HK1262485B - Method and composition for treating neuronal hyper-excitability - Google Patents

Description

Methods and compositions for treating neuronal hyperexcitability

Cross-references to related literature

This application claims priority under 35 U.S.C. §119(e) to U.S. Serial No. 62/314,128, filed on March 28, 2016, the entire contents of which are incorporated herein by reference.

Funding Information

This invention was made with government support under Grant No. NS051644-02A2 awarded by the National Institutes of Health. The U.S. Government has certain rights in this invention.

Technical Field

The present invention relates generally to treating spinal cord injury, and more particularly to combined therapeutic regimens for regulating chronic spasticity in patients following traumatic or ischemic injury to the spinal cord.

Background Art

Spinal cord injury (traumatic or ischemic) can lead to the development of clinically defined spasticity and rigidity. One of the potential mechanisms leading to the development of spasticity after spinal cord injury is believed to be the loss of local segmental inhibition, resulting in: i) increased firing of tonic motor neurons, ii) increased primary afferent input during muscle stretch, and/or iii) exacerbated responses to peripheral sensory stimuli (i.e., allodynia). Loss of gamma-aminobutyric acid (GABA)-mediated presynaptic, reciprocal, and reciprocal postsynaptic inhibition, as well as its inhibitory effect in flexor afferent pathways, has been shown to represent one of the key mechanisms.

Interestingly, however, previous studies have shown a significant increase in spinal cord parenchyma GAD67 expression in lumbar spinal cord segments of Th12 transected cats. Similarly, in adult rats that underwent a thoracic median spinal cord transection on postnatal day 5, an increase in the density of inhibitory boutons apposing the membrane of α-motoneurons has been shown. These data suggest that a static increase in GABA synthase in spinal interneurons, or an increase in the number of inhibitory contacts with α-motoneurons after spinal cord trauma in the absence of specific inhibitory neuronal driver activity, is insufficient to prevent the development of spasticity/hyperreflexia. In addition to the effects of reduced inhibition, several other potential mechanisms have been suggested to contribute to the development of spasticity after spinal cord trauma, including: i) a progressive increase in α-motoneuron 5- _HT2C receptor activity, which spontaneously becomes active in the absence of brain-derived serotonin, or ii) downregulation of the potassium chloride cotransporter KCC2 in motor neurons, leading to GABA-mediated depolarization. Collectively, these data suggest that the mechanisms leading to the development of spasticity following spinal cord injury (traumatic or ischemic) are complex and can vary depending on the model used and the age of the experimental animal when the injury is induced.

Clinical drug treatment studies have shown that the use of systemic or spinal administration of baclofen (GABA _B receptor agonist) represents the most effective antispasticity drug treatment. Although it is effective for regulating spasticity of different causes (including spinal cord trauma, amyotrophic lateral sclerosis or central stroke), major side effects such as general sedation and progressive tolerance development often limit its long-term use. The use of systemic administration of GABA-like compounds such as tiagabine (GABA reuptake inhibitor) shows only weak or no antispasticity effect in clinically acceptable doses, which is associated with a relatively moderate enhancement of GABA release in the brain or spinal cord parenchyma after systemic delivery. In addition, currently available spinal drug delivery systems (such as epidural or intrathecal delivery) do not allow spinal cord segment-restricted therapeutic effects. This is because the origin of spasticity affecting individual muscle groups can be mapped to specific spinal cord segments by the body, so by reducing unwanted side effects, the development of segment-targeted antispasticity treatments will have significant advantages over current treatment methods. Therefore, there is a demand for novel antispasticity treatments.

Summary of the Invention

The present invention is based on the observation that combination treatment consisting of spinal segment-specific upregulation of GAD65 (glutamate decarboxylase) and VGAT (vesicular GABA transporter) in rats with ischemia-induced spasticity induces an antispastic effect and that such combination treatment results in a reduction of muscle spasticity.

Therefore, the invention provides a method for treating a spasm in a subject. The method includes raising GAD65 (glutamic acid decarboxylase) gene and VGAT (vesicle GABA transporter) gene, so as to treat a spasm in a subject. The raising of GAD65 gene and VGAT gene can be the spinal cord specific raising of GAD65 gene and VGAT gene, by administering a viral vector comprising a polynucleotide encoding GAD65 and VGAT to the subject, wherein GAD65 and VGAT are expressed, so as to reduce spasm. GAD65 gene and VGAT gene may be overexpressed. Carrier can be a lentiviral vector, an adenoviral vector or an adeno-associated vector (AAV). AAV can be AAV 9 types (AAV9). In various embodiments, the viral vector is directly administered to the spinal cord parenchyma of the subject, to the intrathecal space of the subject, to the spinal cord subpial space of the subject or to the peripheral spastic muscles of the subject.

On the other hand, the present invention provides a method for treating spasm in a subject. The method includes administering a viral vector of a therapeutically effective amount to a subject in need thereof, comprising a polynucleotide encoding GAD65 gene and VGAT gene, thereby treating spasm in the subject. The vector can be a lentiviral vector, an adenoviral vector or an adeno-associated vector (AAV), and can be directly administered to the spinal cord of the subject. AAV can be an AAV9 type (AAV9). In various embodiments, the vector is administered directly to the spinal cord parenchyma of the subject, to the intrathecal space of the subject, to the spinal cord subpial space of the subject, or to the peripheral spastic muscles of the subject.

On the other hand, the invention provides a therapeutic regimen for treating a subject suffering from spinal cord injury.The therapeutic regimen includes administering a viral vector comprising the polynucleotides encoding GAD65 and VGAT, wherein GAD65 and VGAT are expressed, thereby reducing spasm.The increase of GAD65 and VGAT includes administering a viral vector encoding GAD65 and VGAT, wherein GAD65 and VGAT are expressed and reduce spasm.Carrier can be a lentiviral vector, an adenoviral vector or an adeno-associated vector, and can be directly administered to the spinal cord parenchyma of the subject, to the intrathecal space of the subject, to the spinal cord subpial space of the subject or to the peripheral spastic muscles of the subject.In various embodiments, carrier is directly administered to the spinal cord parenchyma of the subject, to the intrathecal space of the subject, to the spinal cord subpial space of the subject or to the peripheral spastic muscles of the subject.

In another aspect, the present invention provides an expression cassette comprising a promoter or regulatory sequence functionally linked to a polynucleotide encoding GAD65 and VGAT. A vector such as AAV9 is also provided, comprising a regulatory sequence such as a promoter functionally linked to a polynucleotide encoding GAD65 and VGAT.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic diagram showing exemplary methodology for practicing the methods of the present invention.

Figure 2 is a schematic diagram showing the distribution of transgene expression achieved after subpial AAV9-UBI-GFP delivery in the lumbar spine. Extensive GFP expression can be seen in interneurons throughout the gray matter. AAV9 viruses encoding GAD65 (glutamate decarboxylase 65) and VGAT (vesicular GABA transporter) were injected into the targeted segment using a subpial delivery method.

3A-3D are graphs showing the potent anti-spasticity and anti-nociceptive effects of AAV9-UBI-GAD65+VGAT delivered subpially to the lumbar spine of rats with chronic spinal cord transection-induced spasticity.

Figure 4A-Figure 4D is a schematic diagram showing the induction of mixed inhibitory-excitatory neurotransmitter phenotypes in spinal cord excitatory interneurons by lumbar subpial AAV9-UBI-GAD65+VGAT delivery. At week 8, immunofluorescence analysis of segments injected with GAD65/VGAT genes showed significant upregulation of both genes and the appearance of mixed inhibitory/excitatory neurotransmitter phenotypes (co-expression of GAD65 or VGAT with VGLUT2 (vesicular glutamate transporter)) (Figures 4A and 4B). Co-expression was not found in animals injected with control AAV9 (Figures 4C and 4D). These data confirm the effective induction of inhibitory drive in neurons overexpressing GAD65/VGAT, which may mediate the reduction of muscle spasms.

Figure 5 is a graph showing that the number of mixed inhibitory-excitatory interneurons and projecting DRG neurons in the lumbar spinal cord of spastic rats increased significantly after lumbar subpial AAV9-UBI-GAD65+VGAT delivery. The table shows the quantitative analysis of GAD65 and VGAT expression.

DETAILED DESCRIPTION

The present invention is based on the observation that combination therapy consisting of spinal segment-specific upregulation of the GAD65 (glutamate decarboxylase) gene and the VGAT (vesicular GABA transporter) gene in rats with ischemia-induced spasticity induces an antispastic effect and that this combination therapy results in a reduction of muscle spasticity.

Before describing the compositions and methods of the present invention, it should be understood that the present invention is not limited to the specific compositions, methods and experimental conditions described, as such compositions, methods and conditions may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the present invention is limited only by the appended claims.

As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "the method/the method" includes one or more methods, and/or steps of the type described herein, that will become apparent to one skilled in the art upon reading this disclosure and the like. The term "comprising," which is used interchangeably with "including," "containing," or "characterized by," is inclusive or open-ended language and does not exclude additional, unrecited elements or method steps. The phrase "consisting of excludes any element, step, or ingredient not specified in the claim. The phrase "consisting essentially of limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claimed invention. The present invention contemplates embodiments of the compositions and methods of the present invention that correspond to the scope of each of these phrases. Thus, a composition or method that includes a recited element or step contemplates a specific embodiment in which the composition or method consists essentially of those elements or steps.

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 the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.

As used herein, the term "subject" refers to any individual or patient who performs the methods of the present invention. Typically, the subject is a human, but as will be appreciated by those skilled in the art, the subject may be an animal. The definition of subject therefore also includes other animals, including mammals such as rodents (including mice, rats, hamsters, and guinea pigs), cats, dogs, rabbits, farm animals including cattle, horses, goats, sheep, pigs, and the like, and primates (including monkeys, chimpanzees, orangutans, and gorillas).

As used herein, "therapeutic effect" includes therapeutic benefit and/or prophylactic benefit as described herein.

As used herein, the terms "reduce" and "inhibit" are used together because it is recognized that in some cases, reduction can be reduced to below the detection level of a particular assay. Therefore, it may not always be clear whether the expression level or activity is "reduced" to below the detection level of the assay, or is completely "inhibited." However, after treatment according to the present invention, it will be clearly determinable.

As used herein, "treatment" or "treating" means administering a composition to a subject or system having an undesirable condition. The condition may include a disease or disorder. "Prevention" or "preventing" refers to administering a composition to a subject or system at risk of having a condition. The condition may include a susceptibility to a disease or disorder. The effect of administering a composition to a subject (treating and/or preventing) can be, but is not limited to, cessation of one or more symptoms of the condition, reduction or prevention of one or more symptoms of the condition, reduction in the severity of the condition, complete elimination of the condition, stabilization or delay in the development or progression of a particular event or characteristic, or minimization of the chance that a particular event or characteristic will occur.

The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

The term "amino acid" refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a similar manner to naturally occurring amino acids. Naturally occurring amino acids are the amino acids encoded by the genetic code, as well as those that are later modified, such as hydroxyproline, α-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., an α-carbon bound to a hydrogen, a carboxyl group, an amino group, and an R group), such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid but function in a similar manner to naturally occurring amino acids.

Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Likewise, nucleotides may be referred to by their commonly accepted single-letter codes.

As used herein, a "regulatory gene" or "regulatory sequence" is a nucleic acid sequence that encodes a product (eg, a transcription factor) that controls the expression of other genes.

As used herein, a "protein coding sequence" or a sequence encoding a specific protein or polypeptide is a nucleic acid sequence that is placed under the control of appropriate regulatory sequences and transcribed into mRNA (in the case of DNA) and translated into a polypeptide (in the case of mRNA) in vitro or in vivo. The boundaries of the coding sequence are determined by a start codon at the 5' end (N-terminus) and a translation termination nonsense codon at the 3' end (C-terminus). Coding sequences may include, but are not limited to, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic DNA, and synthetic nucleic acids. A transcription termination sequence is typically located at the 3' end of the coding sequence.

As used herein, "promoter" is defined as a regulatory DNA sequence generally located upstream of a gene that mediates transcription initiation by guiding RNA polymerase to bind DNA and initiate RNA synthesis. A promoter can be a constitutively active promoter (i.e., a promoter that is continuously active/"ON" state); It can be an inducible promoter (i.e., a promoter in an active/"ON" or inactive/"OFF" state that is controlled by an external stimulus (e.g., the presence of a specific compound or protein); It can be a spatially restricted promoter (i.e., transcription control element, enhancer, etc.) (e.g., tissue-specific promoter, specific cell type promoter, etc.); And it can be a time-restricted promoter (i.e., at a specific stage of embryonic development or during a specific stage of a biological process, the promoter is in an "ON" state or an "OFF" state).

As used herein, the term "gene" refers to the deoxyribonucleotide sequence comprising the coding region of a structural gene. A "gene" may also include non-translated sequences located near the coding region at the 5' and 3' ends, such that the gene corresponds to the length of the full-length mRNA. Sequences located at the 5' end of the coding region and present on the mRNA are referred to as 5' non-translated sequences. Sequences located at the 3' end or downstream of the coding region and present on the mRNA are referred to as 3' non-translated sequences. The term "gene" includes both cDNA and genomic forms of a gene. Genomic forms or clones of a gene contain coding regions interrupted by non-coding sequences known as "introns," "insertion regions," or "insertion sequences." Introns are gene segments that are transcribed into heterologous nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or "spliced out" from the nuclear or primary transcript; therefore, they are not present in messenger RNA (mRNA) transcripts. mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.

As used herein, the terms "functionally linked" and "operably linked" are used interchangeably and refer to the functional relationship between two or more DNA segments, particularly a gene sequence to be expressed and those sequences that control its expression. For example, a promoter/enhancer sequence (including any combination of cis-acting transcriptional control elements) is operably linked to a coding sequence if it stimulates or regulates the transcription of the coding sequence in a suitable host cell or other expression system. A promoter regulatory sequence that is operably linked to a transcribed gene sequence is physically contiguous to the transcribed sequence.

"Conservatively modified variants" apply to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, a conservatively modified variant refers to a nucleic acid encoding the same or substantially the same amino acid sequence, or, when the nucleic acid does not encode an amino acid sequence, to a substantially identical sequence. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at each position where a codon specifies alanine, the codon can be changed to any of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid variations are "silent variations," which are a type of conservatively modified variation. Each nucleic acid sequence encoding a polypeptide herein also describes each possible silent variation of the nucleic acid. The skilled artisan will appreciate that each codon in the nucleic acid (except AUG, which is typically the only codon for methionine; and except TGG, which is typically the only codon for tryptophan) can be modified to produce functionally identical molecules. Thus, each silent variation of a nucleic acid encoding a polypeptide is implicit in each described sequence.

With respect to amino acid sequences, the skilled artisan will recognize that individual alterations, deletions, or additions to a nucleic acid, peptide, polypeptide, or protein sequence that alter, add, or delete a single amino acid or a small percentage of amino acids in the encoded sequence are "conservatively modified variants," where the alteration results in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables of functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to, and do not exclude, the polymorphic variants, interspecies homologs, and alleles of the present invention.

The following eight groups each contain amino acids that are conservative substitutions for each other:

1) Alanine (A), glycine (G);

2) Aspartic acid (D), glutamic acid (E);

3) Asparagine (N), glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

6) Phenylalanine (F), tyrosine (Y), tryptophan (W);

7) serine (S), threonine (T); and

8) Cysteine (C), methionine (M) (see, for example, Creighton, Proteins (1984)).

Conservative substitutions may include substitutions such as basic for basic, acidic for acidic, polar for polar, and the like. Due to structural reasons, groups of amino acids derived therefrom may be conservative. These groups can be described in the form of Venn diagrams (Livingstone C.D. and Barton G.J. (1993) "Protein sequence alignments: a strategy for the hierarchical analysis of residue conservation" Comput. Appl Biosci. 9:745-756; Taylor W.R. (1986) "The classification of amino acid conservation" J. Theor. Biol. 119:205-218). For example, conservative substitutions can be described according to the following table, which describes the generally accepted Venn diagram groupings of amino acids.

Table 1: Amino acid groups

"Percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, wherein for optimal alignment of the two sequences, the portion of the polynucleotide sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to a reference sequence (e.g., a polypeptide of the invention), while the reference sequence does not contain the additions or deletions. The ratio is calculated by determining the number of positions at which identical nucleic acid bases or amino acid residues appear in the two sequences to produce the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity.

In the case of two or more nucleic acid or peptide sequences, the term "consistent" or percentage "consistency" refers to two or more sequences or subsequences of the same sequence. When comparing and comparing in a comparison window to obtain maximum correspondence, or using one of the following sequence alignment algorithms or by manual comparison and visual inspection, if two sequences have the identical amino acid residues or nucleotides of a specified percentage (that is, on a specified region, or when not specified, there is 60% on the entire sequence, alternatively 65%, 70%, 75%, 80%, 85%, 90% or 95% identity), then two sequences are "substantially identical". The present invention provides polypeptides and uses thereof substantially identical to the polypeptides exemplified herein, including but not limited to for treating or preventing neurological diseases or disorders (such as neurodegenerative diseases or disorders), and/or treating SCI. Alternatively, consistency is present in a region having a length of at least about 50 nucleotides, or more preferably in a region having a length of 100 to 500 or 1000 or more nucleotides, or in the entire length of a reference sequence.

For sequence comparison, one sequence typically serves as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, the test and reference sequences are entered into a computer, subsequence coordinates are specified, if necessary, and the sequence algorithm program parameters are specified. Default program parameters can be used, or alternative parameters can be specified. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the program parameters.

As used herein, a "comparison window" includes a segment involving any number of contiguous positions selected from the group consisting of 20 to 600, typically about 50 to about 200, more typically about 100 to about 150, wherein a sequence can be compared to a reference sequence after the sequence has been optimally aligned with a reference sequence having the same number of contiguous positions. Methods of sequence alignment for comparison are well known in the art. Optimal alignment of sequences for comparison can be achieved, for example, by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, by computerized versions of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)).

Two examples of algorithms suitable for determining sequence identity percentages and sequence similarity are BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25: 3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215: 403-410, respectively. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. The algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or meet some positive threshold score T when aligned with words of the same length in the database sequence. T is referred to as the adjacent word score threshold (Altschul et al., supra). These initial adjacent word hits serve as seeds for initiating searches to find longer HSPs containing them. As long as the cumulative alignment score can be increased, the word hits are extended in both directions along each sequence. For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatched residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hit in each direction is stopped when: the cumulative alignment score drops by the amount X from its maximum achieved value; the cumulative score is zero or below zero due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both chains as defaults. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915), an alignment (B) of 50, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

" nucleic acid " refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single-stranded or double-stranded form, and their complements. The term encompasses backbone residues or connections containing known nucleotide analogs or modifications, which are synthetic, naturally occurring, and non-naturally occurring, and have binding properties similar to reference nucleic acids, and are metabolized in a manner similar to reference nucleic acids. The example of such analogs includes but is not limited to phosphorothioate, phosphoramidate, methylphosphonate, chiral-methylphosphonate, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNA). In various embodiments, nucleic acids are separated when purified from other cellular components or other contaminants (for example, other nucleic acids or proteins present in cells) by standard techniques, including alkaline/SDS treatment, CsCl bands, column chromatography, agarose gel electrophoresis, and other techniques well known in the art. See, for example, F.Ausubel, et al., ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. In various embodiments, nucleic acid is, for example, DNA or RNA, and may or may not include intron sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

As used herein, "pharmaceutically acceptable carrier" encompasses any standard pharmaceutical carrier, such as phosphate-buffered saline solution, water, and emulsions (such as oil/water or water/oil emulsions), and various types of wetting agents.

As used herein, term " neuron " includes neuron and a part or multiple parts thereof (for example, neuron cell body, axon or dendrite).Term " neuron " used herein represents nervous system cell, and it includes central cell body or soma, and two types of extension or prominence: dendrite, usually most neuron signals are transmitted to cell body by dendrite, and axon, usually most neuron signals are transmitted to effector cell (such as target neuron or muscle) from cell body by axon. Neuron can pass information to central nervous system (afferent neuron or sensory neuron) from tissue and organ, and pass signal to effector cell (efferent neuron or motor neuron) from central nervous system.Other neurons, referred to as interneurons, connect neurons in central nervous system (brain and spinal cord).Some instantiations of the neuron type that can carry out treatment according to the present invention or method include cerebellar granule neurons, dorsal root ganglion neurons and cortical neurons.

The term "neuronal degeneration" is widely used and refers to any pathological change in neuronal cells, including but not limited to the death or loss of neuronal cells, any change before cell death, and any reduction or loss of the activity or function of nerve cells. Pathological change can be spontaneous or can be induced by any event, and includes, for example, the pathological change associated with apoptosis. Neuron can be any neuron, including but not limited to sensory, sympathetic, parasympathetic or enteric, such as dorsal root ganglion neurons, motor neurons and central neurons (for example, neurons from the spinal cord). Neuronal degeneration or cell loss are the features of multiple neurological diseases or disorders (for example, neurodegenerative diseases or disorders). In some embodiments, the neuron is a sensory neuron. In some embodiments, the neuron is a motor neuron. In some embodiments, the neuron is an injured spinal cord neuron.

As used herein, "spasticity" refers to a condition in which certain muscles contract continuously. This contraction causes muscle stiffness or tightness and may interfere with normal movement, speech, and gait. Spasticity primarily occurs in disorders of the central nervous system (CNS), affecting upper motor neurons in the form of lesions such as spastic diplegia or upper motor neuron syndrome, and may also be present in various types of multiple sclerosis, where symptoms appear as a progressively worsening attack on the myelin sheath, and are therefore unrelated to the type of spasticity present in neuromuscular cerebral palsy radicular spasm disorder. Without being bound by theory, spasticity occurs when an imbalance in the excitatory and inhibitory inputs to alpha motor neurons occurs due to damage to the spinal cord and/or central nervous system. The damage causes a change in the signal balance between the nervous system and the muscles, resulting in increased excitability of the muscles. Spasticity occurs when the brain and/or spinal cord are damaged or fail to develop normally; these include cerebral palsy, multiple sclerosis, spinal cord injury, and acquired brain injury, including stroke.

As used herein, "neurodegenerative disorder" or "neurological disorder" refers to a disorder that causes the morphology and/or functional abnormalities of a nerve cell or nerve cell group. A neurodegenerative disorder can result in the impairment or absence of normal neural function in a subject, or the presence of abnormal neural function. For example, a neurodegenerative disorder may be the result of disease, injury and/or aging. Non-limiting examples of morphological and functional abnormalities include physical degeneration and/or death of nerve cells, abnormal growth patterns of nerve cells, abnormalities in the physical connections between nerve cells, insufficient or excessive generation of substance or material (e.g., neurotransmitters) produced by nerve cells, failure of nerve cells to produce the substance or material that is normally produced, production of substances (e.g., neurotransmitters) and/or transmission of electrical impulses in abnormal patterns or at abnormal times. Neurodegeneration can occur in any area of the brain of a subject and is found in many diseases, including, for example, head trauma, stroke, ALS, multiple sclerosis, Huntington's disease, Parkinson's disease and Alzheimer's disease.

As used herein, the term "nociception" refers to the response of the sensory nervous system to certain noxious or potentially noxious stimuli. In nociception, strong chemical (e.g., chili powder in the eyes), mechanical (e.g., cutting, crushing), or thermal (heat and cold) stimuli to sensory nerve cells called nociceptors generate signals that are conducted along chains of nerve fibers from the spinal cord to the brain. Nociception triggers a variety of physiological and behavioral responses and generally results in the subjective experience of felt pain.

Gamma-aminobutyric acid (GABA) and glutamate are the main inhibitory and excitatory neurotransmitters in mammals. The balance between GABA and glutamate controls various processes, such as neurogenesis, movement, circadian rhythm, tissue development, and blood sugar regulation. GABA is synthesized from glutamate by the 65 kDa and 67 kDa pyridoxal phosphate (PLP)-dependent enzymes glutamate decarboxylase (GAD65 and GAD67). Human GAD65 and GAD67 have been isolated and cloned in Bu et al. (1992) Proc Natl Acad Sci 89:2115-2119. Human GAD65 cDNA encodes a polypeptide with a molecular weight of 65,000 and 585 amino acid residues (Genbank Accession Nos. NM000818; M81882), and human GAD67 encodes a polypeptide with a molecular weight of 67,000 and 594 amino acid residues (Genbank Accession Nos. NM013445; M81883); each of which is incorporated herein by reference. See also U.S. Publication No. 2016/0081956, which is incorporated herein by reference.

Other nucleic acid and amino acid sequences of human GAD65 are known in the art. See, for example, GenBank Accession No. Q05329, human glutamate decarboxylase 2 (GAD2/GAD65), which provides an amino acid sequence (SEQ ID NO: 3):

See also, e.g., GenBank Accession No.: X69936, Homo sapiens mRNA for glutamate decarboxylase (GAD2/GAD65), which provides the nucleic acid sequence (SEQ ID NO: 2):

GABA acts at inhibitory synapses in the brain by binding to specific transmembrane receptors in the plasma membrane of presynaptic and postsynaptic neuronal processes. This binding causes the opening of ion channels, allowing negatively charged chloride ions to flow into the cell or positively charged potassium ions to flow out of the cell. This action causes a negative change in the transmembrane potential, generally resulting in hyperpolarization. Two general classes of GABA receptors are known: GABA _A , which is part of a ligand-gated ion channel complex, and GABA _B metabotropic receptors, which are G protein-coupled receptors that open or close ion channels via an intermediate (G protein).

Loss of GABA-mediated presynaptic inhibition after spinal cord injury plays a key role in the progressive increase in spinal reflexes and the development of spasticity. Clinical studies have shown that the use of baclofen (a GABA _B receptor agonist), while effective in modulating spasticity, is associated with major side effects such as general sedation and progressive development of tolerance. This study evaluated whether combination therapy consisting of spinal segment-specific upregulation of the GAD65 (glutamic acid decarboxylase) and VGAT (vesicular GABA transporter) genes would elicit antispasticity effects.

VGAT (vesicular GABA transporter) (also known as vesicular inhibitory amino acid transporter (VIAAT)) is a protein encoded by the SLC32A1 gene (also known as the VGAT gene) in humans. VGAT is highly concentrated in the nerve endings of GABAergic neurons in the brain and spinal cord, but also in glycinergic nerve endings. Caudhry, et al., J. Neurosci., 18(23):9733-9750 (1998), incorporated herein by reference. The nucleic acid and amino acid sequences of human VGAT are known in the art. See, for example, GenBank Accession No.: Q9H598, human vesicular inhibitory amino acid transporter (VIAAT/VGAT), which provides the amino acid sequence (SEQ ID NO: 3):

See also, e.g., GenBank Accession No.: NM_080552, Homo sapiens solute carrier family 32, member 1 (SLC32A1), mRNA, which provides the nucleic acid sequence (SEQ ID NO:4):

Accordingly, in one aspect, the present invention provides vectors comprising nucleotide sequences encoding GAD65 and VGAT. Polypeptides encoded by nucleotide sequences having at least 60% homology to GAD65 or its functional fragments are also within the scope of the present invention. Polypeptides encoded by nucleotide sequences having approximately 70% homology, approximately 75% homology, approximately 80% homology, approximately 85% homology, approximately 90% homology, approximately 95% homology, or approximately 99% homology to GAD65 or its functional fragments are also within the scope of the present invention. Polypeptides encoded by nucleotide sequences having at least 60% homology to VGAT or its functional fragments are also within the scope of the present invention. Polypeptides encoded by nucleotide sequences having approximately 70% homology, approximately 75% homology, approximately 80% homology, approximately 85% homology, approximately 90% homology, approximately 95% homology, or approximately 99% homology to VGAT or its functional fragments are also within the scope of the present invention.

Reduced activity or complete loss of facilitatory spinal inputs to spinal GABAergic inhibitory interneurons and the resulting reduction in local segmental inhibition have been postulated to be one of the key mechanisms leading to the development of muscle spasticity in patients with spinal cord injury (SCI). In contrast, the loss of spinal inhibitory interneurons seen after brief episodes of spinal cord ischemia leads to the development of functionally defined muscle spasticity and stiffness. Independent of the nature of the injury (e.g., spinal cord trauma or local ischemia), clinical and experimental animal pharmacology studies have demonstrated comparable and potent antispastic effects following systemic or spinal treatment with baclofen, a GABA _B receptor agonist, the most commonly used antispastic agent. The primary site of baclofen-mediated hyperpolarization is thought to be at presynaptic Ia afferents.

However, one of the major limitations of systemic baclofen therapy is the lack of a localized spinal segment-restricted effect, and the relatively high doses required to achieve clinically relevant spasticity relief often produce undesirable systemic side effects such as sedation. Direct spinal delivery of baclofen using a chronic intrathecal catheter offers a more site-restricted effect with less pronounced systemic activity, but it requires surgical intervention and the subsequent complications associated with chronic intrathecal catheterization (such as cerebrospinal fluid leaks or infections, which have been described). More importantly, limitations to the effective long-term use of IT baclofen include the development of baclofen tolerance (i.e., gradual dose escalation to achieve consistent antispasticity) and withdrawal following abrupt discontinuation of baclofen therapy.

The preferential expression of the GAD65 gene in infected astrocytes (control of neurons) seems to provide a specific advantage about the expected GABA-mediated anticonvulsant effect (see, for example, WO2014/116652, incorporated herein by reference). As shown in vitro, the infection of primary astrocytes results in a Ca2 ⁺ independent increase in extracellular GABA concentration. Therefore, it is expected that astrocyte-mediated GABA release in the spinal cord parenchyma will be independent of the functionality and connectivity of the local neuronal inhibitory circuits, and will specifically exert its hyperpolarizing effect on the GABA _B receptors expressed on Ia afferent neurons and/or α-motor neurons. The biological activity of GABA produced by astrocytes is confirmed by its depolarization induction on hNT neurons cultured in preferentially expressing GABA _A receptors.

The use of dual-gene therapy with GAD65 and VGAT represents a novel approach aimed at increasing regional neuronal inhibition, an approach not previously tested in the context of spinal cord or brain delivery. Central to this finding is the need for both genes to be upregulated to achieve functionally relevant inhibition of otherwise hyperexcitable neurons. The potency of this therapeutic effect suggests that sufficient amounts of releasable GABA are available in the synaptic cleft to induce inhibition at the postsynaptic membrane, leading to a decrease in α-motor neuron excitability and resulting in inhibition of muscle spasticity.

Therefore, in another aspect, the present invention provides a method for treating spasticity in a subject by spinal cord-specific upregulation of the GAD65 gene and the VGAT gene. In various embodiments, upregulation of GAD65 and VGAT comprises administering a viral vector encoding GAD65 and VGAT and expressing GAD65 and VGAT in the spinal cord of the subject, thereby reducing spasticity in the subject.

Viral vectors are very useful for introducing the polynucleotides implementing the methods of the present invention into target cells. Viral vectors for specific host systems, particularly mammalian systems, have been developed, including, for example, retroviral vectors, other lentiviral vectors such as those based on human immunodeficiency virus (HIV), adenoviral vectors (AV), adeno-associated viral vectors (AAV), herpes virus vectors, vaccinia virus vectors, etc. (see Miller and Rosman, BioTechniques 7:980-990, 1992; Anderson et al., Nature 392:25-30 Suppl., 1998; Verma and Somia, Nature 389:239-242, 1997; Wilson, New Engl. J. Med. 334:1185-1187 (1996), each of which is incorporated herein by reference). In one aspect of the invention, lentivirus, AV or AAV are used. Adenovirus represents the largest non-enveloped virus because it has the maximum size that can be transported through endosomes (i.e., without the need for envelope fusion). The virion also has unique "spikes" or fibers associated with each penton matrix of the capsid that facilitate attachment to the host cell. AAV is a dependent parvovirus that, by definition, requires co-infection with another virus (usually an adenovirus or herpes virus) to initiate and maintain a productive infectious cycle. In the absence of such a helper virus, AAV is still able to infect or transduce target cells through receptor-mediated binding and internalization, penetrating the nucleus in both non-dividing and dividing cells.

Once inside the cell nucleus, the virus uncoats and the transgene is expressed in a variety of forms—the most persistent of which is a circular monomer. AAV will integrate into the genome of 1-5% of stably transduced cells (Nakai et al., J. Virol. 76:11343-349, 2002). Transgene expression can be exceptionally stable. Because progeny virus is not produced from AAV infection in the absence of a helper virus, the extent of transduction is limited to the original cells infected by the virus. It is this characteristic that makes AAV a suitable gene therapy vector for the present invention.

Other references describing adenoviral vectors and other viral vectors that can be used in the methods of the present invention include the following: Horwitz, M.S., Adenoviridae and Their Replication, in Fields, B., et al. (eds.) Virology, Vol. 2, Raven Press New York, pp. 1679-1721, 1990); Graham, F., et al., pp. 109-128 in Methods in Molecular Biology, Vol. 7: Gene Transfer and Expression Protocols, Murray, E. (ed.), Humana Press, Clifton, N.J. (1991); Miller, N., et al., FASEB Journal 9: 190-199, 1995; Schreier, H, Pharmaceutical Acta Helvetiae 68: 145-159, 1994; Schneider and French, Circulation 88:1937-1942,1993; Curiel D.T., et al., Human Gene Therapy 3:147-154, 1992; Graham, F.L., et al., WO 95/00655 (5Jan.1995); Falck-Pedersen, E.S., WO 95/16772(22Jun.1995); Denefle,P.et al.,WO 95/23867(8Sep.1995); Haddada,H.et al.,WO 94/26914(24Nov.1994);Perricaudet,M.etal.,WO 95/02697(26Jan.1995); Zhang,W.,et al.,WO 95/25071 (12 Oct. 1995). A variety of adenovirus plasmids are also available from commercial sources, including, for example, Microbix Biosystems of Toronto, Ontario (see, for example, Microbix Product Information Sheet: Plasmids for Adenovirus Vector Construction, 1996).

Other references describing AAV vectors useful in the methods of the present invention include the following: Carter, B., Handbook of Parvoviruses, vol. 1, pp. 169-228, 1990; Berns, Virology, pp. 1743-1764 (Raven Press 1990); Carter, B., Curr. Opin. Biotechnol., 3: 533-539, 1992; Muzyczka, N., Current Topics in Microbiology and Immunology, 158: 92-129, 1992; Flotte, T. R., et al., Am. J. Respir. Cell Mol. Biol. 7: 349-356, 1992; Chatterjee et al., Ann. NY Acad. Sci., 770: 79-90, 1995; Flotte, T. R., et al., WO 95/13365 (18 May 1995); Trempe, J.P., et al., WO 95/13392 (18 May 1995); Kotin, R., Human Gene Therapy, 5:793-801, 1994; Flotte, T.R., et al., Gene Therapy 2:357-362, 1995; Allen, J.M., WO 96/17947(13Jun.1996); and Du et al., Gene Therapy 3:254-261, 1996.

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 3A 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, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, and any other AAV now known. In one embodiment, the AAV is AAV type 2. In another embodiment, the AAV is AAV type 9.

In the expression vector, any of the transcription and translation elements of the present invention can be used, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. (Bitter et al., Meth. Enzymol. 153: 516 544, 1987). As defined above, reference to "promoter" or "promoter sequence" should be carried out in its broadest context, and include DNA regulatory regions that can bind RNA polymerase in cells and initiate transcription of polynucleotides or polypeptide coding sequences (such as messenger RNA, ribosomal RNA, small nuclear nucleolar RNA, or any type of RNA transcribed by any RNA polymerase of any class). "Promoter" considered herein can also include the transcriptional regulatory sequences of classical genomic genes, including Goldberg-Hogness boxes, which are necessary for accurate transcription initiation in eukaryotic cells, with or without CAT box sequences and additional regulatory elements (i.e., upstream activation sequences, enhancers, and silencers).

Sequence is placed under the regulation control of promoter sequence and means to place described molecule so that expression is subject to promoter sequence control.Promoter is usually positioned at the 5 ' end (upstream) of the gene that it controls.In the structure of heterologous promoter/structural gene combination, usually promoter position can be roughly the same as the distance between the distance of gene transcription start site and this promoter and the gene (i.e. the gene that this promoter comes from) that it controls in its natural environment.As known in the art, some variations of this distance can be adjusted and do not lose promoter function.Similarly, regulating sequence element is defined by the positioning of element in its natural environment (i.e. the gene that it comes from) with respect to the positioning of the heterologous gene to be placed under its control.Equally, as known in the art, some variations of this distance can also occur.

Exemplary promoters useful for the methods and treatment regimens of the present invention include, but are not limited to, the human ubiquitin promoter and the human synapsin promoter. However, other known tissue-specific or cell-specific promoters may be used.

Suitable host cells for producing recombinant AAV particles include, but are not limited to, microorganisms, yeast cells, insect cells, and mammalian cells that can be or have been used as recipients of exogenous nucleic acid molecules. Thus, as used herein, "host cell" generally refers to a cell that has been transfected with an exogenous nucleic acid molecule. Host cells include any eukaryotic cell or cell line, so long as the cell or cell line is compatible with the protein to be expressed, the selection system selected, or the fermentation system used.

The AAV vector can be formulated into an injection or administration formulation by dissolving, suspending, or emulsifying the AAV vector in a suitable pharmaceutically acceptable carrier or diluent. Examples of such pharmaceutically acceptable carriers or diluents include aqueous or non-aqueous solvents, such as oils, synthetic fatty acid glycerides, higher fatty acid esters, or propylene glycol; if necessary, conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives can be used.

If it is not possible to obtain a viral vector specific to cell type, then the carrier can be modified to express a receptor (or ligand) specific to the ligand (or receptor) expressed on target cell, or it can be encapsulated in liposomes, it can also be modified to include such a ligand (or receptor). Peptide reagents can be introduced into cells by various methods, including, for example, by transforming peptides to include protein transduction domains such as human immunodeficiency virus TAT protein transduction domains, it can promote the transfer of peptides to cells. In addition, there are also various technologies based on biomaterials, such as nano cages and drug delivery wafers (such as for brain cancer chemotherapeutics), which can also be modified to adapt to the technology.

In addition to cells that integrate gene transfer after using lentiviral vectors, there are reports of successful overexpression of the GAD65 gene after AAV-GAD65 injection into the subthalamic nucleus. In those studies, sustained GAD65 expression was observed 4-5 months after AAV-GAD65 injection. More importantly, recent systematic data demonstrate that even after a limited number of AAV injections (1-2 injections), the efficiency of AAV-based gene delivery to the striatum of rats or minipigs is high. Therefore, in another embodiment, the present invention uses AAV-based, non-integrated genome-encoding GAD65 and VGAT encoding vectors to achieve segment-specific GAD65 and VGAT expression.

By combining the spinal delivery of GAD65 and VGAT, a significant and functionally relevant increase in spinal cord spasticity inhibition was achieved. The efficacy of spinal cord inhibition was tested in a well-characterized rat spinal cord injury-induced muscle spasticity model. This animal model is characterized by the presence of highly developed spinal hyperreflexia and results in significant muscle spasticity in the chronic stage after spinal cord injury. Chronic spastic animals that received subpial injection of GAD65+VGAT (delivered in an AAV9-UBI vector) showed significant inhibition of spasticity reactions observed 5 weeks after gene delivery, and this significant therapeutic effect lasted for at least the 8th week. Immunofluorescence analysis showed the appearance of a mixed inhibitory-excitatory neurotransmitter phenotype in spinal interneurons, as demonstrated by the colocalization of GAD65 and VGAT expression with glutamatergic markers VGLUT1 and VGLUT2. No antispasmodic effect was observed in animals injected with a control GFP vector, and no colocalization of GAD65 and VGAT expression with glutamatergic markers VGLUT1 and VGLUT2 was detected.

Any of various methods and delivery systems known to those skilled in the art can be used to realize or carry out the administration of this combined treatment. As used herein, the term "administration" or "administering" is defined as including when carrying out the method of the present invention, providing the subject with the behavior of the compound of the present invention or pharmaceutical composition. Exemplary routes of administration include, but are not limited to, intravenous, intraarticular, intracisternal, intraocular, intraventricular, intrathecal, subpial, intramuscular, intraperitoneal, intradermal, intracavitary, and any two or more combinations thereof. In certain embodiments, AAV can be delivered directly to the spinal cord parenchyma, the intrathecal space of the spinal cord, into the spinal cord subpial space of the subject, and/or into peripheral spastic muscles, to achieve the spinal cord upregulation of GAD65 gene and VGAT gene. See, for example, WO2016/122791, which is incorporated herein by reference.

The term "therapeutically effective amount" or "effective amount" refers to the amount of a compound or composition that causes a biological or medical response (such as spinal upregulation of the GAD65 gene and VGAT gene) in a tissue, system, animal or human that a researcher, veterinarian, doctor or other clinician is seeking. Therefore, the term "therapeutically effective amount" is used herein to refer to any amount of a preparation that causes a significant improvement in the disease condition when repeatedly applied to the diseased area over a period of time. This amount will vary with the condition being treated, the stage of progression of the condition, and the type and concentration of the preparation used. In any given example, the appropriate amount is obvious to those skilled in the art or can be determined by routine experiments. For example, with respect to subject treatment methods, a "therapeutically effective amount" of an AAV encoding the GAD65 gene and the VGAT gene or a composition comprising an AAV encoding the GAD65 gene and the VGAT gene refers to the amount of AAV in the preparation that, when applied as part of a desired treatment regimen, causes upregulation of the GAD65 gene and the VGAT gene.

Conventional calculation methods can be used to determine the therapeutic or preventive effective amount of the delivery vector based on animal data. Among other factors, the appropriate dosage will depend on the specificity of the selected transfer vector, the route of administration, the mammal being treated (e.g., a human or non-human primate or other mammal), the age, weight and general condition of the subject to be treated, the severity of the disorder to be treated, the position of the region in the treated subject and the mode of administration. Therefore, the appropriate dosage can vary from patient to patient. Those skilled in the art can easily determine a suitable effective amount.

Dosage therapy can be a single dose regimen or a multiple dose regimen. In addition, the subject can be administered multiple appropriate doses. Those skilled in the art can easily determine the appropriate number of doses. However, it may be necessary to consider alternative routes of administration, or to balance the therapeutic benefits with any side effects to adjust the dosage. These dosages can vary depending on the therapeutic application using the recombinant vector.

Alternatively, the delivery of AAV mediation according to the present invention can be combined with the delivery by other viruses and non-viral vectors. Such other viral vectors can be easily selected and produced according to methods known in the art, including but not limited to, adenoviral vectors, retroviral vectors, lentiviral vectors, herpes simplex virus (HSV) vectors and baculovirus vectors. Similarly, non-viral vectors can be easily selected and produced according to methods known in the art, including but not limited to liposomes, lipid-based vectors, composite vectors, molecular conjugates, polyamines and polycationic vectors. When administered by these alternative approaches, the dosage is ideal within the above range.

On the other hand, the present invention also provides a treatment regimen for treating a subject suffering from spinal cord injury. The treatment regimen includes administering spinal cord-specific upregulation of the GAD65 gene and the VGAT gene. As discussed in detail above, the upregulation of GAD65 and VGAT can include administering a viral vector encoding GAD65 and VGAT, wherein GAD65 and VGAT are expressed and treat spinal cord injury.

In addition, the methods of the present invention can be used to treat nerve damage such as peripheral neuropathy, which is caused by exposure to toxic compounds, including heavy metals (e.g., lead, arsenic, and mercury) and industrial solvents, as well as drugs, including chemotherapeutic agents (e.g., vincristine and cisplatin), dapsone, HIV drugs (e.g., zidovudine, didanosine, stavudine, zaluxetine, ritonavir, and amprenavir), cholesterol-lowering drugs (e.g., lovastatin, indapamilide, and gemfibrozil), heart or blood pressure drugs (e.g., amiodarone, hydralazine, perhexiline), and metronidazole.

The methods of the present invention can also be used to treat damage to the nervous system caused by physical, mechanical, or chemical trauma. Thus, the methods can be used to treat peripheral nerve damage caused by physical injury (e.g., associated with burns, gunshot wounds, surgery, and accidents), ischemia, prolonged exposure to cold temperatures (e.g., frostbite), and damage to the central nervous system due to, for example, stroke or intracranial hemorrhage (e.g., cerebral hemorrhage). Similarly, the methods of the present invention can be used to treat chronic pain/nociception caused by these traumas.

The following examples are intended to illustrate but not to limit the present invention.

Example 1

AAV9 viruses encoding GAD65 (glutamate decarboxylase 65) and VGAT (vesicular GABA transporter) were injected into the targeted segment using a subpial delivery method (Figure 1). Animals (rats) with spinal cord injury-induced muscle spasm were used. The distribution of transgene expression achieved after lumbar subpial AAV9-UBI-GFP delivery is shown in Figure 2. Extensive GFP expression can be seen in interneurons throughout the gray matter.

After gene delivery, the spastic response was measured for up to 8 weeks after GAD65 and VGAT gene delivery. In the control spastic animals, the control AAV9-UBI-GFP was used. Figures 3A-3D show that the spastic response of animals injected with AAV9-UBI-GAD65+VGAT gradually decreased. The significant anti-spastic effect lasted for at least 8 weeks after gene delivery (Figures 3A and 3B). If compared with animals injected with control AAV9, the measurement of rate-dependent depression (representing an indicator of altered spinal cord inhibition) showed a significant recovery (Figure 3C).

At week 8, immunofluorescence analysis of segments injected with the GAD65/VGAT gene showed significant upregulation of both genes and the emergence of a mixed inhibitory/excitatory neurotransmitter phenotype (co-expression of GAD65 or VGAT with VGLUT2 (vesicular glutamate transporter)) (Figures 4A and 4B). No co-expression was observed in animals injected with control AAV9 (Figures 4C and 4D). These data confirm the effective induction of inhibitory drive in GAD65/VGAT overexpressing neurons, which may mediate the reduction of muscle spasticity.

Although the present invention has been described with reference to the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the present invention is limited only by the following claims.

Claims (15)

1. A composition for treating spasms in a subject, wherein: (i) The composition comprises a viral vector containing a polynucleotide encoding glutamate decarboxylase GAD65 and vesicle GABA transporter (VGAT) to express the GAD65 gene and the VGAT gene. (ii) The viral vector is directly administered to the subpiaural space of the subject. (iii) The composition upregulates GAD65 and VGAT, and (iv) The expression and upregulation of GAD65 and VGAT produced spinal interneurons with a mixed inhibitory-excitatory neurotransmitter phenotype, thereby treating the spasticity of the subject. 2. The composition for the purpose of claim 1, wherein the upregulation of the GAD65 gene and the VGAT gene is a spinal cord-specific upregulation of the GAD65 gene and the VGAT gene. 3. The composition for the said use according to claim 1, wherein the GAD65 and VGAT are overexpressed. 4. The composition for the said use according to claim 1, wherein the viral vector is a lentiviral vector, an adenovirus vector (AV), or an adeno-associated vector (AAV). 5. The composition for the said use according to claim 4, wherein the gland-associated carrier is AAV9. 6. The composition of claim 1 for the stated purpose, wherein the viral vector is administered directly to the spinal cord parenchyma of the subject, to the intrathecal space of the subject, or to the peripheral spastic muscles of the subject. 7. A viral vector for treating a subject with spinal cord injury, the viral vector comprising polynucleotides encoding GAD65 and VGAT to express GAD65 and VGAT, the viral vector upregulating GAD65 and VGAT, wherein the viral vector is administered directly to the subpiaural space of the subject, and the expression of GAD65 and VGAT produces spinal cord interneurons with a mixed inhibitory-excitatory neurotransmitter phenotype, thereby treating the spinal cord injury. 8. The viral vector for the said purpose as claimed in claim 7, wherein GAD65 and VGAT are overexpressed. 9. The viral vector for the said purpose as described in claim 7, wherein the viral vector is a lentiviral vector, AV, or AAV. 10. The viral vector of claim 9 for the said purpose, wherein the AAV is AAV9. 11. The viral vector for the said purpose as claimed in claim 7, wherein the viral vector is administered directly to the spinal cord parenchyma of the subject, to the intrathecal space of the subject, or to the peripheral spastic muscles of the subject. 12. A carrier for treating spasticity in a subject, the carrier comprising a promoter functionally linked to a multinucleotide encoding GAD65 and VGAT to express and upregulate GAD65 and VGAT, wherein the carrier is administered directly to the subpiacular space of the subject, and the expression and upregulation of GAD65 and VGAT produces spinal interneurons with a mixed inhibitory-excitatory neurotransmitter phenotype. 13. The vector of claim 12, wherein the vector is a viral vector selected from the group consisting of lentivirus, adenovirus, and AAV vectors. 14. The carrier of claim 13, wherein the carrier is AAV9-UBI-GAD65+VGAT. 15. An isolated mammalian host cell containing the vector according to claim 12.