEQ ID NO: 8 and the antisense strand set forth in SEQ ID NO: 78; Group 9), the sense strand set forth in SEQ ID NO: 9 and the antisense strand set forth in SEQ ID NO: 79; Group 10), the sense strand set forth in SEQ ID NO: 10 and the antisense strand set forth in SEQ ID NO: 80, or the sense strand set forth in SEQ ID NO: 35 and the antisense strand set forth in SEQ ID NO: 105; Group 11), the sense strand set forth in SEQ ID NO: 11 and the antisense strand set forth in SEQ ID NO: 81; Group 12), the sense strand as shown in SEQ ID NO: 12 and the antisense strand as shown in SEQ ID NO: 82, or the sense strand as shown in SEQ ID NO: 20 and the antisense strand as shown in SEQ ID NO:

90.

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

5. The RNAi agent of claim 4, wherein: The three consecutive nucleotides in the sense strand are 2'-fluoro-modified nucleotides, Preferably, in the direction from the 5' end to the 3' end, the 7th, 8th and 9th nucleotides of the sense strand are each independently a 2'-fluoro-modified nucleotide; and / or, 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; 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; 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 antisense strand is a phosphodiester group having a modified group, preferably a phosphorothioate diester group.

7. The RNAi agent of claim 6, wherein the phosphodiester group having a modifying group is present in at least one of the following positions: between the first and second nucleotides at the 5' end of the sense strand; between the second and third nucleotides at the 5' end of the sense strand; between the first and second nucleotides at the 3' end of the sense strand; between the second and third nucleotides at the 3' end of the sense strand; between the first and second nucleotides at the 5' end of the antisense strand; between the second and third nucleotides at the 5' end of the antisense strand; between the first and second nucleotides at the 3' end of the antisense strand; between the second and third nucleotides at the 3' end of the antisense strand; Preferably, the sense strand and / or antisense strand comprises a plurality of phosphorothioate diester groups, wherein the phosphorothioate diester groups are present in: between the first nucleotide and the second nucleotide at the 5' end of the sense strand; and between the second and third nucleotides at the 5' end of the sense strand; and between the first nucleotide and the second nucleotide at the 3' end of the sense strand; and between the second and third nucleotides at the 3' end of the sense strand; and between the first nucleotide and the second nucleotide at the 5' end of the antisense strand; and between the second and third nucleotides from the 5' end of the antisense strand; and between the first nucleotide and the second nucleotide at the 3' end of the antisense strand; 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 to 7, wherein The sense strand comprises a nucleotide sequence as shown in any one of SEQ ID NO: 141 to SEQ ID NO: 164, SEQ ID NO: 287 to SEQ ID NO: 290; and / or The antisense strand comprises a nucleotide sequence as shown in any one of SEQ ID NO: 214 to SEQ ID NO: 237 and SEQ ID NO: 291 to SEQ ID NO:

294.

9. The RNAi agent according to any one of claims 1 to 8, wherein The sense strand and / or the antisense strand further comprises one or more lipophilic groups; Preferably, the lipophilic group contains a saturated or unsaturated C16 hydrocarbon chain; More preferably, the RNAi agent contains a lipophilic group, and the lipophilic group is n-hexadecyl.

10. The RNAi agent according to claim 9, wherein The lipophilic group is connected to the sense chain; Preferably, the lipophilic group is linked to the 1st, 2nd, 6th, 7th, 19th, 20th or 21st nucleoside from the 5' end of the sense strand; More preferably, the lipophilic group is linked to the 2nd, 7th or 20th nucleoside from the 5' end of the sense strand.

11. The RNAi agent according to claim 9 or 10, wherein The nucleoside connected to the lipophilic group has a structure as shown in formula (II): Here, Base represents a base.

12. A pharmaceutical composition comprising: The RNAi agent according to any one of claims 1 to 11, and Pharmaceutically acceptable carrier.

13. A cell comprising the RNAi agent of any one of claims 1-11.

14. A kit comprising the RNAi agent according to any one of claims 1 to 11 and / or the pharmaceutical composition according to claim 12.

15. A method for reducing LRRK2 gene expression, comprising administering to a subject an effective amount or effective dose of the RNAi agent according to any one of claims 1 to 11 and / or the pharmaceutical composition according to claim 12.

16. A method for treating and / or preventing a disease in a subject, comprising administering to the subject an effective amount or effective dose of the RNAi agent according to any one of claims 1 to 11 and / or the pharmaceutical composition according to claim 12; Preferably, the disease is a neurodegenerative disease; More preferably, the neurodegenerative disease is selected from the group consisting of Parkinson's disease, amyotrophic lateral sclerosis, Alzheimer's disease, Huntington's disease, schizophrenia, progressive myoclonic epilepsy, and Hallevorden-Spatz disease.

17. A method for in vivo delivery of an RNAi agent that inhibits LRRK2 expression and / or replication, the method comprising administering to a subject an effective amount or effective dose of the RNAi agent according to any one of claims 1 to 11 and / or the pharmaceutical composition according to claim 12.

18. A method for preparing an RNAi agent, comprising: Synthesize the RNAi agent of any one of claims 1-11.

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