WO2024056902A2 - Compositions et méthodes de traitement de maladies neurologiques - Google Patents

Compositions et méthodes de traitement de maladies neurologiques Download PDF

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WO2024056902A2
WO2024056902A2 PCT/EP2023/075536 EP2023075536W WO2024056902A2 WO 2024056902 A2 WO2024056902 A2 WO 2024056902A2 EP 2023075536 W EP2023075536 W EP 2023075536W WO 2024056902 A2 WO2024056902 A2 WO 2024056902A2
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nucleic acid
atxn2
acid sequence
seq
inhibitory nucleic
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WO2024056902A3 (fr
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Christopher Shaw
Youn Bok Lee
Romain JOUBERT
Do Young Lee
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Christopher Shaw
Youn Bok Lee
Joubert Romain
Do Young Lee
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • Ataxin-2 is a protein involved in several functions, such as formation of stress granules, P- bodies, and regulation of mRNA translation.
  • Ataxin-2 protein is encoded by the ATXN2 gene and is associated with diseases such as spinocerebellar ataxia-2 (SCA2), as CAG trinucleotide repeat mutations in the ATXN2 gene can give rise to neuronal degeneration.
  • SCA2 spinocerebellar ataxia-2
  • Ataxin-2 protein generally has a sequence of glutamine residues, referred to as the polyglutamine repeat.
  • Wild-type Ataxin-2 genes typically contain from about 13 to about 31 CAG trinucleotide repeats, with 22 repeats being among the most common.
  • the length of the sequence in healthy individuals is approximately 22 amino acids; yet expansions of this sequence have been observed.
  • glutamine repeats of 34 residues or more have been found in individuals with SCA2 and repeats of from 27 to 40 residues have been reported in individuals with amyotrophic lateral sclerosis (ALS). Both ALS and SCA2 are progressive, neurodegenerative, can be highly debilitating, and are often fatal in nature.
  • compositions and methods useful for treating diseases associated with expression of wild-type or mutant Ataxin-2 include inhibitory nucleic acid constructs, for example, interfering RNA constructs, that suppress the expression of wild-type or mutant mRNA transcripts.
  • inhibitory nucleic acids of the disclosure are, without limitation, microRNA (miRNA), short hairpin RNA (shRNA), and short interfering RNA (siRNA) constructs. Without being limited by mechanism, these inhibitory nucleic acids may anneal to portions of wild-type or mutant ATXN2 mRNA and promote the degradation of pathological transcripts by way of various cellular processes.
  • the present disclosure additionally features vectors, such as viral vectors, encoding such inhibitory nucleic acid constructs.
  • Exemplary viral vectors described herein that encode inhibitory nucleic acid constructs are adeno-associated viral (AAV) vectors, such as pseudotyped AAV2/8 and AAV2/9 vectors.
  • AAV adeno-associated viral
  • a patient diagnosed as having a disease associated with wild-type or mutant ATXN2 can be administered an inhibitory nucleic acid, such as an interfering RNA construct, or a vector encoding the same, so as to reduce the expression of wild-type or mutant mRNA transcripts.
  • an inhibitory nucleic acid such as an interfering RNA construct, or a vector encoding the same, so as to reduce the expression of wild-type or mutant mRNA transcripts.
  • compositions and methods described herein can be used to treat patients having SCA2, as such patients may be administered an inhibitory nucleic acid construct or a viral vector, such as an AAV vector, encoding such a construct, thereby reducing the expression of mRNA transcripts encoding wild-type or mutated Ataxin-2 protein.
  • Wild-type Ataxin-2 gene typically contains from about 13 to about 31 CAG trinucleotide repeats, with 22 repeats being the most common.
  • ATXN2 mRNA transcripts transcribed from a mutant version of the gene that contains 34 or more CAG repeats have been reported in patients with SCA2
  • ATXN2 mRNA transcripts transcribed from a mutant version of the gene that contains between 27 to 40 CAG repeats have been reported in patients with ALS (e.g., at least 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 CAG repeats).
  • compositions and methods described herein can be used to treat patients expressing wild-type or mutant ATXN2 mRNA (e.g., wild-type or mutant human ATXN2 mRNA), e.g., by using inhibitory nucleic acid constructs to suppress the expression of the wild-type or mutant ATXN2 mRNA.
  • the disclosure features an inhibitory nucleic acid comprising a guide strand (and, optionally, a passenger strand having complementarity to the guide strand).
  • the guide strand has complementarity sufficient to hybridize to a region within an ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.
  • 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 ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153. In some embodiments, the guide strand has at least 75% complementarity to a segment of 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides within the region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.
  • the guide strand has at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementarity to a segment of 15, 16, 17, 18, 19, 20, 21, or more contiguous nucleotides within the region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.
  • the guide strand has at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementarity to a segment of 15 contiguous nucleotides within the region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.
  • the guide strand has at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementarity to a segment of 16 contiguous nucleotides within the region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.
  • the guide strand has at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementarity to a segment of 17 contiguous nucleotides within the region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.
  • the guide strand has at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementarity to a segment of 18 contiguous nucleotides within the region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.
  • the guide strand has at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementarity to a segment of 19 contiguous nucleotides within the region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.
  • the guide strand has at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementarity to a segment of 20 contiguous nucleotides within the region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.
  • the guide strand has at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementarity to a segment of 21 contiguous nucleotides within the region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.
  • the guide strand comprises at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleotides that are fully complementary to a contiguous polynucleotide of equal length within the region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153. In some embodiments, the guide strand comprises at least 10 contiguous nucleotides that are fully complementary to a contiguous polynucleotide of equal length within the region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.
  • the guide strand comprises at least 11 contiguous nucleotides that are fully complementary to a contiguous polynucleotide of equal length within the region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153. In some embodiments, the guide strand comprises at least 12 contiguous nucleotides that are fully complementary to a contiguous polynucleotide of equal length within the region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.
  • the guide strand comprises at least 13 contiguous nucleotides that are fully complementary to a contiguous polynucleotide of equal length within the region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153. In some embodiments, the guide strand comprises at least 14 contiguous nucleotides that are fully complementary to a contiguous polynucleotide of equal length within the region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.
  • the guide strand comprises at least 15 contiguous nucleotides that are fully complementary to a contiguous polynucleotide of equal length within the region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153. In some embodiments, the guide strand comprises at least 16 contiguous nucleotides that are fully complementary to a contiguous polynucleotide of equal length within the region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.
  • the guide strand comprises at least 17 contiguous nucleotides that are fully complementary to a contiguous polynucleotide of equal length within the region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153. In some embodiments, the guide strand comprises at least 18 contiguous nucleotides that are fully complementary to a contiguous polynucleotide of equal length within the region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.
  • the guide strand comprises at least 19 contiguous nucleotides that are fully complementary to a contiguous polynucleotide of equal length within the region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153. In some embodiments, the guide strand comprises at least 20 contiguous nucleotides that are fully complementary to a contiguous polynucleotide of equal length within the region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.
  • the guide strand comprises 21 contiguous nucleotides that are fully complementary to a contiguous polynucleotide of equal length within the region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153. In some embodiments, the guide strand comprises from 10 to 21 contiguous nucleotides that are fully complementary to a contiguous polynucleotide of equal length within the region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.
  • the guide strand comprises from 12 to 21 contiguous nucleotides that are fully complementary to a contiguous polynucleotide of equal length within the region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153. In some embodiments, the guide strand comprises from 15 to 21 contiguous nucleotides that are fully complementary to a contiguous polynucleotide of equal length within the region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.
  • the guide strand comprises from 18 to 21 contiguous nucleotides that are fully complementary to a contiguous polynucleotide of equal length within the region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153. In some embodiments, the guide strand comprises 19, 20, or 21 contiguous nucleotides that are fully complementary to a contiguous polynucleotide of equal length within the region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.
  • the guide strand comprises 9 or fewer nucleotide mismatches relative to a segment of 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides within the region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153, 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 ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.
  • the region of the ATXN2 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 103, 104, 105, 106, 121, 122, 126, 127, 139, 140, 141, 147, 149, and 153.
  • the guide strand has a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence of any one of SEQ ID NOs: 1-51 (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence of any one of SEQ ID NOs: 1-51).
  • the guide strand has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of any one of SEQ ID NOs: 1-51 (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence of any one of SEQ ID NOs: 1-51).
  • the guide strand has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NOs: 1-51 (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence of any one of SEQ ID NOs: 1-51).
  • the guide strand has the nucleic acid sequence of any one of SEQ ID NOs: 1-51. In some embodiments, the nucleic acid sequence is any one of SEQ ID NOs: 1, 2, 3, 4, 19, 20, 24, 25, 37, 38, 39, 45, 47, and 51.
  • the inhibitory nucleic acid comprises a hairpin having a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence of any one of SEQ ID NOs: 52- 102 (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence of any one of SEQ ID NOs: 52-102).
  • the inhibitory nucleic acid comprises a hairpin having a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of any one of SEQ ID NOs: 52-102 (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence of any one of SEQ ID NOs: 52-102).
  • the hairpin has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of any one of SEQ ID NOs: 52- 102 (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence of any one of SEQ ID NOs: 52-102).
  • the hairpin strand has the nucleic acid sequence of any one of SEQ ID NOs: 52-102.
  • the nucleic acid sequence is any one of SEQ ID NOs: 52, 53, 54, 55, 70, 71, 75, 76, 88, 89, 90, 96, 98, and 102.
  • the inhibitory nucleic acid is an interfering RNA molecule.
  • the interfering RNA molecule is a microRNA (miRNA), short hairpin RNA (shRNA), or short interfering RNA (siRNA).
  • the inhibitory nucleic acid is a miRNA.
  • the disclosure features a viral vector comprising a transgene encoding the inhibitory nucleic acid of any of the above aspects or embodiments of the disclosure.
  • the viral vector comprises a plurality of the transgenes (e.g., 2, 3, 4, 5, or more of the transgenes).
  • 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 a synthetic virus.
  • AAV adeno-associated virus
  • the viral vector is an AAV.
  • the AAV is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, or AAVrh74 serotype.
  • the viral vector is a pseudotyped AAV.
  • the pseudotyped AAV has the ITRs of one AAV serotype (e.g., AAV2) and the VP1, VP2, and/or VP3 capsid proteins from a different AAV serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, or AAVrh74).
  • AAV1 AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, or AAVrh74
  • the pseudotyped AAV is AAV2/9.
  • the pseudotyped AAV is AAV2/8.
  • the AAV comprises a recombinant capsid protein.
  • the AAV comprises a capsid disclosed, e.g., in WO 2017/218842, the disclosure of which is incorporated herein by reference.
  • 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.
  • the AAV comprises an AAV2-retro or an AAV9-retro capsid protein.
  • the synthetic virus is chimeric virus, mosaic virus, or pseudotyped virus, and/or comprises a foreign protein, synthetic polymer, nanoparticle, or small molecule.
  • the disclosure features a pharmaceutical composition comprising the inhibitory nucleic acid or the viral vector of any of the above aspects or embodiments of the disclosure, along with a pharmaceutically acceptable excipient, carrier, or diluent.
  • the disclosure features a method of treating a neurological disease 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.
  • the neurological disease is associated with a TDP-43 (TAR DNA binding protein 43) proteinopathy.
  • the neurological disease is caused by, or associated with, expression of a wild-type or mutant form of ATXN2.
  • the neurological disease is ALS, frontotemporal lobar dementia, primary lateral sclerosis, progressive muscular atrophy, limbic-predominant age-related TDP-43 encephalopathy, chronic traumatic encephalopathy, dementia with Lewy bodies, corticobasal degeneration, progressive supranuclear palsy, dementia Parkinsonism ALS complex of guam, Pick’s disease, Perry syndrome, cerebral age- related TDP-43 with sclerosis, hippocampal sclerosis, Huntington’s disease, Parkinson’s disease, Alzheimer’s disease, or SCA2.
  • the neurological disease is SCA2, and the subject may be one that has a plurality of CAG trinucleotide repeat mutations in the ATXN2 locus, e.g., at least 34, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, or 100 CAG repeats.
  • the neurological disease is ALS, and the subject may be one that has a plurality of CAG trinucleotide repeat mutations in the ATXN2 locus, e.g., at least 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 CAG repeats.
  • the neurological disease is Huntington’s disease
  • the subject may be one that has upregulated ATXN2.
  • the inhibitory nucleic acid, viral vector, or pharmaceutical composition is administered to the subject by a route selected from intrathalamic, intrathecal, subpial, intraparenchymal, intrastriatal, intracranial, intracisternal, intracerebral, intracerebroventricular, intraocular (e.g., intravitreal), intraventricular, intralumbar, intravenous, intramuscular, subcutaneous, intraperitoneal, intradermal, transdermal, parenteral, intranasal, percutaneous, intratracheal, intraarterial, intravascular, and oral administration, inhalation, perfusion, lavage, or any combination thereof.
  • the subject is a mammal (e.g., a human).
  • 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 include a package insert instructing the 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 as having a neurological disease described herein).
  • a subject e.g., a mammal, such as a human, diagnosed as having a neurological disease described herein.
  • FIG.1 is a bar graph showing the knockdown efficacy of a series of ATXN2-specific miRNA constructs.
  • FIG.1 abbreviations: ATXN2, Ataxin-2; A2, Ataxin-2; HEK, Human embryonic kidney.
  • FIG.2A is a bar graph showing the knockdown efficacy of several ATXN2-specifc miRNA constructs from FIG.1.
  • FIG.2A abbreviations: ATXN2, Ataxin-2; HEK, Human embryonic kidney.
  • FIG.2B is a bar graph showing the knockdown efficacy of several ATXN2-specific miRNA constructs from FIG.1.
  • FIG.2B abbreviations: ATXN2, Ataxin-2; HEK, Human embryonic kidney; GAPDH, Glyceraldehyde-3-phosphate dehydeogenase; qPCR, quantitative polymerase chain reaction.
  • FIG.2C is a bar graph showing the knockdown efficacy of several ATXN2-specific miRNA constructs from FIG.1.
  • FIG.2C is a bar graph showing the knockdown efficacy of several ATXN2-specific miRNA constructs from FIG.1. Data were obtained from a qPCR assay performed in HEK293 cells after viral transduction and are presented as a ratio of ATXN2 to GAPDH mRNA signal.
  • Control is a non-targeting miRNA.
  • Data represent 3-4 independent experiments with biological triplicates.
  • FIG.3 abbreviations: ATXN2, Ataxin-2; HEK, Human embryonic kidney; GAPDH, Glyceraldehyde-3- phosphate dehydeogenase; qPCR, quantitative polymerase chain reaction.
  • FIG.4A is a series of bar graphs showing the knockdown efficacy of an ATXN2-specific miRNA construct from FIG.1. Data were obtained from a qPCR assay performed using homogenized mouse tissue after AAV/PBS injections and quantified as percentage of human ATXN2 copy/ ⁇ g RNA expression relative to the control group (PBS).
  • FIG.4A abbreviations: ATXN2, Ataxin-2; PBS, Phosphate-buffered saline.
  • FIG.4B is a series of bar graphs showing the knockdown efficacy of an ATXN2-specific miRNA construct from FIG.1.
  • PBS group represents no silencing of the endogenous ATXN2 mRNA and serves as a baseline.
  • the experimental groups received AAV injections with doses of 1e8 viral genomes/hemisphere (vg/hem) or 1e9 vg/hem.
  • the left and right panels show expression of mouse Atxn2 mRNA in the mouse thalamus and sub-cortex, respectively.
  • FIG.4B abbreviations: Atxn2, Ataxin-2; PBS, Phosphate-buffered saline.
  • FIG.5 abbreviations: ATXN2, Ataxin-2; PBS, Dulbecco’s phosphate buffered saline; neg, negative. Definitions As used herein, the term “about” refers to a value that is within 10% above or below the value being described. For example, the phrase “about 100 nucleic acid residues” refers to a value of from 90 to 110 nucleic acid residues.
  • the term "anneal” refers to the formation of a stable duplex of nucleic acids by way of hybridization mediated by inter-strand hydrogen bonding, for example, according to Watson- Crick base pairing.
  • the nucleic acids of the duplex may be, for example, at least 50% complementary to one another (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 one another.
  • the "stable duplex" formed upon the annealing of one nucleic acid to another is a duplex structure that is not denatured by a stringent wash.
  • exemplary stringent wash conditions include temperatures of about 5° C less than the melting temperature of an individual strand of the duplex and low concentrations of monovalent salts, such as monovalent salt concentrations (e.g., NaCl concentrations) 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).
  • monovalent salt concentrations e.g., NaCl concentrations
  • the terms “conservative mutation,” “conservative substitution,” or “conservative amino acid substitution” refer to a substitution of one or more amino acids for one or more different amino acids that exhibit similar physicochemical properties, such as polarity, electrostatic charge, and steric volume. These properties are summarized for each of the twenty naturally occurring amino acids in Table 1 below. Table 1.
  • conservative amino acid families include, e.g., (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.
  • a conservative mutation or substitution is therefore one that substitutes one amino acid for a member of the same amino acid family (e.g., a substitution of Ser for Thr or Lys for Arg).
  • the “length” of a nucleic acid refers to the linear size of the nucleic acid as assessed by measuring the quantity of nucleotides from the 5’ to the 3’ end of the nucleic acid. Exemplary molecular biology techniques that may be used to determine the length of a nucleic acid of interest are known in the art.
  • the term “operably linked” refers to a first molecule (e.g., a first nucleic acid) joined to a second molecule (e.g., a second nucleic acid), wherein the molecules are so arranged that the first molecule affects the function of the second molecule.
  • the two molecules may or may not be part of a single contiguous molecule and may or may not be adjacent to one another.
  • a promoter is operably linked to a transcribable polynucleotide molecule if the promoter modulates transcription of the transcribable polynucleotide molecule of interest in a cell.
  • two portions of a transcription regulatory element are operably linked to one another if they are joined such that the transcription-activating functionality of one portion is not adversely affected by the presence of the other portion.
  • Two transcription regulatory elements may be operably linked to one another by way of a linker nucleic acid (e.g., an intervening non-coding nucleic acid) or may be operably linked to one another with no intervening nucleotides present.
  • one segment of a nucleic acid molecule is considered to “overlap with” another segment of the same nucleic acid molecule if the two segments share one or more constituent nucleotides.
  • two segments of the same nucleic acid molecule are considered to “overlap with” one another if the two segments share 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, or more, constituent nucleotides.
  • the two segments are not considered to “overlap with” one another if the two segments have zero constituent nucleotides in common.
  • Percent (%) sequence complementarity with respect to a reference polynucleotide sequence is defined as the percentage of nucleic acids in a candidate sequence that are complementary to the nucleic acids in the reference polynucleotide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence complementarity.
  • a given nucleotide is considered to be “complementary” to a reference nucleotide as described herein if the two nucleotides form canonical Watson-Crick base pairs.
  • Watson-Crick base pairs in the context of the present disclosure include adenine-thymine, adenine-uracil, and cytosine-guanine base pairs.
  • a proper Watson-Crick base pair is referred to in this context as a “match,” while each unpaired nucleotide, and each incorrectly paired nucleotide, is referred to as a “mismatch.”
  • Alignment for purposes of determining percent nucleic acid sequence complementarity can be achieved in various ways that are within the capabilities of one of skill 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 appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal complementarity over the full length of the sequences being compared.
  • the percent sequence complementarity of a given nucleic acid sequence, A, to a given nucleic acid sequence, B, is calculated as follows: 100 multiplied by (the fraction X/Y) where X is the number of complementary base pairs in an alignment (e.g., as executed by computer software, such as BLAST) in that program’s alignment of A and B, and where Y is the total number of nucleic acids in B.
  • nucleic acid sequence A is not equal to the length of nucleic acid sequence B
  • percent sequence complementarity of A to B will not equal the percent sequence complementarity of B to A.
  • a query nucleic acid sequence is considered to be “completely complementary” to a reference nucleic acid sequence if the query nucleic acid sequence has 100% sequence complementarity to the reference nucleic acid sequence.
  • Percent (%) sequence identity with respect to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to the nucleic acids or amino acids in the reference polynucleotide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid or amino acid sequence identity can be achieved in various ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software.
  • percent sequence identity values may be generated using the sequence comparison computer program BLAST.
  • percent sequence identity of a given nucleic acid or amino acid sequence, A, to, with, or against a given nucleic acid or amino acid sequence, B, (which can alternatively be phrased as a given nucleic acid or amino acid sequence, A that has a certain percent sequence identity to, with, or against a given nucleic acid or amino acid sequence, B) is calculated as follows: 100 multiplied by (the fraction X/Y) where X is the number of nucleotides or amino acids scored as identical matches by a sequence alignment program (e.g., BLAST) in that program’s alignment of A and B, and where Y is the total number of nucleic acids in B.
  • sequence alignment program e.g., BLAST
  • the term “pharmaceutical composition” refers to a mixture containing a therapeutic agent, such as a nucleic acid or vector described herein, 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 affecting or that may affect the subject.
  • the term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms, which are suitable for contact with the tissues of a subject, such as a mammal (e.g., a human) without excessive toxicity, irritation, allergic response and other problem complications commensurate with a reasonable benefit/risk ratio.
  • a mammal e.g., a human
  • wild-type or non-mutant 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, such as a repeat region expansion that results in higher risk of developing, onset, or progression of a neurodegenerative disease).
  • mutation refers to any change in the structure of a gene, e.g., gene sequence, resulting in an altered form of the gene, which may be passed onto subsequent generations (hereditary mutation) or not (somatic mutation).
  • Gene mutations include the substitution, insertion, or deletion of a single base in DNA or the substitution, insertion, deletion, or rearrangement of multiple bases or larger sections of genes or chromosomes, including repeat expansions.
  • Ataxin 2 or “Ataxin-2” or “ATXN2” refers to a protein encoded by the ATXN2 gene, which contains a polyglutamine (polyQ, CAG repeat) tract.
  • ATXN2 gene or transcript may refer to normal alleles of ATXN2, which usually have 22 or 23 repeats, or mutated alleles having intermediate ( ⁇ 24-32 repeats) or longer repeat expansions ( ⁇ 33 to >100 repeats).
  • ATXN2 refers to mammalian ATXN2, including human ATXN2.
  • Exemplary ATXN2 proteins that may be targeted using the compositions and methods of the disclosure include a protein having the amino acid sequence represented by NCBI ID NP_001297050.1, as well as naturally occurring variants thereof.
  • an exemplary ATXN2 gene has the nucleic acid sequence of NCBI ID NC_000012.12:c111599673-111452214, or a naturally occurring variant thereof.
  • Exemplary ATXN2 mRNA transcripts include those having the nucleic acid sequence of NCBI ID NM_002973.4, as well as naturally occurring variants thereof.
  • the term “inhibitory nucleic acid” refers to a nucleic acid that comprises a guide strand sequence that hybridizes to at least a portion of a target nucleic acid, e.g., ATXN2 RNA, mRNA, pre-mRNA, or mature mRNA, and inhibits its expression or activity.
  • An inhibitory nucleic acid may target a protein coding region (e.g., exon) or non-coding region (e.g., 5’UTR, 3’UTR, intron, etc.) of a target nucleic acid.
  • an inhibitory nucleic acid is a single stranded or double stranded molecule.
  • An inhibitory nucleic acid may further comprise a passenger strand sequence on a separate strand (e.g., double stranded duplex) or in the same strand (e.g., single stranded, self-annealing duplex structure).
  • an inhibitory nucleic acid is an interfering RNA molecule, such as short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), or double-stranded RNA (dsRNA).
  • the term “interfering RNA” refers to an RNA, such as an siRNA, miRNA, or shRNA that suppresses the expression of a target RNA transcript by way of (i) annealing to the target RNA transcript, thereby forming a nucleic acid duplex; and (ii) promoting the nuclease-mediated degradation of the RNA transcript and/or (iii) slowing, inhibiting, or preventing the translation of the RNA transcript, such as by sterically precluding the formation of a functional ribosome-RNA transcript complex or otherwise attenuating formation of a functional protein product from the target RNA transcript.
  • an RNA such as an siRNA, miRNA, or shRNA that suppresses the expression of a target RNA transcript by way of (i) annealing to the target RNA transcript, thereby forming a nucleic acid duplex; and (ii) promoting the nuclease-mediated degradation of the RNA transcript and/or (iii) slowing, inhibit
  • Interfering RNAs as described herein may be provided to a patient, such as a human patient having myotonic dystrophy, in the form of, for example, a single- 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) containing a transgene encoding the interfering RNA.
  • a patient such as a human patient having myotonic dystrophy
  • a vector e.g., a viral vector, such as an adeno-associated viral vector described herein
  • RNA platforms are described, for example, 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.
  • a “microRNA” or “miRNA” refers to a small non-coding RNA molecule capable of mediating silencing of a target gene by cleavage of the target mRNA, translational repression of the target mRNA, target mRNA degradation, or a combination thereof.
  • miRNA is transcribed as a hairpin or stem-loop (e.g., having a self-complementary, single-stranded backbone) duplex structure, referred to as a primary miRNA (pri-miRNA), which is enzymatically processed (e.g., by Drosha, DGCR8, Pasha, etc.) into a pre-miRNA.
  • pri-miRNA primary miRNA
  • Pre-miRNA is exported into the cytoplasm, where it is enzymatically processed by Dicer to produce a miRNA duplex with the passenger strand and then a single- stranded mature miRNA molecule, which is subsequently loaded into the RNA-induced silencing complex (RISC).
  • RISC RNA-induced silencing complex
  • Reference to a miRNA may include synthetic or artificial miRNAs.
  • a “synthetic miRNA” or “artificial miRNA” or “amiRNA” refers to an endogenous, modified, or synthetic pri-miRNA or pre-miRNA (e.g., miRNA backbone or scaffold) in which the endogenous miRNA guide sequence and passenger sequence within the stem sequence have been replaced with a miRNA guide sequence and a miRNA passenger sequence that direct highly efficient RNA silencing of the targeted gene (see, e.g., Eamens et al. (2014), Methods Mol. Biol.1062:211-224).
  • the nature of the complementarity of the guide and passenger sequences can be similar or different from the nature of complementarity of the guide and passenger sequences in the endogenous miRNA backbone upon which the synthetic miRNA is constructed.
  • miRNA backbone e.g., number of bases, position of mismatches, types of bulges, etc.
  • the term “microRNA backbone,” “miR backbone,” “microRNA scaffold,” or “miR scaffold” refers to a pri-miRNA or pre-miRNA scaffold, with the stem sequence replaced by a miRNA of interest, and is capable of producing a functional, mature miRNA that directs RNA silencing at the gene targeted by the miRNA of interest.
  • a miR backbone comprises a 5’ flanking region (also referred to 5’ miR context, ⁇ 9 nucleotides), a stem region comprising the miRNA duplex (guide strand sequence and passenger strand sequence) and basal stem (5’ and 3’, each about 4-13 nucleotides), at least one loop motif region including the terminal loop ( ⁇ 10 nucleotides for terminal loop), a 3’ flanking region (also referred to 3’ miR context, ⁇ 9 nucleotides), and optionally one or more bulges in the stem.
  • a miR backbone may be derived completely or partially from a wild type miRNA scaffold or be a completely artificial sequence.
  • 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 for silencing.
  • the antisense sequence is sufficiently complementary to the target mRNA sequence to direct target-specific silencing, e.g., to trigger the destruction of the target mRNA by the RNAi machinery or process.
  • the antisense sequence or guide strand sequence refers to the mature sequence remaining following cleavage by Dicer.
  • the term “sense sequence” or “passenger strand sequence” of an inhibitory nucleic acid refers to a sequence that is homologous to the target mRNA and partially or completely complementary to the antisense strand sequence or guide strand sequence of an inhibitory nucleic acid.
  • the antisense strand sequence and sense strand sequence of an inhibitory nucleic acid are hybridized to form a duplex structure (e.g., forming a double-stranded duplex or single-stranded self- annealing duplex structure).
  • the sense sequence or passenger strand sequence refers to the mature sequence remaining following cleavage by Dicer.
  • a “duplex,” when used in reference to an inhibitory nucleic acid refers to two nucleic acid strands (e.g., a guide strand and passenger strand) hybridizing together to form a duplex structure.
  • a duplex may be formed by two separate nucleic acid strands or by a single nucleic acid strand having a region of self-complementarity (e.g., hairpin or stem-loop).
  • expression construct refers to any type of genetic construct containing a nucleic acid (e.g., transgene) in which part or all the nucleic acid encoding sequence is capable of being transcribed.
  • expression includes transcription of the nucleic acid, for example, to generate a biologically active polypeptide product or inhibitory RNA (e.g., siRNA, shRNA, miRNA) from a transcribed gene.
  • inhibitory RNA e.g., siRNA, shRNA, miRNA
  • the transgene is operably linked to expression control sequences.
  • the term “transgene” refers to an exogenous nucleic acid that has been transferred naturally or by genetic engineering means into another cell and is capable of being transcribed, and optionally translated.
  • the term “gene expression” refers to the process by which a nucleic acid is transcribed from a nucleic acid molecule, and often, translated into a peptide or protein. The process can include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post translational modification, or any combination thereof. Reference to a measurement of “gene expression” may refer to measurement of the product of transcription (e.g., RNA or mRNA), the product of translation (e.g., peptides or proteins). As used herein, the term “inhibit expression of a gene” means to reduce, down-regulate, suppress, block, lower, or stop expression of the gene.
  • the expression product of a gene can be an RNA molecule transcribed from the gene (e.g., an mRNA) or a polypeptide translated from an mRNA transcribed from the gene. Typically, a reduction in the level of an mRNA results in a reduction in the level of a polypeptide translated therefrom.
  • the level of expression may be determined using standard techniques for measuring mRNA or protein.
  • “neurodegenerative disease” or “neurodegenerative disorder” refers to diseases or disorders that exhibit neural cell death as a pathological state.
  • a neurodegenerative disease may exhibit chronic neurodegeneration, e.g., slow, progressive neural cell death over a period of several years, or acute neurodegeneration, e.g., sudden onset or neural cell death.
  • Chronic neurodegenerative diseases include Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, spinocerebellar ataxia type 2 (SCA2), frontotemporal lobar dementia (FTLD), and amyotrophic lateral schlerosis (ALS).
  • Chronic neurodegenerative diseases include diseases that feature TDP-43 proteinopathy, which is characterized by nucleus to cytoplasmic mislocalization, deposition of ubiquitinated and hyper-phosphorylated TDP-43 into inclusion bodies, protein truncation leading to formation of toxic C-terminal TDP-43 fragments, and protein aggregation.
  • TDP-43 proteinopathy diseases include ALS, FTLD, primary lateral sclerosis, progressive muscular atrophy, limbic-predominant age-related TDP-43 encephalopathy, chronic traumatic encephalopathy, dementia with Lewy bodies, corticobasal degeneration, progressive supranuclear palsy (PSP), dementia Parkinsonism ALS complex of guam (G-PDC), Pick’s disease, hippocampal sclerosis, Huntington’s disease, Parkinson’s disease, and Alzheimer’s disease.
  • Acute neurodegeneration may be caused by ischemia (e.g., stroke, traumatic brain injury), axonal transection by demyelination or trauma (e.g., spinal cord injury or multiple sclerosis).
  • a neurodegenerative disease may exhibit death of mainly one type of neuron or of multiple types of neurons.
  • the term “repeat region” refers to segments within a gene of interest or an RNA transcript thereof containing nucleic acid repeats, such as the poly CAG sequence in the ATXN2 gene.
  • a repeat region is considered to be an “expanded repeat region,” a “repeat expansion,” or the like, if the number of nucleotide repeats in the repeat region exceeds the quantity of repeats ordinarily found in the repeat region of a wild-type form of the gene or RNA transcript thereof.
  • the wild-type human ATXN2 genes typically contain from 13 to 31 CAG repeats.
  • “Expanded repeat regions” and “repeat expansions” in the context of the ATXN2 gene or an RNA transcript thereof thus include repeat regions containing greater than 31 repeats, among others.
  • the term “sample” refers to a specimen (e.g., blood, blood component (e.g., serum or plasma), urine, saliva, amniotic fluid, cerebrospinal fluid, tissue (e.g., placental or dermal), pancreatic fluid, chorionic villus sample, or cells) isolated from a subject.
  • the subject may be, for example, a patient suffering from a disease described herein, such as a disease associated with expression of wild-type or mutant ATXN2 (e.g., SCA2, ALS, Huntington’s disease, Parkinson’s disease.).
  • a disease described herein such as a disease associated with expression of wild-type or mutant ATXN2 (e.g., SCA2, ALS, Huntington’s disease, Parkinson’s disease.).
  • the phrases “specifically binds” and “binds” refer to a binding reaction which is determinative of the presence of a particular molecule, such as an RNA transcript, in a heterogeneous population of ions, salts, small molecules, and/or proteins that is recognized, e.g., a mutant ATXN2 RNA transcript.
  • a ligand that specifically binds to a species (e.g., an RNA transcript) may bind to the species, e.g., with a KD of less than 1 mM.
  • a ligand that specifically binds to a species may bind to the species with a KD of up to 100 ⁇ M (e.g., between 1 pM and 100 ⁇ M).
  • a ligand that does not exhibit specific binding to another molecule may exhibit a K D of greater than 1 mM (e.g., 1 ⁇ M, 100 ⁇ M, 500 ⁇ M, 1 mM, or greater) for that particular molecule or ion.
  • a variety of assay formats may be used to determine the affinity of a ligand for a specific protein.
  • solid-phase ELISA assays are routinely used to identify ligands that specifically bind a target protein. See, e.g., Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1988) and Harlow & Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1999), for a description of assay formats and conditions that can be used to determine specific protein binding.
  • the terms “subject” and “patient” refer to an organism that receives treatment for a particular disease or condition as described herein (such as a disease associated with expression of an ATXN2 mutant, e.g., SCA2). Examples of subjects and patients include mammals, such as humans, receiving treatment for a disease or condition described herein.
  • the term “transcription regulatory element” refers to a nucleic acid that controls, at least in part, the transcription of a gene of interest. Transcription regulatory elements may include promoters, enhancers, and other nucleic acids (e.g., polyadenylation signals) that control or help to control gene transcription.
  • transcription regulatory elements are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, CA, 1990).
  • treatment refers to therapeutic treatment, in which the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the progression of a disease associated with wild-type or mutant ATXN2, for example, SCA2, ALS, Huntington’s disease, and Parkinson’s disease.
  • beneficial or desired clinical results that are indicative of successful treatment include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • Treatment of a patient having SCA2 may manifest in one or more detectable changes, such as a decrease in the expression of mutant ATXN2 RNA transcripts (e.g., a decrease in the expression of ATXN2 RNA transcripts that contain expanded CAG trinucleotide repeat regions).
  • the term “vector” refers to a nucleic acid, e.g., DNA or RNA, that may function as a vehicle for the delivery of a gene of interest into a cell (e.g., a mammalian cell, such as a human cell), tissue, organ, or organism, such as a patient undergoing treatment for a disease or condition described herein, for purposes of expressing an encoded transgene.
  • a cell e.g., a mammalian cell, such as a human cell
  • tissue e.g., a mammalian cell, such as a human cell
  • exemplary vectors useful in conjunction with the compositions and methods described herein are plasmids, DNA vectors, RNA vectors, virions, or other suitable replicon (e.g., viral vector).
  • vectors have been developed for the delivery of polynucleotides encoding exogenous proteins into a prokaryotic or eukaryotic cell. Examples of such expression vectors are disclosed in, e.g., WO 1994/11026, the disclosure of which is incorporated herein by reference.
  • Expression vectors described herein contain a polynucleotide sequence as well as, e.g., additional sequence elements used for the expression of proteins and/or the integration of these polynucleotide sequences into the genome of a mammalian cell.
  • Certain vectors that can be used for the expression of transgenes described herein include plasmids that contain regulatory sequences, such as promoter and enhancer regions, which direct gene transcription.
  • kits for expression of transgenes contain polynucleotide sequences that enhance the rate of translation of these genes or improve the stability or nuclear export of the mRNA that results from gene transcription. These sequence elements include, e.g., 5’ and 3’ untranslated regions, an internal ribosomal entry site (IRES), and polyadenylation signal site in order to direct efficient transcription of the gene carried on the expression vector.
  • the expression vectors described herein may also contain a polynucleotide encoding a marker for selection of cells that contain such a vector. Examples of a suitable marker include genes that encode resistance to antibiotics, such as ampicillin, chloramphenicol, kanamycin, or nourseothricin.
  • compositions and methods described herein are useful for treating disorders associated with expression of wild-type or mutant Ataxin-2 (ATXN2), such as spinocerebellar ataxia type 2 or spinocerebellar ataxia-2 (SCA2), amyotrophic lateral sclerosis (ALS), Huntington’s disease, FTD (Frontotemporal dementia), TDP-43 (TAR DNA binding protein 43) proteinopathies, and others.
  • Ataxin-2 Ataxin-2
  • ATXN2 wild-type or mutant Ataxin-2
  • SCA2 spinocerebellar ataxia type 2 or spinocerebellar ataxia-2
  • ALS amyotrophic lateral sclerosis
  • FTD Ferontotemporal dementia
  • TDP-43 TAR DNA binding protein 43 proteinopathies
  • compositions described herein include inhibitory nucleic acid constructs such as interfering RNA constructs, for example, short interfering RNA (siRNA), short hairpin RNA (shRNA), or microRNA (miRNA) that suppress the expression of wild-type or mutant mRNA transcripts transcribed from wild- type or mutant genes.
  • interfering RNA constructs for example, short interfering RNA (siRNA), short hairpin RNA (shRNA), or microRNA (miRNA) that suppress the expression of wild-type or mutant mRNA transcripts transcribed from wild- type or mutant genes.
  • shRNA short hairpin RNA
  • miRNA microRNA
  • Ataxin-2 and Disorders Associated with Ataxin-2 ATXN2 protein is a cytoplasmic protein that is a component of stress granules. Stress granules are transient, subcellular compartments induced by arrest of protein translation and include a number of proteins known to be mutated in subjects with neurodegenerative disease (Brown and Al- Chalabi, N Engl J Med (2017) 377:162-172).
  • ATXN2 contains a sequence of glutamine residues, known as a polyglutamine repeat (polyQ), that in normal individuals is about 22 amino acids in length. Expansions of this polyglutamine repeat to a length of 34 or longer is found in individuals with neurodegenerative diseases, including spinocerebellar ataxia type 2 (SCA2). This disease is characterized by progressive death of Purkinje neurons in the cerebellum and other neuronal cell types. Patients with SCA2 develop ataxia, sensory problems, and other clinical features, which worsen over time.
  • polyQ polyglutamine repeat
  • SCA2 spinocerebellar ataxia type 2
  • Ataxin-2 polyglutamine repeat e.g., between 27 and 40 glutamine residues
  • ALS amyotrophic lateral sclerosis
  • the pathogenic functions of polyQ disease proteins that occur with polyQ expansion may be attributed to the gain of toxicity associated with the development of intranuclear inclusion bodies or with soluble toxic oligomers (Lajoie et al., PLoS One, 2011, 5: e15245). While SCA2 patient brains are characterized by loss of Purkinje cells, SCA2 Purkinje cells lack inclusion bodies indicating polyQ- expanded ataxin-2 may cause toxicity that is unrelated to inclusion body formation (Huynh et al., Ann. Neurol., 1999, 45: 232–241). Functions gained in polyQ-expanded ataxin-2 may include anomalous accumulation in Golgi bodies (Huynh et al., Hum. Mol.
  • Ataxin-2 is present in stress granules and P-bodies suggesting functions in sequestering mRNAs and protein translation regulation during stress (Nonhoff et al., Mol. Biol. Cell, 2007, 18: 1385–1396). Ataxin-2 overexpression interfered with the P-body assembly, while underexpression interfered with stress granule assembly (Nonhoff et al., Mol. Biol. Cell, 2007, 18: 1385–1396). Interactions with polyA-binding protein 1, the RNA splicing factor A2BP1/Fox1 and polyribosomes further support roles for ataxin-2 in RNA metabolism (Shibata et al., Hum. Mol.
  • Ataxin-2 is a regulator of EGF receptor internalization and signaling by the way of its interactions with SRC kinase and the endocytic protein CIN85 (Nonis et al., Cell Signal., 2008, 20: 1725– 1739).
  • Ataxin-2 also interacts with the ALS-related protein TDP-43 in an RNA-dependent manner and familial and sporadic ALS associates with the occurrence of long normal CAG repeat expansion ATXN2 (Elden et al., Nature, 2010, 466: 1069–1075; Van Damme et al., Neurology, 2011, 76: 2066–2072).
  • SCA2 is an autosomal dominant neurodegenerative disease characterized by progressive functional and cell loss of neurons in the cerebellum, brain stem and spinal cord.
  • the cause of SCA2 is CAG expansion in the ATXN2 gene resulting in polyglutamine (polyQ) expansion in the ataxin-2 protein.
  • SCA2 Patients with SCA2 are characterized by progressive cerebellar ataxia, slow saccadic eye movements and other neurologic features such as neuropathy (Pulst, S.M. (ed.), Genetics of Movement Disorders. Elsevier, Inc., Amsterdam, 2003, pp.19–34.). Moderate CAG expansion in the ATXN2 gene is also associated with parkinsonism or ALS indistinguishable from the idiopathic forms of these diseases (Kim et al., Arch. Neurol., 2007, 64: 1510–1518; Ross et al., Hum. Mol. Genet., 2011, 20: 3207–3212; Corrado et al., Hum.
  • ATXN2 polyglutamine repeat to a length of 34 or longer causes SCA2.
  • intermediate length polyglutamine expansions in ATXN2 increases the risk of ALS.
  • Reduction of ATXN2 levels has been demonstrated to have therapeutic benefit in animal models of spinocerebellar ataxia-2 and ALS.
  • Knocking down the ATXN2 protein using nucleic acid-based therapies alleviates the progressive neurodegeneration that occurs in animal models expressing a variant of the human ATXN2 containing an expanded polyglutamine repeat.
  • TDP-43 proteinopathy has also been observed in a number of neurodegenerative diseases, including ALS, frontotemporal lobar dementia (FTLD), primary lateral sclerosis, progressive muscular atrophy, limbic-predominant age-related TDP-43 encephalopathy, chronic traumatic encephalopathy, dementia with Lewy bodies, corticobasal degeneration, progressive supranuclear palsy (PSP), dementia Parkinsonism ALS complex of guam (G-PDC), Pick’s disease, hippocampal sclerosis, Huntington’s disease, Parkinson’s disease, and Alzheimer’s disease.
  • ALS frontotemporal lobar dementia
  • FTLD frontotemporal lobar dementia
  • primary lateral sclerosis progressive muscular atrophy
  • limbic-predominant age-related TDP-43 encephalopathy chronic traumatic encephalopathy
  • dementia with Lewy bodies corticobasal degeneration
  • PSP progressive supranuclear palsy
  • G-PDC dementia Parkinsonism ALS complex of
  • ATXN2 is a causative agent (e.g., SCA2)
  • reducing ATXN2 levels may be useful for treating neurodegenerative diseases where ATXN2 is a causative agent (e.g., SCA2)
  • ATXN2 is not the causative agent but modifies TDP-43 pathological aggregation.
  • Aspects of the disclosure relate to inhibitory nucleic acids, such as interfering RNA molecules (e.g., short interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), micro RNAs (miRNAs), including artificial miRNAs), that when administered to a subject reduce the expression or activity of Ataxin-2 in the subject.
  • interfering RNA molecules e.g., short interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), micro RNAs (miRNAs), including artificial miRNAs
  • compositions and methods provided in the present disclosure are useful for the treatment of neurodegenerative diseases, including SCA2, ALS, Huntington’s disease, Alzheimer’s disease, FTLD, parkinsonism, and conditions associated with TDP-43 proteinopathies.
  • Inhibitory Nucleic Acids In one aspect, the disclosure provides isolated inhibitory nucleic acids that inhibit expression or activity of Ataxin 2 (ATXN2).
  • the inhibitory nucleic acid is a nucleic acid that specifically binds (e.g., hybridizes to) at least a portion of the ATXN2 nucleic acid, such as an ATXN2 RNA, pre-mRNA, or mRNA, and inhibits its expression or activity.
  • the inhibitory nucleic acid is complementary to a protein coding region or non-coding region (e.g., 5’UTR, 3’UTR, intron, etc.) of ATXN2.
  • the inhibitory nucleic acid is complementary to a wild type ATXN2 nucleic acid or a naturally occurring variant thereof.
  • the ATXN2 allele contains approximately 22 CAG trinucleotide repeats.
  • the ATXN2 allele has at least 22 CAG trinucleotide repeats, at least 23 CAG trinucleotide repeats, at least 24 CAG trinucleotide repeats, at least 25 CAG trinucleotide repeats, at least 26 CAG trinucleotide repeats, at least 27 CAG trinucleotide repeats, at least 28 CAG trinucleotide repeats, at least 29 CAG trinucleotide repeats, at least 30 CAG trinucleotide repeats, at least 31 CAG trinucleotide repeats, at least 32 CAG trinucleotide repeats, at least 33 CAG trinucleotide repeats, at least 34 CAG trinucleotide repeats, at least 35 CAG trinucleotide repeats, at least 36 CAG trinucleotide repeats, at least 37 CAG trinucleotide repeats, at least
  • the inhibitory nucleic acid is single stranded or double-stranded.
  • the inhibitory nucleic acid is an interfering RNA molecule, such as short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), or double-stranded RNA (dsRNA).
  • the inhibitory nucleic acid is a miRNA.
  • a miRNA may be a pri-mRNA, a pre-mRNA, mature miRNA, or artificial miRNA.
  • a miRNA is comprised of a guide strand and passenger strand.
  • the guide strand and passenger strand are within the same nucleic acid strand, where the guide strand and passenger strand hybridize together to form a self-annealing duplex structure.
  • MiRNA is initially transcribed as a pri-mRNA, which is processed by nuclear nuclease (e.g., Drosha-DGCR8 complex) into pre-mRNA.
  • a pri-mRNA is a single-stranded molecule having a stem-loop structure.
  • Pre-miRNA is also a single-stranded molecule having 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 inducing silencing complex (RISC), mediated by TRBP (HIV transactivating response RNA-binding protein).
  • RISC RNA inducing silencing complex
  • TRBP HIV transactivating response RNA-binding protein
  • the passenger strand is generally released and cleaved, while the guide strand remains in RISC and binds to the target mRNA and mediates silencing.
  • a mature miRNA refers to the guide strand of a mature miRNA duplex.
  • Artificial miRNA refers to an endogenous, modified, or synthetic pri-mRNA or pre-mRNA scaffold or backbone capable of producing a functional mature miRNA, where the guide strand sequence and passenger strand sequence of the miRNA duplex within the stem region have been replaced with a guide strand sequence and passenger strand sequence of interest that directs silencing of the target mRNA of interest.
  • 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).
  • the inhibitory nucleic acid constructs such as the interfering RNA constructs, described herein may be in any of a variety of forms, such as siRNA, shRNA, or miRNA.
  • the interfering RNAs described herein may additionally be encoded by a vector, such as a viral vector.
  • a vector such as a viral vector.
  • AAV adeno-associated viral
  • pseudotyped AAV vectors e.g., AAV2/8 and AAV2/9 vectors
  • transgenes encoding interfering RNA constructs that attenuate the expression of wild-type or mutant RNA transcripts, for example, RNA transcripts harboring expanded nucleotide repeats.
  • compositions and methods described herein provide, among other benefits, the advantageous feature of being able to selectively suppress the expression of wild-type or pathologic RNA transcripts among other RNAs that contain expanded nucleotide repeat regions. This property is particularly beneficial in view of the prevalence of nucleotide repeats in mammalian genomes, such as in the genomes of human patients.
  • the expression of wild-type RNA transcripts or mutant RNA transcripts that contain pathological nucleotide repeat expansions can be diminished, while preserving the expression of important healthy RNA transcripts and their encoded protein products.
  • This advantageous feature is based, in part, on the surprising discovery that inhibitory nucleic acid constructs that anneal to wild-type or repeat-expanded RNA targets can be used to suppress the expression of these RNA transcripts.
  • the compositions and methods described herein can thus attenuate the expression of wild-type or pathological RNA transcripts.
  • the sections that follow provide a description of exemplary inhibitory nucleic acid constructs, such as interfering RNA constructs, that may be used in conjunction with the compositions and methods described herein, as well as a description of vectors encoding such constructs and procedures that may be used to treat diseases associated with expression of wild-type or mutant ATXN2.
  • a patient having a disease characterized by expression of wild-type or mutant ATXN2 may be administered an interfering RNA molecule, a composition containing the same, or a vector encoding the same, so as to suppress the expression of an RNA transcript.
  • interfering RNA molecules that may be used in conjunction with the compositions and methods described herein for the treatment of diseases associated with expression of wild-type or mutant ATXN2, such as SCA2, ALS, Huntington’s disease, FTD, TDP-43 proteinopathies, Parkinson’s disease and others, are siRNA molecules, miRNA molecules, and shRNA molecules, among others.
  • the siRNA may be single stranded or double stranded.
  • miRNA molecules in contrast, are single-stranded molecules that form a hairpin, thereby adopting a hydrogen-bonded structure reminiscent of a nucleic acid duplex.
  • the interfering RNA may contain an antisense or “guide” strand that anneals (e.g., by way of complementarity) to the repeat-expanded mutant RNA target.
  • the interfering RNA may also contain a “passenger” strand that is complementary to the guide strand and, thus, may have the same nucleic acid sequence as the RNA target.
  • Exemplary interfering RNA molecules that anneal to ATXN2 RNA may be used in conjunction with the compositions and methods described herein for the treatment of diseases associated with expression of wild-type or mutant ATXN2 shown in Table 2, below. Table 2.
  • Exemplary inhibitory nucleic acids useful for suppressing ATXN2 expression Methods of Treating Diseases Characterized by Expression of Wild-type or Mutant ATXN2 Using the compositions and methods described herein, a patient experiencing and/or having a disease associated with expression of wild-type or mutant ATXN2, such as SCA2, ALS, Huntington’s disease, FTD, TDP-43 proteinopathies, Parkinson’s disease, among others, can be administered an inhibitory nucleic acid construct, such as an interfering RNA construct, for example, short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), or a vector encoding the same, so as to reduce the expression of wild-type or mutant RNA transcripts.
  • siRNA short inter
  • the present disclosure provides methods for inhibiting the expression or activity of ATXN2 in a cell, comprising administering a composition of the present disclosure (e.g., inhibitory nucleic acid, isolated nucleic acid comprising an expression construct encoding an inhibitory nucleic acid, vector, rAAV particle, pharmaceutical composition) to a cell, thereby inhibiting the expression or activity of ATXN2 in the cell.
  • a composition of the present disclosure e.g., inhibitory nucleic acid, isolated nucleic acid comprising an expression construct encoding an inhibitory nucleic acid, vector, rAAV particle, pharmaceutical composition
  • the cell is a CNS cell.
  • the cell is a non-neuronal cell or neuronal cell of the CNS.
  • the non-neuronal cell of the CNS is a glial cell, astrocyte, or microglial cell.
  • the cell is in vitro.
  • the cell is from a subject having one or more symptoms of a neurodegenerative disease or suspected of having a neurodegenerative disease.
  • the cell expresses an ATXN2 having at least 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, 100, or more CAG trinucleotide (polyglutamine) repeats.
  • the cell expresses an ATXN2 having about 22 or 23 repeats, 24-32 repeats, or 33-100 or more repeats.
  • the present disclosure provides methods for inhibiting the expression or activity of ATXN2 in the central nervous system of a subject, comprising administering a composition of the present disclosure (e.g., inhibitory nucleic acid, isolated nucleic acid comprising an expression construct encoding an inhibitory nucleic acid, vector, rAAV particle, pharmaceutical composition) to the subject, thereby inhibiting the expression or activity of ATXN2 in the subject.
  • a composition of the present disclosure e.g., inhibitory nucleic acid, isolated nucleic acid comprising an expression construct encoding an inhibitory nucleic acid, vector, rAAV particle, pharmaceutical composition
  • the present disclosure provides methods for treating a subject having or suspected of having a neurodegenerative disease, comprising administering a composition of the present disclosure (e.g., inhibitory nucleic acid, isolated nucleic acid comprising an expression construct encoding an inhibitory nucleic acid, vector, rAAV particle, pharmaceutical composition) to the subject, thereby treating the subject.
  • a composition of the present disclosure e.g., inhibitory nucleic acid, isolated nucleic acid comprising an expression construct encoding an inhibitory nucleic acid, vector, rAAV particle, pharmaceutical composition
  • the term "treat” refers to preventing or delaying onset of neurodegenerative disease (e.g., SCA2, ALS/FTLD, Huntington’s disease, Alzheimer's disease, Parkinson's disease, etc.); reducing severity of neurodegenerative disease; reducing or preventing development of symptoms characteristic of neurodegenerative disease; preventing worsening of symptoms characteristic of neurodegenerative disease, or any combination thereof.
  • neurodegenerative disease e.g., SCA2, ALS/FTLD, Huntington’s disease, Alzheimer's disease, Parkinson's disease, etc.
  • reducing severity of neurodegenerative disease reducing or preventing development of symptoms characteristic of neurodegenerative disease
  • preventing worsening of symptoms characteristic of neurodegenerative disease or any combination thereof.
  • Neurodegenerative diseases that may be treated in a subject using the compositions of the present disclosure include neurodegenerative diseases where ATXN2 is a causative agent (e.g., SCA2), as well as neurodegenerative diseases where ATXN2 is not the causative agent (e.g., directly causative) but modifies TDP-43 pathological aggregation.
  • ATXN2 is a causative agent (e.g., SCA2)
  • ATXN2 is not the causative agent (e.g., directly causative) but modifies TDP-43 pathological aggregation.
  • Neurodegenerative diseases associated with TDP-43 proteinopathy include ALS, FTLD, primary lateral sclerosis, progressive muscular atrophy, limbic-predominant age-related TDP-43 encephalopathy, chronic traumatic encephalopathy, dementia with Lewy bodies, corticobasal degeneration, progressive supranuclear palsy (PSP), dementia Parkinsonism ALS complex of guam (G-PDC), Pick’s disease, Perry syndrome, cerebral age-related TDP-43 with sclerosis (CARTS), hippocampal sclerosis, Huntington’s disease, Parkinson’s disease, and Alzheimer’s disease.
  • the neurodegenerative disease is SCA2.
  • the subject with SCA2 may be one that has a plurality of CAG trinucleotide repeat mutations in the ATXN2 locus, e.g., at least 34, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, or 100 CAG repeats.
  • ATXN2 is a validated target for treating SCA2 as disclosed in Giunti et al. Brain 121, 459-467 (1998).
  • the neurodegenerative disease is ALS.
  • the subject with ALS may be one that has a plurality of CAG trinucleotide repeat mutations in the ATXN2 locus, e.g., at least 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 CAG repeats.
  • ATXN2 is a validated target for treating ALS as disclosed in Wang et al. PLoS ONE 9(8): e105534 (2014).
  • the neurodegenerative disease is Huntington’s disease.
  • the subject with Huntington’s disease may be one that has upregulated ATXN2.
  • ATXN2 is a validated target for treating Huntington’s disease as disclosed in Xu et al.
  • the subject is characterized as having an ATXN2 allele having at least 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, 100, or more CAG trinucleotide (polyglutamine) repeats.
  • the subject is characterized as having an ATXN2 allele having about 22 or 23 repeats, 24-32 repeats, or 33-100 or more repeats.
  • the methods for treatment of the present disclosure reduces, prevents, or slows development or progression of one or more symptom characteristic of a neurodegenerative disease.
  • Examples of symptoms characteristic of neurodegenerative disease include motor dysfunction, cognitive dysfunction, emotional/behavioral dysfunction, or any combination thereof. Paralysis, shaking, unsteadiness, rigidity, twitching, muscle weakness, muscle cramping, muscle stiffness, muscle atrophy, difficulty swallowing, difficulty breathing, speech and language difficulties (e.g., slurred speech), slowness of movement, difficulty with walking, dementia, depression, anxiety, or any combination thereof.
  • the methods for treatment of the present disclosure of the present disclosure comprise administration as a monotherapy or in combination with one or more additional therapies for the treatment of the neurodegenerative disease.
  • Combination therapy may mean administration of the compositions of the present disclosure (e.g., inhibitory nucleic acid, isolated nucleic acid comprising an expression construct encoding an inhibitory nucleic acid, vector, rAAV particle, pharmaceutical composition) to the subject concurrently, prior to, subsequent to one or more additional therapies.
  • Concurrent administration of combination therapy may mean that the compositions of the present disclosure (e.g., inhibitory nucleic acid, isolated nucleic acid comprising an expression construct encoding an inhibitory nucleic acid, vector, rAAV particle, pharmaceutical composition) and additional therapy are formulated for administration in the same dosage form or administered in separate dosage forms.
  • the one or additional therapies that may be used in combination with the inhibitory nucleic acids of the present disclosure include: inhibitory nucleic acids or antisense oligonucleotides that target neurodegenerative disease related genes or transcripts, gene editing agents (e.g., CRISPR, TALEN, ZFN based systems) that target neurodegenerative related genes, agents that reduce oxidative stress, such as free radical scavengers (e.g., Radicava (edaravone), bromocriptine); antiglutamate agents (e.g., Riluzole, Topiramate, Lamotrigine, Dextromethorphan, Gabapentin and AMPA receptor antagonist (e.g., Talampanel)); Anti-apoptosis agents (e.g., Minocycline, Sodium phenylbutyrate and Arimoclomol); Anti-inflammatory agents (e.g., ganglioside, Celecoxib, Cyclosporine, Nimesulide, Azathi
  • a subject treated in any of the methods described herein is a mammal (e.g., mouse, rat), preferably a primate (e.g., monkey, chimpanzee), or human.
  • a composition of the present disclosure e.g., inhibitory nucleic acid, isolated nucleic acid comprising an expression construct encoding an inhibitory nucleic acid, vector, rAAV particle, pharmaceutical composition
  • a composition of the present disclosure is directly injected into the CNS of the subject.
  • direct injection into the CNS is intrathalamic injection, intracerebral injection, intraparenchymal injection, intrathecal injection, intrastriatal injection, subpial injection, or any combination thereof.
  • direct injection into the CNS is direct injection into the cerebrospinal fluid (CSF) of the subject, optionally wherein the direct injection is intracisternal injection, intraventricular injection, intralumbar injection, or any combination thereof.
  • CSF cerebrospinal fluid
  • Viral genomes provide a rich source of vectors that can be used for the efficient delivery of a gene of interest into the genome of a target cell in a patient (e.g., a mammalian cell, such as a human cell).
  • Viral genomes are particularly useful vectors for gene delivery because the polynucleotides contained within such genomes are typically incorporated into the genome of a target cell by generalized or specialized transduction. These processes occur as part of the natural viral replication cycle, and do not require added proteins or reagents in order to induce gene integration.
  • viral vectors examples include AAV, retrovirus, adenovirus (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvovirus (e.g., adeno- associated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g.
  • RNA viruses such as picornavirus and alphavirus
  • double stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein- Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox and canarypox).
  • herpesvirus e.g., Herpes Simplex virus types 1 and 2, Epstein- Barr virus, cytomegalovirus
  • poxvirus e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox and canarypox
  • Other viruses that may be used in conjunction with the compositions and methods described herein include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example.
  • retroviruses examples include: avian leukosis-sarcoma, mammalian C-type, B-type viruses, D-type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).
  • murine leukemia viruses include murine leukemia viruses, murine sarcoma viruses, 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 lentiviruses.
  • vectors are described, for example, in US Patent No.5,801,030, the disclosure of which is incorporated herein by reference as it pertains to viral vectors for use in gene therapy.
  • inhibitory nucleic acid constructs such as interfering RNA constructs (for example, short interfering RNA (siRNA), short hairpin RNA (shRNA), or microRNA (miRNA)) described herein are incorporated into recombinant AAV (rAAV) vectors in order to facilitate their introduction into a cell.
  • interfering RNA constructs for example, short interfering RNA (siRNA), short hairpin RNA (shRNA), or microRNA (miRNA)
  • rAAV recombinant AAV
  • rAAV vectors useful in the conjunction with the compositions and methods described herein include recombinant nucleic acid constructs that contain (1) a transgene encoding an inhibitory nucleic acid construct, such as an interfering RNA construct described herein (such as an siRNA, shRNA, or miRNA described herein), and (2) one or more nucleic acids that facilitate and expression of the heterologous genes.
  • the viral nucleic acids may include those sequences of AAV that are required in cis for replication and packaging (e.g., functional ITRs) of the DNA into a virion.
  • Such rAAV vectors may also contain marker or reporter genes.
  • Useful rAAV vectors include those having one or more of the naturally-occurring AAV genes deleted in whole or in part, but retain functional flanking ITR sequences.
  • the AAV ITRs may be of any serotype (e.g., derived from serotype 2) suitable for a particular application. Methods for using rAAV vectors are described, for example, in Tal et al., J. Biomed. Sci.7:279-291 (2000), and Monahan and Samulski, Gene Delivery 7:24-30 (2000), the disclosures of each of which are incorporated herein by reference as they pertain to AAV vectors for gene delivery.
  • the nucleic acids and vectors described herein can be incorporated into a rAAV virion in order to facilitate introduction of the nucleic acid or vector into a cell.
  • the capsid proteins of AAV compose the exterior, non-nucleic acid portion of the virion and are encoded by the AAV cap gene.
  • the cap gene encodes three viral coat proteins, VP1, VP2 and VP3, which are required for virion assembly.
  • the construction of rAAV virions has been described, for example, in US Patent Nos. 5,173,414; 5,139,941; 5,863,541; 5,869,305; 6,057,152; and 6,376,237; as well as in Rabinowitz et al., J.
  • rAAV virions useful in conjunction with the compositions and methods described herein include those derived from a variety of AAV serotypes including AAV 1, 2, 3, 4, 5, 6, 7, 8 and 9. Construction and use of AAV vectors and AAV proteins of different serotypes are described, for example, in Chao et al., Mol. Ther.2:619-623 (2000); Davidson et al., Proc. Natl. Acad. Sci.
  • Pseudotyped vectors include AAV vectors of a given serotype (e.g., AAV2) pseudotyped with a capsid gene derived from a serotype other than the given serotype (e.g., AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9, among others).
  • a representative pseudotyped vector is an AAV2 vector encoding a therapeutic protein pseudotyped with a capsid gene derived from AAV serotype 8 or AAV serotype 9.
  • the pseudotyped AAV has the ITRs of one AAV serotype (e.g., AAV2) and the VP1, VP2, and/or VP3 capsid proteins from a different AAV serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, or AAVrh74).
  • AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, or AAVrh74 AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, or AAVrh74.
  • the AAV comprises a capsid disclosed, e.g., in WO 2017/218842, the disclosure of which is incorporated herein by reference.
  • 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.
  • the AAV comprises an AAV2-retro or an AAV9-retro capsid protein.
  • AAV virions that have mutations within the virion capsid may be used to infect particular cell types more effectively than non-mutated capsid virions.
  • suitable AAV mutants may have ligand insertion mutations for the facilitation of targeting AAV to specific cell types.
  • the construction and characterization of AAV capsid mutants including insertion mutants, alanine screening mutants, and epitope tag mutants is described in Wu et al., J. Virol.74:8635-45 (2000).
  • Other rAAV virions that can be used in methods of the invention include those capsid hybrids that are generated by molecular breeding of viruses as well as by exon shuffling. See, e.g., Soong et al., Nat.
  • a transgene such as a transgene encoding an inhibitory nucleic acid described herein
  • a target cell e.g., a target cell from or within a human patient suffering from RNA dominance
  • electroporation can be used to permeabilize mammalian cells (e.g., human target cells) by the application of an electrostatic potential to the cell of interest.
  • Mammalian cells such as human cells, subjected to an external electric field in this manner are subsequently predisposed to the uptake of exogenous nucleic acids. Electroporation of mammalian cells is described in detail, e.g., in Chu et al., Nucleic Acids Research 15:1311 (1987), the disclosure of which is incorporated herein by reference. A similar technique, Nucleofection TM , utilizes an applied electric field in order to stimulate the uptake of exogenous polynucleotides into the nucleus of a eukaryotic cell.
  • Nucleofection TM and protocols useful for performing this technique are described in detail, e.g., in Distler et al., Experimental Dermatology 14:315 (2005), as well as in US 2010/0317114, the disclosures of each of which are incorporated herein by reference.
  • Additional techniques useful for the transfection of target cells include the squeeze-poration methodology. This technique induces the rapid mechanical deformation of cells in order to stimulate the uptake of exogenous DNA through membranous pores that form in response to the applied stress. This technology is advantageous in that a vector is not required for delivery of nucleic acids into a cell, such as a human target cell.
  • Lipofection represents another technique useful for transfection of target cells. This method involves the loading of nucleic acids into a liposome, which often presents cationic functional groups, such as quaternary or protonated amines, towards the liposome exterior. This promotes electrostatic interactions between the liposome and a cell due to the anionic nature of the cell membrane, which ultimately leads to uptake of the exogenous nucleic acids, for example, by direct fusion of the liposome with the cell membrane or by endocytosis of the complex.
  • cationic functional groups such as quaternary or protonated amines
  • Lipofection is described in detail, for example, in US Patent No.7,442,386, the disclosure of which is incorporated herein by reference. Similar techniques that exploit ionic interactions with the cell membrane to provoke the uptake of foreign nucleic acids include contacting a cell with a cationic polymer-nucleic acid complex.
  • Exemplary cationic molecules that associate with polynucleotides so as to impart a positive charge favorable for interaction with the cell membrane are activated dendrimers (described, e.g., 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:I:9.2:9.2.1 (1997), the disclosure of which is incorporated herein by reference.
  • activated dendrimers described, e.g., in Dennig, Topics in Current Chemistry 228:227 (2003), the disclosure of which is incorporated herein by reference
  • DEAE diethylaminoethyl
  • Magnetic beads are another tool that can be used to transfect target cells in a mild and efficient manner, as this methodology utilizes an applied magnetic field in order to direct 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.
  • Another useful tool for inducing the uptake of exogenous nucleic acids by target cells is laserfection, a technique that involves exposing a cell to electromagnetic radiation of a particular wavelength in order to gently permeabilize the cells and allow polynucleotides to penetrate the cell membrane. This technique is described in detail, e.g., in Rhodes et al., Methods in Cell Biology 82:309 (2007), the disclosure of which is incorporated herein by reference.
  • Microvesicles represent another potential vehicle that can be used to modify the genome of a target cell according to the methods described herein.
  • microvesicles that have been induced by the co-overexpression of the glycoprotein VSV-G with, e.g., a genome-modifying protein, such as a nuclease can be used to efficiently deliver proteins into a cell that subsequently catalyze the site-specific cleavage of an endogenous polynucleotide sequence so as to prepare the genome of the cell for the covalent incorporation of a polynucleotide of interest, such as a gene or regulatory sequence.
  • vesicles also referred to as Gesicles
  • Gesicles for the genetic modification of eukaryotic cells is described in detail, e.g., in Quinn et al., Genetic Modification of Target Cells by Direct Delivery of Active Protein [abstract].
  • Methylation changes in early embryonic genes in cancer [abstract] in: Proceedings of the 18th Annual Meeting of the American Society of Gene and Cell Therapy; 2015 May 13, Abstract No.122.
  • a transgene such as a transgene encoding an inhibitory nucleic acid construct, such as an interfering RNA construct (for example, short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA) described herein), into a target cell, and particularly into a human cell.
  • an interfering RNA construct for example, short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA) described herein
  • siRNA short interfering RNA
  • shRNA short hairpin RNA
  • miRNA microRNA
  • Transposons are polynucleotides that encode transposase enzymes and contain a polynucleotide sequence or gene of interest flanked by 5’ and 3’ excision sites. Once a transposon has been delivered into a cell, expression of the transposase gene commences and results in active enzymes that cleave the gene of interest from the transposon. This activity is mediated by the site-specific recognition of transposon excision sites by the transposase. In some instances, these excision sites may be terminal repeats or inverted terminal repeats.
  • the transgene of interest can be integrated into the genome of a mammalian cell by transposase-catalyzed cleavage of similar excision sites that exist within the nuclear genome of the cell. This allows the transgene of interest to be inserted into the cleaved nuclear DNA at the complementary excision sites, and subsequent covalent ligation of the phosphodiester bonds that join the gene of interest to the DNA of the mammalian cell genome completes the incorporation process.
  • the transposon may be a retrotransposon, such that the gene encoding the target gene is first transcribed to an RNA product and then reverse- transcribed to DNA before incorporation in the mammalian cell genome.
  • transposon systems are the piggybac transposon (described in detail in, e.g., WO 2010/085699) and the sleeping beauty transposon (described in detail in, e.g., US 2005/0112764), the disclosures of each of which are incorporated herein by reference as they pertain to transposons for use in gene delivery to a cell of interest.
  • Another tool for the integration of target transgenes into the genome of a target cell is the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system, a system that originally evolved as an adaptive defense mechanism in bacteria and archaea against viral infection.
  • CRISPR clustered regularly interspaced short palindromic repeats
  • the CRISPR/Cas system includes palindromic repeat sequences within plasmid DNA and an associated Cas9 nuclease. This ensemble of DNA and protein directs site specific DNA cleavage of a target sequence by first incorporating foreign DNA into CRISPR loci. Polynucleotides containing these foreign sequences and the repeat-spacer elements of the CRISPR locus are in turn transcribed in a host cell to create a guide RNA, which can subsequently anneal to a target sequence and localize the Cas9 nuclease to this site.
  • CRISPR/Cas to modulate gene expression has been described in, for example, US Patent No.8,697,359, the disclosure of which is incorporated herein by reference as it pertains to the use of the CRISPR/Cas system for genome editing.
  • Alternative methods for site-specifically cleaving genomic DNA prior to the incorporation of a transgene of interest in a target cell include the use of zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs).
  • ZFNs zinc finger nucleases
  • TALENs transcription activator-like effector nucleases
  • these enzymes do not contain a guiding polynucleotide to localize to a specific target sequence. Target specificity is instead controlled by DNA binding domains within these enzymes.
  • ZFNs and TALENs in genome editing applications is described, e.g., in Urnov et al., Nature Reviews Genetics 11:636 (2010); and in Joung et al., Nature Reviews Molecular Cell Biology 14:49 (2013), the disclosure of each of which are incorporated herein by reference as they pertain to compositions and methods for genome editing.
  • Additional genome editing techniques that can be used to incorporate polynucleotides encoding target transgenes into the genome of a target cell include the use of ARCUS TM meganucleases that can be rationally designed so as to site-specifically cleave genomic DNA.
  • Single chain meganucleases can be modified at certain amino acid positions in order to create nucleases that selectively cleave DNA at desired locations, enabling the site-specific incorporation of a target transgene into the nuclear DNA of a target cell.
  • These single- chain nucleases have been described extensively in, for example, US Patent Nos.8,021,867 and US 8,445,251, the disclosures of each of which are incorporated herein by reference as they pertain to compositions and methods for genome editing.
  • RNA transcript expression level of a wild-type or pathological RNA transcript can be ascertained, for example, by a variety of nucleic acid detection techniques. Additionally, or alternatively, RNA transcript expression can be inferred by evaluating the concentration or relative abundance of an encoded protein produced by translation of the RNA transcript. Protein concentrations can also be assessed, for example, using functional assays. Using these techniques, a reduction in the concentration of wild-type or pathological RNA transcripts in response to the compositions and methods described herein can be observed, while monitoring the expression of the encoded protein.
  • RNA transcript expression can be evaluated by a number of methodologies known in the art, including, but not limited to, nucleic acid sequencing, microarray analysis, proteomics, in-situ hybridization (e.g., fluorescence in-situ hybridization (FISH)), amplification-based assays, in situ hybridization, fluorescence activated cell sorting (FACS), northern analysis and/or PCR analysis of RNAs.
  • FISH fluorescence in-situ hybridization
  • FACS fluorescence activated cell sorting
  • Nucleic Acid-based methods for detection of RNA transcript expression include imaging- based techniques (e.g., Northern blotting or Southern blotting), which may be used in conjunction with cells obtained from a patient following administration of, for example, a vector encoding an inhibitory nucleic acid construct (such as an interfering RNA, for example, short interfering RNA (siRNA), short hairpin RNA (shRNA), or microRNA (miRNA) described herein) or a composition containing such an inhibitory nucleic acid construct.
  • an inhibitory nucleic acid construct such as an interfering RNA, for example, short interfering RNA (siRNA), short hairpin RNA (shRNA), or microRNA (miRNA) described herein
  • a composition containing such an inhibitory nucleic acid construct such as an interfering RNA, for example, short interfering RNA (siRNA), short hairpin RNA (shRNA), or microRNA (miRNA) described herein
  • 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 evaluating the status of genes and gene products are found, for example in Ausubel et al., eds., 1995, Current Protocols In Molecular Biology, Units 2 (Northern Blotting), 4 (Southern Blotting), 15 (Immunoblotting) and 18 (PCR Analysis).
  • RNA detection techniques that may be used in conjunction with the compositions and methods described herein to evaluate the expression level of RNA transcripts, such as the ATXN2 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, without limitation, Illumina sequencing, Ion Torrent sequencing, 454 sequencing, SOLiD sequencing, and nanopore sequencing platforms. Additional methods of sequencing known in the art can also be used.
  • transgene expression at the mRNA level may be determined using RNA-Seq (e.g., as described in Mortazavi et al., Nat.
  • RNA-Seq is a robust technology for monitoring expression by direct sequencing the RNA molecules in a sample. Briefly, this methodology may involve fragmentation of RNA to an average length of 200 nucleotides, conversion to cDNA by random priming, and synthesis of double-stranded cDNA (e.g., using the Just cDNA DoubleStranded cDNA Synthesis Kit from Agilent Technology®). Then, the cDNA is converted into a molecular library for sequencing by addition of sequence adapters for each library (e.g., from Illumina®/Solexa), and the resulting 50-100 nucleotide reads are mapped onto the genome.
  • sequence adapters for each library e.g., from Illumina®/Solexa
  • RNA expression levels may be determined using microarray-based platforms (e.g., single- nucleotide polymorphism arrays), as microarray technology offers high resolution. Details of various microarray methods can be found in the literature. See, for example, U.S. Pat. No.6,232,068 and Pollack et al., Nat. Genet.23:41-46 (1999), the disclosures of each of which are incorporated herein by reference in their entirety.
  • nucleic acid microarrays mRNA samples are reverse transcribed and labeled to generate cDNA. The probes can then hybridize to one or more complementary nucleic acids arrayed and immobilized on a solid support.
  • the array can be configured, for example, such that the sequence and position of each member of the array is known.
  • Hybridization of a labeled probe with a particular array member indicates that the sample from which the probe was derived expresses that gene.
  • Expression level may be quantified according to the amount of signal detected from hybridized probe-sample complexes.
  • a typical microarray experiment involves the following steps: 1) preparation of fluorescently labeled target from RNA isolated from the sample, 2) hybridization of the labeled target to the microarray, 3) washing, staining, and scanning of the array, 4) analysis of the scanned image and 5) generation of gene expression profiles.
  • Amplification-based assays also can be used to measure the expression level of a particular RNA transcript, such as a wild-type or mutant ATXN2 transcript.
  • the nucleic acid sequence of the transcript acts as a template in an amplification reaction (for example, PCR, such as qPCR).
  • PCR PCR, such as qPCR
  • the amount of amplification product is proportional to the amount of template in the original sample.
  • RNA transcript expression as described herein can be determined, for example, by RT-PCR technology.
  • Probes used for PCR may be labeled with a detectable marker, such as, for example, a radioisotope, fluorescent compound, bioluminescent compound, a chemiluminescent compound, metal chelator, or enzyme.
  • a detectable marker such as, for example, a radioisotope, fluorescent compound, bioluminescent compound, a chemiluminescent compound, metal chelator, or enzyme.
  • Protein Detection Expression of an RNA construct may also be inferred by analyzing expression of the protein encoded by the construct. Protein levels can be assessed using standard detection techniques known in the art.
  • Protein expression assays suitable for use with the compositions and methods described herein include proteomics approaches, immunohistochemical and/or western blot analysis, immunoprecipitation, molecular binding assays, ELISA, enzyme-linked immunofiltration assay (ELIFA), mass spectrometry, mass spectrometric immunoassay, and biochemical enzymatic activity assays.
  • proteomics methods can be used to generate large-scale protein expression datasets in multiplex.
  • Proteomics methods may utilize mass spectrometry to detect and quantify polypeptides (e.g., proteins) and/or peptide microarrays utilizing capture reagents (e.g., antibodies) specific to a panel of target proteins to identify and measure expression levels of proteins expressed in a sample (e.g., a single cell sample or a multi-cell population).
  • exemplary peptide microarrays have a substrate-bound plurality of polypeptides, the binding of an oligonucleotide, a peptide, or a protein to each of the plurality of bound polypeptides being separately detectable.
  • the peptide microarray may include a plurality of binders, including, but not limited to, monoclonal antibodies, polyclonal antibodies, phage display binders, yeast two-hybrid binders, aptamers, which can specifically detect the binding of specific oligonucleotides, peptides, or proteins.
  • binders including, but not limited to, monoclonal antibodies, polyclonal antibodies, phage display binders, yeast two-hybrid binders, aptamers, which can specifically detect the binding of specific oligonucleotides, peptides, or proteins.
  • Examples of peptide arrays may be found in U.S. Patent Nos. 6,268,210, 5,766,960, and 5,143,854, the disclosures of each of which are incorporated herein by reference in their entirety.
  • Mass spectrometry may be used in conjunction with the methods described herein to identify and characterize transgene expression in a cell from a patient (e.g., a human patient) following delivery of the transgene. Any method of MS known in the art may be used to determine, detect, and/or measure a protein or peptide fragment of interest, e.g., LC-MS, ESI-MS, ESI-MS/MS, MALDI-TOF-MS, MALDI-TOF/TOF-MS, tandem MS, and the like.
  • Mass spectrometers generally contain an ion source and optics, mass analyzer, and data processing electronics.
  • Mass analyzers include scanning and ion-beam mass spectrometers, such as time-of-flight (TOF) and quadruple (Q), and trapping mass spectrometers, such as ion trap (IT), Orbitrap, and Fourier transform ion cyclotron resonance (FT-ICR), may be used in 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 in its entirety.
  • TOF time-of-flight
  • Q quadruple
  • trapping mass spectrometers such as ion trap (IT), Orbitrap, and Fourier transform ion cyclotron resonance (FT-ICR)
  • proteins in a sample obtained from the patient can be first digested into smaller peptides by chemical (e.g., via cyanogen bromide cleavage) or enzymatic (e.g., trypsin) digestion.
  • Complex peptide samples also benefit from the use of front-end separation techniques, e.g., 2D-PAGE, HPLC, RPLC, and affinity chromatography.
  • the digested, and optionally separated, sample is then ionized using an ion source to create charged molecules for further analysis.
  • Ionization of the sample may be performed, e.g., by 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/plasmaspray ionization, and particle beam ionization. Additional information relating to the choice of ionization method is known to those of skill in the art. After ionization, digested peptides may then be fragmented to generate signature MS/MS spectra.
  • ESI electrospray ionization
  • APCI atmospheric pressure chemical ionization
  • FAB fast atom bombardment
  • LIMS liquid secondary ionization
  • MALDI matrix assisted laser desorption/ionization
  • field ionization field desorption
  • Tandem MS also known as MS/MS
  • Tandem MS may be particularly useful for analyzing complex mixtures. Tandem MS involves multiple steps of MS selection, with some form of ion fragmentation occurring in between the stages, which may be accomplished with individual mass spectrometer elements separated in space or using a single mass spectrometer with the MS steps separated in time.
  • spatially separated tandem MS the elements are physically separated and distinct, with a physical connection between the elements to maintain high vacuum.
  • separation is accomplished with ions trapped in the same place, with multiple separation steps taking place over time.
  • Signature MS/MS spectra may then be compared against a peptide sequence database (e.g., SEQUEST).
  • SEQUEST a peptide sequence database
  • Post-translational modifications to peptides may also be determined, for example, by searching spectra against a database while allowing for specific peptide modifications.
  • the inhibitory nucleic acid constructs such as interfering RNA constructs (for example, short interfering RNA (siRNA), short hairpin RNA (shRNA), or microRNA (miRNA)), as well as the vectors and compositions encoding or containing such constructs, may be incorporated into a vehicle for administration into a patient, such as a human patient suffering from a disorder, as described herein.
  • Pharmaceutical compositions containing vectors, such as viral vectors, that encode an inhibitory nucleic acid construct described herein can be prepared using methods known in the art.
  • compositions can be prepared using, e.g., physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980); incorporated herein by reference), and in a desired form, e.g., in the form of lyophilized formulations or aqueous solutions.
  • Mixtures of the nucleic acids and viral vectors described herein may be prepared in water suitably mixed with one or more excipients, carriers, or diluents.
  • Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (described in US 5,466,468, the disclosure of which is incorporated herein by reference).
  • the formulation may be sterile and may be fluid to the extent that easy syringability exists.
  • Formulations may be stable under the conditions of manufacture and storage and may be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils.
  • polyol e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • suitable mixtures thereof e.g., vegetable oils
  • vegetable oils e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • suitable mixtures thereof e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • vegetable oils e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • a solution containing a pharmaceutical composition described herein may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for administration by a route selected from intrathalamic, intrathecal, subpial, intraparenchymal, intrastriatal, intracranial, intracisternal, intracerebral, intracerebroventricular, intraocular (e.g., intravitreal), intraventricular, intralumbar, intravenous, intramuscular, subcutaneous, intraperitoneal, intradermal, transdermal, parenteral, intranasal, percutaneous, intratracheal, intraarterial, intravascular, and oral administration, inhalation, perfusion, lavage, or any combination thereof.
  • sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations may meet sterility, pyrogenicity, general safety, and purity standards as required by FDA Office of Biologics standards.
  • a pharmaceutical composition containing, for example, an inhibitory nucleic acid described herein, typically includes a pharmaceutically acceptable diluent or carrier.
  • a pharmaceutical composition may include (e.g., consist of), e.g., a sterile saline solution and a nucleic acid.
  • the sterile saline is typically a pharmaceutical grade saline.
  • a pharmaceutical composition may include (e.g., consist of), e.g., sterile water and a nucleic acid.
  • the sterile water is typically a pharmaceutical grade water.
  • a pharmaceutical composition may include (e.g., consist of), e.g., phosphate-buffered saline (PBS) and a nucleic acid.
  • PBS phosphate-buffered saline
  • pharmaceutical compositions include one or more composition or nucleic acid molecule and one or more excipients.
  • excipients are selected from water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose and polyvinylpyrrolidone.
  • nucleic acid molecules may be admixed with pharmaceutically acceptable active and/or inert substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of pharmaceutical compositions depend on a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.
  • compositions including a nucleic acid molecule encompass any pharmaceutically acceptable salts of the inhibitor, esters of the inhibitor, or salts of such esters.
  • pharmaceutical compositions including a nucleic acid molecule upon administration to a subject (e.g., a human), are capable of providing (directly or indirectly) the biologically active metabolite or residue thereof.
  • a subject e.g., a human
  • pharmaceutically acceptable salts of inhibitors, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents include, but are not limited to, sodium and potassium salts.
  • prodrugs include one or more conjugate group attached to a nucleic acid molecule, wherein the conjugate group is cleaved by endogenous nucleases within the body.
  • Lipid moieties have been used in nucleic acid therapies in a variety of methods.
  • the nucleic acid is introduced into preformed liposomes or lipoplexes made of mixtures of cationic lipids and neutral lipids.
  • DNA complexes with mono- or poly-cationic lipids are formed without the presence of a neutral lipid.
  • a lipid moiety is selected to increase distribution of a pharmaceutical agent to a particular cell or tissue.
  • a lipid moiety is selected to increase distribution of a pharmaceutical agent to fat tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to muscle tissue.
  • pharmaceutical compositions include a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems are useful for preparing certain pharmaceutical compositions including those including hydrophobic compounds. In certain embodiments, certain organic solvents such as dimethylsulfoxide are used.
  • pharmaceutical compositions include one or more tissue-specific delivery molecules designed to deliver the one or more pharmaceutical agents of the present invention to specific tissues or cell types. For example, in certain embodiments, pharmaceutical compositions include liposomes coated with a tissue-specific antibody.
  • compositions include a co-solvent system.
  • co-solvent systems include, for example, benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase.
  • co-solvent systems are used for hydrophobic compounds.
  • a non-limiting example of such a co-solvent system is the VPD co- solvent system, which is a solution of absolute ethanol including 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80TM and 65% w/v polyethylene glycol 300.
  • the proportions of such co-solvent systems may be varied considerably without significantly altering their solubility and toxicity characteristics.
  • co-solvent components may be varied: for example, other surfactants may be used instead of Polysorbate 80TM; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.
  • pharmaceutical compositions are prepared for oral administration.
  • pharmaceutical compositions are prepared for buccal administration.
  • a pharmaceutical composition is prepared for administration by injection (e.g., intraocular (e.g., intravitreal), intravenous, subcutaneous, intramuscular, intrathalamic, subpial, intrathecal, intracerebroventricular, etc.).
  • a pharmaceutical composition includes a carrier and is formulated in aqueous solution, such as water or physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
  • aqueous solution such as water or physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
  • other ingredients are included (e.g., ingredients that aid in solubility or serve as preservatives).
  • injectable suspensions are prepared using appropriate liquid carriers, suspending agents and the like.
  • Certain pharmaceutical compositions for injection are presented in unit dosage form, e.g., in ampoules or in multi-dose containers.
  • Certain pharmaceutical compositions for injection are suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • Certain solvents suitable for use in pharmaceutical compositions for injection 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.
  • Routes of Administration and Dosing Viral vectors, such as AAV vectors and others described herein, containing a transgene encoding an inhibitory nucleic acid of the disclosure may be administered to a patient (e.g., a human patient) by a variety of routes of administration.
  • the route of administration may vary, for example, with the onset and severity of disease, and may be selected from, e.g., intrathalamic, intrathecal, subpial, intraparenchymal, intrastriatal, intracranial, intracisternal, intracerebral, intracerebroventricular, intraocular (e.g., intravitreal), intraventricular, intralumbar, intravenous, intramuscular, subcutaneous, intraperitoneal, intradermal, transdermal, parenteral, intranasal, percutaneous, intratracheal, intraarterial, intravascular, and oral administration, inhalation, perfusion, lavage, or any combination thereof.
  • Intravascular administration includes delivery into the vasculature of a patient.
  • the administration is into a vessel considered to be a vein (intravenous), and in some administration, the administration is into a vessel considered to be an artery (intraarterial).
  • Veins include, but are not limited to, the internal jugular vein, a peripheral vein, a coronary vein, a hepatic vein, the portal vein, great saphenous vein, the pulmonary vein, superior vena cava, inferior vena cava, a gastric vein, a splenic vein, inferior mesenteric vein, superior mesenteric vein, cephalic vein, and/or femoral vein.
  • Arteries include, but are not limited to, coronary artery, pulmonary artery, brachial artery, internal carotid artery, aortic arch, femoral artery, peripheral artery, and/or ciliary artery. It is contemplated that delivery may be through or to an arteriole or capillary. Treatment regimens may vary, and often depend on disease severity and the age, weight, and sex of the patient. Treatment may include administration of vectors (e.g., viral vectors) or other agents described herein as useful for the introduction of a transgene into a target cell in various unit doses. Each unit dose will ordinarily contain a predetermined quantity of the therapeutic composition.
  • vectors e.g., viral vectors
  • Example 1 Determining the knockdown efficacy of ATXN2-specific miRNA constructs in HEK293 cells via dual luciferase reporter assay Objective The objective of this study was to evaluate the knockdown efficacy of several ATXN2 miRNA candidates in HEK293 cells via the dual luciferase reporter assay. This study was designed to determine the efficiency of the miRNA constructs in silencing ATXN2 mRNA.
  • HEK293 cells were transduced with AAV vectors incorporated with ATXN2 miRNA candidates for miRNA delivery and subsequent knockdown of the ATXN2 mRNA.
  • a dual luciferase reporter assay was then performed to measure the efficacy of the knockdown obtained with the miRNA constructs in comparison with a negative control, which effectuates no significant silencing.
  • a dual luciferase reporter assay was then performed to measure the efficacy of the knockdown achieved by each miRNA construct in comparison to a negative control, which effectuates no significant silencing.
  • the construct numbering in this example is the same as Example 1. Results In result, we observed that the miRNA constructs chosen for this study were able to silence ATXN2. Percentage of luciferase activity relative to negative control ranged from 20% to 54% (FIG. 2A), with miRNA construct 1 showing the best knockdown efficacy of those tested. Knockdown efficacy is inverse to the percentage luciferase activity relative to negative control, therefore, the lower the percentage value of the percentage luciferase activity relative to negative control, the higher the knockdown efficacy.
  • Control is a non-targeting miRNA and shows no knockdown.
  • Example 3 Determining the knockdown efficacy of ATXN2-specific miRNA constructs in HEK293 cells via a probe-based quantitative PCR (qPCR) assay after plasmid transfection Objective The objective of this study was to evaluate the knockdown efficacy of several enodgenous ATXN2 miRNA constructs in HEK293 cells via a probe-based qPCR assay after plasmid transfection. This study was designed to determine the efficiency of ATXN2-specific miRNA constructs in silencing the ATXN2 mRNA. Materials and Methods HEK293 cells were transfected with plasmids encoding ATXN2-specific miRNA constructs.
  • a probe-based qPCR assay was performed to measure the efficacy of the knockdown achieved by each miRNA construct as compared to a negative control. This experiment was performed twice. The first round of experiments tested a higher number of ATXN2-specific miRNA constructs compared to the second round. The construct numbering in this example is the same as Example 1. Results In result, we observed that the miRNA constructs chosen for this study in the first round were able to silence ATXN2. ATXN2 to GAPDH mRNA signals generally ranged from 25% to 74% (FIG. 2B). miRNA construct 2 showed the best knockdown efficacy of those tested.
  • Knockdown efficacy is inverse to the ratio of ATXN2 to GAPDH mRNA signal, therefore, the lower the percentage value of the ATXN2 to GAPDH mRNA signal, the higher the knockdown efficacy.
  • Control is a non- targeting miRNA and shows no knockdown.
  • the miRNA constructs chosen in the second round were also able to silence ATXN2 (FIG.2C).
  • miRNA construct 2 showed the best knockdown efficacy of those tested as evident from the lowest percentage of mRNA expression relative to control (CONT).
  • CONT is a non- targeting miRNA and shows no knockdown.
  • mice In these mice, a 169 kb human BAC (RP11-798L5) containing the entire 150 kb human ATXN2 locus including regulatory regions was engineered to replace the endogenous ATXN2 exon-1 CAG22 with CAG72 repeats.
  • the thalamus and sub-cortex from one hemisphere were harvested 6 weeks post-intrathalamic administration and analysed for mouse and human ATXN2 mRNA expression using qPCR. Following tissue harvesting, a probe-based qPCR assay was performed using homogenized mouse tissue to measure the efficacy of the knockdown achieved by each ATXN2-specific miRNA construct in comparison with a negative control.
  • Vector expression was confirmed by measuring the quantity of viral genomes (vg) using qPCR in the thalamus (data not shown).
  • the experiments in this study were performed twice, starting with a small-scale pilot study with construct 2 followed by a large-scale study including multiple ATXN2-specific miRNA constructs.
  • Both mouse and human ATXN2 mRNA were measured in the mouse thalamus and sub-cortex in the small-scale pilot study whereas only human ATXN2 mRNA was measured in the large-scale study.
  • the experimental groups received AAV injections with doses of 1e8 viral genomes/hemisphere (vg/hem) or 1e9 vg/hem in the pilot study whereas in the large-scale study only AAV doses of 1e9 vg/hem were injected.
  • the PBS group was used as a control.
  • PBS group represented no silencing of the endogenous ATXN2 mRNA and served as a baseline.
  • the large- scale study there were 3 control groups: PBS - mice injected with Dulbecco’s phosphate buffered saline; na ⁇ ve - non-injected mice; and neg - mice injected with miRNA cassette not specific for ATXN2 miRNA. All 3 control groups represented negligible silencing of the endogenous ATXN2 mRNA, with the PBS group serving as a baseline.
  • the construct numbering in this example is the same as Example 1.

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Abstract

L'invention concerne des compositions et des méthodes pour le traitement de troubles associés à l'expression de transcrits d'ARN d'ataxine-2 (ATXN2) de type sauvage ou mutants, comprenant des troubles caractérisés par des gènes contenant des régions de répétition étendues de manière aberrante qui peuvent conduire à un phénotype pathologique. L'invention concerne des constructions d'ARN inhibitrices qui suppriment l'expression d'ATXN2, ainsi que des vecteurs viraux, tels que des vecteurs viraux adéno-associés, codant pour de telles molécules d'ARN inhibitrices.
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