WO2023034966A1

WO2023034966A1 - Compositions and methods of using the same for treating disorders associated with thymosin βeta 4

Info

Publication number: WO2023034966A1
Application number: PCT/US2022/075904
Authority: WO
Inventors: Bin Xiao; Xiao Xiao; Juan Li
Original assignee: The University Of North Carolina At Chapel Hill
Priority date: 2021-09-03
Filing date: 2022-09-02
Publication date: 2023-03-09

Abstract

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 β4 (Tβ4), 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).

Description

COMPOSITIONS AND METHODS OF USING THE SAME FOR TREATING DISORDERS ASSOCIATED WITH THYMOSIN BETA 4

STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING

A Sequence Listing in XML format, entitled 5470-912WO_ST26.xml, 14,892 bytes in size, generated on September 1, 2022 and filed herewith, is hereby incorporated by reference in its entirety for its disclosures.

FIELD OF THE INVENTION

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).

BACKGROUND OF THE INVENTION

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.

SUMMARY OF THE INVENTION

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).

Additionally provided are vectors, transformed cells, transgenic animals, compositions, and pharmaceutical compositions 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.

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.

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.

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.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C show data graphs of miR-1 and miR-206 expression in the CNS of ALS mice. FIG. 1A shows a bar graph of miR-1 expression in the CNS of ALS mice, where miR-1 in the spinal cord was detected by real-time PCR, miR-26b was an internal control. n=6 * <0.05, compared with age-matched WT mice, /-test. 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. Tp4- 3' UTR and its mutant were inserted to the 3 '-terminal of the report gene luciferase. FIG. 2C shows a bar graph of luciferase expression level as detected in 293 cells 48h after shRNA-206/206mut transfection. pEMBL-CMV-Lacz plasmid was an internal conference. n=4, *** < 0.001, /-test. FIG. 2D shows a bar graph of luciferase expression level as detected in 293 cells 48h after pre-miR- 1/206 transfection. pEMBL-CMV-Lacz plasmid was an internal conference. n=4,

0.001, /-test;

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. 4B shows a bar graph quantifying Tp4 expression analyzed by Western blot. n=3 * < 0.05.

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. 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. * P <0.05, ** P <0.01, *** P <0.001, t-test. 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. 7A shows a data plot of survival curves of C57BL/6-backgroud ALS, B6.Cg- Tg(SODl*G93A)lGur/J mice, treated by PBS (156.5 days; n=18, half male and half female) and AAV9-CMV-anti-miR-l (171 days; n=9, male=5 and female=4). P <0.001, Log-rank test. FIG. 7B shows a data plot of survival curves of C57BL/6-backgroud ALS mice treated with PBS (156.5 days; n=18, half male and half female) and AAV9-CMV-anti-miR-206 (168 days, n=9, male=5 and female=4). P <0.01, Log-rank test. FIG. 7C shows a data plot of disease onset percentage comparing PBS-treated group (120.5 days; n=18, half male and half female) and AAV9-CMV-anti-miR-l treated group (134 days; n=9, male=5 and female=4), P <0.01. Log-rank test. FIG. 7D shows a data plot of disease onset percentage comparing with disease onset between PBS-treated group (120.5 days; n=18, half male and half female) and AAV9-CMV-anti-miR-206 (133 days; n=9, male=5 and female=4), P <0.01. Log-rank test. FIG. 7E shows a bar graph quantifying the average survival of C57BL/6-backgroud ALS mice treated with PBS (155.5±2.0 days; n=18, half male and half female), AAV9-CMV-anti- miR-l(170.2±4.8 days, n=9, male=5 and female=4), and AAV9-CMV-anti-miR-206 (168.1±3.7 days, n=9, male=5 and female=4). ** <0.01, /-test. 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. 7H shows a bar graph quantifying the data of Tp4 expression in the spinal cord. n=4; * P <0.05, ** P <0.01, /-test. 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. 7J shows a bar graph quantifying of the number of motor neurons in the spinal cord. n=6; ** P <0.01, /-test.

FIG. 8A shows a data graph plotting weight over weeks of treatment in the Tp4 IV treatment group. Body weight loss was delayed in TP4-IV group from 17-week old. PBS (n=30 ); TP4-IV (n=20 ). P <0.05, /-test. FIG. 8B shows a data graph plotting weight over weeks of treatment in Tp4 IT treatment groups. Body weight loss was delayed in TP4-IT group from 14-week old. PBS (n=30 ); TP4-IT (n=14). P <0.05, /-test.

FIG. 9A shows a data graph of survival curves for a PBS-treated group (mean=130 days; n=20, half male and half female), and a TP4-IV treatment group (mean=147.5 days; n=16, half male and half female, P <0.001, Log-rank test). FIG. 9B shows a data graph of survival curves for a PBS-treated group (mean=130 days; n=20, half male and half female ) and a TP4-IT treatment group (mean=145.5 days; n=14, half male and half female; P < 0.001, Log-rank test). FIG. 9C shows a bar graph quantifying the average days of survival of FIGS. 9A and 9B. PBS-treated group (128.9±2.6 days, n=20, half male and half female), TP4-IV treatment group (148.6±2.5days; n=16, half male and half female), and TP4-IT treatment group (145.9±3.5 days; n=14, half male and half female). ***P <0.001, /-test. FIG. 9D shows a data plot of % disease onset for the PBS-treated group (mean=101.5 days; n=20, half male and half female), and the TP4-IV treatment group (mean=l 17.5 days; n=16, half male and half female; P <0.001, Log-rank test). FIG. 9E shows a data plot of % disease onset for the PBS-treated group (mean=101.5 days; n=20, half male and half female) and the TP4-IT treatment group (mean=l 12.5 days, n=14, half male and half female; P <0.001, Logrank test). FIG. 9F shows a bar graph quantifying disease progression in days. No significant differences were found between the groups.

FIG. 10A shows a data graph of grip force ability from the PBS-treated group (n=20 before 15 weeks, half male and half female; n=19, 19, 17, 14 at the 15th, 16th, 17th, 18th week, respectively), and the TP4-IV treatment group (n=16 mice, half male and half female). *P <0.05, /-test. FIG. 10B shows a data graph of grip force ability from the PBS-treated group (n=20 before 15 weeks, half male and half female; n=19, 19, 17, 14 at the 15th, 16th, 17th, 18th week, respectively) and the TP4-IT treatment group (n=14 mice, half male and half female). *P <0.05, /-test.

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. 11B shows a bar graph quantifying the data of Tp4 expression in the spinal cord in FIG. 11 A. n=4; * <0.05, ** P <0.01, /-test. 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). FIG. HE shows a bar graph quantifying the number of motor neurons in the spinal cord. n=6 per group, **P<0.01, /-test.

FIGS. 12A-12E show ALS-like symptom induction by shRNA-206. FIG. 12A shows a data plot of ShRNA-206-treated mice weight (18.5-21.6% less) as compared with shRNA- 206mut-treated mice. n=10 mice per group, ** P <0.01 vs shRNA-206mut group, /-test. 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). Some axons in shRNA-206 treated group are undergoing demyelination (white arrow), and black arrow heads show vacuoles in the axon, while the axons in the shRNA-206mut group appear normal. Scale bar 4 pm (left), 1 pm (right). 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. Pathological TDP-43 formed short neurite-like or global inclusions, which located in the outsides of nucleus in the spinal cord of sh206-treated mice (white arrow). Left: TDP-43; Middle: DAPI; Right: merge. Scale bar: 100pm. 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.

DETAILED DESCRIPTION

The present invention now will be described hereinafter with reference to the accompanying drawings and examples, in which embodiments of the invention are shown. This description is not intended to be a detailed catalog of all the different ways in which the invention may be implemented, or all the features that may be added to the instant invention. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. Thus, the invention contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure, which do not depart from the instant invention. Hence, the following descriptions are intended to illustrate some particular embodiments of the invention, and not to exhaustively specify all permutations, combinations, and variations thereof.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

All publications, patent applications, patents and other references cited herein are incorporated by reference in their entireties for the teachings relevant to the sentence and/or paragraph in which the reference is presented.

Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a composition comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

As used in the description of the invention and the appended claims, the singular forms "a," "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

Also as used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").

The term "about," as used herein when referring to 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. For example, "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.

As used herein, phrases such as "between X and Y" and "between about X and Y" should be interpreted to include X and Y. As used herein, 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."

The term "comprise," "comprises" and "comprising" as used herein, specify the presence of the stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.

As used herein, the transitional phrase "consisting essentially of means that the scope of a claim is to be interpreted to encompass the specified materials or steps recited in the claim and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. Thus, the term "consisting essentially of when used in a claim of this invention is not intended to be interpreted to be equivalent to "comprising."

Except as otherwise indicated, standard methods known to those skilled in the art may be used for cloning genes, amplifying and detecting nucleic acids, and the like. Such techniques are known to those skilled in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd Ed. (Cold Spring Harbor, NY, 1989); Ausubel et al. Current Protocols in Molecular Biology (Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York).

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.

To illustrate further, if, for example, the specification indicates that a particular amino acid can be selected from A, G, I, L and/or V, this language also indicates that the 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. Moreover, such language also indicates that one or more of the specified amino acids can be disclaimed (e.g., by negative proviso). For example, in particular embodiments 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.

As used 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.

As used herein, 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.

The term "parvovirus" as used herein 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).

As used herein, the term "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.

FIELDS et al., VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers). A number of additional AAV serotypes and clades have been identified (see, e.g., Gao et al., (2004) J. Virology 78:6381-6388; Mori et al., (2004) Virology 33-:375-383; and Table 1).

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. These 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). See in addition, e.g., GenBank Accession Numbers NC_002077, NC_001401, NC_001729, NC_001863, NC_001829, NC_001862, NC_000883, NC_001701, NC_001510, NC_006152, NC_006261, AF063497, U89790, AF043303, AF028705, AF028704, J02275, J01901, J02275, X01457, AF288061, AH009962, AY028226, AY028223, NC_001358, NC_001540, AF513851, AF513852, AY530579; the disclosures of which are incorporated by reference herein for teaching parvovirus and AAV nucleic acid and amino acid sequences. See also, e.g., Srivistava et al. (1983) J. Virology 45:555; Chiorini et al. (1998) J. Virology 71 :6823; Chiorini et al. (1999) J. Virology 73: 1309; Bantel-Schaal et al. (1999) J. Virology 73:939; Xiao et al. (1999) J. Virology 73:3994; Muramatsu et al. (1996) Virology 221 :208; Shade et al. (1986) J. Virol. 58:921; Gao et al. (2002) Proc. Nat. Acad. Sci. USA 99: 11854; Mori et al. (2004) Virology 33-:375-383; international patent publications WO 00/28061, WO 99/61601, WO 98/11244; and U.S. Patent No. 6,156,303; the disclosures of which are incorporated by reference herein for teaching parvovirus and AAV nucleic acid and amino acid sequences. See also Table 1.

The capsid structures of autonomous parvoviruses and AAV are described in more detail in BERNARD N. FIELDS et al. VIROLOGY, volume 2, chapters 69 & 70 (4th ed., Lippincott-Raven Publishers). See also, description of the crystal structure of AAV2 (Xie et al. (2002) Proc. Nat. Acad. Sci. 99: 10405-10); AAV4 (Padron et al. (2005) J. Virol. 79: 5047- 58); AAV5 (Walters et al. (2004) J. Virol. 78:3361-71); and CPV (Xie et al. (1996) J. Mol. Biol. 6:497-520 and Tsao et al. (1991) Science 251 : 1456-64).

The term "tropism" as used herein 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.

As used herein, "transduction" of a cell by a virus vector (e.g., an AAV 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.

Unless indicated otherwise, "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).

As used herein, the term "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.

As used herein, 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. In representative embodiments 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.

Likewise, 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. In representative embodiments 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.

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. The term 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.

As used herein, 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.

As used herein, the terms "express," "expresses," "expressed" or "expression," and the like, with respect to a nucleotide sequence (e.g., RNA or DNA) indicates that the nucleotide sequence is transcribed and, optionally, translated. Thus, 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.

The terms "complementary" or "complementarity," as used herein, refer to the natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing. For example, the 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. Thus, 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. In particular embodiments, 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. The term " anti -microRNA oligonucleotide," "anti-miRNA," "anti-miR," or "AMO" as used herein interchangeably refer to antisense oligonucleotides (ASOs) that target microRNAs. 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. While not wishing to be bound to theory, complementary binding of the ASO and/or AMO to the complementary target (e.g., the miRNA) 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. 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.

As used herein, 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.

Likewise, 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.

The term "codon-optimized," as used herein, 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. In this manner, the protein encoded by the gene is identical, but the underlying nucleobase sequence of the gene or corresponding mRNA is different. In some embodiments, 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. For example, in human codon-optimization one or more codons in a coding sequence are replaced by codons that occur more frequently in human cells for the same amino acid. 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. Desirably, 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.

The term "sequence identity," as used herein, 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. USA 55:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, WI), the Best Fit sequence program described by Devereux et al., Nucl. Acid Res. 12 :387 (1984), preferably using the default settings, or by inspection.

An example of a useful algorithm is PILEUP. 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).

Another example of a useful algorithm is the BLAST algorithm, described in Altschul et al., J. Mol. Biol. 215 :403 (1990) and Karlin et al., Proc. Natl. Acad. Sci. USA 90:5873 (1993). A particularly useful BLAST program is the WU-BLAST-2 program which was obtained from Altschul et aL, Meth. EnzymoL, 266:460 (1996); blast. wustl/edu/blast/README.html. 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.

An additional useful algorithm is gapped BLAST as reported by Altschul et al., Nucleic Acids Res. 25:3389 (1997). A percentage amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the "longer" sequence in the aligned region. The "longer" sequence is the one having the most actual residues in the aligned region (gaps introduced by WU-Blast-2 to maximize the alignment score are ignored).

In a similar manner, 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. In addition, for sequences which contain either more or fewer nucleotides than the polynucleotides specifically disclosed herein, it is understood that in one embodiment, the percentage of sequence identity will be determined based on the number of identical nucleotides in relation to the total number of nucleotides. Thus, for example, 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. In percent identity calculations relative weight is not assigned to various manifestations of sequence variation, such as insertions, deletions, substitutions, etc.

In one embodiment, only 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. For example, an isolated cell can be a cell in culture medium and/or a cell in a pharmaceutically acceptable carrier of this invention. Thus, an isolated cell can be delivered to and/or introduced into a subject. In some embodiments, 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.

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.

The term "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.

As used herein, the term "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.

A "heterologous nucleotide sequence" or "heterologous nucleic acid" is a sequence that is not naturally occurring in the virus. Generally, 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.

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.

By "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.

The terms "prevent," "preventing" and "prevention" (and grammatical variations thereof) refer 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. Alternatively stated, 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. Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject.

A "prevention effective" amount as used herein 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. Those skilled in the art will appreciate that the level of prevention need not be complete, as long as some preventative benefit is provided to the subject.

The terms "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). Generally, the NOI, 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).

As used herein, the term "modified," as applied to a polynucleotide or polypeptide sequence, refers to a sequence that differs from a wild-type sequence due to one or more deletions, additions, substitutions, or any combination thereof.

As used herein, the terms "virus vector," "vector" or "gene delivery 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. Alternatively, in some contexts, the term "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. Thus, 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. Alternatively, 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). Typically, 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.

The term "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. For example, 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. Further, 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.

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). Thus, in some embodiments, double stranded (duplex) genomes can be packaged into the virus capsids of the invention.

Further, the 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. In some embodiments, complete or partial domains, functional regions, epitopes, etc., from one AAV serotype can replace the corresponding wildtype domain, functional region, epitope, etc. of a different AAV serotype, in any combination, to produce 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.

As used herein, the term "amino acid" or "amino acid residue" encompasses any naturally occurring amino acid, modified forms thereof, and synthetic amino acids.

Naturally occurring, levorotatory (L-) amino acids are shown in Table 2. Conservative amino acid substitutions are known in the art. In particular embodiments, 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.

Alternatively, 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).

Further, the 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.

As used herein, the term "binding site" refers to any general structural feature that acts as a location for binding between components. As applied to nucleic acids or polynucleotides, the term "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. As applied to peptides, polypeptides, or proteins, the term "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.

As used herein, the term "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. The terms "seed" or "seed sequence" refer 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. In general, 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.

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).

MicroRNAs (miRs) 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.

Thymosin beta 4 (Tp4) 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.

Thus, 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.

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. In some embodiments, 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. For example, in some embodiments, 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. In some embodiments, 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. In some embodiments, 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). In some embodiments, 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). In some embodiments, 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.

SEQ ID NO:1 human TB4 3’ UTR sequence targeted by miR-l/miR-206 seed sequence ACAUUCC

In some embodiments, 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.

SEQ ID NO:2 miR-l/miR-206 shared seed sequence

UGUAAGG

In some embodiments, 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.

SEQ ID NO:3 AMO anti-miRNA-miR-1-2

ATACATACTTCTTTACATTCCA

In some embodiments, 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.

SEO ID NO:4 AMO anti-miRNA-miR-206

CCACACACTTCCTTACATTCCA

In some embodiments, 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.

SEO ID NO: 5 polynucleotide with 4 copies of AMO anti-miRNA-miR-1-2

CTGCGCGGCCGCACTAGTGACCTCGAGGACAATACATACTTCTTTACATTCCATA GATATCAATACATACTTCTTTACATTCCATATGCGTCGAGGACAATACATACTTC TTTACATTCCATAGATATCAATACATACTTCTTTACATTCCATATGCGTCGACTGC AGATCTGCGGCCGC

In some embodiments, 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.

SEO ID NO:6 polynucleotide with 4 copies of AMO anti-miRNA-miR-206

CTGCGCGGCCGCACTAGTGACCTCGAGGACGCCACACACTTCCTTACATTCCAGA TATCGCCACACACTTCCTTACATTCCATGCGTCGAGGACGCCACACACTTCCTTA CATTCCAGATATCGCCACACACTTCCTTACATTCCATGCGTCGACTGCAGATCTG CGGCCGCGCAG

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. In some embodiments, the polynucleotide may be operably linked to one or more expression elements that may enhance expression of the polynucleotide products, e.g., the AMO. In some embodiments, 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. In some embodiments, 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.

In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

Another aspect of the present invention provides 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. In some embodiments, a coding region encoding a Tp4 gene product of the present invention may be codon-optimized, e.g., a codon-optimized coding region.

SEQ ID NO:7 synthetic human TB4

MSDKPDMAEIEKFDKSKLKKTETQEKNPLPSKETIEQEKQAGES

In some embodiments, 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. In some embodiments, 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. In some embodiments, the polynucleotide may be further linked to one or more molecular tags (e.g., GFP), such as to indicate gene expression.

Those skilled in the art will further appreciate that a variety of 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. In representative embodiments, 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.

In embodiments wherein the AMO and/or Tp4 coding region is transcribed and then translated in the target cells, specific initiation signals may be employed for efficient translation of inserted coding sequences. These 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). For example, 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). In some embodiments, 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.

In some embodiments, 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. In some embodiments, the AAV vector may comprise wildtype capsid proteins. In other embodiments, 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.

In some embodiments, the vector is a single-stranded AAV (ssAAV) vector. In some embodiments, 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.

In some embodiments, 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.

SEQ ID NO:8 pEMBL-CMV-human

cagcagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggaattccagacgattgagcgtcaaaatgt aggtatttccatgagcgtttttcctgttgcaatggctggcggtaatattgttctggatattaccagcaaggccgatagtttgagttcttctactcaggcaagtgatgttatta ctaatcaaagaagtattgcgacaacggttaatttgcgtgatggacagactcttttactcggtggcctcactgattataaaaacacttctcaggattctggcgtaccgttcc tgtctaaaatccctttaatcggcctcctgtttagctcccgctctgattctaacgaggaaagcacgttatacgtgctcgtcaaagcaaccatagtacgcgccctgtagcg gcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgt tcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacg tagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggt ctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttacaatttaaa tatttgcttatacaatcttcctgtttttggggcttttctgattatcaaccggggtacatatgattgacatgctagttttacgattaccgttcatcgccctgcgcgctcgctcgct cactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggaattcacgcgt ggatcttaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacc cccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagt acatcaagtgtatcatatgccaagtccgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttacgggactttcctacttgg cagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacaccaatgggcgtggatagcggtttgactcacggggatttccaagtctccacc ccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaataaccccgccccgttgacgcaaatgggcggtaggcgtgtacgg tgggaggtctatataagcagagctcgtttagtgaaccgtcagatcactagaagctttattgcggtagtttatcacagttaaattgctaacgcagtcagtgcttctgacac aacagtctcgaacttaagctgcagaagttggtcgtgaggcactgggcaggtaagtatcaaggttacaagacaggtttaaggagaccaatagaaactgggcttgtcg agacagagaagactcttgcgtttctgataggcacctattggtcttactgacatccactttgcctttctctccacaggtgtccactcccagttcaattacagctcttaaggct agagtacttaatacgactcactataggctagcctcgacggtaccgcgggcccgggatccgccaccatgtctgacaaacccgatatggctgagatcgagaaattcg ataagtcgaaactgaagaagacagagacgcaagagaaaaatccactgccttccaaagaaacgattgaacaggagaagcaagcaggcgaatcgtaattgagcgg ccgcgactctagatcataatcagccataccacatttgtagaggttttacttgctttaaaaaacctcccacacctccccctgaacctgaaacataaaatgaatgcaattgtt gttgttaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcat caatgtatcttaaggcgggaattgatctaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccg ggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaaccccccccccccccccccggcgattctcttgtttgc tccagactctcaggcaatgacctgatagcctttgtagagacctctcaaaaatagctaccctctccggcatgaatttatcagctagaacggttgaatatcatattgatggt gatttgactgtctccggcctttctcacccgtttgaatctttacctacacattactcaggcattgcatttaaaatatatgagggttctaaaaatttttatccttgcgttgaaataa aggcttctcccgcaaaagtattacagggtcataatgtttttggtacaaccgatttagctttatgctctgaggctttattgcttaattttgctaattctttgccttgcctgtatgat ttattggatgttggaattcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatatggtgcactctcagtacaatctgctctgatgccgcatagttaa gccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatg tgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcagg tggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatatt gaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaaga tgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagca cttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcacc agtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcg gaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagc gtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcg gataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactg gggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactg attaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgac caaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaac aaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtcct tctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtc gtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacct acaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaaca ggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcagg ggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtgga taaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacg caaaccgcctctccccgcgcgttggccgattcattaatg

An additional aspect of the invention relates to a transformed cell comprising the polynucleotide, expression cassette, and/or vector of the invention. In some embodiments, 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.

Another aspect of the invention relates to a transgenic animal comprising the polynucleotide, expression cassette, vector, and/or the transformed cell of the invention. In some embodiments, 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. In some embodiments, 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. For injection, the carrier will typically be a liquid. For other methods of administration, the carrier may be either solid or liquid. For inhalation administration, the carrier will be respirable, and will preferably be in solid or liquid particulate form.

By "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.

In some embodiments, the polynucleotide, expression cassette, vector, and/or transformed cell of the invention is isolated.

In some embodiments, the polynucleotide, expression cassette, vector, and/or transformed cell of the invention is purified.

Methods of use.

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.

Thus, 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.

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, spleen cells, keratinocytes, fibroblasts, endothelial cells, prostate cells, joint cells (including, e.g., cartilage, meniscus, synovium and bone marrow), germ cells, and the like. Alternatively, the cell may be any progenitor cell. As a further alternative, the cell can be a stem cell (e.g., neural stem cell, liver stem cell). As still a further alternative, the cell may be a cancer or tumor cell. Moreover, the cells can be from any species of origin, as indicated above.

The polynucleotide, expression cassette, and/or vector of the invention, e.g., virus vector and/or plasmid expression vector, may be introduced to cells in vitro for the purpose of administering the modified cell to a subject. In particular embodiments, 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). Alternatively, the polynucleotide, expression cassette, and/or vector of the invention, e.g., virus vector and/or plasmid expression vector, 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 10² to about 10⁸ or about 10³ to about 10⁶ 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. In some embodiments, 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. Optionally, 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.

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.

The miR-1 regulated gene may be any gene or gene product known to be regulated to miR-1 or later discovered. In some embodiments, the miR-1 regulated gene may be Tp4.

The methods of the present invention may be used for the treatment of any disorder associated with dysregulation (e.g., aberrant overexpression and/or activity) of miR-1 and/or miR-260, and/or dysregulation of Tp4. In some embodiments, a disorder of the present invention may be amyotrophic lateral sclerosis (ALS). In some embodiments, the disorder may be familial ALS. In some embodiments, the disorder may be sporadic ALS.

Thus, 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.

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.

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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, the encoded Tp4 of the present invention may be codon-optimized, e.g., a codon-optimized coding region.

The compositions and methods of the present invention find use in both veterinary and medical applications. Suitable subjects include both avians and mammals. The term "avian" as used herein includes, but is not limited to, chickens, ducks, geese, quail, turkeys, pheasant, parrots, parakeets. The term "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. Optionally, 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. As a further option, the subject can be a laboratory animal and/or an animal model of disease. Preferably, the subject is a human.

In some embodiments of the methods of the present invention, 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.

In some embodiments, 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.

In some embodiments, 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. In some embodiments, the polynucleotide, expression cassette, vector, transformed cell, and/or composition is delivered intravenously. In some embodiments, 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. For example, in some embodiments, 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 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵, 10¹⁶ transducing units or more, e.g., about 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, or 10¹⁵ transducing units, yet more preferably about 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, or 10¹⁵ transducing units. Doses and virus titer transducing units may be calculated as vector or viral genomes (vg).

In particular embodiments, more than one administration (e.g., two, three, four or more administrations) 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 and severity of the condition being treated and on the nature of the particular vector that is being used.

In some embodiments, the vector (e.g., virus vector and/or plasmid expression vector) is administered to the CNS, the peripheral nervous system, or both.

In some embodiments, the vector (e.g., virus vector and/or plasmid expression vector) is administered directly to 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.

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.

Typically, the vector (e.g., virus vector and/or plasmid expression 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. In some embodiments, the vector can be delivered via a reservoir and/or pump. In other embodiments, 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. As a further alternative, 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. Alternatively, one may administer the virus vector in a local rather than systemic manner, for example, in a depot or sustained-release formulation. Further, 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).

Pharmaceutical 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). In general, 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. For example, 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.

Pharmaceutical 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.

Pharmaceutical 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. Examples of 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.

The 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.

Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets of the kind previously described. For example, 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. When the composition is substantially waterinsoluble, a sufficient amount of emulsifying agent, which is physiologically acceptable, can be included in sufficient quantity to emulsify the composition in an aqueous carrier. One such useful emulsifying agent is phosphatidyl choline.

Pharmaceutical 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.

Pharmaceutical 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. In some embodiments, for example, 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.

Pharmaceutical 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 (e.g., virus vectors and/or plasmid expression vectors) disclosed herein 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.

The invention will now be described with reference to the following examples. It should be appreciated that these examples are not intended to limit the scope of the claims to the invention but are rather intended to be exemplary of certain embodiments. Any variations in the exemplified methods that occur to the skilled artisan are intended to fall within the scope of the invention.

EXAMPLES

Example 1: Enhanced miR-1 in the spinal cord of ALS.

Expression of mir-1 and miR-206 was 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.

Example 2: Tp4 is a possible target gene of miR-1.

The TargetScan.org database was used to screen possible target genes, among of which TP4 gene was considered to be the most promising (FIG. 2A). To confirm that Tp4 was a target of miR-1/206 in the invention, the 3'-UTR sequences of Tp4 (Tp4-3'-UTR) or a mutant sequence (TP4-3¹ UTR-mut, from ACATTCCA to ACAAAGGA) were inserted into the 3 '-terminus of a luciferase reporter gene (FIG. 2B). Over-expression of shRNA206 in vitro reduced the levels of luciferase containing the TP4-3 TR target sequence by about 67%, but did not inhibit the expression of the luciferase with the mutated TP4-3' UTR (FIG. 2C). Similarly, in treatment groups comprising either pEMBL-CMV-pre-miR-1 or pEMBL- CMV-pre-miR-206, the expression of luciferase with Tp4-3'UTR was inhibited by about 37.55% and 30.62%, respectively, compared to control. The expression of luciferase with Tp4-3'UTR-mut could not only be downregulated by pEMBL-CMV-pre-miR- 1/206 treatment, but inversely increased from losing the inhibition of endogenous miR- 1/206 in cells (FIG. 2D). These data confirm that Tp4 is a specific target of miR- 1/206.

Example 3: Tp4 is deficient in ALS spinal cord.

This study found that 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). However, Tp4 mRNA levels did not decrease accordingly (FIG. 3B). These data indicate that TP4 is curbed by miR- 1/206 through post- transcriptional regulation.

To further support this relationship, 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 4: Anti-miR-1 down-regulates miR-1 level.

Since miR-1 increased in the spinal cord of ALS during the presymptomatic and symptomatic stage of disease, it was hypothesized that downregulation of miR-1 would be therapeutic in ALS. Two constructs of anti-miR-1/206 were developed in order to reduce the level of miR- 1/206 both in vitro and in vivo. First, 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. Addition of two anti-miR plasmids pEMBL-CMV-GFP-4><anti-miR-l and pEMBL-CMV-GFP-4xanti-miR-206 individually rescued the luciferase expression, but the plasmid with four copies of anti-miR-138 did not (FIG. 6A and FIG. 6B). These data suggested that the two anti-miR plasmids pEMBL-CMV-GFP-4><anti-miR-l and pEMBL- CMV-GFP-4xanti-miR-206 could antagonize the mature miR-1 and miR-206. When applied to Neuro-2a (N2a) cells, a mouse neuroblastoma cell line, infection with AAV2-CMV-GFP- 4xanti-miR-l/206 vectors could rescue the expression level of Tp4 inhibited by the treatment of AAV2-CMV-G93A-SOD1 (FIG. 6C), suggesting that Tp4 was regulated by miR- 1/206.

Example 5: Anti-miR-1 protects neurons and delays the survival of ALS.

To identify therapeutic effects of antagonizing miR-1/206, neonates of C57BL/6J- background ALS (B6.Cg-Tg(SODl*G93A)lGur/J ) were treated with AAV9-CMV-GFP- 4><anti-miR-l and AAV9-CMV-GFP-4xanti-miR-206 vectors via superficial temporal vein injection at 1 day post-natal. The observed survival ratio was increased up to 14.5 days by anti-miR-1 treatment and 11.5 days by anti-miR-206 (FIG. 7A and FIG. 7B). The average survival days reached up to 14.7 and 12.6 days in the two treated groups (FIG. 7E). Further analysis showed 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).

After samples were retrieved from the ALS mice at the 16th week, western blot analysis showed that the expression of TP4 in the spinal cord was rescued by anti-miR-1 and anti-miR-206 treatment (FIG. 7G and FIG. 7H). Nissl staining discovered the number of motor neurons at the anterior horn was enhanced by 1.8 and 2.0 fold, respectively, in the anti- miR-1 and anti-miR-206 treatment group compared with control group (FIG. 71 and FIG. 7 J). Thus, the delivery of anti-miR-1 and anti-miR-206 was shown to be able to effectively counteract downregulation by miR-1 on Tp4 expression in the spinal cord, restore the number of motor neurons, and postpone the progression of ALS.

Example 6: Over-expression of Tp4 postpones ALS progression.

Following the discovery that deficiency of Tp4 in ALS plays an important role in the progression of ALS, it was determined whether in vivo over-expression of Tp4 could alleviate ALS symptoms. First, 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). In another group, 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).

In addition, 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). At the same time, 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.

Western blot results showed IV and IT injection with AAV9-CMV-TP4 significantly enhanced the expression of TP4 in the spinal cord (FIG. 11A and FIG. 11B). Immunostaining showed Tp4 expressed well in the surviving neuron cells of the anterior horn in both IV- and IT-treated groups compared with control group (FIG. 11C). Nissl staining further confirmed PBS-treated ALS group lost most of motor neuron cells in the anterior horn, but Tp4 treatment by IV or IT prevented neuron cell death and increased the number of surviving motor neurons about 2.1 and 2.4 fold, as compared with PBS control group (FIG. 11D and FIG. HE). Thus, AAV9-mediated exogenous expression of Tp4, by either IV treatment in newborn mice or IT treatment in young adult mice, could prevent the death of motor neuron cells, improve the function of skeletal muscles, and delay the survival ratio of ALS mice.

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). Upon diagnosis of ALS, the expected survival time is only 5 years, and current pharmacological treatments such as Riluzole may only increase survival time by 3 to 5 months. Thus, understanding the pathological mechanism of ALS to identify more effective therapies is imperative. MicroRNAs (miRNAs) 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. In a previous study, whole-body knockout of miR-206 decreased compensatory muscle innervation and worsened ALS pathology; however, exogenous expression of miR-206 inhibited cell proliferation, and promoted cell apoptosis (Williams et al 2009; Wang et al. 2012 Cell Physiol Biochem 29:381-390; Singh et al. 2013 J Clin Invest 123:2921-2934). The results of these studies suggest miR-206 has different roles in developmental and pathological processes. Thus far, the function of miR- 1/206 and their role in ALS pathology remain elusive.

In the present study, 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. It was also shown that miR-1, but not miR-206, is highly upregulated in the spinal cord of G93A-SOD1 ALS mice. It was further demonstrated that 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. Both miR-1 antagonism and AAV9-TP4 treatment were shown to increase the expression of TP4 in the spinal cord, improve the active function of limb muscles and extend the survival time of G93A-SOD1 ALS mice.

To determine the role of miR-1/206, 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. To serve as a control, the 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. Purified vectors were then injected into 1 -day-old C57BL/6J neonates by temporal vein injection. After 1 to 2 months, the shRNA-206-treated mice began to exhibit abnormal gait and limb grasping reflex with upside-down hanging. Eventually, shRNA-206-treated mice progressed to full paralysis. The control shRNA-206mut-treated mice did not exhibit any of the aforementioned symptoms. The mice in the shRNA-206 treatment group also had consistently lower bodyweights on average compared to the shRNA-206mut group (FIG. 12A). In long-term observation, the shRNA-206-treated mice had a higher mortality rate than shRNA-206mut controls. (FIG. 12B)

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. 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. Furthermore, 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. In addition, transmission electron microscopy (TEM) showed that the branched nerves were undergoing demyelination, and other pathological signs such as numerous vacuoles were present within the axons in the shRNA-206-treated group (FIG. 12C). Consistently, the hindlimb muscles exhibited atrophy after shRNA-206 treatment, as evidenced by decreasing muscle mass and fiber size (FIG. 12D) Thus, over-expression of shRNA-206 resulted in the loss of motor neurons, followed by nerve degeneration and muscle atrophy.

In ALS patients and animal models, 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.

In order to imitate the natural processing of miRNAs, 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. 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). The results showed pEMBL-CMV-pre- miR-1 and pre-miR-206 efficiently downregulated GFP expression of the reporter plasmid pEMBL-CMV-GFP-4xanti-miR-l/206. No difference in GFP expression was seen after transfection with the control plasmid containing the empty hCG introns or the four copies of the miR-138 precursor. 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. The results of real-time PCR analysis showed that the precursor of miR-1/206 was processed correctly into mature miR-1/206 in the spinal cord from AAV9-CMV-pre-miR-l/206-treated groups. In order to discern pathological morphology, Nissl staining was used to detect the neuronal cells in the spinal cord. It was found that the size and number of motor neurons were decreased in the anterior horn of AAV9-CMV-pre-miR-l/206-treated mice in comparison to the control group. Also, short neuritis-like inclusions of TDP-43 were observed in the spinal cord of AAV9-CMV-pre-miR-l and pre-miR-206-treated mice but not in the control group. Skeletal muscle mass and hematoxylin and eosin (H&E) staining in muscle sections revealed signs of atrophy in the pre-miR-1/206 treatment groups. These pathological changes in morphology indicated that overexpression of miR-1/206 in the spinal cord also causes the death of motor neurons, followed by the atrophy of the involved skeletal muscles, which is similar to the mechanisms of ALS disease.

It remained to be determined whether 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). However, 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). In the brain cortex, however, there is no significant difference in miR- 1 or miR-206 expression levels between WT and ALS mice. In skeletal muscle, 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. Thus, 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.

The TargetScan database was used to screen possible target genes of miR-1, and the TP4 gene was determined to be the most promising (FIG. 2A). To confirm that Tp4 was a target of miR-1/206, the 3'-UTR sequences of Tp4 (Tp4-3'-UTR) or its mutant sequence (TP4-3¹ UTR-mut, from ACATTCCA to ACAAAGGA) were inserted into the 3 '-terminus of a luciferase reporter gene (FIG. 2B). Over-expression of shRNA206 in vitro reduced the levels of luciferase containing the Tp4-3'UTR target sequence by about 67%, but did not inhibit the expression of the luciferase with a mutated TP4-3' UTR (FIG. 2C). Similarly, in the pEMBL-CMV-pre-miR-1 and pEMBL-CMV-pre-miR-206-treated groups, the expression of luciferase with Tp4-3'UTR was inhibited by about 37.6% and 30.6%, respectively, compared to the control. The expression of luciferase with Tp4-3'UTR-mut could not be downregulated by pEMBL-CMV-pre-miR- 1/206 treatment, but instead was increased due to the loss inhibition of endogenous miR-1/206 in cells (FIG. 2D). These data confirm that Tp4 is a specific target of miR-1/206.

Because miR-1 was overexpressed in the spinal cord of G93A-SOD1 ALS mouse model, it was next examined whether the expression level of 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). However, Tp4 mRNA levels did not decrease accordingly. These data indicated that 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. In vivo, overexpression of the mutated gene G93A-SOD1 in the spinal cord was responsible for Tp4 deficiency. To confirm these findings in vitro, human G93A-SOD1 was over-expressed by adding AAV2-CMV-G93A-SOD1 vectors into SH-SY5Y cells, a human- derived neuronal cell line. Western blot showed that, following AAV2-CMV-G93A-SOD1 treatment, Tp4 expression was inhibited by 41.1% and 41.5% compared with GFP and wild type SOD1 treatment groups, respectively (FIGS. 4A and 4B). Thus, these results indicated that the overexpression of G93A-SOD1 negatively regulated the expression level of Tp4 in vivo and in vitro.

Since 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. First, 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. The addition of two anti-miR plasmids pEMBL-CMV-GFP- 4><anti-miR-l and pEMBL-CMV-GFP-4><anti-miR-206 individually rescued the luciferase expression, but the plasmid with four copies of anti-miR-138 did not (FIGS. 6A and 6B). This suggests that the two anti-miR plasmids pEMBL-CMV-GFP-4><anti-miR-l and pEMBL-CMV-GFP-4xanti-miR-206 can antagonize the mature miR-1 and miR-206. In mouse neuroblastoma Neuro-2a (N2a) cells, infection with AAV2-CMV-GFP-4xanti-miR- 1/206 vectors could rescue TP4 expression inhibited by human G93A-SOD1 overexpression of AAV2-CMV-G93A-SOD1 (FIG. 6C), which confirmed that Tp4 was regulated by miR- 1/206 and that miR-1/206 could be blocked by the anti-miR- 1/206.

To identify the therapeutic effect of miR-1/206 antagonism, w 1-day-old neonates on a C 57BL/6J-b ackground ALS (B6.Cg-Tg(SODl*G93A)lGur/J) were treated with AAV9- CMV-GFP-4xanti-miR-l and AAV9-CMV-GFP-4xanti-miR-206 vectors via temporal vein injection. On survival analysis, the survival ratio was increased up to 14.5 days by anti-miR-1 treatment and 11.5 days by anti-miR-206 (FIGS. 7A and 7B). The average survival time reached up to 14.7 and 12.6 days in the two treated groups (FIG. 7E). Further analysis showed 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). After sacrificing the ALS mice at the 16th week, western blot showed that expression of Tp4 in the spinal cord was rescued by anti-miR-1 and anti-miR-206 treatment (FIGS. 7G and 7H) Nissl staining revealed the number of motor neurons in the anterior horn was increased by 1.8- and 2-fold in the anti-miR-1 and anti-miR-206 treatment group compared with control group, respectively (FIGS. 71 and 7 J). Thus, the delivery of anti-miR-1 and anti-miR-206 effectively counteracted miR-1 -induced downregulation of Tp4 expression in the spinal cord leading to restoration of motor neurons and extension of survival time in the ALS mice.

Given the role of TP4 in the motor neurons of ALS mice, it was next determined whether in vivo overexpression of Tp4 could directly alleviate the progression of ALS. First, 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. In another group, 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). In particular, 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). IT injection at the early stages, before the appearance of ALS symptoms, extended lifespan up to 15.5 days (FIG. 9B). The average time to death was delayed up to 19.7 and 17 days by TP4-IV and IT treatment, respectively (FIG. 9C, TP4-IV group 148.6±2.5 days and TP4-IT group 145.9±3.5 days vs PBS group 128.9±2.6 days, P <0.001). Further analysis showed Tp4 injection by IV and IT postponed the disease onset of ALS mice 16 days and 11 days, individually (FIGS. 9D and 9E), but the disease progression was not significantly extended (FIG. 9F). At the same time, the volume and mass of hind limb skeletal muscle were increased by both Tp4 IV and IT treatment compared with PBS. Consequently, forelimb grip force was also enhanced significantly in both of Tp4-treatment groups (FIGS. 10A and 10B) Western blot results showed IV and IT injection with AAV9- CMV-TP4 significantly enhanced the expression of Tp4 in the spinal cord (FIGS. 11A and 11B). Immunostaining showed Tp4 expressed well in the surviving neuron cells of the anterior horn in both IV- and IT-treated groups compared with control group (FIG. 11C). Nissl staining further confirmed PBS-treated ALS group did lose the majority of motor neuron cells in the anterior horn, but Tp4 treatment by IV and IT administration prevented motor neuron death and increased the number of the survived motor neurons approximately 2.1- and 2.4- fold compared with PBS group, respectively (FIG. 11D). Thus, AAV9- mediated exogenous overexpression of Tp4, by either IV treatment in neonatal mice or IT treatment in young adult mice, prevented the death of motor neuron cells, improved the function of skeletal muscles and extended the survival time of ALS mice.

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.

In this study, G93A-SOD1 ALS mice (6-, 10- and 14-week-old, half male and half female) receive AAV viruses (l x l0ⁿvg/mouse, including AAV9-Syn-GFP, AAV9-Syn-anti- miR-1 and AAV9-Syn-Opti-Tp4) by IT injection, individually. To observe the curve of body weight and the motor function of ALS mice treated by anti-miR-1 and Tp4, all the mice are weighed every week after AAV virus injection. The grip force, latency time on rotated rod and treadmill are performed each month. To analyze survival curves, disease onset and average survival days are tracked. The ALS disease onset is set at the time point when the ALS mouse begins to lose body weight from the peak. The endpoint is the time when the mouse cannot right itself in 20 seconds after it is laid on one side.

To identify whether the enhanced miR-1 could be inhibited by AAV-mediated anti- miR-1, 4~6 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. The level of miR-1 is detected by NCodeTM miRNA qRT-PCR kit.

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. In the two-in-one construct, 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.

Evaluation of enhanced miR-1 and deficient Tp4 in CSF may be an early diagnostic indicator in the clinic. The diagnosis of ALS is currently primarily based on patient symptoms and signs, and a series of tests to exclude other diseases (Campos-Melo et al. 2013 Mol Brain 6:26). 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.

To evaluate safety, the vectors are administered in non-human primates for their clinical application. 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) are used in the study. Nine monkeys without pre-existing anti-AAV9 antibody are divided into 3 groups (n=3 monkeys/group), including AAV9-Syn-GFP (control), AAV9-Syn-anti-miR-l, and AAV9-Syn-Opti-Tp4 treatment groups. Each monkey receives 3.0* 10¹³ vg of the virus vector injected by IT. After the animals are under anesthesia by inhalation of 1% to 2% isoflurane in oxygen, the animals are placed in lateral recumbence. 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.

Table 3.

The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims

THAT WHICH IS CLAIMED IS:

1. A synthetic polynucleotide encoding one or more anti-miRNA oligonucleotide (AMO) directed to microRNA-1 (miR-1) and/or miR-206, wherein the AMO is about 20 to 25 nucleotides.

2. The synthetic polynucleotide of claim 1, encoding four AMOs directed to miR-1 and/or miR-206.

3. The synthetic polynucleotide of claim 1 or 2, wherein the AMO binds to a shared seed sequence of miR-1 and miR-206.

4. The synthetic polynucleotide of claim 3, wherein the seed sequence comprises a nucleotide sequence which binds to the nucleotide sequence ACAUUCC or a nucleotide sequence having at least about 90% identity thereto.

5. The synthetic polynucleotide of claim 3 or 4, wherein the seed sequence comprises the nucleotide sequence UGUAAGG or a nucleotide sequence having at least about 90% identity thereto.

6. The synthetic polynucleotide of claim 1 or 2, wherein the AMO binds to a portion of miR-1 outside of the shared seed sequence.

7. The synthetic polynucleotide of claim 1 or 2, wherein the AMO binds to a portion of miR-206 outside of the shared seed sequence.

8. The synthetic polynucleotide of any one of claims 1-7, wherein the AMO comprises the nucleotide sequence of SEQ ID NO: 3 or a nucleotide sequence having at least about 90% identity thereto.

9. The synthetic polynucleotide of claim 8, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 5 or a nucleotide sequence having at least about 90% identity thereto.

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10. The synthetic polynucleotide of any one of claims 1-7, wherein the AMO comprises the nucleotide sequence of SEQ ID NO:4 or a nucleotide sequence having at least about 90% identity thereto.

11. The synthetic polynucleotide of claim 10, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 6 or a nucleotide sequence having at least about 90% identity thereto.

12. An expression cassette comprising a synthetic polynucleotide encoding one or more copies of an anti-miRNA oligonucleotide (AMO) directed to microRNA-1 (miR-1) and/or miR-206.

13. The expression cassette of claim 12, wherein the synthetic polynucleotide is the polynucleotide of any one of claims 1-11.

14. The expression cassette of claim 12 or 13, wherein the one or more AMO is operably linked to a promoter and/or enhancer.

15. The expression cassette of claim 14, wherein the promoter is a cytomegalovirus (CMV) promoter or a synapsin (e.g., syn-1) promoter.

16. An expression cassette comprising a synthetic polynucleotide comprising a coding region encoding a human thymosin P4 (Tp4), wherein the encoded human Tp4 comprises SEQ ID NO: 7 or a sequence at least about 90% identical thereto.

17. The expression cassette of claim 16, wherein the human Tp4 coding region is codon- optimized.

18. The expression cassette of claim 16 or 17, wherein the polynucleotide is operably linked to a promoter and/or enhancer.

19. The expression cassette of claim 18, wherein the promoter is a cytomegalovirus (CMV) promoter or a synapsin (e.g., syn-1) promoter.

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20. A vector comprising the synthetic polynucleotide of any one of claims 1-11 or the expression cassette of any one of claims 12-19.

21. The vector of claim 20, wherein the vector is a plasmid (e.g., pEMBL).

22. The vector of claim 21, comprising the nucleotide sequence of SEQ ID NO:8 or a sequence at least about 90% identical thereto.

23. The vector of claim 20, wherein the vector is a viral vector.

24. The vector of claim 23, wherein the viral vector is an AAV vector.

25. The vector of claim 24, wherein the AAV vector is an AAV2 vector or AAV9 vector.

26. A transformed cell comprising the polynucleotide of any one of claims 1-11, the expression cassette of any one of claims 12-19, and/or the vector of any one of claims 20-25.

27. A transgenic animal comprising the polynucleotide of any one of claims 1-11, the expression cassette of any one of claims 12-19, the vector of any one of claims 20-25, and/or the transformed cell of claim 26.

28. A pharmaceutical composition comprising the polynucleotide of any one of claims 1- 11, the expression cassette of any one of claims 12-19, the vector of any one of claims 20-25, and/or the transformed cell of claim 26 in a pharmaceutically acceptable carrier.

29. A method of reducing miR-1 expression in a cell, comprising contacting the cell with the polynucleotide of any one of claims 1-11, the expression cassette of any one of claims 12- 15, and/or the vector of any one of claims 20, 21, or 23-25.

30. A method of enhancing thymosin beta 4 (Tp4) protein expression in a cell, comprising contacting the cell with the polynucleotide of any one of claims 1-11, the expression cassette of any one of claims 12-19, and/or the vector of any one of claims 20-25.

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31. A method of reducing miR-1 expression in a subject, comprising delivering to the subject the polynucleotide of any one of claims 1-11, the expression cassette of any one of claims 12-19, the vector of any one of claims 20-25, and/or the transformed cell of claim 26.

32. A method of enhancing thymosin beta 4 (Tp4) protein expression in a subject, comprising delivering to the subject the polynucleotide of any one of claims 1-11, the expression cassette of any one of claims 12-19, the vector of any one of claims 20-25, and/or the transformed cell of claim 26.

33. 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 the polynucleotide of any one of claims 1-11, the expression cassette of any one of claims 12-15, the vector of any one of claims 20, 21, or 23-25, and/or the transformed cell of claim 26, and/or the pharmaceutical composition of claim 28.

34. The method of claim 33, wherein the miR-1 regulated gene is Tp4.

35. The method of claim 33 or 34, wherein the disorder is amyotrophic lateral sclerosis

(ALS).

36. The method of claim 35, wherein the disorder is familial ALS.

37. The method of claim 35, wherein the disorder is sporadic ALS.

38. 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 the polynucleotide of any one of claims 1-11, the expression cassette of any one of claims 12-19, the vector of any one of claims 20-25, the transformed cell of claim 26, and/or the pharmaceutical composition of claim 28.

39. The method of claim 38, wherein the disorder is amyotrophic lateral sclerosis (ALS).

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40. The method of claim 39, wherein the disorder is familial ALS.

41. The method of claim 39, wherein the disorder is sporadic ALS.

42. A method of treating amyotrophic lateral sclerosis (ALS) (e.g., familial or sporadic ALS) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the polynucleotide of any one of claims 1-11, the expression cassette of any one of claims 12-19, the vector of any one of claims 20-25, the transformed cell of claim 26, and/or the pharmaceutical composition of claim 28.

43. A method of treating sporadic amyotrophic lateral sclerosis (ALS) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the polynucleotide of any one of claims 1-11, the expression cassette of any one of claims 12-19, the vector of any one of claims 20-25, the transformed cell of claim 26, and/or the pharmaceutical composition of claim 28.

44. A method of treating familial amyotrophic lateral sclerosis (ALS) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the polynucleotide of any one of claims 1-11, the expression cassette of any one of claims 12-19, the vector of any one of claims 20-25, the transformed cell of claim 26, and/or the pharmaceutical composition of claim 28.

45. A method of treating amyotrophic lateral sclerosis (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 anti-miRNA oligonucleotide (AMO) directed to microRNA-1 (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 thymosin P4 (Tp4), wherein the encoded human Tp4 comprises SEQ ID NO:5 or a sequence at least about 90% identical thereto, and/or an expression cassette, vector, transformed cell, and/or pharmaceutical composition comprising the same.

46. A method of postponing disease progression of amyotrophic lateral sclerosis (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:

47. A method of reducing disease severity of amyotrophic lateral sclerosis (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:

48. The method of any one of claims 45-47, wherein the synthetic polynucleotide of step

(a) comprises the synthetic polynucleotide of any one of claims 1-11.

49. The method of any one of claims 45-48, wherein the synthetic polynucleotide of step

(b) comprises the synthetic polynucleotide comprised in the expression cassette of any one of claims 16-19.

50. The method of any one of claims 30-49, wherein the subject exhibits symptoms of the disease prior to delivery of the polynucleotide, expression cassette, vector, transformed cell, and/or pharmaceutical composition.

51. The method of any one of claims 30-49, wherein the subject is 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.

52. The method of any one of claims 30-51, wherein the subject is a human.

53. The method of any one of claims 30-52, wherein the subject is an adult.

54. The method of any one of claims 33-53, wherein the polynucleotide, expression cassette, vector, transformed cell, and/or pharmaceutical composition is delivered to the nervous system of the subject.

55. The method of any one of claims 30-54, wherein the polynucleotide, expression cassette, vector, transformed cell, and/or pharmaceutical composition is delivered intravenously.

56. The method of any one of claims 30-54, wherein the polynucleotide, expression cassette, vector, transformed cell, and/or pharmaceutical composition is delivered by intrathecal, intracerebral, intraparenchymal, intracerebroventricular, intranasal, intra-aural, intra-ocular, or peri-ocular delivery, or any combination thereof.

57. The method of claim 56, wherein the polynucleotide, expression cassette, vector, transformed cell and/or pharmaceutical composition is delivered intrathecally.

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