WO2023034966A1 - Compositions et procédés d'utilisation de celles-ci pour traiter les troubles associés à la thymosine βeta 4 - Google Patents

Compositions et procédés d'utilisation de celles-ci pour traiter les troubles associés à la thymosine βeta 4 Download PDF

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WO2023034966A1
WO2023034966A1 PCT/US2022/075904 US2022075904W WO2023034966A1 WO 2023034966 A1 WO2023034966 A1 WO 2023034966A1 US 2022075904 W US2022075904 W US 2022075904W WO 2023034966 A1 WO2023034966 A1 WO 2023034966A1
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mir
vector
polynucleotide
subject
expression cassette
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Bin Xiao
Xiao Xiao
Juan Li
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The University Of North Carolina At Chapel Hill
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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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • 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
    • 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
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/113—Antisense targeting other non-coding nucleic acids, e.g. antagomirs
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/51—Physical structure in polymeric form, e.g. multimers, concatemers
    • 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
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • This invention relates to polynucleotides comprising anti-microRNA oligonucleotides (AMO) directed to miR-1 and/or miR-206, and/or a coding region encoding a thymosin P4 (Tp4), as well as expression cassettes, vectors, and compositions comprising the same, and methods of using the same for delivery of the polynucleotides and/or expression cassettes to a cell or a subject and to treat disorders associated with aberrant expression of a miR-1 and/or miR-206 regulated gene in the subject, such as amyotrophic lateral sclerosis (ALS).
  • AMO anti-microRNA oligonucleotides
  • Tp4 thymosin P4
  • ALS Amyotrophic lateral sclerosis
  • ALS is a severe neuromuscular disease, which is characterized by skeletal muscle dystrophy, limb and respiratory muscle paralysis (Kiernan et al. 2011 Lancet 377:942-955). After ALS is diagnosed, the expected survival time is only 5 years and current treatments such as Riluzole, increase survival by merely 3 to 5 months (Bruijn et al. 2004 Annu Rev Neurosci 27:723-749; Bensimon et al. 1994 N Engl J Med 330:585-591). Thus, understanding the pathological mechanism of ALS and finding therapeutic methods are imperative.
  • the present invention overcomes shortcomings in the art by providing synthetic polynucleotides and expression cassettes comprising anti-miR oligonucleotides (AMO) and/or thymosin P4 coding regions as well as compositions and methods comprising the same for treating ALS.
  • AMO anti-miR oligonucleotides
  • thymosin P4 thymosin P4 coding regions
  • One aspect of the present invention comprises a synthetic polynucleotide encoding one or more anti-miRNA oligonucleotide (AMO) directed to microRNA-1 (miR-1) and/or miR-206.
  • An additional aspect of the present invention comprises an expression cassette comprising a synthetic polynucleotide encoding one or more anti-miRNA oligonucleotide (AMO) directed to microRNA-1 (miR-1) and/or miR-206.
  • Another aspect of the present invention provides an expression cassette comprising a synthetic polynucleotide comprising a coding region encoding a human thymosin P4 (Tp4).
  • vectors comprising a synthetic polynucleotide, expression cassette, vector, transformed cell, and/or composition of the present invention.
  • Another aspect of the present invention comprises a method of reducing miR-1 expression in a cell, comprising contacting the cell with a synthetic polynucleotide, expression cassette, vector, and/or composition of the present invention.
  • Another aspect of the present invention provides a method of enhancing Tp4 protein expression in a cell, comprising contacting the cell with a synthetic polynucleotide, expression cassette, vector, and/or composition of the present invention.
  • Another aspect of the present invention provides a method of reducing miR-1 expression in a subject, comprising delivering to the subject a synthetic polynucleotide, expression cassette, vector, transformed cell and/or composition of the present invention.
  • Another aspect of the present invention provides a method of enhancing Tp4 protein expression in a subject, comprising delivering to the subject a synthetic polynucleotide, expression cassette, vector, transformed cell and/or composition of the present invention.
  • Another aspect of the present invention provides a method of treating a disorder associated with aberrant overexpression and/or activity of miR-1 or aberrant activity of a miR-1 regulated gene in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a synthetic polynucleotide, expression cassette, vector, transformed cell and/or composition of the present invention.
  • Another aspect of the present invention provides a method of treating a disorder associated with aberrant overexpression and/or activity of Tp4 gene and/or a Tp4 gene product (e.g., TP4 protein) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a synthetic polynucleotide, expression cassette, vector, transformed cell and/or composition of the present invention.
  • a Tp4 gene product e.g., TP4 protein
  • Another aspect of the present invention provides a method of treating ALS (e.g., familial or sporadic ALS) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a synthetic polynucleotide, expression cassette, vector, transformed cell and/or composition of the present invention.
  • Another aspect of the present invention provides a method of treating sporadic ALS in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a synthetic polynucleotide, expression cassette, vector, transformed cell and/or composition of the present invention.
  • Another aspect of the present invention provides a method of treating familial ALS in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a synthetic polynucleotide, expression cassette, vector, transformed cell and/or composition of the present invention.
  • Another aspect of the present invention provides a method of treating ALS (e.g., familial or sporadic ALS) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of: (a) a synthetic polynucleotide encoding one or more copies of an AMO directed to miR-1 and/or miR-206, and/or an expression cassette, vector, transformed cell, and/or pharmaceutical composition comprising the same; and/or (b) a synthetic polynucleotide comprising a coding region encoding a human Tp4, wherein the encoded human Tp4 comprises SEQ ID NO:5 or a sequence at least about 70% identical thereto, and/or an expression cassette, vector, transformed cell, and/or pharmaceutical composition comprising the same.
  • ALS e.g., familial or sporadic ALS
  • Another aspect of the present invention provides a method of postponing disease progression of ALS (e.g., familial or sporadic ALS) in a subject having ALS or a subject at risk for or suspected to have or develop ALS comprising administering to the subject a therapeutically effective amount of: (a) a synthetic polynucleotide encoding one or more copies of an AMO directed to miR-1 and/or miR-206, and/or an expression cassette, vector, transformed cell, and/or pharmaceutical composition comprising the same; and/or (b) a synthetic polynucleotide comprising a coding region encoding a human Tp4, wherein the encoded human Tp4 comprises SEQ ID NO:5 or a sequence at least about 70% identical thereto, and/or an expression cassette, vector, transformed cell, and/or pharmaceutical composition comprising the same.
  • ALS e.g., familial or sporadic ALS
  • Another aspect of the present invention provides a method of reducing disease severity of ALS (e.g., familial or sporadic ALS) in a subject having ALS or a subject at risk for or suspected to have or develop ALS, comprising administering to the subject a therapeutically effective amount of: (a) a synthetic polynucleotide encoding one or more copies of an AMO directed to miR-1 and/or miR-206, and/or an expression cassette, vector, transformed cell, and/or pharmaceutical composition comprising the same; and/or (b) a synthetic polynucleotide comprising a coding region encoding a human Tp4, wherein the encoded human Tp4 comprises SEQ ID NO:5 or a sequence at least about 70% identical thereto, and/or an expression cassette, vector, transformed cell, and/or pharmaceutical composition comprising the same.
  • ALS e.g., familial or sporadic ALS
  • FIGS. 1A-1C show data graphs of miR-1 and miR-206 expression in the CNS of ALS mice.
  • FIG. IB is a bar graph of miR- 206 in the spinal cord, as detected by real-time PCR. No significant differences between groups was found.
  • FIG. 1C shows an image of a northern blot analysis and a bar graph quantifying mature miR-1 expression level in ALS spinal cord at 8- and 16-week old. * ⁇ 0.05.
  • FIGS. 2A-2D show illustrations and data graphs of Tp4 targeting by miR-1/206 and related studies.
  • FIG. 2A shows a sequence alignment from the database TargetScan.org forecasting Tp4 as one of the most probable target genes of miR-1/206. The sequences indicated with matches are seed-sequences, and correspond to SEQ ID NOs: 1 and 2. The full sequence of Tp4 can be found at GenBank Accession No. BC151215.1, incorporated herein by reference. The sequences shown in FIG. 2A correspond to SEQ ID NOs:9-l 1.
  • FIG. 2B shows a cartoon for the construction of the plasmid pEMBL-CMV-Luc-Tp4-3' UTR and its mutant.
  • FIG. 2C shows a bar graph of luciferase expression level as detected in 293 cells 48h after shRNA-206/206mut transfection.
  • FIG. 2D shows a bar graph of luciferase expression level as detected in 293 cells 48h after pre-miR- 1/206 transfection.
  • FIGS. 3A-3B show images of a western blot and a northern blot examining Tp4 protein and mRNA expression.
  • FIG. 3A shows an image of a western blot for Tp4 protein level in the spinal cord of ALS mice and age-matched WT at 8 and 16-week old.
  • FIG. 3B shows an image of a northern blot of Tp4 mRNA expression in the spinal cord.
  • FIG. 4A shows an image of a western blot analysis of downregulation of TP4 by G93A-SOD-1 treatment.
  • Western blot shows TP4 expression in SH-SY5Y cells treated with AAV2, GAPDH as control.
  • FIG. 5A shows an image comparing thymuses from WT and ALS mice at 16-weeks of age.
  • FIG. 5B shows a bar graph quantifying thymus weight from WT and ALS mice. **P ⁇ 0.01, t-test.
  • FIG. 6A shows a bar graph of luciferase expression of the plasmid pEMBL-CMV- Luciferase inserted with Tp4-3'UTR target sequences.
  • pEMBL-CMV-LacZ was cotransfected at the same time as an internal control.
  • Anti -miR- 1/206 rescued the expression of luciferase inhibited by the plasmid pre-miR-1.
  • n 4 per group. * P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001, t-test.
  • FIG. 6A shows a bar graph of luciferase expression of the plasmid pEMBL-CMV- Luciferase inserted with Tp4-3'UTR target sequences.
  • pEMBL-CMV-LacZ was cotransfected at the same time as an internal control.
  • Anti -miR- 1/206 rescued the expression of luciferase inhibited by the
  • FIG. 6B shows a bar graph of luciferase expression of the plasmid pEMBL-CMV-Luciferase, which was inserted with Tp4-3'UTR target sequences.
  • pEMBL- CMV-LacZ was co-transfected at the same time as an internal conference.
  • Anti-miR- 1/206 rescued the expression of luciferase inhibited by the plasmid pre-miR-206.
  • n 4 per group.
  • n 4 per group.
  • FIG. 6C shows an image of a western blot analysis of the expression of TP4 in N2a cells.
  • AAV2-CMV-GFP-4*anti- miR-1/206 rescued the expression of Tp4 inhibited by AAV2-CMV-G93A-SOD1 treatment.
  • the bands of human SOD1 (hSODl) are above that of mouse SOD1 (mSODl).
  • FIG. 7C shows a data plot of disease onset percentage comparing P
  • FIG. 7F shows a bar graph quantifying disease progression time between the groups. No significant difference was found between the groups.
  • FIG. 7G shows an image of a western blot analyzing Tp4 expression in the spinal cord of ALS mice treated by AAV9-CMV-anti-miR-l and AAV9-CMV-anti-miR- 206.
  • FIG. 71 shows microscopy images of Nissl staining for motor neurons in the anterior horn. The pictures at the top-right corner are high power images from the area of the white dashed frames. Scale bar: 400pm (left), 50pm (top right).
  • FIG. 9C shows a bar graph quantifying the average days of survival of FIGS. 9A and 9B.
  • FIG. 9F shows a bar graph quantifying disease progression in days. No significant differences were found between the groups.
  • FIG. 11A shows an image of a western blot analysis of TP4 expression in the spinal cord of ALS mice treated with TP4 by IV and IT injection.
  • FIG. 11C shows images of immunostaining of Tp4. Tp4 was found mainly expressed in neuronal cells. TP4 treatment by IV and IT injection increased its expression in neuronal cells. The pictures at the top-right corner are high power images from the area of the white dashed frames. Red: Tp4; Green: NeuN; Blue: DAPI; Purple: Merge. Scar bar: 200pm, 50pm (top right).
  • FIG.11D shows microscopy images of Nissl staining for motor neurons in two TP4 treatment groups by IV and IT injection.
  • the high-power pictures at the top-right corner present the area of the white dashed frames.
  • Scar bar 400pm, 50pm (top right).
  • FIGS. 12A-12E show ALS-like symptom induction by shRNA-206.
  • FIG. 12B shows a data plot of the survival curves of the mice from the shRNA-206/206mut groups. P ⁇ 0.05. Log-rank test.
  • FIG. 12C shows images of the branched nerve ultrastructure taken by transmission electron microscopy (TEM).
  • TEM transmission electron microscopy
  • FIG. 12D shows images of Masson's trichrome staining for tibialis anterior muscle. The myofiber size variation in the shRNA-206 treated muscle is increased compared with shRNA-206mut treatment. White arrows show angular and small round myofibers in the shRNA-206 treatment group. Scale bar: 100 pm.
  • FIG. 12E shows images of immunostaining for TDP-43. TDP-43 is normally located in the nucleus.
  • FIG. 13 shows a schematic of the plasmid construction of pEMBL-CMV-GFP-4*- anti-miRl/206, obtained by inserting four copies of anti-miR of miR-1/206 into the 3'-UTR of the plasmid pEMBL-CMV-GFP.
  • FIG. 14 shows a schematic of mechanisms of miR-1 and Tp4 in ALS pathology. While not wishing to be bound to theory, alternative pathogenic factors such as G93A-SOD1, oxidative stress, and pathological TDP-43 may enhance miR-1, which downregulates the expression of Tp4. The deficiency of Tp4 results in loss of functions such as angiogenesis, anti-apoptosis, anti-inflammation and neuroprotection before finally leading to motor neuron death.
  • alternative pathogenic factors such as G93A-SOD1, oxidative stress, and pathological TDP-43 may enhance miR-1, which downregulates the expression of Tp4.
  • the deficiency of Tp4 results in loss of functions such as angiogenesis, anti-apoptosis, anti-inflammation and neuroprotection before finally leading to motor neuron death.
  • a measurable value such as an amount or concentration and the like, is meant to encompass variations of ⁇ 10%, ⁇ 5%, ⁇ 1%, ⁇ 0.5%, or even ⁇ 0.1% of the specified value as well as the specified value.
  • "about X" where X is the measurable value is meant to include X as well as variations of ⁇ 10%, ⁇ 5%, ⁇ 1%, ⁇ 0.5%, or even ⁇ 0.1% of X.
  • a range provided herein for a measurable value may include any other range and/or individual value therein.
  • phrases such as “between X and Y” and “between about X and Y” should be interpreted to include X and Y.
  • phrases such as “between about X and Y” mean “between about X and about Y” and phrases such as “from about X to Y” mean “from about X to about Y.”
  • Nucleotide sequences are presented herein by single strand only, in the 5' to 3' direction, from left to right, unless specifically indicated otherwise. Nucleotides and amino acids are represented herein in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or (for amino acids) by either the one-letter code, or the three letter code, both in accordance with 37 C.F.R. ⁇ 1.822 and established usage.
  • amino acid can be selected from any subset of these amino acid(s) for example A, G, I or L; A, G, I or V; A or G; only L; etc. as if each such sub combination is expressly set forth herein.
  • amino acid can be disclaimed (e.g., by negative proviso).
  • the amino acid is not A, G or I; is not A; is not G or V; etc. as if each such possible disclaimer is expressly set forth herein.
  • the terms “reduce,” “reduces,” “reduction,” “diminish,” “inhibit” and similar terms mean a decrease of at least about 5%, 10%, 15%, 20%, 25%, 35%, 50%, 75%, 80%, 85%, 90%, 95%, 97% or more.
  • the terms “enhance,” “enhances,” “enhancement” and similar terms indicate an increase of at least about 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500% or more.
  • parvovirus encompasses the family Parvoviridae, including autonomously replicating parvoviruses and dependoviruses.
  • the autonomous parvoviruses include members of the genera Parvovirus, Erythrovirus, Densovirus, Iteravirus, and Contravirus.
  • Exemplary autonomous parvoviruses include, but are not limited to, minute virus of mouse, bovine parvovirus, canine parvovirus, chicken parvovirus, feline panleukopenia virus, feline parvovirus, goose parvovirus, Hl parvovirus, Muscovy duck parvovirus, B19 virus, and any other autonomous parvovirus now known or later discovered.
  • Other autonomous parvoviruses are known to those skilled in the art. See, e.g., BERNARD N. FIELDS et al., VIROLOGY, Volume 2, Chapter 69 (4th ed., Lippincott-Raven Publishers).
  • AAV adeno-associated virus
  • AAV includes but is not limited to, AAV type 1, AAV type 2, AAV type 3 (including types 3 A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, AAV type 12, AAV type 13, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, and any other AAV now known or later discovered. See, e.g., BERNARD N.
  • the genomic sequences of various serotypes of AAV and the autonomous parvoviruses, as well as the sequences of the native terminal repeats (TRs), Rep proteins, and capsid subunits are known in the art. Such sequences may be found in the literature or in public databases such as GenBank.
  • sequences include known amino acid sequences of the serotype capsid proteins, including but not limited to, AAD27757.1 (AAV1), YP 068409.1 (AAV5), AAC03780.1 (AAV2), AAC58045.1 (AAV4), NP_043941.1 (AAV3), AAB95450.1 (AAV6), YP_077178.1 (AAV7), YP_077180.1 (AAV8), AAS99264.1 (AAV9), and AAO88201.1 (AAVrhlO).
  • tropism refers to preferential entry of the virus into certain cells or tissues, optionally followed by expression (e.g., transcription and, optionally, translation) of a sequence(s) carried by the viral genome in the cell, e.g., for a recombinant virus, expression of a heterologous nucleic acid(s) of interest.
  • transduction of a cell by a virus vector means entry of the vector into the cell and transfer of genetic material into the cell by the incorporation of nucleic acid into the virus vector and subsequent transfer into the cell via the virus vector.
  • efficient transduction or “efficient tropism,” or similar terms, can be determined by reference to a suitable positive or negative control (e.g., at least about 50%, 60%, 70%, 80%, 85%, 90%, 95% or more of the transduction or tropism, respectively, of a positive control or at least about 110%, 120%, 150%, 200%, 300%, 500%, 1000% or more of the transduction or tropism, respectively, of a negative control).
  • a suitable positive or negative control e.g., at least about 50%, 60%, 70%, 80%, 85%, 90%, 95% or more of the transduction or tropism, respectively, of a positive control or at least about 110%, 120%, 150%, 200%, 300%, 500%, 1000% or more of the transduction or tropism, respectively, of a negative control.
  • polypeptide encompasses both peptides and proteins, unless indicated otherwise.
  • a "polynucleotide” is a sequence of nucleotide bases, and may be RNA, DNA or DNA-RNA hybrid sequences (including both naturally occurring and non-naturally occurring nucleotides), but in representative embodiments are either single or double stranded DNA sequences.
  • an "isolated" polynucleotide e.g., an "isolated DNA” or an “isolated RNA" means a polynucleotide at least partially separated from at least some of the other components of the naturally occurring organism or virus, for example, the cell or viral structural components or other polypeptides or nucleic acids commonly found associated with the polynucleotide.
  • an "isolated" nucleotide is enriched by at least about 10-fold, 100-fold, 1000-fold, 10,000-fold or more as compared with the starting material.
  • an "isolated" polypeptide means a polypeptide that is at least partially separated from at least some of the other components of the naturally occurring organism or virus, for example, the cell or viral structural components or other polypeptides or nucleic acids commonly found associated with the polypeptide.
  • an "isolated" polypeptide is enriched by at least about 10-fold, 100-fold, 1000-fold, 10,000-fold or more as compared with the starting material.
  • nucleic acid As used herein, “nucleic acid,” “nucleotide sequence,” and “polynucleotide” are used interchangeably and encompass both RNA and DNA, including cDNA, genomic DNA, mRNA, synthetic (e.g., chemically synthesized) DNA or RNA and chimeras of RNA and DNA.
  • polynucleotide, nucleotide sequence, or nucleic acid refers to a chain of nucleotides without regard to length of the chain.
  • the nucleic acid can be double-stranded or single-stranded. Where single-stranded, the nucleic acid can be a sense strand or an antisense strand.
  • the term "gene” refers to a nucleic acid molecule capable of being used to produce mRNA, antisense RNA, miRNA, anti-microRNA antisense oligonucleotides (AMO) and the like. Genes may or may not be capable of being used to produce a functional protein or gene product. Genes can include both coding and non-coding regions (e.g., introns, regulatory elements, promoters, enhancers, termination sequences and/or 5' and 3' untranslated regions (UTRs).
  • a gene may be "isolated” by which is meant a nucleic acid that is substantially or essentially free from components normally found in association with the nucleic acid in its natural state. Such components include other cellular material, culture medium from recombinant production, and/or various chemicals used in chemically synthesizing the nucleic acid.
  • RNA or DNA indicates that the nucleotide sequence is transcribed and, optionally, translated.
  • a nucleotide sequence may express a polypeptide of interest or a functional untranslated RNA.
  • a "functional" RNA includes any untranslated RNA that has a biological function in a cell, e.g., regulation of gene expression.
  • Such functional RNAs include but are not limited to RNAi (e.g., siRNA, shRNA, antisense RNA), miRNA, ribozymes, RNA aptamers, and the like.
  • complementarity refers to the natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing.
  • sequence "A-G-T” binds to the complementary sequence "T-C-A.”
  • Complementarity between two single-stranded molecules may be “partial,” in which only some of the nucleotides bind, or it may be complete when total complementarity exists between the single stranded molecules.
  • the degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.
  • a nucleic acid (e.g., polynucleotide, oligonucleotide, and the like) of the invention can be about 70% to about 100% complementary to a target nucleic acid (e.g., about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or any range or value therein) and therefore hybridizes to that target nucleic acid.
  • a target nucleic acid e.g., about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,
  • an oligonucleotide of the invention can be about 80 to about 100%, about 85% to about 100%, about 90% to about 100%, about 95% to about 100%, and the like, complementary to a target nucleic acid.
  • anti -microRNA oligonucleotide “anti-miRNA,” “anti-miR,” or “AMO” as used herein interchangeably refer to antisense oligonucleotides (ASOs) that target microRNAs.
  • ASOs antisense oligonucleotides
  • An ASO and/or AMO of the present invention may be of any length, and minimally comprise a complementary sequence which directs the ASO and/or AMO to its target and allows complementary binding thereto.
  • complementary binding of the ASO and/or AMO to the complementary target may promote degradation of the bound target, for example, via endogenous pathways such as RNAses (e.g., RNAse H), and/or silence functionality of the bound target via steric blocking.
  • RNAses e.g., RNAse H
  • silence functionality of the bound target via steric blocking.
  • ASOs, AMOs, their design and their use are further described in Lima et al., 2018 RNA Biology 15(3):338-352, incorporated herein by reference.
  • an “isolated” nucleic acid or nucleotide sequence e.g., an “isolated DNA” or an “isolated RNA” means a nucleic acid or nucleotide sequence separated or substantially free from at least some of the other components of the naturally occurring organism or virus, for example, the cell or viral structural components or other polypeptides or nucleic acids commonly found associated with the nucleic acid or nucleotide sequence.
  • an "isolated" polypeptide means a polypeptide that is separated or substantially free from at least some of the other components of the naturally occurring organism or virus, for example, the cell or viral structural components or other polypeptides or nucleic acids commonly found associated with the polypeptide.
  • codon-optimized refers to a gene coding sequence that has been optimized to increase expression by substituting one or more codons normally present in a coding sequence (for example, in a wild-type sequence, including, e.g., a coding sequence for FIG4) with a codon for the same (synonymous) amino acid.
  • the protein encoded by the gene is identical, but the underlying nucleobase sequence of the gene or corresponding mRNA is different.
  • the optimization substitutes one or more rare codons (that is, codons for tRNA that occur relatively infrequently in cells from a particular species) with synonymous codons that occur more frequently to improve the efficiency of translation.
  • Codon optimization can also increase gene expression through other mechanisms that can improve efficiency of transcription and/or translation.
  • Strategies include, without limitation, increasing total GC content (that is, the percent of guanines and cytosines in the entire coding sequence), decreasing CpG content (that is, the number of CG or GC dinucleotides in the coding sequence), removing cryptic splice donor or acceptor sites, and/or adding or removing ribosomal entry sites, such as Kozak sequences.
  • a codon-optimized gene exhibits improved protein expression, for example, the protein encoded thereby is expressed at a detectably greater level in a cell compared with the level of expression of the protein provided by the wild-type gene in an otherwise similar cell.
  • sequence identity has the standard meaning in the art. As is known in the art, a number of different programs can be used to identify whether a polynucleotide or polypeptide has sequence identity or similarity to a known sequence. Sequence identity or similarity may be determined using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the sequence identity alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 45:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Natl. Acad. Sci.
  • PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. Mol. Evol. 35:351 (1987); the method is similar to that described by Higgins & Sharp, CABIOS 5: 151 (1989).
  • BLAST BLAST algorithm
  • WU-BLAST-2 WU-BLAST-2 uses several search parameters, which are preferably set to the default values. The parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity.
  • percent nucleic acid sequence identity is defined as the percentage of nucleotide residues in the candidate sequence that are identical with the nucleotides in the polynucleotide specifically disclosed herein.
  • the alignment may include the introduction of gaps in the sequences to be aligned.
  • the percentage of sequence identity will be determined based on the number of identical nucleotides in relation to the total number of nucleotides.
  • sequence identity of sequences shorter than a sequence specifically disclosed herein will be determined using the number of nucleotides in the shorter sequence, in one embodiment.
  • percent identity calculations relative weight is not assigned to various manifestations of sequence variation, such as insertions, deletions, substitutions, etc.
  • identities are scored positively (+1) and all forms of sequence variation including gaps are assigned a value of "0," which obviates the need for a weighted scale or parameters as described below for sequence similarity calculations.
  • Percent sequence identity can be calculated, for example, by dividing the number of matching identical residues by the total number of residues of the "shorter" sequence in the aligned region and multiplying by 100. The "longer" sequence is the one having the most actual residues in the aligned region.
  • an "isolated cell” refers to a cell that is separated from other components with which it is normally associated in its natural state.
  • an isolated cell can be a cell in culture medium and/or a cell in a pharmaceutically acceptable carrier of this invention.
  • an isolated cell can be delivered to and/or introduced into a subject.
  • an isolated cell can be a cell that is removed from a subject and manipulated as described herein ex vivo and then returned to the subject.
  • virus vector or virus particle or population of virus particles As used herein, by “isolate” or “purify” (or grammatical equivalents) a virus vector or virus particle or population of virus particles, it is meant that the virus vector or virus particle or population of virus particles is at least partially separated from at least some of the other components in the starting material. In representative embodiments an “isolated” or “purified” virus vector or virus particle or population of virus particles is enriched by at least about 10-fold, 100-fold, 1000-fold, 10,000-fold or more as compared with the starting material.
  • endogenous refers to a component naturally found in an environment, i.e., a gene, nucleic acid, miRNA, protein, cell, or other natural component expressed in the subject, as distinguished from an introduced component, i.e., an "exogenous” component.
  • heterologous refers to a nucleotide/polypeptide that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention.
  • heterologous nucleotide sequence or “heterologous nucleic acid” is a sequence that is not naturally occurring in the virus.
  • the heterologous nucleic acid or nucleotide sequence comprises an open reading frame that encodes a polypeptide and/or a nontranslated RNA.
  • a “therapeutic polypeptide” is a polypeptide that can alleviate, reduce, prevent, delay and/or stabilize symptoms that result from an absence or defect in a protein in a cell or subject and/or is a polypeptide that otherwise confers a benefit to a subject, e.g., anti-cancer effects or improvement in transplant survivability or induction of an immune response.
  • treat By the terms “treat,” “treating” or “treatment of' (and grammatical variations thereof) it is meant that the severity of the subject's condition is reduced, at least partially improved or stabilized and/or that some alleviation, mitigation, decrease or stabilization in at least one clinical symptom is achieved and/or there is a delay in the progression of the disease or disorder.
  • substantially retain a property and/or to maintain a property “substantially the same” as a comparison (e.g., a control), it is meant that at least about 75%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the property (e.g., activity or other measurable characteristic) is retained.
  • prevent refers to prevention and/or delay of the onset of a disease, disorder and/or a clinical symptom(s) in a subject and/or a reduction in the severity of the onset of the disease, disorder and/or clinical symptom(s) relative to what would occur in the absence of the methods of the invention.
  • the prevention can be complete, e.g., the total absence of the disease, disorder and/or clinical symptom(s).
  • the prevention can also be partial, such that the occurrence of the disease, disorder and/or clinical symptom(s) in the subject and/or the severity of onset are substantially less than what would occur in the absence of the present invention.
  • a “treatment effective” or “effective” amount as used herein is an amount that is sufficient to provide some improvement or benefit to the subject.
  • a “treatment effective” or “effective” amount is an amount that will provide some alleviation, mitigation, decrease or stabilization in at least one clinical symptom in the subject.
  • prevention effective amount is an amount that is sufficient to prevent and/or delay the onset of a disease, disorder and/or clinical symptoms in a subject and/or to reduce and/or delay the severity of the onset of a disease, disorder and/or clinical symptoms in a subject relative to what would occur in the absence of the methods of the invention.
  • level of prevention need not be complete, as long as some preventative benefit is provided to the subject.
  • nucleotide sequence of interest (NOI)," “heterologous nucleotide sequence” and “heterologous nucleic acid molecule” are used interchangeably herein and refer to a nucleic acid sequence that is not naturally occurring (e.g., engineered).
  • NOI nucleotide sequence of interest
  • heterologous nucleic acid molecule or heterologous nucleotide sequence comprises an open reading frame that encodes a polypeptide and/or nontranslated RNA of interest (e.g., for delivery to a cell and/or subject).
  • modified refers to a sequence that differs from a wild-type sequence due to one or more deletions, additions, substitutions, or any combination thereof.
  • virus vector can refer to a virus (e.g., AAV) particle that functions as a nucleic acid delivery vehicle, and which comprises a viral genome (e.g., viral DNA [vDNA]) and/or replicon nucleic acid molecule packaged within a virus particle.
  • virus e.g., AAV
  • vDNA viral DNA
  • vector may be used to refer to the vector genome/vDNA alone.
  • the term "vector,” as used herein, can also mean any nucleic acid entity capable of amplification in a host cell.
  • the vector may be an autonomously replicating vector, /. ⁇ ., a vector, which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid.
  • the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated. The choice of vector will often depend on the host cell into which it is to be introduced.
  • Vectors include, but are not limited to plasmid vectors, phage vectors, viruses or cosmid vectors. Vectors usually contain a replication origin and at least one selectable gene, i.e., a gene which encodes a product which is readily detectable or the presence of which is essential for cell growth
  • a "rAAV vector genome” or “rAAV genome” is an AAV genome i.e., vDNA) that comprises at least one terminal repeat (e.g., two terminal repeats) and one or more heterologous nucleotide sequences.
  • rAAV vectors generally require only the 145 base terminal repeat(s) (TR(s)) in cis to generate virus. All other viral sequences are dispensable and may be supplied in trans (Muzyczka, (1992) Curr. Topics Microbiol. Immunol. 158:97).
  • the rAAV vector genome will only retain the minimal TR sequence(s) so as to maximize the size of the transgene that can be efficiently packaged by the vector.
  • the structural and non- structural protein coding sequences may be provided in trans (e.g., from a vector, such as a plasmid, or by stably integrating the sequences into a packaging cell).
  • the rAAV vector genome optionally comprises two AAV TRs, which generally will be at the 5' and 3' ends of the heterologous nucleotide sequence(s), but need not be contiguous thereto.
  • the TRs can be the same or different from each other.
  • a "rAAV particle” comprises a rAAV vector genome packaged within an AAV capsid.
  • terminal repeat or "TR” or “inverted terminal repeat (ITR)” includes any viral terminal repeat or synthetic sequence that forms a hairpin structure and functions as an inverted terminal repeat (i.e., mediates the desired functions such as replication, virus packaging, integration and/or provirus rescue, and the like).
  • the TR can be an AAV TR or a non- AAV TR.
  • a non- AAV TR sequence such as those of other parvoviruses (e.g., canine parvovirus (CPV), mouse parvovirus (MVM), human parvovirus B-19) or any other suitable virus sequence (e.g., the SV40 hairpin that serves as the origin of SV40 replication) can be used as a TR, which can further be modified by truncation, substitution, deletion, insertion and/or addition.
  • the TR can be partially or completely synthetic, such as the "double-D sequence" as described in United States Patent No. 5,478,745 to Samulski et al., which is hereby incorporated by reference in its entirety.
  • An "AAV terminal repeat” or “AAV TR” may be from any AAV, including but not limited to serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or any other AAV now known or later discovered (see, e.g., Table 1).
  • An AAV terminal repeat need not have the native terminal repeat sequence (e.g., a native AAV TR sequence may be altered by insertion, deletion, truncation and/or missense mutations), as long as the terminal repeat mediates the desired functions, e.g., replication, virus packaging, integration, and/or provirus rescue, and the like.
  • the virus vectors of the invention can further be "targeted” virus vectors (e.g., having a directed tropism) and/or a "hybrid” parvovirus (i.e., in which the viral TRs and viral capsid are from different parvoviruses) as described in international patent publication WO 00/28004 and Chao et al., (2000) Molecular Therapy 2:619, which is hereby incorporated by reference in its entirety.
  • targeted virus vectors e.g., having a directed tropism
  • a “hybrid” parvovirus i.e., in which the viral TRs and viral capsid are from different parvoviruses
  • the virus vectors of the invention can further be duplexed parvovirus particles as described in international patent publication WO 01/92551 (the disclosure of which is incorporated herein by reference in its entirety).
  • double stranded (duplex) genomes can be packaged into the virus capsids of the invention.
  • viral capsid or genomic elements can contain other modifications, including insertions, deletions and/or substitutions.
  • a “chimeric” capsid protein and/or “chimeric” or “modified” capsid as used herein means an AAV capsid protein or capsid that has been modified by substitutions in one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid residues in the amino acid sequence of a capsid protein relative to wild type, as well as insertions and/or deletions of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid residues in the amino acid sequence relative to wild type.
  • complete or partial domains, functional regions, epitopes, etc., from one AAV serotype can replace the corresponding wildtype domain, functional region, epitope, etc.
  • a chimeric capsid protein or modified capsid of this invention Production of a chimeric capsid protein or modified capsid can be carried out according to protocols well known in the art and a large number of chimeric capsid proteins are described in the literature as well as herein that can be included in the capsid of this invention.
  • amino acid or “amino acid residue” encompasses any naturally occurring amino acid, modified forms thereof, and synthetic amino acids.
  • a conservative amino acid substitution includes substitutions within one or more of the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and/or phenylalanine, tyrosine.
  • the amino acid can be a modified amino acid residue (nonlimiting examples are shown in Table 3) and/or can be an amino acid that is modified by post- translation modification (e.g., acetylation, amidation, formylation, hydroxylation, methylation, phosphorylation or sulfatation).
  • post- translation modification e.g., acetylation, amidation, formylation, hydroxylation, methylation, phosphorylation or sulfatation.
  • non-naturally occurring amino acid can be an "unnatural" amino acid as described by Wang et al., Annu Rev Biophys Biomol Struct. 35 :225-49 (2006)). These unnatural amino acids can advantageously be used to chemically link molecules of interest to the AAV capsid protein.
  • binding site refers to any general structural feature that acts as a location for binding between components.
  • binding site can refer to, though is not limited to, a nucleotide sequence in a specific motif of primary, secondary, or tertiary structure wherein that motif provides a binding location for an interacting molecule, which may comprise other nucleic acids or proteins.
  • binding site can refer to, though is not limited to, a sequence of amino acids in a specific motif of primary, secondary, tertiary or quaternary structure wherein that motif provides a binding location for an interacting molecule, which may comprise other nucleic acids or proteins.
  • seed match specifically refers to a subset of nucleotides within a longer endogenous mRNA sequence empirically identified, validated, or putatively predicted to be the relevant target nucleotide sequence for recognition by, and complementary binding of, a microRNA (miR) species to the corresponding mRNA containing said seed match.
  • miR microRNA
  • seed match refers to a subset of nucleotides within the longer endogenous miRNA sequence empirically identified, validated, or putatively predicted to be the relevant nucleotide sequence for recognition of, and complementary binding to, a target seed match of an mRNA species by that miRNA species.
  • the seed match of an mRNA is encoded within its respective 3 prime (3') untranslated region (3' UTR), but may be present in other locations.
  • a “validated” or “empirically identified” seed match is defined as a seed match currently known in the art and those identified in the future.
  • a “putative” or “predicted” seed match is defined as a seed match not yet empirically known or defined.
  • compositions of the invention are provided.
  • the present invention is based, in part, on the unexpected discovery that correcting dysregulation of microRNA (miR) miR-1 via anti-miR- 1/206 and/or over-expressing exogenous thymosin beta 4 (Tp4) in the central nervous system (CNS) provided novel therapeutic methods for treatment of amyotrophic lateral sclerosis (ALS).
  • miR microRNA
  • Tp4 exogenous thymosin beta 4
  • MicroRNAs are short non-coding sequences that cleave or inhibit messenger RNAs (mRNAs) by targeting their 3 '-untranslated region (3 '-UTR), and play important roles in the development and progression of diseases such as ALS (Haramati et al. 2010 PNAS 107: 13111-13116; Morel et al. 2013 J Biol Chem 288:7105-7116; Campos-Melo et al. 2013 Mol Brain 6:26; Koval et al. 2013 Hum Mol Genet 22:4127-4135; Goodall et al. 2013 Frontiers Cell Neuro 7: 178; Kye and Goncalves, 2014 Frontiers Cell Neuro 8: 15; Cunha et al. 2017 Mol Neuro 55:4207-7224.
  • ALS Garamati et al. 2010 PNAS 107: 13111-13116; Morel et al. 2013 J Biol Chem 288:7105-7116; Campos-Melo et al. 2013 Mol Brain 6:
  • Thymosin beta 4 is a peptide of 43 amino acids, originally isolated from thymic extract and highly expressed in heart and CNS.
  • the peptide has many roles including angiogenesis (Smart et al. 2007 Nature 445: 177-182), anti-apoptosis (Kumar and Gupta, 2011 PLoS One 6:e26912), anti-inflammation (Zhang et al. 2016 Cell Physiol Biochem 38:2230-2238) and neuroprotection (Zhang et al. 2017 J Neurosurg 126:782-795). These protective functions are lacking or weak in ALS patients or ALS animal models.
  • the present invention is based, in part, on the discovery that protein levels of Tp4 are down-regulated in the ALS mouse model, especially in the neuronal cells of anterior horn. While not wishing to be bound to theory, deficiency of Tp4 in the spinal cord may decrease angiogenesis and increase neuronal apoptosis, and may further deteriorate functionality in the development of ALS.
  • the inventors of the present invention discovered that, in a mouse model of ALS, miR-1 but not miR-206 was continually upregulated in the spinal cord, and that enhanced miR-1 targeted the 3 '-untranslated region (3'-UTR) on the mRNA of Tp4, and thereby negatively regulates Tp4 expression, leading to progression of ALS. Conversely, downregulation of miR-1 with the introduction of anti-microRNA oligonucleotides (also known as anti-miRs or AMOs) and/or exogenous Tp4 expression, prevented the death of motor neurons and improved skeletal muscle function, as well as extended survival of ALS mice.
  • anti-microRNA oligonucleotides also known as anti-miRs or AMOs
  • one aspect of the present invention provides a synthetic polynucleotide encoding one or more anti-miRNA oligonucleotide (AMO) directed to microRNA-1 (miR-1) and/or miR-206.
  • AMO anti-miRNA oligonucleotide
  • the AMO may be of any length of nucleotides which retains the functionality of binding to the directed target, e.g., miR-1 and/or miR-206.
  • the AMO may be about 15 to about 30 nucleotides, e.g., about 115, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides or any value or range therein.
  • the AMO may be about 20 to about 25 nucleotides, about 15 to about 22 nucleotides, about 18 to about 30 nucleotides, or about 20 nucleotides, about 22 nucleotides, or about 25 nucleotides.
  • a synthetic polynucleotide of the present invention may comprise any number of copies of an AMO, for example such as needed to have a therapeutically effective dose of said polynucleotide in suppressing the directed target thereof, e.g., miR-1 and/or miR-206.
  • a synthetic polynucleotide of the present invention may comprise one or more AMO, e.g., about one, two, three, four, five, six, seven, eight, nine, or ten or more AMO, or any value or range therein, e.g., about one to about four AMO, about two to about six AMO, about one to about 10 AMO, etc.
  • An AMO of the present invention may be directed to any nucleotide sequence of miR- 1 and/or miR-2.
  • the AMO of the present invention may bind to a shared sequence of miR-1 and miR-206 such as but not limited to, the shared seed sequence of miR-1 and miR-206 targeting the mRNA encoding thymosin beta 4 (Tp4).
  • the AMO may bind to a portion of miR-1 and/or miR-260 outside of the shared seed sequence of miR-1 and miR-206 targeting the mRNA encoding thymosin beta 4 (Tp4).
  • the seed sequence may comprise, consist essentially of, or consist of a nucleotide sequence which binds to SEQ ID NO: 1 or a nucleotide sequence having at least about 70% (e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identity thereto.
  • 70% e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%
  • the seed sequence may comprise, consist essentially of, or consist of the nucleotide sequence of SEQ ID NO:2 or a nucleotide sequence having at least about 70% (e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identity thereto.
  • 70% e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%
  • an AMO of the present invention may comprise, consist essentially of, or consist of the nucleotide sequence of SEQ ID NO:3 or a nucleotide sequence having at least about 70% (e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identity thereto.
  • 70% e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%
  • SEQ ID NO:3 AMO anti-miRNA-miR-1-2
  • an AMO of the present invention may comprise, consist essentially of, or consist of the nucleotide sequence of SEQ ID NO:4 or a nucleotide sequence having at least about 70% (e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identity thereto.
  • 70% e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%
  • a polynucleotide of the present invention may comprise, consist essentially of, or consist of the nucleotide sequence of SEQ ID NO:5 or a nucleotide sequence having at least about 70% (e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identity thereto.
  • 70% e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%
  • a polynucleotide of the present invention may comprise, consist essentially of, or consist of the nucleotide sequence of SEQ ID NO:6 or a nucleotide sequence having at least about 70% (e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identity thereto.
  • 70% e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%
  • Another aspect of the invention relates to an expression cassette comprising a synthetic polynucleotide of the present invention, e.g., a synthetic polynucleotide encoding one or more copies of an AMO directed to miR-1 and/or miR-206.
  • the polynucleotide may be operably linked to one or more expression elements that may enhance expression of the polynucleotide products, e.g., the AMO.
  • the polynucleotide is operably linked to a promoter and/or an enhancer, e.g., a cytomegalovirus (CMV) promoter and/or a neuron-specific (e.g., synapsin (e.g., syn-1)) promoter.
  • CMV cytomegalovirus
  • a neuron-specific e.g., synapsin (e.g., syn-1)
  • the polynucleotide may be further linked to one or more molecular tags, such as to indicate gene expression.
  • Example molecular tags of the present invention include, but are not limited to, GFP.
  • a synthetic polynucleotide of the present invention may comprise, consist essentially of, or consist of, e.g., one or more copies of an AMO directed to miR-1 (i.e., an anti-miR-1 AMO) and/or miR-206 (i.e., an anti-miR-206 AMO), a promoter, and/or a molecular tag.
  • a synthetic polynucleotide of the present invention may comprise, consist essentially of, or consist of, e.g., four or more copies of an AMO directed to miR-1 (i.e., an anti-miR-1 AMO) and/or miR-206 (i.e., an anti-miR-206 AMO), a promoter, and/or a molecular tag.
  • a synthetic polynucleotide of the present invention may comprise, consist essentially of, or consist of, e.g., four or more copies of an AMO directed to miR-1 (i.e., an anti-miR-1 AMO) and/or miR-206 (i.e., an anti- miR-206 AMO), a CMV promoter, and/or a molecular tag.
  • a synthetic polynucleotide of the present invention may comprise, consist essentially of, or consist of, e.g., four or more copies of an AMO directed to miR-1 (i.e., an anti-miR-1 AMO) and/or miR-206 (i.e., an anti-miR-206 AMO), a CMV promoter, and/or a GFP molecular tag.
  • an expression cassette comprising a synthetic polynucleotide comprising a coding region encoding a thymosin P4 (Tp4) gene product, e.g., an encoded human TP4 comprising, consisting essentially of, or consisting of SEQ ID NO:7 or a sequence at least about 70% (e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identity thereto.
  • a coding region encoding a Tp4 gene product of the present invention may be codon-optimized, e.g., a codon-optimized coding region.
  • the polynucleotide may be operably linked to one or more expression elements that may enhance expression of the polynucleotide products, e.g., the encoded Tp4.
  • the polynucleotide is operably linked to a promoter and/or an enhancer, e.g., a cytomegalovirus (CMV) promoter or a neuron-specific (e.g., synapsin (e.g., syn-1)) promoter.
  • CMV cytomegalovirus
  • synapsin e.g., syn-1
  • the polynucleotide may be further linked to one or more molecular tags (e.g., GFP), such as to indicate gene expression.
  • GFP molecular tags
  • promoter/enhancer elements may be used depending on the level and tissue-specific expression desired.
  • the promoter/enhancer may be constitutive or inducible, depending on the pattern of expression desired.
  • the promoter/enhancer may be native or foreign and can be a natural or a synthetic sequence. By foreign, it is intended that the transcriptional initiation region is not found in the wildtype host into which the transcriptional initiation region is introduced.
  • Promoter/enhancer elements can be native to the target cell or subject to be treated and/or native to the heterologous nucleic acid sequence.
  • the promoter/enhancer element is generally chosen so that it will function in the target cell(s) of interest.
  • the promoter/enhancer element is a viral promoter/enhancer element.
  • the promoter/enhance element may be constitutive or inducible.
  • Inducible expression control elements are generally used in those applications in which it is desirable to provide regulation over expression of the heterologous synthetic polynucleotides.
  • Inducible promoters/enhancer elements for gene delivery can be tissuespecific or tissue-preferred promoter/enhancer elements, and include muscle specific or preferred (including cardiac, skeletal and/or smooth muscle), neural tissue specific or preferred (including brain-specific), eye (including retina-specific and cornea-specific), liver specific or preferred, bone marrow specific or preferred, pancreatic specific or preferred, spleen specific or preferred, and lung specific or preferred promoter/enhancer elements.
  • Other inducible promoter/enhancer elements include hormone-inducible and metal-inducible elements.
  • Exemplary inducible promoters/enhancer elements include, but are not limited to, a Tet on/off element, a RU486-inducible promoter, an ecdysone-inducible promoter, a rapamycin-inducible promoter, and a metallothionein promoter.
  • specific initiation signals may be employed for efficient translation of inserted coding sequences.
  • exogenous translational control sequences which may include the ATG initiation codon and adjacent sequences, can be of a variety of origins, both natural and synthetic.
  • a further aspect of the invention relates to a vector comprising the polynucleotide or the expression cassette of the invention.
  • Suitable vectors include, but are not limited to, a plasmid (e.g., a pEMBL expression plasmid), phage, viral vector (e.g., an AAV vector, an adenovirus vector, a herpesvirus vector, an alphavirus vector, or a baculovirus vector), bacterial artificial chromosome (BAC), or yeast artificial chromosome (YAC).
  • the nucleic acid can comprise, consist of, or consist essentially of an AAV vector comprising a 5' and/or 3' terminal repeat (e.g., 5' and/or 3' AAV terminal repeat).
  • the vector is a delivery vehicle such as a particle (e.g., a microparticle or nanoparticle) or a liposome to which the expression cassette is attached or in which the expression cassette is embedded.
  • the vector may be any delivery vehicle suitable to carry the expression cassette into a cell.
  • the vector is a viral vector, e.g., an AAV vector.
  • the AAV vector may be any AAV serotype, e.g., AAV2, e.g., AAV9.
  • the AAV vector may comprise wildtype capsid proteins.
  • the AAV vector may comprise a modified capsid protein with altered tropism compared to a wildtype capsid protein, e.g., a modified capsid protein is targeted and/or detargeted for a particular tissue, and/or has enhanced tropism for particular cells.
  • the vector is a single-stranded AAV (ssAAV) vector.
  • the vector is a self-complementary or duplexed AAV (scAAV) vector.
  • scAAV vectors are described in international patent publication WO 01/92551 (the disclosure of which is incorporated herein by reference in its entirety). Use of scAAV to express the coding regions of the present invention may provide an increase in the number of cells transduced, the copy number per transduced cell, or both.
  • a vector of the present invention may comprise, consist essentially of, or consist of the nucleotide sequence of SEQ ID NO:8 or a nucleotide sequence having at least about 70% (e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identity thereto.
  • 70% e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%
  • An additional aspect of the invention relates to a transformed cell comprising the polynucleotide, expression cassette, and/or vector of the invention.
  • the polynucleotide, expression cassette, and/or vector is stably incorporated into the cell genome.
  • the cell may be an in vitro, ex vivo, or in vivo cell.
  • transgenic animal comprising the polynucleotide, expression cassette, vector, and/or the transformed cell of the invention.
  • the animal is a laboratory animal, e.g., a mouse, rat, rabbit, dog, monkey, or non-human primate.
  • a further aspect of the invention relates to a pharmaceutical formulation comprising the polynucleotide, expression cassette, vector, and/or transformed cell of the invention in a pharmaceutically acceptable carrier.
  • the present invention provides a pharmaceutical composition comprising a polynucleotide, expression cassette, vector, and/or transformed cell of the invention in a pharmaceutically acceptable carrier and, optionally, other medicinal agents, pharmaceutical agents, stabilizing agents, buffers, carriers, adjuvants, diluents, etc.
  • the carrier will typically be a liquid.
  • the carrier may be either solid or liquid.
  • the carrier will be respirable, and will preferably be in solid or liquid particulate form.
  • pharmaceutically acceptable it is meant a material that is not toxic or otherwise undesirable, /. ⁇ ., the material may be administered to a subject without causing any undesirable biological effects.
  • the polynucleotide, expression cassette, vector, and/or transformed cell of the invention is isolated.
  • the polynucleotide, expression cassette, vector, and/or transformed cell of the invention is purified.
  • the present invention also relates to methods for delivering a synthetic polynucleotide, expression cassette, vector, composition, and/or transformed cell to a cell or a subject to produce the encoded products thereof, e.g., for therapeutic or research purposes in vitro, ex vivo, or in vivo.
  • one aspect of the invention relates to a method of reducing miR-1 expression in a cell, comprising contacting the cell with a polynucleotide, expression cassette, and/or vector of the present invention.
  • Another aspect of the present invention relates to a method of enhancing thymosin beta 4 (TP4) protein expression in a cell, comprising contacting the cell with a polynucleotide, expression cassette, and/or vector of the present invention.
  • TP4 thymosin beta 4
  • the cell(s) into which the polynucleotide, expression cassette, and/or vector of the invention, e.g., virus vector and/or plasmid expression vector, can be introduced may be of any type, including but not limited to neural cells (including cells of the peripheral and central nervous systems, in particular, brain cells such as neurons, oligodendrocytes, glial cells, astrocytes), lung cells, cells of the eye (including retinal cells, retinal pigment epithelium, and corneal cells), epithelial cells (e.g., gut and respiratory epithelial cells), skeletal muscle cells (including myoblasts, myotubes and myofibers), diaphragm muscle cells, dendritic cells, pancreatic cells (including islet cells), hepatic cells, a cell of the gastrointestinal tract (including smooth muscle cells, epithelial cells), heart cells (including cardiomyocytes), bone cells (e.g., bone marrow stem cells), hematopoietic stem cells, sple
  • the cell may be any progenitor cell.
  • the cell can be a stem cell (e.g., neural stem cell, liver stem cell).
  • the cell may be a cancer or tumor cell.
  • the cells can be from any species of origin, as indicated above.
  • the polynucleotide, expression cassette, and/or vector of the invention may be introduced to cells in vitro for the purpose of administering the modified cell to a subject.
  • the cells have been removed from a subject, the polynucleotide, expression cassette, and/or vector of the invention, e.g., virus vector and/or plasmid expression vector, is introduced therein, and the cells are then replaced back into the subject.
  • Methods of removing cells from subject for treatment ex vivo, followed by introduction back into the subject are known in the art (see, e.g., U.S. patent No. 5,399,346).
  • the polynucleotide, expression cassette, and/or vector of the invention e.g., virus vector and/or plasmid expression vector
  • the polynucleotide, expression cassette, and/or vector of the invention is introduced into cells from another subject, into cultured cells, or into cells from any other suitable source, and the cells are administered to a subject in need thereof.
  • Suitable cells for ex vivo gene therapy are as described above. Dosages of the cells to administer to a subject will vary upon the age, condition and species of the subject, the type of cell, the nucleic acid being expressed by the cell, the mode of administration, and the like. Typically, at least about 10 2 to about 10 8 or about 10 3 to about 10 6 cells will be administered per dose in a pharmaceutically acceptable carrier. In particular embodiments, the cells transduced with the virus vector ex vivo are administered to the subject in an effective amount in combination with a pharmaceutical carrier.
  • a further aspect of the invention is a method of administering the polynucleotide, expression cassette, vector (e.g., virus vector and/or plasmid vector), and/or composition (e.g., pharmaceutical composition) of the invention to a subject.
  • the method comprises a method of delivering a polynucleotide, expression cassette, vector (e.g., virus vector and/or plasmid vector), and/or composition (e.g., pharmaceutical composition) of the invention to an animal subject, the method comprising: administering an effective amount of a virus vector and/or plasmid expression vector according to the invention to an animal subject.
  • Administration of the vectors of the present invention to a human subject or an animal in need thereof can be by any means known in the art.
  • the vector is delivered in an effective dose in a pharmaceutically acceptable carrier.
  • Another aspect of the present invention relates to a method of reducing miR-1 expression in a subject, comprising delivering to the subject a polynucleotide, expression cassette, vector, transformed cell, and/or composition (e.g., pharmaceutical composition) of the present invention.
  • Another aspect of the present invention relates to a method of enhancing thymosin beta 4 (Tp4) protein expression in a subject, comprising delivering to the subject a polynucleotide, expression cassette, vector, transformed cell, and/or composition (e.g., pharmaceutical composition) of the present invention.
  • Tp4 thymosin beta 4
  • Another aspect of the present invention relates to a method of treating a disorder associated with aberrant overexpression and/or activity of miR-1 or aberrant activity of a miR-1 regulated gene in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polynucleotide, expression cassette, vector, transformed cell, and/or composition (e.g., pharmaceutical composition) of the present invention.
  • a polynucleotide, expression cassette, vector, transformed cell, and/or composition e.g., pharmaceutical composition
  • the miR-1 regulated gene may be any gene or gene product known to be regulated to miR-1 or later discovered.
  • the miR-1 regulated gene may be Tp4.
  • a disorder of the present invention may be amyotrophic lateral sclerosis (ALS).
  • ALS amyotrophic lateral sclerosis
  • the disorder may be familial ALS.
  • the disorder may be sporadic ALS.
  • another aspect of the present invention relates to a method of treating a disorder associated with aberrant overexpression and/or activity of thymosin beta 4 (Tp4) gene and/or a thymosin beta 4 (Tp4) gene product (e.g., Tp4 protein) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polynucleotide, expression cassette, vector, transformed cell, and/or composition (e.g., pharmaceutical composition) of the present invention.
  • Tp4 gene product e.g., Tp4 protein
  • Another aspect of the present invention relates to a method of treating ALS (e.g., familial or sporadic ALS) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polynucleotide, expression cassette, vector, transformed cell, and/or composition (e.g., pharmaceutical composition) of the present invention.
  • ALS e.g., familial or sporadic ALS
  • composition e.g., pharmaceutical composition
  • Another aspect of the present invention relates to a method of treating sporadic ALS in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polynucleotide, expression cassette, vector, transformed cell, and/or composition (e.g., pharmaceutical composition) of the present invention.
  • Another aspect of the present invention relates to a method of treating familial ALS in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polynucleotide, expression cassette, vector, transformed cell, and/or composition (e.g., pharmaceutical composition) of the present invention.
  • Another aspect of the present invention relates to a method of treating ALS (e.g., familial or sporadic ALS) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of: (a) a synthetic polynucleotide encoding one or more copies of an AMO directed to miR-1 and/or miR-206, and/or an expression cassette, vector, transformed cell, and/or pharmaceutical composition comprising the same (e.g., a synthetic polynucleotide, expression cassette, vector, transformed cell, and/or pharmaceutical composition of the present invention); and/or (b) a synthetic polynucleotide comprising a coding region encoding a Tp4 (e.g., a human Tp4) and/or an expression cassette, vector, transformed cell, and/or pharmaceutical composition comprising the same.
  • a synthetic polynucleotide encoding one or more copies of an AMO directed to miR-1 and/or miR-206, and/or an expression
  • the encoded Tp4 may comprise SEQ ID NO:7 or a sequence at least about 70% (e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identity thereto.
  • the encoded Tp4 of the present invention may be codon-optimized, e.g., a codon-optimized coding region.
  • Another aspect of the present invention relates to a method of postponing disease progression of ALS (e.g., familial or sporadic ALS) in a subject having ALS or a subject at risk for or suspected to have or develop ALS comprising administering to the subject a therapeutically effective amount of: (a) a synthetic polynucleotide encoding one or more copies of an AMO directed to miR-1 and/or miR-206, and/or an expression cassette, vector, transformed cell, and/or pharmaceutical composition comprising the same (e.g., a synthetic polynucleotide, expression cassette, vector, transformed cell, and/or pharmaceutical composition of the present invention); and/or (b) a synthetic polynucleotide comprising a coding region encoding a Tp4 (e.g., a human Tp4) and/or an expression cassette, vector, transformed cell, and/or pharmaceutical composition comprising the same.
  • a synthetic polynucleotide encoding one or more copies of an A
  • the encoded Tp4 may comprise SEQ ID NO:7 or a sequence at least about 70% (e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identity thereto.
  • the encoded Tp4 of the present invention may be codon-optimized, e.g., a codon-optimized coding region.
  • Another aspect of the present invention relates to a method of reducing disease severity of ALS (e.g., familial or sporadic ALS) in a subject having ALS or a subject at risk for or suspected to have or develop ALS, comprising administering to the subject a therapeutically effective amount of: (a) a synthetic polynucleotide encoding one or more copies of an AMO directed to miR-1 and/or miR-206, and/or an expression cassette, vector, transformed cell, and/or pharmaceutical composition comprising the same (e.g., a synthetic polynucleotide, expression cassette, vector, transformed cell, and/or pharmaceutical composition of the present invention); and/or (b) a synthetic polynucleotide comprising a coding region encoding a Tp4 (e.g., a human Tp4) and/or an expression cassette, vector, transformed cell, and/or pharmaceutical composition comprising the same.
  • a synthetic polynucleotide encoding one or more copies of an A
  • the encoded Tp4 may comprise SEQ ID NO:7 or a sequence at least about 70% (e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identity thereto.
  • the encoded Tp4 of the present invention may be codon-optimized, e.g., a codon-optimized coding region.
  • compositions and methods of the present invention find use in both veterinary and medical applications. Suitable subjects include both avians and mammals.
  • avian as used herein includes, but is not limited to, chickens, ducks, geese, quail, turkeys, pheasant, parrots, parakeets.
  • mammal as used herein includes, but is not limited to, humans, primates, non-human primates (e.g., monkeys and baboons), cattle, sheep, goats, pigs, horses, cats, dogs, rabbits, rodents (e.g., rats, mice, hamsters, and the like), etc.
  • Human subjects include neonates, infants, juveniles, and adults.
  • the subject is "in need of the methods of the present invention, e.g., because the subject has or is believed at risk for a disorder including those described herein or that would benefit from the delivery of a polynucleotide and/or expression vector including those described herein.
  • the subject can be a laboratory animal and/or an animal model of disease.
  • the subject is a human.
  • the subject may exhibit symptoms of disease (e.g., ALS) prior to delivery of the polynucleotide, expression cassette, vector, transformed cell, and/or pharmaceutical composition.
  • symptoms of disease e.g., ALS
  • the subject may be pre-symptomatic (e.g., does not exhibit symptoms of the disease) prior to delivery of the polynucleotide, expression cassette, vector, transformed cell, and/or pharmaceutical composition.
  • the polynucleotide, expression cassette, vector, transformed cell, and/or composition (e.g., pharmaceutical composition) is delivered to the subject, e.g., systemically (e.g., intravenously) or directly to the central nervous system (e.g., to the cerebrospinal fluid by intrathecal or intraventricular injection) of the subject.
  • the polynucleotide, expression cassette, vector, transformed cell, and/or composition is delivered intravenously.
  • the polynucleotide, expression cassette, vector, transformed cell, and/or composition is delivered by intrathecal, intracerebral, intraparenchymal, intracerebroventricular, intranasal, intra-aural, intra-ocular, or peri-ocular delivery, or any combination thereof.
  • the polynucleotide, expression cassette, vector, transformed cell, and/or composition is delivered intrathecally.
  • Dosages of the vectors to be administered to a subject will depend upon the mode of administration, the disease or condition to be treated, the individual subject's condition, the particular vector, and the polynucleotide to be delivered, and can be determined in a routine manner.
  • Exemplary doses for achieving therapeutic effects are virus titers of at least about 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 16 transducing units or more, e.g., about 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , or 10 15 transducing units, yet more preferably about 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , or 10 15 transducing units.
  • Doses and virus titer transducing units may be calculated as vector or viral genomes (vg).
  • more than one administration may be employed to achieve the desired level of polynucleotide product expression over a period of various intervals, e.g., daily, weekly, monthly, yearly, etc.
  • Exemplary modes of administration include oral, rectal, transmucosal, topical, intranasal, inhalation (e.g., via an aerosol), buccal (e.g., sublingual), vaginal, intrathecal, intraocular, transdermal, in utero (or in ovo), parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular [including administration to skeletal, diaphragm and/or cardiac muscle], intradermal, intrapleural, intracerebral, and intraarticular), topical (e.g., to both skin and mucosal surfaces, including airway surfaces, and transdermal administration), intro- lymphatic, and the like, as well as direct tissue or organ injection (e.g., to liver, skeletal muscle, cardiac muscle, diaphragm muscle or brain).
  • Administration can also be to a tumor (e.g., in or a near a tumor or a lymph node). The most suitable route in any given case will depend on the nature
  • the vector e.g., virus vector and/or plasmid expression vector
  • the vector is administered to the CNS, the peripheral nervous system, or both.
  • the vector e.g., virus vector and/or plasmid expression vector
  • the CNS e.g., the brain or the spinal cord.
  • Direct administration can result in high specificity of transduction of CNS cells, e.g., wherein at least 80%, 85%, 90%, 95% or more of the transduced cells are CNS cells. Any method known in the art to administer vectors directly to the CNS can be used.
  • the vector may be introduced into the spinal cord, brainstem (medulla oblongata, pons), midbrain (hypothalamus, thalamus, epithalamus, pituitary gland, substantia nigra, pineal gland), cerebellum, telencephalon (corpus striatum, cerebrum including the occipital, temporal, parietal and frontal lobes, cortex, basal ganglia, hippocampus and amygdala), limbic system, neocortex, corpus striatum, cerebrum, and inferior colliculus.
  • the vector may also be administered to different regions of the eye such as the retina, cornea or optic nerve.
  • the vector may be delivered into the cerebrospinal fluid (e.g., by lumbar puncture) for more disperse administration of the vector.
  • the delivery vector may be administered to the desired region(s) of the CNS by any route known in the art, including but not limited to, intrathecal, intracerebral, intraventricular, intranasal, intra-aural, intra-ocular (e.g., intra-vitreous, sub-retinal, anterior chamber) and peri-ocular (e.g., sub-Tenon's region) delivery or any combination thereof.
  • intrathecal intracerebral
  • intraventricular intranasal
  • intra-aural intra-ocular
  • intra-ocular e.g., intra-vitreous, sub-retinal, anterior chamber
  • peri-ocular e.g., sub-Tenon's region
  • the delivery vector may be administered in a manner that produces a more widespread, diffuse transduction of tissues, including the CNS, the peripheral nervous system, and/or other tissues.
  • the vector e.g., virus vector and/or plasmid expression vector
  • the vector will be administered in a liquid formulation by direct injection (e.g., stereotactic injection) to the desired region or compartment in the CNS and/or other tissues.
  • the vector can be delivered via a reservoir and/or pump.
  • the vector may be provided by topical application to the desired region or by intra-nasal administration of an aerosol formulation. Administration to the eye or into the ear, may be by topical application of liquid droplets.
  • the vector may be administered as a solid, slow- release formulation. Controlled release of parvovirus and AAV vectors is described by international patent publication WO 01/91803.
  • Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
  • the virus vector can be delivered dried to a surgically implantable matrix such as a bone graft substitute, a suture, a stent, and the like (e.g., as described in U.S. Patent 7,201,898).
  • compositions suitable for oral administration can be presented in discrete units, such as capsules, cachets, lozenges, or tablets, each containing a predetermined amount of the composition of this invention; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil emulsion.
  • Oral delivery can be performed by complexing a virus vector of the present invention to a carrier capable of withstanding degradation by digestive enzymes in the gut of an animal. Examples of such carriers include plastic capsules or tablets, as known in the art.
  • Such formulations are prepared by any suitable method of pharmacy, which includes the step of bringing into association the composition and a suitable carrier (which may contain one or more accessory ingredients as noted above).
  • a suitable carrier which may contain one or more accessory ingredients as noted above.
  • the pharmaceutical composition according to embodiments of the present invention are prepared by uniformly and intimately admixing the composition with a liquid or finely divided solid carrier, or both, and then, if necessary, shaping the resulting mixture.
  • a tablet can be prepared by compressing or molding a powder or granules containing the composition, optionally with one or more accessory ingredients.
  • Compressed tablets are prepared by compressing, in a suitable machine, the composition in a free-flowing form, such as a powder or granules optionally mixed with a binder, lubricant, inert diluent, and/or surface active/dispersing agent(s). Molded tablets are made by molding, in a suitable machine, the powdered compound moistened with an inert liquid binder.
  • compositions suitable for buccal (sub-lingual) administration include lozenges comprising the composition of this invention in a flavored base, usually sucrose and acacia or tragacanth; and pastilles comprising the composition in an inert base such as gelatin and glycerin or sucrose and acacia.
  • compositions suitable for parenteral administration can comprise sterile aqueous and non-aqueous injection solutions of the composition of this invention, which preparations are optionally isotonic with the blood of the intended recipient. These preparations can contain anti-oxidants, buffers, bacteriostats and solutes, which render the composition isotonic with the blood of the intended recipient.
  • Aqueous and non-aqueous sterile suspensions, solutions and emulsions can include suspending agents and thickening agents.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • compositions can be presented in unit/dose or multi-dose containers, for example, in sealed ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or water-for- inj ection immediately prior to use.
  • sterile liquid carrier for example, saline or water-for- inj ection immediately prior to use.
  • Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets of the kind previously described.
  • an injectable, stable, sterile composition of this invention in a unit dosage form in a sealed container can be provided.
  • the composition can be provided in the form of a lyophilizate, which can be reconstituted with a suitable pharmaceutically acceptable carrier to form a liquid composition suitable for injection into a subject.
  • the unit dosage form can be from about 1 pg to about 10 grams of the composition of this invention.
  • a sufficient amount of emulsifying agent which is physiologically acceptable, can be included in sufficient quantity to emulsify the composition in an aqueous carrier.
  • emulsifying agent is phosphatidyl choline.
  • compositions suitable for rectal administration can be presented as unit dose suppositories. These can be prepared by admixing the composition with one or more conventional solid carriers, such as for example, cocoa butter and then shaping the resulting mixture.
  • compositions of this invention suitable for topical application to the skin can take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil.
  • Carriers that can be used include, but are not limited to, petroleum jelly, lanoline, polyethylene glycols, alcohols, transdermal enhancers, and combinations of two or more thereof.
  • topical delivery can be performed by mixing a pharmaceutical composition of the present invention with a lipophilic reagent (e.g., DMSO) that is capable of passing into the skin.
  • a lipophilic reagent e.g., DMSO
  • compositions suitable for transdermal administration can be in the form of discrete patches adapted to remain in intimate contact with the epidermis of the subject for a prolonged period of time.
  • Compositions suitable for transdermal administration can also be delivered by iontophoresis (see, for example, Pharm. Res. 3:318 (1986)) and typically take the form of an optionally buffered aqueous solution of the composition of this invention.
  • Suitable formulations can comprise citrate or bis ⁇ tris buffer (pH 6) or ethanol/water and can contain from 0.1 to 0.2M active ingredient.
  • the vectors may be administered to the lungs of a subject by any suitable means, for example, by administering an aerosol suspension of respirable particles comprised of the virus vectors, which the subject inhales.
  • the respirable particles may be liquid or solid.
  • Aerosols of liquid particles comprising the virus vectors may be produced by any suitable means, such as with a pressure-driven aerosol nebulizer or an ultrasonic nebulizer, as is known to those of skill in the art. See, e.g., U.S. Patent No. 4,501,729. Aerosols of solid particles comprising the virus vectors may likewise be produced with any solid particulate medicament aerosol generator, by techniques known in the pharmaceutical art.
  • Example 1 Enhanced miR-1 in the spinal cord of ALS.
  • mir-1 and miR-206 were detected in the spinal cord of G93A-SOD1 (superoxide dismutase-1) ALS model mice (B6SJL-Tg(SODl*G93A)lGur/J ) by real-time PCR. It was found that miR-1, but not miR-206, was elevated 2.7 times at the presymptomatic stage at 8-weeks old, and about to 2.3 times at the symptomatic stage at 16- weeks old, as compared with age-matched WT mice (FIG. 1A and FIG. IB). Northern Blot analysis further confirmed these results (FIG. 1C). Thus, this study identified that enhanced expression of miR-1 in CNS may be a factor in ALS pathogenesis.
  • Tp4 is a possible target gene of miR-1.
  • Tp4 was a target of miR-1/206 in the invention
  • Tp4-3'-UTR 3'-UTR sequences of Tp4
  • TP4-3 1 UTR-mut a mutant sequence
  • Example 3 Tp4 is deficient in ALS spinal cord.
  • Tp4 is down-regulated in vivo in the G93A-SOD1 ALS mouse model.
  • Western blot data showed Tp4 expression reduced markedly in the spinal cord of G93A-SOD1 ALS mice from 8-week old to the symptomatic stage at 16 weeks, compared with wild-type C57B6 mice (FIG. 3A).
  • Tp4 mRNA levels did not decrease accordingly (FIG. 3B).
  • Tp4 expression after AAV2-CMV-G93A-SOD1 treatment in vitro was analyzed and found to be inhibited by 41.1% and 41.5% respectively, compared with GFP and SOD 1 -treated groups (FIG. 4A, FIG. 4B). It was also found that thymus size and weight of ALS mice was less than those of WT mice (FIG. 5A and FIG. 5B), in agreement with other studies (Seksenyan et al. 2010 J Cell Mol Med 14:2470-2482) which suggested a thymic defect was a co-pathological factor in both ALS patients and animal model. Thus, the deficiency of TP4 in CNS is accompanied with a thymic defect which may promote ALS progression.
  • Example 5 Anti-miR-1 protects neurons and delays the survival of ALS.
  • anti-miR-1 and anti-miR-206 treatment could delay disease onset from 120.5 days to 134 and 133 days, respectively (FIG. 7C and FIG. 7D), but disease progression could not be efficaciously inhibited when the ALS symptoms became worse (FIG. 7F).
  • Example 6 Over-expression of Tp4 postpones ALS progression.
  • AAV9-CMV-TP4 was injected into the neonates of the B6SJL-background ALS (B6SJL- Tg(SODl*G93A)lGur/J, Num. 002726, Jackson Lab) via superficial temporal vein at 1 day post-natal (IV injection).
  • B6SJL- Tg(SODl*G93A)lGur/J Num. 002726, Jackson Lab
  • IV injection 1 day post-natal mice
  • 6-week-old ALS mice were treated with AAV9-CMV-TP4 by intrathecal injection (IT injection). Tp4- treated mice showed a delay in body weight loss (FIG. 8A and FIG. 8B).
  • Tp4 over-expression of Tp4 by IV injection increased the life span time of ALS mice up to 17.5 days (from 130 days to 147.5 days) compared with control group (FIG. 9 A).
  • IT injection at the early stages, before the appearance of ALS symptoms, extended lifespan up to 15.5 days (FIG. 9B).
  • the averages of survival days were delayed up to 19.7 and 17 days by TP4-IV and IT treatment, respectively (FIG. 9C, TP4-IV group 148.6 ⁇ 2.5days and TP4-IT group 145.9 ⁇ 3.5 days versus PBS group 128.9 ⁇ 2.6 days, P ⁇ 0.001).
  • Further analysis showed Tp4 injection by IV and IT postponed disease onset of ALS mice 16 days and 11 days, respectively (FIG. 9D and FIG. 9E), but disease progression was not significantly extended (FIG. 9F).
  • grip force was also enhanced significantly in both Tp4-treatment groups (FIG.10A and FIG. 10B).
  • Example 7 AAV-mediated Tp4 overexpression improves the survival of neurons in the spinal cord.
  • Example 8 Increased miR-1 expression contributes to the ALS phenotype via negative regulation of thymosin beta 4.
  • ALS is a severe neuromuscular disease characterized by muscle atrophy and eventual paralysis due to the progressive death of motor neurons.
  • the main cause of hereditary familial ALS is mutations in genes including, but not limited to, superoxide dismutase- 1 (SOD1), transactive response DNA binding protein 43 (TDP-43, TARDBP) or the DNA/RNA-binding protein fused in sarcoma (FUS).
  • SOD1 superoxide dismutase- 1
  • TDP-43, TARDBP transactive response DNA binding protein 43
  • FUS DNA/RNA-binding protein fused in sarcoma
  • MicroRNAs are short non-coding sequences that cleave or inhibit messenger RNAs (mRNAs) by targeting their 3 '-untranslated regions (3'-UTR). Dysregulation of miRNAs such as miR-9, miR-124, miR-146a*, and miR-155 have been associated with ALS development and progression; the first miRNA implicated in ALS pathology was miR-206, which was found to be highly expressed in the skeletal muscle of an ALS mouse model after disease onset (Williams et al. 2009 Science 326: 1549-1554). miR-1 belongs to the same miRNA family as miR-206, and both miR-1 and miR-206 have the same seed sequences.
  • adeno-associated virus (AAV)-mediated overexpression of miR- 1/206 in the spinal cord of C57BL/6J mice caused a loss of motor neurons, leading to abnormal gait and limb paralysis, which are hallmark symptoms observed in ALS.
  • miR-1 but not miR-206, is highly upregulated in the spinal cord of G93A-SOD1 ALS mice.
  • miR-1 targets the mRNA of the thymosin beta 4, X-linked (Tp4) gene and negatively regulates its expression, leading to the loss of motor neurons and accelerating ALS progression.
  • Tp4 X-linked
  • short hairpin RNA-206 (shRNA-206) was used to mimic miR-1/206 function.
  • the pEMBL-U6-shRNA-206 plasmid was constructed by inserting the 22 base pairs (bp) sequences of the mature miR-206 into a pEMBL AAV expression vector.
  • shRNA-206 mutant (shRNA-206mut) plasmid was constructed by converting the first five seed sequences of the mature miR-206 from GGAAT to TTACC, rendering the miRNA non-functional. Then, the plasmids were packaged into AAV serotype 9 (AAV9) by triple plasmids transfection.
  • AAV9-mediated target gene is easily delivered to central nervous system (CNS) when the vector was injected into neonates in 1 day post-natal via temporal vein.
  • CNS central nervous system
  • Northern blot analysis showed that shRNA driven by a U6 promoter were successfully expressed in the spinal cord except for skeletal muscles, which indicates that the shRNA-206 could be processed to mature miR-206.
  • overexpression of miR-206 in the shRNA-206- treated mice caused the severe symptoms observed in nervous system, which strongly suggested that the pathologic changes arose from the CNS.
  • shRNA-206-treated mice also had fewer intact motor neurons than the shRNA-206mut group.
  • TEM transmission electron microscopy
  • insoluble mutated SOD1 or TARDBP causes delocalization or aggregation of TDP-43, which is normally distributed within the nucleus.
  • Abnormal TDP-43 trafficking or aggregation is thought to be one of the main features of neurodegeneration diseases (Neumann et al. 2006 Science 314: 130-133; Dewey et al. 2012 Brain Research 1462: 16-25).
  • Immunostaining of the spinal cord showed that TDP-43 aggregated outside of the nucleus in shRNA-206-treated mice but not in the shRNA-206mut group (FIG. 12E). Therefore, exogenous overexpression of shRNA-206 appeared to reproduce many pathological signs of ALS or similar motor neuron diseases.
  • the plasmids pEMBL-CMV- pre-miR-1 and pre-miR-206 were constructed via insertion of two copies of the miR-1 or miR-206 precursor, which was flanked by about 150 bp sequences from both upstream and downstream. The copies were inserted into human chorionic gonadotropin (hCG) introns, which could cut the inserters to form the miRNA precursors. These miRNA precursors were then further processed into mature miRNA-1/206 by the ribonuclease III enzyme Drosha in the nucleus and Dicer in cytoplasm.
  • hCG human chorionic gonadotropin
  • the pEMBL-CMV-pre-miR-1 and pre-miR-206 plasmids were individually co-transfected into 293 cells in vitro with the reporter plasmid pEMBL-CMV-GFP-4xanti-miR-l/206, in which four copies of a complementary sequence of miR-1/206 (anti-miR-1/206) were inserted at the 3' terminus of a green fluorescent protein (GFP) sequence as its artificial 3'-UTR (FIG. 13).
  • GFP green fluorescent protein
  • MiR-138 is also enriched in the spinal cord and has a completely different seed sequence than that of miR-1/206. This specific inhibition indicated that pEMBL-CMV-pre-miR-1/206 was processed into the mature miR-1/206, which bound the 3'- UTR of the plasmids pEMBL-CMV-GFP-4xanti-miR-l/206 leading to inhibit GFP expression.
  • the precursor plasmids pEMBL-CMV-pre-miR-1/206 were next packaged into AAV9 vectors, which were administered into 1 -day-old C57BL/6J neonates via temporal vein injection. After 2 months, the C57BL/6J mice treated with AAV9-CMV-pre-miR- 1/206 began to exhibit abnormal gait and limb grasping reflex, which was similar to what was observed in the shRNA-206 treated mice. These conditions also deteriorated over time. Treadmill running distance, rotarod latency time, and forelimb grip force measured at 5 months old were significantly decreased in pre-miR-l/206-treated groups. Treatment with the control vector containing empty hCG introns did not cause any functional impairment.
  • H&E hematoxylin and eosin
  • MiR-1 and miR-206 were increased in the spinal cord and brain of ALS mice.
  • MiR-1 and miR-206 expression were detected in the spinal cord of G93A-SOD1 ALS model mice (B6SJL-Tg(SODl*G93A)lGur/J) by real-time PCR and it was found that miR-1 was elevated 2.7-fold in the spinal cord at the pre- symptomatic 8 weeks old, and 2.3-fold at the symptomatic 16 weeks old, compared with age- matched wild type (WT) mice (FIG. 1A).
  • miR-206 expression was normally present at 10- to 100-fold lower levels than miR-1 in the spinal cord, and furthermore did not appear to be significantly increased in ALS groups compared with WT groups (FIG. IB).
  • Northern blot analysis further confirmed the increased expression levels of miR-1 in the spinal cord (FIG. 1C).
  • miR- 1 or miR-206 expression levels In the brain cortex, however, there is no significant difference in miR- 1 or miR-206 expression levels between WT and ALS mice.
  • miR-206 was increased about 6-fold in 16 weeks old ALS mice compared with age-matched WT mice, but there were no significant differences in miR-1 between WT and ALS groups.
  • the enhanced expression of miR-1 in the spinal cord from presymptomatic to the symptomatic stage may be one of pathogenic factors within the CNS in ALS.
  • Tp4 was a target of miR-1/206
  • Tp4-3'-UTR 3'-UTR sequences of Tp4
  • TP4-3 1 UTR-mut mutant sequence
  • Tp4 was downregulated in the ALS mice.
  • Western blot data showed a marked reduction in Tp4 expression in the spinal cord of G93A-SOD1 ALS mice from 8 weeks old to the symptomatic stage at 16 weeks, compared to wild-type C57BL/6 mice (FIG. 3A).
  • Tp4 mRNA levels did not decrease accordingly.
  • TP4 may be curbed by miR-1/206 through post- transcriptional regulation.
  • Immunostaining showed Tp4 mainly expressed in neurons and its expression level was reduced in the anterior horn of the spinal cord from ALS mice at 16 weeks compared to WT mice.
  • miR-1 was shown to be increased in the spinal cord of ALS mice during the presymptomatic and symptomatic stage of disease, it was determined whether downregulation of miR-1 would have therapeutic benefit.
  • Two constructs with anti-miR- 1/206 were developed to reduce the level of miR- 1/206 both in vitro and in vivo.
  • in vitro application of two plasmids pEMBL-CMV-pre-miR-1 and pEMBL-CMV-pre-miR-206 decreased the luciferase expression of the pEMBL-CMV-luciferase plasmid containing the Tp4-3'UTR target sequence.
  • anti-miR-1 and anti-miR-206 treatment could delay the disease onset from 120.5 days to 134 and 133 days, respectively (FIGS. 7C and 7D), but the disease progression could not be efficaciously inhibited by the anti-miRs when the condition of the ALS mice deteriorated (FIG. 7F).
  • western blot showed that expression of Tp4 in the spinal cord was rescued by anti-miR-1 and anti-miR-206 treatment (FIGS.
  • AAV9-CMV-TP4 vectors were administered into 1 -day-old neonates on a B6SJL- background ALS, a more serious ALS model than C 57BL/6J-b ackground ALS, via superficial temporal vein injection.
  • 6-week-old ALS mice were treated with AAV9-CMV-TP4 by intrathecal injection (IT injection group). The mice in the two Tp4- treated groups had a lower rate of limb grasping reflex (and a delay in bodyweight loss (FIGS. 8A and 8B).
  • Tp4 overexpression of Tp4 by IV injection increased the lifespan of ALS mice up to 17.5 days (from 130 days to 147.5 days) compared to control (FIG. 9A).
  • FIG. 14 A summary schematic of the function of miR-1 and Tp4 is shown in FIG. 14.
  • Example 9 anti-miRl and Tp4 constructs for ALS treatment.
  • the anti-miR-1 and Tp4 polynucleotides and expression cassettes of the present invention are optimizable for increased target gene expression and tropism, such as but not limited to, neuron-specific promoters to limit target gene expression in neuronal cells (e.g., the neuron-specific synapse- 1 (syn-1) promoter) and to reduce off-target effects, codonoptimization for enhanced gene product expression, and/or insertion of a secretory signal peptide to enhance secretion of the gene products (e.g., Tp4) from the cell.
  • neuron-specific promoters to limit target gene expression in neuronal cells
  • codonoptimization for enhanced gene product expression e.g., codonoptimization for enhanced gene product expression
  • a secretory signal peptide e.g., Tp4 from the cell.
  • G93A-SOD1 ALS mice (6-, 10- and 14-week-old, half male and half female) receive AAV viruses (l x l0 n vg/mouse, including AAV9-Syn-GFP, AAV9-Syn-anti- miR-1 and AAV9-Syn-Opti-Tp4) by IT injection, individually.
  • AAV viruses l x l0 n vg/mouse, including AAV9-Syn-GFP, AAV9-Syn-anti- miR-1 and AAV9-Syn-Opti-Tp4
  • mice/group are sacrificed at 16 weeks of age, spinal cords are sectioned for Nissl staining and immunofluorescence staining with anti-Choline Acetyltransferase (anti-ChAT), by which motor neurons are accounted and analyzed.
  • anti-ChAT anti-Choline Acetyltransferase
  • the level of miR-1 is detected by NCodeTM miRNA qRT-PCR kit.
  • Tp4 To identify whether motor neurons are protected by overexpression of Tp4, the expression of Tp4 is detected by western blot and motor neurons counted by Nissl staining and anti-ChAT immunostaining.
  • Two vectors, anti-miR-1 and Tp4 are cloned into one to comprise AAV9-Syn-Opti-Tp4- hCGin-anti-miR-1.
  • the sequence of anti-miR-1 is inserted into 3'- terminal of Tp4 linked with hCG intron, which may cut off the inserted sequence of anti- miR-1 to form anti-miRs.
  • the two-in-one construct and the combination injection of two vectors anti-miR-1 and Tp4 are compared in the ALS mouse model. 10 mice/group are used for survival analysis.
  • Enhanced miR-1 may be a potential early diagnostic indicator for ALS.
  • MiR-1 level in the CSF of ALS patients and healthy volunteers is measured by rt- PCR, and confirm miR-1 is increased in ALS patient.
  • Tp4 is detected in the CSF by ELISA kit, and the relationship between Tp4 and severity of patient's symptom and life span, and evaluate whether deficient Tp4 be a predictive factor for ALS prognosis is analyzed.
  • AAV is used in the field as a safe tool for gene delivery. It has been used for clinical trial in some diseases such as muscular dystrophy and hemophilia.
  • Rhesus monkeys 2-4 years old
  • pre-screened for pre-existing anti-AAV9 antibody ⁇ l/50
  • a catheter (needle 21G) is introduced via a cannula into the intrathecal space L3-L4. Placement is verified by the presence of CSF. 1-1.5 mL of CSF is systematically removed in order to decrease the pressure of subarachnoid space before intrathecal injection of the AAV vectors. The catheter is slowly ascended to the cervical vertebrae under radioscopic control, and a solution of 1 mL of vector is then infused at a rate of 0.5 mL/min. The catheter is removed from 8 cm to be opposite of the thoracic vertebrae, and another dose of vector (1 ml) is administered.
  • Regular indexes are recorded and evaluated after IT injection, including the body weight, limbs movement and muscle force, etc.
  • the nervous system is examined including nerve responses, activity, emotion, etc.
  • MiR-1 expression is analyzed via rt-PCR in CSF, spinal cord, and brain.
  • Tp4 expression is analyzed in CSF by ELISA kit, and in spinal cord and brain by western blot and immunostaining. Table 1. Table 2.

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Abstract

La présente invention concerne des polynucléotides comprenant des oligonucléotides anti-microARN (AMO) dirigés contre miR-1 et/ou miR-206, et/ou une région codante codant pour une thymosine β4 (Tβ4), ainsi que des cassettes d'expression, des vecteurs et des compositions les comprenant, et des procédés d'utilisation de ceux-ci pour l'administration des polynucléotides et/ou des cassettes d'expression à une cellule ou un sujet et pour traiter des troubles associés à l'expression aberrante d'un gène régulé par miR-1 et/ou miR-206 chez le sujet, comme la sclérose latérale amyotrophique (SLA).
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060264360A1 (en) * 2002-04-12 2006-11-23 Yale University Office Of Cooperstive Research Anti-inflammatory and wound healing effects of lymphoid thymosin beta-4
WO2007070483A2 (fr) * 2005-12-12 2007-06-21 The University Of North Carolina At Chapel Hill Micro-arn regulant la proliferation et la differenciation des cellules musculaires
US20140234274A1 (en) * 2008-04-30 2014-08-21 University Of North Carolina At Chapel Hill Directed Evolution and In Vitro Panning of Virus Vectors

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060264360A1 (en) * 2002-04-12 2006-11-23 Yale University Office Of Cooperstive Research Anti-inflammatory and wound healing effects of lymphoid thymosin beta-4
WO2007070483A2 (fr) * 2005-12-12 2007-06-21 The University Of North Carolina At Chapel Hill Micro-arn regulant la proliferation et la differenciation des cellules musculaires
US20140234274A1 (en) * 2008-04-30 2014-08-21 University Of North Carolina At Chapel Hill Directed Evolution and In Vitro Panning of Virus Vectors

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE Protein GenBank; ANONYMOUS : "TMSB4X protein, partial [Homo sapiens] ", XP093043178, retrieved from NCBI *
GUI-HONG ZHANG, KRISHNA DILIP MURTHY, RAHMAWATI BINTI PARE, YI-HUA QIAN: "Protective effect of Tβ4 on central nervous system tissues and its developmental prospects ", EUROPEAN JOURNAL OF INFLAMMATION, vol. 18, 1 January 2020 (2020-01-01), pages 1 - 11, XP093043177 *

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