Viral vectors encoding GAD for treating spasticity

Abstract

The present invention provides a method for treating spasticity in a subject, comprising administering a herpes simplex virus 1 (HSV-1) vector carrying a glutamic acid decarboxylase (GAD) gene (preferably GAD67) directly to one or more dermatomes of the subject.

Description

[Technical Field]

[0001] Related Applications This application claims priority to French Patent Application No. 2212771, filed December 5, 2022, the entire contents of which are incorporated herein by reference.

[0002] The present application relates to methods of treating spasticity. [Background technology]

[0003] Spasticity is a condition in which muscles become stiff or tense, preventing normal fluid movement. The muscles remain contracted and resist stretching, thus affecting movement, speech, and walking. Spasticity commonly occurs with central nervous system (CNS) disorders, usually after spinal cord injury (SCI), and is part of the upper motor neuron (UMN) syndrome because it affects the upper motor neurons. The impact of spasticity on patients ranges from subtle neurological symptoms to severe spasticity (e.g., tightly clenched fists, twisted wrist and elbow joints, and arms fixed in a flexed position) that causes extreme discomfort, pain, spasms, and contractures. These symptoms can be exacerbated by fatigue, stress, infection, and lesions. Furthermore, patients with spasticity may experience increased fatigue on a daily basis, typically due to the need to expend extra energy to overcome muscle tension during daily activities.

[0004] Spasticity often requires both pharmacological and non-pharmacological interventions. Non-pharmacological interventions, such as physical therapy (i.e., muscle stretching and strengthening exercises), can serve as adjunctive treatments. Baclofen has been used as a common pharmacological intervention to treat spasticity. Baclofen is a muscle relaxant that acts on spinal nerves. Clinical trials have shown that baclofen is the most potent antispasticity pharmacological treatment; however, baclofen is often associated with side effects, including drowsiness, dizziness, headache, fatigue, muscle weakness, and progressive tolerance. Baclofen can be administered orally or intrathecally using a pump implanted under the skin. Intrathecal administration is often preferred for treating patients with spasticity because intrathecal administration requires a much lower dose of baclofen and therefore reduces side effects. However, implanted pumps can cause post-implant complications, including pump failure, infection, and lead migration. Meanwhile, injections of botulinum toxin (Botox) and neurolytic drugs (phenol) have also been used to reduce spasticity, either alone or in combination with each other or with baclofen. Botulinum toxin and neurolytic injections typically require highly trained physicians and relatively long injection times. Botulinum toxin may need to be injected into multiple muscles to demonstrate therapeutic efficacy. Neurolytic drugs must be injected directly into the nerve, for example, to locate the nerve to be blocked in order to send messages to the muscle to contract. The subject is sedated while a specialist locates the nerve using mild electrical impulses. The therapeutic effects of botulinum toxin and neurolytic injections in reducing spasticity, alone or in combination with other treatments, are short-term, requiring repeated administration every 3 to 6 months. Neurolytic injections impair nerve conduction by destroying part of the nerve, often causing additional necrosis of adjacent sensory nerves, skin, muscles, blood vessels, and other soft tissues. Additionally, although the origin of spasticity affecting individual muscle groups can be somatosensorily mapped to a specific spinal segment, currently available intrathecal delivery methods may not specifically target the designated spinal segment, thereby reducing side effects on other spinal segments that would otherwise not be affected by spasticity.

[0005] In severe cases, surgery may be performed to sever nerves and reduce spasticity, such as with dorsal rhizotomy. Selective dorsal rhizotomy consists of spinal cord surgery to reduce spasticity by selectively cutting sensory nerves. Sensory nerves are the primary source of excitation to the spinal cord and, together with other components (spinal interneurons, motor neurons, and muscles), form a closed-loop neuromuscular system that naturally generates and regulates movement. However, after SCI, affected sensory nerves generate, amplify, and propagate neural activity that triggers sustained, involuntary muscle contractions. Interruption of this closed-loop system with dorsal rhizotomy has been shown to be beneficial in reducing spasticity after SCI. Nevertheless, this procedure is highly invasive and does not allow for the modulation or preservation of sensory function in SCI patients. While these surgical procedures typically reduce spasticity in the upper limbs, they are also associated with severe long-term adverse effects, such as sensory impairment and reduced motor function in the affected area.

[0006] Therefore, there is a need for non-surgical, minimally invasive, and region-specific therapeutic approaches to treat spasticity. Summary of the Invention

[0007] The present application provides a method for treating spasticity in a subject, the method comprising upregulating the GAD (glutamic acid decarboxylase) gene. The upregulation of the GAD gene can be region-specific upregulation of the GAD gene. In some embodiments, the upregulation of the GAD gene comprises administering to the subject a viral vector comprising a polynucleotide encoding GAD, wherein GAD is expressed and converts excitatory neurotransmitters into inhibitory neurotransmitters, thereby reducing spasticity. In one aspect, the GAD gene is overexpressed. The polynucleotide encoding GAD can include the GAD67 gene (GenBank: M81883.1; SEQ ID NO: 1) encoding GAD67 (SEQ ID NO: 2) and the GAD65 gene (GenBank: M81882.1; SEQ ID NO: 3). In a preferred embodiment, the GAD is GAD67.

[0008] In some embodiments, the viral vector used in the methods of the present application is an adeno-associated viral (AAV) vector or a herpes simplex viral (HSV) vector, preferably an HSV-1 vector or an HSV-2 vector, and more preferably a defective viral vector derived from HSV-1, such as a recombinant HSV-1 vector, an amplicon HSV-1 vector, or an HSV-1 vector comprising a pre-HSV-1 vector and an inserted GAD expression cassette. In a preferred embodiment, the viral vector used in the methods of the present application is a defective HSV-1 vector, and a polynucleotide encoding GAD is inserted into the LAT (latency-associated transcript) locus of the defective HSV-1 vector.

[0009] In some embodiments, the viral vector comprises a promoter for promoting long-term expression of the GAD gene. In some embodiments, promoters useful in the present invention may be selectively active in afferent neurons. Such promoters may be selected from promoters of genes encoding sensory neuroreceptors, promoters of genes encoding sensory neuromodulators or sensory neurotransmitters, and promoters of genes involved in neurite outgrowth and stress responses in sensory neurons. In some embodiments, the promoter of a gene encoding a sensory neuroreceptor according to the present invention is selected from promoters of the TRP gene family, more preferably promoters TRPV1 or TRPM8. In some embodiments, the promoter of a gene encoding a sensory neuromodulator or sensory neurotransmitter according to the present invention is selected from promoters of substance P, PACAP, and calcitonin gene-related peptide (CGRP). In some embodiments, the promoter of a gene involved in neurite outgrowth and stress responses in sensory neurons is the promoter of a gene encoding advillin (ADVL).

[0010] In some embodiments, the promoter useful in the present invention is a ubiquitous promoter selected from the group consisting of the human cytomegalovirus (HCMV) promoter, human elongation factor 1 alpha (hEF-1 alpha) promoter, β-actin promoter, Rous sarcoma virus (RSV) promoter, human ubiquitin C (hUBC) promoter, ubiquitin B promoter, simian vacuolar virus 40 (SV40) promoter, phosphoglycerate kinase (PGK) promoter, β-globin promoter, NF-κB promoter, EGR1 promoter, eIF4A1 promoter, FerL promoter, GAPDH promoter, β-Kin promoter, ROSA26 promoter, and human surfactant protein C (hSP-C) promoter. Preferably, the promoter used in the present invention is hEF-1 alpha (SEQ ID NO: 5).

[0011] In some embodiments, the viral vector is administered directly into the spinal parenchyma of the subject, into the intrathecal space of the subject, into the spinal subpial space of the subject, or into a peripheral spastic muscle of the subject, or into one or more dermatomes of the subject. In preferred embodiments, the viral vector is administered directly into one or more dermatomes of the subject via one or more injections.

[0012] The present application also provides a method for treating spasticity in a subject, comprising administering to the subject a therapeutically effective amount of a viral vector comprising a polynucleotide encoding GAD, thereby treating the subject's spasticity. In some embodiments, the polynucleotide encoding GAD may comprise a GAD67 gene (SEQ ID NO: 1) encoding GAD67 (SEQ ID NO: 2) and a GAD65 gene (SEQ ID NO: 3) encoding GAD65. Preferably, the GAD is GAD67.

[0013] In some embodiments, the viral vector is an adeno-associated viral (AAV) vector or a herpes simplex viral (HSV) vector, preferably an HSV-1 or HSV-2 vector, more preferably a defective viral vector derived from HSV-1, such as a recombinant HSV-1 vector, an amplicon HSV-1 vector, or an HSV-1 vector comprising a pre-HSV-1 vector and an inserted GAD expression cassette. In a preferred embodiment, the viral vector used in the methods of the present application is a defective viral vector derived from HSV-1, and a polynucleotide encoding GAD is inserted into the LAT (latency-associated transcript) locus of the defective viral vector derived from HSV-1.

[0014] In some embodiments, the viral vector is administered directly into the spinal parenchyma of the subject, into the intrathecal space of the subject, into the spinal subpial space of the subject, or into a peripheral spastic muscle of the subject, or into one or more dermatomes of the subject. In a preferred embodiment, the viral vector is administered directly into one or more dermatomes of the subject.

[0015] In some embodiments, the viral vector comprises a promoter for promoting long-term expression of the polynucleotide. In some embodiments, promoters useful in the present invention may be selectively active in afferent neurons. Such promoters may be selected from promoters of genes encoding sensory neuroreceptors, promoters of genes encoding sensory neuromodulators or sensory neurotransmitters, and promoters of genes involved in neurite outgrowth and stress responses in sensory neurons. In some embodiments, the promoter of a gene encoding a sensory neuroreceptor according to the present invention is selected from promoters of the TRP gene family, more preferentially promoter TRPV1 or TRPM8. In some embodiments, the promoter of a gene encoding a sensory neuromodulator or sensory neurotransmitter according to the present invention is selected from promoters of substance P, PACAP, and calcitonin gene-related peptide (CGRP). In some embodiments, the promoter of a gene involved in neurite outgrowth and stress responses in sensory neurons is the promoter of a gene encoding advillin (ADVL).

[0016] In some embodiments, the promoter useful in the present invention is a ubiquitous promoter selected from the group consisting of the human cytomegalovirus (HCMV) promoter, human elongation factor 1 alpha (hEF-1 alpha) promoter, β-actin promoter, Rous sarcoma virus (RSV) promoter, human ubiquitin C (hUBC) promoter, ubiquitin B promoter, simian vacuolar virus 40 (SV40) promoter, phosphoglycerate kinase (PGK) promoter, β-globin promoter, NF-κB promoter, EGR1 promoter, eIF4A1 promoter, FerL promoter, GAPDH promoter, β-Kin promoter, ROSA26 promoter, and human surfactant protein C (hSP-C) promoter. Preferably, the promoter used in the present invention is hEF-1 alpha (SEQ ID NO: 5).

[0017] The present application also provides a therapeutic regimen for treating a subject with spasticity or a spasticity-related condition, comprising administering a viral vector containing a polynucleotide encoding GAD, thereby expressing GAD and thereby treating spasticity or a spasticity-related condition. The polynucleotide encoding GAD may include the GAD67 gene (SEQ ID NO: 1) encoding GAD67 (SEQ ID NO: 2) and the GAD65 gene (SEQ ID NO: 3) encoding GAD65. Preferably, the GAD is GAD67. The viral vector is an adeno-associated virus (AAV) vector or a herpes simplex virus (HSV) vector, preferably an HSV-1 vector or an HSV-2 vector, more preferably a defective viral vector derived from HSV, such as a recombinant HSV-1 vector, an amplicon HSV-1 vector, or an HSV-1 vector comprising a pre-HSV-1 vector and an inserted GAD expression cassette. In a preferred embodiment, the viral vector used in the method of the present application is a defective viral vector derived from HSV-1, and a polynucleotide encoding GAD is inserted into the LAT (latency-associated transcript) gene locus of the defective viral vector derived from HSV-1.

[0018] In some embodiments, the viral vector can be administered directly into the spinal cord parenchyma of a subject, into the spinal cavity of a subject, into the spinal subpial space of a subject, or into a peripheral spastic muscle of a subject, or into one or more dermatomes of a subject. Preferably, the viral vector can be administered directly into one or more dermatomes of a subject.

[0019] The viral vector may comprise a promoter. In some embodiments, a promoter useful in a therapeutic regimen may be selectively active in afferent neurons. Such a promoter may be selected from promoters of genes encoding sensory neuroreceptors, promoters of genes encoding sensory neuromodulators or sensory neurotransmitters, and promoters of genes involved in neurite outgrowth and stress responses in sensory neurons. In some embodiments, the promoter of a gene encoding a sensory neuroreceptor according to the present invention is selected from promoters of the TRP gene family, more preferentially promoter TRPV1 or TRPM8. In some embodiments, the promoter of a gene encoding a sensory neuromodulator or sensory neurotransmitter according to the present invention is selected from promoters of substance P, PACAP, and calcitonin gene-related peptide (CGRP). In some embodiments, the promoter of a gene involved in neurite outgrowth and stress responses in sensory neurons is the promoter of the gene encoding advillin (ADVL).

[0020] In some embodiments, the promoter useful in the treatment regimen is a ubiquitous promoter selected from the human cytomegalovirus (HCMV) promoter, human elongation factor 1 alpha (hEF-1α) promoter, β-actin promoter, Rous sarcoma virus (RSV) promoter, human ubiquitin C (hUBC) promoter, ubiquitin B promoter, simian vacuolar virus 40 (SV40) promoter, phosphoglycerate kinase (PGK) promoter, β-globin promoter, NF-κB promoter, EGR1 promoter, eIF4A1 promoter, FerL promoter, GAPDH promoter, β-Kin promoter, ROSA26 promoter, and human surfactant protein C (hSP-C) promoter. [Brief explanation of the drawings]

[0021] [Figure 1A] Figure 1A shows a statistically significant reduction in tonic muscle hyperactivity when mice were treated with the hEF-1α::GAD67 vector. Figure 1A shows a representative image of the mouse tail from a lesioned control animal in a chronic state of SCI 3 weeks after injection of the vector. [Figure 1B] Figure 1B shows a statistically significant reduction in tonic muscle hyperactivity when mice were treated with the hEF-1α::GAD67 vector. Figure 1B shows a representative image of the mouse tail from a treated animal in a chronic state of SCI 3 weeks after injection of the vector. [Figure 1C] Figure 1C shows a statistically significant reduction in tonic muscle hyperactivity when mice were treated with the hEF-1α::GAD67 vector. Figure 1C shows a severity index characterizing the increased tonic hyperactivity resulting in abnormal postural quantification. [Figure 1D] Figure 1D shows a statistically significant reduction in tonic muscle hyperactivity when mice were treated with the hEF-1α::GAD67 vector. The severity index for each group (N = 10 per group) is shown. Each dot represents one animal, and the bars represent the mean ± standard deviation. Statistical analysis by unpaired t-test showed a significant difference (p < 0.05) between treated mice compared to lesioned controls, indicating that the severity index is significantly reduced after hEF1α-hGAD67 treatment. [Figure 1E] Figure 1E shows a statistically significant reduction in tonic muscle hyperactivity when mice were treated with the hEF-1α::GAD67 vector. When the composite index was divided into three contributing factors, only the first curvature of the tail (θ1) showed a statistically significant difference between groups. These results tend to indicate a local effect of the hEF-1α::GAD67 vector on the first muscle segment (proximal to the base of the tail). [Figure 2A] The mean EMG change during tactile stimulation of the tail (tip or base) compared to the pre-stimulation baseline is shown in Figure 2A. Figure 2A shows the EMG recording of a muscle at the base of the tail, which is assumed to be associated with the same somitic level as the injected dermatome (S1-S2). [Figure 2B] Figure 2B shows the mean EMG change during tactile stimulation of the tail (tip or base) compared to the pre-stimulation baseline. Figure 2B shows the effect in two other tail muscles: one in the middle of the tail and one at the tip of the tail (closer to the posterior end). [Figure 2C] Figure 2C shows the mean EMG change during tactile stimulation of the tail (tip or base) compared to the pre-stimulation baseline. Figure 2C shows the effect in two other tail muscles: one in the middle of the tail and one at the tip of the tail (closer to the posterior end). [Figure 3A] Figure 3 shows EMG assessment of the effect of a combination of vector and a GABA reuptake inhibitor such as tiagabine on tail twitches. Figure 3A shows the change in EMG activity of the caudal muscles upon tactile stimulation at the base and tip of the tail. [Figure 3B] Figure 3B shows EMG assessment of the effect of a combination of vector and a GABA reuptake inhibitor such as tiagabine on tail twitches. Figure 3B shows the effect on two other tail muscles: one in the middle of the tail and one at the tip of the tail (closer to the posterior end). [Figure 3C] Figure 3C shows EMG assessment of the effect of a combination of vector and a GABA reuptake inhibitor such as tiagabine on tail twitches in two other tail muscles: one in the middle of the tail and one at the tip of the tail (closer to the posterior end). DETAILED DESCRIPTION OF THE INVENTION

[0022] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to a "method" includes one or more methods, and / or steps, of the type described herein that will become apparent to those skilled in the art upon reading this disclosure, etc.

[0023] The term "comprising," used interchangeably with "including," "containing," or "characterized by," is inclusive or open-ended language and does not exclude additional, unrecited elements or method steps.

[0024] The phrase "consisting of" excludes any element, step, or ingredient not specified in the claim. The phrase "consisting essentially of" limits the claim to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claimed invention. This disclosure contemplates embodiments of the compositions and methods of the invention that fall within the scope of each of these phrases. Thus, a composition or method that comprises recited elements or steps contemplates specific embodiments in which the composition or method consists essentially of or consists of those elements or steps.

[0025] 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. 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.

[0026] The term "subject" as used herein refers to any individual or patient on whom the method is performed. Generally, the subject is a human, but as will be understood by those skilled in the art, the subject may be an animal. Thus, mammals such as rodents (including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits, and other livestock, including cows, horses, goats, sheep, pigs, and other animals, as well as primates (including monkeys, chimpanzees, orangutans and gorillas), are included within the definition of a subject.

[0027] As used herein, "drug resistance" is meant to describe a decreased response of a subject to a drug, usually after repeated use of the drug. Increasing the dosage of the drug may again amplify the drug's effect, which may accelerate tolerance and further reduce the drug's effectiveness. In some embodiments, the terms "tolerance," "resistance," and "insensitivity" may be used interchangeably to describe a decrease in the effectiveness of a drug.

[0028] As used herein, a "therapeutic benefit" encompasses the therapeutic benefits and / or prophylactic benefits described herein.

[0029] As used herein, the terms "reduce" and "inhibit" are used together because it is recognized that in some cases, the reduction can be below the detection level of a particular assay. Thus, it is not always clear whether the expression level or activity is "reduced" below the detection level of the assay or completely "inhibited." Nevertheless, after treatment with the present method, it will be clearly determinable.

[0030] As used herein, "treatment" or "treating" means administering a composition to a subject or system having an undesired condition. The condition may include a disease or disorder. "Prevention" or "preventing" means administering a composition to a subject or system at risk for a condition. The condition may include a predisposition to a disease or disorder. The effect of administering a composition to a subject (either therapeutic and / or preventative) may be, but is not limited to, arresting one or more symptoms of the condition, reducing or preventing one or more symptoms of the condition, reducing the severity of the condition, eliminating the condition entirely, stabilizing or delaying the occurrence or progression of a particular event or characteristic, or minimizing the likelihood of a particular event or characteristic occurring.

[0031] 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 are artificial chemical mimetics of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and to unnatural amino acid polymers.

[0032] The term "active fragment" refers to an amino acid fragment that is less than the entire amino acid sequence of the molecule and that retains substantially the same or a corresponding biological activity, e.g., greater than 50%, such as 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% activity.

[0033] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids include those 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 a naturally occurring amino acid, i.e., a carbon bonded 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 a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function similarly to a naturally occurring amino acid.

[0034] 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. Similarly, nucleotides may be referred to by their commonly accepted single-letter codes.

[0035] 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.

[0036] As used herein, a "protein coding sequence," or a sequence encoding a specific protein or polypeptide, is a nucleic acid sequence that is transcribed into mRNA (in the case of DNA) and translated into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences (in the case of mRNA). The boundaries of the coding sequence are determined by a start codon at the 5' (N-) terminus and a translation stop nonsense codon at the 3' (C-) terminus. Coding sequences can 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 3' to the coding sequence.

[0037] The term "transgene" refers to a specific nucleic acid sequence that encodes an RNA and / or a polypeptide or portion of a polypeptide that is expressed in a cell into which the nucleic acid sequence is introduced. The term "transgene" includes (1) a nucleic acid sequence that is not naturally found in a cell (i.e., a heterologous nucleic acid sequence), (2) a nucleic acid sequence that is a mutant form of a nucleic acid sequence that is naturally found in the cell into which it is introduced, (3) a nucleic acid sequence that serves to add additional copies of the same (i.e., homologous) or similar nucleic acid sequence that is naturally present in the cell into which it is introduced, or (4) a silent native or homologous nucleic acid sequence whose expression is induced in the cell into which it is introduced. "Mutant form" refers to a nucleic acid sequence that contains one or more nucleotides that differ from the wild-type or native sequence; i.e., a mutant nucleic acid sequence contains one or more nucleotide substitutions, deletions, and / or insertions. In some cases, the transgene may also contain a sequence encoding a leader peptide, or a signal sequence, so that the transgene product is secreted from the cell, or the transgene may contain both a leader peptide or signal sequence and a membrane anchor peptide so that the transgene remains anchored in the cell membrane, or even may be a fusion protein between two naturally occurring proteins or portions thereof, or a sequence that allows the protein to accumulate in a specific region of the cell, such as a nuclear localization signal.

[0038] As used herein, the term "expression cassette" or "transcription cassette" refers to the distinct components of vector DNA consisting of genes and regulatory sequences expressed by transfected cells. With each successful transfection, the expression cassette directs the cellular machinery to make RNA and protein. Some expression cassettes are designed for modular cloning of protein-coding sequences so that the same cassette can be easily altered to make different proteins. An expression cassette can consist of one or more genes and sequences that control their expression. An expression cassette contains at least three components: a promoter sequence, an open reading frame, and a 3' untranslated region, which in eukaryotes usually contains a polyadenylation site.

[0039] As used herein, a "promoter" is defined as a regulatory DNA sequence typically located upstream of a gene that mediates transcription initiation by directing RNA polymerase to bind to DNA and initiate RNA synthesis. A promoter may be a constitutively active promoter (i.e., a promoter that is constitutively active / "ON" state), an inducible promoter (i.e., a promoter whose state, active / "ON" or inactive / "OFF," is controlled by an external stimulus, e.g., the presence of a specific compound or protein), a spatially restricted promoter (i.e., a transcriptional control element, enhancer, etc.; e.g., a tissue-specific promoter, a cell-type-specific promoter, etc.), or a temporally restricted promoter (i.e., a promoter that is "ON" or "OFF" during a specific stage of embryonic development or a specific stage of a biological process). For purposes of the present invention, a promoter sequence includes at least the minimum number of bases or elements necessary to initiate transcription of a gene of interest at a level detectable above background. Within the promoter sequence is a transcription initiation site and an RNA polymerase binding domain. Eukaryotic promoters often, but not always, contain "TATA" boxes and other DNA motifs, such as "CAT" boxes or "SP1" boxes.

[0040] As used herein, the term "gene" refers to a deoxyribonucleotide sequence comprising the coding region of a structural gene. A "gene" may also include untranslated sequences located adjacent to the coding region at both the 5' and 3' ends, such that the length of the gene corresponds to the full-length mRNA. Sequences located 5' of the coding region and present on the mRNA are referred to as 5' untranslated sequences. Sequences located 3' or downstream of the coding region and present on the mRNA are referred to as 3' untranslated sequences. The term "gene" encompasses both cDNA and genomic forms of a gene. Genomic forms or clones of a gene contain coding regions interrupted by non-coding sequences called "introns" or "intervening regions" or "intervening sequences." Introns are segments of a gene that are transcribed into heterologous nuclear RNA (hnRNA). Introns may contain regulatory elements, such as enhancers. Introns are removed or "spliced ​​out" from nuclear or primary transcripts. Thus, introns are absent in messenger RNA (mRNA) transcripts. mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.

[0041] As used herein, the terms "functionally linked" and "operably linked" are used interchangeably and refer to a functional relationship between two or more DNA segments, particularly gene sequences to be expressed and sequences that control their 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 an appropriate host cell or other expression system. A promoter regulatory sequence operably linked to a transcribed gene sequence is physically contiguous to the transcribed sequence.

[0042] The term "conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, conservatively modified variants refer to nucleic acids encoding identical or essentially identical amino acid sequences, or, if the nucleic acid does not encode an amino acid sequence, essentially identical sequences. 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 every position where alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one type of conservatively modified variation. Every nucleic acid sequence herein that encodes a polypeptide also represents every possible silent variation of the nucleic acid. Those skilled in the art will recognize that each codon within a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be modified to obtain a functionally identical molecule. Thus, each silent variation of a nucleic acid that encodes a polypeptide is encompassed within each described sequence.

[0043] With respect to amino acid sequences, those skilled in the art will recognize that individual substitutions, 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," in which the alteration replaces an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to, and do not exclude, polymorphic variants, interspecies homologs, and alleles of the invention.

[0044] The following eight groups each contain amino acids that are conservative substitutions for one another: 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, e.g., Creighton, Proteins (1984)).

[0045] Conservative substitutions (also called conservative replacements or conservative mutations) can include basic-to-basic, acidic-to-acidic, polar-to-polar, etc. Sets of amino acids derived in this way are likely to be conserved for structural reasons. These sets can be represented in the form of Venn diagrams, which are incorporated herein by reference (Livingstone CD and Barton GJ, "Protein sequence alignments: a strategy for the hierarchical analysis of residue conservation", Comput. Appl. Biosci. 1993, 9, 745-756; Taylor WR, "The classification of amino acid conservation", J. Theor. Biol. 1986, 119, 205-218).

[0046] "Percent sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, and a portion of the polynucleotide sequence within the comparison window may contain additions or deletions (i.e., gaps) compared to a reference sequence (e.g., a polypeptide of the invention) that does not contain additions or deletions in order to optimally align the two sequences. The percentage is calculated by determining the number of positions in both sequences where the same nucleic acid base or amino acid residue appears to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percent sequence identity.

[0047] The terms "identical" or "percent identity" in the context of two or more nucleic acid or polypeptide sequences refer to two or more sequences or subsequences that are the same. Two sequences are "substantially identical" if, when compared and aligned for maximum correspondence over a comparison window or designated region, they have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity over a designated region, or, if not specified, over the entire sequence), as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. The present invention provides polypeptides each substantially identical to the polypeptides exemplified herein, and uses thereof, including, but not limited to, for treating or preventing a neurological disease or disorder, e.g., a neurodegenerative disease or disorder, and / or for treating SCI. Optionally, identity exists over a region that is at least about 50 nucleotides in length, or more preferably over a region that is 100-500, or 1000 or more nucleotides in length, or over the entire length of the reference sequence.

[0048] For sequence comparison, typically, one sequence acts as a reference sequence to which test sequence is compared.When using sequence comparison algorithm, test sequence and reference sequence are input into computer, and partial sequence coordinates are designated as necessary, and sequence algorithm program parameters are designated.Default program parameters can be used, or alternative parameters can be designated.The sequence comparison algorithm then calculates the percent sequence identity of test sequence compared with reference sequence based on program parameters.

[0049] As used herein, a "comparison window" includes reference to any segment of a number of contiguous positions selected from the group consisting of 20 to 600, usually about 50 to about 200, and more usually about 100 to about 150, within which a sequence can be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be carried out, for example, by the local homology algorithm of Smith and Waterman, Adv. Appl. Math., 1970, 2:482c, by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol., 1970, 48:443, by the search for similarity method of Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA, 1988, 85:2444, by computerized implementations 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 (e.g., Ausubel et al., Current Protocols in Molecular Biology, 1995, see supplement).

[0050] Two examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms described in Altschul et al., Nuc. Acids Res., 1977, 25, 3389-3402; and Altschul et al., J. Mol. Biol., 1990, 215, 403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This 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 a database sequence. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of word hits in each direction is stopped when the cumulative alignment score falls below its maximum achieved value by an amount X, when the cumulative score falls below 0 due to the accumulation of one or more negative-scoring residue alignments, or when 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 as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands.The BLASTP program uses as defaults, for amino acid sequences, a word length of 3, and an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignment (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands.

[0051] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 1993, 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 to be similar to a reference sequence if the smallest sum probability in the comparison of the test nucleic acid with 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.

[0052] "Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and their polymers in single- or double-stranded form, as well as their complements. The term encompasses synthetic, natural, and non-natural nucleic acids containing known nucleotide analogs or modified backbone residues or linkages that have similar binding properties as the reference nucleic acid and are metabolized similarly to the reference nucleotide. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2-O-methyl ribonucleotides, and peptide nucleic acids (PNAs). In various embodiments, nucleic acids are isolated when purified from other cellular components or other contaminants (e.g., other nucleic acids or proteins present in the cell) by standard techniques, including alkali / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and others well known in the art. See, e.g., F. Ausubel, et al., ed., Current Protocols in Molecular Biology, 1987, Greene Publishing and Wiley Interscience, New York. In various embodiments, the nucleic acid is, for example, DNA or RNA, and may or may not contain intron sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

[0053] As used herein, "pharmaceutically acceptable carrier" includes any of the standard pharmaceutical carriers, such as phosphate buffered saline, water and emulsions, e.g., oil / water emulsions or water / oil emulsions, and various types of wetting agents.

[0054] As used herein, the term "neuron" includes neurons and one or more parts thereof (e.g., neuronal cell body, axon, or dendrites). As used herein, the term "neuron" refers to a nervous system cell that includes a central cell body or nerve cell body and two types of extensions or processes: dendrites (which generally transmit the majority of the neural signal to the cell body) and axons (which generally transmit the majority of the neural signal from the cell body to target neurons or effector cells, such as muscles). Neurons can transmit information from tissues and organs to the central nervous system (afferent neurons or sensory neurons) and transmit signals from the central nervous system to effector cells (efferent neurons or motor neurons). Other neurons, called interneurons, connect neurons within the central nervous system (brain and spinal column). Specific examples of neuron types that may be subjected to treatments or methods according to the present invention include cerebellar granule neurons, dorsal root ganglion neurons, and cortical neurons.

[0055] The term "neurodegeneration" is used broadly and refers to any pathological change in neurons, including, but not limited to, death or loss of neurons, any changes preceding cell death, and any decline or loss of neuronal activity or function. Pathological changes can be spontaneous or induced by any event, including, for example, pathological changes associated with apoptosis. The neuron can be any neuron, including, but not limited to, a sensory neuron, a sympathetic neuron, a parasympathetic neuron, or an enteric neuron, e.g., a dorsal root ganglion neuron, a motor neuron, and a central neuron, e.g., an interneuron, from the spinal cord. Neurodegeneration or cell loss is a characteristic of various neurological diseases or disorders, e.g., 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.

[0056] As used herein, the term "dorsal root ganglia," also known as "DRG" ​​or "spinal ganglion" or "posterior root ganglion," refers to a cluster (ganglion) of neurons in the dorsal root of a spinal nerve. The dorsal root is the afferent sensory root, which carries sensory information to the brain. The cell bodies of sensory neurons, known as primary neurons, are located in the dorsal root ganglia. The axons of dorsal root ganglion neurons are known as afferent nerves. In the peripheral nervous system, afferent nerves refer to axons that relay sensory information to the central nervous system (i.e., the brain and spinal cord).

[0057] As used herein, the term "afferent neurons" carries information from sensory receptors in the skin and other organs to the central nervous system (i.e., the brain and spinal cord), while "efferent neurons" carry motor information from the central nervous system to the muscles and glands of the body. "Afferent" in this application refers to carrying inward to a central organ or central part, as in nerves that conduct impulses from the periphery of the body to the brain or spinal cord.

[0058] As used herein, the term "systemic" refers to relating to or affecting the entire body rather than a portion thereof. As used herein, the term "region-specific" refers to affecting only a specific region of the body (specific cell types, specific dermatomes, and specific spinal nerves) with no or minimal effect on the rest of the body.

[0059] As used herein, the term "sensory neuron," also known as "afferent neuron," refers to a neuron in the nervous system that converts specific types of stimuli into action potentials or graded potentials through its receptors. This process is called sensory transduction. The cell bodies of sensory neurons are located in the dorsal root ganglia of the spinal cord. In some embodiments, the terms "sensory neuron," "sensory fiber," and "afferent neuron" are used interchangeably in this application. Sensory neurons can be classified in various ways, for example, by their morphology, location, and the stimuli they are involved in detecting. For example, considering the stimuli that sensory neurons are involved in detecting, sensory neurons are classified into olfactory neurons for detecting odors, gustatory receptors for detecting tastes, photoreceptors for converting light into electrical signals, thermoreceptors for detecting temperature changes, mechanoreceptors for detecting changes in pressure or mechanical stress, proprioceptors (also called position detectors) for sensing the position of body parts relative to other body parts, and nociceptors for processing pain and temperature sensations. As another example, sensory neurons can also be classified as A, B, and C, according to their different sizes and degrees of myelination (and therefore different conduction velocities), and type A can be further classified as alpha, beta, gamma, and delta.

[0060] spasticity As used herein, "spasticity" refers to a condition in which certain muscles are hypersensitive to the stretch reflex. The term "spastic muscle" can also be used to describe such continuously contracted muscles resulting from spasticity. This contraction can cause muscle stiffness or tension, interfering with normal movement, speech, and walking. Furthermore, symptoms of spasticity can range from mild muscle stiffness or tension to painful, uncontrollable spasms. Joint pain or tension is also common with spasticity. Spasticity most often occurs in central nervous system disorders affecting upper motor neurons in the form of lesions, such as spastic diplegia or upper motor neuron syndrome, and can also be present in various types of multiple sclerosis, where spasticity occurs as a symptom of progressively worsening attacks on the myelin sheath and is therefore unrelated to the type of spasticity present in spasticity disorders rooted in neuromuscular cerebral palsy. Without being bound by theory, spasticity develops when an imbalance between excitatory and inhibitory inputs to motor neurons occurs due to damage to the spinal cord and / or central nervous system. The damage causes a change in the balance of signals between the nervous system and muscles, resulting in increased excitability of motor neurons and inducing muscle overactivity. Pharmacological interventions such as baclofen attempt to reduce muscle excitability and thus alleviate spasticity. Spasticity is seen in conditions in which the brain and / or spinal cord are damaged or unable to develop normally. These include cerebral palsy, multiple sclerosis, spinal cord injury, and acquired brain injuries, including stroke and traumatic brain injury.

[0061] In the case of spasticity due to spinal cord injury (SCI), the maladaptive state of the spinal cord below the injury results in increased spinal reflexes, increased muscle tone, and the appearance of involuntary tonic muscle contractions or spasms. These clinical symptoms represent a human clinical condition called spasticity. Regardless of the type and level of lesion, more than 70% of people with SCI experience spasticity one year after the traumatic event, which results in a lifelong, permanent motor disability.

[0062] Gamma-aminobutyric acid (GABA) Gamma-aminobutyric acid (GABA) and glutamate are the primary inhibitory and excitatory neurotransmitters in mammals. Neurotransmitters are signaling molecules secreted by neurons to affect other cells across synapses. Specifically, excitatory neurotransmitters have an excitatory effect on neurons. This means that they increase the likelihood that a neuron will fire a signal called an action potential within the receiving cell. Neurotransmitters can act in a predictable manner but can also be affected by drugs, diseases, and interactions with other chemical messengers. The balance between GABA and glutamate controls diverse processes such as neurogenesis, movement, the circadian clock, tissue development, and blood glucose regulation. Spasticity is thought to result from an imbalance between excitatory and inhibitory inputs to alpha motor neurons caused by damage to the spinal cord and / or central nervous system. Loss of GABA-mediated inhibition may play an important role in the gradual increase in spinal reflexes and the emergence of spasticity.

[0063] GABA acts at inhibitory synapses in the brain by binding to specific transmembrane receptors in the plasma membrane of both presynaptic and postsynaptic neuronal processes. This binding opens ion channels, allowing the flow of negatively charged chloride ions into the cell or positively charged potassium ions out of the cell. This action results in a negative change in the transmembrane potential, usually resulting in hyperpolarization. There are two general classes of GABA receptors: GABA receptors in which the receptor is part of a ligand-gated ion channel complex; A , and GABA, a G protein-coupled receptor that gates ion channels via an intermediate (G protein). B The mechanism of action of baclofen for treating spasticity is that it acts on GABA receptors. B It is believed that they act as receptor agonists to modulate ion channels.

[0064] Glutamic acid decarboxylase (GAD) GABA is synthesized from glutamate by the 67 kDa and 65 kDa isoforms of the enzyme glutamic acid decarboxylase (GAD67 and GAD65) with pyridoxal phosphate (PLP) as a cofactor. This process converts glutamate into GABA, i.e., the primary excitatory neurotransmitter, into the primary inhibitory neurotransmitter, thus reducing neuronal excitability.

[0065] Human GAD67 and GAD65 are encoded by the GAD1 gene (chromosome 2) and the GAD2 gene (chromosome 10), respectively, and were isolated and cloned by Bu et al. (1992) Proc Natl Acad Sci 89:2115-2119. Human GAD67 cDNA (GenBank: M81883.1; SEQ ID NO: 1) encodes a Mr 67,000 polypeptide with 594 amino acid residues (GenBank accession number NM_000817; SEQ ID NO: 2). Human GAD65 cDNA (GenBank: M81882.1; SEQ ID NO: 3) encodes a Mr 65,000 polypeptide with 585 amino acid residues (GenBank accession number NM_000818). Each of these is incorporated herein by reference.

[0066] In the literature, GAD67 and GAD1 are often used interchangeably to describe the gene encoding GAD67, and GAD65 and GAD2 are often used interchangeably to describe the gene encoding GAD65. In some embodiments, GAD67 and GAD65 may be referred to as two different enzymes, glutamic acid decarboxylase 67 and glutamic acid decarboxylase 65, respectively. In some embodiments, GAD67 and GAD65 may be referred to as two different genes encoding different enzymes, glutamic acid decarboxylase 67 and glutamic acid decarboxylase 65, respectively. In some embodiments, GAD1 and GAD2 may be referred to as two different genes encoding different enzymes, glutamic acid decarboxylase 67 and glutamic acid decarboxylase 65, respectively. In some embodiments, GAD1 may be used interchangeably with GAD67, and GAD2 may be used interchangeably with GAD65.

[0067] In some embodiments, "glutamic acid decarboxylase" or "GAD" as used herein can include wild-type or modified GAD67, wild-type or modified GAD65, and active fragments thereof. For example, a "polynucleotide encoding a GAD" can refer to a polynucleotide encoding wild-type or modified GAD67, wild-type or modified GAD65, or an active fragment thereof.

[0068] The present application provides a method for treating spasticity in a subject, comprising upregulating GAD gene, thereby treating spasticity in a subject. In some embodiments, GAD can include wild-type GAD67 or modified GAD67, wild-type GAD65 or modified GAD65, or an active fragment thereof. In some embodiments, GAD is wild-type GAD67 or modified GAD67, or an active fragment thereof.

[0069] Herpes simplex virus (HSV) Gene therapy capable of upregulating GAD genes (including GAD1 / GAD67 and GAD2 / GAD65) may provide a therapeutic approach for treating spasticity, using viral vectors to deliver therapeutic gene products such as wild-type or modified GAD, or active segments thereof.

[0070] As used herein, the term "viral vector" or "viral expression vector" refers to a nucleic acid vector that contains at least one element of a viral genome and can be packaged into a viral particle. In the context of the present invention, the term "viral vector" should be broadly understood to include a nucleic acid vector (e.g., a DNA viral vector) and the resulting viral particle. In the present application, the viral expression vector is an adeno-associated viral (AAV) vector or a herpes simplex viral (HSV) vector, preferably an HSV-1 vector or an HSV-2 vector, and even more preferably a defective viral vector derived from HSV-1. As used herein, the term "defective viral vector" refers to a viral vector that is missing a gene or part of a gene necessary to successfully complete the viral life cycle for replication.

[0071] The term "AAV" refers to the adeno-associated virus itself or its derivatives, including recombinant AAV vector particles. Furthermore, as used herein, the term "AAV" encompasses many different serotypes isolated from both human and non-human primate samples. Preferred AAV serotypes are human serotypes, more preferably human AAV serotypes 2, 5, and 9, and most preferably human AAV serotype 5, which is the serotype that exhibits the highest level of neurotropism.

[0072] The term "herpes simplex virus (HSV)" refers to a complex, non-integrating DNA virus that can infect a wide range of human and animal cells. HSV encompasses two serotypes: herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2). The genome of HSV-1 is approximately 153 kbp in size. It contains approximately 90 protein-coding genes and more than 12 microRNAs. The HSV-1 genome is composed of two unique segments, UL and US, each flanked by inverted repeats that encode important diploid genes.

[0073] The term "defective viral vector derived from HSV" includes defective recombinant HSV vectors, amplicon HSV vectors, and "pre-HSV-1 vectors" as defined herein. The term "defective recombinant HSV," as used herein, refers to a helper-independent vector whose genome contains at least a complete deletion of the genes encoding two essential proteins known as ICP4 and ICP27. The ICP4 gene is present in two copies located in the inverted repeat sequences known as c and c' of the viral genome; both copies of this gene are deleted. The gene encoding ICP27 is located in the unique long (UL) sequence of the viral genome.

[0074] Preferably, helper-independent vectors according to the invention carry a therapeutic transcription cassette integrated into the LAT (latency-associated transcript) locus, a repetitive locus contained in the inverted repeat sequences known as b and b' of the viral genome (see Berthomme et al., "Evidence for bidirectional element located downstream from the herpes virus simplex type 1 latency-associated promoter that increases its activity during latency", JOURNAL OF VIROLOGY, 2000, 74, 3613-3622; and Berthomme et al., "Enhancer and long-term expression functions of herpes simplex virus type 1 latency-associated promoter are both located in the same region", JOURNAL OF VIROLOGY, 2001, 75, 4386-4393, the contents of which are incorporated by reference).

[0075] More preferentially, the transcription cassette is located either between the latency-associated promoter (LAP) and the long-term expression (LTE) region (site 1) or between the LTE region and the DNA insulator (INS) sequence downstream of the LTE (site 2). The defective recombinant HSV-1 vector of the present invention expresses the GAD gene described above to restore GABA-mediated inhibition and balance in excitatory and inhibitory inputs, e.g., carries a transcription cassette expressing wild-type or modified GAD67 and / or wild-type or modified GAD65, or active fragments thereof, driven by a promoter. The b and b' sequences of the viral genome are also known as TRL (terminal repeat long) and IRL (internal repeat short), respectively, and the c' and c sequences are also known as IRS (internal repeat short) and TRS (terminal repeat short), where L and S refer to the unique long (L) and unique short (S) sequences of the HSV-1 genome, respectively.

[0076] Furthermore, helper-independent vectors according to the invention can contain additional deletions in genes encoding non-essential proteins, such as the ICP34.5, UL55, UL56 and UL41 proteins. These defective HSV vectors are propagated in cell lines co-expressing the proteins ICP4 and ICP27 (Marconi et al., "HSV-1-derived helper-independent defective vectors, replicating vectors and amplicon vectors, for the treatment of brain diseases", CURRENT OPINION IN DRUG DISCOVERY AND DEVELOPMENT, 13, 2010, 169-183, the contents of which are incorporated by reference).

[0077] WO 2006 / 050211 discloses the use of defective HSV-1 vectors for gene therapy to treat pain. However, the vectors according to the present invention differ from those described in WO 2006 / 050211 in several significant respects that are important for the usefulness and efficacy of the vectors according to the present invention. The most important transgenic transcription cassettes according to the present invention were introduced into the LAT locus because this region contains an LTE and a DNA insulator sequence (INS) that confers long-term expression to the promoter driving transgene expression in the transcription cassettes according to the present invention, whereas the vectors described in WO 2006 / 050211 were conceived and validated for their short duration of action and therefore the transcription cassettes were not introduced into the LAT region.

[0078] "Amplicon or amplicon vector" refers to a helper-dependent vector whose genome lacks most or all HSV genes encoding viral proteins. The genome of an amplicon vector is a concatameric DNA composed of multiple copies in tandem of a plasmid known as an amplicon plasmid, which carries, in addition to the transgenic DNA of interest (i.e., the transcription cassette), a single origin of DNA replication and a single packaging signal from the HSV-1 genome. Amplicon plasmids according to the present invention express the GAD genes described above to restore GABA-mediated inhibition and balance in excitatory and inhibitory inputs, e.g., carry a transcription cassette expressing wild-type or modified GAD67 and / or wild-type or modified GAD65, or active fragments thereof, driven by a promoter. In some embodiments, the promoter can be a DRG-specific promoter as described herein. In some embodiments, the promoter can be a ubiquitous promoter.

[0079] In a preferred embodiment, the vector according to the invention is a defective recombinant vector lacking at least the genes encoding the essential proteins ICP4 and ICP27, preferentially a vector lacking both ICP4 and ICP27, which vector may lack other genes encoding non-essential proteins such as the ICP34.5, UL55, UL56 and / or UL41 proteins, and which carries a transcription cassette integrated into the LAT region of the vector genome.

[0080] In any of the embodiments described herein, the defective recombinant vector lacks one copy of the ICP0 gene. In a preferred embodiment, one copy of the ICP0 gene is deleted from the IRL region of the HSV vector within the LAT, ICP0, UL34.5 cluster.

[0081] In some embodiments, the vector of the present invention is an amplicon vector carrying a transcription cassette driven by a promoter, as described elsewhere herein. In a preferred embodiment, the transcription cassette of the present invention is introduced into the LAT locus. The term "recombinant DNA," as used herein, refers to a nucleic acid molecule, i.e., a polynucleotide of genomic, cDNA, viral, semisynthetic, and / or synthetic origin, that is unrelated, by virtue of its origin or manipulation, to all or part of the polynucleotide with which it is associated in nature. The term "recombinant," as used with respect to viruses, refers to viruses carrying a modified genome or a genome engineered to introduce one or more heterologous polynucleotides containing mutations, deletions, or genes. The term "recombinant," as used with respect to proteins or polypeptides, refers to a polypeptide produced by expression of a recombinant nucleic acid. The term "recombinant," as used with respect to host cells, refers to a recombinant vector carrying recombinant DNA within a host cell or a cell containing recombinant DNA inserted into its genome. The term "infection" refers to the ability of a viral vector to enter a host cell, organ, or subject, or the ability of a gene product of the viral vector to enter a host cell.

[0082] Defective vectors derived from HSV can infect adjacent sensory neurons and establish a latent infection in the nuclei of these neurons located in the trigeminal ganglion or dorsal root ganglion (DRG), depending on the site of infection. In particular, HSV-1 naturally infects sensory neurons and establishes a lifelong latent infection in the nuclei of these neurons. For example, subcutaneous inoculation of an HSV vector encoding GAD67 in the foot allows infection of DRG neurons, resulting in the constitutive production of GAD and the release of GABA to treat spinal cord injury pain. (See Liu et al., MOLECULAR THERAPY, 2004, Vol. 10, No. 1, 57-66.) Following injection into a dermatome, the defective vectors derived from HSV-1 disclosed herein can reach the sensory DRG innervating the dermatome stably expressing therapeutic transgenes, provided that an appropriate promoter drives their long-term expression. In some embodiments, promoters can include both afferent neuron-specific and ubiquitous promoters. In some embodiments, the promoter is a ubiquitous promoter, preferably EF-lα. In some embodiments, the promoter is an afferent neuron-specific promoter as disclosed in WO2017220800.

[0083] Pre-HSV-1 vector In some embodiments, the viral vector used in the methods of the present application may comprise a pre-HSV-1 vector.

[0084] As used herein, a "pre-HSV-1 vector" is a mini-HSV-1 backbone (also known as a "mini-HSV-1" or "pre-HSV-1 vector") from which non-essential genes, essential genes, or a combination thereof have been deleted to arrive at a genome containing less than 130 kbp and more than 75 kbp. Pre-vector or mini-vector "backbones" embodying the present invention are described with the understanding that, as "backbones," it is contemplated that a polynucleotide encoding a GAD may be inserted therein, with or without exogenous regulatory elements. In some embodiments, the GAD may be wild-type or modified GAD67, wild-type or modified GAD65, or an active fragment thereof.

[0085] As used herein, the modifier "essential" in the phrases "essential gene" or "non-essential gene" means that a given gene is required (or not) to achieve multiplication and packaging of the viral genome and thus produce infectious progeny viral particles. HSV-1 essential genes include UL1, UL5-UL9, UL12, UL14, UL15, UL17-UL19, UL22, UL25-UL38, UL42, UL48, UL49, UL52-UL54, US6, and ICP4 (2 copies). HSV-1 non-essential genes include ICP34.5 (2 copies), ICP0 (2 copies), LAT (2 copies), UL2 to UL4, UL10, UL11, UL13, UL16, UL20, UL21, UL23, UL24, UL39, UL40, UL41, UL43 to UL47, UL50, UL51, UL55, UL56, US1 to US5, and US7 to US12.

[0086] In some embodiments, clusters of genes that may be deleted include, but are not limited to, genes UL2, UL3, UL4 (10.200-12.600); genes UL10, UL11 (23.200-25.200); gene UL16 (30.200-31.400); genes UL20, UL21 (40.800-43.700); genes UL23, UL24 (46.700-48.600); genes UL39, UL40, UL41 (86.400-92.700); genes UL43-UL47 (94.700-103. 200); genes UL50, UL51 (107.700 to 109.100); genes UL55, UL56 (115.400 to 117.100); one copy of genes LAT, ICP0, UL34.5 (IRL) (118.700 to 126.100); genes US2 to US5 (134.000 to 138.200); genes US7 to US12 (139.700 to 145.600); and / or a second copy of gene ICP0 if the first copy has already been removed from the LAT, ICP0, UL34.5 cluster.

[0087] In some embodiments, the miniHSV-1 comprises a genome with at least 30 kbp deleted. In some embodiments, the miniHSV-1 comprises a genome with at least 40 kbp deleted. In some embodiments, the miniHSV-1 comprises a genome with at least 45 kbp deleted. In some embodiments, the miniHSV-1 comprises a genome with at least 50 kbp deleted. In some embodiments, the miniHSV-1 comprises a genome with at least 55 kbp deleted. In some embodiments, the miniHSV-1 comprises a genome with at least 60 kbp deleted. In some embodiments, the miniHSV-1 comprises a genome with at least 65 kbp deleted. In some embodiments, the miniHSV-1 comprises a genome with at least 75 kbp deleted.

[0088] In some embodiments, the mini-HSV-1 comprises a genome with a 25 kbp to 80 kbp deletion. In some embodiments, the mini-HSV-1 comprises a genome with a 30 kbp to 75 kbp deletion. In some embodiments, the mini-HSV-1 comprises a genome with a 35 kbp to 70 kbp deletion. In some embodiments, the mini-HSV-1 comprises a genome with a 40 kbp to 60 kbp deletion.

[0089] Pre-HSV-1 in which nonessential genes, essential genes, or a combination thereof have been deleted to arrive at a genome containing less than 130 kbp or more than 75 kbp can allow for the insertion therein of a polynucleotide encoding GAD, with or without exogenous regulatory elements. As a non-limiting example, the recombinant viral vectors used in the present invention may use recombination techniques based on the markerless Red recombination system to generate scarless point mutations, deletions, and insertions of relatively small and large sequences (e.g., Tischer et al., En Passant Mutagenesis: A Two-Step Markerless Red Recombination System. Chapter 30, In Vitro Mutagenesis Protocols: Third Edition, Methods in Molecular Biology, vol. 634. DOI 10.1007 / 978-1-60761-652-8_30, © Springer Science+Business Media, LLC 2010). In some embodiments, a pre-HSV-1 vector as used in the present invention comprises a modified HSV-1 genome in which non-essential genes, essential genes, or a combination thereof have been deleted to arrive at a genome comprising less than 130 kbp and more than 75 kbp.

[0090] In some embodiments, a transgene of interest can be introduced into the LAT (latency associated transcript) locus, a repetitive locus contained in inverted repeat sequences known as b and b' of the viral genome. The b and b' sequences of the viral genome are also known as TRL (Terminal Repeat Long) and IRL (Internal Repeat Long), respectively. In some embodiments, the viral genome contains both LAT regions, one in the TRL and the other in the IRL. In some embodiments, one of the LAT regions, either in the TRL or the IRL, is deleted. In some embodiments, if the vector genome contains two LAT loci, a transgene of interest can be introduced into both the TRL and IRL loci. In some embodiments, if the LAT locus in the IRL region is deleted, a transgene of interest can be introduced into the LAT locus in the TRL region only. In some embodiments, if the LAT locus in the TRL region is deleted, a transgene of interest can be introduced into the LAT locus in the IRL region only.

[0091] The LAT locus comprises an upstream DNA insulator (INS) sequence, a latency-associated promoter (LAP), a region conferring long-term expression (LTE), and a downstream DNA insulator (INS). In some embodiments, a transgene of interest is introduced either between the latency-associated promoter (LAP) and the long-term expression (LTE) region, or between the LTE region and a DNA insulator (INS) sequence located downstream of the LTE.

[0092] Importantly, the LAT locus contains an LTE and a DNA insulator sequence (INS) that confers long-term expression to a polynucleotide encoding GAD that is introduced at this site.

[0093] "Long-term expression sequence" or "long-term expression element (LTE)" means a nucleotide sequence that, when operably linked to a foreign DNA of interest, enables sustained expression of a gene product for more than 15-45 days, or more than 30-45 days, or 45-90 days, or 90-365 days, or 365 days to several years, or even the lifetime of the patient. The long-term expression (LTE) sequence was identified in HSV-1 as a region of the latency-associated transcript (LAT) that is derived from the LAT-associated promoter (LAP). The LTE is located downstream of the LAT transcription start site.

[0094] Indeed, viruses carrying a DNA fragment 3' of the LAT promoter maintained detectable promoter expression throughout latency (Lokensgard et al., "The latency-associated promoter of herpes simplex virus type 1 requires a region downstream of the transcription start site for long-term expression during latency," Journal of Virology, 1997, 71, 6714-6719; Berthomme et al., "Evidence for a bidirectional element located downstream from the herpes virus simplex type 1 latency-associated promoter that increases its activity during latency," Journal of Virology, 74, 2000, 3613-3622; and Berthomme et al., "Enhancer and long-term expression functions of herpes simplex virus type 1 latency-associated promoter are both located in the same region," Journal of Virology, 75, 2001, 4386-4393, the contents of which are incorporated by reference). Preferably, the LTE is contained approximately 1.5 kb to approximately 3 kb downstream of the LAT transcription start site (Perng et al., "The spontaneous reactivation function of the herpes simplex virus type 1 LAT gene resides completely within the first 1.5 kilobases of the 8.3-kilobase primary transcript", JOURNAL OF VIROLOGY, 1996, 70, 976-984, the contents of which are incorporated by reference).More recently, additional sequences known as DNA insulators have been described both upstream and downstream of LTE regions (Amelio et al., "A chromatin insulator-like element in the herpes simplex virus type 1 latency-associated transcription region binds CCCTC-binding factor and displays enhancer-blocking and silencing activities," JOURNAL OF VIROLOGY, 2006, 80, 2358-2368, the contents of which are incorporated by reference). These sequences also contribute to providing long-term expression to a given transcription cassette, presumably by inhibiting epigenetic silencing, and are incorporated into the present invention as part of an LTE element to confer long-term expression to an expression cassette. Interestingly, sequences conferring long-term expression to a transcription cassette (both LTE sequences and DNA insulator sequences) can be placed either upstream and / or downstream of the GAD expression cassette.

[0095] Those skilled in the art will recognize that other LTE-like sequences and other DNA insulator sequences have been described and continue to be discovered, and all such LTE-like sequences and DNA insulator sequences are encompassed by the present invention.

[0096] In some embodiments, one or more exogenous genes of interest are introduced into the pre-HSV-1 vector. In some embodiments, a combination of one or more HSV-1 essential genes, one or more HSV-1 nonessential genes, and one or more exogenous genes of interest are introduced into the pre-HSV-1 vector. In some embodiments, a combination of one or more HSV-1 essential genes and one or more exogenous genes of interest are introduced into the pre-HSV-1 vector. In some embodiments, a combination of one or more HSV-1 nonessential genes and one or more exogenous genes of interest are introduced into the pre-HSV-1 vector.

[0097] It is important that the pre-HSV-1 vector used herein maintains sufficient HSV-1 genome so as not to become an HSV-1 amplicon. "Amplicon or amplicon vector" refers to a helper-dependent vector whose genome lacks most or all HSV genes encoding viral proteins. The genome of an amplicon vector is a concatemeric DNA composed of multiple copies in tandem of a plasmid (known as an amplicon plasmid) that carries a single origin of DNA replication and a single packaging signal from the HSV-1 genome. In cells expressing the complete set of structural, replicative, and DNA packaging functions from HSV-1 due to the presence of the HSV-1 genome acting as a helper, the amplicon plasmid is amplified by a rolling circle mechanism into long head-to-tail concatemers, which are then cleaved and packaged into HSV-1 virions to the size of a single genome (Kwong and Frenkel, 1985; Bataille and Epstein, 1997). Thus, amplicon vectors are concatameric plasmid DNAs packaged into HSV-1 particles.

[0098] In contrast to amplicons, the pre-HSV-1 vectors of the present invention are helper-independent vector platforms, meaning that they do not require the presence of the HSV-1 genome to act as a helper virus for vector replication and packaging.

[0099] HSV-1 vector As explained above, it is contemplated that the pre-vector or mini-vector "backbone" used in the methods of the present invention is a vector template into which a polynucleotide encoding GAD, with or without exogenous regulatory elements, may be inserted. For example, in some embodiments, a polynucleotide encoding GAD may be introduced into the LAT region of a pre-HSV-1 vector. In embodiments according to the present invention, a transcription cassette expressing the GAD gene described above to restore GABA-mediated inhibition and balance in excitatory and inhibitory inputs, e.g., promoter-driven expression of wild-type or modified GAD67 and / or wild-type or modified GAD65, or active fragments thereof, may be introduced into the LAT region of a pre-HSV-1 vector.

[0100] In one embodiment, the methods and treatment regimens of the present invention use HSV-1 vectors, including pre-HSV-1 vectors described herein, into which a polynucleotide encoding GAD can be introduced, enabling the HSV-1 vector to persistently express GAD. In some embodiments, the LAT region of the HSV-1 vector can be used to introduce the polynucleotide encoding GAD. GAD, as defined above, can include wild-type or modified GAD67, wild-type or modified GAD65, and active fragments thereof. In a preferred embodiment, the methods and treatment regimens of the present invention use HSV-1 vectors, including pre-HSV-1 vectors described herein, into which a polynucleotide encoding GAD67 (SEQ ID NO: 1) can be introduced, enabling the HSV-1 vector to persistently express the protein product GAD67 (SEQ ID NO: 2). In some embodiments, the LAT region of the HSV-1 vector can be used to introduce the polynucleotide encoding GAD67.

[0101] In a preferred embodiment, the HSV-1 vector comprises a polynucleotide encoding GAD inserted in operably linked relation to one or more LTE and / or DNA insulator sequences within the HSV vector genome. By "operably linked," it is understood that the one or more LTE and / or DNA insulator sequences allow the polynucleotide encoding GAD to be expressed in a cellular environment in which genetic elements (i.e., "genes") otherwise present within the HSV genome are transcriptionally silent.

[0102] As described herein, the pre-HSV-1 vectors of the present invention contain deletions in the HSV genome such that the pre-HSV-1 vectors contain genomes having less than 130 kbp and more than 75 kbp. These deletions create genomic space that allows for the introduction and delivery of very large pieces of foreign DNA. Introduction of a GAD expression cassette into the LAT region increases the genome size (i.e., number of base pairs) of the resulting HSV-1 vector.

[0103] As used herein, the term "one or more exogenous genes of interest" may include, but is not limited to, a reporter gene (e.g., GFP, RFP, luciferase, or fusion protein) driven by a transient promoter that acts as an internal expression control or for biodistribution studies; a recombinase driven by an inducible promoter that allows cellular or viral genes to be modified in vivo; an antibiotic resistance gene such as chloramphenicol; a tetracycline inducible element (TRE); a GAD gene, or a combination thereof. The use of a reporter gene, such as, but not limited to, cherry, RFP, GFP, or CFP, can facilitate identification of recombinant genomes and scoring of both infectious particles (PFU) and transducing units (TU).

[0104] In some embodiments, the modified HSV-1 vector may optionally include a DNA sequence introduced into a gene region within the genome, such as an endogenous and / or foreign DNA sequence encoding a cell-targeting protein. The cell-targeting protein can retarget viral entry to any tissue or cell type of interest. Cell-targeting genes for insertion into the HSV-1 vectors used in the present invention may include, but are not limited to, HER-2, IL13α2, or modified versions thereof. Examples of HSV-modified glycoprotein D can be found, for example, in European Patent No. 3469071, which is incorporated herein by reference.

[0105] promoter At least a promoter sequence is present in the GAD expression cassette inserted into the viral vector described herein, and expression of the polynucleotide encoding GAD can be controlled by the promoter.

[0106] The promoter may comprise DNA sequences starting at least 2 kb, preferably 3 kb, and more preferably 4 kb upstream of the start site of the polynucleotide encoding GAD. These sequences preferably contain known promoter sequence elements, such as specific transcription binding sites, and distal sequences upstream of the gene that contain additional regulatory elements.

[0107] The promoter useful in the present invention can be any promoter that is desirable for controlling / regulating the expression of a polynucleotide encoding GAD. Exemplary promoters useful in the methods and treatment regimens of the present application include, but are not limited to, the human ubiquitin promoter and the human synaptic promoter. Other known tissue-specific or cell-specific promoters can also be used.

[0108] In some embodiments, contemplated promoters are constitutive mammalian promoters such as those known in the art (e.g., EF-lα, UBC, β-actin, PGK, etc.).

[0109] In some embodiments, promoters useful in the present invention may be selectively active in afferent neurons. "Selectively active in afferent neurons," as used herein, means that the promoter is primarily or only active in afferent neurons and promotes RNA transcription. Promoters for use in afferent neurons may be selected from, but are not limited to, promoters of genes encoding sensory neuroreceptors, such as transient receptor potential vanilloid 1 (TRPV1) or transient receptor potential cation channel subfamily M member 8 (TRPM8); and promoters of genes encoding sensory neuromodulators or sensory neurotransmitters, such as promoters of substance P, PACAP, and calcitonin gene-related peptide (CGRP). In some embodiments, the promoter of a gene encoding a sensory neuroreceptor according to the present invention is a promoter of the TRP gene family, more preferably the promoter TRPV1 or TRPM8. In some embodiments, the promoter of a gene encoding a sensory neuromodulator or sensory neurotransmitter according to the present invention is a promoter of CGRP, or a promoter of a gene involved in neurite outgrowth and stress response in sensory neurons, preferably a promoter of the gene encoding advillin (ADVL). In other embodiments, the promoter in the GAD expression cassette inserted into the viral vectors described herein can be an inducible promoter.

[0110] Those skilled in the art will also recognize that many such mammalian afferent neuron-specific promoters are known, and that additional afferent neuron-specific promoters continue to be discovered. All such afferent neuron-specific promoters are encompassed by the present invention. By way of non-limiting example, contemplated afferent neuron-specific promoters can be selected from those disclosed in WO2017220800 and Joussain et al. Int. J. Mol. Sci. 2022, 23, 8474.

[0111] In some embodiments, promoters useful in the present invention may be ubiquitous promoters, such as, but not limited to, the human cytomegalovirus (HCMV) promoter, human elongation factor 1 alpha (hEF-1α) promoter, β-actin promoter, Rous sarcoma virus (RSV) promoter, human ubiquitin C (hUBC) promoter, ubiquitin B promoter, simian vacuolar virus 40 (SV40) promoter, phosphoglycerate kinase (PGK) promoter, β-globin promoter, NF-κB promoter, EGR1 promoter, eIF4A1 promoter, FerL promoter, GAPDH promoter, β-Kin promoter, ROSA26 promoter, and human surfactant protein C (hSP-C) promoter. As used herein, the term "ubiquitous promoter" or "non-specific promoter" means that the promoter is active in a wide range of cells, tissues, and / or organs.

[0112] In some embodiments, the non-specific promoter according to the present invention is selected from the hEF-1α promoter of SEQ ID NO: 5, the HCMV promoter of SEQ ID NO: 6, the RSV promoter of SEQ ID NO: 7, the hUBC promoter of [[SEQ ID NO: 8]], the SV40 promoter of SEQ ID NO: 9, the PGK promoter of SEQ ID NO: 10, the β-globin promoter, the NF-κB promoter, and the hSP-C promoter.

[0113] In a preferred embodiment, the non-specific promoter useful in the method is the hEF-1α promoter (SEQ ID NO: 5).

[0114] The viral expression vector of the present invention is more particularly intended for vertebrates, preferably mammals, more preferably primates and humans.Therefore, those skilled in the art will recognize that such promoters are species-specific and can select the homologous sequence of the specific species of interest.For example, the promoter of the present invention is, inter alia, the human homolog of rat TRPV1, or human TRPM8, or rat CGRP or human CGRP, or rat advilin or human advilin.

[0115] dermatome A "dermatome" is a skin area primarily supplied by afferent nerve fibers from the dorsal root of any given spinal nerve. In other words, a dermatome is a distinct skin area defined by its connection to one of the 30 spinal nerves. There are eight cervical nerves (C1-C8, with C1 being the exception, as it does not have a dermatome), 12 thoracic nerves (T1-T12), five lumbar nerves (L1-L5), and five sacral nerves (S1-S5). Each of these nerves relays sensation (including pain) from a specific skin area (i.e., a specific dermatome) to the brain. Dermatomes are clinically important because they can help diagnose various conditions. For example, symptoms occurring along a specific dermatome may indicate a condition related to a specific spinal nerve.

[0116] In some embodiments, the viral vectors described herein for upregulating the GAD gene to treat spasticity may be administered by one or more injections into one or more dermatomes. In some embodiments, the viral vectors described herein for upregulating the GAD gene to treat spasticity may be administered by multiple injections, preferably subcutaneous injection, into multiple dermatomes. To achieve sufficient expression of the GAD gene, multiple injections may be necessary to recruit as many afferent nerves as possible. In some embodiments, the viral vectors described herein for upregulating the GAD gene to treat spasticity may be administered by 1 to 30 injections, preferably subcutaneous injection, into multiple dermatomes. In some embodiments, the viral vectors described herein for upregulating the GAD gene to treat spasticity may be administered by 5 to 20 injections, preferably subcutaneous injection, into multiple dermatomes. In some embodiments, the viral vectors described herein for upregulating the GAD gene to treat spasticity may be administered by 10 to 15 injections, preferably subcutaneous injection, into multiple dermatomes. In some embodiments, the viral vectors described herein for upregulating the GAD gene to treat spasticity may be administered by 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 injections, preferably subcutaneous injection, into multiple dermatomes. In some embodiments, the viral vectors described herein for upregulating the GAD gene to treat spasticity may be administered by 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 injections, preferably subcutaneous injection, into 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 dermatomes, with each dermatome receiving 1 to 20 injections. In some embodiments, the dermatome into which the viral vector for upregulating the GAD gene is injected can be determined according to the spastic muscle that the viral vector targets for treatment.

[0117] In some embodiments, the viral vectors described herein for upregulating the GAD gene to treat spasticity can be administered by multiple injections, preferably subcutaneous injections, into a single dermatome. In some embodiments, the viral vectors described herein for upregulating the GAD gene to treat spasticity can be administered by multipoint subcutaneous injections into a single dermatome. In some embodiments, contemplated multipoint subcutaneous injections include 1 to 20 injections at 1 to 20 sites in the target dermatome to cover the entire or most of the surface of the target dermatome. In some embodiments, contemplated multipoint subcutaneous injections include 5 to 15 injections at 5 to 15 sites in the target dermatome to cover the entire or most of the surface of the target dermatome. In some embodiments, contemplated multi-point subcutaneous injections include 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 injections at 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 sites in the target dermatome, respectively, to cover the entire or most of the surface of the target dermatome.

[0118] In some embodiments, a medical device for administering multiple subcutaneous injections can be used to provide multiple injections of a viral vector described herein at multiple sites in a target dermatome at once, covering the entire or most of the surface of the target dermatome. In some embodiments, the medical device can provide 1 to 10 injections of a viral vector described herein at multiple sites in a target dermatome at once, covering at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% of the surface of the target dermatome. In some embodiments, the medical device can provide 2 to 9 injections of a viral vector described herein at multiple sites in a target dermatome at once, covering the entire or most of the surface of the target dermatome. In some embodiments, the medical device can provide 3, 4, 5, 6, 7, or 8 injections of the viral vector described herein at multiple sites in the target dermatome at once, covering the entire or most of the surface of the target dermatome. Medical devices for administering multiple subcutaneous injections can include any known or currently discovered medical device capable of providing subcutaneous injections at multiple sites in a minimally invasive manner (e.g., multi-injectors, circular, 7-needle connections, Mesoram®). In a preferred embodiment, the viral vector for upregulating the GAD gene is a defective viral vector derived from HSV described herein. Depending on the severity of spasticity or a particular spastic muscle, in some embodiments, a single injection into one particular dermatome is required to be therapeutically effective. In some embodiments, multiple injections into one particular dermatome over a period of time are required to be therapeutically effective. In some embodiments, multiple injections into each of multiple specific dermatomes, whether adjacent to each other or separated from each other, are required to be therapeutically effective.After injection into the dermatome, the viral vector, e.g., a vector derived from HSV-1, reaches the sensory DRGs innervating the dermatome where it stably expresses the therapeutic transgene, provided that an appropriate promoter drives their expression. The therapeutic transgene described herein can be a polynucleotide encoding wild-type or modified GAD or an active fragment thereof, e.g., wild-type or modified GAD67 or an active fragment thereof, or wild-type or modified GAD65 or an active fragment thereof.

[0119] Pharmaceutical Composition The viral vectors described in the present application can be administered in pharmaceutical compositions that can include a pharmaceutically acceptable carrier. The carrier of the composition can be any carrier suitable for the vector. The carrier is typically liquid, but can also be solid, or a combination of liquid and solid components. The carrier is desirably a pharmaceutically acceptable (e.g., physiologically or pharmacologically acceptable) carrier (e.g., an excipient or diluent). The composition can further include any other suitable ingredients, particularly to enhance the stability of the composition and / or its end use. Accordingly, there are a wide variety of suitable formulations of viral vector compositions. The following formulations and methods are merely exemplary and are in no way limiting.

[0120] Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions which may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives. The formulations may be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials, and may be stored in a freeze-dried (lyophilized) state requiring only the addition of a sterile liquid vehicle for injection, e.g., water, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets of the kind described above.

[0121] In addition, the composition can include additional therapeutic or biologically active agents. For example, therapeutic agents useful for treating specific indications can be present. Inflammation-controlling agents, such as ibuprofen or steroids, can be part of the composition to reduce swelling and inflammation and physiological distress associated with in vivo administration of the viral vector. Immune system suppressants can be administered with the composition to reduce immune responses to the vector itself or immune responses associated with the disorder. Alternatively, immune enhancers can be included in the composition to upregulate the body's natural defenses against disease. Antibiotics, i.e., bactericides and fungicides, can be present to reduce the risk of infection associated with gene transfer procedures and other disorders.

[0122] The method for treating spasticity may further include the administration (i.e., pre-administration, co-administration, and / or post-administration) of other treatments and / or agents to modify (e.g., enhance) the effectiveness of the method. The method of the present invention may further include the administration of other substances that locally or systemically alter (i.e., reduce or enhance) the effect of the composition on the host. For example, substances that reduce any systemic effect of a protein produced by expression of the nucleic acid sequence of the vector in the host can be used to control the level of systemic toxicity in the host. Similarly, substances that enhance the local effect of a protein produced by expression of the nucleic acid sequence of the vector in the host can be used to reduce the level of the protein necessary to provide a prophylactic or therapeutic effect in the host. Such substances include antagonists, e.g., soluble receptors or antibodies against the protein produced by expression of the nucleic acid sequence of the vector, and agonists of the protein.

[0123] Dermatome-specific administration In some embodiments, the application also provides methods of administering a viral vector to a subject for the treatment of spasticity.

[0124] As used herein, the terms "administration" or "administering" are defined to include the act of providing a viral vector pharmaceutical composition described herein to a subject when performing the methods of the present invention. Exemplary routes of administration include, but are not limited to, intravenous, intraarticular, intracisternal, intraocular, intraventricular, intrathecal, subcutaneous, subpial, intramuscular, intraperitoneal, intradermal, intracavitary, etc., and combinations of any two or more thereof. In some embodiments, the defective viral vectors derived from HSV described herein can be delivered to the spinal cord parenchyma, the spinal cavity of the spine, the spinal subpial space of a subject, and / or directly to peripheral spastic muscles to achieve spinal upregulation of the GAD gene. (See, e.g., WO 2016 / 122791.) In some embodiments, the defective viral vectors derived from HSV described herein can be delivered peripherally to any skin region to transfect connected afferent neurons. (See, e.g., Liu et al., MOLECULAR THERAPY, 2004, Vol. 10, No. 1, 57-66.)

[0125] The term "therapeutically effective amount" or "effective amount" refers to the amount of viral vector that induces the biological or medical response of a tissue, system, animal, or human that is desired by a researcher, veterinarian, physician, or other clinician, e.g., upregulation of the GAD genes in afferent nerve fibers supplying a dermatome, including the GAD67 and GAD65 genes, preferably the GAD67 gene. Thus, the term "therapeutically effective amount" is used herein to refer to any amount of formulation that, when applied repeatedly to the affected area over a period of time, causes substantial improvement in a condition associated with spasticity. The amount will vary depending on the condition being treated, the stage of progression of the condition, and the type and concentration of formulation applied.

[0126] Determination of a therapeutically or prophylactically effective amount of a delivery vector can be performed based on animal data using routine calculation methods. The appropriate dose depends, among other factors, on the specifics of the transfer vector selected, the route of administration, the number of injection sites, the mammal (e.g., human or non-human primate or other mammal) being treated, the age, weight, and general condition of the subject being treated, the severity of the disorder being treated, the location of the area within the heart being treated, and the mode of administration. Thus, the appropriate dosage may vary from patient to patient.

[0127] Dosage treatment can be a single dose schedule or a multiple dose schedule. Furthermore, subjects can receive multiple doses as needed. Dosage may need to be adjusted to take into account alternative routes of administration, to account for decreased efficacy over time, or to balance therapeutic benefit against any side effects.

[0128] HSV-1 vectors can efficiently infect cells and resist immune clearance, which may be due to the innate immune evasion properties of HSV envelope proteins. The natural immune evasion function, combined with the deletion of IE genes (such as ICP4, ICP22, and ICP27) from the HSV-1 vector backbone, allows for multiple administration of defective viral vectors derived from HSV-1 vectors, improving transduction efficiency and making the vectors particularly well suited for gene therapy. (See Heldwein et al., Cell. Mol. Life Sci., 2008, 65, 1653-1668; Tognarelli et al., Front. Cell. Infect. Microbiol., 2019, 9, 127; Yang et al., Front. Immunol., 2019, 10, 2196; Gurevich et al., Nature Medicine, 2022, 28, 780-788.)

[0129] Also provided herein are methods for treating spasticity in a subject, comprising administering multiple doses of an HSV-1 vector described herein to the subject. Over the course of spasticity treatment, multiple doses of the HSV-1 vector described herein may be administered based on the severity of the spasticity symptoms, with 1 to 10 doses typically administered at intervals of about 21 days (3 weeks) to about 3 years. For example, if the treatment schedule needs to be modified to improve therapeutic efficacy or reduce side effects, intervals of about 3 months to about 12 months may be used. Over the course of spasticity treatment, 2 to 10 treatments (each treatment comprising, for example, one or more subcutaneous injections at a time, with or without a multi-injection device described herein) of the vector described herein may be administered, with 2 to 4 treatments typically administered at intervals of about 3 months, about 6 months, about 12 months, about 24 months, or about 36 months. In some embodiments, the vector described herein is administered at a dose of about 1 x 10 6 vg / kg weight ~ approx. 1×10 15 In some embodiments, the vectors described herein may be administered to a subject at a dose of about 1 x 10 vg / kg body weight. 7 vg / kg weight ~ approx. 1×10 12 The dose may be administered to a subject at a dose of 0.05 mg / kg body weight. Over the course of treatment for spasticity, the dose may remain constant over time, or the dose may be decreased or increased over time to optimize therapeutic efficacy and minimize side effects.

[0130] Optionally, HSV-1-mediated delivery according to the present invention can be combined with delivery by other viral and non-viral vectors. Such other viral vectors can include, but are not limited to, adenoviral vectors, adeno-associated viral (AAV) vectors, retroviral vectors, lentiviral vectors, and baculoviral vectors. Non-viral vectors can include, but are not limited to, liposomes, lipid-based vectors, polyplex vectors, molecular conjugates, polyamine, and polycation vectors.

[0131] In a preferred embodiment, the present application provides a method for treating spasticity in a subject, the method comprising upregulating a GAD gene, thereby treating spasticity in the subject. Upregulating the GAD gene comprises administering to the subject a viral vector comprising a polynucleotide encoding GAD, whereby GAD is expressed, thereby reducing spasticity. The viral vector is a defective viral vector derived from HSV as described herein. The polynucleotide may encode wild-type GAD67 or modified GAD67 and / or wild-type GAD65 or modified GAD65, or an active fragment thereof, more preferably wild-type GAD67 or modified GAD67, or an active fragment thereof. The defective viral vector derived from HSV as described herein is administered directly to one or more dermatomes of the subject. In one aspect, the defective viral vector derived from HSV as described herein is administered directly to one or more dermatomes of the subject by subcutaneous inoculation. As described herein, depending on the severity of spasticity or a particular spastic muscle, in some embodiments, a single injection of the vector into one particular dermatome is required for therapeutic efficacy. In some embodiments, multiple injections of the vector into one specific dermatome over a period of time are required to be therapeutically effective. In some embodiments, multiple injections of the vector into each of multiple specific dermatomes, regardless of whether the dermatomes are adjacent to one another or whether the dermatomes are distant from one another, are required to be therapeutically effective. In some embodiments, the dermatome selected for administration of the viral vector is connected to a spastic muscle group. Administration of the viral vector into the dermatome allows infection of sensory neurons associated with the spastic muscle group, causing the sensory neurons to produce and release GABA and become inhibitory neurons, thereby reducing spasticity.

[0132] In a preferred embodiment, a method for treating spasticity in a subject comprises administering to the subject a therapeutically effective amount of a viral vector comprising a polynucleotide encoding GAD67, thereby treating the spasticity in the subject. The viral vector is a defective viral vector derived from HSV as described herein. The polynucleotide may encode wild-type GAD67 or modified GAD67 and / or wild-type GAD65 or modified GAD65, or an active fragment thereof, more preferably wild-type GAD67 or modified GAD67, or an active fragment thereof. The defective viral vector derived from HSV as described herein is administered directly to one or more dermatomes of the subject. In one aspect, the defective viral vector derived from HSV as described herein is administered directly to one or more dermatomes of the subject by subcutaneous inoculation. As described herein, depending on the severity of the spasticity or the particular spastic muscle, one or more injections of the vector into one or more dermatomes may be required to be therapeutically effective.

[0133] In another aspect, the present invention also provides a therapeutic regimen for treating a subject suffering from spasticity or a spasticity-related condition. The therapeutic regimen comprises afferent neuron-specific upregulation of GAD genes, including the GAD67 gene and the GAD65 gene, preferably the GAD67 gene. As described in detail above, upregulation of GAD (preferably GAD67) can include administering a viral vector encoding a GAD gene (preferably the GAD67 gene), such that GAD (preferably GAD67) is expressed and neurons produce and release GABA, i.e., excitatory neurotransmitters are converted into inhibitory neurotransmitters, thereby treating spasticity or a spasticity-related condition. As described herein, depending on the severity of spasticity or a particular spastic muscle, one or more injections of the vector into one or more dermatomes may be required to be therapeutically effective.

[0134] Mechanism of action In some embodiments, the present application proposes a mechanism of action for using the viral vectors described herein (preferably, defective viral vectors derived from HSV-1) for the treatment of spasticity. The disclosed methods for treating spasticity are region-specific (i.e., specifically affecting the spinal microcircuits generating abnormal muscle activity while avoiding widespread attenuation of spinal cord function (or even other brain functions, as does baclofen)) and tunable (e.g., being able to modulate neural activity based on the severity of spasticity symptoms), resulting in long-term antispastic effects.

[0135] Injection of the viral vector described herein into a dermatome results in the vector infecting the afferent nerve fibers connecting to the dermatome, resulting in stable expression of the GAD gene (preferably the GAD67 gene) in sensory neurons, driven by the promoter described herein. GAD (preferably GAD67) functions as an enzyme that induces and promotes the synthesis of the inhibitory neurotransmitter GABA from the excitatory neurotransmitter glutamate. Sensory neurons infected with the vector can produce and release increased levels of GABA. Therefore, muscle excitability is reduced, and spasticity is treated or alleviated.

[0136] The novel combination of HSV-1-mediated gene therapy that upregulates the GAD gene and dermatome-specific administration allows for targeted targeting of specific spinal levels associated with specific dermatomes, thus providing a therapeutic role for specific spastic muscles. Due to the neurotropic nature of HSV-1 vectors and dermatome-specific administration, the therapeutically effective dose required to treat spasticity is significantly reduced, minimizing systemic toxicity and side effects. The present invention provides a non-surgical, minimally invasive, and region-specific therapeutic approach for treating spasticity.

[0137] The following examples are intended to illustrate, but not limit, the present invention. All citations throughout this disclosure are expressly incorporated herein by reference. [Example]

[0138] Experiments can be performed to evaluate the effect of the disclosed methods on the in vivo conversion of glutamatergic sensory neurons into GABAergic neurons to modulate hyperexcitability of spinal circuits and thus treat spasticity.

[0139] The HSV-GAD67 viral vector can be directly injected into a mouse model of chronic sacral SCI to infect sensory afferent neurons and reduce spasticity. The behavioral effects of direct injection in the mouse model can be evaluated and compared with the effects of baclofen. Anatomical evaluation can be performed to further evaluate the effects of direct injection of the HSV vector described herein, an effective and minimally invasive therapeutic approach for treating spasticity.

[0140] Neuromodulatory HSV-mediated gene therapy for treating spasticity after spinal cord injury To determine the effect of in vivo conversion of glutamatergic sensory neurons to GABAergic neurons to modulate hyperexcitability of spinal circuits typical of spasticity, the following experiments were performed.

[0141] In the following experiments, mice (n=20) all underwent complete spinal cord transection at the sacral level between the S1 and S2 levels. Such animals developed a tail spastic phenotype. More specifically, their tails exhibited abnormal postures with increased tonic stretch reflexes and spontaneous or evoked spasms (associated with increased phasic reflexes).

[0142] To develop a spastic phenotype, mice were randomly assigned to either a treatment or control group (n = 10 each) 8 weeks after spinal cord transection. The "treatment" group received subcutaneous injections of 20 microliters of a non-replicating HSV-1 vector expressing GAD67 (described below), the key enzyme responsible for converting glutamate to GABA, at six sites distributed around the base of the tail. The "control" group received similar injections of the UV-inactivated product. The injection sites at the base of the tail were located within a dermatome in the sacral region.

[0143] Vector Overview. The vector (referred to herein as hEF-1α::GAD67) is a non-replicating recombinant herpes simplex virus type 1 (HSV-1)-derived vector expressing human glutamic acid decarboxylase (hGAD67) driven by the human elongation factor 1 alpha (hEF-1α) promoter. A transcription cassette was inserted into the latency-associated transcript locus of the HSV-1 genome. The vector was deleted within the immediate early (IE) viral genes for ICP4 and ICP27 to block its replication ability. In addition, one copy of the ICP0 gene was also deleted to reduce the toxicity of the vector. 1.3 x 10 5 Provide the hEF-1α::GAD67 vector as purified viral particles in phosphate buffered saline (PBS) at a concentration of 100 PFU (plaque forming units) / μl (titration by plaque assay on a monolayer of cells).

[0144] Evaluating treatment for tonic activity by characterizing tail posture As described by Marcantoni et al. (Sci. Transl. Med. 12, eaay0167 (2020)), this model defines a severity index (Figure 1C) that characterizes the increased tonic extensor deterioration resulting in abnormal postural quantification. Figures 1A and 1B show representative images of mouse tails from lesioned control (Figure 1A) and treated (Figure 1B) animals in a chronic state of SCI 3 weeks after vector injection. Figure 1D shows the severity index for each group (N = 10 / group). Each dot represents one animal, and the bar graphs represent the mean ± standard deviation. Statistical analysis by unpaired t-test showed significant differences (p < 0.05) between treated mice compared to lesioned controls, and the severity index is significantly reduced after hEF1α::GAD67 treatment. When the overall index was divided into three contributing factors (Figure 1E), only the first curvature of the tail θ1 showed statistically significant differences between groups. These results demonstrate a localized effect of the hEF-1α::GAD67 vector on the first muscle segment (proximal to the base of the tail), thus demonstrating a statistically significant reduction in tonic muscle hyperactivity when mice were treated with the hEF-1α::GAD67 vector.

[0145] Electromyographic assessment of vector effect on tail spasms In lesioned mice, similar to humans, spasms could occur spontaneously in the muscles beneath the lesion or be induced by sensory stimulation. To assess the effect of the vector on such spasms, muscle activity was measured using electromyography (EMG). Animals were evaluated at least 3 weeks after injection (3–13 weeks after injection) to allow the vector to reach a stable pseudolatent stage within the infected cells. The area under the curve was calculated for each 2-second EMG recording with 10 repetitions for each animal, either without stimulation (no contact with the tail except with the EMG electrodes, and the animal remained motionless) or during tactile stimulation of the skin. Tactile stimulation was performed at two different levels: the base of the tail, where the vector had been previously injected, and the tip of the tail. Figure 2 shows the average EMG change during stimulation (tip or base) compared to a baseline without stimulation for each animal. Figure 2(A) shows EMG recordings from the muscles at the base of the tail, which are associated with the same somitic level as the injected dermatome (S1–S2). The control group shows increased activity upon both tip and base stimulation. The treatment group shows increased activity upon stimulation of the tip of the tail (not statistically significant from the control group), but a statistically significant decrease in activity upon stimulation of the base of the tail (p<0.05 - two-way anova with Tukey's multiple comparisons). Thus, the hEF-1α::GAD67 vector has the effect of limiting tail-induced twitching upon stimulation, and this activity appears to be localized to the infected neurons upon stimulation.

[0146] To further investigate the effects of the hEF-1α::GAD67 vector, we simultaneously evaluated two other tail muscles using similar EMG recordings: one in the middle of the tail and one at the tip of the tail (also closer to the posterior end), which are thought to be primarily driven by somitic levels closer to the posterior end. No statistically significant effect on twitch intensity was achieved, suggesting that the vector's effects may be limited to muscle activity driven by the same somitic level as the injected dermatome (see Figures 2B and 2C).

[0147] EMG evaluation of the combination of vector and tiagabine for tail spasms. Next, we determined whether the effect of GAD67 overexpression, which leads to increased GABA release at the spinal cord level, could be ameliorated by a GABA reuptake inhibitor such as tiagabine. The same set of EMG assessments was performed using control and treatment groups, both of which were injected with 15 mg / kg tiagabine prior to assessment.

[0148] Figure 3A shows the change in EMG activity of the caudal muscles upon tactile stimulation at the base and tip of the tail. As observed above with treatment without tiagabine, the treatment group alone did not show an increase in EMG activity and, therefore, did not show induction of caudal muscles spasms upon tactile stimulation of the base of the tail alone. Notably, the difference between the treatment group and the control group was highly significant (p<0.001 - two-way anova with Tukey's multiple comparisons). As can be seen from the activity induced by distal tail stimulation, tiagabine alone had no effect in preventing spasms. Furthermore, in 5 out of 10 animals, stimulation of the base of the tail induced a decrease in muscle activity. These experiments support the modulatory effect of tiagabine in combination with the vector.

[0149] When muscle activity in the mid-tail and near-posterior muscles was assessed after base stimulation in the presence of tiagabine (Fig. 3B and 3C), no statistically significant differences in evoked muscle activity were observed compared to control animals.

[0150] conclusion Overall, these experiments support the conclusion that local subcutaneous injection of the hEF-1α::GAD67 vector has a local effect on muscle tone and spasms, which can be synergistically increased by tiagabine.

[0151] Without wishing to be bound by any particular theory, it is believed that vector-infected sensory nerves in the injected dermatome release not only glutamate but also GABA from their axon terminals at the spinal cord level, and this release of GABA is thought to affect the overall excitability of motor neurons, which are known to be hyperactive in spasticity.

[0152] Tactile stimulation of the skin at the injection site, which elicits sensory nerve firing, results in a decrease in local muscle activity instead of the well-known induction of seizures. This effect appears to be restricted to muscles whose activity is primarily driven by the same somitic level as the injected dermatome, suggesting that GABA release at the spinal cord level may not have a diffuse effect.

[0153] SEQ ID NO: 1 1785 DNA Human glutamic acid decarboxylase 67

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[0154] SEQ ID NO: 2 594 AA Human glutamic acid decarboxylase 67

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[0155] SEQ ID NO: 3 2400 DNA Human glutamic acid decarboxylase 65

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[0156] SEQ ID NO:5 1179 DNA hEF-1a promoter

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[0157] SEQ ID NO:6 508 DNA Cytomegalovirus promoter

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[0158] SEQ ID NO:7 552 DNA Rous sarcoma virus promoter

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[0159] SEQ ID NO:8 1212 DNA Ubiquitin C promoter

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[0160] SEQ ID NO:9 331 DNA Simian vacuolar virus 40 promoter

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[0161] SEQ ID NO: 10 500 DNA Phosphoglycerate kinase promoter

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Claims

1. 1. A method of treating spasticity in a subject, comprising upregulating the GAD (glutamic acid decarboxylase) gene, thereby treating spasticity in said subject.

2. 2. The method of claim 1, wherein said upregulation of the GAD gene is a region-specific upregulation of the GAD gene.

3. 2. The method of claim 1, wherein said upregulation of the GAD gene comprises administering to said subject a viral vector comprising a polynucleotide encoding GAD, such that GAD is expressed, thereby reducing spasticity.

4. The method of claim 3 , wherein the GAD gene is overexpressed.

5. 2. The method of claim 1, wherein the viral vector is an adeno-associated viral (AAV) vector or a herpes simplex viral (HSV) vector, preferably an HSV-1 vector or an HSV-2 vector, more preferably a defective viral vector derived from HSV, such as a recombinant HSV vector, an amplicon HSV vector, or an HSV-1 vector comprising a pre-HSV-1 vector and a GAD expression cassette.

6. 6. The method of claim 5, wherein the viral vector is a defective viral vector derived from HSV-1, and the polynucleotide encoding GAD is inserted into the LAT (latency-associated transcript) gene locus of the defective viral vector derived from HSV-1.

7. 4. The method of claim 3, wherein the GAD gene is the GAD67 gene (SEQ ID NO: 1) or the GAD65 gene (SEQ ID NO: 3), preferably GAD67.

8. The method of claim 5 , wherein the viral vector comprises a promoter.

9. 9. The method of claim 8, wherein the promoter is an afferent neuron-specific promoter selected from the group consisting of promoters of genes encoding sensory neuroreceptors, preferably promoters of the TRP gene family, more preferably the promoter of TRPV1 or TRPM8; or promoters of genes encoding sensory neuromodulators or sensory neurotransmitters, preferably the promoters of substance P, PACAP, CGRP, ADVL, more preferably the promoter of CGRP or ADVL.

10. 9. The method of claim 8, wherein the promoter is a non-specific promoter selected from the group consisting of hEF-1α promoter (SEQ ID NO: 5), cytomegalovirus (CMV) promoter (SEQ ID NO: 6), Rous sarcoma virus (RSV) promoter (SEQ ID NO: 7), human ubiquitin C (hUBC) promoter (SEQ ID NO: 8), simian vacuolar virus 40 (SV40) promoter (SEQ ID NO: 9), phosphoglycerate kinase (PGK) promoter (SEQ ID NO: 10), β-globin promoter, NF-kB promoter, EGR1 promoter, eIF4A1 promoter, FerL promoter, GAPDH promoter, β-Kin promoter, ROSA26 promoter, and human surfactant protein C (hSP-C) promoter, preferably the promoter is hEF-1α promoter (SEQ ID NO: 5).

11. 4. The method of claim 3, wherein the viral vector is administered directly into the spinal cord parenchyma of the subject, into the spinal cavity of the subject, into the spinal subpial space of the subject, or into a peripheral spastic muscle of the subject, or into one or more dermatomes of the subject.

12. 12. The method of claim 11, wherein the viral vector is administered directly to one or more dermatomes of the subject via one or more injections.

13. A method of treating spasticity in a subject, comprising administering to said subject a therapeutically effective amount of a viral vector comprising a polynucleotide encoding GAD, thereby treating spasticity in said subject.

14. 14. The method of claim 13, wherein the viral vector is an adeno-associated viral (AAV) vector or a herpes simplex viral (HSV) vector, preferably an HSV-1 vector or an HSV-2 vector, more preferably a defective viral vector derived from HSV, such as a recombinant HSV vector, an amplicon HSV vector, or an HSV-1 vector comprising a pre-HSV-1 vector and a GAD expression cassette.

15. 15. The method of claim 14, wherein the viral vector is a defective viral vector derived from HSV-1, and the polynucleotide encoding GAD is inserted into the LAT (latency-associated transcript) gene locus of the defective viral vector derived from HSV-1.

16. 14. The method of claim 13, wherein the GAD is GAD67 or GAD65, preferably GAD67.

17. The method of claim 12 , wherein the viral vector comprises a promoter.

18. 18. The method of claim 17, wherein the promoter is an afferent neuron-specific promoter selected from the group consisting of promoters of genes encoding sensory neuroreceptors, preferably promoters of the TRP gene family, more preferably promoters of TRPV1 or TRPM8; or promoters of genes encoding sensory neuromodulators or sensory neurotransmitters, preferably promoters of substance P, PACAP, CGRP, ADVL, more preferably promoters of CGRP, ADVL.

19. 18. The method of claim 17, wherein the promoter is a non-specific promoter selected from the group consisting of hEF-1 alpha promoter, cytomegalovirus (CMV) promoter, Rous sarcoma virus (RSV) promoter, human ubiquitin C (hUBC) promoter, simian vacuolar virus 40 (SV40) promoter, phosphoglycerate kinase (PGK) promoter, β-globin promoter, NF-kB promoter, EGR1 promoter, eIF4A1 promoter, FerL promoter, GAPDH promoter, β-Kin promoter, ROSA26 promoter, and human surfactant protein C (hSP-C) promoter, preferably the promoter is hEF-1 alpha promoter.

20. 14. The method of claim 13, wherein the viral vector is administered directly into the spinal cord parenchyma of the subject, into the spinal cavity of the subject, into the spinal subpial space of the subject, or into a peripheral spastic muscle of the subject, or into one or more dermatomes of the subject.

21. 21. The method of claim 20, wherein the viral vector is administered directly to one or more dermatomes of the subject via one or more injections.

22. A therapeutic regimen for treating a subject having spasticity or a condition associated with spasticity, comprising administering a viral vector comprising a polynucleotide encoding GAD, wherein GAD is expressed, thereby treating said spasticity or said condition associated with spasticity.

23. 23. The therapeutic regimen of claim 22, wherein the viral vector is an adeno-associated viral (AAV) vector or a herpes simplex viral (HSV) vector, preferably an HSV-1 vector or an HSV-2 vector, more preferably a defective viral vector derived from HSV, such as a recombinant HSV vector, an amplicon HSV vector, or an HSV-1 vector comprising a pre-HSV-1 vector and a GAD expression cassette.

24. 24. The therapeutic regimen of claim 23, wherein the viral vector is a defective viral vector derived from HSV-1, and the polynucleotide encoding GAD is inserted into the LAT (latency-associated transcript) locus of the defective viral vector derived from HSV-1.

25. 23. The therapeutic regimen of claim 22, wherein the GAD is GAD67 or GAD65, preferably GAD67.

26. 24. The therapeutic regimen of claim 23, wherein the viral vector comprises a promoter.

27. 27. The therapeutic regimen of claim 26, wherein the promoter is an afferent neuron-specific promoter selected from the group consisting of promoters of genes encoding sensory neuroreceptors, preferably promoters of the TRP gene family, more preferably promoters of TRPV1 or TRPM8; or promoters of genes encoding sensory neuromodulators or sensory neurotransmitters, preferably promoters of substance P, PACAP, CGRP, ADVL, more preferably promoters of CGRP, ADVL.

28. 27. The therapeutic regimen of claim 26, wherein the promoter is a non-specific promoter selected from the group consisting of hEF-1α promoter, cytomegalovirus (CMV) promoter, Rous sarcoma virus (RSV) promoter, human ubiquitin C (hUBC) promoter, simian vacuolar virus 40 (SV40) promoter, phosphoglycerate kinase (PGK) promoter, β-globin promoter, NF-kB promoter, EGR1 promoter, eIF4A1 promoter, FerL promoter, GAPDH promoter, β-Kin promoter, ROSA26 promoter, and human surfactant protein C (hSP-C) promoter, preferably wherein the promoter is hEF-1α promoter.

29. 23. The therapeutic regimen of claim 22, wherein the viral vector is administered directly into the spinal cord parenchyma of the subject, into the spinal cavity of the subject, into the spinal subpial space of the subject, or into a peripheral spastic muscle of the subject, or into one or more dermatomes of the subject.

30. 30. The treatment regimen of claim 29, wherein the viral vector is administered directly to one or more dermatomes of the subject via one or more injections.

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