Compositions and methods for treating neurological diseases

Abstract

The present disclosure features compositions and methods for treating a condition associated with the expression of a wild-type or mutant superoxide dismutase 1 (SOD1) RNA transcript, which can result in a pathological phenotype. Disclosed herein are inhibitory RNA constructs that inhibit the expression of SOD1, as well as viral vectors, such as adeno-associated viral vectors, encoding such inhibitory RNA molecules.

Description

[0001] Sequence Listing

[0002] This application contains a sequence listing that has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy was created on November 14, 2023, is named "51585-013WO2_Sequence_Listing_11_14_23" and is 43,593 bytes in size. Technical Field

[0003] The present invention relates to the field of nucleic acid biotechnology and provides compositions and methods for treating diseases associated with superoxide dismutase 1 (SOD1) expression. Background Art

[0004] Superoxide dismutase 1 (SOD1) is an enzyme that destroys free superoxide radicals in the body. It is one of the oxide dismutases in the human body and binds to copper and zinc. The SOD1 enzyme is encoded by the SOD1 gene and is associated with diseases such as amyotrophic lateral sclerosis (ALS). It is associated with familial and sporadic ALS, and mutations in this gene are the second most common cause of familial ALS. The most common mutations found in the SOD1 gene include D90A, A4V, H46R, and G93A. A4V is the most common mutation in ALS patients in the United States. Mutations in the SOD1 gene can cause oxidative damage to the mitochondrial DNA of motor neurons (such as spinal motor neurons). ALS is progressive, neurodegenerative, can be highly debilitating, and is often fatal. It primarily affects upper and lower motor neurons. Currently, there is a lack of strategies that can successfully treat and ameliorate the symptoms of ALS, Parkinson's disease, Huntington's disease, and other diseases or conditions associated with wild-type or mutant SOD1. Therefore, there is still a need for effective treatments for these pathologies. Summary of the Invention

[0005] Described herein are compositions and methods useful for treating diseases associated with expression of wild-type or mutant superoxide dismutase 1 (SOD1). Compositions useful for treating such conditions, as described herein, include inhibitory nucleic acid constructs, such as interfering RNA constructs, that inhibit expression of wild-type or mutant mRNA transcripts. Exemplary inhibitory nucleic acids disclosed herein include, but are not limited to, microRNA (miRNA) constructs, short hairpin RNA (shRNA) constructs, and short interfering RNA (siRNA) constructs. Without being limited by mechanism, these inhibitory nucleic acids can anneal to various portions of wild-type or mutant SOD1 mRNA and promote degradation of pathological transcripts through various cellular processes. The present disclosure further features vectors, such as viral vectors, encoding such inhibitory nucleic acid constructs. Exemplary viral vectors encoding inhibitory nucleic acid constructs, such as interfering RNA constructs (e.g., miRNA), described herein are adeno-associated virus (AAV) vectors, such as pseudotyped AAV2 / 8 and AAV2 / 9 vectors.

[0006] Using the compositions and methods described herein, inhibitory nucleic acids (such as interfering RNA constructs) or vectors encoding the same can be administered to patients diagnosed with diseases associated with wild-type or mutant SOD1, such as amyotrophic lateral sclerosis (ALS), Huntington's disease, and Parkinson's disease, 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 ALS, as inhibitory nucleic acid constructs or viral vectors encoding such constructs (such as AAV vectors) can be administered to such patients to reduce the expression of mRNA transcripts encoding wild-type or mutant SOD1 proteins. The most common mutations found in the SOD1 gene include D90A, A4V, H46R, and G93A. A4V is the most common mutation in amyotrophic lateral sclerosis (ALS) patients in the United States. The compositions and methods described herein can be used to treat patients expressing wild-type or mutant SOD1 mRNA (e.g., wild-type or mutant human SOD1 mRNA), for example, by inhibiting the expression of the wild-type or mutant SOD1 mRNA using an inhibitory nucleic acid construct.

[0007] In a first aspect, the disclosure features an inhibitory nucleic acid comprising a guide strand (and, optionally, a passenger strand that is complementary to the guide strand). In some embodiments, the guide strand has sufficient complementarity to hybridize to a region within a SOD1 mRNA transcript having a nucleic acid sequence of any one of SEQ ID NO: 33 to SEQ ID NO: 48. In some embodiments, the guide strand has at least 70% complementarity to a segment of 15, 16, 17, 18, 19, 20, 21 or more contiguous nucleotides within the region of the SOD1 mRNA transcript having a nucleic acid sequence of any one of SEQ ID NO: 33 to SEQ ID NO: 48.

[0008] In some embodiments, the guide strand is at least 75% complementary to a segment of 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides within the region of the SOD1 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NO: 33 to SEQ ID NO: 48. In some embodiments, the guide strand is 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% complementary to a segment of 15, 16, 17, 18, 19, 20, 21 or more consecutive nucleotides within a region of the SOD1 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NO: 33 to SEQ ID NO: 48.

[0009] In some embodiments, the guide strand is 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% complementary to a segment of 15 contiguous nucleotides within the region of the SOD1 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NO: 33 to SEQ ID NO: 48.

[0010] In some embodiments, the guide strand is 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% complementary to a segment of 16 contiguous nucleotides within the region of the SOD1 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NO: 33 to SEQ ID NO: 48.

[0011] In some embodiments, the guide strand is 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% complementary to a segment of 17 contiguous nucleotides within the region of the SOD1 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NO: 33 to SEQ ID NO: 48.

[0012] In some embodiments, the guide strand is 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% complementary to a segment of 18 contiguous nucleotides within the region of the SOD1 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NO: 33 to SEQ ID NO: 48.

[0013] In some embodiments, the guide strand is 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% complementary to a segment of 19 contiguous nucleotides within the region of the SOD1 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NO: 33 to SEQ ID NO: 48.

[0014] In some embodiments, the guide strand is 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% complementary to a segment of 20 contiguous nucleotides within a region of the SOD1 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NO: 33 to SEQ ID NO: 48.

[0015] In some embodiments, the guide strand is 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% complementary to a segment of 21 contiguous nucleotides within a region of the SOD1 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NO: 33 to SEQ ID NO: 48.

[0016] 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 consecutive nucleotides that are fully complementary to an equal length of consecutive polynucleotides within the region of the SOD1 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NO: 33 to SEQ ID NO: 48.

[0017] In some embodiments, the guide strand comprises at least 10 consecutive nucleotides that are fully complementary to a continuous polynucleotide of equal length within the region of the SOD1 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NO: 33 to SEQ ID NO: 48.

[0018] In some embodiments, the guide strand comprises at least 11 consecutive nucleotides that are fully complementary to a consecutive polynucleotide of equal length within the region of the SOD1 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NO: 33 to SEQ ID NO: 48.

[0019] In some embodiments, the guide strand comprises at least 12 consecutive nucleotides that are fully complementary to a consecutive polynucleotide of equal length within the region of the SOD1 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NO: 33 to SEQ ID NO: 48.

[0020] In some embodiments, the guide strand comprises at least 13 consecutive nucleotides that are fully complementary to a continuous polynucleotide of equal length within the region of the SOD1 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NO: 33 to SEQ ID NO: 48.

[0021] In some embodiments, the guide strand comprises at least 14 consecutive nucleotides that are fully complementary to a consecutive polynucleotide of equal length within the region of the SOD1 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NO: 33 to SEQ ID NO: 48.

[0022] In some embodiments, the guide strand comprises at least 15 consecutive nucleotides that are fully complementary to a continuous polynucleotide of equal length within the region of the SOD1 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NO: 33 to SEQ ID NO: 48.

[0023] In some embodiments, the guide strand comprises at least 16 consecutive nucleotides that are fully complementary to a consecutive polynucleotide of equal length within the region of the SOD1 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NO: 33 to SEQ ID NO: 48.

[0024] In some embodiments, the guide strand comprises at least 17 consecutive nucleotides that are fully complementary to a consecutive polynucleotide of equal length within the region of the SOD1 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NO: 33 to SEQ ID NO: 48.

[0025] In some embodiments, the guide strand comprises at least 18 consecutive nucleotides that are fully complementary to a continuous polynucleotide of equal length within the region of the SOD1 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NO: 33 to SEQ ID NO: 48.

[0026] In some embodiments, the guide strand comprises at least 19 consecutive nucleotides that are fully complementary to a continuous polynucleotide of equal length within the region of the SOD1 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NO: 33 to SEQ ID NO: 48.

[0027] In some embodiments, the guide strand comprises at least 20 consecutive nucleotides that are fully complementary to a continuous polynucleotide of equal length within the region of the SOD1 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NO: 33 to SEQ ID NO: 48.

[0028] In some embodiments, the guide strand comprises 21 consecutive nucleotides that are fully complementary to a consecutive polynucleotide of equal length within the region of the SOD1 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NO: 33 to SEQ ID NO: 48.

[0029] In some embodiments, the guide strand comprises 10 to 21 consecutive nucleotides that are fully complementary to a contiguous polynucleotide of equal length within the region of the SOD1 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NO: 33 to SEQ ID NO: 48. In some embodiments, the guide strand comprises 12 to 21 consecutive nucleotides that are fully complementary to a contiguous polynucleotide of equal length within the region of the SOD1 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NO: 33 to SEQ ID NO: 48. In some embodiments, the guide strand comprises 15 to 21 consecutive nucleotides that are fully complementary to a contiguous polynucleotide of equal length within the region of the SOD1 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NO: 33 to SEQ ID NO: 48. In some embodiments, the guide strand comprises 18 to 21 consecutive nucleotides that are fully complementary to a continuous polynucleotide of equal length within the region of the SOD1 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NO: 33 to SEQ ID NO: 48.

[0030] In some embodiments, the guide strand comprises 19, 20, or 21 consecutive nucleotides that are fully complementary to a continuous polynucleotide of equal length within the region of the SOD1 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NO: 33 to SEQ ID NO: 48.

[0031] In some embodiments, the guide strand comprises 9 or fewer nucleotide mismatches relative to a stretch of 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides within a region of the SOD1 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NO: 33 to SEQ ID NO: 48, optionally wherein 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 relative to the region of the SOD1 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NO: 33 to SEQ ID NO: 48.

[0032] In some embodiments, the region of the SOD1 mRNA transcript has a nucleic acid sequence of any one of SEQ ID NO:33, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, and SEQ ID NO:45. In some embodiments, the region of the SOD1 mRNA transcript has a nucleic acid sequence of any one of SEQ ID NO:33 and SEQ ID NO:41. In some embodiments, the region of the SOD1 mRNA transcript has a nucleic acid sequence of any one of SEQ ID NO:33, SEQ ID NO:41, and SEQ ID NO:42. In some embodiments, the region of the SOD1 mRNA transcript has a nucleic acid sequence of any one of SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, and SEQ ID NO:45. In some embodiments, the region of the SOD1 mRNA transcript has a nucleic acid sequence of any one of SEQ ID NO:41, SEQ ID NO:42, and SEQ ID NO:45.

[0033] In some embodiments, the guide strand has a nucleic acid sequence that is at least 85% identical (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 NO: 1 to SEQ ID NO: 16. In some embodiments, the guide strand has a nucleic acid sequence that is at least 90% identical (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 NO: 1 to SEQ ID NO: 16. In some embodiments, the guide strand has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 1 to SEQ ID NO: 16 (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 16). In some embodiments, the guide strand has a nucleic acid sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 16. In some embodiments, the nucleic acid sequence is any one of SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 13. In some embodiments, the nucleic acid sequence is any one of SEQ ID NO: 1 and SEQ ID NO: 9. In some embodiments, the nucleic acid sequence is any one of SEQ ID NO: 1, SEQ ID NO: 9, and SEQ ID NO: 10. In some embodiments, the nucleic acid sequence is any one of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 13. In some embodiments, the nucleic acid sequence is any one of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 13.

[0034] In some embodiments, the inhibitory nucleic acid comprises a hairpin having a nucleic acid sequence that is at least 85% identical (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 NO: 17 to SEQ ID NO: 32. In some embodiments, the inhibitory nucleic acid comprises a hairpin having a nucleic acid sequence that is at least 90% identical (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 NO: 17 to SEQ ID NO: 32. 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 NO: 17 to SEQ ID NO: 32 (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence of any one of SEQ ID NO: 17 to SEQ ID NO: 32). In some embodiments, the hairpin has a nucleic acid sequence of any one of SEQ ID NO: 17 to SEQ ID NO: 32. In some embodiments, the nucleic acid sequence is any one of SEQ ID NO: 17, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 29. In some embodiments, the nucleic acid sequence is any one of SEQ ID NO: 17 and SEQ ID NO: 25. In some embodiments, the nucleic acid sequence is any one of SEQ ID NO: 17, SEQ ID NO: 25, and SEQ ID NO: 26. In some embodiments, the nucleic acid sequence is any one of SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 29. In some embodiments, the nucleic acid sequence is any one of SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 29.

[0035] 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 a miRNA.

[0036] In another aspect, the disclosure features a viral vector comprising a transgene encoding an inhibitory nucleic acid of any of the above aspects or embodiments of the disclosure. In some embodiments, the viral vector comprises multiple transgenes (e.g., 2, 3, 4, 5, or more transgenes).

[0037] 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 virus. In some embodiments, the viral vector is AAV.

[0038] In some embodiments, the viral vector is an AAV1 serotype, an AAV2 serotype, an AAV3 serotype, an AAV4 serotype, an AAV5 serotype, an AAV6 serotype, an AAV7 serotype, an AAV8 serotype, an AAV9 serotype, an AAVrh10 serotype, or an AAVrh74 serotype. In some embodiments, the viral vector is a pseudotyped AAV. In some embodiments, the pseudotyped AAV has an ITR of an AAV serotype (e.g., AAV2) and VP1 capsid protein, VP2 capsid protein, and / or VP3 capsid protein from a different AAV serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, or AAVrh74). In some embodiments, the pseudotyped AAV is AAV2 / 9. In some embodiments, the pseudotyped AAV is AAV2 / 8. In some embodiments, the AAV comprises a recombinant capsid protein.

[0039] In some embodiments, the AAV comprises a capsid disclosed, for example, in WO 2017 / 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-retroviral capsid protein or an AAV9-retroviral capsid protein. In some embodiments, the AAV comprises a capsid protein conjugated to a ligand or aptamer.

[0040] In some embodiments, the synthetic virus is a chimeric virus, a mosaic virus, or a pseudotyped virus, and / or comprises exogenous proteins, synthetic polymers, nanoparticles, or small molecules.

[0041] In yet another aspect, the disclosure features a pharmaceutical composition comprising the inhibitory nucleic acid or viral vector of any of the above aspects or embodiments of the disclosure, and a pharmaceutically acceptable excipient, carrier, or diluent.

[0042] In another aspect, the disclosure features a method of treating a neurological disorder in a subject in need thereof by administering to the subject a therapeutically effective amount of the inhibitory nucleic acid, viral vector, or pharmaceutical composition of any of the above aspects or embodiments of the disclosure.

[0043] In some embodiments, the nervous system disorder is a neurodegenerative disorder. In some embodiments, the nervous system disorder is caused by or associated with the expression of wild-type or mutant SOD1. In some embodiments, the nervous system disorder is amyotrophic lateral sclerosis (ALS), Huntington's disease, Parkinson's disease, frontotemporal dementia, primary lateral sclerosis, progressive muscular atrophy, limbic-based age-related TDP-43 encephalopathy, chronic traumatic encephalopathy, Lewy body dementia, cortical basal degeneration, progressive supranuclear palsy, Parkinson's dementia ALS syndrome, Pick's disease, Perry syndrome, brain age-related TDP-43 and sclerosis, hippocampal sclerosis or Alzheimer's disease. In some embodiments, the nervous system disorder is ALS.

[0044] In another aspect, the disclosure features a method of treating a cell proliferative disorder in a subject in need thereof by administering to the subject a therapeutically effective amount of the inhibitory nucleic acid, viral vector, or pharmaceutical composition of any of the above aspects or embodiments of the disclosure.

[0045] In some embodiments, the cell proliferative disorder is cancer. In some embodiments, the cell proliferative disorder is leukemia, lymphoma, liver cancer, bone cancer, lung cancer, brain cancer, bladder cancer, gastrointestinal cancer, breast cancer, cardia cancer, cervical cancer, uterine cancer, head and neck cancer, gallbladder cancer, laryngeal cancer, lip and oral cancer, eye cancer, melanoma, pancreatic cancer, prostate cancer, colorectal cancer, testicular cancer, laryngeal cancer, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, adrenocortical carcinoma, acquired immunodeficiency syndrome-associated lymphoma, primary central nervous system lymphoma, anal cancer, appendix cancer, astrocytoma, atypical teratoma, Fetal rhabdoid tumor, basal cell carcinoma, bile duct cancer, extrahepatic cancer, Ewing's sarcoma, osteosarcoma, malignant fibrous histiocytoma, central nervous system embryonal tumor, central nervous system germ cell tumor, craniopharyngioma, ependymoma, bronchial tumor, Burkitt's lymphoma, carcinoid tumor, primary lymphoma, chordoma, chronic myeloproliferative neoplasms, colon cancer, extrahepatic bile duct cancer, ductal carcinoma in situ, endometrial cancer, ependymoma, esophageal cancer, olfactory neuroblastoma, extracranial germ cell tumor, extragonadal germ cell tumor, fallopian tube cancer, fibrous histiocytoma of bone, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, testicular germ cell Tumors, gestational trophoblastic disease, gliomas, childhood brainstem gliomas, hairy cell leukemia, hepatocellular carcinoma, Langerhans cell histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, islet cell tumors, pancreatic neuroendocrine tumors, Wilms tumor, childhood kidney tumors, nephroblastoma, small cell lung cancer, cutaneous T-cell lymphoma, intraocular melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer, midline tract cancer, multiple endocrine neoplasia syndrome, multiple myeloma, plasma cell neoplasms, plasmacytoma, myelodysplastic syndrome, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer Lung cancer, epithelial ovarian cancer, germ cell ovarian cancer, low malignant potential ovarian cancer, pancreatic neuroendocrine tumors, papilloma, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary peritoneal cancer, rectal cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Kaposi sarcoma, Sézary syndrome, small intestine cancer, soft tissue sarcoma, laryngeal cancer, thymoma and thymic cancer, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, urethral cancer, endometrial cancer, uterine sarcoma, vaginal cancer, vulvar cancer, or Waldenstrom's macroglobulinemia.

[0046] In some embodiments, the subject is a mammal (eg, a human).

[0047] In some embodiments, the inhibitory nucleic acid, viral vector, or pharmaceutical composition of any of the foregoing aspects or embodiments of the present disclosure is administered to the subject by a route selected from the group consisting of intrathalamic, intrathecal, subpial, intraparenchymal, intrastriatal, intracranial, intracisternal, intracerebral, intracerebroventricular, intraocular (e.g., intravitreal), intraventricular, intralumbar spinal cord, intravenous, intramuscular, subcutaneous, intraperitoneal, intradermal, transdermal, parenteral, intranasal, transdermal, intratracheal, intraarterial, intravascular, and oral administration, inhalation, infusion, lavage, or any combination thereof.

[0048] In another aspect, the disclosure features a kit comprising the inhibitory nucleic acid, viral vector, or pharmaceutical composition of any of the above aspects or embodiments of the disclosure. The kit may further comprise 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, such as a human, diagnosed with a neurological disease described herein). BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1A Is a bar graph showing the knockdown efficacy of a series of SOD1-specific miRNA constructs. Data were obtained from a dual-luciferase reporter gene (DLR) assay performed in HEK cells after standard plasmid transfection. Data represent 3 independent experiments using biological replicates. Figure 1A Abbreviation: SD, superoxide dismutase 1.

[0050] Figure 1B Figure 2 is a bar graph showing the knockdown efficacy of a series of SOD1-specific miRNA constructs. Data were obtained from a probe-based qPCR assay performed in HEK cells after standard plasmid transfection. Data are representative of three independent experiments using biological replicates. Figure 1B Abbreviations: SD, superoxide dismutase 1; KD, knockdown.

[0051] Figure 2A Is a bar graph showing the knockdown efficacy of a series of SOD1-specific miRNA constructs. Data are obtained from a dual-luciferase reporter gene (DLR) assay performed in HEK cells after viral plasmid transfection. The control (CONT) is a non-targeted miRNA. Data represent 3 independent experiments using biological replicates. Figure 2A Abbreviation: SD, superoxide dismutase 1.

[0052] Figure 2BFigure 2 is a bar graph showing the knockdown efficacy of a series of SOD1-specific miRNA constructs. Data were obtained from probe-based qPCR assays performed in HEK cells after viral plasmid transfection. The control (CONT) was a non-targeting miRNA. Data are representative of 3-5 independent experiments using biological replicates. Figure 2B Abbreviations: SD, superoxide dismutase 1; qPCR, quantitative polymerase chain reaction.

[0053] Figure 3A Figure 2 is a bar graph showing the knockdown efficacy of a series of SOD1-specific miRNA constructs. Data were obtained from a probe-based qPCR assay performed in HEK293 cells after viral transduction. The control (neg) was a non-targeting miRNA. Data are representative of three independent experiments using biological replicates. Figure 3A Abbreviations: SOD1, superoxide dismutase 1; HEK, human embryonic kidney.

[0054] Figure 3B Figure 2 is a bar graph showing the knockdown efficacy of a series of SOD1-specific miRNA constructs. Data were obtained from a probe-based qPCR assay performed in SH-SY5Y cells after viral transduction. The control (neg) was a non-targeting miRNA. Data represent three independent experiments using biological replicates. Figure 3B Abbreviation: SOD1, superoxide dismutase 1.

[0055] definition

[0056] As used herein, the term "about" refers to a value that is within 10% of the stated value. For example, the phrase "about 100 nucleic acid residues" refers to a value of 90 to 110 nucleic acid residues.

[0057] As used herein, the term "annealing" refers to the formation of a stable nucleic acid duplex by hybridization mediated by interstrand hydrogen bonding, for example, according to Watson-Crick base pairing. The nucleic acids in 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 of one nucleic acid to another is a duplex structure that is not denatured by stringent washes. Exemplary stringent wash conditions are known in the art and include a temperature about 5°C below the melting temperature of the individual strands of the duplex and a low monovalent salt concentration, such as a monovalent salt concentration (e.g., NaCl concentration) of less than 0.2 M (e.g., 0.2 M, 0.19 M, 0.18 M, 0.17 M, 0.16 M, 0.15 M, 0.14 M, 0.13 M, 0.12 M, 0.11 M, 0.1 M, 0.09 M, 0.08 M, 0.07 M, 0.06 M, 0.05 M, 0.04 M, 0.03 M, 0.02 M, 0.01 M or less).

[0058] As used herein, the term "conservative mutation," "conservative substitution," or "conservative amino acid substitution" refers to the substitution of one or more amino acids with one or more different amino acids exhibiting similar physicochemical properties (such as polarity, net charge, and steric bulk). These properties for each of the twenty naturally occurring amino acids are summarized in Table 1 below.

[0059] Table 1. Representative physicochemical properties of naturally occurring amino acids

[0060]

[0061] Based on A 3 Medium volume: 50-100 is small, 100-150 is medium,

[0062] 150-200 is large, and >200 is huge

[0063] It will be appreciated from this table 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, a conservative mutation or substitution is one in which an amino acid is substituted with a member of the same amino acid family (e.g., Ser for Thr or Lys for Arg).

[0064] As used herein, the "length" of a nucleic acid refers to the linear size of the nucleic acid as assessed by measuring the number of nucleotides from the 5' end to the 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.

[0065] As used herein, the term "operably linked" refers to a first molecule (e.g., a first nucleic acid) being linked to a second molecule (e.g., a second nucleic acid), wherein the molecules are arranged so that the first molecule affects the function of the second molecule. The two molecules may or may not be part of a single continuous molecule and may or may not be adjacent to each other. For example, if a promoter modulates the transcription of a transcribable polynucleotide molecule of interest in a cell, the promoter is operably linked to the transcribable polynucleotide molecule. Additionally, if the two parts of a transcriptional regulatory element are linked so that the transcriptional activation functionality of one part is not adversely affected by the presence of the other part, the two parts are operably linked to each other. Two transcriptional regulatory elements can be operably linked to each other by means of a linker nucleic acid (e.g., an intervening non-coding nucleic acid) or can be operably linked to each other in the absence of an intervening nucleotide.

[0066] As used herein, a 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 they 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 two segments do not have common constituent nucleotides, the two segments are not considered to "overlap" with each other.

[0067] " Percentage sequence complementarity (%) " with respect to a reference polynucleotide sequence is defined as the percentage of nucleic acids complementary to the nucleic acids in the candidate sequence with respect to the reference polynucleotide sequence after the sequences are aligned and room (if necessary) is introduced to achieve maximum sequence complementarity. If two nucleotides form a standard Watson-Crick base pair, a given nucleotide is considered to be "complementary" to a reference nucleotide as described herein. For the avoidance of doubt, the Watson-Crick base pair in the context of the present disclosure includes adenine-thymine, adenine-uracil and cytosine-guanine base pairs. In this article, correct Watson-Crick base pairs are referred to as "matching", while each unpaired nucleotide and each wrongly paired nucleotide are referred to as "mispairing". Comparison for determining nucleic acid sequence identity percentage can be achieved in various ways within the capabilities of those skilled in the art, for example, using publicly available computer software, such as BLAST, BLAST-2 or Megalign software. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithm required for achieving maximum complementarity over the full length of the compared sequences. For example, the sequence complementarity percentage between a given nucleic acid sequence A and a given nucleic acid sequence B (which can also be expressed as a certain complementarity percentage between a given nucleic acid sequence A and a given nucleic acid sequence B) is calculated as follows:

[0068] 100×(fraction X / Y)

[0069] where X is the number of complementary base pairs in the alignment (e.g., as performed by computer software such as BLAST) in a program alignment of A and B, and where Y is the total number of nucleic acids in B. It will be understood that where 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 "fully complementary" to a reference nucleic acid sequence if they have 100% sequence complementarity.

[0070] " Percentage (%) of sequence identity relative to a reference polynucleotide or peptide sequence" is defined as after comparing sequences and introducing room (if necessary) to realize maximum sequence identity percentage, the percentage of nucleic acid or amino acid consistent with the nucleic acid in the reference polynucleotide or peptide sequence in the candidate sequence. The comparison for determining nucleic acid or amino acid sequence identity percentage can be realized in a variety of ways within the capabilities of those skilled in the art, for example, using publicly available computer software, such as BLAST, BLAST-2 or Megalign software. Those skilled in the art can determine the appropriate parameters for comparing sequences, including any algorithm required for realizing maximum comparison on the full length of the compared sequences. For example, sequence comparison computer program BLAST can be used to generate sequence identity percentage values. For example, given nucleic acid or amino acid sequence A to, with or for given nucleic acid or amino acid sequence B sequence identity percentage (also can be alternatively stated as given nucleic acid or amino acid sequence A to, with or for given nucleic acid or amino acid sequence B certain sequence identity percentage) is calculated as follows:

[0071] 100×(fraction X / Y)

[0072] 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 where Y is the total number of nucleic acids in B. It will be appreciated that where the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, the percent sequence identity of A to B will not equal the percent sequence identity of B to A.

[0073] As used herein, the term "pharmaceutical composition" refers to a mixture containing a therapeutic agent, optionally in combination with one or more pharmaceutically acceptable excipients, diluents and / or carriers, to be administered to a subject (such as a mammal, e.g., a human) in order to prevent, treat or control a particular disease or condition that affects or may affect the subject.

[0074] As used herein, the term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms that are suitable for contact with the tissues of subjects, such as mammals (e.g., humans) without excessive toxicity, irritation, allergic response, and other problematic complications, and are commensurate with a reasonable benefit / risk ratio.

[0075] As used herein, the term "wild-type" or "non-mutated" form of a gene refers to a nucleic acid that encodes a protein associated with normal or non-pathogenic activity (e.g., a protein lacking a mutation that results in a higher risk of development, onset, or progression of a neurodegenerative disease).

[0076] As used herein, the term "mutation" refers to any change in gene structure (e.g., gene sequence) that results in a change in the form of the gene that can be passed on to offspring (inherited mutation) or not (somatic mutation). Genetic mutations include substitutions, insertions, or deletions of single bases in DNA, or substitutions, insertions, deletions, or rearrangements of multiple bases or larger segments of a gene or chromosome, including repeat amplification.

[0077] As used herein, the term "superoxide dismutase 1" or "SOD1" refers to a protein encoded by the SOD1 gene. The SOD1 gene or transcript may refer to a normal allele of SOD1, or a mutant allele having a mutation, such as D90A, A4V, H46R, and G93A. In some embodiments, SOD1 refers to mammalian SOD1, including human SOD1. Exemplary SOD1 proteins that can be targeted using the compositions and methods of the present disclosure include proteins having an amino acid sequence represented by NCBI ID NP_000445.1, as well as naturally occurring variants thereof. In some embodiments, an exemplary SOD1 gene has a nucleic acid sequence of NCBI ID NC_000021.9:31659693-31668931, or a naturally occurring variant thereof. Exemplary SOD1 mRNA transcripts include transcripts having a nucleic acid sequence of NCBI ID NM_000454.5, as well as naturally occurring variants thereof.

[0078] As used herein, the term "inhibitory nucleic acid" refers to a nucleic acid comprising a guide strand sequence that hybridizes with at least a portion of a target nucleic acid (e.g., SOD1 RNA, mRNA, pre-mRNA, or mature mRNA) and inhibits its expression or activity. The inhibitory nucleic acid can target the protein coding region (e.g., exon) or non-coding region (e.g., 5'UTR, 3'UTR, intron, etc.) of the target nucleic acid. In some embodiments, the inhibitory nucleic acid is a single-stranded molecule or a double-stranded molecule. The inhibitory nucleic acid may further comprise a passenger strand sequence on a separate strand (e.g., a double-stranded duplex) or in the same strand (e.g., a single-stranded, self-annealing duplex structure). 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 (dsRNA).

[0079] As used herein, the term "interfering RNA" refers to an RNA, such as an siRNA, miRNA, or shRNA, that inhibits the expression of a target RNA transcript by: (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, inhibiting, or preventing translation of the RNA transcript, such as by sterically preventing the formation of a functional ribosome-RNA transcript complex or otherwise attenuating the formation of a functional protein product from the target RNA transcript. Interfering RNAs as described herein can be provided to a patient, such as a human patient with myotonic dystrophy, in the form of, for example, a single-stranded or double-stranded oligonucleotide or in the form of a vector (e.g., a viral vector, such as an adeno-associated viral vector described herein) comprising a transgene encoding the interfering RNA. Exemplary interfering RNA platforms are described, e.g., in Lam et al., 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 entirety.

[0080] As used herein, "microRNA" or "miRNA" refers to a small non-coding RNA molecule that can mediate target gene silencing by cutting target mRNA, inhibiting the translation of target mRNA, degrading target mRNA or a combination thereof. Typically, miRNA is transcribed as a hairpin or stem-loop (e.g., a single-stranded backbone with self-complementarity) duplex structure, referred to as primary miRNA (pri-miRNA), which is enzymatically processed (e.g., by Drosha, DGCR8, Pasha, etc.) into pre-miRNA. Pre-miRNA is exported to the cytoplasm, where it is enzymatically processed by Dicer to produce a miRNA duplex with a passenger chain, followed by a single-stranded mature miRNA molecule, which is then loaded into an RNA-induced silencing complex (RISC). Reference to miRNA may include synthetic miRNA or artificial miRNA.

[0081] As used herein, "synthetic miRNA" or "artificial miRNA" or "amiRNA" refers to an endogenous, modified or synthetic pri-miRNA or pre-miRNA (e.g., a miRNA backbone or scaffold) in which the endogenous miRNA guide sequence and passenger sequence within the backbone sequence have been replaced by miRNA guide sequences and miRNA passenger sequences that direct efficient target gene RNA silencing (see, e.g., Eamens et al. (2014), Methods Mol. Biol. 1062: 211-224). In some embodiments, the complementary properties of the guide sequence and passenger sequence (e.g., number of bases, mismatch positions, bulge type, etc.) can be similar or different to the complementary properties of the guide sequence and passenger sequence in the endogenous miRNA backbone from which the synthetic miRNA is constructed.

[0082] As used herein, the terms "microRNA backbone," "miR backbone," "microRNA scaffold," or "miR scaffold" refer to a pri-miRNA or pre-miRNA scaffold in which the backbone sequence is replaced by the miRNA of interest and capable of producing a functional mature miRNA that directs RNA silencing of the gene targeted by the miRNA of interest. The miR backbone comprises a 5' flanking region (also referred to as a 5' miR context, ≥9 nucleotides), a stem region comprising the miRNA duplex (guide and passenger strand sequences) and a base stem (5' and 3', approximately 4-13 nucleotides each), at least one loop motif region including a terminal loop (terminal loop ≥10 nucleotides), a 3' flanking region (also referred to as a 3' miR context, ≥9 nucleotides), and optionally one or more protrusions in the stem. The miR backbone can be derived entirely or partially from a wild-type miRNA scaffold, or be a completely artificial sequence.

[0083] 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 the gene targeted by silencing. The antisense sequence is sufficiently complementary to the target mRNA sequence to guide target-specific silencing, for example, to trigger the destruction of the target mRNA by an RNAi mechanism or process. In some embodiments, the antisense sequence or guide strand sequence refers to the mature sequence remaining after being cut by Dicer.

[0084] 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 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., to 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.

[0085] As used herein, "duplex" when used with respect to an inhibitory nucleic acid refers to two nucleic acid strands (e.g., a guide strand and a passenger strand) hybridized together to form a duplex structure. A duplex can be formed by two separate nucleic acid strands, or by a single nucleic acid strand with a self-complementary region (e.g., a hairpin or stem-loop).

[0086] As used herein, "expression construct" refers to any type of genetic construct containing a nucleic acid (e.g., a transgene) in which part or all of a nucleic acid coding sequence can be transcribed. In some embodiments, expression comprises transcribing the nucleic acid, e.g., to generate a biologically active polypeptide product or inhibitory RNA (e.g., siRNA, shRNA, miRNA) from the transcribed gene. In some embodiments, the transgene is operably linked to an expression control sequence.

[0087] As used herein, the term "transgene" refers to an exogenous nucleic acid that has been transferred into another cell, either naturally or by genetic engineering means, and is capable of being transcribed and optionally translated.

[0088] As used herein, the term "gene expression" refers to the process by which nucleic acids are transcribed from nucleic acid molecules and typically 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 measurement of "gene expression" can refer to measurement of transcription products (e.g., RNA or mRNA), translation products (e.g., peptides or proteins).

[0089] As used herein, the term "inhibit gene expression" means to reduce, downregulate, inhibit, block, lower or stop gene expression. 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 reduction in the level of mRNA results in a reduction in the level of the polypeptide translated from the mRNA. Standard techniques for measuring mRNA or protein can be used to determine expression levels.

[0090] As used herein, "neurological disease" or "neurological disorder" refers to a disease or disorder that affects the nerves in the brain, spinal cord, and other parts of the human body. A neurological disorder may be caused by, among other things, electrical, structural, or biochemical abnormalities in neurons. As used herein, a neurological disorder includes neurodegenerative disorders. As used herein, a "neurodegenerative disease" or "neurodegenerative disorder" refers to a disease or disorder that exhibits nerve cell death as a pathological condition. A neurodegenerative disease may exhibit chronic neurodegeneration, such as slow, progressive nerve cell death over a period of several years, or acute neurodegeneration, such as sudden onset or death of nerve cells. As used herein, the term "neurodegenerative disorder" refers to a disorder, disease, or condition caused by the degeneration of cellular and tissue components of the nervous system. Some non-limiting examples of neurodegenerative disorders include stroke, Alzheimer's disease, Parkinson's disease, Huntington's disease, periventricular leukomalacia (PVL), amyotrophic lateral sclerosis (ALS, "Lou Gehrig's disease"), ALS-Parkinson-dementia syndrome of Guam, Friedreich's ataxia, Wilson's disease, multiple sclerosis, cerebral palsy, progressive supranuclear palsy (Stills-Richardson syndrome), bulbar and pseudobulbar palsy, diabetic retinopathy, multi-infarct dementia, macular degeneration, Pick's disease, diffuse Lewy body disease, prion diseases such as Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker disease, and leukoencephalopathy. disease), kuru and fatal familial insomnia, primary lateral sclerosis, degenerative ataxias, Machado-Joseph disease / spinocerebellar ataxia type 3 and olivopontocerebellar degeneration, spinal and spinobulbar muscular atrophy (Kennedy disease), familial spastic paraplegia, Wolff-Kugelberg-Weiland disease, Tay-Sachs disease, multisystem degeneration (Schay-Drager syndrome), Gilles de la Tourette disease, familial autonomic dysfunction Dementia (including Reye-Dieter syndrome), Kugelberg-Weiland disease, subacute sclerosing panencephalitis, Werdnig-Hoffmann disease, synucleinopathies (including multiple system atrophy), Sandhoff disease, corticobasal degeneration, spastic paraplegia, primary progressive aphasia, progressive multifocal leukoencephalopathy, striatonigral degeneration, familial spastic disorders, chronic epilepsy associated with neurodegeneration, Binswanger disease, and dementia (including all potential causes of dementia). 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 those characterized by TDP-43 proteinopathies, which are characterized by nuclear to cytoplasmic mislocalization, deposition of ubiquitinated and hyperphosphorylated TDP-43 into inclusion bodies, protein truncation leading to the formation of toxic C-terminal TDP-43 fragments, and protein aggregation.TDP-43 proteinopathy diseases include ALS, FTLD, primary lateral sclerosis, progressive amyotrophic lateral sclerosis, limbic-dominant age-related TDP-43 encephalopathy, chronic traumatic encephalopathy, Lewy body dementia, corticobasal degeneration, progressive supranuclear palsy (PSP), Guam dementia-Parkinson disease ALS syndrome (G-PDC), Pick's disease, hippocampal sclerosis, Huntington's disease, Parkinson's disease and Alzheimer's disease. Acute neurodegeneration may be caused by axonal transection caused by ischemia (e.g., stroke, traumatic brain injury), demyelination or trauma (e.g., spinal cord injury or multiple sclerosis). Neurodegenerative diseases may be primarily manifested by one or more types of neuronal death.

[0091] As used herein, the term "cell proliferative disorder" refers to a disease or disorder that causes unnatural and uncontrolled cell proliferation or division in any area of ​​the human body, which can spread to other parts of the body. Cell proliferative disorders can be caused by genetic defects as well as external factors such as radiation. In some embodiments, the cell proliferative disorder is cancer. In some embodiments, the cell proliferative disorder can result in a tumor or cyst. In some embodiments, the tumor or cyst can be benign in nature. In some embodiments, the tumor or cyst can be malignant in nature. Abnormal cells such as tumors or cysts that arise from a cell proliferative disorder have the ability to infiltrate and destroy normal body tissues.In some embodiments, the cell proliferative disorder is leukemia, lymphoma, liver cancer, bone cancer, lung cancer, brain cancer, bladder cancer, gastrointestinal cancer, breast cancer, cardia cancer, cervical cancer, uterine cancer, head and neck cancer, gallbladder cancer, laryngeal cancer, lip and oral cancer, eye cancer, melanoma, pancreatic cancer, prostate cancer, colorectal cancer, testicular cancer, laryngeal cancer, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, adrenocortical carcinoma, acquired immunodeficiency syndrome-associated lymphoma, primary central nervous system lymphoma, anal cancer, appendix cancer, astrocytoma, atypical teratoma, Fetal rhabdoid tumor, basal cell carcinoma, bile duct cancer, extrahepatic cancer, Ewing's sarcoma, osteosarcoma, malignant fibrous histiocytoma, central nervous system embryonal tumor, central nervous system germ cell tumor, craniopharyngioma, ependymoma, bronchial tumor, Burkitt's lymphoma, carcinoid tumor, primary lymphoma, chordoma, chronic myeloproliferative neoplasms, colon cancer, extrahepatic bile duct cancer, ductal carcinoma in situ, endometrial cancer, ependymoma, esophageal cancer, olfactory neuroblastoma, extracranial germ cell tumor, extragonadal germ cell tumor, fallopian tube cancer, fibrous histiocytoma of bone, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, testicular germ cell Tumors, gestational trophoblastic disease, gliomas, childhood brainstem gliomas, hairy cell leukemia, hepatocellular carcinoma, Langerhans cell histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, islet cell tumors, pancreatic neuroendocrine tumors, Wilms tumor, childhood kidney tumors, nephroblastoma, small cell lung cancer, cutaneous T-cell lymphoma, intraocular melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer, midline tract cancer, multiple endocrine neoplasia syndrome, multiple myeloma, plasma cell neoplasms, plasmacytoma, myelodysplastic syndrome, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer Lung cancer, epithelial ovarian cancer, germ cell ovarian cancer, low malignant potential ovarian cancer, pancreatic neuroendocrine tumors, papilloma, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary peritoneal cancer, rectal cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Kaposi sarcoma, Sézary syndrome, small intestine cancer, soft tissue sarcoma, laryngeal cancer, thymoma and thymic cancer, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, urethral cancer, endometrial cancer, uterine sarcoma, vaginal cancer, vulvar cancer, or Waldenstrom's macroglobulinemia.

[0092] As used herein, the term "sample" refers to a specimen isolated from a subject (e.g., blood, a blood component (e.g., serum or plasma), urine, saliva, amniotic fluid, cerebrospinal fluid, tissue (e.g., placenta or skin), pancreatic juice, chorionic villus sample, or cells). The subject can be, for example, a patient suffering from a disease described herein, such as a disease associated with expression of a SOD1 mutant (e.g., ALS).

[0093] As used herein, the phrases "specifically bind" and "bind" refer to a binding reaction that determines the presence of a particular molecule (such as an RNA transcript, e.g., a mutant SOD1 RNA transcript) in a heterogeneous population of recognized ions, salts, small molecules, and / or proteins. A ligand (e.g., an RNA binding protein as described herein) that specifically binds to a substance (e.g., an RNA transcript) can bind to the substance, e.g., at a concentration of less than 1 mM. KD For example, a ligand that specifically binds to a substance may have a K of at most 100 μM (e.g., between 1 pM and 100 μM). D A ligand that does not exhibit specific binding to another molecule may exhibit a K for that particular molecule or ion that is greater than 1 mM (e.g., 1 μM, 100 μM, 500 μM, 1 mM, or greater). 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, for example, Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1988) and Harlow and Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1999).

[0094] As used herein, the terms "subject" and "patient" refer to an organism that is being treated for a particular disease or condition as described herein, such as a disease associated with expression of a SOD1 mutant, e.g., ALS. Examples of subjects and patients include mammals, such as humans, that are being treated for a disease or condition as described herein.

[0095] As used herein, the term "transcriptional regulatory element" refers to a nucleic acid that at least partially controls the transcription of a gene of interest. A transcriptional regulatory element can include promoters, enhancers, and other nucleic acids (e.g., polyadenylation signals) that control or help control gene transcription. Examples of transcriptional regulatory elements are described in, for example, Goeddel, Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, CA, 1990).

[0096] As used herein, the term "treat" or "treatment" refers to therapeutic treatment in which the object is to prevent or slow (lessen) an undesirable physiological change or condition, such as the progression of a disease (e.g., ALS) associated with the expression of a SOD1 mutant. In the context of ALS treatment, beneficial or desirable clinical outcomes indicative of successful treatment include, but are not limited to, alleviation of symptoms, reduction in disease extent, a stable (i.e., non-worsening) disease state, a delay or slowing of disease progression, an improvement or alleviation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Treatment of a patient with a neurological disorder (e.g., ALS) may be manifested as one or more detectable changes, such as a decrease in expression of mutant SOD1 RNA transcripts.

[0097] As used herein, the term "vector" refers to a vector that can be used to deliver a gene of interest to a cell (e.g., a mammalian cell, such as a human cell), a tissue, an organ, or an organism (such as a patient undergoing treatment for a disease or condition described herein) for the purpose of expressing the encoded transgene. Exemplary vectors that can be used in conjunction with the compositions and methods described herein are plasmids, DNA vectors, RNA vectors, virions, or other suitable replicons (e.g., viral vectors). A variety of vectors have been developed for delivering polynucleotides encoding exogenous proteins into 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 other sequence elements, for example, for expressing proteins and / or integrating these polynucleotide sequences into the genome of mammalian cells. Certain vectors that can be used to express the transgenes described herein include plasmids containing regulatory sequences (e.g., promoter regions and enhancer regions) that direct gene transcription. Other vectors that can be used to express transgenes contain polynucleotide sequences that enhance the translation rate of these genes or improve the stability or nuclear export of mRNA transcribed from the genes. These sequence elements include, for example, 5' and 3' untranslated regions, internal ribosome entry sites (IRES), and polyadenylation signal sites to guide efficient transcription of the genes carried on the expression vector. The expression vectors described herein may also contain polynucleotides encoding markers for selecting cells containing the vector. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, or nourseothricin. DETAILED DESCRIPTION

[0098] The compositions and methods described herein can be used to treat disorders associated with expression of wild-type or mutant superoxide dismutase 1 (SOD1), such as amyotrophic lateral sclerosis (ALS), Parkinson's disease, Huntington's disease, and the like. The compositions described herein include inhibitory nucleic acid constructs, such as interfering RNA constructs, e.g., short interfering RNA (siRNA), short hairpin RNA (shRNA), or microRNA (miRNA), that inhibit the expression of wild-type or mutant mRNA transcripts transcribed from wild-type or mutant genes. Without being limited by mechanism, the compositions described herein can improve neuropathology by reducing the expression of wild-type or mutant mRNA transcripts, thereby preventing the expression of disease phenotypes.

[0099] The following sections provide descriptions of exemplary inhibitory nucleic acids of the present disclosure and vectors (e.g., viral vectors) encoding the inhibitory nucleic acids, as well as methods of using such inhibitory nucleic acids and vectors to treat neurological diseases.

[0100] Superoxide dismutase 1 (SOD1) and SOD1-related diseases

[0101] "SOD1" refers to the enzyme superoxide dismutase 1, which is one of the three superoxide dismutases involved in converting harmful superoxide radicals into water. Approximately 10% of all ALS cases are dominantly inherited, and about 20% of these are due to defects in cytosolic superoxide dismutase 1 (SOD1). In addition, SOD1 is associated with non-familial (e.g., sporadic) forms of ALS. (Jones, CT, Brock, DJH, Chancellor, AM, Warlow, CP, Swingler, RJ. Cu / Zn superoxide dismutase (SOD1) mutations and sporadic amyotrophic lateral sclerosis. Lancet 342: 1050-1051, 1993). Without wishing to be bound by any theory, multiple studies have shown that mutations in SOD1 do not cause ALS by loss of the dismutase activity of this enzyme. In contrast, mutant SOD1 is neurotoxic through a variety of alternative mechanisms, many of which involve conformational instability and abnormal binding and aggregation of the mutant protein. Although the specific details of mutant SOD1 toxicity have not yet been fully determined, it is clear that reducing the burden of mutant SOD1 protein in animal models significantly delays death. This has been achieved through the use of antisense oligonucleotides (ASOs) (Smith et al. 2004) and siRNA (Maxwell, Pasinelli et al. 2006) (Xia, Zhao et al. 2006) (Wang, Ghosh et al. 2008). These studies demonstrate the potential of siRNA-based drugs to be a major therapeutic advance for the treatment of ALS and many other CNS diseases. In both cases, efficacy is achieved by delivering large amounts of material over long periods of time; these studies suggest that the main limitation of current forms of ASO and siRNA therapy is the lack of the most efficient and non-toxic in vivo delivery system (Smith, Miller et al. 2006; Wang, Ghosh et al. 2008). ALS is a progressive neurodegenerative disease that affects motor neurons in the central nervous system. Degeneration of motor neurons leads to paralysis and ultimately death, often due to respiratory failure. In a subset of cases, ALS is caused by dominantly transmitted mutations in the gene encoding the cytosolic superoxide dismutase (SOD1). Transgenic expression of mutant SOD1 causes ALS in mice.

[0102] Aspects of the present disclosure relate to inhibitory nucleic acids, such as interfering RNA molecules (e.g., short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), including artificial miRNA), which, when administered to a subject, reduce the expression or activity of SOD1 in the subject. Accordingly, the compositions and methods provided in the present disclosure can be used to treat neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), Parkinson's disease, and Huntington's disease.

[0103] Inhibitory nucleic acids

[0104] In one aspect, the present disclosure provides isolated inhibitory nucleic acids that inhibit the expression or activity of SOD1. The inhibitory nucleic acid is a nucleic acid that specifically binds (e.g., hybridizes) to at least a portion of a SOD1 nucleic acid (such as, SOD1 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 non-coding region (e.g., 5'UTR, 3'UTR, intron, etc.) of SOD1. In some embodiments, the inhibitory nucleic acid is complementary to a wild-type SOD1 nucleic acid or a naturally occurring variant thereof. In some embodiments, the SOD1 allele has a mutation, such as D90A, A4V, H46R, and G93A. 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 (dsRNA).

[0105] In some embodiments, the inhibitory nucleic acid is a miRNA. The miRNA can be a pri-mRNA, a pre-mRNA, a mature miRNA, or an 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 located within the same nucleic acid chain, wherein the guide strand and the passenger strand hybridize together to form a self-annealing duplex structure. The miRNA is initially transcribed as a pri-mRNA, which is processed into a pre-mRNA by a ribonuclease (e.g., the Drosha-DGCR8 complex). The pri-mRNA is a single-stranded molecule with a stem-loop structure. The pre-mRNA is 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 produce a mature double-stranded miRNA duplex comprising a guide strand and a passenger strand. The mature miRNA duplex is then incorporated into the RNA-induced silencing complex (RISC) mediated by TRBP (HIV transcription activation response RNA binding protein). The passenger strand is typically released and cleaved, while the guide strand remains in RISC and binds to the target mRNA and mediates silencing. In some embodiments, the mature miRNA refers to the guide strand of the mature miRNA duplex.

[0106] Artificial miRNA refers to an endogenous, modified or synthetic pri-mRNA or pre-mRNA scaffold or backbone capable of producing a functional mature miRNA, wherein the guide strand sequence and the passenger strand sequence of the miRNA duplex in the stem region have been replaced by the guide strand sequence and the passenger strand sequence of interest, which guide the silencing of the 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 backbones are described in U.S. Patent Publication 2008 / 0313773 (incorporated by reference in its entirety).

[0107] The inhibitory nucleic acid constructs described herein (such as interfering RNA constructs) can be any of a variety of forms, such as siRNA, shRNA or miRNA. The interfering RNA described herein can also be encoded by a vector (such as a viral vector). For example, described herein are adeno-associated virus (AAV) vectors, such as pseudotyped AAV vectors (e.g., AAV2 / 8 and AAV2 / 9 vectors), containing a transgene encoding an interfering RNA construct that attenuates the expression of wild-type or mutant RNA transcripts.

[0108] Among other benefits, the compositions and methods described herein provide the advantageous feature of being able to selectively inhibit the expression of wild-type or pathological RNA transcripts. Using the compositions and methods described herein, the expression of wild-type or mutant RNA transcripts can be reduced while preserving the expression of important healthy RNA transcripts and their encoded protein products.

[0109] This advantageous feature is based in part on the surprising discovery that inhibitory nucleic acid constructs that anneal to wild-type or mutant RNA targets can be used to inhibit the expression of these RNA transcripts. Thus, the compositions and methods described herein can attenuate the expression of wild-type or pathological RNA transcripts.

[0110] The following sections provide descriptions of exemplary inhibitory nucleic acid constructs, such as interfering RNA constructs, that can be used in conjunction with the compositions and methods described herein, as well as descriptions of vectors encoding such constructs and procedures that can be used to treat diseases associated with expression of wild-type or mutant SOD1.

[0111] interfering RNA

[0112] Using the compositions and methods described herein, interfering RNA molecules, compositions containing the interfering RNA molecules, or vectors encoding the interfering RNA molecules can be administered to patients suffering from diseases characterized by wild-type or mutant SOD1 expression to inhibit expression of RNA transcripts.

[0113] Exemplary interfering RNA molecules that can be used in conjunction with the compositions and methods described herein to treat diseases associated with expression of SOD1 mutants (such as ALS, etc.) are siRNA molecules, miRNA molecules, and shRNA molecules, among others. In the case of siRNA molecules, siRNA can be single-stranded or double-stranded. In contrast, miRNA molecules are single-stranded molecules that form hairpins, thereby adopting a hydrogen-bonded structure similar to a nucleic acid duplex. In either case, the interfering RNA may contain an antisense strand or "guide" strand that anneals (e.g., by complementation) to the mutant RNA target. The interfering RNA may also contain a "passenger" strand that is complementary to the guide strand and, therefore, may have the same nucleic acid sequence as the RNA target.

[0114] Exemplary interfering RNA molecules that anneal to SOD1 RNA can be used in conjunction with the compositions and methods described herein to treat diseases associated with wild-type or mutant SOD1 expression as shown in Table 2 below.

[0115] Table 2. Exemplary inhibitory nucleic acids for inhibiting SOD1 expression

[0116]

[0117]

[0118] Methods for treating diseases characterized by wild-type or mutant SOD1 expression

[0119] Using the compositions and methods described herein, inhibitory nucleic acid constructs (such as interfering RNA constructs, e.g., short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA)) or vectors encoding the inhibitory nucleic acid constructs can be administered to patients experiencing and / or suffering from diseases associated with wild-type or mutant SOD1 (such as ALS, Huntington's disease, Parkinson's disease, etc.) to reduce the expression of wild-type or mutant RNA transcripts.

[0120] In another aspect, the present disclosure provides a method for inhibiting the expression or activity of SOD1 in a cell, 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, rAAV particles, a pharmaceutical composition) to a cell, thereby inhibiting the expression or activity of SOD1 in the cell. In some embodiments, the cell is a CNS cell. In some embodiments, the cell is a non-neuronal cell or a neuronal cell of the CNS. In some embodiments, the non-neuronal cell of the CNS is a glial cell, an astrocyte, or a microglia. In some embodiments, the cell is in vitro. In some embodiments, the cell is from a subject with one or more symptoms of a neurodegenerative disease or a suspected neurodegenerative disease. In some embodiments, the cell expresses SOD1 with a mutation (such as D90A, A4V, H46R, and G93A, etc.).

[0121] In another aspect, the present disclosure provides a method for inhibiting the expression or activity of SOD1 in the central nervous system of a subject, 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, a rAAV particle, a pharmaceutical composition), thereby inhibiting the expression or activity of SOD1 in the subject.

[0122] 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, rAAV particles, a pharmaceutical composition), thereby treating the subject. As used herein, the term "treating" refers to preventing or delaying the onset of a neurodegenerative disease (e.g., ALS / FTLD, Alzheimer's disease, Parkinson's disease, etc.); reducing the severity of a neurodegenerative disease; reducing or preventing the development of characteristic symptoms of a neurodegenerative disease; preventing the worsening of characteristic symptoms of a neurodegenerative disease, or any combination thereof.

[0123] Neurodegenerative diseases that can be treated in a subject using the compositions of the present disclosure include neurodegenerative diseases in which SOD1 is a pathogen (eg, ALS), as well as neurodegenerative diseases in which SOD1 is not a pathogen (eg, directly pathogenic).

[0124] Neurodegenerative diseases associated with mutant SOD1 include ALS, FTLD, primary lateral sclerosis, progressive amyotrophic lateral sclerosis, limbic predominant age-related TDP-43 encephalopathy, chronic traumatic encephalopathy, dementia with Lewy bodies, corticobasal degeneration, progressive supranuclear palsy (PSP), Guam dementia-Parkinson disease-ALS syndrome (G-PDC), Pick's disease, Perry syndrome, brain age-related TDP-43 and sclerosis (CARTS), hippocampal sclerosis, Huntington's disease, Parkinson's disease, and Alzheimer's disease.

[0125] In some embodiments, the treatment methods disclosed herein reduce, prevent, or slow the development or progression of one or more characteristic symptoms of a neurodegenerative disease. Characteristic symptoms of a neurodegenerative disease include motor dysfunction, cognitive dysfunction, emotional / behavioral dysfunction, or any combination thereof. Paralysis, tremors, instability, stiffness, convulsions, muscle weakness, muscle spasms, muscle rigidity, muscle atrophy, difficulty swallowing, difficulty breathing, speech and language difficulties (e.g., slurred speech), slow movements, difficulty walking, dementia, depression, anxiety, or any combination thereof.

[0126] In some embodiments, the methods of treatment of the present invention include administration as a monotherapy or co-administration with one or more additional therapies for the treatment of neurodegenerative diseases. Conjunctive therapy can mean administering a composition of the present invention (e.g., inhibitory nucleic acid, an isolated nucleic acid comprising an expression construct encoding an inhibitory nucleic acid, a vector, rAAV particles, a pharmaceutical composition) to a subject while, before, or after one or more additional therapies. Conjunctive therapy can mean administering a composition of the present invention (e.g., inhibitory nucleic acid, an isolated nucleic acid comprising an expression construct encoding an inhibitory nucleic acid, a vector, rAAV particles, a pharmaceutical composition) and an additional therapy to be formulated for administration with the same dosage form or with a separate dosage form.

[0127] In some embodiments, a therapy or additional therapy that can be used in combination with the inhibitory nucleic acids of the present disclosure includes: inhibitory nucleic acids or antisense oligonucleotides targeting neurodegenerative disease-associated genes or transcripts, gene editing agents targeting neurodegenerative disease-associated genes (e.g., CRISPR, TALEN, ZFN-based systems), agents that reduce oxidative stress (such as free radical scavengers (e.g., Radicava (edaravone), bromocriptine)); anti-glutamate agents (e.g., riluzole, topiramate, lamotrigine, dextromethorphan, gabapentin, and AMPA receptor antagonists (e.g., talampanel); )); anti-apoptotic agents (e.g., minocycline, sodium phenylbutyrate, and arilomol); anti-inflammatory agents (e.g., gangliosides, celecoxib, cyclosporine, nimesulide, azathioprine, cyclophosphamide, plasma exchange, glatiramer acetate, and thalidomide); β-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, Xaliproden, thyrotropin-releasing hormone, and ADNF).

[0128] In some embodiments, the subject treated with any of the methods described herein is a mammal (eg, mouse, rat), preferably a primate (eg, monkey, chimpanzee) or a human.

[0129] In any of the therapeutic methods described herein, a composition of the 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) can be administered to a subject by intrathecal, subpial, intraparenchymal, intrastriatal, intracranial, intracisternal, intracerebral, intraventricular, intraocular, intraventricular, intralumbar spinal administration, or any combination thereof.

[0130] In some embodiments, the compositions of the present disclosure (e.g., inhibitory nucleic acids, isolated nucleic acids comprising expression constructs encoding the inhibitory nucleic acids, vectors, rAAV particles, pharmaceutical compositions) are injected directly into the CNS of a subject. In some embodiments, direct injection into the CNS is intracerebral injection, intraparenchymal injection, intrathecal injection, intrastriatal injection, subpial injection, or any combination thereof. In some embodiments, direct injection into the CNS is direct injection into the cerebrospinal fluid (CSF) of a subject, optionally wherein direct injection is intracisternal injection, intraventricular injection, intralumbar spinal injection, or any combination thereof.

[0131] Methods for treating cell proliferative disorders

[0132] Using the compositions and methods described herein, an inhibitory nucleic acid construct (such as an interfering RNA construct, e.g., short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA)) or a vector encoding the inhibitory nucleic acid construct can be administered to a patient experiencing and / or suffering from a cell proliferative disorder to reduce the expression of wild-type or mutant RNA transcripts (such as wild-type or mutant SOD1 transcripts). In some embodiments, the cell proliferative disorder is cancer. SOD1 is an intracellular enzyme that converts superoxide anions into hydrogen peroxide, which is then converted by catalase into oxygen and water. Therefore, SOD1 plays an important role in resisting oxidative damage. Resistance to platinum-based therapies such as cisplatin and disease recurrence are major obstacles in cancer treatment. SOD1 may be responsible for conferring resistance to oxidative stress against platinum compounds. Silencing or knocking down SOD1 helps enhance cisplatin-based cytotoxicity to inhibit tumor growth. The compositions described herein are useful therapeutic agents for treating a variety of cell proliferative disorders and can be administered to a mammalian subject, such as a human, suffering from a cell proliferative disorder, such as cancer.

[0133] Compositions as described herein can be applied to mammalian subjects (for example, humans) suffering from cell proliferation disorders, to improve the patient's illness. Compositions as described herein can be applied to subjects, for example, by any route of administration as described herein. In some embodiments, compositions as described herein can be applied to the subject by being selected from the following approach: intrathalamic, intrathecal, subpial, intracerebral, intrastriatal, intracranial, intracisternal, intracerebral, intraventricular, intraocular (for example, intravitreal), intraventricular, intraspinal, intralumbar, intravenous, intramuscular, subcutaneous, intraperitoneal, intradermal, transdermal, parenteral, intranasal, transdermal, intratracheal, intraarterial, intravascular and oral administration, suction, perfusion, lavage or any combination thereof. Compositions as described herein can also be formulated together with excipients, biologically acceptable carriers, and can optionally be conjugated, blended or administered alone (for example, sequentially) with additional therapeutic agents (such as anticancer agents).Cell proliferative disorders that can be treated by the compositions and methods described herein include leukemias, lymphomas, liver cancer, bone cancer, lung cancer, brain cancer, bladder cancer, gastrointestinal cancer, breast cancer, cardia cancer, cervical cancer, uterine cancer, head and neck cancer, gallbladder cancer, laryngeal cancer, lip and oral cancer, eye cancer, melanoma, pancreatic cancer, prostate cancer, colorectal cancer, testicular cancer, laryngeal cancer, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, adrenocortical carcinoma, acquired immunodeficiency syndrome-associated lymphoma, primary central nervous system lymphoma, anal cancer, appendix cancer, astrocytoma, Atypical teratoid rhabdoid tumor, basal cell carcinoma, bile duct cancer, extrahepatic cancer, Ewing's sarcoma, osteosarcoma, malignant fibrous histiocytoma, central nervous system embryonal tumor, central nervous system germ cell tumor, craniopharyngioma, ependymoma, bronchial tumor, Burkitt's lymphoma, carcinoid tumor, primary lymphoma, chordoma, chronic myeloproliferative neoplasms, colon cancer, extrahepatic bile duct cancer, ductal carcinoma in situ, endometrial cancer, ependymoma, esophageal cancer, olfactory neuroblastoma, extracranial germ cell tumor, extragonadal germ cell tumor, fallopian tube cancer, fibrous histiocytoma of bone, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, testicular cancer Germ cell tumor, gestational trophoblastic disease, glioma, childhood brainstem glioma, hairy cell leukemia, hepatocellular carcinoma, Langerhans cell histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, islet cell tumor, pancreatic neuroendocrine tumor, Wilms tumor, childhood kidney tumor, nephroblastoma, small cell lung cancer, cutaneous T-cell lymphoma, intraocular melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer, midline tract cancer, multiple endocrine neoplasia syndrome, multiple myeloma, plasma cell neoplasms, plasmacytoma, myelodysplastic syndrome, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma, Non-small cell lung cancer, epithelial ovarian cancer, germ cell ovarian cancer, low malignant potential ovarian cancer, pancreatic neuroendocrine tumors, papilloma, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary peritoneal cancer, rectal cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Kaposi sarcoma, Sézary syndrome, small intestine cancer, soft tissue sarcoma, laryngeal cancer, thymoma and thymic cancer, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, urethral cancer, endometrial cancer, uterine sarcoma, vaginal cancer, vulvar cancer, or Waldenstrom's macroglobulinemia.

[0134] Vectors for inhibitory nucleic acid delivery

[0135] Viral vectors for inhibitory nucleic acid delivery

[0136] Viral genomes provide a rich source of vectors that can be used to effectively deliver a gene of interest to a target cell (e.g., mammalian cell, such as a human cell) in a patient's body. Viral genomes are particularly useful for gene delivery because the polynucleotides contained within such genomes are typically incorporated into the genome of the target cell by generalized or specialized transduction. These processes occur as part of the natural viral replication cycle and do not require the addition of proteins or reagents to induce gene integration. Exemplary of viral vectors that can be used in conjunction with the compositions and methods described herein are AAV, retroviruses, adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvoviruses (e.g., adeno-associated virus), coronaviruses, negative-strand RNA viruses such as orthomyxoviruses (e.g., influenza virus), rhabdoviruses (e.g., rabies virus and vesicular stomatitis virus), paramyxoviruses (e.g., measles virus and Sendai virus), positive-strand RNA viruses (such as picornaviruses and alphaviruses), and 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, modified vaccinia Ankara (MVA), fowlpox, and canarypox). Other viruses that can be used in conjunction with the compositions and methods described herein include, for example, Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, and hepacivirus. Examples of retroviruses include: avian leukosis sarcoma, mammalian C-type, B-type, D-type viruses, HTLV-BLV group, lentivirus, foamy virus (Coffin, JM, Retroviridae: The viruses and their replication, Fundamental Virology, 3rd 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 ape leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus, and lentivirus. Other examples of vectors are described, for example, in U.S. Pat. No. 5,801,030, the disclosure of which relating to viral vectors for gene therapy is incorporated herein by reference.

[0137] AAV vectors for inhibitory nucleic acid delivery

[0138] In some embodiments, an inhibitory nucleic acid construct, such as an interfering RNA construct described herein (e.g., a short interfering RNA (siRNA), a short hairpin RNA (shRNA), or a microRNA (miRNA)), is incorporated into a recombinant AAV (rAAV) vector to facilitate its introduction into cells. rAAV vectors that can be used in conjunction with the compositions and methods described herein include recombinant nucleic acid constructs comprising (1) a transgene encoding an inhibitory nucleic acid construct, such as an interfering RNA construct described herein (e.g., an siRNA, shRNA, or miRNA described herein); and (2) one or more nucleic acids that promote expression of a heterologous gene. The viral nucleic acid can include those sequences of AAV required for DNA replication in cis and packaging (e.g., functional ITRs) into viral particles. Such rAAV vectors can also include marker genes or reporter genes. Useful rAAV vectors include one or more of the naturally occurring AAV genes with a total or partial deletion but retaining functional flanking ITR sequences. AAV ITRs can be of any serotype suitable for a particular application (e.g., derived from serotype 2). Methods of 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), each of which is incorporated herein by reference for its disclosure of AAV vectors for gene delivery.

[0139] The nucleic acids and vectors described herein can be incorporated into rAAV virions to facilitate the introduction of nucleic acids or vectors into cells. The capsid protein of AAV constitutes the external 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. The construction of rAAV virions has been described in, for example, U.S. Patent Nos. 5,173,414; 5,139,941; 5,863,541; 5,869,305; 6,057,152; and 6,376,237; and Rabinowitz et al., J. Virol. 76: 791-801 (2002) and Bowles et al., J. Virol. 77: 423-432 (2003), each of which is incorporated herein by reference for its disclosure of AAV vectors for gene delivery.

[0140] rAAV virions useful in conjunction with the compositions and methods described herein include those derived from various AAV serotypes, including AAV 1, 2, 3, 4, 5, 6, 7, 8, and 9. The construction and use of AAV vectors and AAV proteins of different serotypes are described in, for example, 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), each of which is incorporated herein by reference for its disclosure relating to AAV vectors for gene delivery.

[0141] Also useful in conjunction with the compositions and methods described herein are pseudotyped rAAV vectors. Pseudotyped vectors include AAV vectors of a given serotype (e.g., AAV2) that are pseudotyped with a capsid gene derived from a serotype other than a given serotype (e.g., AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9, etc.). For example, a representative pseudotyped vector is an AAV2 vector that encodes a therapeutic protein pseudotyped with a capsid gene derived from AAV serotype 8 or AAV serotype 9. In some embodiments, the pseudotyped AAV has an ITR of an AAV serotype (e.g., AAV2) and VP1 capsid protein, VP2 capsid protein, and / or VP3 capsid protein from different AAV serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, or AAVrh74). Techniques involving the construction and use of pseudotyped rAAV 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).

[0142] In some embodiments, the AAV comprises a capsid disclosed, for example, in WO 2017 / 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-retroviral capsid protein or an AAV9-retroviral capsid protein. In some embodiments, the AAV comprises a capsid protein conjugated to a ligand or aptamer.

[0143] AAV virions with mutations in the virion capsid can be used to infect specific cell types more efficiently than non-mutated capsid virions. For example, suitable AAV mutants may have ligand insertion mutations that promote AAV targeting of specific cell types. The 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 those capsid hybrids generated by molecular breeding of viruses and by exon shuffling. See, for example, Soong et al., Nat. Genet., 25:436-439 (2000) and Kolman and Stemmer, Nat. Biotechnol. 19:423-428 (2001).

[0144] Additional Methods for Delivery of Inhibitory Nucleic Acids

[0145] Transfection technology

[0146] Techniques that can be used to introduce transgenes (such as transgenes encoding inhibitory nucleic acids described herein) into target cells (e.g., target cells from or located in human patients with RNA dominance) are known in the art. For example, mammalian cells (e.g., human target cells) can be permeabilized using electroporation by applying an electrostatic potential to the cells of interest. Mammalian cells (such as human cells) subjected to an external electric field in this way are subsequently susceptible to uptake of exogenous nucleic acids. Electroporation of mammalian cells is described in detail in, for example, Chu et al., Nucleic Acids Research 15: 1311 (1987), the disclosure of which is incorporated herein by reference. Similar techniques Nucleofection TM Nucleofection utilizes an applied electric field to stimulate the uptake of exogenous polynucleotides into the nucleus of eukaryotic cells. TMProtocols that can be used to perform this technique are described in detail in, for example, Distler et al., Experimental Dermatology 14:315 (2005) and US 2010 / 0317114, the disclosures of each of which are incorporated herein by reference.

[0147] Other techniques that can be used to transfect target cells include extrusion perforation. This technique induces rapid mechanical deformation of the cell to stimulate the uptake of exogenous DNA through membrane pores formed in response to the applied stress. The advantage of this technique is that a vector is not required to deliver the nucleic acid to cells (such as human target cells). Extrusion perforation 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.

[0148] Lipofection represents another technology that can be used to transfect target cells. This method involves loading nucleic acid into liposomes, which generally have cationic functional groups (such as quaternary amines or protonated amines) toward the outside of the liposomes. This promotes electrostatic interactions between liposomes and cells due to the anionic nature of the cell membrane, ultimately causing the uptake of exogenous nucleic acids, such as by direct fusion of liposomes with cell membranes or by endocytosis of complexes. Lipofection is described in detail in, for example, U.S. Patent No. 7,442,386, the disclosure of which is incorporated herein by reference. Similar techniques for causing exogenous nucleic acid uptake using ionic interactions with cell membranes include contacting cells with cationic polymer-nucleic acid complexes. Exemplary cationic molecules that associate with polynucleotides to impart a positive charge that facilitates interaction with cell membranes are activated dendrimers (described, for example, 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 a transfection agent 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 because this method utilizes an applied magnetic field to guide the uptake of nucleic acids. This technology is described in detail, for example, in US 2010 / 0227406, the disclosure of which is incorporated herein by reference.

[0149] Another tool that can be used to induce the uptake of exogenous nucleic acids by target cells is laser transfection, a technique that involves exposing cells to electromagnetic radiation of a specific wavelength to gently permeabilize the cells and allow the polynucleotide 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.

[0150] Microvesicle represents another potential medium that can be used to modify the target cell genome according to the method described herein.For example, protein can be effectively delivered to cells using microvesicles caused by co-expression of glycoprotein VSV-G and, for example, genome modification proteins (such as nucleases), followed by catalysis of site-specific cleavage of endogenous polynucleotide sequences to prepare the genome of cells for covalent inclusion of related polynucleotides (such as genes or regulatory sequences). The use of such vesicles (also referred to as nanovesicles (Gesicle)) in the genetic modification of eukaryotic cells is described in detail in, for example, Quinn et al., Genetic Modification of Target Cells by Direct Delivery of Active Protein [Abstract]., Methylation changes in early embryonic genes in cancer [Abstract], American Society of Gene and Cell Therapy 18th Annual Meeting Record (Proceedings of the 18th Annual Meeting of the American Society of Gene and Cell Therapy); May 13, 2015, in Abstract No. 122.

[0151] Incorporation of genes encoding inhibitory nucleic acids through gene editing

[0152] In addition to the above, a variety of tools have been developed that can be used to incorporate transgenes, such as transgenes encoding inhibitory nucleic acid constructs, such as interfering RNA constructs (e.g., short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA) as described herein), into target cells, particularly human cells. One such method that can be used to incorporate polynucleotides encoding inhibitory nucleic acids into target cells involves the use of transposons. A transposon is a polynucleotide that encodes a transposase and contains a polynucleotide sequence or gene of interest flanked by 5' and 3' excision sites. Once the transposon is delivered into the cell, expression of the transposase gene begins immediately and produces an active enzyme that cleaves the gene of interest from the transposon. This activity is mediated by site-specific recognition of the transposon excision sites by the transposase. In some cases, these excision sites can be terminal repeats or inverted terminal repeats. Once excised from the transposon, the gene of interest can be integrated into the genome of the mammalian cell by transposase-catalyzed cleavage at similar excision sites present in the nuclear genome of the cell. This allows the gene of interest to be inserted into the cut nuclear DNA at the complementary excision site, and subsequent covalent attachment of phosphodiester bonds linking the gene of interest to the DNA of the mammalian cell genome completes the incorporation process. In some cases, the transposon may be a retrotransposon, so that the gene encoding the target gene is first transcribed into an RNA product and then reverse transcribed into DNA before being incorporated into the mammalian cell genome. Exemplary transposon systems are the piggybac transposon (described in detail in, for example, WO 2010 / 085699) and the Sleeping Beauty transposon (described in detail in, for example, US 2005 / 0112764), each of which is incorporated herein by reference for its disclosure of the use of transposons for gene delivery to cells of interest.

[0153] Another tool for integrating target transgenes into the genome of target cells is the clustered regularly interspaced short palindromic repeats (CRISPR) / Cas system, a system that originally evolved as an adaptive defense mechanism for bacteria and archaea against viral infection. The CRISPR / Cas system comprises palindromic repeats within plasmid DNA and the associated Cas9 nuclease. This DNA and protein ensemble guides site-specific DNA cleavage of a target sequence by first incorporating exogenous DNA into the CRISPR locus. Polynucleotides containing these exogenous sequences and the repeat spacer elements of the CRISPR locus are then transcribed in the host cell to produce guide RNAs, which can then anneal to the target sequence and localize the Cas9 nuclease to this site. In this way, highly site-specific cas9-mediated DNA cleavage can be induced in exogenous polynucleotides because the interaction that brings cas9 into close proximity with the target DNA molecule is governed by RNA:DNA hybridization. Therefore, the CRISPR / Cas system can be designed to cleave any relevant target DNA molecule. This technology has been used to edit eukaryotic genomes (Hwang et al., Nature Biotechnology 31: 227 (2013)) and can be used as an effective way to site-specifically edit the genome of a target cell to cleave DNA before incorporating a gene encoding a target gene. Using CRISPR / Cas to regulate gene expression has been described in, for example, U.S. Patent No. 8,697,359, the disclosure of which is incorporated herein by reference for genome editing using the CRISPR / Cas system. Alternative methods for site-specific cutting of genomic DNA before incorporating a transgene of interest into a target cell include the use of zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Unlike the CRISPR / Cas system, these enzymes do not contain guide polynucleotides for locating at a specific target sequence. Target specificity is controlled by the 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), each of which is incorporated herein by reference for its disclosure of compositions and methods for genome editing.

[0154] Additional genome editing technologies that can be used to incorporate polynucleotides encoding target transgenes into the genome of target cells include the use of ARCUS, which can be rationally designed to site-specifically cleave genomic DNA. TMMeganucleases. In view of the defined structure-activity relationship established for such enzymes, it is advantageous to use these enzymes to incorporate genes encoding target genes into the genome of mammalian cells. Single-chain meganucleases can be modified at certain amino acid positions to produce nucleases that selectively cut DNA at the desired position, thereby allowing the target transgenic site to be specifically incorporated into the nuclear DNA of the target cell. These single-chain nucleases have been widely described in, for example, U.S. Patent Nos. 8,021,867 and US8,445,251, each of which is incorporated herein by reference for its disclosure of compositions and methods for genome editing.

[0155] Methods for detecting RNA transcript expression

[0156] The expression level of wild-type or pathological RNA transcripts (such as wild-type or mutant SOD1 RNA transcripts) can be determined, for example, by various nucleic acid detection techniques. Additionally or alternatively, RNA transcript expression can be inferred 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. Using these techniques, while monitoring the expression of the encoded protein, a decrease in the concentration of the wild-type or pathological RNA transcript in response to the compositions and methods described herein can be observed. The following sections describe exemplary techniques that can be used to measure the expression level of wild-type or pathological RNA transcripts and their downstream protein products. RNA transcript expression can be assessed by various methods known in the art, including, but not limited to, nucleic acid sequencing, microarray analysis, proteomics, in situ hybridization (e.g., fluorescence in situ hybridization (FISH)), amplification-based assays, in situ hybridization, fluorescence-activated cell sorting (FACS), Northern analysis of RNA, and / or PCR analysis.

[0157] Nucleic acid amplification testing

[0158] Nucleic acid-based methods for detecting RNA transcript expression include imaging-based techniques (e.g., Northern blotting or Southern blotting), which can be used in conjunction with cells obtained from a patient after administration of, for example, a vector encoding an inhibitory nucleic acid construct (such as an interfering RNA, e.g., a short interfering RNA (siRNA), a short hairpin RNA (shRNA), or a microRNA (miRNA) as described herein) or a composition containing such an inhibitory nucleic acid construct. Northern blot analysis is a conventional technique well 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. Typical protocols for assessing the status of genes and gene products are found in, for example, Ausubel et al., eds., 1995, Current Protocols In Molecular Biology, Units 2 (Northern Blotting), Unit 4 (Southern Blotting), Unit 15 (Immunoblotting), and Unit 18 (PCR Analysis).

[0159] RNA detection technologies that can be used in conjunction with the compositions and methods described herein to assess the expression level of RNA transcripts (such as SOD1 RNA transcripts) further include microarray sequencing experiments (e.g., Sanger sequencing and next-generation sequencing methods, also known as high-throughput sequencing or deep 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, transgenic expression at the mRNA level can be determined 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 in its entirety). RNA-Seq is a powerful technology for monitoring expression by directly sequencing RNA molecules in a sample. Briefly, this method may involve fragmenting RNA to an average length of 200 nucleotides, converting to cDNA by random priming, and synthesizing double-stranded cDNA (e.g., using a cDNA fragment from Agilent Technologies). Then, by adding sequence adapters (e.g., from / Solexa) converts the cDNA into molecular libraries for sequencing and maps the resulting 50-100 nucleotide reads to the genome.

[0160] RNA expression levels can be determined using a microarray-based platform (e.g., a single nucleotide polymorphism array) because microarray technology provides high resolution. Detailed information on 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 disclosure of each of which is incorporated herein by reference in its entirety. Using a nucleic acid microarray, an mRNA sample is reverse transcribed and labeled to generate cDNA. The probe can then hybridize with one or more complementary nucleic acids arranged and fixed on a solid support. The array can be configured so that, for example, the sequence and position of each member of the array are known. Hybridization of the labeled probe with a specific array member indicates that the sample from which the probe originates expresses the gene. Expression levels can be quantified based on the amount of signal detected from the hybridized probe-sample complex. A typical microarray experiment includes the following steps: 1) preparing a fluorescently labeled target from RNA isolated from the sample; 2) hybridizing the labeled target with the microarray; 3) cleaning, staining, and scanning the array; 4) analyzing the scanned image; and 5) generating a gene expression profile. An example of a microarray processor is Affymetrix Systems are commercially available and comprise arrays made by direct synthesis of oligonucleotides on a glass surface. Other systems known to those skilled in the art may be used.

[0161] Amplification-based assays can also be used to measure the expression level of specific RNA transcripts (such as wild-type or mutant SOD1 transcripts). In such assays, the nucleic acid sequence of the transcript 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. According to the principles described herein, comparison with appropriate controls provides a measure of the expression level of the transcript of interest corresponding to the specific probe used. Methods for real-time qPCR using TaqMan probes are well 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 level of RNA transcripts as described herein can be determined, for example, by RT-PCR techniques. Probes used in PCR can 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.

[0162] Protein detection

[0163] The expression of RNA construct can also be inferred by analyzing the expression of the protein encoded by the construct. Protein level can be assessed using standard detection techniques known in the art. Protein expression assays applicable to compositions and methods as herein described include proteomic methods, immunohistochemistry and / or western blot analysis, immunoprecipitation, molecular binding assays, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunosorbent assay (ELIFA), mass spectrometry, mass spectrometry immunoassays, and biochemical enzymatic activity assays. Specifically, proteomic methods can be used to generate multiple large-scale protein expression data sets. Proteomic methods can utilize mass spectrometry to detect and quantify polypeptide (e.g., protein) and / or peptide microarrays, utilize capture reagents (e.g., antibodies) specific to target protein groups to identify and measure the expression level of proteins expressed in samples (e.g., single cell samples or multicellular populations).

[0164] An exemplary peptide microarray has a plurality of polypeptides bound to a substrate, and the binding of an oligonucleotide, peptide, or protein to each of the plurality of binding polypeptides is individually detectable. Alternatively, the peptide microarray can comprise a plurality of binding agents, including but not limited to monoclonal antibodies, polyclonal antibodies, phage-displayed binding agents, yeast two-hybrid binding agents, aptamers, that can specifically detect the binding of 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 which are incorporated herein by reference in their entirety.

[0165] Mass spectrometry (MS) can be used in combination with the methods described herein to identify and characterize transgene expression in cells from patients (e.g., human patients) 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. A mass spectrometer typically includes an ion source and optical devices, a mass analyzer, and data processing electronics. Mass analyzers include scanning and ion beam mass spectrometers, such as time of flight (TOF) and quadrupole (Q); and capture mass spectrometers, such as ion traps (IT), Orbitrap, and Fourier transform ion cyclotron resonance (FT-ICR), which can be used for the methods described herein. Details of various MS methods can be found in the literature. See, for example, Yates et al., Annu. Rev. Biomed. Eng. 11: 49-79, 2009, the disclosure of which is incorporated herein by reference as a whole.

[0166] Before performing MS analysis, the proteins in the sample obtained from the patient can be digested into smaller peptides by chemical (e.g., via cyanogen bromide cleavage) or enzymatic (e.g., trypsin) digestion. Complex peptide samples also benefit from front-end separation techniques, such as the use of 2D-PAGE, HPLC, RPLC, and affinity chromatography. The digested and optionally separated sample is then ionized using an ion source to produce charged molecules for further analysis. The ionization of the sample 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 relevant to the selection of the ionization method is known to those skilled in the art.

[0167] After ionization, the digested peptides may then be fragmented to generate a signature MS / MS spectrum. Tandem MS, also known as MS / MS, may be particularly useful for analyzing complex mixtures. Tandem MS involves multiple steps of MS selection, where some form of ion fragmentation occurs between the stages, which can be achieved using individual mass spectrometer elements that are spatially separated or using a single mass spectrometer and MS steps that are separated in time. In spatially separated tandem MS, the elements are physically separated and distinct, with physical connections between the elements to maintain a high vacuum. In temporally separated tandem MS, separation is achieved by trapping ions in the same location, and multiple separation steps occur over time. The signature MS / MS spectrum can then be compared to a peptide sequence database (e.g., SEQUST). Post-translational modifications of the peptide can also be determined, for example by searching the spectrum against a database while allowing for specific peptide modifications.

[0168] Pharmaceutical composition

[0169] Inhibitory nucleic acid constructs, such as interfering RNA constructs (e.g., short interfering RNA (siRNA), short hairpin RNA (shRNA), or microRNA (miRNA)), and vectors and compositions encoding or containing such constructs can be incorporated into vectors for administration to patients (e.g., human patients suffering from a disease), as described herein. Pharmaceutical compositions containing vectors encoding inhibitory nucleic acid constructs as described herein (e.g., viral vectors) can be prepared using methods known in the art. For example, such compositions can be prepared using, for example, physiologically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences 16th ed., Osol, A., ed. (1980); incorporated herein by reference) and in a desired form, for example, as a lyophilized formulation or an aqueous solution.

[0170] The mixture of nucleic acid and viral vector described herein can be prepared in water mixed with one or more excipients, carriers or diluents. Dispersions can also be prepared in glycerol, liquid polyethylene glycol and their mixtures and in oil. Under normal storage and use conditions, these preparations may contain preservatives to prevent microbial growth. Pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions and sterile powders for the temporary preparation of sterile injectable solutions or dispersions (described in US 5,466,468, the disclosure of which is incorporated herein by reference). In either case, the preparation can be sterile and can be fluid to the extent that there is easy injectability. The preparation can be stable under manufacturing and storage conditions and can be preserved under conditions that prevent the contamination of microorganisms (such as bacteria and fungi). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.), suitable mixtures thereof and / or vegetable oils. Suitable fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by using a surfactant. The prevention of the action of microorganisms can be achieved 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 the injectable compositions can be achieved by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0171] For example, if necessary, the solution comprising the pharmaceutical composition as herein described can be appropriately buffered, and first the liquid diluent can be made isotonic with enough saline or glucose. These specific aqueous solutions are particularly suitable for use by being selected from the following approach: intrathalamic, intrathecal, subpial, intracerebral, intrastriatal, intracranial, intracisternal, intracerebral, intraventricular, intraocular (for example, intravitreal), intraventricular, intraspinal, intralumbar, intravenous, intramuscular, subcutaneous, intraperitoneal, intradermal, transdermal, parenteral, intranasal, transdermal, intratracheal, intraarterial, intravascular and oral administration, suction, perfusion, lavage or any combination thereof. In this regard, according to the disclosure, those skilled in the art will know the sterile aqueous medium that can be used. For example, a dose can be dissolved in 1ml isotonic NaCl solution and added to 1000ml subcutaneous infusion fluid or injected at the infusion site of suggestion. According to the disease of the subject being treated, certain dosage changes will necessarily occur. In either case, the person responsible for using will determine the dosage that is applicable to individual subjects. Moreover, for human administration, preparations may meet sterility, pyrogenicity, general safety, and purity standards as required by FDA Office of Biologics standards.

[0172] Pharmaceutical compositions containing, for example, inhibitory nucleic acids as described herein, typically comprise a pharmaceutically acceptable diluent or carrier. Pharmaceutical compositions can comprise, for example, sterile saline solution and nucleic acid (e.g., consisting thereof). Sterile saline is typically pharmaceutical grade saline. Pharmaceutical compositions can comprise, for example, sterile water and nucleic acid (e.g., consisting thereof). Sterile water is typically pharmaceutical grade water. Pharmaceutical compositions can comprise, for example, phosphate buffered saline (PBS) and nucleic acid (e.g., consisting thereof). Sterile PBS is typically pharmaceutical grade PBS.

[0173] In certain embodiments, pharmaceutical composition comprises one or more compositions or nucleic acid molecules and one or more excipients.In certain embodiments, excipient is selected from water, saline solution, alcohol, polyethylene glycol, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethyl cellulose and polyvinyl pyrrolidone.

[0174] In certain embodiments, the nucleic acid molecules can be mixed with pharmaceutically acceptable active and / or inert substances to prepare pharmaceutical compositions or formulations. The composition and the method for preparing the pharmaceutical composition depend on many criteria, including but not limited to the route of administration, the extent of the disease, or the dose to be administered.

[0175] In certain embodiments, the pharmaceutical composition comprising nucleic acid molecules encompasses any pharmaceutically acceptable salt of an inhibitor, an ester of an inhibitor, or a salt of such an ester. In certain embodiments, the pharmaceutical composition comprising nucleic acid molecules can provide (directly or indirectly provide) its biologically active metabolite or residue after being administered to a subject (e.g., a person). Therefore, for example, the disclosure further relates to pharmaceutically acceptable salts, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents of inhibitors. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium salts and potassium salts. In certain embodiments, prodrugs include one or more conjugate groups attached to nucleic acid molecules, wherein the conjugate groups are cleaved by endogenous nucleases in vivo.

[0176] Lipid moieties have been used in nucleic acid therapies in various methods. In some such methods, nucleic acids are introduced into preformed liposomes or lipid complexes made from a mixture of cationic lipids and neutral lipids. In some methods, DNA complexes with monocationic lipids or polycationic lipids are formed in the absence of neutral lipids. In certain embodiments, lipid moieties are selected to increase the distribution of the agent to specific cells or tissues. In certain embodiments, lipid moieties are selected to increase the distribution of the agent to adipose tissue. In certain embodiments, lipid moieties are selected to increase the distribution of the agent to muscle tissue.

[0177] 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 can be used to prepare certain pharmaceutical compositions, including those comprising hydrophobic compounds. In certain embodiments, certain organic solvents, such as dimethyl sulfoxide, are used.

[0178] In certain embodiments, the pharmaceutical composition comprises one or more tissue-specific delivery molecules designed to deliver one or more pharmaceutical agents of the present invention to a specific tissue or cell type. For example, in certain embodiments, the pharmaceutical composition comprises liposomes coated with tissue-specific antibodies.

[0179] In certain embodiments, the pharmaceutical composition comprises a co-solvent system. Certain such co-solvent systems comprise, for example, benzyl alcohol, a non-polar surfactant, a water-immiscible organic polymer, and an aqueous phase. In certain embodiments, the co-solvent system is for hydrophobic compounds. A non-limiting example of such a co-solvent system is the VPD co-solvent system, which comprises 3% w / v benzyl alcohol, 8% w / v of the non-polar surfactant polysorbate 80, and 1% w / v of the non-polar surfactant polysorbate 80. TM and 65% w / v polyethylene glycol 300 in absolute ethanol. The proportions of such cosolvent systems can be varied significantly without significantly changing their solubility and toxicity characteristics. In addition, the characteristics of the cosolvent components can be varied: for example, other surfactants can be used instead of polysorbate 80. TM ; The fraction size of polyethylene glycol can be varied; other biocompatible polymers can replace polyethylene glycol, such as polyvinyl pyrrolidone; and other sugars or polysaccharides can replace dextrose.

[0180] 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, intrathecal, intracerebroventricular, etc.). In certain embodiments of such embodiments, the pharmaceutical composition includes a carrier and is formulated in an aqueous solution, such as water or a physiologically compatible buffer such as Hanks solution, Ringer's solution or physiological saline buffer. In certain embodiments, other ingredients (e.g., ingredients that help dissolve or act as preservatives) are included. In certain embodiments, injectable suspensions are prepared using appropriate liquid carriers, suspending agents, etc. Some pharmaceutical compositions for injection are in unit dosage form, for example, provided in ampoules or in multidose containers. Some pharmaceutical compositions for injection are suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain preparatons, such as suspending agents, stabilizers and / or dispersants. Certain suitable solvents for 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.

[0181] Route of administration and dosing

[0182] A transgenic viral vector (such as an AAV vector and other vectors described herein) containing an inhibitory nucleic acid encoding the present invention can be administered to a patient (e.g., a human patient) by a variety of administration routes. The administration route can, for example, vary with the onset and severity of the disease, and can be selected from, for example, the following: intrathalamic, intrathecal, subpial, intracerebral, intrastriatal, intracranial, intracisternal, intracerebral, intraventricular, intraocular (e.g., intravitreal), intraventricular, intraspinal, intravenous, intramuscular, subcutaneous, intraperitoneal, intradermal, transcutaneous, parenteral, intranasal, transdermal, intratracheal, intraarterial, intravascular, and oral administration, inhalation, perfusion, lavage, or any combination thereof. Intravascular administration includes delivery to the patient's vascular system. In some embodiments, administration is administration into a blood vessel considered as a vein (intravenous), and in some administrations, administration is administration into a blood vessel considered as an artery (intraarterial). Veins include, but are not limited to, the internal jugular vein, peripheral vein, coronary vein, hepatic vein, portal vein, great saphenous vein, pulmonary vein, 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 arch, femoral arteries, peripheral arteries, and / or ciliary arteries. It is contemplated that delivery may be through or to arterioles or capillaries.

[0183] Treatment regimens can vary and generally depend on the severity of the disease and the age, weight, and sex of the patient. Treatment can include administration of vectors (e.g., viral vectors) or other agents described herein that can be used to introduce a transgene into target cells at varying unit doses. Each unit dose will generally contain a predetermined amount of the therapeutic composition.

[0184] Reagent test kit

[0185] The compositions and methods described herein can be provided in a kit for use in treating disorders associated with expression of wild-type or mutant superoxide dismutase 1 (SOD1) RNA transcripts (e.g., Parkinson's disease), etc. In some embodiments, the kit can include a pharmaceutical composition of the present disclosure. The kit can include a package insert that instructs the user of the kit (such as a physician with skills in the art) to perform any of the therapeutic methods described herein. The kit can optionally include a syringe or other device for administering a composition of the present disclosure. In some embodiments, the kit can include one or more additional therapeutic agents.

[0186] Example

[0187] The following examples are put forth so as to provide one of ordinary skill in the art with a description of how the compositions and methods described herein might be used and evaluated and are intended to be purely illustrative of the invention and are not intended to limit the scope of what the inventors regard as their invention.

[0188] Example 1. Determination of knockdown efficacy of SOD1-specific miRNA constructs in HEK cells by dual-luciferase reporter assay and probe-based qPCR assay after standard plasmid (pMix) transfection

[0189] Purpose

[0190] The aim of this study was to evaluate the knockdown efficacy of several SOD1 miRNA candidates in HEK cells using a dual-luciferase reporter assay and a probe-based qPCR assay. This study was designed to determine the efficiency of the miRNA constructs in silencing SOD1 mRNA.

[0191] Materials and methods

[0192] HEK cells were transfected with a standard plasmid (pMix) encoding a SOD1-specific miRNA construct. Dual-luciferase reporter assays and probe-based qPCR assays were then performed to measure the knockdown efficacy achieved by each miRNA construct.

[0193] The miRNA constructs tested are shown in the table below:

[0194]

[0195]

[0196] result

[0197] As a result, we observed that SOD1 miRNA candidates were able to silence SOD1 to varying degrees ( Figure 1A 、 Figure 1B ), some of the miRNA constructs (miRNA8, 9, 10, and 13) achieved higher knockdown efficiency compared with other miRNA constructs.

[0198] Example 2. Determination of knockdown efficacy of SOD1-specific miRNA constructs in HEK cells by dual luciferase reporter assay and probe-based qPCR assay after viral plasmid (iMix) transfection

[0199] Purpose

[0200] The aim of this study was to evaluate the knockdown efficacy of several SOD1-specific miRNA candidates in HEK cells following plasmid transfection using a dual-luciferase reporter assay and a probe-based qPCR assay. This study was designed to determine the efficiency of the miRNA constructs in silencing SOD1 mRNA.

[0201] Materials and methods

[0202] HEK cells were transfected with viral plasmids encoding SOD1-specific miRNA constructs (iMix). Dual-luciferase reporter assays and probe-based qPCR assays were then performed to measure the knockdown efficacy achieved by each miRNA construct compared to a negative control that did not achieve significant silencing.

[0203] result

[0204] As a result, we observed that SOD1 miRNA candidates were able to silence SOD1 to varying degrees ( Figure 2A 、 Figure 2B ), some of which (miRNA1, 6, 8, 9, 10, and 13) achieved higher knockdown efficiency than other miRNA constructs. The control (CONT) was a non-targeting miRNA and showed no knockdown.

[0205] Example 3. Determination of knockdown efficacy of SOD1-specific miRNA constructs in HEK293 cells and SH-SY5Y cells by probe-based qPCR assay after viral transduction

[0206] Purpose

[0207] The aim of this study was to evaluate the knockdown efficacy of several homologous SOD1 miRNA constructs in HEK293 and SH-SY5Y cells following viral transduction by a probe-based qPCR assay. This study was designed to determine the efficiency of SOD1-specific miRNA constructs in silencing SOD1 mRNA.

[0208] Materials and methods

[0209] HEK293 and SH-SY5Y cells were transduced with AAV vectors (vMix) incorporated with SOD1 miRNA candidates for miRNA delivery and subsequent knockdown of SOD1 mRNA. Probe-based qPCR assays were then performed to measure the knockdown efficacy achieved with the miRNA constructs compared to a negative control that did not achieve significant silencing.

[0210] result

[0211] As a result, we observed that SOD1 miRNA candidates were able to silence SOD1 to varying degrees in HEK293 cells ( Figure 3A ), some of the miRNA constructs (miRNA1, 9, and 10) achieved higher knockdown efficiency compared with other miRNA constructs. As a result, we observed that the SOD1 miRNA candidates were able to silence SOD1 to varying degrees in SH-SY5Y cells ( Figure 3B ), some of the miRNA constructs (miRNA1 and 9) achieved higher knockdown efficiency compared to other miRNA constructs. The control (neg) is a non-targeting miRNA and shows no knockdown.

[0212] Other implementation plans

[0213] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.

[0214] Although the invention has been described in conjunction with specific embodiments thereof, it will be appreciated that it is capable of further modifications and this application is intended to cover any changes, uses or adaptations of the invention that are basically based on the principles of the invention, including changes that are not part of the invention but are known or customary implementations in the field to which the invention pertains and that fall within the essential features described above and that fall within the scope of the claims.

[0215] Other embodiments are within the 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 a superoxide dismutase 1 (SOD1) mRNA transcript having a nucleic acid sequence of any one of SEQ ID NO: 33 to SEQ ID NO:

48.

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 consecutive nucleotides within the region of the SOD1 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NO: 33 to SEQ ID NO:

48.

3. The inhibitory nucleic acid of claim 2, wherein the guide strand has at least 75% complementarity to a segment of 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides within the region of the nucleic acid sequence of any one of SEQ ID NO: 33 to SEQ ID NO: 48 of the SOD1 mRNA transcript, optionally wherein the guide strand has at least 75% complementarity to a segment of 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides within the region of the nucleic acid sequence of any one of SEQ ID NO: 33 to SEQ ID NO: 48 of the SOD1 mRNA transcript. NO:

48. A segment of 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides within the region of the nucleic acid sequence of any one of NO:48 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.

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 consecutive nucleotides that are fully complementary to an equal length of consecutive polynucleotides within the region of the SOD1 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NO: 33 to SEQ ID NO:

48.

5. The inhibitory nucleic acid of claim 4, wherein the guide strand comprises 10 to 21 consecutive nucleotides that are fully complementary to a continuous polynucleotide of equal length within the region of the SOD1 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NO: 33 to SEQ ID NO:

48.

6. The inhibitory nucleic acid of claim 5, wherein the guide strand comprises 12 to 21 consecutive nucleotides that are fully complementary to a continuous polynucleotide of equal length within the region of the SOD1 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NO: 33 to SEQ ID NO:

48.

7. The inhibitory nucleic acid of claim 6, wherein the guide strand comprises 15 to 21 consecutive nucleotides that are fully complementary to a continuous polynucleotide of equal length within the region of the SOD1 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NO: 33 to SEQ ID NO:

48.

8. The inhibitory nucleic acid of claim 7, wherein the guide strand comprises 18 to 21 consecutive nucleotides that are fully complementary to a continuous polynucleotide of equal length within the region of the SOD1 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NO: 33 to SEQ ID NO:

48.

9. The inhibitory nucleic acid of claim 8, wherein the guide strand comprises 19, 20, or 21 consecutive nucleotides that are fully complementary to a continuous polynucleotide of equal length within the region of the SOD1 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NO: 33 to SEQ ID NO:

48.

10. The inhibitory nucleic acid of any one of claims 1 to 9, wherein the guide strand comprises 9 or fewer nucleotide mismatches relative to a stretch of 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides within the region of the SOD1 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NO: 33 to SEQ ID NO: 48, optionally wherein 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 relative to the region of the SOD1 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NO: 33 to SEQ ID NO:

48.

11. The inhibitory nucleic acid of any one of claims 1 to 10, wherein the region of the SOD1 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NO:33, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, and SEQ ID NO:

45.

12. The inhibitory nucleic acid of any one of claims 1-11, 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 NO: 1 to SEQ ID NO:

16.

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 NO: 1 to SEQ ID NO:

16.

14. The inhibitory nucleic acid of claim 13, wherein 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 NO: 1 to SEQ ID NO: 16, optionally wherein the guide strand 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 NO: 1 to SEQ ID NO:

16.

15. The inhibitory nucleic acid molecule of claim 14, wherein the guide strand has a nucleic acid sequence of any one of SEQ ID NO: 1 to SEQ ID NO:

16.

16. The inhibitory nucleic acid of any one of claims 12-15, wherein the guide strand has a nucleic acid sequence of any one of SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO:

13.

17. The inhibitory nucleic acid of any one of claims 1-16, 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 NO: 17 to SEQ ID NO:

32.

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 NO: 17 to SEQ ID NO:

32.

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 NO: 17 to SEQ ID NO: 32, optionally wherein 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 NO: 17 to SEQ ID NO:

32.

20. The inhibitory nucleic acid of claim 19, wherein the hairpin has a nucleic acid sequence of any one of SEQ ID NO: 17 to SEQ ID NO:

32.

21. The inhibitory nucleic acid of any one of claims 17-20, wherein the hairpin has a nucleic acid sequence of any one of SEQ ID NO: 17, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO:

29.

22. The inhibitory nucleic acid of any one of claims 1-21, 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. A viral vector comprising a transgene encoding the inhibitory nucleic acid of any one of claims 1-23, optionally wherein the viral vector comprises a plurality of said transgenes (e.g., 2, 3, 4, 5 or more said transgenes).

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 AAV.

27. The viral vector of claim 26, wherein: (i) the AAV is of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, or AAVrh74, or wherein the AAV comprises an AAV2-retroviral capsid protein or an AAV9-retroviral capsid protein; and / or (ii) The AAV comprises a capsid protein conjugated to a ligand or aptamer.

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

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

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

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

32. The viral vector of claim 25, wherein the synthetic virus is a chimeric virus, a mosaic virus, or a pseudotyped virus, and / or comprises exogenous proteins, synthetic polymers, nanoparticles, or small molecules.

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

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

35. The method of claim 34, wherein the nervous system disorder is a neurodegenerative disorder.

36. The method of claim 34 or 35, wherein the nervous system disorder is caused by or associated with the expression of wild-type or mutant SOD1.

37. The method of any one of claims 34-36, wherein the nervous system disorder is amyotrophic lateral sclerosis (ALS), Huntington's disease, Parkinson's disease, frontotemporal dementia, primary lateral sclerosis, progressive muscular dystrophy, limbic predominant age-related TDP-43 encephalopathy, chronic traumatic encephalopathy, dementia with Lewy bodies, corticobasal degeneration, progressive supranuclear palsy, Parkinson's disease-ALS syndrome, Pick's disease, Perry syndrome, brain age-related TDP-43 and sclerosis, hippocampal sclerosis, or Alzheimer's disease.

38. The method of claim 34, wherein the neurological disorder is ALS.

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

40. The method of claim 39, wherein the cell proliferative disorder is cancer.

41. The method of claim 40, wherein the cancer is leukemia, lymphoma, liver cancer, bone cancer, lung cancer, brain cancer, bladder cancer, gastrointestinal cancer, breast cancer, cardia cancer, cervical cancer, uterine cancer, head and neck cancer, gallbladder cancer, laryngeal cancer, lip and oral cancer, eye cancer, melanoma, pancreatic cancer, prostate cancer, colorectal cancer, testicular cancer, laryngeal cancer, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, adrenocortical carcinoma, acquired immunodeficiency syndrome-associated lymphoma, primary central nervous system lymphoma, anal cancer, appendix cancer, astrocytoma, non Typical teratoid rhabdoid tumor, basal cell carcinoma, bile duct cancer, extrahepatic cancer, Ewing's sarcoma, osteosarcoma, malignant fibrous histiocytoma, central nervous system embryonal tumor, central nervous system germ cell tumor, craniopharyngioma, ependymoma, bronchial tumor, Burkitt's lymphoma, carcinoid tumor, primary lymphoma, chordoma, chronic myeloproliferative tumor, colon cancer, extrahepatic bile duct cancer, ductal carcinoma in situ, endometrial cancer, ependymoma, esophageal cancer, olfactory neuroblastoma, extracranial germ cell tumor, extragonadal germ cell tumor, fallopian tube cancer, bone fibrous histiocytoma, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, testicular cancer Germ cell tumor, gestational trophoblastic disease, glioma, childhood brainstem glioma, hairy cell leukemia, hepatocellular carcinoma, Langerhans cell histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, islet cell tumor, pancreatic neuroendocrine tumor, Wilms tumor, childhood kidney tumor, nephroblastoma, small cell lung cancer, cutaneous T-cell lymphoma, intraocular melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer, midline tract cancer, multiple endocrine neoplasia syndrome, multiple myeloma, plasma cell neoplasms, plasma cell neoplasms, myelodysplastic syndrome, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma, non Small cell lung cancer, epithelial ovarian cancer, germ cell ovarian cancer, low malignant potential ovarian cancer, pancreatic neuroendocrine tumors, papilloma, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary peritoneal cancer, rectal cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Kaposi sarcoma, Sézary syndrome, small intestine cancer, soft tissue sarcoma, laryngeal cancer, thymoma and thymic cancer, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, urethral cancer, endometrial cancer, uterine sarcoma, vaginal cancer, vulvar cancer, or Waldenstrom's macroglobulinemia.

42. The method of any one of claims 34-41, wherein the inhibitory nucleic acid, viral vector, or pharmaceutical composition is administered to the subject by a route selected from the group consisting of intrathalamic, intrathecal, subpial, intraparenchymal, intrastriatal, intracranial, intracisternal, intracerebral, intraventricular, intraocular, intravitreal, intraventricular, intralumbar spinal cord, intravenous, intramuscular, subcutaneous, intraperitoneal, intradermal, transdermal, parenteral, intranasal, transdermal, intratracheal, intraarterial, intravascular, and oral administration, inhalation, infusion, lavage, or any combination thereof.

43. The method of any one of claims 34 to 42, wherein the subject is a mammal.

44. The method of claim 43, wherein the mammal is a human.

45. A kit comprising an inhibitory nucleic acid as described in any one of claims 1-23, a viral vector as described in any one of claims 24-32, or a pharmaceutical composition as described in claim 33, wherein the kit further comprises 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 wherein the subject is a human.

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