Dsrna molecule for regulating ATXN2 expression

Abstract

A double-stranded RNA for inhibiting the expression of the ATXN2 gene, a cell comprising same, and a method using the dsRNA or the cell for treating a disease or symptom mediated or associated with ATXN2 in a subject.

Description

dsRNA molecules that regulate ATXN2 expression

[0001] Cross-reference to related applications

[0002] This application claims priority to Chinese patent application CN202311286990.5, filed on September 28, 2023, entitled “dsRNA molecules for regulating ATXN2 expression,” the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to the field of RNA interference. Background Art

[0004] Ataxin 2 (ATXN2) is a protein encoded by the ATXN2 gene. Ataxin 2 is expressed throughout the body and is localized in the cytoplasm. It is an RNA-binding protein and regulator of stress granule assembly, interacting with the endoplasmic reticulum and involved in the regulation of RNA stability.

[0005] Ataxin 2 has been implicated in the pathogenesis of various neurodegenerative diseases. The ATXN2 gene contains a CAG trinucleotide repeat expansion. Normally, the CAG trinucleotide repeat is approximately 22 times. CAG expansions exceeding 22 repeats have been associated with certain neurodegenerative diseases.

[0006] Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant neurodegenerative disease characterized by progressive functional and cellular loss of neurons in the cerebellum, brainstem, and spinal cord. It is caused by 31 or more CAG repeats in the ATXN2 gene, resulting in a glutamine repeat expansion of the ATXN2 protein. SCA2 patients are characterized by progressive cerebellar ataxia, slow saccadic eye movements, and other neurological features, such as neuropathy.

[0007] Furthermore, expanded CAG repeats are a genetic risk factor for amyotrophic lateral sclerosis (ALS) through RNA-dependent interactions with TAR DNA-binding protein 43 (TDP-43). ATXN2 polyQ expansion mutations are found in ALS patients. This gene sequence alteration promotes TDP43 nuclear deletion and translocation, increasing caspase-3 activity, thereby promoting TDP43 cleavage and phosphorylation, ultimately leading to its accumulation in the cytoplasm. ATXN2 polyQ expansion mutations also induce endoplasmic reticulum stress and Golgi fragmentation, leading to neuronal cell death.

[0008] Moderate CAG expansions in the ATXN2 gene have also been associated with frontotemporal dementia (FTD) and Parkinson's disease.

[0009] There is a need in the art for compositions and methods for treating neurodegenerative diseases associated with ATXN2.

[0010] One approach to treat ATXN2-related neurodegenerative diseases is to reduce ATXN2 expression through RNA interference mechanism using small interfering RNA (siRNA).

[0011] Summary of the Invention

[0012] The present invention provides novel double-stranded RNA (dsRNA) for inhibiting ATXN2 gene expression, cells, and pharmaceutical compositions and kits comprising the dsRNA or cells, as well as methods of using the dsRNA, cells, pharmaceutical compositions and kits to inhibit or reduce ATXN2 gene expression or treat diseases or conditions that benefit from reduced ATXN2 gene expression.

[0013] In a first aspect, the present invention provides a double-stranded RNA (dsRNA) for inhibiting the expression of the ATXN2 gene, the dsRNA comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand and the antisense strand are each independently 15-30 nucleotides in length, and the antisense strand comprises a nucleotide sequence of at least 15 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 25 to 48. In some embodiments, the sense strand comprises a nucleotide sequence of at least 15 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 1 to 24.

[0014] In some embodiments, a hairpin loop is formed between the sense strand and the antisense strand in the dsRNA. In other embodiments, the dsRNA is siRNA.

[0015] In some embodiments, the sense strand and the antisense strand are each independently 15-27 nucleotides in length, preferably 18-25 nucleotides, more preferably 19-21 nucleotides in length. In some embodiments, the sense strand is 15-27 nucleotides in length, preferably 17-25 nucleotides, more preferably 18-23 nucleotides, more preferably 19-21 nucleotides, and most preferably 19 amino acids in length. In some embodiments, the antisense strand is 15-27 nucleotides in length, preferably 17-25 nucleotides, more preferably 18-23 nucleotides, more preferably 19-22 nucleotides, and most preferably 21 amino acids in length.

[0016] In some embodiments, the double-stranded region has a length of 15-25 nucleotide pairs, preferably 16-23 nucleotide pairs, more preferably 18-20 nucleotide pairs, and most preferably 19 nucleotide pairs.

[0017] In some embodiments, one or both of the sense strand and the antisense strand comprises a 3' overhang and / or a 5' overhang having at least 1 nucleotide, for example, one or both of the sense strand and the antisense strand comprises a 3' overhang and / or a 5' overhang having at least 2 nucleotides. In some embodiments, the antisense strand has a 3' overhang and / or a 5' overhang having at least 1 nucleotide, preferably, the antisense strand comprises a 3' overhang and / or a 5' overhang having 2 nucleotides. In some specific embodiments, the dsRNA has two nucleotide overhangs at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0018] In some embodiments, the antisense strand comprises a nucleotide sequence of at least 16 consecutive nucleotides, a nucleotide sequence of at least 17 consecutive nucleotides, a nucleotide sequence of at least 18 consecutive nucleotides, a nucleotide sequence of at least 19 consecutive nucleotides, or a nucleotide sequence of at least 20 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 25-48. In a preferred embodiment, the antisense strand comprises the nucleotide sequence shown in any one of SEQ ID NOs: 25-48.

[0019] In some embodiments, the sense strand comprises a nucleotide sequence of at least 16 consecutive nucleotides, a nucleotide sequence of at least 17 consecutive nucleotides, or a nucleotide sequence of at least 18 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 1-24. In a preferred embodiment, the sense strand comprises a nucleotide sequence shown in any one of SEQ ID NOs: 1-24.

[0020] In some embodiments, the dsRNA comprises any pair of paired sense strand sequence and antisense strand sequence as shown in Table 3 of the specification.

[0021] In some embodiments, substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, or all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides.

[0022] In some embodiments, the sense strand and the antisense strand each independently comprise one or more modified nucleotides selected from the group consisting of 2'-O-alkyl modified nucleotides (e.g., 2'-O-methyl modified nucleotides), 2'-methoxyethyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, inosine ribonucleotides, abasic nucleotides, inverted abasic deoxyribonucleotides, nucleotides containing a phosphorothioate group, vinylphosphonate modified nucleotides, locked nucleotides, unlocked nucleotides, 2'-amino-modified nucleotides, 2'-C-alkyl-modified Nucleotides, 2'-O-allyl modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides containing unnatural bases, terminal nucleotides attached to a cholesteryl derivative or a dodecyldiamide group, deoxyribonucleotides, 3'-terminal deoxythymidine (dT) nucleotides, conformationally restricted nucleotides, restricted ethyl nucleotides, 2'-hydroxyl modified nucleotides, nucleotides containing a methylphosphonic acid group, nucleotides containing a 5'-phosphate, nucleotides containing a 5'-phosphate mimetic, diol-modified nucleotides (GNA), and 2-O-(N-methylacetamide) modified nucleotides.

[0023] In some embodiments, the sense strand and the antisense strand each independently comprise one or more nucleotide modifications selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, and nucleotides comprising a phosphorothioate group.

[0024] In some embodiments, the sense strand and / or the antisense strand comprises at least two 2'-fluoro-modified nucleotides. In some embodiments, the sense strand and / or the antisense strand comprises at least eight 2'-O-methyl-modified nucleotides. In some embodiments, the 3' end and / or the 5' end of the sense strand and / or the antisense strand comprises 1-5 phosphorothioate internucleotide linkages, preferably 2-4 phosphorothioate internucleotide linkages.

[0025] In some embodiments, the antisense strand of the dsRNA is 21 nucleotides in length and has

[0026] (i) (counting from the 5′ end) 2′-O-methyl modified nucleotides at nucleotide positions 1, 3 to 5, 7 to 13, 15, 17 to 21, and 2′-fluoro modified nucleotides at nucleotide positions 2, 6, 14 and 16; and / or

[0027] (ii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21 (counting from the 5' end).

[0028] In some embodiments, the sense strand of the dsRNA has a length of 19 nucleotides and has:

[0029] (i) 2′-O-methyl modified nucleotides at positions 1 to 6 and 10 to 19 (counting from the 5′ end), and 2′-fluoro modified nucleotides at positions 7 to 9; and / or

[0030] (ii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3, between nucleotide positions 17 and 18, and between nucleotide positions 18 and 19 (counting from the 5′ end).

[0031] In some embodiments, the antisense strand of the dsRNA comprises any one of the modified nucleotide sequences shown in Table 5, and / or the sense strand comprises any one of the modified nucleotide sequences shown in Table 4. In some embodiments, the dsRNA comprises any one of the paired modified sense strand sequences and modified antisense strand sequences shown in Table 6.

[0032] In some preferred embodiments, the dsRNA of the present invention comprises a sense strand and an antisense strand, wherein

[0033] (1) the sense strand comprises UmUmGmUmUmGmUfGfGfUmAmCmAmGmUmUmUmAmAm (SEQ ID NO: 125), and the antisense strand comprises UmUfAmAmAmCfUmGmUmAmCmCmAmCfAmAfCmAmAmAmGm (SEQ ID NO: 126);

[0034] (2) the sense strand comprises CmAmAmUmGmAmUfAfUfGmUmUmUmCmGmAmUmAmUm (SEQ ID NO: 127), and the antisense strand comprises AmUfAmUmCmGfAmAmAmCmAmUmAmUfCmAfUmUmGmGmGm (SEQ ID NO: 128);

[0035] (3) the sense strand comprises CmCmUmCmCmUmGfGfAfCmAmAmAmGmAmAmAmUmAm (SEQ ID NO: 129), and the antisense strand comprises UmAfUmUmUmCfUmUmUmGmUmCmCmAfGmGfAmGmGmAmAm (SEQ ID NO: 130);

[0036] (4) the sense strand comprises AmGmAmCmCmAmAfAfGfAmGmUmAmGmUmUmAmAmUm (SEQ ID NO: 131), and the antisense strand comprises AmUfUmAmAmCfUmAmCmUmCmUmUmUfGmGfUmCmUmGmAm (SEQ ID NO: 132);

[0037] (5) the sense strand comprises CmUmGmAmAmUmCfUfAfUmGmGmAmUmCmAmAmCmUm (SEQ ID NO: 133), and the antisense strand comprises AmGfUmUmGmAfUmCmCmAmUmAmGmAfUmUfCmAmGmAmAm (SEQ ID NO: 134);

[0038] (6) the sense strand comprises AmAmUmCmUmAmUfGfGfAmUmCmAmAmCmUmAmCmUm (SEQ ID NO: 135), and the antisense strand comprises AmGfUmAmGmUfUmGmAmUmCmCmAmUfAmGfAmUmUmCmAm (SEQ ID NO: 136);

[0039] (7) the sense strand comprises GmCmCmAmAmAmGfCfCfUmUmCmUmAmCmUmAmCmAm (SEQ ID NO: 137), and the antisense strand comprises UmGfUmAmGmUfAmGmAmAmGmGmCmUfUmUfGmGmCmUmGm (SEQ ID NO: 138);

[0040] (8) the sense strand comprises GmAmAmUmCmAmAfGfCfCmAmAmGmAmCmAmUmAmUm (SEQ ID NO: 139), and the antisense strand comprises AmUfAmUmGmUfCmUmUmGmGmCmUmUfGmAfUmUmCmAmCm (SEQ ID NO: 140);

[0041] (9) the sense strand comprises GmCmUmAmUmUmCfCfAfUmAmAmUmUmCmCmAmUmAm (SEQ ID NO: 141), and the antisense strand comprises UmAfUmGmGmAfAmUmUmAmUmGmGmAfAmUfAmGmCmUmAm (SEQ ID NO: 142);

[0042] (10) the sense strand comprises AmCmUmGmGmAmAfGfUfUmAmUmUmUmAmUmUmUmUm (SEQ ID NO: 143), and the antisense strand comprises AmAfAmAmUmAfAmAmUmAmAmCmUmUfCmCfAmGmUmUmUm (SEQ ID NO: 144);

[0043] (11) the sense strand comprises CmCmAmUmCmAmAfGfAfUmCmCmAmCmUmUmCmUmAm (SEQ ID NO: 145), and the antisense strand comprises UmAfGmAmAmGfUmGmGmAmUmCmUmUfGmAfUmGmGmCmAm (SEQ ID NO: 146);

[0044] (12) the sense strand comprises AmCmAmGmCmCmAfAfGfUmUmCmUmAmCmUmUmCmUm (SEQ ID NO: 147), and the antisense strand comprises AmGfAmAmGmUfAmGmAmAmCmUmUmGfGmCfUmGmUmAmAm (SEQ ID NO: 148);

[0045] (13) the sense strand comprises GmUmUmCmUmAmCfUfUfCmUmGmAmAmUmCmUmAmUm (SEQ ID NO: 149), and the antisense strand comprises AmUfAmGmAmUfUmCmAmGmAmAmGmUfAmGfAmAmCmUmUm (SEQ ID NO: 150);

[0046] (14) the sense strand comprises UmUmCmUmAmCmUfUfCfUmGmAmAmUmCmUmAmUmAm (SEQ ID NO: 151), and the antisense strand comprises UmAfUmAmGmAfUmUmCmAmGmAmAmGfUmAfGmAmAmCmUm (SEQ ID NO: 152);

[0047] (15) the sense strand comprises CmAmAmGmUmGmCfUfAfAmGmGmAmUmUmCmUmUmUm (SEQ ID NO: 153), and the antisense strand comprises AmAfAmGmAmAfUmCmCmUmUmAmGmCfAmCfUmUmGmGmUm (SEQ ID NO: 154);

[0048] (16) the sense strand comprises AmGmUmGmCmUmAfAfGfGmAmUmUmCmUmUmUmCmAm (SEQ ID NO: 155), and the antisense strand comprises UmGfAmAmAmGfAmAmUmCmCmUmUmAfGmCfAmCmUmUmGm (SEQ ID NO: 156);

[0049] (17) the sense strand comprises CmCmAmCmGmUmUfCfCfUmUmCmUmCmUmCmAmGmAm (SEQ ID NO: 157), and the antisense strand comprises UmCfUmGmAmGfAmGmAmAmGmGmAmAfCmGfUmGmGmGmUm (SEQ ID NO: 158);

[0050] (18) the sense strand comprises AmUmAmCmUmCmAfGfCfCmUmGmUmUmUmGmUmUmUm (SEQ ID NO: 159), and the antisense strand comprises AmAfAmCmAmAfAmCmAmGmGmCmUmGfAmGfUmAmUmAmAm (SEQ ID NO: 160);

[0051] (19) the sense strand comprises UmAmCmUmCmAmGfCfCfUmGmUmUmUmGmUmUmUmUm (SEQ ID NO: 161), and the antisense strand comprises AmAfAmAmCmAfAmAmCmAmGmGmCmUfGmAfGmUmAmUmAm (SEQ ID NO: 162);

[0052] (20) the sense strand comprises CmAmCmCmAmAmAfUfAfUmGmAmUmGmUmAmUmCmAm (SEQ ID NO: 163), and the antisense strand comprises UmGfAmUmAmCfAmUmCmAmUmAmUmUfUmGfGmUmGmCmAm (SEQ ID NO: 164);

[0053] (21) the sense strand comprises CmCmAmGmCmUmUfAfCfUmCmCmAmCmGmCmAmUm (SEQ ID NO: 165), and the antisense strand comprises AmUfUmGmCmGfUmGmGmAmGmUmAmAfGmCfUmGmGmUmGm (SEQ ID NO: 166);

[0054] (22) the sense strand comprises CmUmUmAmCmUmCfCfAfCmGmCmAmAmUmAmUmGmUm (SEQ ID NO: 167), and the antisense strand comprises AmCfAmUmAmUfUmGmCmGmUmGmGmAfGmUfAmAmGmCmUm (SEQ ID NO: 168);

[0055] (23) the sense strand comprises GmUmGmCmCmAmCfAfUfUmAmUmCmAmGmUmCmUmAm (SEQ ID NO: 169), and the antisense strand comprises UmAfGmAmCmUfGmAmUmAmAmUmGmUfGmGfCmAmCmAmUm (SEQ ID NO: 170);

[0056] (24) the sense strand comprises UmCmAmGmCmAmUfCfCfUmCmAmUmGmUmCmUmAmUm (SEQ ID NO: 171), and the antisense strand comprises AmUfAmGmAmCfAmUmGmAmGmGmAmUfGmCfUmGmAmGmAm (SEQ ID NO: 172);

[0057] (25) the sense strand comprises nan (SEQ ID NO: 173), and the antisense strand comprises nan (SEQ ID NO: 174); or

[0058] (26) The sense strand comprises nan (SEQ ID NO: 175), and the antisense strand comprises nan (SEQ ID NO: 176), wherein the 5' end and the 3' end of the sense strand and the antisense strand may independently comprise one or two phosphorothioate modifications.

[0059] In one embodiment, the dsRNA of the present invention is further conjugated to a ligand moiety comprising N-acetylgalactosamine, preferably the sense strand is conjugated to the ligand moiety, more preferably the 3' end of the sense strand is conjugated to the ligand moiety via phosphorothioate.

[0060] In a second aspect, the present invention provides a cell comprising the dsRNA according to the first aspect of the present invention.

[0061] In a third aspect, the present invention provides a pharmaceutical composition comprising the dsRNA according to the first aspect of the present invention or the cell according to the second aspect of the present invention, and optionally a pharmaceutically acceptable carrier or excipient.

[0062] In a fourth aspect, the present invention provides a kit comprising the dsRNA of the first aspect of the present invention, the cell of the second aspect of the present invention, or the pharmaceutical composition of the third aspect of the present invention.

[0063] In a fifth aspect, the present invention provides a method for inhibiting ATXN2 gene expression in a cell, the method comprising contacting the cell with the dsRNA of the first aspect of the present invention or the pharmaceutical composition of the third aspect of the present invention. In some embodiments, the method is performed in vitro.

[0064] In a sixth aspect, the present invention provides a method of inhibiting ATXN2 gene expression in cells in a subject, the method comprising administering to the subject the dsRNA of the first aspect of the invention, the cells of the second aspect of the invention, or the pharmaceutical composition of the third aspect of the invention.

[0065] The present invention also provides a method for treating a disease or condition in a subject that benefits from reduced ATXN2 gene expression, the method comprising administering to the subject the dsRNA of the first aspect of the invention, the cell of the second aspect of the invention, or the pharmaceutical composition of the third aspect of the invention.

[0066] The present invention also provides a method for preventing at least one symptom in a subject suffering from a disease or condition that benefits from reduced ATXN2 gene expression, the method comprising administering to the subject the dsRNA of the first aspect of the invention, the cell of the second aspect of the invention, or the pharmaceutical composition of the third aspect of the invention.

[0067] The present invention also provides a method for preventing the progression of a disease or condition that benefits from reduced ATXN2 gene expression in a subject, the method comprising administering to the subject the dsRNA of the first aspect of the invention, the cell of the second aspect of the invention, or the pharmaceutical composition of the third aspect of the invention.

[0068] In some embodiments, the disease or disorder that benefits from reduced ATXN2 gene expression is an Ataxin2-related disease. In some preferred embodiments, the Ataxin2-related disease is selected from the group consisting of progressive cerebellar ataxia, blindness, spinocerebellar ataxia (SCA), such as spinocerebellar ataxia 2 (SCA2), and amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Parkinson's disease.

[0069] In some embodiments, the dsRNA, cells, or pharmaceutical composition is administered intrathecally.

[0070] In some embodiments, the subject is a human.

[0071] Detailed Description of the Invention

[0072] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different perspectives and applications without departing from the spirit of the present invention.

[0073] It should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific embodiments rather than for limiting the scope of protection of the present invention.

[0074] Throughout the specification and claims of the present invention, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0075] When the embodiment provides a numerical range, it should be understood that, unless otherwise specified in the present invention, the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the examples, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any method, equipment, and material of the prior art similar or equivalent to the methods, equipment, and materials described in the examples of the present invention can also be used to realize the present invention, and all belong to the protection scope of the present invention. The following describes embodiments of the present invention in more detail.

[0076] definition

[0077] "Double-stranded region" herein refers to a region comprising two nucleic acid strands that are antiparallel and complementary or substantially complementary.

[0078] As used herein, the term "double-stranded RNA" or "dsRNA" refers to a ribonucleic acid molecule or a ribonucleic acid molecule complex comprising a double-stranded region as defined above. The two parts forming the double-stranded region can be two different parts of a larger RNA molecule, or they can be separate RNA molecules.

[0079] When the two parts are separate RNA molecules, the dsRNA herein is referred to as small interfering RNA (siRNA) or short interfering RNA (siRNA).

[0080] When the two moieties are two distinct parts of a larger molecule, that is, when the 3' end of one moiety is linked to the 5' end of the other moiety by one or more uninterrupted nucleotides in between to form a double-stranded region, the uninterrupted nucleotides used to connect the two moieties are called a "hairpin." When the two moieties are covalently linked to form a double-stranded region by a method other than a hairpin, the connection structure is called a "linker." When such dsRNA is introduced into cells, it is cleaved into siRNA by an intracellular endoribonuclease called Dicer.

[0081] The term "siRNA" herein refers to a class of double-stranded RNA molecules comprising a sense strand and an antisense strand that can mediate the silencing of a target RNA (e.g., mRNA, e.g., a transcript of a gene encoding a protein) that is complementary or substantially complementary to the antisense strand. siRNA is typically double-stranded, comprising an antisense strand that is complementary to the target RNA, and a sense strand that is complementary or substantially complementary to the antisense strand. For convenience, such mRNA is also referred to herein as mRNA to be silenced. Such a gene is also referred to as a target gene. Typically, the RNA to be silenced is an endogenous gene or a pathogen gene. In addition, RNA (e.g., tRNA) and viral RNA other than mRNA can also be targeted.

[0082] As used herein, the term "antisense strand" refers to the strand of a dsRNA, particularly a siRNA, that contains a region that is fully complementary or substantially complementary to a target sequence.

[0083] As used herein, the term "complementary region" refers to a region on the antisense strand that is completely or substantially complementary to the target mRNA sequence. In the case where the complementary region is not completely complementary to the target sequence, mispairing can be located in the interior or terminal regions of the molecule. Typically, the most tolerated mispairing is located in the terminal regions, for example, within 5, 4, 3, 2 or 1 nucleotides at 5' and / or 3' ends. The antisense strand portion that is most sensitive to mispairing is referred to as a "seed region." For example, in a siRNA comprising a 19nt chain, the 19th position (from 5' to 3') can tolerate some mispairing.

[0084] As used herein, the term "complementary" refers to the ability of a first polynucleotide to hybridize to a second polynucleotide under certain conditions, such as stringent conditions. For example, stringent conditions may include 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA at 50° C. or 70° C. for 12-16 hours.

[0085] As used herein, "complementary" sequences, insofar as they meet the above requirements for their ability to hybridize, may also include or be formed entirely from non-Watson-Crick base pairs and / or base pairs formed from non-natural and modified nucleotides. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble base pairing or Hoogstein base pairing.

[0086] As used herein, a polynucleotide that is "at least partially complementary" or "substantially complementary" to a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of an mRNA of interest (e.g., an mRNA encoding apolipoprotein (a)). For example, a polynucleotide is complementary to at least a portion of an mRNA encoding apolipoprotein (a) if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding apolipoprotein (a).

[0087] As used herein, the terms "complementary," "fully complementary," and "substantially complementary" can be used with respect to base pairing between the sense and antisense strands of a dsRNA, particularly an siRNA, or between the antisense strand of a dsRNA, particularly an siRNA, and a target sequence.

[0088] As used herein, the term "sense strand" refers to the strand of an siRNA that includes a region that is substantially complementary to a region defined herein as the antisense strand.

[0089] A "nucleoside" is a compound composed of a purine or pyrimidine base and ribose or deoxyribose; a "nucleotide" is a compound composed of a purine or pyrimidine base, ribose or deoxyribose, and phosphate; an "oligonucleotide" is a nucleic acid molecule (RNA or DNA) having, for example, fewer than 100, 200, 300, or 400 nucleotides in length.

[0090] "Bases" are the fundamental building blocks of nucleosides, nucleotides, and nucleic acids. They contain nitrogen and are also called "nitrogenous bases." Unless otherwise specified, the capital letters A, U, T, G, and C represent the bases of nucleotides, representing adenine, uracil, thymine, guanine, and cytosine, respectively.

[0091] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the double-stranded region of an siRNA. For example, when the 3'-end of a chain of an siRNA extends beyond the 5'-end of another chain or vice versa, there is a nucleotide overhang. The siRNA can comprise an overhang with at least one nucleotide, an overhang with at least two nucleotides, an overhang with at least three nucleotides, an overhang with at least four nucleotides, an overhang with at least five nucleotides or more. The nucleotide overhang can comprise or consist of nucleotides / modified nucleotides (including deoxynucleotides / nucleosides). One or more overhangs can be on a sense strand or an antisense strand or any combination thereof. An overhang with one or more nucleotides can be present on the 5'-end, the 3'-end or both ends of the antisense or sense strand of the siRNA.

[0092] "Blunt end" or "blunt end" or "blunt end" means that there are no unpaired nucleotides at that end of the double-stranded siRNA, i.e., no nucleotide overhangs. A "blunt-ended siRNA" is a siRNA that is double-stranded throughout the entire length of the siRNA, i.e., there are no nucleotide overhangs at either end of the molecule.

[0093] Substantially all of the nucleotides of the dsRNA (particularly siRNA) of the present invention are modified.For example, substantially all of the nucleotides of the sense strand are modified nucleotides, or substantially all of the nucleotides of the antisense strand are modified nucleotides, or substantially all of the nucleotides of both the sense strand and the antisense strand are modified nucleotides. In other embodiments of the present invention, all of the nucleotides of the dsRNA (particularly siRNA) of the present invention are modified nucleotides.For example, all of the nucleotides of the sense strand are modified nucleotides, or all of the nucleotides of the antisense strand are modified nucleotides, or all of the nucleotides of both the sense strand and the antisense strand are modified nucleotides. As used herein, "substantially all of the nucleotides are modified" means that most of the nucleotides of the dsRNA (particularly siRNA) of the present invention are modified, but not all of the nucleotides are modified, and can include no more than 5,4,3,2 or 1 unmodified nucleotides.

[0094] As used herein, "modified nucleotides" include, but are not limited to, 2'-O-alkyl modified nucleotides (e.g., 2'-O-methyl modified nucleotides, 2'-methoxyethyl modified nucleotides), 2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, inosine ribonucleotides, abasic nucleotides, inverted abasic deoxyribonucleotides, nucleotides containing a phosphorothioate group, phosphorothioate internucleotide linkages, vinylphosphonate modified nucleotides, locked nucleotides, unlocked nucleotides, 2'-amino-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-O -allyl modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides containing non-natural bases, terminal nucleotides attached to a cholesterol derivative or a dodecanoic acid didecylamide group, deoxyribonucleotides, 3'-terminal deoxythymidine (dT) nucleotides, conformationally restricted nucleotides, restricted ethyl nucleotides, 2'-hydroxyl modified nucleotides, nucleotides containing a methylphosphonic acid group, nucleotides containing a 5'-phosphate, nucleotides containing a 5'-phosphate mimetic, diol-modified nucleotides (GNA), and 2-O-(N-methylacetamide) modified nucleotides, etc.

[0095] For example, a "2'-fluoro modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2' position of the ribose group is replaced by fluorine. A "2'-O-methyl modified nucleotide" refers to a nucleotide in which the 2'-hydroxyl group at the ribose group is replaced by a methoxy group.

[0096] A "phosphorothioate-containing nucleotide" refers to a nucleotide in which one or more oxygen atoms on the phosphate group of the nucleotide are replaced by a sulfur atom. A "phosphorothioate internucleotide linkage modification" refers to a modification in which two adjacent nucleotides are linked by a phosphorothioate.

[0097] In some embodiments, the sense strand of the dsRNA (particularly siRNA) of the present disclosure has a modification of one or two phosphorothioate internucleotide linkages located at positions 1 to 5 (counting from the 5' end) and / or has a modification of one or two phosphorothioate internucleotide linkages located at positions 1 to 5 (counting from the 3' end), and / or the antisense strand of the dsRNA (particularly siRNA) of the present disclosure has a modification of one or two phosphorothioate internucleotide linkages located at positions 1 to 5 (counting from the 5' end) and / or has a modification of one or two phosphorothioate internucleotide linkages located at positions 1 to 5 (counting from the 3' end).

[0098] In some embodiments, the sense strand of the dsRNA (particularly siRNA) of the present disclosure has a modification of one or two phosphorothioate internucleotide linkages at positions 1 to 4 (counting from the 5' end) and / or has a modification of one or two phosphorothioate internucleotide linkages at positions 1 to 4 (counting from the 3' end), and / or the antisense strand of the dsRNA (particularly siRNA) of the present disclosure has a modification of one or two phosphorothioate internucleotide linkages at positions 1 to 4 (counting from the 5' end) and / or has a modification of one or two phosphorothioate internucleotide linkages at positions 1 to 4 (counting from the 3' end).

[0099] In some embodiments, the sense strand of the dsRNA (particularly siRNA) of the present disclosure has a modification of one or two phosphorothioate internucleotide linkages at positions 1 to 3 (counting from the 5' end) and / or has a modification of one or two phosphorothioate internucleotide linkages at positions 1 to 3 (counting from the 3' end), and / or the antisense strand of the dsRNA (particularly siRNA) of the present disclosure has a modification of one or two phosphorothioate internucleotide linkages at positions 1 to 3 (counting from the 5' end) and / or has a modification of one or two phosphorothioate internucleotide linkages at positions 1 to 3 (counting from the 3' end).

[0100] In some embodiments, the sense strand of the dsRNA (particularly siRNA) of the present disclosure has a modification of one or two phosphorothioate internucleotide linkages at positions 1 and 2 (counting from the 5' end) and / or has a modification of one or two phosphorothioate internucleotide linkages at positions 1 and 2 (counting from the 3' end), and / or the antisense strand of the dsRNA (particularly siRNA) of the present disclosure has a modification of one or two phosphorothioate internucleotide linkages at positions 1 to 2 (counting from the 5' end) and / or has a modification of one or two phosphorothioate internucleotide linkages at positions 1 to 2 (counting from the 3' end).

[0101] As used herein, the term "inhibit" is used interchangeably with "reduce," "silence," "downregulate," and other similar terms and includes any level of inhibition.

[0102] The phrase "inhibiting the expression of an ATXN2 gene" refers to inhibiting the expression of any ATXN2 gene, as well as variants or mutants of an ATXN2 gene. Thus, the ATXN2 gene can be a wild-type ATXN2 gene, a mutant ATXN2 gene, or a transgenic ATXN2 gene in the context of a genetically manipulated cell, cell group, or organism.

[0103] "Inhibiting the expression of the ATXN2 gene" includes any level of inhibition of the ATXN2 gene, such as at least partial inhibition of ATXN2 gene expression. ATXN2 gene expression can be assessed based on the level or change in the level of any variable associated with ATXN2 gene expression, such as the level of mRNA encoding apolipoprotein (a) or the level of apolipoprotein (a) protein. This level can be assessed in a single cell or in a group of cells (including, for example, a sample derived from a subject).

[0104] Inhibition can be assessed by a decrease in the absolute or relative level of one or more variables associated with ATXN2 gene expression compared to a control level. The control level can be any type of control level utilized in the art, such as a pre-dose baseline level or a level determined from a similar untreated or control (e.g., a buffer-only control or an inert agent control) treated subject, cell, or sample.

[0105] As used herein, the terms "treat", "treat", and the like refer to the administration of an agent or the performance of a procedure in order to obtain an effect. These effects may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or may be therapeutic in terms of affecting a partial or complete cure of a disease and / or disease symptoms. As used herein, "treatment" may include treating a disease or condition in a mammal, particularly a human, and includes: (a) preventing the occurrence of the disease or disease symptoms in a subject susceptible to the disease but not yet diagnosed as having the disease (e.g., including diseases that may be associated with or caused by the primary disease); (b) inhibiting the disease, i.e., preventing its development; (c) alleviating the disease, i.e., causing regression of the disease. Treatment may refer to any reference to success in treating, ameliorating, or preventing cancer, including any objective or subjective parameter, such as elimination; alleviation; reducing symptoms or making the disease condition more tolerable for the patient; slowing the rate of deterioration or decline; or reducing the end point of deterioration. Treatment or improvement of symptoms is based on one or more objective or subjective parameters; including the results of a doctor's examination. Thus, the term "treatment" includes administering the dsRNA, cells, or pharmaceutical compositions disclosed herein to prevent, delay, alleviate, arrest, or inhibit the development of symptoms or conditions associated with a disease. The term "therapeutic effect" refers to the reduction, elimination, or prevention of a disease, disease symptom, or disease side effect in a subject.

[0106] The term "effective amount" as used herein refers to an amount that, when administered to a subject for treating a disease, is sufficient to effect such treatment.

[0107] As used herein, the term "subject" refers to any mammalian subject for whom diagnosis, treatment, or therapy is desired. "Mammal" for therapeutic purposes refers to any animal classified as a mammal, including humans, livestock, and laboratory, zoo, sport, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, and the like.

[0108] I.dsRNA

[0109] The present invention provides a double-stranded RNA (dsRNA) for inhibiting the expression of the ATXN2 gene, wherein the dsRNA comprises a sense strand and an antisense strand forming a double-stranded region, wherein the length of the sense strand and the antisense strand are each independently 15-30 nucleotides, and the antisense strand comprises a nucleotide sequence of at least 15 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 25-48.

[0110] In some embodiments, the double-stranded region formed by the sense strand and the antisense strand is fully complementary. In other embodiments, the double-stranded region formed by the sense strand and the antisense strand is substantially complementary and may comprise 1, 2, 3, 4 or 5 non-complementary sites.

[0111] In some specific embodiments, the sense strand comprises a nucleotide sequence of at least 15 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 1-24.

[0112] In some embodiments, the sense strand and the antisense strand are each independently 15-27 nucleotides in length, preferably 18-25 nucleotides, and more preferably 19-21 nucleotides in length.

[0113] In some embodiments, the length of the double-stranded region is 15-25 nucleotide pairs, preferably 16-23 nucleotide pairs, and more preferably 18-20 nucleotide pairs.

[0114] In some embodiments, the dsRNA of the present invention is siRNA.In other embodiments, a hairpin loop is formed between the sense strand and the antisense strand of the dsRNA of the present invention.

[0115] One or both of the sense strand and the antisense strand comprise a 3' overhang and / or a 5' overhang having at least 1 nucleotide. In some embodiments, one or both of the sense strand and the antisense strand comprise a 3' overhang and / or a 5' overhang having at least 1 nucleotide. In some specific embodiments, one or both of the sense strand and the antisense strand comprise a 3' overhang and / or a 5' overhang having 1 nucleotide. In some specific embodiments, one or both of the sense strand and the antisense strand comprise a 3' overhang and / or a 5' overhang having 2 nucleotides. In some specific embodiments, one or both of the sense strand and the antisense strand comprise a 3' overhang and / or a 5' overhang having 3 nucleotides. In some specific embodiments, one or both of the sense strand and the antisense strand comprise a 3' overhang and / or a 5' overhang having 4 nucleotides.

[0116] In some specific embodiments, the antisense strand comprises a 3' overhang and / or a 5' overhang of 1 nucleotide. In some embodiments, the antisense strand comprises a 3' overhang and / or a 5' overhang of 2 nucleotides. In some embodiments, the antisense strand comprises a 3' overhang and / or a 5' overhang of 3 nucleotides. In some embodiments, the antisense strand comprises a 3' overhang and / or a 5' overhang of 4 nucleotides. In some preferred embodiments, the antisense strand has a 3' overhang and / or a 5' overhang of at least 2 nucleotides.

[0117] Preferably, the antisense strand comprises a 3' overhang and / or a 5' overhang of 2 nucleotides.

[0118] In some embodiments, the sense strand and the antisense strand are the same length.

[0119] In some embodiments, the full length of the sense strand is complementary to the full length of the antisense strand to form a double strand, ie, has blunt ends.

[0120] In other embodiments, the sense strand and the antisense strand are the same length, and a portion of the sense strand is complementary to a portion of the antisense strand, i.e., both the sense strand and the antisense strand have a 5' overhang. In some embodiments, the sense strand and the antisense strand are different lengths. In a preferred embodiment, the 5' end of the antisense strand has an overhang of at least 1 nucleotide, more preferably an overhang of 2 or 3 nucleotides.

[0121] The dsRNA of the present invention is included in a dsRNA having a nucleotide overhang (that is, a reagent with an overhang and a blunt end) at one end or at both ends. For example, the 5'-end of the sense strand of the dsRNA comprises an overhang with one or more nucleotides and the 3'-end of the sense strand comprises an overhang with one or more nucleotides. For example, the 5'-end of the antisense strand of the dsRNA comprises an overhang with one or more nucleotides and the 3'-end of the antisense strand comprises an overhang with one or more nucleotides. For example, the 5'-end of the sense strand of the dsRNA comprises an overhang with one or more nucleotides and the 5'-end of the antisense strand comprises an overhang with one or more nucleotides. For example, the 3'-end of the sense strand of the dsRNA comprises an overhang with one or more nucleotides and the 3'-end of the antisense strand comprises an overhang with one or more nucleotides. For example, the 5'-end of the sense strand of the dsRNA comprises an overhang with one or more nucleotides and the 3'-end of the antisense strand comprises an overhang with one or more nucleotides. For example, the 5'-end of the sense strand of the dsRNA comprises an overhang with one or more nucleotides and the 3'-end of the antisense strand comprises an overhang with one or more nucleotides. For example, the 3'-end of the sense strand of a dsRNA comprises an overhang having one or more nucleotides and the 5'-end of the sense strand comprises a blunt end. For example, the 5'-end of the antisense strand of a dsRNA comprises an overhang having one or more nucleotides and the 3'-end of the antisense strand comprises a blunt end. For example, the 3'-end of the antisense strand of a dsRNA comprises an overhang having one or more nucleotides and the 5'-end of the antisense strand comprises a blunt end.

[0122] In some preferred embodiments, the 3'-end of the antisense strand of the dsRNA of the invention comprises an overhang of one or more nucleotides and the 5'-end of the antisense strand comprises a blunt end. In some more preferred embodiments, the 3'-end of the antisense strand of the dsRNA of the invention comprises an overhang of 1, 2, 3, or 4 nucleotides and the 5'-end of the antisense strand comprises a blunt end. In some more preferred embodiments, the 3'-end of the antisense strand of the dsRNA of the invention comprises an overhang of 2 nucleotides and the 5'-end of the antisense strand comprises a blunt end.

[0123] In some embodiments, the antisense strand comprises a nucleotide sequence of at least 16 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 25-48, a nucleotide sequence of at least 17 consecutive nucleotides, a nucleotide sequence of at least 18 consecutive nucleotides, a nucleotide sequence of at least 19 consecutive nucleotides, or a nucleotide sequence of at least 20 consecutive nucleotides. Preferably, the antisense strand comprises the nucleotide sequence shown in any one of SEQ ID NOs: 25-48.

[0124] In some embodiments, the sense strand comprises a nucleotide sequence of at least 16 consecutive nucleotides, a nucleotide sequence of at least 17 consecutive nucleotides, or a nucleotide sequence of at least 18 consecutive nucleotides of any one of the nucleotide sequences shown in SEQ ID NOs: 1-24. Preferably, the sense strand comprises the nucleotide sequence shown in any one of SEQ ID NOs: 1-24.

[0125] In some embodiments, the dsRNA comprises any pair of paired sense strand sequences and antisense strand sequences shown in Table 3.

[0126] II. Modification of Nucleotides

[0127] In some embodiments, substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides. In some embodiments, at least 80% of the nucleotides of the sense strand are modified nucleotides, and / or at least 80%, at least 85%, at least 90%, at least 92%, at least 95% of the nucleotides of the antisense strand are modified nucleotides. In some embodiments, at least 80% of the nucleotides of the antisense strand are modified nucleotides, and / or at least 80%, at least 85%, at least 90%, at least 92%, at least 95% of the nucleotides of the sense strand are modified nucleotides.

[0128] In some embodiments, all nucleotides of the sense strand are modified nucleotides and / or all nucleotides of the antisense strand are modified nucleotides.

[0129] The nucleotide modification described in the present invention may be a modification on the phosphate group, ribose group and / or base group of the nucleotide.

[0130] In some specific embodiments, the sense strand and the antisense strand each independently comprise one or more nucleotide modifications selected from the group consisting of 2'-O-alkyl modified nucleotides (e.g., 2'-O-methyl modified nucleotides), 2'-methoxyethyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, inosine ribonucleotides, abasic nucleotides, inverted abasic deoxyribonucleotides, nucleotides containing a thiophosphate group, vinylphosphonate modified nucleotides, locked nucleotides, unlocked nucleotides, 2'-amino-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-O-allyl-modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides containing unnatural bases, terminal nucleotides attached to a cholesteryl derivative or a dodecyldiamide group, deoxyribonucleotides, 3'-terminal deoxythymidine (dT) nucleotides, conformationally restricted nucleotides, restricted ethyl nucleotides, 2'-hydroxyl-modified nucleotides, nucleotides containing a methylphosphonic acid group, nucleotides containing a 5'-phosphate, nucleotides containing a 5'-phosphate mimetic, diol-modified nucleotides (GNA), and 2-O-(N-methylacetamide)-modified nucleotides.

[0131] In some preferred embodiments, the sense strand and the antisense strand each independently comprise one or more nucleotide modifications selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, and nucleotides comprising thiophosphate groups. In some preferred embodiments, the sense strand and / or the antisense strand comprise at least 2 2'-fluoro modified nucleotides. In some preferred embodiments, the sense strand and / or the antisense strand comprise at least 8 2'-O-methyl modified nucleotides. In some preferred embodiments, the 3' end and / or the 5' end of the sense strand and / or the antisense strand comprise 1-5 phosphorothioate internucleotide linkages, preferably 2-3 phosphorothioate internucleotide linkages.

[0132] In some preferred embodiments, the antisense strand comprises any one of the modified nucleotide sequences shown in Table 5, and / or the sense strand comprises any one of the modified nucleotide sequences shown in Table 4. In some preferred embodiments, the dsRNA comprises any one of the paired modified sense strand sequences and modified antisense strand sequences shown in any one of Table 6.

[0133] IV. Inhibition of ATXN2 gene expression

[0134] The dsRNA of the invention is capable of inhibiting ATXN2 gene expression by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0135] Inhibition of ATXN2 gene expression can be manifested by a decrease in the amount of mRNA expressed by a first cell or group of cells (such cells can be present, for example, in a sample derived from a subject) in which the ATXN2 gene is transcribed and which cell or cells have been treated (e.g., by contacting the cell or cells with a dsRNA of the invention, or by administering a dsRNA of the invention to a subject in which the cells are or were present) such that ATXN2 gene expression is inhibited compared to a second cell or group of cells (one or more control cells) that is substantially identical to the first cell or group of cells but has not been so treated.

[0136] In preferred embodiments, inhibition is assessed by expressing the level of mRNA in treated cells as a percentage of the level of mRNA in control cells using the formula: -△△Ct The difference between the experimental group and the control group was compared by ΔΔCt, where ΔΔCt = [(Ct target gene of experimental group - Ct internal reference of experimental group) - (Ct target gene of control group - Ct internal reference of control group)].

[0137] Control cells or cell groups that can be used to assess inhibition of ATXN2 gene expression include cells or cell groups that have not been contacted with the dsRNA of the invention. For example, the control cells or cell groups can be derived from an individual subject (e.g., a human or animal subject) before the subject is treated with the dsRNA.

[0138] V. Cells

[0139] The present invention provides cells containing the dsRNA of the present invention.

[0140] VI. Pharmaceutical Compositions

[0141] The present invention provides a pharmaceutical composition comprising the dsRNA or cells of the present invention, and optionally a pharmaceutically acceptable carrier or excipient.

[0142] As used herein, "pharmaceutically acceptable" refers to those compounds, materials, compositions, and / or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human and animal subjects without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0143] As used herein, a pharmaceutically acceptable carrier refers to a pharmaceutical carrier that facilitates administration of dsRNA or cells containing the same into the human body and / or facilitates its absorption or function. Examples include diluents and excipients such as water; fillers such as starch and sucrose; binders such as cellulose derivatives, alginates, gelatin, and polyvinyl pyrrolidone; humectants such as glycerol; disintegrants such as agar, calcium carbonate, and sodium bicarbonate; absorption enhancers such as quaternary ammonium compounds; surfactants such as cetyl alcohol; adsorption carriers such as kaolin and bentonite; and lubricants such as talc, calcium / magnesium stearate, and polyethylene glycol. Other adjuvants such as flavoring agents and sweeteners may also be added to the composition.

[0144] The pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable diluent or a sustained-release matrix, in which the dsRNA or cells of the present invention are embedded.

[0145] Pharmaceutical composition of the present invention can include the drug delivery system for sending dsRNA.Drug delivery system of the present invention includes but is not limited to nano-particle (such as lipid nano-particle, nano-particle based on polymer), polymer, PEG or cationic delivery system, polylactic acid (PLA) microsphere, poly(lactic-co-glycolic acid) (PLGA) microsphere, liposome, micelle, reverse micelle (inverse micelles), lipid helix (lipid cochleates), lipid microtubule (lipid microtubules), cholesterol, PEG lipid PEG-2000-C-DMG, PEG-2000-DMG (Moderna), ALC-0159 or DSPC.

[0146] In some embodiments, the dsRNA in the pharmaceutical compositions of the present invention may be contained within polymers and polymer-based nanoparticles.

[0147] In some specific embodiments, the polymer is a poly(lactic-co-glycolic acid) (PLGA)-based polymer. In some specific embodiments, the PLGA-based polymer is modified to contain a single cationic group.

[0148] In some specific embodiments, the polymer contains an amine group that can become cationic, such as polyethyleneimine (PEI) and poly (L-lysine) (PLL), which can form a complex with dsRNA through electrostatic interactions and deliver dsRNA into cells. In some embodiments, PEG and PLL are chemically modified to improve in vivo efficacy and tolerability.

[0149] In some embodiments, siRNA or cells in the pharmaceutical compositions of the present invention can be delivered via the cationic polymer poly(β-amino ester) (PBAE).

[0150] VII. Kit

[0151] The present invention provides a kit comprising the dsRNA or cells of the present invention.

[0152] The present invention also provides kits for using the dsRNA or cells described herein and / or performing the methods described herein. Such kits include one or more dsRNAs or cells described herein and may further include instructions for use. The instructions may include instructions for inhibiting ATXN2 gene expression in cells by contacting the cells with the dsRNA described herein in an amount effective to inhibit ATXN2 gene expression.

[0153] When the dsRNA of the present invention is contacted with cells in vitro, the kit of the present invention may optionally further include a means for contacting cells with the dsRNA of the present invention (e.g., an injection device) or a means for measuring the inhibitory effect of the ATXN2 gene (e.g., a device for measuring the inhibition of ATXN2 mRNA or protein). Such a device for measuring the inhibition of the ATXN2 gene may include a device for obtaining a sample from a subject.

[0154] When the dsRNA of the present invention or cells into which the dsRNA has been introduced in vitro are administered in vivo, the kit of the present invention may optionally further comprise a device for administering the dsRNA or cells of the present invention to a subject or a device for determining a therapeutically effective amount or a prophylactically effective amount.

[0155] VIII. Methods of treatment and pharmaceutical uses

[0156] The present invention provides methods for inhibiting ATXN2 gene expression in a cell, comprising contacting the cell with a dsRNA or pharmaceutical composition of the present invention. In some embodiments, the method is performed in vitro. In some embodiments, the method is performed in vitro. The present invention provides dsRNA or pharmaceutical compositions of the present invention for inhibiting ATXN2 gene expression in a cell.

[0157] The present invention provides a method for inhibiting ATXN2 gene expression in a cell in a subject, the method comprising administering to the subject a dsRNA, cell, or pharmaceutical composition of the present invention. The present invention provides a dsRNA, cell, or pharmaceutical composition of the present invention for inhibiting ATXN2 gene expression in a cell in a subject. The present invention provides use of a dsRNA, cell, or pharmaceutical composition of the present invention in the preparation of a medicament for inhibiting ATXN2 gene expression in a cell in a subject.

[0158] The present invention also provides a method for treating a disease or condition that would benefit from reduced ATXN2 gene expression in a subject, the method comprising administering to the subject a dsRNA, cell, or pharmaceutical composition of the present invention. The present invention also provides a dsRNA, cell, or pharmaceutical composition of the present invention for use in treating a disease or condition that would benefit from reduced ATXN2 gene expression in a subject. The present invention also provides use of a dsRNA, cell, or pharmaceutical composition of the present invention in the preparation of a medicament for treating a disease or condition that would benefit from reduced ATXN2 gene expression in a subject.

[0159] The present invention also provides a method for preventing at least one symptom in a subject suffering from a disease or condition that would benefit from reduced ATXN2 gene expression, the method comprising administering to the subject a dsRNA, cell, or pharmaceutical composition of the present invention. The present invention also provides a dsRNA, cell, or pharmaceutical composition of the present invention for use in preventing a subject suffering from a disease or condition that would benefit from reduced ATXN2 gene expression. The present invention also provides use of a dsRNA, cell, or pharmaceutical composition of the present invention in the preparation of a medicament for preventing a subject suffering from a disease or condition that would benefit from reduced ATXN2 gene expression.

[0160] The present invention also provides a method for preventing the progression of a disease or condition that benefits from reduced ATXN2 gene expression in a subject, the method comprising administering to the subject a dsRNA, cell, or pharmaceutical composition of the present invention. The present invention also provides a dsRNA, cell, or pharmaceutical composition of the present invention for preventing the progression of a disease or condition that benefits from reduced ATXN2 gene expression in a subject. The present invention also provides use of a dsRNA, cell, or pharmaceutical composition of the present invention in the preparation of a medicament for preventing the progression of a disease or condition that benefits from reduced ATXN2 gene expression in a subject.

[0161] In some embodiments, the disease or disorder that benefits from reduced ATXN2 gene expression is an Ataxin2-related disease. In some preferred embodiments, the Ataxin2-related disease is selected from the group consisting of progressive cerebellar ataxia, blindness, spinocerebellar ataxia (SCA), such as spinocerebellar ataxia 2 (SCA2), and amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Parkinson's disease.

[0162] In some embodiments, the dsRNA, cells, or pharmaceutical composition is administered intrathecally.

[0163] In some embodiments, the subject is a mammal. In some embodiments, the subject is a primate mammal. In some embodiments, the subject is a human.

[0164] sequence

[0165] The RNA sequence provided by the present invention targets the human ATXN2 gene (or target gene, target mRNA sequence, target sequence). The target ATXN2 mRNA sequence is the gene shown in Genbank accession number NM_002973.4.

[0166] Table 3. Nucleotide sequences of the sense and antisense strands targeting ATXN2 mRNA (where the sense and antisense strands in the same row are paired)

[0167] Table 4 and Table 5 respectively show the modified RNA sequences used in the present invention.

[0168] In this article, the meanings of the abbreviations are as follows:

[0169] A, U, G and C represent the natural ribonucleotides adenine, uracil, guanine and cytosine, respectively.

[0170] d indicates that the nucleotide adjacent to its right is a deoxyribonucleotide. For example, dA, dT, dG, and dC represent adenine deoxyribonucleotide, thymine deoxyribonucleotide, guanine deoxyribonucleotide, and cytosine deoxyribonucleotide, respectively.

[0171] i represents inosine ribonucleotide.

[0172] m indicates that the nucleotide adjacent to its left is a 2'-OCH3-modified nucleotide. For example, Am, Um, Gm, and Cm represent 2'-OCH3-modified A, U, G, and C, respectively.

[0173] f indicates that the nucleotide adjacent to its left is a 2'-fluoro-modified nucleotide. For example, Af, Uf, Gf, and Cf represent 2'-fluoro-modified A, U, G, and C, respectively.

[0174] "s" or s- indicates that the two adjacent nucleotides and / or delivery vectors are linked by phosphorothioate.

[0175] VP indicates that the adjacent nucleotide on its right side is a vinylphosphonate-modified nucleotide, which is well known in the art, and can be found in, for example, PCT Publication Nos. WO2011139702, WO2013033230, and WO2019105419.

[0176] IB represents an inverted abasic deoxyribonucleotide, which can include the following three structures depending on its location / connection method in the siRNA (used for the 5' end, middle and 3' end of the nucleic acid chain respectively):

[0177] IB is well known in the art, see, for example, F. Czauderna, Nucleic Acids Res., 2003, 31(11), 2705-16 and PCT Publication Nos. WO2016011123 and WO2019051402.

[0178] L96 represents a GalNAc delivery vector of the following structure well known in the art, wherein The position of the siRNA is indicated by a phosphate group or a phosphorothioate group, as described in, for example, PCT Publication Nos. WO2009073809 and WO2009082607.

[0179] Table 4. Sense strand sequences of modified siRNAs targeting ATXN2 mRNA

[0180] Table 5. Antisense strand sequences of modified siRNA targeting ATXN2 mRNA

[0181] Table 6. Paired siRNA sense and antisense strands targeting ATXN2 mRNA (adjacent sense and antisense strands in the same row are paired)

[0182] The present invention will be further described below with reference to the following examples. It should be understood that the following examples are merely illustrative and should not be considered as limiting the scope of the present invention. Example

[0183] Unless otherwise specified, the sources of materials used in the examples are as follows:

[0184] The human osteosarcoma cell line U2OS was purchased from Wuhan Punosai Life Science Technology Co., Ltd., catalog number CL-0236;

[0185] The human neuroblastoma cell line SK-N-BE (2) was purchased from Wuhan Punosai Life Science Technology Co., Ltd., catalog number CL-0441;

[0186] Mouse brain neuroma cell line Neuro-2a was purchased from Wuhan Punosai Life Science Technology Co., Ltd., catalog number CL-0168;

[0187] Monkey primary hepatocytes PCH were purchased from Miaoshun (Shanghai) Biotechnology Co., Ltd.

[0188] Example 1 Preparation of siRNA

[0189] The siRNA of the present invention is prepared using the solid phase phosphoramidite method well known in the art. Specific methods can be found in, for example, PCT Publication Nos. WO2016081444 and WO2019105419, and are briefly described below.

[0190] 1.1 Synthesis of the positive sense chain (SS chain)

[0191] Using solid-phase phosphoramidite synthesis, a blank CPG solid support is used as the starting cycle, and nucleoside monomers are linked one by one in the 3'-5' direction according to the order of the positive strand nucleotides. Each linking step involves four steps: deprotection, coupling, capping, and oxidation or thiolation. The synthesis conditions for a 5μmol oligonucleotide are as follows:

[0192] Commercially available phosphoramidites with 2'-F, 2'-O-methyl, and other modifications were used. Nucleoside monomers were provided in 0.05 mol / L acetonitrile. The reaction conditions for each step were identical: 25°C, three deprotection cycles with 3% trichloroacetic acid in dichloromethane, two coupling reactions with 0.25 mol / L ETT in acetonitrile as the activator, and two capping reactions with 10% acetic anhydride in acetonitrile and pyridine / N-methylimidazole / acetonitrile (10:14:76, v / v / v). Oxidation reactions were performed twice with 0.05 mol / L iodine / tetrahydrofuran / pyridine / water (70 / 20 / 10, v / v / v), and two thiolation reactions with 0.2 mol / L PADS in acetonitrile / 3-methylpyridine (1 / 1, v / v).

[0193] 1.2 Synthesis of antisense strand (AS strand)

[0194] Using solid-phase phosphoramidite synthesis, a blank CPG solid support is used as the starting cycle. Nucleoside monomers are linked one by one in the 3'-5' direction according to the nucleotide arrangement of the antisense strand. Each ligation step involves four steps: deprotection, coupling, capping, and oxidation or thiolation. The synthesis conditions for a 5 μmol oligonucleotide of the antisense strand are the same as those for the sense strand.

[0195] 1.3 Oligonucleotide purification and annealing

[0196] 1.3.1 Ammonolysis

[0197] The synthesized solid phase carrier (sense chain or antisense chain) was added to a 5 mL centrifuge tube, 3% diethylamine / ammonia water (v / v) was added, and the reaction was carried out in a constant temperature water bath at 35°C for 16 hours (or in a constant temperature water bath at 55°C for 8 hours). After filtering, the solid phase carrier was washed three times with ethanol / water, each time with 1 mL. The filtrate was centrifuged and concentrated, and the crude product was purified.

[0198] 1.3.2 Purification

[0199] Purification and desalination methods are well known in the art. For example, a column packed with a strong anionic filler can be used, and elution and purification can be performed using a sodium chloride-sodium hydroxide system, with the product collected and stored in a tube. Desalination can also be performed using a gel-filled purification column, with pure water as the elution system.

[0200] 1.3.3 Annealing

[0201] According to Table 6, the sense strand (SS strand) and the antisense strand (AS strand) were mixed at a molar ratio (SS strand / AS strand = 1 / 1.05), heated in a water bath to 70-95° C. for 3-5 min, cooled naturally to room temperature, and the system was lyophilized to obtain the product.

[0202] Example 2 Activity screening of U2OS cell line

[0203] Cell transfection

[0204] On the first day, U2OS human osteosarcoma cell line (purchased from Wuhan Punosai Life Science Technology Co., Ltd., catalog number CL-0236) was digested, resuspended, and counted. The cell resuspension was plated into a 96-well plate at 100 μL / well, and 1×10 4 cells / well, and transfection was performed 18 h later.

[0205] On the second day, 20 μM siRNA stock solution was diluted with Opti-MEM. 198 μL Opti-MEM was added to 2 μL siRNA stock solution. The final siRNA concentration was as shown below. Pipet and mix thoroughly. Set aside.

[0206] On the next day, take 14.1 μL Opti-MEM and dilute 0.9 μL Lipofectamine TM Gently pipette RNAiMAX (Thermo, 13778150) to mix thoroughly, and let it stand at room temperature for 5 minutes. Then, take 15 μL of the prepared RNAi-MAX mixture and 15 μL of the diluted siRNA and gently pipette to mix thoroughly, avoiding the introduction of bubbles. Let it stand at room temperature for 10 minutes, then add 10 μL / well to a 96-well plate. Incubate at 37°C, 5% CO2 for 24 hours (no siRNA was added to the control group).

[0207] RNA extraction

[0208] Cell RNA was extracted using a nucleic acid extractor (Auto-pure96, Hangzhou Aosheng) according to the operating instructions of the high-throughput cell RNA extraction kit (Fanzhi Medical, FG0417-L).

[0209] RNA reverse transcription

[0210] Denaturation reaction mixture preparation reference PrimeScript TM The single-well preparation volume of II 1st Strand cDNA Synthesis Kit (Takara, 6210B) is as follows: Oligo dT Primer 1 μL, dNTP Mixture 1 μL, template RNA 12.5 μL, incubate at 65°C in a conventional PCR instrument for 5 minutes, and then quickly cool on ice for 2 minutes.

[0211] Reverse transcription reaction solution preparation reference PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, 6210B). Each well contains 4 μL of 5× PrimeScript II Buffer, 0.5 μL of RNase Inhibitor, and 1 μL of PrimeScript II RTase.

[0212] Slowly mix 14.5 μL of the denatured reaction solution with the reverse transcription reaction solution, incubate at 42°C for 45 minutes for reverse transcription using a conventional PCR instrument, incubate at 95°C for 5 minutes to inactivate the enzyme, and cool the reverse transcription product (cDNA) at 4°C.

[0213] After inversion, 30 μL of DNase- and RNase-free distilled water was added to the cDNA sample in each well.

[0214] Fluorescence quantitative PCR

[0215] ReferenceTaqMan TMQuantitative PCR (ABI, QuantStudio3) was performed using 20 μL of Fast Advanced Master Mix (ABI, 4444965). The reaction schedule was: (50°C, 2 minutes) × 1 cycle; (95°C, 20 seconds) × 1 cycle; (95°C, 1 second; 60°C, 24 seconds) × 40 cycles.

[0216] Table 7. Primer information

[0217] Statistics

[0218] Calculation 2 -△△Ct The values ​​were converted into percentages to obtain the residual inhibition rate;

[0219] △△Ct=[(target gene of Ct experimental group-internal reference of Ct experimental group)-(target gene of Ct control group-internal reference of Ct control group)].

[0220] The target gene was hATXN2, and the internal reference was hGAPDH.

[0221] The final concentration of siRNA was 10 nM for siRNA compound cell line activity screening. The experimental screening results are shown in Table 8.

[0222] Table 8. U2OS human osteosarcoma cell line activity screening results

[0223] Example 3 Activity screening of SK-N-BE (2) human neuroblastoma cell line

[0224] Cell transfection

[0225] On the first day, SK-N-BE (2) human neuroblastoma cell line (purchased from Wuhan Punosai Life Science Technology Co., Ltd., catalog number CL-0441) was digested, resuspended, and counted. The cell resuspension was plated into a 96-well plate at 100 μL / well, and 1×10 4 cells / well, and transfection was performed 18 h later.

[0226] On the second day, 20 μM siRNA stock solution was diluted with Opti-MEM. 198 μL Opti-MEM was added to 2 μL siRNA stock solution. The final siRNA concentration was as shown below. Pipet and mix thoroughly. Set aside.

[0227] On the next day, take 14.1 μL Opti-MEM and dilute 0.9 μL Lipofectamine TMGently pipette RNAiMAX (Thermo, 13778150) to mix thoroughly, and let it stand at room temperature for 5 minutes. Then, take 15 μL of the prepared RNAi-MAX mixture and 15 μL of the diluted siRNA and gently pipette to mix thoroughly, avoiding the introduction of bubbles. Let it stand at room temperature for 10 minutes, then add 10 μL / well to a 96-well plate. Incubate at 37°C, 5% CO2 for 24 hours (no siRNA was added to the control group).

[0228] RNA extraction

[0229] Cell RNA was extracted using a nucleic acid extractor (Auto-pure96, Hangzhou Aosheng) according to the operating instructions of the high-throughput cell RNA extraction kit (Fanzhi Medical, FG0417-L).

[0230] RNA reverse transcription

[0231] Denaturation reaction mixture preparation reference PrimeScript TM The single-well preparation volume of II 1st Strand cDNA Synthesis Kit (Takara, 6210B) is as follows: Oligo dT Primer 1 μL, dNTP Mixture 1 μL, template RNA 12.5 μL, incubate at 65°C in a conventional PCR instrument for 5 minutes, and then quickly cool on ice for 2 minutes.

[0232] Reverse transcription reaction solution preparation reference PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, 6210B). Each well contains 4 μL of 5× PrimeScript II Buffer, 0.5 μL of RNase Inhibitor, and 1 μL of PrimeScript II RTase.

[0233] Slowly mix 14.5 μL of the denatured reaction solution with the reverse transcription reaction solution, incubate at 42°C for 45 minutes for reverse transcription using a conventional PCR instrument, incubate at 95°C for 5 minutes to inactivate the enzyme, and cool the reverse transcription product (cDNA) at 4°C.

[0234] After inversion, 30 μL of DNase- and RNase-free distilled water was added to the cDNA sample in each well.

[0235] Fluorescence quantitative PCR

[0236] ReferenceTaqMan TMUsing the Fast Advanced Master Mix (ABI, 4444965), a 20 μL fluorescence quantitative PCR reaction (ABI, QuantStudio3) was performed. The reaction schedule was: (50°C, 2 minutes) × 1 cycle; (95°C, 20 seconds) × 1 cycle; (95°C, 1 second; 60°C, 24 seconds) × 40 cycles. The primers used were the same as those in Table 7 of Example 2.

[0237] Statistics

[0238] Calculation 2 -△△Ct The values ​​were converted into percentages to obtain the residual inhibition rate;

[0239] △△Ct=[(target gene of Ct experimental group-internal reference of Ct experimental group)-(target gene of Ct control group-internal reference of Ct control group)].

[0240] The final concentration of siRNA was 10 nM or 0.1 nM for siRNA compound cell line activity screening. The experimental screening results are shown in Table 9.

[0241] Table 9. Activity screening results of SK-N-BE (2) human neuroblastoma cell line

[0242] Example 4 Neuro-2a cell line activity screening

[0243] Cell transfection

[0244] On the first day, Neuro-2a mouse brain neuroma cell line (purchased from Wuhan Punosai Life Science Technology Co., Ltd., catalog number CL-0168) was digested, resuspended, and counted. The cell resuspension was plated into a 96-well plate at 100 μL / well, and 1×10 4 cells / well, and transfection was performed 18 h later.

[0245] On the second day, 20 μM siRNA stock solution was diluted with Opti-MEM. 198 μL Opti-MEM was added to 2 μL siRNA stock solution. The final siRNA concentration was as shown below. Pipet and mix thoroughly. Set aside.

[0246] On the next day, take 14.1 μL Opti-MEM and dilute 0.9 μL Lipofectamine TMGently pipette RNAiMAX (Thermo, 13778150) to mix thoroughly, and let it stand at room temperature for 5 minutes. Then, take 15 μL of the prepared RNAi-MAX mixture and 15 μL of the diluted siRNA and gently pipette to mix thoroughly, avoiding the introduction of bubbles. Let it stand at room temperature for 10 minutes, then add 10 μL / well to a 96-well plate. Incubate at 37°C, 5% CO2 for 24 hours (no siRNA was added to the control group).

[0247] RNA extraction

[0248] Cell RNA was extracted using a nucleic acid extractor (Auto-pure96, Hangzhou Aosheng) according to the operating instructions of the high-throughput cell RNA extraction kit (Fanzhi Medical, FG0417-L).

[0249] RNA reverse transcription

[0250] Denaturation reaction mixture preparation reference PrimeScript TM The single-well preparation volume of II 1st Strand cDNA Synthesis Kit (Takara, 6210B) is as follows: Oligo dT Primer 1 μL, dNTP Mixture 1 μL, template RNA 12.5 μL, incubate at 65°C in a conventional PCR instrument for 5 minutes, and then quickly cool on ice for 2 minutes.

[0251] Reverse transcription reaction solution preparation reference PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, 6210B). Each well contains 4 μL of 5× PrimeScript II Buffer, 0.5 μL of RNase Inhibitor, and 1 μL of PrimeScript II RTase.

[0252] Slowly mix 14.5 μL of the denatured reaction solution with the reverse transcription reaction solution, incubate at 42°C for 45 minutes for reverse transcription using a conventional PCR instrument, incubate at 95°C for 5 minutes to inactivate the enzyme, and cool the reverse transcription product (cDNA) at 4°C.

[0253] After inversion, 30 μL of DNase- and RNase-free distilled water was added to the cDNA sample in each well.

[0254] Fluorescence quantitative PCR

[0255] ReferenceTaqMan TMQuantitative PCR (ABI, QuantStudio3) was performed using 20 μL of Fast Advanced Master Mix (ABI, 4444965). The reaction schedule was: (50°C, 2 minutes) × 1 cycle; (95°C, 20 seconds) × 1 cycle; (95°C, 1 second; 60°C, 24 seconds) × 40 cycles.

[0256] Table 10. Primer information

[0257] Statistics

[0258] Calculation 2 -△△Ct The values ​​were converted into percentages to obtain the residual inhibition rate;

[0259] △△Ct=[(target gene of Ct experimental group-internal reference of Ct experimental group)-(target gene of Ct control group-internal reference of Ct control group)].

[0260] The target gene was mATXN2, and the internal reference was mGAPDH.

[0261] The final concentration of siRNA was 10 nM for siRNA compound cell line activity screening. The experimental screening results are shown in Table 11.

[0262] Table 11. Activity screening results of Neuro-2a mouse brain neuroma cell line

[0263] Example 5 Primary rat hepatocyte (PRH) activity screening

[0264] Isolation of primary rat hepatocytes

[0265] Rat primary hepatic cells were isolated and cultured according to the method in the literature (Isolation and Primary Culture of Rat Hepatic Cells. J Vis Exp. 2012; (64): 3917.) Rat primary stem cells were counted and plated into 96-well cell plates at 90 μL / well, 1×10 4 cells / well.

[0266] Cell transfection

[0267] Compound dilution: Dilute 20 μM siRNA stock solution with Opti-MEM. Take 198 μL Opti-MEM and add 2 μL siRNA stock solution. Mix by pipetting. This is used as the first concentration point. Perform corresponding dilution operations according to the concentrations used in Table 14.

[0268] Transfection process: dilute 14.1 μL Opti-MEM with 0.9 μL Lipofectamine TM Gently pipette RNAiMAX (Thermo, 13778150) to mix thoroughly. Let stand at room temperature for 5 minutes. Then, take 15 μL of the prepared RNAi-MAX mixture and 15 μL of the diluted siRNA and gently pipette to mix thoroughly, avoiding the introduction of bubbles. Let stand at room temperature for 10 minutes. Add the mixture to a 96-well plate at a rate of 10 μL per well. Incubate overnight in a 37°C, 5% CO2 incubator. A control group was not treated with siRNA.

[0269] On the second day, RNA was extracted using the same method as in Example 2 and fluorescence quantitative PCR was performed using the primers in Table 12. The RNA extraction and fluorescence quantitative PCR methods were the same as in Example 2.

[0270] Table 12: Primer information:

[0271] Statistics

[0272] Calculation 2 -△△Ct The values ​​were converted into percentages to obtain the residual inhibition rate;

[0273] △△Ct=[(target gene of Ct experimental group-internal reference of Ct experimental group)-(target gene of Ct control group-internal reference of Ct control group)].

[0274] The target gene was rATXN2, and the internal reference was rGAPDH.

[0275] The starting concentration of siRNA was 10 nM, and the activity of 10 siRNA compounds on rat primary hepatocytes was tested at 11 concentration points with 3-fold dilution. The experimental results are shown in Table 13.

[0276] Table 13. IC50 results of 11 concentrations of siRNA in rat primary hepatocytes

[0277] Example 6 Activity screening of primary cynomolgus monkey hepatocytes (PCH)

[0278] Cell transfection-reverse transfection

[0279] 100 μL of coating medium was added to each well of a 96-well cell culture plate and coated at 37° C. for 30 minutes. After the coating was completed, the coating medium was aspirated.

[0280] Monkey primary hepatocytes (PCH, from Miaoshun (Shanghai) Biotechnology Co., Ltd.) were revived at 37°C, resuspended in thawing medium, and counted after centrifugation. PCH were plated in 96-well plates at 90 μL / well and 2×10 4 cells / well.

[0281] siRNA dilution: Take 198 μL of Opti-MEM and add 2 μL of 20 μM siRNA stock solution. Mix by pipetting as the first concentration point. Perform corresponding dilution operations according to the final siRNA concentration in Table 15 (no siRNA was added to the control group).

[0282] Transfection procedure: Dilute 0.9 μL RNAiMAX (Thermo, 13778150) in 14.1 μL Opti-MEM. Gently pipette to mix thoroughly, and let stand at room temperature for 5 minutes. Then, take 15 μL of the prepared RNAi-MAX mixture and 15 μL of the diluted siRNA. Gently pipette to mix thoroughly, let stand at room temperature for 10 minutes, and then add 10 μL / well to a 96-well plate. Incubate in a 37°C, 5% CO2 incubator for 4 hours, then replace the culture medium. RNA will be extracted the next day.

[0283] RNA extraction

[0284] Cell RNA was extracted using a nucleic acid extractor (Hangzhou Aosheng, Auto-pure96) according to the operating procedures of a high-throughput cell RNA extraction kit (Fanzhi Medical, FG0412).

[0285] RNA reverse transcription

[0286] Refer to PrimeScript for the preparation of denaturation reaction mixture TM II 1st Strand cDNA Synthesis Kit (Takara, 6210B). Each well contains 1 μL of Oligo dT Primer, 1 μL of dNTP Mixture, and 12.5 μL of template RNA. Incubate at 65°C for 5 minutes in a conventional PCR instrument to perform the denaturation reaction. Rapidly cool the mixture on ice for 2 minutes.

[0287] The preparation of reverse transcription reaction solution refers to PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, 6210B). Each well contains 4 μL of 5× PrimeScript II Buffer, 0.5 μL of RNase Inhibitor, and 1 μL of PrimeScript II RTase.

[0288] 14.5 μL of the denatured reaction solution was slowly mixed with 5.5 μL of the reverse transcription reaction solution, and the mixture was incubated at 42°C for 45 minutes in a conventional PCR instrument for reverse transcription. The enzyme was inactivated by incubation at 95°C for 5 minutes, and the reverse transcription product (cDNA) was cooled at 4°C.

[0289] After inversion, add 30 μL of DNase- and RNase-free distilled water to the cDNA sample in each well.

[0290] Fluorescence quantitative PCR

[0291] ReferenceTaqMan TM Quantitative PCR (ABI, QuantStudio3) was performed using 20 μL of Fast Advanced Master Mix (ABI, 4444965). The reaction schedule was: (50°C, 2 minutes) × 1 cycle; (95°C, 20 seconds) × 1 cycle; (95°C, 1 second; 60°C, 24 seconds) × 40 cycles.

[0292] Table 14. Primer information

[0293] The activity of 21 siRNA compounds in cynomolgus monkey primary hepatocytes was tested at a starting concentration of 10 nM and 10-fold dilutions at five concentration points. The experimental results are shown in Table 15. The percentage values ​​in Table 15 represent the residual inhibition rate, which was calculated in the same way as in Example 5. The target gene was mkATXN2, and the internal reference was mkGAPDH.

[0294] Table 15. IC50 results of siRNA activity screening at five concentrations on primary cynomolgus monkey hepatocytes

Claims

1. A double-stranded RNA (dsRNA) for inhibiting the expression of the ATXN2 gene, the dsRNA comprising a sense strand and an antisense strand forming a double-stranded region, wherein the lengths of the sense strand and the antisense strand are each independently 15-30 nucleotides, and the antisense strand comprises a nucleotide sequence of at least 15 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 25-48.

2. The dsRNA of claim 1, wherein the sense strand comprises a nucleotide sequence of at least 15 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 1-24.

3. The dsRNA of claim 1 or 2, wherein the dsRNA is siRNA.

4. The dsRNA according to any one of claims 1 to 3, wherein the length of the sense strand and the antisense strand are each independently 15-27 nucleotides, preferably 18-25 nucleotides, more preferably 19-21 nucleotides.

5. The dsRNA of any one of claims 1 to 4, wherein the antisense strand comprises a nucleotide sequence of at least 16 consecutive nucleotides, a nucleotide sequence of at least 17 consecutive nucleotides, a nucleotide sequence of at least 18 consecutive nucleotides, a nucleotide sequence of at least 19 consecutive nucleotides, or a nucleotide sequence of at least 20 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 25 to 48, preferably the antisense strand comprises the nucleotide sequence shown in any one of SEQ ID NOs: 25 to 48.

6. The dsRNA of any one of claims 1 to 5, wherein the sense strand comprises a nucleotide sequence of at least 16 consecutive nucleotides, a nucleotide sequence of at least 17 consecutive nucleotides, or a nucleotide sequence of at least 18 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 1 to 24, preferably the sense strand comprises the nucleotide sequence shown in any one of SEQ ID NOs: 1 to 24.

7. The dsRNA according to any one of claims 1 to 6, wherein the siRNA comprises any pair of paired sense strand sequence and antisense strand sequence as shown in Table 3 of the specification.

8. The dsRNA of any one of claims 1 to 7, wherein substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, or all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides.

9. The dsRNA of claim 8, wherein the sense strand and the antisense strand each independently comprise one or more modified nucleotides selected from the group consisting of 2'-O-alkyl modified nucleotides (e.g., 2'-O-methyl modified nucleotides), 2'-methoxyethyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, inosine ribonucleotides, abasic nucleotides, inverted abasic deoxyribonucleotides, nucleotides containing a phosphorothioate group, vinylphosphonate modified nucleotides, locked nucleotides, unlocked nucleotides, 2'-amino-modified nucleotides, 2'-C-alkyl- Modified nucleotides, 2'-O-allyl modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides containing non-natural bases, terminal nucleotides attached to a cholesterol derivative or a dodecanoic acid didecylamide group, deoxyribonucleotides, 3'-terminal deoxythymine (dT) nucleotides, conformationally restricted nucleotides, restricted ethyl nucleotides, 2'-hydroxyl modified nucleotides, nucleotides containing a methylphosphonic acid group, nucleotides containing a 5'-phosphate, nucleotides containing a 5'-phosphate mimetic, diol-modified nucleotides (GNA), and 2-O-(N-methylacetamide) modified nucleotides.

10. The dsRNA of claim 9, wherein the sense strand and the antisense strand each independently comprise one or more nucleotide modifications selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, and nucleotides comprising a phosphorothioate group.

11. The dsRNA of any one of claims 8 to 10, wherein the 3' end and / or the 5' end of the sense strand and / or the antisense strand comprises 1 to 5 phosphorothioate internucleotide linkages, preferably 2 to 4 phosphorothioate internucleotide linkages.

12. The dsRNA of any one of claims 1-11, wherein the antisense strand has a length of 21 nucleotides and has (i) (counting from the 5' end) 2'-O-methyl modified nucleotides at positions 1, 3 to 5, 7 to 13, 15, 17 to 21, and 2'-fluoro modified nucleotides at positions 2, 6, 14 and 16; and / or (ii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21 (counting from the 5' end).

13. The dsRNA of any one of claims 1-12, wherein the sense strand has a length of 19 nucleotides and has: (i) (counting from the 5' end) 2'-O-methyl modified nucleotides at positions 1 to 6, 10 to 19, and 2'-fluoro modified nucleotides at positions 7-9; and / or (ii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 17 and 18, and between nucleotide positions 18 and 19 (counting from the 5′ end).

14. The dsRNA of claim 8, wherein the antisense strand comprises any one of the modified nucleotide sequences shown in Table 5 of the specification, and / or the sense strand comprises any one of the modified nucleotide sequences shown in Table 4 of the specification.

15. The dsRNA of claim 8, wherein the dsRNA comprises any pair of the paired modified sense strand sequence and modified antisense strand sequence shown in Table 6 of the specification.

16. The dsRNA of claim 8, wherein: (1) the sense strand comprises UmUmGmUmUmGmUfGfGfUmAmCmAmGmUmUmUmAmAm, and the antisense strand comprises UmUfAmAmAmCfUmGmUmAmCmCmAmCfAmAfCmAmAmAmGm; (2) the sense strand comprises CmAmAmUmGmAmUfAfUfGmUmUmUmCmGmAmUmAmUm, and the antisense strand comprises AmUfAmUmCmGfAmAmAmCmAmUmAmUfCmAfUmUmGmGmGm; (3) the sense strand comprises CmCmUmCmCmUmGfGfAfCmAmAmAmGmAmAmAmUmAm, and the antisense strand comprises UmAfUmUmUmCfUmUmUmGmUmCmCmAfGmGfAmGmGmAmAm; (4) the sense strand comprises AmGmAmCmCmAmAfAfGfAmGmUmAmGmUmUmAmAmUm, and the antisense strand comprises AmUfUmAmAmCfUmAmCmUmCmUmUmUfGmGfUmCmUmGmAm; (5) the sense strand comprises CmUmGmAmAmUmCfUfAfUmGmGmAmUmCmAmAmCmUm, and the antisense strand comprises AmGfUmUmGmAfUmCmCmAmUmAmGmAfUmUfCmAmGmAmAm; (6) the sense strand comprises AmAmUmCmUmAmUfGfGfAmUmCmAmAmCmUmAmCmUm, and the antisense strand comprises AmGfUmAmGmUfUmGmAmUmCmCmAmUfAmGfAmUmUmCmAm; (7) the sense strand comprises GmCmCmAmAmAmGfCfCfUmUmCmUmAmCmUmAmCmAm, and the antisense strand comprises UmGfUmAmGmUfAmGmAmAmGmGmCmUfUmUfGmGmCmUmGm; (8) the sense strand comprises GmAmAmUmCmAmAfGfCfCmAmAmGmAmCmAmUmAmUm, and the antisense strand comprises AmUfAmUmGmUfCmUmUmGmGmCmUmUfGmAfUmUmCmAmCm; (9) the sense strand comprises GmCmUmAmUmUmCfCfAfUmAmAmUmUmCmCmAmUmAm, and the antisense strand comprises UmAfUmGmGmAfAmUmUmAmUmGmGmAfAmUfAmGmCmUmAm; (10) the sense strand comprises AmCmUmGmGmAmAfGfUfUmAmUmUmUmAmUmUmUmUm, and the antisense strand comprises AmAfAmAmUmAfAmAmUmAmAmCmUmUfCmCfAmGmUmUmUm; (11) the sense strand comprises CmCmAmUmCmAmAfGfAfUmCmCmAmCmUmUmCmUmAm, and the antisense strand comprises UmAfGmAmAmGfUmGmGmAmUmCmUmUfGmAfUmGmGmCmAm; (12) the sense strand comprises AmCmAmGmCmCmAfAfGfUmUmCmUmAmCmUmUmCmUm, and the antisense strand comprises AmGfAmAmGmUfAmGmAmAmCmUmUmGfGmCfUmGmUmAmAm; (13) the sense strand comprises GmUmUmCmUmAmCfUfUfCmUmGmAmAmUmCmUmAmUm, and the antisense strand comprises AmUfAmGmAmUfUmCmAmGmAmAmGmUfAmGfAmAmCmUmUm; (14) the sense strand comprises UmUmCmUmAmCmUfUfCfUmGmAmAmUmCmUmAmUmAm, and the antisense strand comprises UmAfUmAmGmAfUmUmCmAmGmAmAmGfUmAfGmAmAmCmUm; (15) the sense strand comprises CmAmAmGmUmGmCfUfAfAmGmGmAmUmUmCmUmUmUm, and the antisense strand comprises AmAfAmGmAmAfUmCmCmUmUmAmGmCfAmCfUmUmGmGmUm; (16) the sense strand comprises AmGmUmGmCmUmAfAfGfGmAmUmUmCmUmUmUmCmAm, and the antisense strand comprises UmGfAmAmAmGfAmAmAmUmCmCmUmUmAfGmCfAmCmUmUmGm; (17) the sense strand comprises CmCmAmCmGmUmUfCfCfUmUmCmUmCmUmCmAmGmAm, and the antisense strand comprises UmCfUmGmAmGfAmGmAmAmGmGmAmAfCmGfUmGmGmGmUm; (18) the sense strand comprises AmUmAmCmUmCmAfGfCfCmUmGmUmUmUmGmUmUmUm, and the antisense strand comprises AmAfAmCmAmAfAmCmAmGmGmCmUmGfAmGfUmAmUmAmAm; (19) the sense strand comprises UmAmCmUmCmAmGfCfCfUmGmUmUmUmGmUmUmUmUm, and the antisense strand comprises AmAfAmAmCmAfAmAmCmAmGmGmCmUfGmAfGmUmAmUmAm; (20) the sense strand comprises CmAmCmCmAmAmAfUfAfUmGmAmUmGmUmAmUmCmAm, and the antisense strand comprises UmGfAmUmAmCfAmUmCmAmUmAmUmUfUmGfGmUmGmCmAm; (21) the sense strand comprises CmCmAmGmCmUmUfAfCfUmCmCmAmCmGmCmAmAmUm, and the antisense strand comprises AmUfUmGmCmGfUmGmGmAmGmUmAmAfGmCfUmGmGmUmGm; (22) the sense strand comprises CmUmUmAmCmUmCfCfAfCmGmCmAmAmUmAmUmGmUm, and the antisense strand comprises AmCfAmUmAmUfUmGmCmGmUmGmGmAfGmUfAmAmGmCmUm; (23) the sense strand comprises GmUmGmCmCmAmCfAfUfUmAmUmCmAmGmUmCmUmAm, and the antisense strand comprises UmAfGmAmCmUfGmAmUmAmAmUmGmUfGmGfCmAmCmAmUm; or (24) the sense strand comprises UmCmAmGmCmAmUfCfCfUmCmAmUmGmUmCmUmAmUm, and the antisense strand comprises AmUfAmGmAmCfAmUmGmAmGmGmAmUfGmCfUmGmAmGmAm, The 5' end and 3' end of the sense strand and the antisense strand may independently contain one or two phosphorothioate modifications.

17. The dsRNA of any one of claims 1-16, wherein the dsRNA is further conjugated to a ligand moiety comprising N-acetylgalactosamine, preferably the sense strand is conjugated to the ligand moiety, more preferably the 3' end of the sense strand is conjugated to the ligand moiety via phosphorothioate.

18. A cell comprising the dsRNA of any one of claims 1 to 17.

19. A pharmaceutical composition comprising the dsRNA of any one of claims 1 to 17, or the cell of claim 18, and optionally a pharmaceutically acceptable carrier or excipient.

20. A kit comprising the dsRNA of any one of claims 1-17, the cell of claim 18, or the pharmaceutical composition of claim 19.

21. A method for inhibiting the expression of the ATXN2 gene in a cell, the method comprising contacting the cell with the dsRNA of any one of claims 1 to 17 or the pharmaceutical composition of claim 19.

22. A method of inhibiting ATXN2 gene expression in a cell in a subject, the method comprising administering to the subject the dsRNA of any one of claims 1-17, the cell of claim 18, or the pharmaceutical composition of claim 19.

23. A method of treating a disease or condition in a subject that would benefit from reduced expression of the ATXN2 gene, the method comprising the step of administering to the subject the dsRNA of any one of claims 1-17, the cell of claim 18, or the pharmaceutical composition of claim 19.

24. The method of claim 23, wherein the disease or condition that would benefit from decreased expression of the ATXN2 gene is an Ataxin2-associated disease.

25. The method of claim 24, wherein the Ataxin2-related disease is selected from the group consisting of progressive cerebellar ataxia, blindness, spinocerebellar ataxia (SCA), such as spinocerebellar ataxia 2 (SCA2), amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Parkinson's disease.

26. The method of any one of claims 22-25, wherein the dsRNA, cell or pharmaceutical composition is administered intrathecally.

27. The method of any one of claims 22-26, wherein the subject is a human.

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