Compositions and methods for treating neurological disorders

Abstract

This disclosure features compositions and methods for treating disorders associated with expression of wild-type or mutant ataxin-2 (ATXN2) RNA transcripts, including disorders characterized by genes containing abnormally expanded repeat regions that can lead to pathological phenotypes. Disclosed herein are inhibitory RNA constructs that suppress the expression of ATXN2, and viral vectors, such as adeno-associated viral vectors, that encode such inhibitory RNA molecules.

Description

[Technical Field]

[0001] The present invention relates to the field of nucleic acid biotechnology and provides compositions and methods for treating diseases associated with the expression of ataxin-2. [Background technology]

[0002] Ataxin-2 is a protein involved in several functions, including the formation of stress granules and P bodies, and the regulation of mRNA translation. Ataxin-2 protein is encoded by the ATXN2 gene. Mutations in the CAG trinucleotide repeat in the ATXN2 gene can cause neuronal degeneration and are associated with diseases such as spinocerebellar ataxia-2 ​​(SCA2). Ataxin-2 protein contains a sequence of glutamine residues commonly referred to as a polyglutamine repeat. Wild-type ataxin-2 genes typically contain about 13 to about 31 CAG trinucleotide repeats, with 22 repeats being the most common. In healthy individuals, the sequence is approximately 22 amino acids in length; however, expansion of this sequence has been observed. For example, glutamine repeats of 34 or more residues have been found in individuals with SCA2, and repeats of 27 to 40 residues have been reported in individuals with amyotrophic lateral sclerosis (ALS). Both ALS and SCA2 are progressive, neurodegenerative, and can be highly debilitating, often essentially fatal. Currently, there are few strategies available to successfully treat and ameliorate the symptoms of SCA2, ALS, Huntington's disease, Parkinson's disease, FTD (frontotemporal dementia), TAR DNA-binding protein 43 (TDP-43) proteopathy, and other diseases or disorders associated with wild-type or mutant ATXN2. Therefore, there remains a need for effective therapies for these conditions. Summary of the Invention [Means for solving the problem]

[0003] Described herein are compositions and methods useful for treating diseases associated with the expression of wild-type or mutant ataxin-2 (ATXN2). The compositions described herein that can be used to treat such disorders include inhibitory nucleic acid constructs, e.g., interfering RNA constructs, that suppress the expression of wild-type or mutant mRNA transcripts. Exemplary inhibitory nucleic acids of the present disclosure include, but are not limited to, microRNA (miRNA), short hairpin RNA (shRNA), and short interfering RNA (siRNA) constructs. Without being limited by mechanism, these inhibitory nucleic acids may anneal to portions of wild-type or mutant ATXN2 mRNA and promote the degradation of the pathological transcript through various cellular processes. The present disclosure also features vectors, such as viral vectors, that encode such inhibitory nucleic acid constructs. Exemplary viral vectors described herein that encode inhibitory nucleic acid constructs (e.g., interfering RNA constructs (e.g., miRNA)) are adeno-associated viral (AAV) vectors, such as pseudotyped AAV2 / 8 and AAV2 / 9 vectors.

[0004] The compositions and methods described herein can be used to administer an inhibitory nucleic acid, such as an interfering RNA construct or a vector encoding the same, to patients diagnosed with a disease associated with wild-type or mutant ATXN2, such as spinocerebellar ataxia type 2 (SCA2), amyotrophic lateral sclerosis (ALS), Huntington's disease, frontotemporal dementia (FTD), and TAR DNA-binding protein 43 (TDP-43) proteopathy, among others, to reduce the expression of wild-type or mutant mRNA transcripts. For example, the compositions and methods described herein can be used to treat patients with SCA2 by administering an inhibitory nucleic acid construct or a viral vector, such as an AAV vector, encoding such a construct to reduce the expression of mRNA transcripts encoding wild-type or mutant ataxin-2 proteins. Wild-type ataxin-2 genes typically contain about 13 to about 31 CAG trinucleotide repeats, with 22 repeats being most common. ATXN2 mRNA transcripts transcribed from mutant forms of the gene containing 34 or more CAG repeats (e.g., at least 34, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, or 100 CAG repeats) have been reported in patients with SCA2, and ATXN2 mRNA transcripts transcribed from mutant forms of the gene containing 27 to 40 CAG repeats have been reported in patients with ALS (e.g., at least 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 CAG repeats). The compositions and methods described herein can be used to treat patients expressing wild-type or mutant ATXN2 mRNA (e.g., wild-type or mutant human ATXN2 mRNA) by suppressing expression of the wild-type or mutant ATXN2 mRNA, for example, using an inhibitory nucleic acid construct.

[0005] In a first aspect, the disclosure features an inhibitory nucleic acid including a guide strand (and optionally, a passenger strand having complementarity to the guide strand). In some embodiments, the guide strand has sufficient complementarity to hybridize to a region within an ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153. In some embodiments, the guide strand has at least 70% complementarity to 15, 16, 17, 18, 19, 20, 21, or more contiguous polynucleotide segments within a region of an ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.

[0006] In some embodiments, the guide strand has at least 70% complementarity to 15, 16, 17, 18, 19, 20, 21, or more contiguous polynucleotide segments within a region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153. In some embodiments, the guide strand has at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementarity to 15, 16, 17, 18, 19, 20, 21, or more contiguous polynucleotide segments within a region of an ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.

[0007] In some embodiments, the guide strand has at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementarity to 15 consecutive polynucleotides within a region of an ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.

[0008] In some embodiments, the guide strand may have at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementarity to 16 contiguous polynucleotides within a region of an ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.

[0009] In some embodiments, the guide strand has at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementarity to 17 contiguous polynucleotides within a region within an ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.

[0010] In some embodiments, for example, the guide strand has at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementarity to 18 contiguous polynucleotides within a region within an ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.

[0011] In some embodiments, the guide strand has at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementarity to 19 contiguous polynucleotides within a region within an ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.

[0012] In some embodiments, the guide strand has at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementarity to 20 contiguous polynucleotides within a region within an ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.

[0013] In some embodiments, the guide strand has at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementarity to 21 contiguous polynucleotides within a region within an ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.

[0014] In some embodiments, the guide strand comprises at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleotides that are completely complementary to a contiguous polynucleotide of equal length within a region of the ATXN2 RNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.

[0015] In some embodiments, the guide strand comprises at least 10 contiguous nucleotides that are perfectly complementary to a contiguous polynucleotide of equal length within a region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.

[0016] In some embodiments, the guide strand comprises at least 11 contiguous nucleotides that are perfectly complementary to a contiguous polynucleotide of equal length within a region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.

[0017] In some embodiments, the guide strand comprises at least 12 consecutive nucleotides that are perfectly complementary to a contiguous polynucleotide of equal length within a region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.

[0018] In some embodiments, the guide strand comprises at least 13 contiguous nucleotides that are perfectly complementary to a contiguous polynucleotide of equal length within a region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.

[0019] In some embodiments, the guide strand comprises at least 14 contiguous nucleotides that are perfectly complementary to a contiguous polynucleotide of equal length within a region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.

[0020] In some embodiments, the guide strand comprises at least 15 consecutive nucleotides that are perfectly complementary to a contiguous polynucleotide of equal length within a region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.

[0021] In some embodiments, the guide strand comprises at least 16 contiguous nucleotides that are perfectly complementary to a contiguous polynucleotide of equal length within a region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.

[0022] In some embodiments, the guide strand comprises at least 17 contiguous nucleotides that are perfectly complementary to a contiguous polynucleotide of equal length within a region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.

[0023] In some embodiments, the guide strand comprises at least 18 consecutive nucleotides that are perfectly complementary to a contiguous polynucleotide of equal length within a region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.

[0024] In some embodiments, the guide strand comprises at least 19 contiguous nucleotides that are perfectly complementary to a contiguous polynucleotide of equal length within a region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.

[0025] In some embodiments, the guide strand comprises at least 20 contiguous nucleotides that are perfectly complementary to a contiguous polynucleotide of equal length within a region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.

[0026] In some embodiments, the guide strand comprises at least 21 contiguous nucleotides that are perfectly complementary to a contiguous polynucleotide of equal length within a region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.

[0027] In some embodiments, the guide strand comprises 10 to 21 contiguous nucleotides that are fully complementary to a contiguous polynucleotide of equal length within a region of an ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153. In some embodiments, the guide strand comprises 12 to 21 contiguous nucleotides that are fully complementary to a contiguous polynucleotide of equal length within a region of an ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153. In some embodiments, the guide strand comprises 15 to 21 contiguous nucleotides that are fully complementary to a contiguous polynucleotide of equal length within a region of an ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153. In some embodiments, the guide strand comprises 18 to 21 contiguous nucleotides that are fully complementary to a contiguous polynucleotide of equal length within a region of an ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.

[0028] In some embodiments, the guide strand comprises 19, 20, or 21 contiguous nucleotides that are fully complementary to a contiguous polynucleotide of equal length within a region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.

[0029] The guide strand comprises 9 or fewer nucleotide mismatches with a segment of 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides within a region of an ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153, and optionally the guide strand comprises 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or only 1 mismatch with a region of an ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.

[0030] In some embodiments, the region of the ATXN2 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 103, 104, 105, 106, 121, 122, 126, 127, 139, 140, 141, 147, 149, and 153.

[0031] In some embodiments, the guide strand has a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence of any one of SEQ ID NOs: 1-51 (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence of any one of SEQ ID NOs: 1-51). In some embodiments, the guide strand has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of any one of SEQ ID NOs: 1-51 (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence of any one of SEQ ID NOs: 1-51). In some embodiments, the guide strand has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of any one of SEQ ID NOs: 1-51 (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence of any one of SEQ ID NOs: 1-51). In some embodiments, the guide strand has the nucleic acid sequence of any one of SEQ ID NOs: 1-51. In some embodiments, the nucleic acid sequence is any one of SEQ ID NOs: 1, 2, 3, 4, 19, 20, 24, 25, 37, 38, 39, 45, 47, and 51.

[0032] In some embodiments, the inhibitory nucleic acid comprises a hairpin having a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence of any one of SEQ ID NOs: 52-102 (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence of any one of SEQ ID NOs: 52-102). In some embodiments, the inhibitory nucleic acid comprises a hairpin having a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of any one of SEQ ID NOs: 52-102 (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence of any one of SEQ ID NOs: 52-102). In some embodiments, the hairpin has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of any one of SEQ ID NOs: 52-102 (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence of any one of SEQ ID NOs: 52-102). In some embodiments, the hairpin strand has the nucleic acid sequence of any one of SEQ ID NOs: 52-102. In some embodiments, the nucleic acid sequence is any one of SEQ ID NOs: 52, 53, 54, 55, 70, 71, 75, 76, 88, 89, 90, 96, 98, and 102.

[0033] In some embodiments, the inhibitory nucleic acid is an interfering RNA molecule. In some embodiments, the interfering RNA molecule is a microRNA (miRNA), a short hairpin RNA (shRNA) or a short interfering RNA (siRNA). In some embodiments, the inhibitory nucleic acid is an miRNA.

[0034] In another aspect, the disclosure features a viral vector including a transgene encoding an inhibitory nucleic acid of any of the above aspects or embodiments of the disclosure. In some embodiments, the viral vector includes multiple transgenes (e.g., 2, 3, 4, 5, or more transgenes).

[0035] In some embodiments, the viral vector is selected from the group consisting of adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, poxvirus, baculovirus, herpes simplex virus, vaccinia virus, and synthetic viruses, hi some embodiments, the viral vector is AAV.

[0036] In some embodiments, the AAV is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhlO, or AAVrhl74 serotype. In some embodiments, the viral vector is a pseudotyped AAV. In some embodiments, the pseudotyped AAV has ITRs from one AAV serotype (e.g., AAV2) and VP1, VP2, and / or VP3 capsid proteins from a different AAV serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhlO, or AAVrhl74). In some embodiments, the pseudotyped AAV is AAV2 / 9. In some embodiments, the pseudotyped AAV is AAV2 / 8. In some embodiments, the AAV comprises recombinant capsid proteins.

[0037] In some embodiments, the AAV comprises a capsid as disclosed, for example, in WO2017 / 218842, the disclosure of which is incorporated herein by reference. In some embodiments, the AAV comprises a capsid as disclosed in Lin et al. Mol Brain 13:138 (2020), the disclosure of which is incorporated herein by reference. In some embodiments, the AAV comprises an AAV2-retro or AAV9-retro capsid protein.

[0038] In some embodiments, the synthetic virus is a chimeric virus, a mosaic virus, or a pseudotyped virus, and / or comprises a foreign protein, synthetic polymer, nanoparticle, or small molecule.

[0039] In a further aspect, the present disclosure provides a pharmaceutical composition comprising an siRNA molecule of any of the foregoing aspects or embodiments of the present disclosure and a pharmaceutically acceptable excipient, carrier, or diluent.

[0040] In another aspect, the present disclosure provides a method of reducing ATXN2 expression in a subject in need thereof by administering a therapeutically effective amount of an siRNA or pharmaceutical composition of any of the above aspects or embodiments of the present disclosure.

[0041] In some embodiments, the neurological disease is associated with TDP-43 (TAR DNA-binding protein 43) proteinopathy. In some embodiments, the neurological disease is caused by or associated with expression of wild-type or mutant forms of ATXN2. In some embodiments, the neurological disease is ALS, frontotemporal lobe dementia, primary lateral sclerosis, progressive muscular atrophy, limbic-predominant senile TDP-43 encephalopathy, chronic traumatic encephalopathy, dementia with Lewy bodies, corticobasal degeneration, progressive supranuclear palsy, Guam Parkinsonism-Dementia Complex, Pick's disease, Perry syndrome, age-related TDP-43 encephalopathy with sclerosis, hippocampal sclerosis, Huntington's disease, Parkinson's disease, Alzheimer's disease, or SCA2. In some embodiments, the neurological disease is SCA2 and the subject can have multiple CAG trinucleotide repeat mutations at the ATXN2 locus, for example, at least 34, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, or 100 CAG repeats. In some embodiments, the neurological disease is ALS and the subject can have multiple CAG trinucleotide repeat mutations at the ATXN2 locus, for example, at least 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 CAG repeats. In some embodiments, the neurological disease is Huntington's disease and the subject can have upregulated ATXN2.

[0042] In some embodiments, the inhibitory nucleic acid, viral vector, or pharmaceutical composition is administered to a subject intrathalamic, intrathecal, subpial, intraparenchymal, intrastriatal, intracranial, intracisternal, intracerebral, intraventricular, intraocular (e.g., intravitreal), intracerebroventricular, intralumbar, intravenous, intramuscular, subcutaneous, intraperitoneal, intradermal, transdermal, parenteral, intranasal, transdermal, intratracheal, intraarterial, intravascular, and oral administration, inhalation, perfusion, lavage, or any combination thereof.

[0043] In some embodiments, the subject comprises a mammal (eg, a human).

[0044] In a further aspect, the disclosure features a kit including an inhibitory nucleic acid, viral vector, or pharmaceutical composition of any of the preceding aspects or embodiments of the disclosure. The kit may further include a package insert directing use of the kit to administer a therapeutically effective amount of the inhibitory nucleic acid, viral vector, or pharmaceutical composition to a subject (e.g., a mammal, e.g., a human, diagnosed with a neurological disorder described herein). [Brief explanation of the drawings]

[0045] [Figure 1] Figure 1 is a bar graph showing the knockdown efficacy of a series of ATXN2-specific miRNA constructs. Data were obtained from a dual-luciferase reporter (DLR) assay performed in HEK293 cells and quantified as the percentage of luciferase activity relative to the negative control. The negative control represents the absence of luciferase reporter silencing. Data are representative of three to four independent experiments, with biological triplicates for each experiment. Figure 1. Abbreviations: ATXN2, Ataxin-2; A2, Ataxin-2; HEK, human embryonic kidney. [Figure 2A]Figure 2A is a bar graph showing the knockdown efficacy of several ATXN2-specific miRNA constructs from Figure 1. Data were obtained from dual luciferase reporter (DLR) assays performed in HEK293 cells after plasmid transfection and quantified as the percentage of luciferase activity relative to the negative control. The control (CONT) is a non-targeting miRNA. Data represent three to four independent experiments, with biological triplicates for each experiment. Figure 2A. Abbreviations: ATXN2, Ataxin-2; HEK, human embryonic kidney. [Figure 2B] Figure 2B is a bar graph showing the knockdown efficacy of several ATXN2-specific miRNA constructs from Figure 1. Data were obtained from qPCR assays performed in HEK293 cells after plasmid transfection and are shown as the ratio of ATXN2 to GAPDH mRNA signals from probe-based qPCR assays. The control (CONT) is a non-targeting miRNA. Data represent three to four independent experiments, with biological triplicates for each experiment. Figure 2B Abbreviations: ATXN2, Ataxin-2; HEK, human embryonic kidney; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; qPCR, quantitative polymerase chain reaction. [Figure 2C] Figure 2C is a bar graph showing the knockdown efficacy of several ATXN2-specific miRNA constructs from Figure 1. Data were obtained from qPCR assays performed in HEK293 cells after plasmid transfection and quantified as a percentage of mRNA expression relative to the negative control (CONT). The negative control lacks silencing of endogenous ATXN2 mRNA and serves as a baseline. Data represent three to four independent experiments, with biological triplicates for each experiment. Figure 2C. Abbreviations: ATXN2, Ataxin-2; HEK, human embryonic kidney. [Figure 3]Figure 3 is a bar graph showing the knockdown efficacy of several ATXN2-specific miRNA constructs from Figure 1. Data were obtained from qPCR assays performed in HEK293 cells after viral transduction and are shown as the ratio of ATXN2 to GAPDH mRNA signals. The control (CONT) is a non-targeting miRNA. Data represent three to four independent experiments, with biological triplicates for each experiment. Figure 3. Abbreviations: ATXN2, Ataxin-2; HEK, human embryonic kidney; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; qPCR, quantitative polymerase chain reaction. [Figure 4A] Figure 4A is a series of bar graphs showing the knockdown efficacy of the ATXN2-specific miRNA constructs from Figure 1. Data were obtained from qPCR assays performed using homogenized mouse tissues after AAV / PBS injection and quantified as the percentage of human ATXN2 copies / µg RNA expression relative to the control group (PBS). The PBS group lacked silencing of endogenous ATXN2 mRNA and served as a baseline. Experimental groups received AAV injections at a dose of 1e8 viral genomes / hemisphere (vg / hem) or 1e9 vg / hem. The left and right panels show human ATXN2 mRNA expression in the mouse thalamus and subcortex, respectively. Experiments were performed with N = 4 samples per group. Figure 4A. Abbreviations: ATXN2, ataxin-2; PBS, phosphate-buffered saline. [Figure 4B]Figure 4B is a series of bar graphs showing the knockdown efficacy of the ATXN2-specific miRNA constructs from Figure 1. Data were obtained from qPCR assays performed using homogenized mouse tissues after AAV / PBS injection and quantified as the percentage of mouse ATXN2 copies / µg RNA expression relative to the control group (PBS). The PBS group lacked silencing of endogenous ATXN2 mRNA and served as a baseline. Experimental groups received AAV injections at a dose of 1e8 viral genomes / hemisphere (vg / hem) or 1e9 vg / hem. The left and right panels show the expression of mouse Atxn2 mRNA in the mouse thalamus and subcortex, respectively. Experiments were performed with N = 4 samples per group. Figure 4B Abbreviations: ATXN2, ataxin-2; PBS, phosphate-buffered saline. [Figure 5] Figure 5 is a series of bar graphs showing the knockdown efficacy of the ATXN2-specific miRNA constructs from Figure 1. Data were obtained from qPCR assays performed using homogenized mouse tissues after AAV / PBS injection and quantified as the percentage of human ATXN2 copies / µg RNA expression relative to the control group (PBS). The PBS group lacked silencing of endogenous ATXN2 mRNA and served as a baseline. Experimental groups received AAV injections at a dose of 1e9 viral genomes / hemisphere (vg / hem). Experiments were performed with N = 7 samples per group. The numbers inserted within the bars (45%, 20%, 12%, and 25%) represent the percentage of knockdown observed for the specific miRNA administered. Figure 5. Abbreviations: ATXN2, Atxin-2; PBS, Dulbecco's phosphate-buffered saline; neg, negative.

[0046] definition As used herein, the term "about" refers to a value within 10% of a stated value. For example, the phrase "about 100 nucleic acid residues" refers to a value between 90 and 110 nucleic acid residues.

[0047] As used herein, the term "anneal" refers to the formation of stable duplexes of nucleic acids, e.g., by hybridization mediated by interstrand hydrogen bonding according to Watson-Crick base pairing. The nucleic acids of the duplex can be, for example, at least 50% complementary to each other (e.g., about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% complementary to each other). A "stable duplex" formed upon annealing one nucleic acid to another is a duplex structure that is not denatured by stringent washing. Exemplary stringent washing conditions are known in the art and include a temperature about 5°C lower than the melting temperature of each strand of the duplex and a low concentration of monovalent salt, such as a monovalent salt concentration (e.g., NaCl concentration) of less than 0.2M (0.2M, 0.19M, 0.18M, ​​0.17M, 0.16M, 0.15M, 0.14M, 0.13M, 0.12M, 0.11M, 0.1M, 0.09M, 0.08M, 0.07M, 0.06M, 0.05M, 0.04M, 0.03M, 0.02M, 0.01M, or lower).

[0048] As used herein, the terms "conservative mutation," "conservative substitution," or "conservative amino acid substitution" refer to the substitution of one or more amino acids with one or more different amino acids that exhibit similar physicochemical properties, such as polarity, electrostatic charge, and steric bulk. These properties are summarized in Table 1 below for each of the 20 naturally occurring amino acids. [Table 1]

[0049] From this table, it can be seen that conservative amino acid families include, for example, (i) G, A, V, L, I, P, and M, (ii) D and E, (iii) C, S, and T, (iv) H, K, and R, (v) N and Q, and (vi) F, Y, and W. Thus, conservative variations or substitutions are those in which one amino acid is replaced with a member of the same amino acid family (e.g., Ser for Thr, or Lys for Arg).

[0050] As used herein, the "length" of a nucleic acid refers to the linear size of the nucleic acid, as determined by measuring the amount of nucleotides from the 5' to 3' end of the nucleic acid. Exemplary molecular biology techniques that can be used to determine the length of a nucleic acid of interest are known in the art.

[0051] As used herein, the term "operably linked" refers to a first molecule (e.g., a first nucleic acid) attached to a second molecule, the molecules being positioned such that the first molecule affects the function of the second molecule (e.g., a second nucleic acid). The two molecules may or may not be part of a single, contiguous molecule, and may or may not be adjacent. For example, a promoter is operably linked to a transcribable polynucleotide molecule if the promoter controls the transcription of the transcribable polynucleotide molecule of interest in a cell. Additionally, two portions of a transcriptional regulatory element are operably linked to each other if they are linked such that the transcriptional activation function of one portion is not adversely affected by the presence of the other portion. Two transcriptional regulatory elements can be operably linked to each other via a linker nucleic acid (e.g., an intervening non-coding nucleic acid) or can be operably linked to each other without any intervening nucleotides.

[0052] As used herein, one segment of a nucleic acid molecule is considered to "overlap" with another segment of the same nucleic acid molecule if the two segments share one or more constituent nucleotides. For example, two segments of the same nucleic acid molecule are considered to "overlap" with each other if the two segments share 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100 or more constituent nucleotides. If the two segments have zero common constituent nucleotides, the two segments are not considered to "overlap" with each other.

[0053] "Percent sequence complementarity (%)" to a reference polynucleotide sequence is defined as the percentage of nucleic acids in a candidate sequence that are complementary to the nucleic acids in the reference polynucleotide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence complementarity. A given nucleotide is considered to be "complementary" to a reference nucleotide described herein if the two nucleotides form a canonical Watson-Crick base pair. For the avoidance of doubt, Watson-Crick base pairs in the context of the present disclosure include adenine-thymine, adenine-uracil, and cytosine-guanine base pairs. In this context, a proper Watson-Crick base pair is referred to as a "match," while each unpaired nucleotide and each incorrectly paired nucleotide is referred to as a "mismatch." Alignment for the purpose of determining nucleic acid sequence complementarity percentage can be achieved in a variety of ways within the capabilities of those skilled in the art, for example, using publicly available computer software (e.g., BLAST, BLAST-2, or Megalign software).Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximum complementarity across the full length of the sequences being compared.By way of example, the sequence complementarity percentage of a given nucleic acid sequence A to a given nucleic acid sequence B (alternatively, it can be expressed as a given nucleic acid sequence A having a specific complementarity percentage to a given nucleic acid sequence B) is calculated as follows: 100×(fraction X / Y) where X is the number of complementary base pairs in the alignment of A and B in the program (e.g., as performed by computer software such as BLAST) and Y is the total number of nucleic acids in B. It should be understood that if the length of nucleic acid sequence A is not equal to the length of nucleic acid sequence B, the percent sequence complementarity of A to B will not equal the percent sequence complementarity of B to A. As used herein, a query nucleic acid sequence is considered to be "fully complementary" to a reference nucleic acid sequence if the query nucleic acid sequence has 100% sequence complementarity to the reference nucleic acid sequence.

[0054] "Percent sequence identity (%)" with respect to a reference polynucleotide or reference polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to those in the reference polynucleotide or reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid or amino acid sequence identity can be achieved in a variety of ways within the capabilities of those skilled in the art using publicly available computer software (e.g., BLAST, BLAST-2, or Megalign software). Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared. For example, percent sequence identity values ​​can be generated using the sequence comparison computer program BLAST. By way of illustration, the percent sequence identity of a given nucleic acid or amino acid sequence A to, with, or against a given nucleic acid or amino acid sequence B (which may alternatively be expressed as a given nucleic acid or amino acid sequence A having a particular percent sequence identity to, with, or against a given nucleic acid or amino acid sequence B) is calculated as follows: 100×(fraction X / Y) where X is the number of nucleotides or amino acids scored as identical matches by a sequence alignment program (e.g., BLAST) in that program's alignment of A and B, and Y is the total number of nucleic acids in B. It will be recognized that if the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, then the percent sequence identity of A to B will not equal the percent sequence identity of B to A.

[0055] As used herein, the term "pharmaceutical composition" refers to a mixture containing a therapeutic agent, e.g., a nucleic acid or vector described herein, optionally in combination with one or more pharmaceutically acceptable excipients, diluents, and / or carriers, administered to a subject, such as a mammal (e.g., a human), to prevent, treat, or control a particular disease or condition that has or may affect the subject.

[0056] As used herein, the term "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are suitable for contact with the tissues of a subject, such as a mammal (e.g., a human), without undue toxicity, irritation, allergic response, and other significant complications, commensurate with a reasonable benefit / risk ratio.

[0057] As used herein, the term "wild-type," or "non-mutated," form of a gene refers to a nucleic acid encoding a protein associated with normal or non-pathogenic activity (e.g., a protein lacking a mutation, such as a recurrent expansion of a region that confers an increased risk for the onset, development, or progression of a neurodegenerative disease).

[0058] As used herein, the term "mutation" refers to any change in the structure of a gene, e.g., in the sequence of a gene, resulting in an altered form of the gene that may or may not be passed on to subsequent generations (genetic mutation). Genetic mutations include the substitution, insertion, or deletion of a single base in DNA, or the substitution, insertion, deletion, or rearrangement of multiple bases, or thus large sections, of a gene or chromosome, including repeat expansions.

[0059] As used herein, the terms "ataxin 2," "ataxin-2," or "ATXN2" refer to the protein encoded by the ATXN2 gene, which contains a polyglutamine (polyQ, CAG repeat) tract. The ATXN2 gene, or transcript, can refer to a normal allele of ATXN2, typically having 22 or 23 repeats, or a mutant allele with an intermediate (approximately 24-32 repeats) or longer repeat expansion (approximately 33-100+ repeats). In some embodiments, ATXN2 refers to mammalian ATXN2, including human ATXN2. Exemplary ATXN2 proteins that can be targeted using the compositions and methods of the present disclosure include the protein having the amino acid sequence represented by NCBI ID NP_001297050.1, as well as naturally occurring variants thereof. In some embodiments, an exemplary ATXN2 gene has the nucleic acid sequence of NCBI ID NC_000012.12:c111599673-111452214, or a naturally occurring variant thereof. Exemplary ATXN2 mRNA transcripts include those having the nucleic acid sequence of NCBI ID NM_002973.4, and naturally occurring variants thereof.

[0060] As used herein, the term "inhibitory nucleic acid" refers to a nucleic acid comprising a guide strand sequence that hybridizes to at least a portion of a target nucleic acid, e.g., ATXN2 RNA, mRNA, pre-mRNA, or mature mRNA, and inhibits its expression or activity. The inhibitory nucleic acid may target a protein-coding region (e.g., an exon) or a non-coding region (e.g., a 5'UTR, a 3'UTR, an intron, etc.) of the target nucleic acid. In some embodiments, the inhibitory nucleic acid is a single-stranded or double-stranded molecule. The inhibitory nucleic acid may further comprise a passenger strand sequence on another strand (e.g., a double-stranded duplex) or the same strand (e.g., a single-stranded, self-annealing duplex structure). In some embodiments, the inhibitory nucleic acid is an interfering RNA molecule, e.g., a short interfering RNA (siRNA), a short hairpin RNA (shRNA), a microRNA (miRNA), or a double-stranded RNA (dsRNA).

[0061] As used herein, the term "interfering RNA" refers to an RNA, such as an siRNA, miRNA, or shRNA, that suppresses the expression of a target RNA transcript by, for example, (i) annealing to the target RNA transcript, thereby forming a nucleic acid duplex, and (ii) promoting nuclease-mediated degradation of the RNA transcript, and / or (iii) slowing down, inhibiting, or preventing the translation of the RNA transcript, such as by sterically excluding the formation of a functional ribosomal RNA-transcript complex or otherwise attenuating the formation of a functional protein product from the target RNA transcript. The interfering RNA described herein can be provided to a patient, for example, a human patient with muscular dystrophy, in the form of, for example, a single-stranded or double-stranded oligonucleotide, or in the form of a vector containing a transgene encoding the interfering RNA, such as an adenovirus-associated vector described herein. Exemplary interfering RNA platforms are described, for example, in Molecular Therapy-Nucleic Acids 4:e252 (2015); Rao et al., Advanced Drug Delivery Reviews 61:746-769 (2009), and Borel et al., Molecular Therapy 22:692-701 (2014), the disclosures of each of which are incorporated herein by reference in their entireties.

[0062] As used herein, "microRNA" or "miRNA" refers to a small non-coding RNA molecule that can mediate the silencing of target genes by cleaving target mRNA, suppressing the translation of target mRNA, degrading target mRNA, or a combination thereof. Typically, miRNA is transcribed as a hairpin or stem-loop (e.g., having a self-complementary single-stranded backbone) duplex structure called primary miRNA (pri-miRNA), which is enzymatically processed into pre-miRNA (e.g., by Drosha, DGCR8, Pasha, etc.). Pre-miRNA is transported to the cytoplasm, where it is enzymatically processed by Dicer to generate a miRNA duplex with a passenger strand, and then a single-stranded mature miRNA molecule, which is then loaded into the RNA-induced silencing complex (RISC). Reference to miRNA may include synthetic or artificial miRNA.

[0063] As used herein, "synthetic miRNA," or "artificial miRNA," or "amiRNA" refers to an endogenous, modified, or synthetic pri-miRNA or pre-miRNA (e.g., miRNA backbone or scaffold) in which the endogenous miRNA guide sequence and passenger sequence within the stem sequence have been replaced with miRNA guide sequences and miRNA passenger sequences that direct highly efficient RNA silencing of target genes (see, e.g., Eamens et al. (2014), Methods Mol. Biol. 1062:211-224). In some embodiments, the nature of complementarity of the guide sequence and passenger sequence (e.g., number of bases, location of mismatches, type of bulge, etc.) can be similar to or different from the nature of complementarity of the guide sequence and passenger sequence in the endogenous miRNA backbone from which the synthetic miRNA is constructed.

[0064] As used herein, the terms "microRNA scaffold," "miR scaffold," "microRNA scaffold," or "miR scaffold" refer to a pri-miRNA or pre-miRNA scaffold in which the stem sequence has been replaced by a miRNA of interest, producing a functional mature miRNA that directs RNA silencing at genes targeted by the miRNA of interest. The miR scaffold includes a 5'-flanking region (also referred to as the 5' miR context, ≥ 9 nucleotides), a stem region containing the miRNA duplex (guide strand sequence and passenger strand sequence), a basic stem (5' and 3', each approximately 4-13 nucleotides), at least one loop motif region (more than 10 nucleotides in the case of a terminal loop) containing a terminal loop, a 3'-flanking region (also referred to as the 3' miR context, ≥ 9 nucleotides), and optionally one or more stem bulges. The miR scaffold may be derived entirely or partially from a wild-type miRNA scaffold, or may be a completely artificial sequence.

[0065] As used herein, the term "antisense strand sequence" or "guide strand sequence" of an inhibitory nucleic acid refers to a sequence that is substantially complementary (e.g., at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary) to a region of about 10-50 nucleotides (e.g., about 15-30, 16-25, 18-23, or 19-22 nucleotides) of the mRNA of a gene targeted for silencing. The antisense sequence is sufficiently complementary to the target mRNA sequence to direct target-specific silencing, e.g., to cause destruction of the target mRNA by the RNAi machinery or process. In some embodiments, the antisense sequence, or guide strand sequence, refers to the mature sequence remaining after cleavage by Dicer.

[0066] As used herein, the term "sense sequence" or "passenger strand sequence" of an inhibitory nucleic acid refers to a sequence that is homologous to a target mRNA and is partially or completely complementary to the antisense strand sequence, or guide strand sequence, of the inhibitory nucleic acid. The antisense strand sequence and the sense strand sequence of the inhibitory nucleic acid hybridize to form a duplex structure (e.g., form a double-stranded duplex or a single-stranded self-annealing duplex structure). In some embodiments, the sense sequence or passenger strand sequence refers to the mature sequence remaining after cleavage by Dicer.

[0067] As used herein, "duplex," when used in reference to an inhibitory nucleic acid, refers to two nucleic acid strands (e.g., a guide strand and a passenger strand) that hybridize together to form a duplex structure. A duplex may be formed by two separate nucleic acid strands, or by a single nucleic acid strand having a region of self-complementarity (e.g., a hairpin or stem-loop).

[0068] As used herein, "expression construct" refers to any type of genetic construct containing a nucleic acid (e.g., a transgene) from which part or all of a nucleic acid coding sequence can be transcribed. In some embodiments, expression includes transcription of a nucleic acid, for example, to produce a biologically active polypeptide product or an inhibitory RNA (e.g., siRNA, shRNA, miRNA) from the transcribed gene. In some embodiments, the transgene is operably linked to an expression control sequence.

[0069] As used herein, the term "transgene" refers to an exogenous nucleic acid that can be introduced, transcribed, and optionally translated into another cell, either naturally or by genetic engineering means.

[0070] As used herein, the term "gene expression" refers to the process by which nucleic acids are transcribed from nucleic acid molecules and often translated into peptides or proteins. The process can include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post-translational modification, or any combination thereof. Reference to measuring "gene expression" can refer to measuring transcription products (e.g., RNA or mRNA), translation products (e.g., peptides or proteins).

[0071] As used herein, the term "inhibiting expression of a gene" means decreasing, down-regulating, suppressing, blocking, reducing, or stopping the expression of a gene. The expression product of a gene can be an RNA molecule (e.g., mRNA) transcribed from the gene or a polypeptide translated from the mRNA transcribed from the gene. Typically, a decrease in the level of mRNA results in a decrease in the level of the polypeptide translated from it. The level of expression can be determined using standard techniques for measuring mRNA or protein.

[0072] As used herein, "neurodegenerative disease" or "neurodegenerative disorder" refers to a disease or disorder that exhibits neuronal cell death as a pathological condition. Neurodegenerative diseases can exhibit chronic neurodegeneration, e.g., slowly progressive neuronal cell death over several years, or acute neurodegeneration, e.g., sudden onset or death of neurons. Examples of chronic neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, Huntington's disease, spinocerebellar ataxia type 2 (SCA2), frontotemporal dementia (FTLD), and amyotrophic lateral sclerosis (ALS). Chronic neurodegenerative diseases include diseases characterized by TDP-43 proteopathy, which is characterized by nuclear-to-cytoplasmic mislocalization, deposition of ubiquitinated and hyperphosphorylated TDP-43 into inclusion bodies, protein cleavage leading to the formation of toxic C-terminal TDP-43 fragments, and protein aggregation. TDP-43 proteopathy diseases include ALS, FTLD, primary lateral sclerosis, progressive muscular atrophy, limb-predominant age-related TDP-43 encephalopathy, chronic traumatic encephalopathy, dementia with Lewy bodies, corticobasal degeneration, progressive supranuclear palsy (PSP), Guam Parkinsonism-Dementia Complex (G-PDC), Pick's disease, hippocampal sclerosis, Huntington's disease, Parkinson's disease, and Alzheimer's disease. Acute neurodegeneration can be caused by ischemia (e.g., stroke, traumatic brain injury), demyelination, or traumatic axonal transection (e.g., spinal cord injury or multiple sclerosis). Neurodegenerative diseases can manifest primarily as the death of one type of neuron or multiple types of neurons.

[0073] As used herein, the term "repeat region" refers to a segment within a gene of interest or its RNA transcript that contains nucleic acid repeats, such as the polyCAG sequence in the ATXN2 gene. A repeat region is considered an "expanded repeat region," "repeat expansion," or the like, if the number of nucleotide repeats within the repeat region exceeds the number of repeats normally found within the repeat region in the wild-type form of the gene or its RNA transcript. For example, the wild-type human ATXN2 gene typically contains 13 to 31 CAG repeats. Thus, "expanded repeat region" and "repeat expansion" in the context of the ATXN2 gene or its RNA transcript include, among others, repeat regions containing more than 31 repeats.

[0074] As used herein, the term "sample" refers to a specimen (e.g., blood, blood components (e.g., serum or plasma), urine, saliva, amniotic fluid, cerebrospinal fluid, tissue (e.g., placental or dermal), pancreatic juice, chorionic villus sample, or cells) isolated from a subject. The subject may be, for example, a patient suffering from a disease described herein, such as a disease associated with expression of wild-type or mutant ATXN2 (e.g., SCA2, ALS, Huntington's disease, Parkinson's disease).

[0075] As used herein, the phrases "specifically bind" and "bind" refer to a binding reaction that is determinative of the presence of a particular molecule, such as an RNA transcript, among a heterogeneous population of recognized ions, salts, small molecules, and / or proteins, e.g., a mutant ATXN2 RNA transcript. A ligand (e.g., an RNA binding protein described herein) that specifically binds to a species (e.g., an RNA transcript) has a K of, e.g., less than 1 mM. D For example, a ligand that specifically binds to a species may have a K of up to 100 nM (e.g., 1 pM to 100 nM). D A ligand that does not exhibit specific binding to another molecule can bind to that species with a K of greater than 1 mM (e.g., greater than 1 μM, 100 μM, 500 μM, or 1 mM) for a particular molecule or ion thereof.D A variety of assay formats can be used to determine the affinity of a ligand for a particular protein. For example, solid-phase ELISA assays are routinely used to identify ligands that specifically bind to a target protein. For a description of assay formats and conditions that can be used to determine specific protein binding, see Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1988), and Harlow & Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1999).

[0076] As used herein, the terms "subject" and "patient" refer to an organism receiving treatment for a particular disease or condition (e.g., cancer, infectious disease, etc.) described herein. Examples of subjects and patients include mammals, such as humans, undergoing treatment for a disease or condition described herein.

[0077] As used herein, the term "transcriptional regulatory element" refers to a nucleic acid that controls, at least in part, the transcription of a gene of interest. Transcriptional regulatory elements can include promoters, enhancers, and other nucleic acids that control or help control gene transcription (e.g., polyadenylation signals). Examples of transcriptional regulatory elements are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, CA, 1990).

[0078] As used herein, the terms "treat" or "treatment" refer to therapeutic procedures aimed at preventing or slowing (alleviating) undesirable physiological changes or disorders, such as the progression of diseases associated with wild-type or mutant ATXN2, e.g., SCA2, ALS, Huntington's disease, and Parkinson's disease. In the context of SCA2, ALS, Huntington's disease, and Parkinson's disease treatment, beneficial or desirable clinical outcomes indicative of successful treatment include, but are not limited to, alleviation of symptoms, disappearance of disease extent, stabilization of disease status (i.e., no worsening), delay or slowing of disease progression, improvement or palliation of disease status, and remission (whether partial or total), whether detectable or undetectable. Treatment of patients with SCA2 may be manifested by one or more detectable changes, such as a decrease in expression of mutant ATXN2 RNA transcripts (e.g., a decrease in expression of ATXN2 RNA transcripts containing an expanded CAG trinucleotide repeat region).

[0079] As used herein, the term "vector" refers to a nucleic acid, e.g., DNA or RNA, that can function as a vehicle to deliver a gene of interest to a cell (e.g., a mammalian cell, such as a human cell), for purposes such as replication and / or expression. Exemplary vectors useful in conjunction with the compositions and methods described herein include plasmids, DNA vectors, RNA vectors, virions, or other suitable replicons (e.g., viral vectors). Various vectors have been developed for delivering polynucleotides encoding exogenous proteins to prokaryotic or eukaryotic cells. Examples of such expression vectors are disclosed, for example, in WO 1994 / 11026, the disclosure of which is incorporated herein by reference. The expression vectors described herein contain polynucleotide sequences and additional sequence elements used, for example, for protein expression and / or integration of these polynucleotide sequences into the genome of a mammalian cell. Particular vectors that can be used to express the transgenes described herein include plasmids containing regulatory sequences, such as promoters and enhancer regions, that direct gene transcription. Other useful vectors for expressing transgenes include polynucleotide sequences that increase the translation rate of these genes or improve the stability or nuclear transport of mRNA resulting from gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions, internal ribosome entry sites (IRES), and polyadenylation signal sites to direct efficient transcription of genes carried on the expression vector. The expression vectors described herein may also contain a polynucleotide encoding a marker for selecting cells containing such a vector. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, or nourseothricin. DETAILED DESCRIPTION OF THE INVENTION

[0080] The compositions and methods described herein are useful for treating disorders associated with wild-type or mutant ataxin-2 (ATXN2) expression, such as spinocerebellar ataxia type 2 (SCA2), amyotrophic lateral sclerosis (ALS), Huntington's disease, frontotemporal dementia (FTD), and TDP-43 (TAR DNA-binding protein 43) proteopathy. The compositions described herein include inhibitory nucleic acid constructs, such as interfering RNA constructs (e.g., small interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), or microRNAs (miRNAs)), that suppress the expression of wild-type or mutant mRNA transcripts transcribed from genes derived from wild-type or mutant genes. Without being limited by mechanism, the compositions described herein may ameliorate neuropathology by reducing the expression of wild-type or mutant mRNA transcripts transcribed from genes bearing nucleotide repeats, thereby preventing the manifestation of disease phenotypes.

[0081] The following sections provide a description of exemplary inhibitory nucleic acids of the present disclosure and vectors (e.g., viral vectors) encoding same, as well as methods of using such inhibitory nucleic acids and vectors for the treatment of neurological disorders.

[0082] Ataxin-2 and ataxin-2-related disorders The ATXN2 protein is a cytoplasmic protein that is a component of stress granules. Stress granules are transient intracellular compartments induced by the cessation of protein translation and contain several proteins known to be mutated in subjects with neurodegenerative diseases (Brown and Al-Chalabi, N Engl J Med (2017) 377:162-172). ATXN2 contains a sequence of glutamine residues known as a polyglutamine repeat (polyQ), which is approximately 22 amino acids long in normal individuals. Expansion of this polyglutamine repeat to a length of 34 or more is found in individuals with neurodegenerative diseases, including spinocerebellar ataxia type 2 (SCA2). This disease is characterized by the progressive death of Purkinje neurons in the cerebellum and other neuronal cell types. SCA2 patients develop ataxia, sensory impairment, and other clinical features that worsen over time. Moderate expansions of ataxin-2 polyglutamine repeats (e.g., 27–40 glutamine residues) longer than those observed in most individuals but shorter than those typically observed in subjects with SCA2 have been reported at substantially higher frequencies in individuals with the motor neuron disease amyotrophic lateral sclerosis (ALS) compared with normal controls (Elden et al., Nature (2010) 466:7310). This suggests that these intermediate-length polyglutamine repeats, i.e., polyglutamine repeats between those found in normal individuals and those found in spinocerebellar ataxia-2 ​​patients, increase the risk of ALS. Currently, treatment options for SCA2 and ALS are limited.

[0083] The pathogenic function of polyQ disease proteins that arise with polyQ expansion may result from increased toxicity associated with the development of intranuclear inclusions or increased toxicity associated with soluble toxic oligomers (Lajoie et al., PLoS One, 2011, 5: e15245). SCA2 brains are characterized by Purkinje cell loss, but SCA2 Purkinje cells lack inclusions, indicating that polyQ-expanded ataxin-2 can cause toxicity independent of inclusion formation (Huynh et al., Ann. Neurol., 1999, 45: 232-241). The functions of polyQ-expanded ataxin-2 include abnormal accumulation in the Golgi apparatus (Uynh et al., Hum. Mol. Genet., 2003, 12: 1485-1496), gain of normal function (Duvick et al., Neuron, 2010, 67: 929-935), and sequestering of transcription factors (TFs) and glyceraldehyde-3-phosphate dehydrogenase, as well as other polyQ proteins (Yamanaka et al., Methods Mol. Biol., 2010, 648, 215-229; Koshy et al., Hum. Mol. Genet., 1996, 5: 1311-1318; Burke et al., Nat. Med., 1996, 2: 347-350). Several normal functions of ataxin-2 have been characterized. Ataxin-2 is present in stress granules and P bodies, suggesting that it functions in sequestration of mRNA and regulation of protein translation during stress (Nonhoff et al., Mol. Biol. Cell, 2007, 18:1385-1396). Overexpression of ataxin-2 prevented P body assembly, whereas underexpression prevented stress granule assembly (Nonhoff et al., Mol. Biol. Cell, 2007, 18:1385-1396).Interactions with poly(A)-binding protein 1, the RNA splicing factor A2BP1 / Fox1, and polyribosomes further support a role for ataxin-2 in RNA metabolism (Shibata et al., Hum. Mol. Genet., 2000, 9: 1303-1313; Ciosk et al., Development, 2004, 131: 4831-4841; Satterfield et al., Hum. Mol. Genet., 2006, 15: 2523-2532). Ataxin-2 is a regulator of EGF receptor internalization and signal transduction through interactions with SRC kinase and the endocytic protein CIN85 (Nonis et al., Cell Signal., 2008, 20: 1725-1739). Ataxin-2 also interacts with the ALS-associated protein TDP-43 in an RNA-dependent manner, and familial and sporadic ALS are associated with the occurrence of a long normal CAG repeat expansion, ATXN2 (Elden et al., Nature, 2010, 466: 1069-1075; Van Damme et al., Neurology, 2011, 76: 2066-2072).

[0084] SCA2 is an autosomal dominant neurodegenerative disease characterized by progressive functional and cellular loss of neurons in the cerebellum, brainstem, and spinal cord. SCA2 is caused by a CAG expansion in the ATXN2 gene, resulting in a polyglutamine (polyQ) expansion in the ataxin-2 protein. SCA2 patients are characterized by other neurological features, such as progressive cerebellar ataxia, slow saccadic eye movements, and neuropathy (Pulst, SM (ed.), Genetics of Movement Disorders. Elsevier, Inc., Amsterdam, 2003, pp. 19-34). Moderate CAG expansions in the ATXN2 gene have also been associated with parkinsonism or ALS indistinguishable from idiopathic forms of these diseases (Kim et al., Arch. Neurol., 2007, 64: 1510-1518; Ross et al., Hum. Mol. Genet., 2011, 20: 3207-3212; Corrado et al., Hum. Genet., 2011, 130: 575-580; Elden et al., Nature, 2010, 466: 1069-1075; Van Damme et al., Neurology, 2011, 76: 2066-2072).

[0085] Expansion of ATXN2 polyglutamine repeats to lengths of 34 or more causes SCA2. Furthermore, moderate-length polyglutamine expansions in ATXN2 increase the risk of ALS. Reduced ATXN2 levels have been shown to have therapeutic benefits in animal models of spinocerebellar ataxia-2 ​​and ALS. Knockdown of ATXN2 protein using nucleic acid-based therapy attenuates the progressive neurodegeneration that occurs in animal models expressing a variant of human ATXN2 containing an expanded polyglutamine repeat. In ALS animal models overexpressing the TDP-43 protein, a component of the most common neuropathology seen in ALS patients, the animals typically develop progressive motor neuron death. However, breeding these animals with ATXN2 knockout mice dramatically increased survival time (Elden et al., Nature (2010) 466:7310). Similarly, reducing ATXN2 protein levels by introducing antisense oligonucleotides increased survival in TDP-43 transgenic mice. Reducing ATXN2 levels significantly extended the lifespan of TDP-43 transgenic mice, improved motor function, and reduced TDP-43 inclusion burden. AXTN2 may regulate TDP-43 toxicity by affecting its aggregation tendency. TDP-43 proteopathy has been observed in numerous neurodegenerative diseases, including ALS, frontotemporal lobe dementia (FTLD), primary lateral sclerosis, progressive muscular atrophy, limbic-predominant senile TDP-43 encephalopathy, chronic traumatic encephalopathy, dementia with Lewy bodies, corticobasal degeneration, progressive supranuclear palsy (PSP), Guam Parkinsonism-Dementia Complex (G-PDC), Pick's disease, hippocampal sclerosis, Huntington's disease, Parkinson's disease, and Alzheimer's disease. Therefore, reducing ATXN2 levels may be useful in treating neurodegenerative diseases in which ATXN2 is a causative agent (e.g., SCA2), as well as neurodegenerative diseases in which ATXN2 is not a causative agent but regulates the pathological aggregation of TDP-43.

[0086] Aspects of the present disclosure relate to interfering RNA molecules (e.g., small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), including artificial miRNA), which, when administered to a subject, inhibit the expression or activity of ataxin-2 in the subject. Accordingly, the compositions and methods provided herein are useful for treating neurodegenerative diseases, including conditions associated with SCA2, ALS, Huntington's disease, Alzheimer's disease, FTLD, Parkinsonism, and TDP-43 proteopathy.

[0087] inhibitory nucleic acid In one aspect, the present disclosure provides an isolated inhibitory nucleic acid that inhibits the expression or activity of ataxin 2 (ATXN2). The inhibitory nucleic acid is a nucleic acid that specifically binds to (e.g., hybridizes with) at least a portion of an ATXN2 nucleic acid, such as ATXN2 RNA, pre-mRNA, or mRNA, and inhibits its expression or activity. In some embodiments, the inhibitory nucleic acid is complementary to a protein-coding region or a non-coding region (e.g., 5'UTR, 3'UTR, intron, etc.) of ATXN2. In some embodiments, the inhibitory nucleic acid is complementary to a wild-type ATXN2 nucleic acid or a naturally occurring variant thereof. In some embodiments, the ATXN2 allele contains approximately 22 CAG trinucleotide repeats. In some embodiments, the ATXN2 allele has at least 22 CAG trinucleotide repeats, at least 23 CAG trinucleotide repeats, at least 24 CAG trinucleotide repeats, at least 25 CAG trinucleotide repeats, at least 26 CAG trinucleotide repeats, at least 27 CAG trinucleotide repeats, at least 28 CAG trinucleotide repeats, at least 29 CAG trinucleotide repeats, at least 30 CAG trinucleotide repeats, at least 31 CAG trinucleotide repeats, at least 32 CAG trinucleotide repeats, at least 33 CAG trinucleotide repeats, at least 34 The inhibitory nucleic acid may have at least 35 CAG trinucleotide repeats, at least 36 CAG trinucleotide repeats, at least 37 CAG trinucleotide repeats, at least 38 CAG trinucleotide repeats, at least 39 CAG trinucleotide repeats, at least 40 CAG trinucleotide repeats, at least 50 CAG trinucleotide repeats, at least 60 CAG trinucleotide repeats, at least 70 CAG trinucleotide repeats, at least 80 CAG trinucleotide repeats, at least 90 CAG trinucleotide repeats, or at least 100 or more CAG trinucleotide repeats. In some embodiments, the inhibitory nucleic acid is single-stranded or double-stranded.In some embodiments, the inhibitory nucleic acid is an interfering RNA molecule, such as a short interfering RNA (siRNA), a short hairpin RNA (shRNA), a microRNA (miRNA), or a double-stranded RNA (shRNA).

[0088] In some embodiments, the inhibitory nucleic acid is an miRNA. The miRNA can be a pri-mRNA, pre-mRNA, mature miRNA, or artificial miRNA. In some embodiments, the miRNA is composed of a guide strand and a passenger strand. In some embodiments, the guide strand and the passenger strand are within the same nucleic acid strand, and the guide strand and the passenger strand hybridize to each other to form a self-annealing duplex structure. The miRNA is initially transcribed as a pri-mRNA, which is processed by nuclear nucleases (e.g., the Drosia-DGCR8 complex) to form a pre-mRNA. The pri-mRNA is a single-stranded molecule with a stem-loop structure. The pre-mRNA is also a single-stranded molecule with a stem-loop structure. The pre-miRNA is transported from the nucleus to the cytoplasm by exportin-5 and further processed by Dicer to generate a mature double-stranded miRNA duplex containing the guide strand and the passenger strand. The mature miRNA duplex is then incorporated into the RNA-induced silencing complex (RISC), which is mediated by TRBP (HIV transactivation response RNA-binding protein). The passenger strand is generally released and cleaved, while the guide strand remains in the RISC, binds to target mRNA, and mediates silencing. In some embodiments, mature miRNA refers to the guide strand of the mature miRNA duplex.

[0089] Artificial miRNA refers to the scaffold or backbone of endogenous, modified, or synthetic pri-mRNA or pre-mRNA, which can produce functional mature miRNA, and the guide strand sequence and passive strand sequence of the miRNA duplex in the stem region are replaced with the desired guide strand sequence and passive strand sequence that directs the silencing of the desired target mRNA.Artificial miRNA design is described in Eamens et al. (2014) Methods Mol Biol. 1062:211-24 (incorporated by reference in its entirety).Synthetic miRNA backbone is described in US Patent Publication No. 2008 / 0313773 (incorporated by reference in its entirety).

[0090] Inhibitory nucleic acid constructs, such as the interfering RNA constructs described herein, can be in any of a variety of forms, such as siRNA, shRNA, or miRNA. The interfering RNAs described herein can also be encoded by vectors, such as viral vectors. For example, adeno-associated virus (AAV) vectors, such as pseudotyped AAV vectors (e.g., AAV2 / 8 and AAV2 / 9 vectors), are described herein, which contain transgenes encoding interfering RNA constructs that attenuate the expression of wild-type or mutant RNA transcripts (e.g., RNA transcripts with expanded nucleotide repeats).

[0091] Among other benefits, the compositions and methods described herein offer the advantageous feature of being able to selectively suppress the expression of wild-type or pathogenic RNA transcripts among other RNAs containing expanded nucleotide repeat regions. This property is particularly beneficial given the prevalence of nucleotide repeats in mammalian genomes, such as those of human patients. The compositions and methods described herein can be used to reduce the expression of wild-type or mutant RNA transcripts containing pathogenic nucleotide repeat expansions while maintaining the expression of important healthy RNA transcripts and their encoded protein products.

[0092] This advantageous feature is based in part on the surprising discovery that inhibitory nucleic acid constructs that anneal to wild-type or repeat-expanded RNA targets can be used to suppress the expression of these RNA transcripts. Thus, the compositions and methods described herein can attenuate the expression of wild-type or pathogenic RNA transcripts.

[0093] The following sections provide a description of exemplary inhibitory nucleic acid constructs, such as interfering RNA constructs, that may be used in combination with the compositions and methods described herein, as well as vectors encoding such constructs and procedures that may be used, including to treat diseases associated with expression of wild-type or mutant ATXN2.

[0094] Interfering RNA The compositions and methods described herein can be used to suppress the expression of RNA transcripts by administering an interfering RNA molecule, a composition containing the same, or a vector encoding the same to a patient having a disease characterized by expression of wild-type or mutant ATXN2.

[0095] Exemplary interfering RNA molecules that may be used in combination with the compositions and methods described herein for the treatment of diseases associated with wild-type or mutant ATXN2 expression, such as SCA2, ALS, Huntington's disease, FTD, TDP-43 proteopathy, and Parkinson's disease, are siRNA molecules, miRNA molecules, and shRNA molecules, among others. In the case of siRNA molecules, the siRNA can be single-stranded or double-stranded. In contrast, miRNA molecules are single-stranded molecules that form hairpins, thereby adopting a hydrogen-bonding structure reminiscent of a nucleic acid duplex. In either case, the interfering RNA can contain an antisense, or "guide," strand that anneals (e.g., via complementarity) to the repeat-expanded mutant RNA target. The interfering RNA can also contain a "passenger" strand that is complementary to the guide strand and thus has the same nucleic acid sequence as the RNA target.

[0096] Exemplary interfering RNA molecules that anneal to ATXN2 RNA can be used in combination with the compositions and methods described herein for the treatment of diseases associated with expression of wild-type or mutant ATXN2, as shown in Table 2 below. [Table 2] TIFF2025531201000003.tif208165TIFF2025531201000004.tif230165TIFF20255312010 00005.tif228165TIFF2025531201000006.tif228165TIFF2025531201000007.tif222165

[0097] Methods for treating diseases characterized by expression of wild-type or mutant ATXN2 Using the compositions and methods described herein, patients experiencing and / or having a disease associated with wild-type or mutant ATXN2 expression, such as SCA2, ALS, Huntington's disease, FTD, TDP-43 proteopathy, Parkinson's disease, among others, can be administered an inhibitory nucleic acid construct, such as an interfering RNA construct, e.g., a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a microRNA (miRNA), or a vector encoding the same, to reduce the expression of the wild-type or mutant RNA transcript.

[0098] In another aspect, the present disclosure provides a method for inhibiting ATXN2 expression or activity in a cell, the method comprising administering to the cell a composition of the present disclosure (e.g., an inhibitory nucleic acid, an isolated nucleic acid comprising an expression construct encoding the inhibitory nucleic acid, a vector, an rAAV particle, a pharmaceutical composition), thereby inhibiting ATXN2 expression or activity in the cell. In some embodiments, the cell is a CNS cell. In some embodiments, the cell is a non-neuronal cell of the CNS or a neuronal cell. In some embodiments, the non-neuronal cell of the CNS is a glial cell, an astrocyte, or a microglial cell. In some embodiments, the cell is in vitro. In some embodiments, the cell is derived from a subject having one or more symptoms of a neurodegenerative disease or suspected of having a neurodegenerative disease. In some embodiments, the cells express ATXN2 with at least 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, 100, or more CAG trinucleotide (polyglutamine) repeats. In some embodiments, the cells express ATXN2 with about 22, or 23 repeats, 24-32 repeats, or 33-100, or more repeats.

[0099] In another aspect, the present disclosure provides a method for inhibiting expression or activity of ATXN2 in the central nervous system of a subject, the method comprising administering a composition of the present disclosure (e.g., an inhibitory nucleic acid, an isolated nucleic acid comprising an expression construct encoding an inhibitory nucleic acid, a vector, an rAAV particle, a pharmaceutical composition) to the subject, thereby inhibiting expression or activity of ATXN2 in the cells.

[0100] In another aspect, the present disclosure provides a method for treating a subject having or suspected of having a neurodegenerative disease, the method comprising administering to the subject a composition of the present disclosure (e.g., an inhibitory nucleic acid, an isolated nucleic acid comprising an expression construct encoding an inhibitory nucleic acid, a vector, an rAAV particle, a pharmaceutical composition) to the subject, thereby treating the subject. As used herein, the term "treating" includes preventing or delaying the onset of a neurodegenerative disease (e.g., SCA2, ALS / FTLD, Huntington's disease, Alzheimer's disease, Parkinson's disease, etc.), reducing the severity of a neurodegenerative disease, reducing or preventing the onset of symptoms characteristic of a neurodegenerative disease, preventing the worsening of symptoms characteristic of a neurodegenerative disease, or any combination thereof.

[0101] Neurodegenerative diseases that can be treated in a subject using the compositions of the present disclosure include neurodegenerative diseases in which ATXN2 is a causative agent (e.g., SCA2), as well as neurodegenerative diseases in which ATXN2 is not a causative agent (e.g., a direct cause) but alters the pathological aggregation of TDP-43.

[0102] Neurodegenerative diseases associated with TDP-43 proteopathy include ALS, FTLD, primary lateral sclerosis, progressive muscular atrophy, limbic-predominant senile TDP-43 encephalopathy, chronic traumatic encephalopathy, dementia with Lewy bodies, corticobasal degeneration, progressive supranuclear palsy (PSP), Guam Parkinsonism-Dementia Complex (G-PDC), Pick's disease, Perry syndrome, cerebral aging-associated TDP-43 sclerosis (CARTS), hippocampal sclerosis, Huntington's disease, Parkinson's disease, and Alzheimer's disease.

[0103] In some embodiments, the neurodegenerative disease is SCA2. In some embodiments, the subject with SCA2 can be the subject with multiple CAG trinucleotide repeat mutations in the ATXN2 locus, for example, at least 34, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90 or 100 CAG repeats. ATXN2 is a confirmed target for treating SCA2, as described in Giunti et al. Brain 121, 459-467 (1998).

[0104] In some embodiments, the neurodegenerative disease is ALS.In some embodiments, the subject with ALS can be the subject with multiple CAG trinucleotide repeat mutations in ATXN2 locus, for example, at least 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 CAG repeats.ATXN2 is a confirmed target for treating ALS, as described in Wang et al.PLoS ONE 9(8): e105534 (2014)).

[0105] In some embodiments, the neurodegenerative disease is Huntington's disease. In some embodiments, the subject with Huntington's disease can be a subject with upregulated ATXN2. ATXN2 is a validated target for treating Huntington's disease, as described in Xu et al. PLoS Genet 15(10): e1008356 (2019).

[0106] In some embodiments, the subject is characterized as having an ATXN2 allele having at least 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, 100, or more CAG trinucleotide (polyglutamine) repeats. In some embodiments, the subject is characterized as having an ATXN2 allele having about 22 or 23 repeats, 24-32 repeats, or 33-100, or more repeats.

[0107] In some embodiments, the therapeutic methods of the present disclosure reduce, prevent, or delay the onset or progression of one or more symptoms characteristic of a neurodegenerative disease. Examples of symptoms characteristic of a neurodegenerative disease include motor dysfunction, cognitive dysfunction, emotional / behavioral dysfunction, or any combination thereof. Paralysis, tremors, dizziness, stiffness, rigidity, convulsions, muscle weakness, muscle spasms, muscle rigidity, muscle atrophy, difficulty swallowing, difficulty breathing, speech and language difficulties (e.g., slurred speech), slowness of movement, difficulty walking, dementia, depression, anxiety, or any combination thereof.

[0108] In some embodiments, the therapeutic methods of the present disclosure include administration as a monotherapy or in combination with one or more additional therapies for the treatment of a neurodegenerative disease. Combination therapy can refer to the administration of a composition of the present disclosure (e.g., an inhibitory nucleic acid, an isolated nucleic acid comprising an expression construct encoding an inhibitory nucleic acid, a vector, a rAAV particle, a pharmaceutical composition) to a subject simultaneously with, before, or after one or more additional therapies. Simultaneous administration of a combination therapy can refer to the administration of a composition of the present disclosure (e.g., an inhibitory nucleic acid, an isolated nucleic acid comprising an expression construct encoding an inhibitory nucleic acid, a vector, a rAAV particle, a pharmaceutical composition) and an additional therapy formulated to be administered in the same dosage form or in separate dosage forms.

[0109] In some embodiments, one or additional therapies that may be used in combination with the inhibitory nucleic acids of the present disclosure include inhibitory nucleic acids or antisense oligonucleotides that target neurodegenerative disease-associated genes or transcripts, gene editing agents that target neurodegeneration-associated genes (e.g., CRISPR, TALEN, ZFN-based systems), agents that reduce oxidative stress, such as free radical scavengers (e.g., Radicava (edaravone), bromocriptine), antiglutamate agents (e.g., riluzole, topiramate, lamotrigine, dextromethorphan, gabapentin, and AMPA receptor antagonists (e.g., talampanel)). , anti-apoptotic agents (e.g., minocycline, sodium phenylbutyrate, arimoclomol), anti-inflammatory agents (e.g., gangliosides, celecoxib, cyclosporine, nimesulide, azathioprine, cyclophosphamide, plasmapheresis, glatimelamer acetate, and thalidomide), beta-lactam antibiotics (penicillin and its derivatives, ceftriaxone, and cephalosporins), dopamine agonists (pramipexole, dexpramipexole), and neurotrophic factors (e.g., IGF-1, GDNF, BDNF, CTNF, VEGF, colivelin, zaliproden, thyrotrophin-releasing hormone, and ADNF).

[0110] In some embodiments, the subject treated with any of the methods described herein is a mammal (e.g., mouse, rat), preferably a primate (e.g., monkey, chimpanzee), or a human.

[0111] In any of the treatment methods described herein, the compositions of the disclosure (e.g., inhibitory nucleic acids, isolated nucleic acids comprising expression constructs encoding inhibitory nucleic acids, vectors, rAAV particles, pharmaceutical compositions) can be administered to a subject by routes including intrathalamic, intrathecal, sublaminar, intraparenchymal, intrastriatal, intracranial, intracisternal, intracerebral, intraventricular, intraocular (e.g., intravitreal), intraventricular, intralumbar, intravenous, intramuscular, subcutaneous, intraperitoneal, intradermal, transdermal, parenteral, intranasal, transdermal, intratracheal, intraarterial, intravascular, and oral administration, inhalation, perfusion, lavage, or any combination thereof.

[0112] In some embodiments, a composition of the present disclosure (e.g., an inhibitory nucleic acid, an isolated nucleic acid comprising an expression construct encoding an inhibitory nucleic acid, a vector, a rAAV particle, a pharmaceutical composition) is directly injected into the CNS of a subject. In some embodiments, the direct injection into the CNS is intrathalamic, intracerebral, intraparenchymal, intrathecal, intrastriatal, subpial, or any combination thereof. In some embodiments, the direct injection into the CNS is direct injection into the cerebrospinal fluid (CSF) of the subject, and optionally, the direct injection is intrapleural, intraventricular, intralumbar, or any combination thereof.

[0113] Vectors for delivery of inhibitory nucleic acids Viral vectors for delivery of inhibitory nucleic acids Viral genomes provide a rich source of vectors that can be used to efficiently deliver genes of interest into the genomes of target cells (e.g., mammalian cells, such as human cells) within a patient. Viral genomes are particularly useful gene delivery vectors because the polynucleotides contained within their genomes are typically integrated into the genomes of target cells by general transduction or specific transduction. These processes occur as part of the natural viral replication cycle and do not require additional proteins or reagents to induce gene integration. Examples of viral vectors that may be used in conjunction with the compositions and methods described herein include AAV, retroviruses, adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvoviruses (e.g., adeno-associated viruses), coronaviruses, negative-strand RNA viruses such as orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies virus and vesicular stomatitis virus), paramyxoviruses (e.g., measles and Sendai), positive-strand RNA viruses such as picornaviruses and alphaviruses, as well as double-stranded DNA viruses, including adenoviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., vaccinia, mutant vaccinia Ankara (MVA), fowlpox, and canarypox). Other viruses that may be used in combination with the compositions and methods described herein include, for example, Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, and hepatitis virus.Examples of retroviruses include avian leukosis sarcoma viruses, mammalian C, B, and D viruses, the HTLV-BLV group, lentiviruses, and spumaviruses (Coffin, JM, Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, BN Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996). Other examples include murine leukemia virus, murine sarcoma virus, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, gibbon leukemia virus, Mason-Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus, and lentiviruses. Other examples of vectors are described, for example, in US Pat. No. 5,801,030, the disclosure of which is incorporated herein by reference with respect to viral vectors for use in gene therapy.

[0114] AAV vectors for nucleic acid delivery In some embodiments, inhibitory nucleic acid constructs, such as the interfering RNA constructs described herein (e.g., small interfering RNA (siRNA), short hairpin RNA (shRNA), or microRNA (miRNA)), are incorporated into recombinant AAV (rAAV) vectors to facilitate their introduction into cells. rAAV vectors useful in combination with the compositions and methods described herein include recombinant nucleic acid constructs containing (1) a transgene encoding an inhibitory nucleic acid construct, such as an interfering RNA construct described herein (e.g., siRNA, shRNA, or miRNA), and (2) one or more nucleic acids that drive expression of a heterologous gene. The viral nucleic acid may include those sequences of AAV required in cis for DNA replication and packaging (e.g., functional ITRs) into virions. Such rAAV vectors may also contain a marker gene or reporter gene. Useful rAAV vectors include those lacking all or part of one or more native AAV genes but retaining functional flanking ITR sequences. The AAV ITRs may be from any serotype (e.g., from serotype 2) suitable for a particular application. Methods for using rAAV vectors are described, for example, in Tal et al., J. Biomed. Sci. 7:279-291 (2000), and Monahan and Samulski, Gene Delivery 7:24-30 (2000), the disclosures of each of which are incorporated herein by reference as they relate to AAV vectors for gene delivery.

[0115] The nucleic acids and vectors described herein can be incorporated into rAAV virions to facilitate the introduction of the nucleic acids or vectors into cells. The AAV capsid protein constitutes the outer, non-nucleic acid portion of the virion and is encoded by the AAV cap gene. The cap gene encodes three viral coat proteins, VP1, VP2, and VP3, required for virion assembly. Construction of rAAV virions is described, for example, in U.S. Pat. Nos. 5,173,414, 5,139,941, 5,863,541, 5,869,305, 6,057,152, and 6,376,237, as well as Rabinowitz et al., J. Virol. 76:791-801 (2002), and Bowles et al., J. Virol. 77:423-432 (2003), the disclosures of each of which are incorporated herein by reference as they relate to AAV vectors for gene delivery.

[0116] rAAV virions useful in conjunction with the compositions and methods described herein include those derived from various AAV serotypes, including AAV1, 2, 3, 4, 5, 6, 7, 8, and 9. The construction and use of AAV vectors of different serotypes and AAV proteins are described, for example, in Chao et al., Mol. Ther. 2:619-623 (2000), Davidson et al., Proc. Natl. Acad. Sci. USA 97:3428-3432 (2000), Xiao et al., J. Virol. 72:2224-2232 (1998), Halbert et al., J. Virol. 74:1524-1532 (2000), Halbert et al., J. Virol. 75:6615-6624 (2001), and Auricchio et al., Hum. Molec. Genet. 10:3075-3081. (2001), the disclosures of each of which are incorporated herein by reference as they relate to AAV vectors for gene delivery.

[0117] Pseudotyped rAAV vectors are also useful in conjunction with the compositions and methods described herein. Pseudotyped vectors include AAV vectors of a given serotype (e.g., AAV9) pseudotyped with a capsid gene from a serotype other than the given serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, etc.). For example, a representative pseudotyped vector is an AAV2 vector encoding a therapeutic protein pseudotyped with a capsid gene from AAV serotype 8 or AAV serotype 9. In some embodiments, the pseudotyped AAV has ITRs from one AAV serotype (e.g., AAV2) and VP1, VP2, and / or VP3 capsid proteins from a different AAV serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhlO, or AAVrhl74). Techniques for constructing and using pseudotyped rAAV viral virions are known in the art and are described, for example, in Duan et al., J. Virol. 75:7662-7671 (2001), Halbert et al., J. Virol. 74:1524-1532 (2000), Zolotukhin et al., Methods, 28:158-167 (2002), and Auricchio et al., Hum. Molec. Genet., 10:3075-3081 (2001).

[0118] In some embodiments, the AAV comprises a capsid disclosed, for example, in WO2017 / 218842, the disclosure of which is incorporated herein by reference. In some embodiments, the AAV comprises a capsid protein disclosed in Lin et al. Mol Brain 13:138 (2020), the disclosure of which is incorporated herein by reference. In some embodiments, the AAV comprises an AAV2-retro or AAV9-retro capsid protein.

[0119] AAV virions with mutations in the virion capsid can be used to effectively infect specific cell types even with non-mutated capsid virions. For example, suitable AAV mutants can have ligand insertion mutations to facilitate targeting of AAV to specific cell types. Construction and characterization of AAV capsid mutants, including insertion mutants, alanine screening mutants, and epitope tag mutants, are described in Wu et al., J. Virol. 74:8635-45 (2000). Other rAAV virions that can be used in the methods of the present invention include capsid hybrids generated by molecular breeding of viruses and exon shuffling. See, for example, Soong et al., Nat. Genet., 25:436-439 (2000), and Kolman and Stemmer, Nat. Biotechnol. 19:423-428 (2001).

[0120] Additional Methods for Delivery of Inhibitory Nucleic Acids Transfection techniques Techniques that can be used to introduce transgenes, such as transgenes encoding the inhibitory nucleic acids described herein, into target cells (e.g., target cells from or within a human patient suffering from RNA dominance) are known in the art. For example, electroporation can be used to permeabilize mammalian cells (e.g., human target cells) by applying an electrostatic potential to the cells of interest. Mammalian cells, such as human cells, subjected to an external electric field in this manner are then susceptible to the uptake of exogenous nucleic acids. Electroporation of mammalian cells is described in detail, for example, in Chu et al., Nucleic Acids Research 15:1311 (1987), the disclosure of which is incorporated herein by reference. A similar technique, Nucleofection®, utilizes an applied electric field to stimulate the uptake of exogenous polynucleotides into the nucleus of eukaryotic cells. Nucleofection® and protocols useful for carrying out this technique are described in detail, for example, in Distler et al., Experimental Dermatology 14:315 (2005), and US 2010 / 0317114, the disclosures of each of which are incorporated herein by reference.

[0121] Another technique useful for transfecting target cells is squeeze-poration, which induces rapid mechanical deformation of cells to stimulate the uptake of exogenous DNA through membrane pores that form in response to applied stress. This technique is advantageous in that it does not require a vector to deliver nucleic acids to cells, such as human target cells. Squeeze-poration is described in detail, for example, in Sharei et al., Journal of Visualized Experiments 81:e50980 (2013), the disclosure of which is incorporated herein by reference.

[0122] Lipofection represents another technique useful for transfecting target cells. This method involves loading nucleic acids into liposomes, which often present cationic functional groups, such as quaternary or protonated amines, toward the exterior of the liposome. Because cell membranes are anionic, this promotes electrostatic interactions between the liposome and the cell, ultimately leading to the uptake of exogenous nucleic acids, for example, by direct fusion of the liposome with the cell membrane or by endocytosis of the complex. Lipofection is described in detail, for example, in U.S. Pat. No. 7,442,386, the disclosure of which is incorporated herein by reference. A similar technique that utilizes ionic interactions with the cell membrane to induce the uptake of exogenous nucleic acids includes contacting cells with cationic polymer-nucleic acid complexes. Exemplary cationic molecules associated with polynucleotides to impart a positive charge favorable for interaction with cell membranes are activated dendrimers (e.g., as described in Dennig, Topics in Current Chemistry 228:227 (2003), the disclosure of which is incorporated herein by reference) and diethylaminoethyl (DEAE)-dextran, the use of which as transfection agents is described in detail, for example, in Gulick et al., Current Protocols in Molecular Biology 40:1:9.2:9.2.1 (1997), the disclosure of which is incorporated herein by reference. Magnetic beads are another tool that can be used to transfect target cells in a gentle and efficient manner, as this method utilizes the application of a magnetic field to induce nucleic acid uptake. This technique is described in detail, for example, in US2010 / 0227406, the disclosure of which is incorporated herein by reference.

[0123] Another useful tool for inducing the uptake of exogenous nucleic acids by target cells is laser infection, a technique that involves exposing cells to electromagnetic radiation of a specific wavelength to gently permeabilize the cells and allow polynucleotides to penetrate the cell membrane. This technique is described in detail, for example, in Rhodes et al., Methods in Cell Biology 82:309 (2007), the disclosure of which is incorporated herein by reference.

[0124] Microvesicles represent another potential vehicle that can be used to modify the genome of target cells according to the methods described herein. For example, microvesicles induced by co-overexpression of glycoprotein VSV-G and a genome-modifying protein, such as a nuclease, can be used to efficiently deliver proteins to cells and then catalyze site-specific cleavage of endogenous polynucleotide sequences to prepare the genome of the cell for covalent integration of a polynucleotide of interest, such as a gene or regulatory sequence. The use of such vesicles, also referred to as gesicles, for genetic modification of eukaryotic cells is described in detail, for example, in Quinn et al., Genetic Modification of Target Cells by Direct Delivery of Active Protein [abstract]. In: Methylation changes in early embryonic genes in cancer [abstract], in: Proceedings of the 18th Annual Meeting of the American Society of Gene and Cell Therapy; 2015 May 13, Abstract No. 122.

[0125] Insertion of a gene encoding an inhibitory nucleic acid by gene editing In addition to the above, various tools have been developed that can be used to integrate transgenes encoding inhibitory nucleic acid constructs, such as interfering RNA constructs (e.g., short interfering RNA (siRNA), short hairpin RNA (shRNA), and microRNA (miRNA) described herein), into target cells, and particularly human cells. One such method that can be used to integrate polynucleotides encoding inhibitory nucleic acids into target cells is the use of transposons. A transposon is a polynucleotide that encodes a transposase enzyme and contains a polynucleotide sequence, or gene, of interest flanked by 5' and 3' cleavage sites. Once the transposon is delivered to a cell, expression of the transposase gene begins, resulting in an active enzyme that cleaves the gene of interest from the transposon. This activity is mediated by site-specific recognition of the transposon cleavage site by the transposase. In some cases, these cleavage sites can be terminal repeat sequences or inverted terminal repeat sequences. Once excised from the transposon, the gene of interest can be integrated into the genome of a mammalian cell by transposase-catalyzed cleavage of a similar cleavage site present in the cell's nuclear genome. This inserts the gene of interest into the complementary cleavage site of the cleaved nuclear DNA, followed by covalent ligation of a phosphodiester bond connecting the gene of interest to the DNA of the mammalian cell genome, completing the integration process. In certain cases, the transposon may be a retrotransposon, in which the gene encoding the target gene is first transcribed into an RNA product and then reverse-transcribed into DNA before being integrated into the mammalian cell genome. Exemplary transposon systems are the piggybac transposon (described in detail, e.g., in WO 2010 / 085699) and the sleeping beauty transposon (described in detail, e.g., in US 2005 / 0112764), the disclosures of each of which are incorporated herein by reference with respect to transposons for use in gene delivery to cells of interest.

[0126] Another tool for integrating target genes into the genome of target cells is the clustered regularly interspaced short palindromic repeats (CRISPR) / Cas system, which originally evolved as an adaptive defense mechanism against bacterial and archaeal viral infections. The CRISPR / Cas system contains short palindromic repeats within plasmid DNA and the associated Cas9 nuclease. This DNA-protein complex first integrates foreign DNA into the CRISPR locus, thereby inducing site-specific DNA cleavage of the target sequence. Polynucleotides containing these foreign sequences and the repeat spacer elements of the CRISPR locus are transcribed in the host cell to generate guide RNAs, which then anneal to the target sequence and localize the Cas9 nuclease to the site. Thus, the proximity of Cas9 to the target DNA molecule is governed by RNA:DNA hybridization, enabling highly site-specific Cas9-mediated DNA cleavage of the foreign polynucleotide. As a result, CRISPR / Cas systems can be designed to cleave any target DNA molecule of interest. This technique has been utilized to edit eukaryotic genomes (Hwang et al., Nature Biotechnology 31:227 (2013)) and can be used as an efficient means of site-specifically editing a target cell genome to integrate a gene encoding a target gene after DNA cleavage. The use of CRISPR / Cas to regulate gene expression is described, for example, in U.S. Patent No. 8,697,359, the disclosure of which is incorporated herein by reference with respect to the use of the CRISPR / Cas system for genome editing. Alternatives for site-specific cleavage of genomic DNA followed by integration of a gene of interest into a target cell include the use of zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Unlike CRISPR / Cas systems, these enzymes do not contain guide polynucleotides that localize them to specific target sequences. Instead, target specificity is controlled by a DNA-binding domain within these enzymes.The use of ZFNs and TALENs in genome editing applications is described, for example, in Urnov et al., Nature Reviews Genetics 11:636 (2010), and Joung et al., Nature Reviews Molecular Cell Biology 14:49 (2013), the disclosures of each of which are incorporated herein by reference with respect to compositions and methods for genome editing.

[0127] An additional genome editing technology that can be used to integrate a polynucleotide encoding a target gene into the genome of a target cell is ARCUS, which can be rationally designed to site-specifically cleave genomic DNA. (商標) The use of meganucleases is included.The use of these enzymes to integrate genes encoding target genes into the genome of mammalian cells is advantageous in view of the clear structure-activity relationship established for such enzymes.By modifying single-chain meganucleases at specific amino acid positions, it is possible to produce nucleases that selectively cut DNA at desired positions, thereby enabling site-specific integration of target genes into the nuclear DNA of target cells.These single-chain nucleases are extensively described in, for example, US Patent No. 8,021,867 and US Patent No. 8,445,251, the disclosures of which are incorporated herein by reference with respect to compositions and methods for genome editing.

[0128] Methods for detecting expression of RNA transcripts The expression level of a wild-type or mutant RNA transcript, such as a wild-type or mutant ATXN2 RNA transcript, can be confirmed, for example, by various nucleic acid detection techniques. Additionally or alternatively, the expression of an RNA transcript can be estimated by assessing the concentration or relative abundance of the encoded protein produced by translation of the RNA transcript. Protein concentration can also be assessed, for example, using functional assays. These techniques can be used to monitor the expression of the encoded protein while observing a decrease in the concentration of a wild-type or pathogenic RNA transcript in response to the compositions and methods described herein. The following section describes exemplary techniques that can be used to measure the expression levels of a wild-type or pathogenic RNA transcript and its downstream protein product. Expression of the transgene can be assessed by a number of methodologies known in the art, including, but not limited to, nucleic acid sequencing, microarray analysis, proteomics, in-situ hybridization (e.g., fluorescent in-situ hybridization (FISH)), amplification-based assays, in-situ hybridization, fluorescence-activated cell sorting (FACS), Northern analysis of mRNA, and / or PCR analysis.

[0129] Nucleic Acid Detection Nucleic acid-based methods for detecting RNA transcript expression include imaging-based techniques (such as Northern blotting or Southern blotting), which can be used in combination with cells obtained from patients, for example, after administering a vector encoding an inhibitory nucleic acid construct (such as the interfering RNA, small interfering RNA (siRNA), short hairpin RNA (shRNA) or microRNA (miRNA) described herein) or after administering a composition containing such an inhibitory nucleic acid construct.Northern blot analysis is a conventional technique known in the art, and is described, for example, in Molecular Cloning, a Laboratory Manual, second edition, 1989, Sambrook, Fritch, Maniatis, Cold Spring Harbor Press, 10 Skyline Drive, Plainview, NY 11803-2500. Exemplary protocols for assessing the status of genes and gene products can be found, for example, in Ausubel et al., eds., 1995, Current Protocols In Molecular Biology, Units 2 (Northern Blotting), 4 (Southern Blotting), 15 (Immunoblotting) and 18 (PCR Analysis).

[0130] RNA detection techniques that can be used with the compositions and methods described herein to assess the expression of RNA transcripts, such as ATXN2 RNA transcripts, can further include microarray sequencing experiments (e.g., Sanger sequencing, also known as high-throughput sequencing or deep sequencing, and next-generation sequencing). Exemplary next-generation sequencing technologies include, but are not limited to, Illumina sequencing, Ion Torrent sequencing, 454 sequencing, SOLiD sequencing, and nanopore sequencing platforms. Additional sequencing methods known in the art can also be used. For example, transgene expression at the mRNA level can be measured using RNA-Seq (e.g., as described in Mortazavi et al., Nat. Methods 5:621-628 (2008), the disclosure of which is incorporated herein by reference). RNA-Seq is a robust technique for monitoring expression by directly sequencing RNA molecules in a sample. Briefly, this methodology involves fragmenting RNA to an average length of 200 nucleotides, converting it to cDNA by random priming, and synthesizing double-stranded cDNA (e.g., using the Just cDNA DoubleStranded cDNA Synthesis Kit from Agilent Technology®). The cDNA is then converted into molecular libraries for sequencing by adding sequence adapters (e.g., from Illumina® / Solexa) for each library, and the resulting 50-100 nucleotide reads are mapped onto the genome.

[0131] Because microarray technology offers high resolution, microarray-based platforms (e.g., single nucleotide polymorphism arrays) can be used to measure RNA expression levels. Details of various microarray methods can be found in the literature. See, for example, U.S. Patent No. 6,232,068 and Pollack et al., Nat. Genet. 23:41-46 (1999), the entire disclosures of each of which are incorporated herein by reference. Using nucleic acid microarrays, mRNA samples are reverse transcribed and labeled to generate cDNA. The probes can then be hybridized to one or more complementary nucleic acids that are aligned and immobilized on a solid support. Arrays can be configured, for example, so that the sequence and location of each element of the array are known. Hybridization of a labeled probe with a specific array member indicates that the sample from which the probe was derived expresses that gene. Expression levels can be quantified according to the amount of signal detected from the hybridized probe-sample complex. A typical microarray experiment involves the following steps: 1) preparation of fluorescently labeled targets from RNA isolated from a sample, 2) hybridization of the labeled targets to the microarray, 3) washing, staining, and scanning the array, 4) analysis of the scanned image, and 5) generation of a gene expression profile. One example of a microarray processor is the Affymetrix GENECHIP® system, which is commercially available and includes arrays manufactured by direct synthesis of oligonucleotides on a glass surface. Other systems known to those skilled in the art may also be used.

[0132] Amplification-based assays can also be used to measure the expression level of a specific RNA transcript, such as a wild-type or mutant ATXN2 transcript. In such assays, the nucleic acid sequence of a gene serves as a template in an amplification reaction (e.g., PCR, such as qPCR). In quantitative amplification, the amount of amplified product is proportional to the amount of template in the original sample. Comparison with an appropriate control results in a measurement of the expression level of the gene of interest transcript corresponding to the specific probe used, according to the principles described herein. Real-time qPCR methods using TaqMan probes are known in the art. Detailed protocols for real-time qPCR are provided, for example, in Gibson et al., Genome Res. 6:995-1001 (1996) and Heid et al., Genome Res. 6:986-994 (1996), the disclosures of each of which are incorporated herein by reference in their entirety. The expression levels of the RNA transcripts described herein can be measured by RT-PCR technology. Probes used in PCR may be labeled with a detectable marker, such as, for example, a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator, or an enzyme.

[0133] Protein detection Expression of an RNA construct can also be inferred by analyzing the expression of the protein encoded by the construct. Protein levels can be assessed using standard detection techniques known in the art. Protein expression assays suitable for use in conjunction with the compositions and methods described herein include proteomic approaches, immunohistochemistry and / or Western blot analysis, immunoprecipitation, molecular binding assays, ELISA, enzyme-linked immunofiltration assays (ELIFA), mass spectrometry, mass spectrometry immunoassays, and biochemical enzyme activity assays. In particular, proteomic methods can be used to generate multiplexed, large-scale protein expression datasets. Proteomic methods can utilize mass spectrometry to detect and quantify polypeptides (e.g., proteins) and / or peptide microarrays that utilize capture reagents (e.g., antibodies) specific for a panel of target proteins to identify and measure the expression levels of proteins expressed in a sample (e.g., a single-cell sample or a multi-cell population).

[0134] An exemplary peptide microarray has multiple polypeptides bound to a substrate, and the binding of an oligonucleotide, peptide, or protein to each of the multiple binding polypeptides is separately detectable. Alternatively, the peptide microarray can include multiple binding agents, including, but not limited to, monoclonal antibodies, polyclonal antibodies, phage display binding agents, yeast two-hybrid binding agents, and aptamers, that are capable of specifically detecting a particular oligonucleotide, peptide, or protein. Examples of peptide arrays can be found in U.S. Patent Nos. 6,268,210, 5,766,960, and 5,143,854, the disclosures of each of which are incorporated herein by reference in their entirety.

[0135] Mass spectrometry (MS) can be used in combination with the methods described herein to identify and characterize transgene expression in cells from a patient (e.g., a human patient) after transgene delivery. Any MS method known in the art can be used to determine, detect, and / or measure proteins or peptide fragments of interest, such as LC-MS, ESI-MS, ESI-MS / MS, MALDI-TOF-MS, MALDI-TOF / TOF-MS, tandem MS, etc. Mass spectrometers generally contain an ion source, optical elements, a mass analyzer, and data processing electronics. Mass analyzers including scanning and ion beam mass analyzers, such as time-of-flight (TOF) and quadrupole (Q), as well as trap-type mass analyzers, such as ion trap (IT) and Orbitrap, and Fourier transform ion cyclotron resonance (FT-ICR), can be used in the methods described herein. Details of various MS methods can be found in the literature. See, e.g., Yates et al., Annu. Rev. Biomed. Eng. 11:49-79, 2009, the disclosure of which is incorporated by reference in its entirety.

[0136] Prior to MS analysis, proteins in samples obtained from patients can first be broken down into smaller peptides by chemical (e.g., cyanogen bromide cleavage) or enzymatic (e.g., trypsin) digestion. Complex peptide samples also benefit from the use of front-end separation techniques, such as 2D-PAGE, HPLC, RPLC, and affinity chromatography. The broken down and optionally separated sample is then ionized using an ion source to generate charged molecules for further analysis. Sample ionization can be performed by, for example, electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), photoionization, electron ionization, fast atom bombardment (FAB) / liquid secondary ionization (LSIMS), matrix-assisted laser desorption / ionization (MALDI), field ionization, field desorption, thermospray / plasma spray ionization, and particle beam ionization. Additional information regarding the selection of ionization methods is known to those skilled in the art.

[0137] After ionization, the resolved peptides are fragmented to generate a signature MS / MS spectrum. Tandem MS, also known as MS / MS, can be used, particularly for analyzing complex mixtures. Tandem MS involves multiple steps of MS selection, with some form of ion fragmentation occurring between steps. This can be achieved using spatially separated, individual mass analyzer elements, or using a single mass analyzer with time-separated MS steps. In spatially separated tandem MS, the elements are physically separated and distinct, and are physically connected to maintain a high vacuum. In time-separated tandem MS, separation is achieved by co-located trapped ions, with multiple separation steps occurring over time. The signature MS / MS spectrum was then compared against a peptide sequence database (e.g., SEQUEST). Post-translational modifications to the peptide can also be determined, for example, by searching the spectrum against the database, allowing for specific peptide modifications.

[0138] Pharmaceutical Composition Inhibitory nucleic acid constructs, such as interfering RNA constructs (e.g., small interfering RNA (siRNA), short hairpin RNA (shRNA), or microRNA (miRNA)), as well as vectors and compositions encoding or containing such constructs, are incorporated into vehicles for administration to patients, e.g., human patients suffering from disorders described herein. Pharmaceutical compositions containing vectors, such as viral vectors, encoding the inhibitory nucleic acid constructs described herein can be prepared using methods known in the art. For example, such compositions can be prepared in a desired form, e.g., a lyophilized formulation or an aqueous solution, using, e.g., physiologically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980); incorporated herein by reference).

[0139] Mixtures of the nucleic acids and viral vectors described herein can be prepared in water, suitably mixed with one or more excipients, carriers, or diluents. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, as well as in oils. These preparations may contain preservatives to prevent the growth of microorganisms under ordinary storage and use conditions. Pharmaceutical forms suitable for injection use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (as described in U.S. Pat. No. 5,466,468, the disclosure of which is incorporated herein by reference). In any case, the formulation may be sterile and may have sufficient fluidity for easy injection. The formulation may be stable under the conditions of manufacture and storage and may be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and / or vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of injectable compositions can be brought about by the use in the composition of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0140] For example, solutions containing the pharmaceutical compositions described herein are suitably buffered, if necessary, and the liquid diluent is first made isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for administration via routes selected from intrathalamic, intrathecal, intrapituitary, intrapleural, intracranial, intracranial, intrasternal, intracerebral, intraventricular, intraocular (e.g., intravitreal), intracerebroventricular, intralumbar, intravenous, intramuscular, subcutaneous, intraperitoneal, intradermal, transdermal, parenteral, intranasal, transdermal, intratracheal, intraarterial, intravascular, and oral administration, inhalation, perfusion, irrigation, or any combination thereof. In this regard, sterile aqueous media that can be employed will be known to those skilled in the art in light of the present disclosure. For example, one dose may be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous infusion solution or injected at the intended injection site. Some variation in dosage may necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for each subject. Moreover, for human administration, preparations can meet sterility, pyrogenicity, general safety, and purity standards as required by FDA Office of Biologics standards.

[0141] A pharmaceutical composition contains, for example, an inhibitory nucleic acid described herein and typically includes a pharmaceutically acceptable diluent or carrier. A pharmaceutical composition may comprise (e.g., consist of) a sterile saline solution and a nucleic acid. The sterile saline is typically pharmaceutical-grade saline. A pharmaceutical composition may comprise (e.g., consist of) sterile water and a nucleic acid. The sterile water is typically pharmaceutical-grade water. A pharmaceutical composition may comprise (e.g., consist of) a phosphate-buffered saline (PBS) and a nucleic acid. The sterile PBS is typically pharmaceutical-grade PBS.

[0142] In certain embodiments, pharmaceutical compositions comprise one or more compositions or nucleic acid molecules and one or more excipients, in certain embodiments, the excipients are selected from water, saline, alcohol, polyethylene glycol, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, and polyvinylpyrrolidone. In certain embodiments, the nucleic acid molecules can be mixed with pharmaceutically acceptable active and / or inactive substances to prepare pharmaceutical compositions or formulations. The compositions and methods for formulating pharmaceutical compositions depend on several criteria, including, but not limited to, the route of administration, the extent of the disease, or the dose to be administered.

[0143] In certain embodiments, the pharmaceutical composition comprises a nucleic acid molecule, including any pharmaceutically acceptable salt of the inhibitor, an ester of the inhibitor, or a salt of such an ester. In certain embodiments, the pharmaceutical composition comprises a nucleic acid molecule and, upon administration to a subject (e.g., a human), can provide (directly or indirectly) a biologically active metabolite or residue thereof. Thus, for example, the present disclosure also encompasses pharmaceutically acceptable salts of the inhibitor, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts. In certain embodiments, the prodrug comprises one or more conjugate groups attached to the oligonucleotide, where the conjugate groups are cleaved by endogenous enzymes in the body.

[0144] Lipid moieties have been used in a variety of ways in nucleic acid therapy. In certain such methods, nucleic acids are introduced into preformed liposomes or lipoplexes made from a mixture of cationic and neutral lipids. In certain methods, DNA complexes with mono- or polycationic lipids are formed without the presence of neutral lipids. In certain embodiments, the lipid moiety is selected to increase distribution of the pharmaceutical agent to specific cells or tissues. In certain embodiments, the lipid moiety is selected to increase distribution of the pharmaceutical agent to adipose tissue. In certain embodiments, the lipid moiety is selected to increase distribution of the pharmaceutical agent to muscle tissue.

[0145] In certain embodiments, the pharmaceutical composition comprises a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems are useful for preparing certain pharmaceutical compositions, such as those containing hydrophobic compounds. In certain embodiments, certain organic solvents, such as dimethyl sulfoxide, are used.

[0146] The pharmaceutical composition may also include one or more tissue-specific delivery molecules designed to deliver one or more pharmaceutical agents of the invention to a particular tissue or cell type, for example, in certain embodiments, the pharmaceutical composition comprises a liposome coated with a tissue-specific antibody.

[0147] In certain embodiments, the pharmaceutical composition includes a cosolvent system. A specific example of such a cosolvent system includes, for example, benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. In certain embodiments, such a cosolvent system is used for hydrophobic compounds. A non-limiting example of such a cosolvent system is the VPD cosolvent system, which is a solution of 3% w / v benzyl alcohol, 8% w / v of the nonpolar surfactant Polysorbate 80™, and 65% w / v polyethylene glycol 300 in absolute ethanol. The proportions of such a cosolvent system may vary significantly without significantly altering its solubility and toxicity characteristics. Furthermore, the identity of the cosolvent components may be varied; for example, other surfactants may be used in place of Polysorbate 80™, the fraction of polyethylene glycol may be varied, other biocompatible polymers, such as polyvinylpyrrolidone, may replace polyethylene glycol, and dextrose may be replaced with other sugars or polysaccharides.

[0148] In certain embodiments, the pharmaceutical composition is prepared for oral administration. In certain embodiments, the pharmaceutical composition is prepared for buccal administration. In certain embodiments, the pharmaceutical composition is prepared for administration by injection (e.g., intraocular (e.g., intravitreal), intravenous, subcutaneous, intramuscular, intrathalamic, intrapituitary, intrathecal, intracerebroventricular, etc.). In certain such embodiments, the pharmaceutical composition includes a carrier and is formulated in an aqueous solution, e.g., water, or a physiologically compatible buffer, e.g., Hank's solution, Ringer's solution, physiological saline buffer, or the like. In certain embodiments, other ingredients (e.g., ingredients that aid in solubility or act as preservatives) are included. In certain embodiments, injectable suspensions are prepared using appropriate liquid carriers, suspending agents, and the like. Certain pharmaceutical compositions for injection are presented in unit dosage form, e.g., in ampoules or in multi-dose containers. Certain pharmaceutical compositions for injection are suspensions, solutions, or emulsions in oily or aqueous vehicles and may contain formulatory agents, such as suspending agents, stabilizing agents, and / or dispersing agents. Particular solvents suitable for use in injectable pharmaceutical compositions include, but are not limited to, lipophilic solvents and fatty oils such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes.

[0149] Route and dosage of administration Viral vectors, e.g., AAV vectors described herein, and other vectors containing a transgene encoding an inhibitory nucleic acid of the present disclosure, can be administered to a patient (e.g., a human patient) by a variety of routes of administration. Routes of administration can vary, for example, depending on the onset and severity of the disease, and can include, for example, intradermal, transdermal, parenteral, intravenous, intramuscular, intranasal, subcutaneous, transdermal, intratracheal, intraperitoneal, intraarterial, intravascular, inhalation, perfusion, lavage, and oral administration. Intravascular administration includes delivery into a patient's vascular system. In some embodiments, administration is into a blood vessel considered a vein (intravenous), and in some administrations, administration is into a blood vessel considered an artery (intraarterial). Veins include, but are not limited to, the internal jugular vein, peripheral veins, coronary veins, hepatic veins, portal vein, saphenous vein, pulmonary veins, superior vena cava, inferior vena cava, gastric vein, splenic vein, inferior mesenteric vein, superior mesenteric vein, cephalic vein, and / or femoral vein. Arteries include, but are not limited to, coronary arteries, pulmonary arteries, brachial arteries, internal carotid arteries, aortic arches, femoral arteries, peripheral arteries, and / or ciliary arteries. It is contemplated that delivery may be via or to arterioles or capillaries.

[0150] Treatment regimens vary and often depend on the severity of the disease, as well as the age, weight, and sex of the patient. Treatment may include the administration of vectors (e.g., viral vectors) or other agents described herein as useful for introducing transgenes into target cells in various unit doses. Each unit dose typically contains a predetermined amount of the therapeutic composition. [Example]

[0151] The following examples are presented to provide one of ordinary skill in the art with a description of how the compositions and methods described herein can be used and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention.

[0152] Example 1. Determining knockdown efficacy of ATXN2-specific miRNA constructs in HEK293 cells via dual-luciferase reporter assay

[0153] the purpose The purpose of this study was to evaluate the knockdown efficacy of several ATXN2 miRNA candidates in HEK293 cells via a dual-luciferase reporter assay. This study was designed to determine the efficiency of miRNA constructs in silencing ATXN2 mRNA.

[0154] Materials and Methods HEK293 cells were transduced with an AAV vector incorporating an ATXN2 miRNA candidate for miRNA delivery and subsequent knockdown of ATXN2 mRNA. Dual-luciferase reporter assays were then performed to measure the efficacy of the knockdown achieved by the miRNA constructs compared with a negative control that did not result in significant silencing. The miRNA constructs tested are shown in the table below: [Table 3] TIFF2025531201000009.tif208165TIFF2025531201000010.tif208165TIFF20255312010 00011.tif208165TIFF2025531201000012.tif230165TIFF2025531201000013.tif143165

[0155] result As a result, we observed that ATXN2 miRNA candidates can silence ATXN2 to various degrees (Figure 1), and some miRNA constructs (miRNA1, 2, 3, 4, 6, 19, 20, 24, 25, 37, 38, 39, 45, 51) thus achieved higher knockdown efficiency compared to other miRNA constructs.

[0156] Example 2. Determination of knockdown efficacy of ATXN2-specific miRNA constructs in HEK293 cells via dual-luciferase reporter assay after plasmid transfection

[0157] the purpose The purpose of this study was to evaluate the knockdown efficacy of several ATXN2 miRNA candidates in HEK293 cells via a dual-luciferase reporter assay after plasmid transfection. This study was designed to determine the efficiency of the miRNA constructs in silencing ATXN2 mRNA.

[0158] Materials and Methods HEK293 cells were transfected with the plasmid encoding ATXN2 specific miRNA construct.Then, dual luciferase reporter assay was carried out to measure the knockdown efficiency obtained by miRNA construct compared with the negative control that does not produce significant silencing.The numbering of constructs in this example is the same as in Example 1.

[0159] result As a result, we observed that the miRNA constructs selected for this study were able to silence ATXN2. The percentage of luciferase activity relative to the negative control ranged from 20% to 54% (Figure 2A), with miRNA construct 1 demonstrating the best knockdown efficacy among those tested. Because knockdown efficacy is the inverse of the percentage of luciferase activity compared to the negative control, the lower the percentage of luciferase activity compared to the negative control, the higher the knockdown efficacy. The control (CONT) was a non-targeting miRNA and showed no knockdown.

[0160] Example 3. Determination of knockdown efficacy of ATXN2-specific miRNA constructs in HEK293 cells via probe-based quantitative PCR (qPCR) assay after plasmid transfection

[0161] the purpose The purpose of this study was to evaluate the knockdown efficacy of several endogenous ATXN2 miRNA constructs in HEK293 cells via a probe-based qPCR assay after plasmid transfection. This study was designed to determine the efficiency of the miRNA constructs in silencing ATXN2 mRNA.

[0162] Materials and Methods HEK293 cells were transfected with a plasmid encoding an ATXN2-specific miRNA construct. A probe-based qPCR assay was performed to measure the knockdown efficacy achieved by each miRNA construct compared with a negative control. This experiment was performed twice. In the first round of experiments, a larger number of ATXN2-specific miRNA constructs were tested compared with the second round. The numbering of constructs in this example is the same as in Example 1.

[0163] result As a result, we observed that the miRNA constructs selected for this study were able to silence ATXN2. The ATXN2-GAPDH mRNA signal generally ranged from 25% to 74% (Figure 2B). miRNA construct 2 showed the best knockdown efficacy among those tested. Since knockdown efficacy is the inverse of the ratio of ATXN2 to GAPDH mRNA signal, the lower the percentage value of ATXN2 to GAPDH mRNA signal, the higher the knockdown efficacy. The control (CONT) was a non-targeting miRNA and showed no knockdown.

[0164] Furthermore, we observed that the miRNA constructs selected in the second round were also able to silence ATXN2 (Figure 2C). miRNA construct 2 showed the best knockdown effect among those tested, as evidenced by the lowest percentage of mRNA expression compared to the control (CONT), a non-targeting miRNA that showed no knockdown.

[0165] Example 4. Determination of knockdown efficacy of ATXN2-specific miRNA constructs in HEK293 cells via a probe-based qPCR assay after viral transfection

[0166] the purpose The aim of this study was to evaluate the knockdown efficacy of several endogenous ATXN2 miRNA constructs in HEK293 cells via a probe-based qPCR assay after viral transfection.

[0167] Materials and Methods HEK293 cells were transduced with AAV vectors encoding several of the ATXN2-specific miRNA constructs described in Examples 1-3 above. Probe-based qPCR assays were performed to measure the efficacy of knockdown achieved by each vector compared to a negative control. The numbering of constructs in this example is the same as in Example 1.

[0168] result As a result, we observed that the miRNA constructs selected for this study were able to silence ATXN2. The ATXN2-GAPDH mRNA signal generally ranged from 54% to 86% (Figure 3). miRNA construct 2 showed the best knockdown efficacy among those tested. Since knockdown efficacy is the inverse of the ratio of ATXN2 to GAPDH mRNA signal, the lower the percentage value of ATXN2 to GAPDH mRNA signal, the higher the knockdown efficacy. The control (CONT) was a non-targeting miRNA and showed no knockdown.

[0169] Example 5. Determination of knockdown efficacy of ATXN2-specific miRNA constructs in mouse tissues following delivery of AAV miR-ATXN2 constructs into the thalamus of BAC-Q72 mice

[0170] the purpose The objective of this study was to evaluate the knockdown efficacy of ATXN2-specific miRNA constructs in mouse tissues after delivery of AAV miR-ATXN2 constructs into the thalamus of BAC-Q72 mice.

[0171] Materials and Methods AAV or phosphate-buffered saline (PBS) was injected into the thalamus of 8- to 12-week-old BAC-Q72 mice by stereotactic injection. BAC-Q72 mice are transgenic mice expressing a CAG72 repeat in the ATXN2 gene. In these mice, a 169-kb human BAC (RP11-798L5) containing the entire 150-kb human ATXN2 locus, including the regulatory region, was engineered to replace CAG22 in endogenous ATXN2 exon-1 with a CAG72 repeat. The thalamus and hypocortex from one hemisphere were harvested 6 weeks after intrathalamic injection and analyzed for mouse and human ATXN2 mRNA expression using qPCR.

[0172] After tissue collection, probe-based qPCR assays were performed using homogenized mouse tissue to measure the efficacy of knockdown achieved by each ATXN2-specific miRNA construct compared to a negative control. Vector expression was confirmed in the thalamus by measuring viral genome quantity (vg) using qPCR (data not shown).

[0173] This study was conducted twice: a small-scale pilot study using construct 2, followed by a large-scale study including multiple ATXN2-specific miRNA constructs. In the small-scale pilot study, both mouse and human ATXN2 mRNA were measured in the mouse thalamus and hypocortex, whereas in the large-scale study, only human ATXN2 mRNA was measured. Experimental groups received AAV injections containing either 1e8 viral genomes per half-day (vg / hem) or 1e9 vg / hem in the pilot study, whereas in the large-scale study, only the 1e9 vg / hem AAV dose was injected. In the small-scale pilot study, a PBS group was used as a control. The PBS group did not silence endogenous ATXN2 mRNA and served as a baseline. In the large-scale study, there were three control groups: PBS-mice injected with Dulbecco's phosphate-buffered saline, naive-uninjected mice, and neg-mice injected with a miRNA cassette not specific for ATXN2 miRNA. Silencing of endogenous ATXN2 mRNA was negligible in all three control groups, and the PBS group served as the baseline. The numbering of the constructs in this example is the same as in Example 1.

[0174] result As a result, we observed that miRNA construct 2, selected for the pilot study, was able to dose-dependently silence both human ATXN2 mRNA (Figure 4A) and mouse Atxn2 mRNA (Figure 4B) in the mouse thalamus (Figure 4A and Figure 4B, left panel) and the hypocortex (Figure 4A and Figure 4B, right panel). Animals injected with miRNA construct 2 showed lower percentages of mouse and human ATXN2 mRNA compared with controls. The PBS group did not silence endogenous ATXN2 mRNA and served as the baseline.

[0175] In a large-scale study involving multiple ATXN2-specific miRNA constructs, we observed that each of the miRNA constructs selected for this study was able to silence ATXN2 compared to the control, with miRNA construct 2 demonstrating the best knockdown efficacy among those tested. It also demonstrated the lowest percentage of human ATXN2 mRNA expression compared to the control (Figure 5). The percentage of knockdown observed for the specific miRNAs administered ranged from 12% to 45%. The PBS group did not silence endogenous ATXN2 mRNA and served as the baseline.

[0176] Other embodiments All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0177] While the invention has been described in connection with particular embodiments thereof, it will be understood that the invention is capable of further modifications, and that this application is intended to cover any variations, uses, or adaptations of the invention which generally follow the principles of the invention and include departures from the invention within known or customary practice in the art to which the invention pertains, as applicable to the essential features described above, and which comply with the scope of the claims. Other embodiments are within the scope of the following claims.

Claims

1. An inhibitory nucleic acid comprising a guide strand and a passenger strand complementary to the guide strand, wherein the guide strand has sufficient complementarity to hybridize to a region within an ataxin-2 (ATXN2) mRNA transcript having a nucleic acid sequence set forth in any one of SEQ ID NOs: 103-153.

2. 2. The inhibitory nucleic acid of claim 1, wherein the guide strand has at least 70% complementarity to a segment of 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides within the region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.

3. the ATXN2 mRNA transcript, wherein the guide strand has at least 75% complementarity to a segment of 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides within said region of the ATXN2 mRNA transcript, wherein the guide strand has the nucleic acid sequence of any one of SEQ ID NOs: 103-153; and optionally, the ATXN2 mRNA transcript, wherein the guide strand has the nucleic acid sequence of any one of SEQ ID NOs: 103-153.

3. The inhibitory nucleic acid of claim 2, having at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementarity to a segment of 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides within said region of the mRNA transcript.

4. 4. The inhibitory nucleic acid of any one of claims 1 to 3, wherein the guide strand comprises at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleotides that are completely complementary to a contiguous polynucleotide of equal length within the region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.

5. 5. The inhibitory nucleic acid of claim 4, wherein the guide strand comprises 10 to 21 contiguous nucleotides that are completely complementary to a contiguous polynucleotide of equal length within the region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103 to 153.

6. 6. The inhibitory nucleic acid of claim 5, wherein the guide strand comprises 12 to 21 contiguous nucleotides that are completely complementary to a contiguous polynucleotide of equal length within the region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103 to 153.

7. 7. The inhibitory nucleic acid of claim 6, wherein the guide strand comprises 15 to 21 contiguous nucleotides that are completely complementary to a contiguous polynucleotide of equal length within the region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103 to 153.

8. 8. The inhibitory nucleic acid of claim 7, wherein the guide strand comprises 18 to 21 contiguous nucleotides that are completely complementary to a contiguous polynucleotide of equal length within the region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103 to 153.

9. 9. The inhibitory nucleic acid of claim 8, wherein the guide strand comprises 19, 20, or 21 consecutive nucleotides that are completely complementary to a contiguous polynucleotide of equal length within the region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.

10. 10. The inhibitory nucleic acid of any one of claims 1-9, wherein the guide strand comprises no more than 9 nucleotide mismatches to a segment of 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides within the region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153, and optionally, the guide strand comprises no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or only 1 mismatch to the region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.

11. 11. The inhibitory nucleic acid of any one of claims 1 to 10, wherein said region of the ATXN2 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 103, 104, 105, 106, 121, 122, 126, 127, 139, 140, 141, 147, 149, and 153.

12. The inhibitory nucleic acid of any one of claims 1 to 11, wherein the guide strand has a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence of any one of SEQ ID NOs: 1 to 51.

13. The inhibitory nucleic acid of claim 12, wherein the guide strand has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of any one of SEQ ID NOs: 1-51.

14. 14. The inhibitory nucleic acid of claim 13, wherein the guide strand has a nucleic acid sequence that is at least 95% identical to a nucleic acid sequence of SEQ ID NOs: 1-51, and optionally, the guide strand has a nucleic acid sequence that is at least 96%, 97%, 98%, or 99% identical to a nucleic acid sequence of any one of SEQ ID NOs: 1-51.

15. The inhibitory nucleic acid of claim 14, wherein the guide strand has a nucleic acid sequence of any one of SEQ ID NOs: 1 to 51.

16. The inhibitory nucleic acid of any one of claims 12 to 15, wherein the guide strand has the nucleic acid sequence of any one of SEQ ID NOs: 1, 2, 3, 4, 19, 20, 24, 25, 37, 38, 39, 45, 47, and 51.

17. 17. The inhibitory nucleic acid of any one of claims 1 to 16, wherein the inhibitory nucleic acid comprises a hairpin having a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence of any one of SEQ ID NOs: 52 to 102.

18. 18. The inhibitory nucleic acid of claim 17, wherein the inhibitory nucleic acid comprises a hairpin having a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of any one of SEQ ID NOs: 52-102.

19. 19. The inhibitory nucleic acid of claim 18, wherein the hairpin has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of any one of SEQ ID NOs: 52-102, and optionally, the hairpin has a nucleic acid sequence that is at least 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of any one of SEQ ID NOs: 52-102.

20. 20. The inhibitory nucleic acid of claim 19, wherein the hairpin strand has the nucleic acid sequence of any one of SEQ ID NOs: 52-102.

21. 21. The inhibitory nucleic acid of any one of claims 17 to 20, wherein the hairpin strand has the nucleic acid sequence of any one of SEQ ID NOs: 52, 53, 54, 55, 70, 71, 75, 76, 88, 89, 90, 96, 98, and 102.

22. 22. The inhibitory nucleic acid of any one of claims 1 to 21, wherein the inhibitory nucleic acid is an interfering RNA molecule, optionally wherein the interfering RNA molecule is a microRNA (miRNA), a short hairpin RNA (shRNA), or a short interfering RNA (siRNA).

23. The inhibitory nucleic acid of claim 22, wherein the inhibitory nucleic acid is a miRNA.

24. 24. A viral vector comprising a transgene encoding an inhibitory nucleic acid according to any one of claims 1 to 23, optionally comprising a plurality of said transgenes (e.g., 2, 3, 4, 5 or more said transgenes).

25. 25. The viral vector of claim 24, wherein the viral vector is selected from the group consisting of adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, poxvirus, baculovirus, herpes simplex virus, vaccinia virus, and synthetic virus.

26. The viral vector of claim 25, wherein the viral vector is an AAV.

27. 27. The viral vector of claim 26, wherein the AAV is of the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhlO, or AAVrh74 serotype, or the AAV comprises an AAV2-retro or AAV9-retro capsid protein.

28. 28. The viral vector of claim 27, wherein the viral vector is a pseudotyped AAV.

29. 29. The viral vector of claim 28, wherein the pseudotyped AAV is AAV2 / 9.

30. 29. The viral vector of claim 28, wherein the pseudotyped AAV is AAV2 / 8.

31. 27. The viral vector of claim 26, wherein the AAV comprises a recombinant capsid protein.

32. 26. The viral vector of claim 25, wherein the synthetic virus is a chimeric virus, a mosaic virus, or a pseudotyped virus, and / or comprises a foreign protein, a synthetic polymer, a nanoparticle, or a small molecule.

33. A pharmaceutical composition comprising: (i) an inhibitory nucleic acid according to any one of claims 1 to 23, or a viral vector according to any one of claims 24 to 32, and (ii) a pharmaceutically acceptable excipient, carrier, or diluent.

34. 32. A method of treating a neurological disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an inhibitory nucleic acid of any one of claims 1 to 23, a viral vector of claims 24 to 32, or a pharmaceutical composition of claim 33.

35. 35. The method of claim 34, wherein the neurological disease is associated with a TAR DNA-binding protein 43 (TDP-43) proteopathy.

36. 36. The method of claim 34 or 35, wherein the neurological disease is caused by or associated with expression of wild-type or mutant form of ATXN2.

37. 37. The method of any one of claims 34-36, wherein the neurological disease is amyotrophic lateral sclerosis (ALS), frontotemporal dementia, primary lateral sclerosis, progressive muscular atrophy, limbic-predominant senile TDP-43 encephalopathy, chronic traumatic encephalopathy, dementia with Lewy bodies, corticobasal degeneration, progressive supranuclear palsy, Guam complex parkinsonism-dementia, Pick's disease, Perry syndrome, age-related TDP-43 encephalopathy with sclerosis, hippocampal sclerosis, Huntington's disease, Parkinson's disease, Alzheimer's disease, or spinocerebellar ataxia type 2 (SCA2), and optionally, the subject has multiple CAG trinucleotide repeat mutations in the endogenous ATXN2 locus.

38. 38. The method of any one of claims 34-37, wherein the inhibitory nucleic acid, viral vector, or pharmaceutical composition is administered to the subject by a route selected from intrathalamic, intrathecal, intrapituitary, intrapleural, intracerebral, intracranial, intrasternal, intracerebral, intraventricular, intraocular, intravitreal, intracerebroventricular, intralumbar, intravenous, intramuscular, subcutaneous, intraperitoneal, intradermal, transdermal, parenteral, intranasal, transdermal, intratracheal, intraarterial, intravascular, oral administration, inhalation, perfusion, lavage, or any combination thereof.

39. 39. The method of any one of claims 34 to 38, wherein the subject is a mammal, optionally wherein the mammal is a human.

40. A kit comprising an inhibitory nucleic acid described in any one of claims 1 to 23, a viral vector described in any one of claims 24 to 32, or a pharmaceutical composition described in claim 33, wherein the kit includes a package insert directing use of the kit for administering a therapeutically effective amount of the inhibitory nucleic acid, viral vector, or pharmaceutical composition to a subject, preferably the subject is a human.

41. 34. An inhibitory nucleic acid according to any one of claims 1 to 23, a viral vector according to any one of claims 24 to 32, or a pharmaceutical composition according to claim 33 for use in a method for treating a neurological disorder in a subject.

42. 42. The inhibitory nucleic acid, viral vector, or pharmaceutical composition for use according to claim 41, wherein the neurological disease is associated with a TDP-43 proteopathy.

43. 43. The inhibitory nucleic acid, viral vector, or pharmaceutical composition for use according to claim 41 or 42, wherein the neurological disease is caused by or associated with expression of wild-type or mutant forms of ATXN2.

44. 44. The inhibitory nucleic acid, viral vector, or pharmaceutical composition for use of any one of claims 41-43, wherein the neurological disease is ALS, frontotemporal dementia, primary lateral sclerosis, progressive muscular atrophy, limbic-predominant senile TDP-43 encephalopathy, chronic traumatic encephalopathy, dementia with Lewy bodies, corticobasal degeneration, progressive supranuclear palsy, Guam Parkinsonism-Dementia Complex, Pick's disease, Perry syndrome, age-related TDP-43 encephalopathy with sclerosis, hippocampal sclerosis, Huntington's disease, Parkinson's disease, Alzheimer's disease, or SCA2, and optionally the subject has multiple CAG trinucleotide repeat mutations in the endogenous ATXN2 locus.

45. The inhibitory nucleic acid, viral vector, or pharmaceutical composition for use according to any one of claims 41 to 44, wherein the subject is a mammal.

46. 46. ​​The inhibitory nucleic acid, viral vector, or pharmaceutical composition for use according to claim 45, wherein the mammal is a human.

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