Treatment of Neuromuscular Diseases via Gene Therapy Expressing Klotho Protein

Abstract

The present disclosure provides a gene construct containing a nucleic acid sequence encoding mammalian s-KL, an isolated cell containing an expression vector, and a method of treating movement disorders and motor neuron diseases, which are delivered by a pharmaceutical composition containing an expression vector and utilize a viral vector and a non-viral vector operably linked to a muscle cell-specific promoter and having muscle cell and motor neuron tropism, for use in the treatment of movement disorders that may develop in, for example, neuromuscular disorders or diseases.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 330,684, filed on April 13, 2022, which is hereby incorporated by reference in its entirety.

[0002] This disclosure relates to the medical field, and more specifically, to a medical approach for treating neuromuscular disorders or neuromuscular diseases characterized by movement disorders and motor neuron diseases such as amyotrophic lateral sclerosis.

[0003] Sequence Listing This application includes a sequence listing that was electronically submitted in ASCII format and is hereby incorporated by reference in its entirety. The XML copy created on April 11, 2023, is named 58578 - 001PCT.xml and is 162 KB in size.

Background Art

[0004] According to the International Neuromodulation Society, movement disorders are associated with partial or complete loss of function of body parts, usually limbs. Diseases that cause movement disorders can result in muscle weakness, insufficient stamina (i.e., fatigue), lack of muscle control, or complete paralysis. Movement disorders are a major cause of physical disability. This is widely caused by peripheral problems affecting the muscles, central nervous system problems affecting the output to the muscles, and sensory problems affecting muscles, movement, and balance. Movement disorders are often apparent in neuromuscular diseases, neurological conditions, and motor neuron diseases, but can also result from cancers of the central and peripheral nervous systems or traumatic injuries.

[0005] Neuromuscular diseases are any diseases that affect the spinal cord or central nervous system (CNS), peripheral nervous system (PNS), neuromuscular junction, or motor neurons of skeletal muscle, all of which are components of the motor unit and thus ultimately affect the subject's motor ability. Damage to the spinal cord, CNS, PNS, neuromuscular junction, or motor neurons of skeletal muscle can cause muscle atrophy and weakness. Sensory problems can also occur. Neuromuscular diseases can be acquired or genetic. Mutations in over 500 genes have been shown to cause neuromuscular diseases. Other causes include nerve or muscle degeneration, autoimmunity, toxins, drugs, nutritional deficiencies, metabolic disorders, hormonal imbalances, infections, nerve compression / striction, the blood supply involved, and trauma.

[0006] Examples of neuromuscular diseases and disorders include amyotrophic lateral sclerosis (ALS), Charcot-Marie-Tooth disease, multiple sclerosis, muscular dystrophy, myasthenia gravis, myopathies, myositis including polymyositis and dermatomyositis, peripheral neuropathy, neuromyotonia, Lambert-Eaton myasthenic syndrome, Friedreich's ataxia, spinal muscular atrophy (SMA), spinal cord injury, peripheral nerve injury, traumatic nerve injury, and muscle metabolic diseases. Some of these neuromuscular diseases are further classified as motor neuron diseases or motor neuron diseases (MND), which are a group of rare neurodegenerative disorders that selectively affect motor neurons, the cells that control the voluntary muscles of the body. Examples of MND include amyotrophic lateral sclerosis (ALS), progressive bulbar palsy (PBP), pseudobulbar palsy, progressive muscular atrophy (PMA), primary lateral sclerosis (PLS), spinal muscular atrophy (SMA), and monomelic amyotrophy (MMA), as well as some rarer variants similar to ALS.

[0007] Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder of the motor system and the most frequent form of motor neuron disease (MND), with an incidence of 1 - 5 per 100,000 person - years. ALS patients suffer from progressive muscle weakness and paralysis and usually die within 2 - 5 years from diagnosis, mainly due to respiratory failure. Any skeletal muscle can be affected, which causes multiple different clinical symptoms among patients. These vary from twitching to fasciculation, marked weakness and muscle atrophy, hyperreflexia and spasticity, and in some cases, mood and memory functions also change. Sporadic ALS (sALS) accounts for most cases, and the peak age of onset is about 60 years. Approximately 5 - 10% of cases are familial (fALS) caused by genetic mutations, most of which are autosomal dominant and onset about 10 years earlier than sporadic cases. Cases in patients under 25 years old are designated as juvenile - onset ALS (jALS) and usually have autosomal recessive inheritance. Some of the most common genetic causes of fALS are mutations in Cu / Zn superoxide dismutase 1 (SOD1), TAR - DNA - binding protein (TDP - 43), fused in sarcoma (FUS), and hexanucleotide repeat expansions in chromosome 9 open reading frame 72 (C9orf72). The neuropathological feature of ALS is that it affects both upper and lower motor neurons (MNs), which distinguishes it from other MNDs. Neurodegeneration occurs in the corticospinal and corticobulbar tracts, with loss of large pyramidal neurons in the primary motor cortex and in the anterior horns of the brainstem and spinal cord.

[0008] Denervation of the end - plate and axonal retraction occur in a "dying - back" pattern and are thought to lead to MN death and subsequent muscle atrophy. A "dying - forward" process has also been discussed, where the primary injury occurs in upper or lower MNs and the degeneration extends forward to the descending axonal processes.

[0009] Unfortunately, as is the case with other neuromuscular diseases, particularly motor neuron diseases, to date, there is no effective treatment for ALS. Despite decades of intensive research and clinical trials, most promising preclinical therapies have failed to translate into success in human trials. Riluzole (Rilutek®) and edaravone (Radicava®) are the only drugs approved for the treatment of ALS. Since both only extend survival by a few months and mildly improve motor function, symptomatic and palliative measures (including feeding and respiratory assistance) are the mainstays of patient management. Interestingly, most of the compounds studied interfere with a single mechanism involved in MN death. Thus, one of the major difficulties in developing effective therapies for ALS is the multiplicity of events contributing to MN death. Considering the multifactorial mechanism of ALS pathogenesis, the use of combination therapies or multiple-target therapies that act simultaneously on several mechanisms and target different cell types may lead to enhanced treatment outcomes and maximize translational efficacy.

[0010] Thus, despite previous efforts, there remains a need for efficient and safe treatments for movement disorders of different etiologies. SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEM

[0011] The present disclosure relates to novel therapies for the prevention and / or treatment of motor and other movement disorders in neuromuscular diseases, based on the administration of specific variants of mammalian Klotho proteins, particularly alternative RNA splicing variants of Klotho (s-KL). To date, there is no evidence that a particular Klotho variant can be safely administered to muscle cells (either in protein form or as gene therapy) to treat movement disorders.

[0012] In one aspect, the present disclosure provides a gene construct comprising a first promoter operably linked to a nucleic acid sequence encoding mammalian s-KL or a functional variant thereof, wherein the first promoter is a muscle cell-specific promoter. In some embodiments, the s-KL amino acid sequence is human s-KL, for example, SEQ ID NO: 1. In some embodiments, the s-KL amino acid sequence is mouse s-KL, for example, SEQ ID NO: 3. Representative functional variants may have at least 85% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 3. In some embodiments, the promoter is a constitutive muscle cell-specific promoter. In some embodiments, the promoter is an inducible muscle cell-specific promoter. In some embodiments, the muscle cell-specific promoter is the human desmin promoter. In some embodiments, the nucleic acid sequence encoding mammalian s-KL or a functional variant thereof is operably linked to at least one additional promoter different from the first promoter, for example, a second promoter. In some embodiments, the second promoter is a muscle cell-specific promoter. In some embodiments, the second promoter is a neuron cell-specific promoter. In some embodiments, the second promoter is inducible. In some embodiments, the second promoter is constitutive. In some embodiments, the second promoter is ubiquitous. In some embodiments, the second promoter is the zinc-driven metallothionein promoter.

[0013] In another aspect, the present disclosure provides a plasmid comprising a gene construct (e.g., integrated or cloned therein) comprising a first promoter operably linked to a nucleic acid sequence encoding mammalian s-KL or a functional variant thereof, wherein the first promoter is a muscle cell-specific promoter and the initiation sequence is operably linked to the first promoter. The plasmid can function as a vector by itself, but may be useful in the preparation of more complex expression vectors, such as AAV vectors and lentiviral vectors. The plasmid may also be useful for obtaining mRNA after linearization of the plasmid and appropriate in vitro transcription, which can be further encapsulated or complexed with cationic lipids if desired.

[0014] In another aspect, the present disclosure provides an expression vector (e.g., integrated or cloned therein) comprising a gene construct or plasmid containing (a) a muscle cell-specific first promoter operably linked to a nucleic acid encoding mammalian s-KL or a functional variant thereof, wherein the expression vector may or may not have muscle cell tropism, or (b) a first promoter that functions in muscle cells, nerve cells, or induced pluripotent stem cells (iPSCs) operably linked to a nucleic acid sequence encoding mammalian s-KL or a functional variant thereof, wherein the expression vector has muscle cell tropism. In some embodiments, the promoter is a muscle cell-specific promoter and the expression vector has nerve cell tropism, which serves to preferentially target the vector to nerve cells. In some embodiments, the vector is a viral expression vector such as a DNA viral expression vector or an RNA viral expression vector. In other embodiments, the expression vector is a non-viral expression vector. In some embodiments, the expression vector is a serotype of adeno-associated virus (AAV) vector having muscle cell tropism, or the AAV vector is of a serotype having nerve cell tropism.

[0015] In some embodiments, the s-KL polypeptide comprises, or consists of, SEQ ID NO: 1, which is human s-KL, or a functional variant thereof. In some embodiments, the variant comprises, or consists of, a sequence having at least 85% sequence identity with SEQ ID NO: 1. In some embodiments, the polypeptide has a sequence that is at least 90% identical to SEQ ID NO: 1. In some embodiments, the polypeptide has a sequence that is at least 95% identical to SEQ ID NO: 1. In some embodiments, the polypeptide has a sequence that is at least 98% identical to SEQ ID NO: 1. In some embodiments, the polypeptide has a sequence that is at least 99% identical to SEQ ID NO: 1. In other embodiments, the polypeptide has the sequence of SEQ ID NO: 2, which is mouse s-KL.

[0016] In some embodiments, the expression vector is a viral vector. In some embodiments, the viral vector is a serotype of adeno-associated virus, and in some embodiments, it is any one of AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9p31, AAVrh10, PHPeB, 9P31, AAVrh74, and AAVMyo.

[0017] In some embodiments, the AAV vector contains a capsid polypeptide of adeno-associated virus having any one of the amino acid sequences of SEQ ID NOs: 17-29. In some embodiments, the AAV vector contains a capsid polypeptide of adeno-associated virus having the amino acid sequence of SEQ ID NO: 17 or 26.

[0018] In some embodiments, the viral vector is a serotype of AAV vector having neurotropism, and in some embodiments, it is any one of AAV1, AAV8, or AAV9.

[0019] In some embodiments, the vector is a lipid-based vector. In some embodiments, the lipid-based vector is a lipid nanoparticle (LNP) or a liposome.

[0020] In another aspect, the present disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of a plasmid or expression vector as defined herein and a pharmaceutically acceptable carrier.

[0021] In another aspect, the present disclosure provides an isolated cell containing a gene construct or plasmid disclosed herein, and the isolated cell is a human muscle cell, a human nerve cell, or a human induced pluripotent stem cell (iPSC) (which can be induced ex vivo to differentiate into muscle cells or nerve cells or can differentiate in vivo). In some embodiments, the isolated cell is a nerve cell, such as a motor neuron. In some embodiments, the isolated cell is a striated muscle cell or a skeletal muscle cell.

[0022] Another aspect of the present disclosure is a method of treating a disease or disorder characterized by a movement disorder in a subject. In some embodiments, the neuromuscular disease is amyotrophic lateral sclerosis (ALS). In some embodiments, the ALS is sporadic ALS (sALS) or familial ALS (fALS). In some embodiments, the neuromuscular disease is multiple sclerosis. In some embodiments, the neuromuscular disease is muscular dystrophy. In some embodiments, the neuromuscular disease is spinal muscular atrophy (SMA). In some embodiments, the neuromuscular disease is a spinal cord injury, a peripheral nerve injury, or a traumatic nerve injury. In some embodiments, the method may involve administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of a plasmid or an expression vector containing a gene construct. In some embodiments, the method may involve continuous administration (e.g., infusion) of the composition to the subject. In some embodiments, the continuous infusion may be performed for one or more days over a period of about 1 hour to about 24 hours per day. In some embodiments, the subject does not exhibit movement disorder symptoms.

[0023] Another aspect of the disclosure is a cell therapy that comprises administering to a subject in need thereof the isolated cells disclosed herein. This method can involve an ex vivo approach in which muscle cells or nerve cells are isolated from the subject, transformed or transfected with a gene construct, plasmid, or expression vector, and then administered to the subject. In some embodiments, the muscle cells or nerve cells are enriched before or after transformation. In other embodiments, partially pluripotent cells or stem cell-like cells are transformed or transfected with an expression vector and induced to differentiate in vitro into a specific cell type (e.g., muscle cells or neurons). When integrated with the expression vector (which can occur before or after differentiation), the cells are administered to the subject. In some embodiments, the method is autologous in the sense that it can involve isolating terminally differentiated cells from the subject, reprogramming (i.e., inducing) pluripotency in those cells to generate iPSCs, integrating an expression vector into the iPSCs, differentiating the iPSCs into nerve cells or muscle cells, and administering the differentiated cells to the subject. In some embodiments, the cells differentiate into muscle cells. In some embodiments, the cells differentiate into nerve cells. In some embodiments, the iPSC cells can differentiate after administration to the subject.

[0024] Without wishing to be bound by theory, the constructs, methods, etc. disclosed herein may increase mammalian s-KL protein levels in skeletal muscle cells, which may protect cells from toxic damage and preserve muscle cell function and / or motor neuron function or improve motor deficits associated with diseases and disorders such as ALS characterized by motor deficits.

[0025] As shown in the examples of this specification, the inventors have found that administration of a mammalian secreted RNA splicing variant of the Klotho (s-KL) protein significantly delays disease progression in a mouse model of ALS disease. Further, administration of the mammalian s-KL protein in gene therapy using an AAVmyo muscle cell-directed expression vector in a mouse model of ALS disease delayed disease progression while reducing the amount of expression vector required compared to other AAV vectors.

[0026] The present disclosure may overcome one or more drawbacks associated with known treatments such as treatment with the Klotho protein. The relatively short half-life of the Klotho protein in vivo, about 7.5 hours, results in an approximate logarithm of decrease every 24 hours. In contrast, the treatment according to the present disclosure may have an effect similar to the administration of mammalian s-Klotho protein via continuous infusion.

Brief Description of the Drawings

[0027]

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Mode for Carrying Out the Invention

[0028] Definitions All terms used in this application and in this specification shall be understood in their ordinary meaning as known in the art, unless otherwise specified. Other more specific definitions for particular terms used in this application are as described below and are intended to be applied uniformly throughout this specification and the claims, unless an explicit definition provides a broader definition.

[0029] As used in this specification, the indefinite articles "a" and "an" are synonymous with "at least one" or "one or more". Unless otherwise indicated, definite articles used in this specification, such as "the", also include plural nouns.

[0030] Throughout the description and the claims, the term "comprising" and its variations are not intended to exclude other technical features, additives, components, elements, or steps. Further, the term "comprise" includes the case of "consisting of".

[0031] Gene construct In one aspect, the present disclosure provides a gene construct (also referred to herein as an expression cassette) comprising a nucleic acid sequence operably linked to a muscle cell-specific promoter. The terms "gene construct" and "expression cassette" are used interchangeably herein and refer to a nucleic acid sequence encoding a mammalian secreted RNA splicing variant of mammalian Klotho (s-KL) or a functional variant thereof operably linked to a muscle cell-specific promoter.

[0032] As used herein, the term "nucleic acid", also referred to herein as "nucleic acid sequence", refers to a polymer of nucleotides, each of which is an organic molecule consisting of a nucleoside (nucleic acid base and 5-carbon sugar) and a phosphate. The term nucleotide includes nucleosides having ribose sugar (i.e., ribonucleotides that form ribonucleic acid, RNA) or 2'-deoxyribose sugar (i.e., deoxyribonucleotides that form deoxyribonucleic acid, DNA), unless specifically recited or apparent from the context. Nucleotides function as monomeric units of nucleic acid polymers or polynucleotides. The four nucleic acid bases in DNA are guanine (G), adenine (A), cytosine (C), and thymine (T). The four nucleic acid bases in RNA are guanine (G), adenine (A), cytosine (C), and uracil (U). A nucleic acid is a linear chain (e.g., at least three nucleotides) of nucleotides chemically linked by a series of ester bonds between the phosphoryl group of one nucleotide and the hydroxyl group of the sugar (i.e., ribose or 2'-deoxyribose) in adjacent nucleotides.

[0033] As used herein, the term "promoter" refers to a nucleic acid sequence that directly or indirectly regulates the transcript of a corresponding nucleic acid sequence to which it is operably linked, and in the context of the present disclosure, is mammalian s-KL or a functional variant thereof. A promoter may function alone to regulate a transcript, or may act in concert with one or more other regulatory sequences (e.g., enhancers or silencers, or regulatory elements that may be present in a gene construct or expression vector). A promoter is located near the transcription start site of a gene, on the same strand, and upstream (towards the 5' region of the sense strand) on the DNA. A promoter is typically in the range of about 100 - 1000 base pairs in length. A "constitutive promoter" is a promoter that is active in all circumstances within a cell, as opposed to other regulated promoters that become active within a cell only in response to a specific stimulus, such as an "inducible promoter".

[0034] As used herein, the term "muscle" encompasses skeletal muscle, which refers to the voluntarily controlled striated muscle type that attaches to the skeleton, and representative examples thereof include the diaphragm, biceps, triceps, quadriceps, tibialis interior, and gastrocnemius muscle.

[0035] The terms "muscle cell-specific" and "muscle-specific" as used herein in connection with a promoter refer to the preferential, selective, or dominant expression of mammalian s-KL (or a functional variant thereof) in muscle cells or muscle tissue as compared to other (i.e., non-muscle) cells and tissues. In some embodiments, at least 50% of the expression, more specifically, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% occurs within muscle cells or muscle tissue. In some embodiments, "muscle cell-specific" refers to substantially no leakage of expression of mammalian s-KL (or a functional variant thereof) to other organs or tissues other than muscle, such as the lung, liver, brain, kidney, and / or spleen.

[0036] As used herein in the context of a promoter, the term "functions in myocytes" refers to a promoter that directly or indirectly regulates the transcript of a mammalian s-KL nucleic acid to which it is operably linked, but is not necessarily muscle cell-specific.

[0037] As used herein, the term "operably linked" should be understood to mean that a nucleic acid sequence encoding s-KL or a functional variant thereof is spatially positioned or arranged within a gene construct relative to a promoter to drive expression of the nucleic acid coding sequence.

[0038] Klotho (KL) is a protein that is expressed mainly in the kidney and brain and to a lesser extent in skeletal muscle, lung, bladder, testis, and ovary. Klotho has two different isoforms, one of which encodes a full-length transmembrane protein (mKL) of approximately 140 kDa, which can be processed into a soluble form (p-KL) of approximately 130 kDa. The other isoform encodes a secreted but shorter isoform (s-KL) of approximately 63 kDa. mKL contains KL1 and KL2 domains and can be released by α / β / γ-secretase to generate p-KL or further cleaved to release p-KL1 and p-KL2 domains (each approximately 70 kDa). s-KL contains a KL1-like domain and an extra tail (15 amino acids at its C-terminus in mouse s-KL and 16 amino acids at its C-terminus in human s-KL). Both isoforms, m-KL and s-KL, have different expression levels as well as spatio-temporal expression profiles, suggesting different roles.

[0039] Soluble and secreted Klotho exists in a form present in the circulation (e.g., blood) and cerebrospinal fluid (CSF), functions as a circulating hormone, binds to cell surface receptors, and exerts biological effects on distant organs and multiple systems by suppressing signals such as insulin and insulin-like growth factor 1 (IGF1) signaling. Similar abbreviations (s-KL) may be used in the prior art to refer to soluble Klotho (a processed version of transmembrane Klotho) and secreted Klotho (an RNA splicing variant), but it should be noted that these two variants exhibit different structures.

[0040] In this specification, the term "secreted RNA splicing variant of mammalian Klotho" abbreviated as "s-KL" refers to a protein resulting from alternative RNA splicing of a transcript encoding the full-length transmembrane form of Klotho protein (m-KL), which generates a cleaved form of the protein formed from the KL1-like domain (i.e., s-KL), has an approximate weight of about 63 kDa, has a specific secretion signal consisting of 15 amino acid tails in mice and 16 amino acid tails in humans not found in the m-KL transcript, and for this reason is also called the secreted isoform of Klotho, s-KL, or the secreted RNA splicing variant of Klotho protein. S-KL is different from other forms of soluble Klotho, namely p-KL, p-KL1, and p-KL2. In the present disclosure, m-KL refers to the full-length transmembrane form, p-KL represents a soluble proteolytic form of Klotho (i.e., KL1-KL2) generated by cleavage of m-KL and having a molecular weight of about 130 kDa, and p-KL1 and p-KL2 refer to soluble Klotho forms consisting of the KL1 domain and the KL2 domain of p-KL, respectively. M-KL refers to a full-length transcript encoding a single-pass transmembrane protein having a molecular weight of about 140 kDa (m-KL). The protein contains three domains: a short transmembrane domain at the C-terminus, an extracellular domain composed of two internal repeat sequences of about 550 amino acids called KL1 and KL2, respectively, and a very short intracellular domain of 10 amino acids. The extracellular domain of the transmembrane form can be cleaved by the metalloproteases ADAM10 and ADAM17, resulting in another form of soluble Klotho of about 130 kDa (abbreviated as p-KL for the proteolytic membrane isoform). Furthermore, the proteases ADAM10 and 17 have a second recognition site located between the KL1 domain and the KL2 domain, which generates two new 70 kDa proteins, one containing only the KL1 domain and the other containing the KL2 domain. s-KL and its functional variants do not include full-length m-KL or p-KL (containing both the KL1 domain and the KL2 domain).The mammalian s-KL polypeptide encoded by the nucleic acids described herein and functional variants thereof exclude the full-length m-KL and p-KL forms of Klotho (containing both the KL1-KL2 domains) and each have a molecular weight of approximately 130 kDa.

[0041] SEQ ID NO: 1 is the amino acid sequence of human s-KL, which, as described above, is a transcript from the alternative splicing of the α-Klotho human gene and contains a KL1 domain of approximately 63 kDa in weight and a specific secretion signal consisting of a 16-amino acid tail not found in m-KL or the KL1 transcript. The α-Klotho human gene is located on chromosome 13 NC_000013.11 (33016063..33066145) of the human genome assembly GRCh38 (24.12.2013) maintained by the Genome Reference Consortium. SEQ ID NO: 1 is derived from the corresponding cDNA of SEQ ID NO: 3, which in turn is derived from an alternatively spliced transcript of an mRNA having 5012 base pairs and version 3 dated May 3, 2014 of GenBank database accession number NM_004795 (the sequence of which is incorporated herein by reference).

[0042] The amino acid sequence of human s-KL is as follows (SEQ ID NO: 1): JPEG2025518626000001.jpg92142

[0043] The nucleic acid sequence of a representative complementary DNA (cDNA) encoding human s-KL (SEQ ID NO: 1) is described below and is designated as SEQ ID NO: 3 (the s-KL specific secretion sequence is shown within the box below): JPEG2025518626000002.jpg45142 JPEG2025518626000003.jpg212142

[0044] Rodent s-KL may also be useful in the present disclosure. SEQ ID NO: 2 is the amino acid sequence of a transcript from alternative splicing of the α-clotho mouse gene, which contains a KL1 domain sequence having an approximate weight of 70 kDa and has a specific secretion signal consisting of a 15-amino acid tail not found in the m-KL transcript. The α-clotho mouse gene is located on chromosome 5 (150,952,607-150,993,809) of the July 2007 mouse UCSC Genome Browser (NCBI37 / mm9) assembly relative to the mouse genome. SEQ ID NO: 2, derived from the corresponding cDNA of SEQ ID NO: 4, is from an alternatively spliced transcript of the mRNA sequence having 5124 base pairs of GenBank database accession number NM_013823, version 2 of February 15, 2015 (the sequence of which is incorporated herein by reference).

[0045] The amino acid sequence of mouse s-KL (SEQ ID NO: 2) is set forth below: JPEG2025518626000004.jpg91142

[0046] The nucleic acid sequence of a representative complementary DNA (cDNA) nucleic acid sequence of mouse s-KL (SEQ ID NO: 4) is set forth below: JPEG2025518626000005.jpg54142 JPEG2025518626000006.jpg202143

[0047] Additional mammalian s-KL may be suitable for use in the present disclosure. Mammalian klotho genes containing KL1 and KL2 domains are known in the art and / or can be readily identified according to standard techniques. If the s-KL isoform from a given mammalian species has not been identified, the s-KL isoform can be readily derived from the identified mammalian klotho gene, particularly the KL1 and / or KL2 domains having an appropriate C-terminal tail, according to standard techniques.

[0048] The amino acid sequences of the natural additional mammalian Klotho genes in mammalian species, bonobo (pygmy chimpanzee, accession number: XP_034792458.1), chimpanzee (chimpanzee, accession number: XP_522655.2), gorilla (western lowland gorilla, accession number: XP_030857339.1), cynomolgus monkey (cynomolgus monkey, accession numbers: AAC77917.1, Q8WP17.1, XP_005586019.2, XP_005586019.3), Sumatran orangutan (Sumatran orangutan, accession numbers: XP_024086695.2, PNJ48590.1), Bornean orangutan (Bornean orangutan, accession number: XP_054302967.1), Ugandan red colobus monkey (Ugandan red colobus monkey, accession number: XP_023064573.2), northern pig-tailed macaque (northern pig-tailed macaque, accession number: XP_030674267.1), Tibetan macaque (Tibetan macaque, accession number: XP_050621876.1), pig-tailed macaque (pig-tailed macaque, accession number: XP_011746941.1), rhesus macaque (rhesus macaque, accession numbers: EHH28941.1, XP_001101127.2), long-tailed macaque (long-tailed macaque, accession number: XP_032005041.1), gelada baboon (gelada baboon, accession number: XP_025220255.1), golden snub-nosed monkey (golden snub-nosed monkey, accession number: XP_030778249.1), François' langur (François' langur, accession number: XP_033091442.1), African green monkey (African green monkey, accession number: XP_007958284.1), and Anubis baboon (Anubis baboon, accession number: XP_031511616.1) are disclosed in the sequence listing attached as SEQ ID NOs: 42-63 (the sequences are incorporated herein by reference). Each mammalian Klotho has at least 85% amino acid sequence identity with SEQ ID NO: 1 and SEQ ID NO: 2.

[0049] This disclosure encompasses functional variants of mammalian s-KL and their use in the disclosed compositions and methods. The functional variants may be non-natural.

[0050] Protein variants are well understood by those skilled in the art and can typically include amino acid modifications that belong to one or more of three classes of substitution, insertion, or deletion variants. In the present disclosure, a variant is "functional" in the sense that it is therapeutically effective in the treatment of movement disorders that can occur in various diseases or disorders such as neuromuscular diseases and disorders.

[0051] As used herein, the term "substitution variant," when referring to a polypeptide, means that at least one amino acid in the native or starting sequence of the polypeptide is removed and a different amino acid is inserted in its place. The substitution may be a single one in which only one amino acid in the polypeptide molecule is substituted, or it may be a plurality in which two or more amino acids are substituted in the same polypeptide molecule.

[0052] As used herein when referring to a polypeptide, the term "conservative substitution" means that at least one amino acid in the native or starting sequence of the polypeptide is replaced with a different amino acid of similar size, charge, or polarity. Examples of conservative substitutions include substituting a nonpolar (hydrophobic) residue with another nonpolar residue, such as exchanging isoleucine, valine, or leucine. Similarly, examples of conservative substitutions include substituting one polar (hydrophilic) residue with another polar (hydrophilic) residue, such as exchanging between arginine and lysine, between glutamine and asparagine, and between glycine and serine. Further examples of conservative substitutions include substitution of a basic amino acid such as lysine, arginine, or histidine with another amino acid, or substitution of one acidic residue such as aspartic acid or glutamic acid with another acidic residue.

[0053] When referring to a polypeptide, as used herein, the term "insertion variant" refers to a variant having one or more amino acids inserted immediately adjacent to an amino acid at a specific position in the native or starting sequence. As used herein, the term "immediately adjacent" to an amino acid refers to an amino acid bond through either the alpha-carboxy functional group or the alpha-amino functional group of the amino acid.

[0054] When referring to a polypeptide as used herein, the term "deletion variant" refers to a variant in which one or more amino acids within the native or starting amino acids have been removed. Typically, deletion variants delete one or more amino acids in a specific region of the molecule.

[0055] In the present disclosure, the term "identity" refers to the percentage of residues that are identical in two sequences when the sequences are optimally aligned. In an optimal alignment, a sequence exhibits identity at a position if that position in the first sequence is occupied by the same amino acid as the corresponding position in the second sequence. The percentage of identity is determined by the number of identical residues over the defined length of a given alignment. Thus, the level of identity between two sequences, or ("percent sequence identity") is measured as the ratio of the number of identical positions shared by the sequences to the number of positions being compared (i.e., percent sequence identity = (number of identical positions / total number of positions being compared) × 100). Gaps, i.e., positions in the alignment where a residue is present in one sequence but not the other, are considered positions with non-identical residues and are counted as positions being compared.

[0056] By way of example, for instance, a polypeptide having an amino acid sequence having at least 95% identity with the reference amino acid sequence of SEQ ID NO: 1, except that the polypeptide sequence may contain up to 5 amino acid changes per 100 amino acids of the reference amino acids of SEQ ID NO: 1, the amino acid sequence of the polypeptide is intended to be identical to the reference sequence. In other words, to obtain a polypeptide having an amino acid sequence that is at least 95% identical to the reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or up to 5% of the number of amino acids of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These changes in the reference sequence can occur anywhere between the amino-terminal position or carboxy-terminal position of the reference amino acid sequence, or between residues in the reference sequence, or between those terminal positions that are individually scattered among any of the residues within the reference sequence or among one or more contiguous groups within the reference sequence.

[0057] Numerous mathematical algorithms are known for rapidly obtaining an optimal alignment between two or more sequences and calculating the identity, and are incorporated into many available software programs. For the purposes of the present disclosure, the sequence identity between two amino acid sequences is preferably determined using a global alignment-based algorithm such as the Needleman-Wunsch algorithm (Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970)) implemented in the Needle program of the EMBOSS package (Rice et al., Trends Genet. 16(6):276-277 (2000)), or the BLAST Global Alignment tool (Altschul et al., J. Mol. Biol. 215(3):403-410 (1990)) using default settings. When the sequences being compared are of substantially the same length, local alignment may also be used.

[0058] Representative functional variant mammalian s-KL, which can be natural or non-natural, has at least 85% amino acid sequence identity to SEQ ID NO: 1 and / or SEQ ID NO: 2. Thus, representative functional s-KL variants can have 85%, 86%, 87%, 88%, 88.5%, 89%, 89.5%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% or more sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2. One representative functional variant of mammalian s-KL having at least 85% amino acid sequence identity to SEQ ID NO: 1 is the KL1 domain (e.g., the cleavage product p-KL1 of p-KL).

[0059] In some embodiments, the polypeptide has an amino acid sequence having at least 88% or 98% identity to SEQ ID NO: 1. In some embodiments, the polypeptide has an amino acid sequence having at least 88% or 98% identity to SEQ ID NO: 2. In some embodiments, the polypeptide is a variant having an amino acid insertion; for example, in one embodiment, the last 15 amino acids of SEQ ID NO: 1 are removed and replaced with SQLTASVSSPPTRALSLASSAFLLGWRSWRILCPEPTR (SEQ ID NO: 17).

[0060] Polypeptides having at least 88% identity to either SEQ ID NO: 1 or SEQ ID NO: 2 include mammalian s-KL other than human and mouse s-KL.

[0061] In another embodiment, optionally in combination with any one of the embodiments provided below, the mammalian s-KL polypeptide or a functional variant thereof has a length of 645 amino acids, 600 amino acids, or 550 amino acids or less. In some embodiments, the polypeptide has the amino acid sequence of SEQ ID NO: 1, or a functional variant thereof having at least 85% sequence identity with SEQ ID NO: 1, and has a length of 645 amino acids, 600 amino acids, or 550 amino acids or less. In some embodiments, the polypeptide has the amino acid sequence of SEQ ID NO: 1, or a functional variant thereof having at least 85% identity with SEQ ID NO: 1, and the variant has a length of 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 584, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, or 600 amino acids, or a length of 545-600 amino acids.

[0062] In some embodiments, optionally in combination with any of the embodiments provided above and below, the s-KL polypeptide encoded by the nucleic acid sequence is linked to a heterologous moiety by a peptide bond.

[0063] As used herein, "heterologous moiety" refers to any molecule that is attached to a polypeptide via a covalent peptide bond. In certain embodiments, the heterologous moiety is located at either the N-terminus or the C-terminus of the polypeptide. In certain embodiments, the heterologous moiety is located at both the N-terminus and the C-terminus of the polypeptide.

[0064] The heterologous moiety may be, for example, a molecule that facilitates the purification of the polypeptide. In certain embodiments, the heterologous moiety is a peptide. In even more specific embodiments, the heterologous moiety is a polyhistidine tag. As will be appreciated by those skilled in the art, small peptides that assist in the purification of a protein can be maintained in the final compound without affecting its functionality.

[0065] In some embodiments, the heterologous moiety is a vehicle agent. As is known in the art, these agents facilitate the absorption, transport, and delivery of polypeptides. Representative examples of vehicle agents include dipalmitoyl-phosphatidyl-choline (DPCC) liposomes, micellar emulsions, dimethylformamide (DMF), and halogenated phenothiazines.

[0066] A promoter operably linked to a nucleic acid encoding mammalian s-KL or a functional variant thereof is a muscle cell-specific promoter. Representative examples of muscle cell-specific promoters that may be suitable for use in the present disclosure include the mammalian desmin (also known as DES, CSM1 or CSM2) promoter, the alpha-2 actinin (also known as ACTN2, CMD1AA) promoter, the filamin-C (also known as FLNC, actin-binding-like protein (ABLP), filamin-2 (FLN2), ABP-280, ABP280A, ABPA, ABPL, MFM5 or MPD4) promoter, the sarcoplasmic / endoplasmic reticulum calcium ATPase 1 (also known as ATP2A1, ATP2A or SERCA1) promoter, the troponin I type 1 (also known as TNNI1, SSTNI or 25TTNI) promoter, the myosin-1 (MYH1) promoter, the phosphorylatable skeletal muscle fast myosin light chain (MYLPF) promoter, the myosin 1 (also known as MYH1, MYHSA1, MYHa, MyC-2X / D or MyHC-2x) promoter, the alpha-3 chain tropomyosin (also known as TPM3, CFTD, NEM1, OK / Scl.5, TM-5, TM3, TM30, TM30nm, TM5, TPMsk3, TRK, h TM5 or hscp30) promoter, the ankyrin repeat domain-containing protein 2 (also known as ANKRD2, ARPP) promoter, the myosin heavy chain (MHC) promoter, the myosin light chain (MLC) promoter, the muscle creatine kinase (MCK) promoter, a synthetic muscle promoter such as, for example, the SPc5-12 promoter as described in Li et al., Nat. Biotechnol. 17(3):241-245 (1999), the muscle creatine kinase (MCK) promoter, the dMCK promoter, and the tMCK promoter consisting of a double tandem or triple tandem of the MCK enhancer to the MCK basal promoter as described in Wang et al., Gene Ther. 15(22):1489-1499(2008), respectively.

[0067] Hybrid (synthetic) muscle cell-specific promoters may also be useful. Such promoter types may include muscle-specific enhancers and / or transcription factor binding sites, including combinations with other promoter elements derived from viral or human sequences (e.g., CMV, CAG, or PGK). Representative examples of hybrid promoters include the hybrid alpha-myosin heavy chain enhancer / MCK enhancer (MHCK7; 770 bp), the MCK-C5-12 promoter described in Wang et al., Gene Ther. 15(22):1489-1499 (2008), and the cardiac and skeletal muscle-specific myosin chaperone Unc45b (195 bp) promoter described in Rudeck et al., Genesis 54(8):431-438 (2016). In some embodiments, the promoter is a mammalian muscle cell promoter such as the human or mouse desmin promoter, examples of which are described, for example, in U.S. Patent Application Publication No. 2020 / 00407746.

[0068] In some embodiments, the promoter is a promoter that is naturally associated with the gene encoding the human desmin protein, and the promoter sequence therefor is set forth below as SEQ ID NO: 5: JPEG2025518626000007.jpg91142 JPEG2025518626000008.jpg63139

[0069] Thus, in some embodiments, the gene construct of the present invention may include the human desmin promoter of SEQ ID NO: 5 operably linked to mouse s-KL of SEQ ID NO: 2 and have the following nucleic acid sequence (SEQ ID NO: 6): JPEG2025518626000009.jpg145141 JPEG2025518626000010.jpg240143 JPEG2025518626000011.jpg26141

[0070] In some embodiments, the gene construct comprises a human desmin gene promoter operably linked to a nucleic acid sequence encoding human s-KL (e.g., the nucleic acid sequence of SEQ ID NO: 3).

[0071] In some embodiments, multiple nucleic acids encoding mammalian s-KL or a functional variant thereof are operably linked to a plurality of, e.g., two, three, or four different promoters, all of which are operably linked to the nucleic acid encoding s-KL. In some embodiments, the additional promoter is also a muscle cell-specific promoter. In some embodiments, the additional promoter is a neuron-specific promoter. In some embodiments, the additional promoter is a ubiquitous promoter. A ubiquitous promoter is a promoter that drives expression in a virtual tissue. In some embodiments, the additional promoter is a constitutive promoter. In some embodiments, the additional promoter is an inducible promoter. In some embodiments, the additional promoter is a neuron-specific promoter. In some embodiments, the gene construct contains three promoters, a muscle cell-specific first promoter, a neuron-specific promoter, and an inducible promoter, all of which are operably linked to the nucleic acid encoding s-KL.

[0072] In some embodiments, the additional promoter is a hybrid (synthetic) neuron-specific promoter, and a muscle-specific promoter may also be useful. Such promoter types may include muscle-specific enhancers and / or transcription factor binding sites, including combinations with other promoter elements from viruses or humans (e.g., CMV, CAG, or PGK). In some embodiments, the neuron-specific promoter is a hybrid promoter, and one of the above-described promoters is fused to a CMV enhancer. Representative examples of hybrid promoters include the cytomegalovirus enhancer (CMV E-380bp) on the 5' side of the PDGF-β promoter described in Liu et al., Gene Ther. 11(1):52-60 (2004), and the CMV enhancer fused to the SYN promoter described in Hioki et al., Gene Ther. 14(11):872-882(2007). Additional hybrid neuron-specific promoters are described, for example, in U.S. Patent Application Publication No. 2009 / 0055941. Still other examples of muscle cell-specific promoters include synthetic neuron-specific promoters having higher activity than natural promoters.

[0073] In other embodiments, the additional promoter is a promoter other than a tissue-specific promoter, and representative types thereof include constitutive promoters and inducible promoters. A constitutive promoter initiates RNA synthesis independent of regulatory influences. An inducible promoter allows for the regulation of gene expression and can be regulated by an exogenously supplied compound, an environmental factor such as temperature, or the presence of a particular physiological state. Representative examples of particular promoters include the CMV promoter (e.g., the cytomegalovirus immediate early (CMV IE) promoter), the beta-actin promoter (e.g., the chicken beta-actin (CAG) promoter), the CASI promoter (e.g., a synthetic promoter described as a combination of the CMV enhancer, the chicken beta-actin promoter, and a splice donor and splice acceptor adjacent to the ubiquitin (UBC) enhancer as described in U.S. Patent No. 8,865,881), the human phosphoglycerate kinase-1 (PGK) promoter, the TBG promoter, the retroviral Rous sarcoma virus LTR promoter, the SV40 promoter, the dihydrofolate reductase promoter, the phosphoglycerol kinase (PGK) promoter, the EF1a promoter, the zinc-inducible murine metallothionein (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system, the ecdysone insect promoter, the tetracycline repressible system, the tetracycline inducible system, the RU486 inducible system, and the rapamycin inducible system.

[0074] In some embodiments, the additional promoter is a zinc-driven inducible promoter. Representative examples of zinc-driven promoters include the zinc-driven metallothionein promoter, the promoter / operator region of the E. coli zntA gene described in Brocklehurst et al., Mol. Microbiol. 31(3):893-902 (1999), and the zinc-regulated promoter described in U.S. Patent No. 8,354,272. In some embodiments, the additional promoter is a zinc-driven metallothionein promoter.

[0075] In some embodiments, the additional promoter operably linked to a nucleic acid encoding mammalian s-KL or a functional variant thereof is a neuron-specific promoter. As used herein, the term "neuron" includes cells of the CNS, PNS, and spinal cord, and representative cell types thereof include neurons (including motor neurons, sensory neurons, and interneurons), glial cells, and astrocytes. Motor neurons emit signals from the brain and spinal cord and control everything from muscle contraction to glandular output. The terms "neuron-specific" and "neuronal-specific" are used interchangeably herein and refer to the preferential, selective, or dominant expression of mammalian s-KL (or a functional variant thereof) in neurons or neural tissue as compared to other (i.e., non-neural) cells and tissues. In some embodiments, at least 50% of the expression, more specifically, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% occurs within neurons or neural tissue. In some embodiments, "neuron-specific" means that there is no leakage of the expression of mammalian s-KL (or a functional variant thereof) to other cells, organs, or tissues other than neurons, such as the lung, liver, brain, kidney, and / or spleen.

[0076] Representative examples of neuronal cell-specific promoters that may be suitable for use in the present disclosure include the synapsin 1 (SYN) promoter, the calcium / calmodulin-dependent protein kinase II promoter, the tubulin alpha I promoter, the neuron-specific enolase (NSE) promoter, the platelet-derived growth factor beta chain (PDGF-β) promoter, the microtubule-associated protein 1B (MAP1B), the dopamine receptor 1 (Drd1a) promoter, the 67 kDa glutamate decarboxylase (GAD67) promoter, the homeobox Dlx5 / 6, the glutamate receptor 1 (GluR1) promoter, the glial fibrillary acidic protein (GFAP) promoter, and the prepro-tachykinin 1 (Tac1) promoter.

[0077] Except for one or more promoters and to the extent not otherwise contained in the expression vector, the gene construct / expression cassette may further include one or more other non-coding regulatory elements, also known as expression control sequences. Representative examples of regulatory elements include appropriate transcription start, termination, promoter sequences and enhancer sequences, efficient RNA processing signals, such as splicing and polyadenylation (polyA) tail sequences, polyA consensus sequences, tetracycline regulatable systems, post-transcriptional regulatory elements, sequences that stabilize cytoplasmic mRNA, sequences that enhance translation efficiency (e.g., Kozak consensus sequences), and sequences that enhance protein stability. As used herein, a "post-transcriptional regulatory element" is a DNA sequence that, when transcribed, enhances the expression of a transgene or a fragment thereof delivered by the viral vectors of the present disclosure. Representative examples of post-transcriptional regulatory elements include the hepatitis B virus post-transcriptional regulatory element (HPRE) and the woodchuck hepatitis post-transcriptional regulatory element (WPRE). WPRE is a tripartite cis-acting element that has been shown to enhance transgene expression driven by certain promoters, but not all promoters.

[0078] In some embodiments, the gene construct comprises a cytomegalovirus immediate early (CMV IE) promoter operably linked to a nucleic acid encoding mammalian s-KL, a post-transcriptional regulatory element of woodchuck hepatitis virus (WPRE), and a polyA.

[0079] In some embodiments, the gene construct comprises a CAG promoter and a polyA tail operably linked to a nucleic acid encoding s-KL (cDNA of mouse or human s-KL).

[0080] The gene construct can be in the form of DNA (e.g., plasmid and various viral vectors such as adenovirus and adeno-associated virus (AAV) vectors), or RNA (e.g., for use in linearized mRNA constructs, or other viral vectors such as retroviral vectors and lentiviral vectors). In some embodiments where the gene construct is in the form of RNA for a linearized mRNA construct, the gene construct is incorporated into a plasmid, which is treated with an enzyme (e.g., a restriction enzyme) to obtain linear DNA, which is transcribed in a bioreactor as an in vitro transcript to produce mRNA. The linearized mRNA is complexed with a positively charged polymer, a cationic lipid, or other complex to form extruded nanoparticles, extruded micron-sized particles, or micelle emulsions.

[0081] In some embodiments where the gene construct is in the form of RNA for retroviral and lentiviral delivery, the gene construct contains the gag (antigen group), pol (reverse transcriptase), and env (envelope) genes, as well as the 5' and 3' long terminal repeats (LTRs). In some embodiments, the 5' LTR is a chimeric 5' LTR that contains a heterologous promoter that does not depend on transactivation by the lentiviral tat protein. The lentiviral LTR may be divided into three elements designated U3, R, and U5. The U3 element is unique to the 3' end of the lentiviral RNA genome. R is repeated at both ends of the lentiviral RNA genome, and U5 is unique to the 5' end of the lentiviral RNA genome. The sizes of the three elements can vary significantly between different viruses. In some embodiments, any part of the LTR is modified, substituted, or deleted, for example, a part of 3' U3 is deleted. In one embodiment, the 3' U5 element is replaced with polyA. In one embodiment, 5' U3 is replaced with a truncated CMV immediate early (IE) enhancer / TATA promoter. Additional lentiviral non-coding regulatory factor elements are known in the art, see, for example, WO2021 / 181108A1, WO2021 / 160993A1, and U.S. Pat. Nos. 6,669,936, 6,924,123, and 10,544,429.

[0082] In some embodiments, this gene construct (or nucleic acid construct) (a) a polyA signal, such as the SV40 polyA tail, (b) a protein translation initiation site consensus nucleic acid, (c) a post-transcriptional regulatory element nucleic acid, (d) 5' and 3' terminal inverted repeat sequences, and (e) further contains one or more of the introns.

[0083] As is known in the art, the term "intron" encompasses any portion of an entire intron that is of sufficient size to be recognized and spliced by a nuclear splicing apparatus. Typically, short functional intron sequences are preferred in order to keep the size of the expression cassette as small as possible to facilitate the construction and manipulation of the expression cassette. In some embodiments, the intron is obtained from a gene that encodes a protein encoded by a coding sequence within the expression cassette. The intron can be located 5' to the coding sequence, 3' to the coding sequence, or within the coding sequence. The advantage of positioning the intron 5' to the coding sequence is to minimize the potential for the intron to interfere with the function of the polyadenylation signal. Representative examples of suitable introns include the murine minute virus (MVM) intron, the beta-globin intron (beta IVS-1), the factor IX (FIX) intron A, the simian virus 40 (SV40) small-t intron, and the beta-actin intron.

[0084] All of these elements (a)-(e) are sequences that enable proper expression of a gene operably linked to a promoter. One of ordinary skill in the art will understand which sequences are being referred to and their function.

[0085] In some embodiments, the gene construct can include muscle-specific regulatory elements other than, for example, a muscle cell-specific promoter, that can enhance the muscle-specific expression of the mammalian s-KL coding sequence. Representative examples of such sequences include the CSk-SH1, CSk-SH2, CSk-SH3, CSk-SH4, CSk-SH51, and CSk-SH6 regulatory elements. See, for example, U.S. Patent Application Publication No. 2020 / 00407746. In some embodiments, such regulatory elements can be located upstream of the promoter.

[0086] The nucleic acid sequence of the gene construct comprising the nucleic acids encoding SEQ ID NO: 6, WPRE, and polyA is set forth below as (SEQ ID NO: 7): JPEG2025518626000012.jpg221142 JPEG2025518626000013.jpg239141 JPEG2025518626000014.jpg240140 JPEG2025518626000015.jpg239141 JPEG2025518626000016.jpg238141 JPEG2025518626000017.jpg25141

[0087] Methods for cloning nucleic acids and gene constructs into vectors are known in the art. In some embodiments, one or more sequences, or the entire gene construct, may be codon-optimized, for example, depleted of CpG dinucleotides. In one embodiment, the promoter of the gene construct is completely depleted of CpG dinucleotides. CpG dinucleotide depletion can be achieved by site-directed mutagenesis, in vitro synthesis of gene constructs, or any suitable molecular biology technique.

[0088] Expression vector An expression vector, also known as an expression construct, is typically a virus or plasmid (e.g., may contain a viral genome or a part thereof) designed for protein expression in cells. An expression vector has features that any vector may have, such as an origin of replication, a selectable marker, and a site suitable for insertion of a gene construct, such as a multiple cloning site (MCS).

[0089] In one aspect, the present disclosure provides an expression vector comprising a nucleic acid construct (a) comprising a muscle cell-specific promoter operably linked to a nucleic acid encoding mammalian s-KL or a functional variant thereof, wherein the vector may or may not have muscle cell tropism, or (b) comprising a first promoter that is functionally operable in muscle cells and operably linked to a nucleic acid encoding mammalian s-KL or a functional variant thereof, wherein the expression vector has muscle cell tropism.

[0090] Viral expression vector In some embodiments, the expression vector is a viral vector, such as a retroviral vector, a lentiviral vector, an adenoviral vector, a herpes viral vector, an adenovirus, or an adeno-associated virus (AAV) vector.

[0091] As used herein, the term "adeno-associated virus" refers to a viral vector that infects both dividing primate (and human) cells and quiescent primate (and human) cells. Lacking pathogenic effects and usually integrating into the same location in the genome (the AAVS1 site on chromosome 19), this viral vector can be safely used to transduce foreign DNA into human cells in gene therapy applications.

[0092] In some embodiments, the expression vector is an adeno-associated virus (AAV). The AAV vector can be derived from any suitable host species, including human (h), baboon, chimpanzee, and rhesus monkey (rh), pig-tailed macaque (pi), and cynomolgus monkey. The genomic structures of all known AAV serotypes are similar. The AAV genome is a linear single-stranded DNA molecule less than about 5,000 nucleotides (nt) in length. The inverted terminal repeats (ITRs) flank the unique coding nucleotide sequences of the non-structural replication (Rep) proteins and the structural (VP) proteins. The VP proteins (VP1, -2, and -3) form the capsid and contribute to the tropism of the virus. The terminal 145 nt of the ITRs are self-complementary and are organized such that an energetically stable intramolecular double-strand that forms a T-shaped hairpin can be formed. These hairpin structures function as the origin of viral DNA replication and as primers for the cellular DNA polymerase complex. After wild-type (wt) AAV infection in mammalian cells, the Rep gene is expressed and functions in the replication of the viral genome.

[0093] Representative examples of AAV expression vectors can be derived from AAV serotypes including AAV1, AAV3, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAVrh.43, AAVrh74, AAVpi.2, AAVrh.8, AAVhu.11, AAVhu.32, and AAVhu.37, PHPeb, 9P31, and AAVmyo. AAV serotype 9 (AAV9) is known to achieve efficient transduction in the heart and skeletal muscle. Thus, in some embodiments, the expression vector is an AAV9 vector, for example, a self-complementary AAV9 vector (scAAV9).

[0094] In some embodiments, the expression vector is a serotype of AAV having myocyte tropism. As used herein, the term "myocyte tropism" refers to a vector that preferentially affects myocytes, e.g., a viral vector that preferentially infects myocytes over other cell types in the body. Representative examples of such AAVs of serotypes having myocyte tropism include AAV1, AAV5, AAV6, AAV8, AAV9(BBB), AAVrh.74, and AAVmyo. Additional AAVs having myocyte tropism are known in the art. See U.S. Patent Application Publication No. 2021 / 0363193, Tabebordbar et al., Cell 184(19):4919-4938 (2021), and Weinmann et al., Nat. Commun. 11(1):5432 (2020). In some embodiments, the expression vector is an adeno-associated virus of a serotype having myocyte tropism selected from AAV8 and AAVmyo. In these embodiments, the promoter need not be a myocyte-specific promoter.

[0095] In some embodiments, the expression vector is an adeno-associated virus containing an AAV capsid polypeptide comprising or consisting of any one of the amino acid sequences of SEQ ID NOs: 18-29, or encoded by a nucleic acid having any one of the sequences of SEQ ID NOs: 30-41. See Grimm et al., U.S. Patent Application Publication No. 2021 / 0363193.

[0096] In some embodiments, the AAV capsid polypeptide comprises, or consists of, the amino acid sequence of SEQ ID NO: 17, which is encoded by the nucleic acid sequence of SEQ ID NO: 30 and corresponds to AAV9P1 disclosed in US Patent Application Publication No. 2021 / 0363193. In some embodiments, the AAV capsid polypeptide comprises, or consists of, the amino acid sequence of SEQ ID NO: 26, which is encoded by the nucleic acid sequence of SEQ ID NO: 38 and corresponds to AAV9S10P1 disclosed in US Patent Application Publication No. 2021 / 0363193. In some embodiments, the AAV capsid polypeptide comprises, or consists of, the amino acid sequence of SEQ ID NO: 27, which is encoded by the nucleic acid sequence of SEQ ID NO: 39 and corresponds to AAVS1P1 disclosed in US Patent Application Publication No. 2021 / 0363193.

[0097] In some embodiments, the expression vector is an adeno-associated virus of serotype AAV8, which comprises, or consists of, the nucleic acid sequence of SEQ ID NO: 7, and this sequence is a gene construct comprising a human desmin promoter, a nucleic acid encoding mouse s-KL, a WPRE sequence, and an SV40 polyA tail.

[0098] In some embodiments, the expression vector is an adeno-associated virus of serotype AAV8, which comprises, or consists of, the nucleic acid sequence of SEQ ID NO: 7, but the gene encodes human s-KL, for example, the nucleic acid sequence of SEQ ID NO: 3.

[0099] Table A shows the positions of different sequences of SEQ ID NO: 7.

[0100] Table A. Sequence legend of SEQ ID NO: 7

Table 1

[0101] In some embodiments, the expression vector is a serotype of AAV having neuron tropism. As used herein, the term "neuron tropic" refers to a vector that preferentially affects neurons, for example, a viral vector that preferentially infects motor neurons over other cell types in the body. Representative examples of serotypes of AAV having neuron tropism include AAV1, AAV6, and AAV7. In some embodiments, the expression vector is a serotype of adeno-associated virus having neuron tropism specific to astrocytes, such as AAV5. In some embodiments, the expression vector is a vector having neuron tropism as described, for example, in U.S. Patent Application Publication Nos. 2019 / 0030138 and 2020 / 0339960. In these embodiments, the promoter need not be a neuron-specific promoter.

[0102] In some embodiments, the expression vector is an AAV delivered by intraspinal parenchymal injection targeting the spinal cord and is of a serotype having neuron tropism. In some of these embodiments, the AAV serotype is AAV1, AAV5, AAV9. In some embodiments, the AAV expression vector is delivered intravenously and has neuron tropism. AAV vector serotypes having neuron tropism well-suited for intravenous injection include AAV9, AAVhr.10, AAVrh.8, and AAVrh43.

[0103] In some embodiments, the expression vector is configured to undergo retrograde axonal transport. In some of these embodiments, the expression vector is an AAV serotype of AAV1, AAV5, AAV8, AAV9, or AAVrh.10. In one embodiment, the AAV1 serotype expression vector is injected into muscle or the sciatic nerve and preferentially targets motor neurons.

[0104] In some embodiments, the viral vector is a recombinant adeno-associated virus (rAAV) comprising at least one capsid protein derived from an AAV serotype. The AAV capsid protein can provide tissue-specific targeting such that the AAV infects the gene construct and delivers it to a desired tissue or organ. In some embodiments, the AAV is a pseudotyped AAV (pAAV) comprising a virus or viral vector having viral envelope proteins from two or more viruses. The pAAV can have changes in host or tissue tropism, or increased or decreased particle stability. In some embodiments, the pAAV contains nucleic acids from two or more different AAVs. For example, the nucleic acid from one AAV source encodes the capsid protein and the nucleic acid from at least one other AAV source encodes other viral proteins and / or the viral genome. In some embodiments, pAAV refers to an AAV comprising the inverted terminal repeats (ITRs) of one AAV serotype and the capsid protein of a different AAV serotype. For example, a pAAV vector containing the ITRs of serotype X encapsulated in the protein of Y is designated as AAVX / Y (e.g., AAV2 / 1 has the ITRs of AAV2 and the capsid of AAV1). In some embodiments, pAAV can be useful in combining the tissue-specific targeting ability of the capsid protein from one AAV serotype with viral DNA (e.g., viral ITRs) from another AAV serotype, thereby enabling targeted delivery of the transgene to the target tissue.

[0105] The production of AAV vectors is most frequently achieved by double plasmid transfection in vitro followed by helper adenovirus infection. The two plasmids for double plasmid transfection are the vector plasmid and the packaging plasmid. The vector plasmid contains the gene construct described herein adjacent to the AAV terminal inverted repeat sequence (ITR), while the packaging plasmid provides the AAV repressor (Rep) and capsid (Cap) genes for vector DNA replication and packaging. When a suitable cell line (e.g., HeLa) is transfected with the two plasmids, it is infected with the helper adenovirus, the AAV genes are expressed, the genes from both plasmids are expressed, and are packaged into AAV particles. The proteins encoded on the packaging plasmid constitute the AAV particles, while the vector plasmid (containing the gene construct) is packaged into the AAV particles. The AAV particles are purified from the helper adenovirus particles, or the adenovirus particles are selectively heat-inactivated. This procedure produces replication-deficient AAV containing the gene construct, which can be further formulated and delivered to a subject.

[0106] Alternatively, instead of helper adenovirus infection in vitro, a third adenovirus-derived plasmid containing the necessary replication genes from adenovirus (e.g., E1A, E1B, E2A, E4, and VA RNA) may also be transfected together with the vector plasmid and the packaging plasmid. In some embodiments, the packaging and adenovirus-derived plasmids may be stably integrated into the cell line.

[0107] Additional methods for producing and delivering AAV, rAAV, and pAAV are known in the art. See, for example, U.S. Patent Publications No. 2015 / 0065560, No. 2013 / 0090374, and No. 2012 / 0309050, and U.S. Patents No. 11,041,171, No. 11,020,443, and No. 7,858,367.

[0108] In some embodiments, the expression vector is a lentiviral vector or a recombinant lentiviral vector. In some embodiments, the expression vector is a non-integrating and non-replicating recombinant lentiviral vector. The construction of lentiviral vectors is described, for example, in U.S. Patent Nos. 5,665,577, 5,981,276, 6,013,516, 7,090,837, 8,119,119, and 10,954,530. Lentiviral vectors contain a defective lentiviral genome, i.e., at least one of the lentiviral genes gag, pol, and env is inactivated or deleted. Additional lentiviral genes and sequences include the Rev response element (RRE), and the central DNA flap of the viral cDNA between the central polypurine tract (cPPT) and the central termination sequence (CTS), often abbreviated as cPPT CTS.

[0109] In some embodiments, the expression vector is a recombinant lentivirus comprising a recombinant genome that includes a lentiviral encapsidation psi sequence, an RNA nuclear export element, a transgene, and a promoter and / or sequences favorable for nuclear import of the RNA, and a mutant integrase that prevents integration of its genome into the genome of the isolated cell, between the LTR 5' lentiviral sequence and the 3' lentiviral sequence. The lentiviral vector can, for example, include the sequence 5'LTR-psi-RRE-cPPT CTS-transgene-LTR3'.

[0110] In some embodiments, the viral vector encoding the nucleic acid sequence of the present disclosure is administered with an empty vector containing a capsid protein that does not contain any of the nucleic acid sequences of the present disclosure. In some embodiments, the empty vector is administered at a ratio of empty vector to viral vector of 5:1 to 3:1, modulating the immune response of the subject to the viral vector.

[0111] In some embodiments, the viral vector is immunomodulated. Immunomodulation can typically be achieved by adding polyethylene glycol (PEG) to the viral vector, typically by pegylation of lysine residues exposed on the surface of the viral vector. See, for example, U.S. Patent Application Publication No. 2021 / 0139860, as well as U.S. Patents Nos. 6,399,385 and 10,022,457. Pegylation results in a longer circulation time of the viral vector particles as well as a decrease in the immune response (i.e., a decrease in immunogenicity).

[0112] Additional viral vectors can be used in combination with the viral vectors disclosed herein. For example, the viral vectors described in U.S. Patent Application Publication No. 2019 / 0030138 may be combined with the viral vectors described herein.

[0113] Non-viral expression vectors In other embodiments, the expression vector is a non-viral vector, and representative examples thereof include plasmids, minicircles, and transposon-based vectors, such as Sleeping Beauty (SB)-based vectors and piggyBac (PB)-based vectors. In still other embodiments, the vector may contain both viral and non-viral elements.

[0114] In some embodiments, the gene construct is incorporated into a plasmid expression vector. The plasmid may contain a sequence encoding the gene construct (promoter and mammalian s-KL sequence), an initiation sequence, a polyA tail sequence, optional regulatory elements, and other optional sequences (e.g., multiple cloning sites). In some embodiments, the mRNA is produced from a circular plasmid. The plasmid may be linearized with a restriction enzyme, transcribed in vitro to produce mRNA, and modified with a 5' cap and a 3' polyA tail.

[0115] In some embodiments, the carrier encapsulates a gene construct or plasmid. The carrier may be a lipid-based system, such as a lipid nanoparticle (LNP), liposome, lipid vesicle, or lipoplex. In some embodiments, the carrier is an LNP. In certain embodiments, the LNP comprises two or more concentric bilayer membranes separated by an aqueous compartment. The lipid bilayers may be functionalized and / or cross-linked to each other. The lipid bilayer may contain one or more ligands, proteins, or channels.

[0116] Lipid carriers, such as LNPs, may include one or more cationic / ionic lipids, one or more polymer-conjugated lipids, one or more structural lipids, and / or one or more phospholipids. "Cationic lipid" refers to a lipid that is positively charged or capable of retaining a positive charge. Cationic lipids contain one or more amine groups that have a positive charge depending on the pH. "Polymer-conjugated lipid" refers to a lipid having a conjugated polymer moiety. Polymer-conjugated lipids include pegylated lipids that are lipids conjugated to polyethylene glycol. "Structural lipid" refers to a non-cationic lipid that has no net charge at physiological pH. Exemplary structural lipids include cholesterol, fucosterol, sitosterol, ergosterol, campesterol, and the like. "Phospholipid" refers to a lipid having a triester of glycerol with two fatty acids and one phosphate ion. The phospholipids in the LNP assemble the lipids into one or more lipid bilayers. Methods for LNPs, their preparation, formulation, and delivery are disclosed, for example, in U.S. Pat. Nos. 9,364,435, 9,518,272, 10,022,435, and 11,191,849, and U.S. Patent Application Publication Nos. 2004 / 0142025, 2007 / 0042031, and 2020 / 0237679.

[0117] Lipoplexes, liposomes, and lipid nanoparticles can include a combination of lipid molecules such as cationic lipids, neutral lipids, anionic lipids, polypeptide-lipid conjugates, and other stabilizing components. Representative stabilizing components include antioxidants, surfactants, and salts. Compositions and methods of preparation of lipoplexes, liposomes, and lipid nanoparticles are known in the art. See, for example, U.S. Pat. Nos. 8,058,069, 8,969,353, 9,682,139, 10,238,754, U.S. Patent Application Publication Nos. 2005 / 0064026 and 2018 / 0291086, and Lasic, Trends Biotechnol. 16(7):307-21 (1998), Lasic et al., FEBS Lett. 312(2-3):255-8 (1992), and Drummond et al., Pharmacol. Rev. 51(4):691-743 (1999).

[0118] Pharmaceutical composition In some embodiments, a pharmaceutical composition containing an expression vector of the present disclosure may be formulated with a pharmaceutically acceptable carrier and optionally a pharmaceutically acceptable excipient (collectively referred to as a pharmaceutically acceptable “vehicle”) for administration to a subject via any suitable and medically acceptable mode of administration. In some embodiments, administration is via parenteral (e.g., intravenous) delivery.

[0119] The terms “pharmaceutically acceptable carrier” and “pharmaceutically acceptable excipient” refer to components that are physiologically inert (e.g., non-immunogenic), non-toxic, compatible with all other components of the pharmaceutical composition, and suitable for contact with a subject's tissue or organ (e.g., mammals including both human and non-human animals) having a reasonable benefit / risk ratio comparable to other methods.

[0120] In some embodiments, the expression vector is formulated for systemic administration, for example, parenteral administration. In some embodiments, the carrier is aqueous, and representative examples thereof include water, saline (e.g., physiological saline, bacteriostatic water, and phosphate buffered saline (PBS)), as well as aqueous solutions of dextrose and glycerol. The aqueous carrier may also include polyols such as glycerol, propylene glycol, and liquid polyethylene glycol. The aqueous composition may contain additional excipients such as solubilizing agents, isotonic agents, suspending agents, emulsifying agents, stabilizing agents, and preservatives. Such liquid-based formulations may be in the form of solutions, suspensions, or dispersions. In some embodiments, the carrier is non-aqueous.

[0121] The pharmaceutical composition may further contain excipients. Similar to the case of the carrier, the choice of excipient is partially determined by the specific vector, as well as by the specific method used to administer the composition. Thus, those skilled in the art will readily understand that there are a variety of suitable formulations for the pharmaceutical compositions of the present disclosure. Representative examples of excipients include coating agents, surfactants and emulsifying agents, antimicrobial agents and other preservatives, solubilizing agents, isotonic agents, absorption blockers, and suspending agents. Lecithin is an exemplary coating agent that establishes appropriate fluidity and maintains the particle size required in the case of dispersions. Surfactants may also assist in maintaining the particle size. Exemplary antimicrobial agents include antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. Exemplary isotonic agents include sugars and polyhydric alcohols (e.g., mannitol, sorbitol, and sodium chloride). Exemplary absorption blockers include aluminum monostearate and gelatin, which extend the absorption of the gene construct in the composition.

[0122] Additional pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions, their formulations, and the delivery of expression vectors such as viral vectors are known in the art. See, for example, U.S. Patent Application Publication Nos. 2013 / 0090374 and 2012 / 0309050, and U.S. Patents Nos. 7,858,367, 11,020,443, and 11,041,171.

[0123] Pharmaceutical compositions containing an expression vector may also be formulated for several other routes of administration or modes of administration, for example, oral (including both liquid and solid dosage forms), inhalation (with pressurized propellants), topical (e.g., lotions, creams, gels, ointments, sticks, sprays, and pastes), and mucosal (e.g., vaginal).

[0124] The composition may be provided in unit dose or multi-dose sealed containers, such as ampoules and vials.

[0125] Method of treatment This method involves treating a subject having a movement disorder resulting from loss of muscle function or from neuron-muscle dysfunction at the level of motor neurons. As understood in the art, "treatment" is an approach for obtaining a beneficial or desirable result, including clinical outcomes. Such results can include, whether detectable or not, reduction or improvement of one or more symptoms of the movement disorder, decrease in the degree of the disease or disorder, stabilization of the state of the disease or disorder, delay or deceleration of the disease or disorder, amelioration or alleviation of the disease or disorder, and remission (partial or complete) of the disease or disorder. As used herein, the term "subject" includes humans and non-human animals (e.g., non-human primates, livestock animals, domestic pets, and laboratory animals such as rodents). As used herein, the term "movement disorder" refers to the loss of motor ability due to loss of muscle function. The loss of function is mainly due to muscle weakness, muscle atrophy, loss of motor neuron function, muscle contractility disorders, and other muscle cell disorders (e.g., metabolic), spinal cord injury, motor neuron disorders, or motor neuron diseases. Examples of movement disorders include total or partial paralysis (e.g., inability to walk or stand, inability to speak, inability to swallow and ultimately inability to breathe (i.e., respiratory failure), the latter leading to death), lack of muscle control, poor stamina, muscle cramps, spasticity, muscle weakness, and muscle atrophy.

[0126] According to the International Neuromodulation Society, movement disorders are associated with partial or complete loss of function of a body part, usually a limb. Diseases that can cause movement disorders can result in muscle weakness, insufficient stamina (i.e., fatigue), lack of muscle control, or complete paralysis. Movement disorders are a major cause of physical disability. This is widely caused by peripheral problems affecting the muscles, central nervous system problems affecting the output to the muscles, and sensory problems affecting the muscles, movement, and balance. Movement disorders are often evident in neuromuscular diseases and neurological conditions, but can also result from cancers of the central and peripheral nervous systems, or traumatic injuries.

[0127] Many neuromuscular and muscle diseases and disorders, including motor neuron diseases such as certain neurodegenerative diseases, can present with one or more symptoms of movement disorders. Diseases and disorders characterized by, or presenting with, symptoms of movement disorders include neuromuscular diseases. As used herein, the term "neuromuscular disease" includes any disease that affects motor neurons of the spinal cord or central nervous system (CNS), peripheral nervous system (PNS), neuromuscular junction, or skeletal muscle, all of which are components of the motor unit and thus ultimately affect the subject's motor ability. Damage to motor neurons in the spinal cord, CNS, PNS, neuromuscular junction, or skeletal muscle can cause muscle atrophy and weakness. Sensory problems can also occur. Neuromuscular diseases can be acquired or genetic. Mutations in over 500 genes have been shown to cause neuromuscular diseases. Other causes include nerve or muscle degeneration, autoimmunity, toxins, drugs, nutritional deficiencies, metabolic disorders, hormonal imbalances, infections, nerve compression / entrapment, compromised blood supply, and trauma.

[0128] Representative examples of diseases or disorders characterized by motor impairment include, more generally or idiopathically (sALS) and familial (fALS) amyotrophic lateral sclerosis (ALS), Charcot-Marie-Tooth disease, multiple sclerosis, muscular dystrophy, Duchenne and Becker muscular dystrophy, myasthenia gravis, muscle diseases, myositis including polymyositis and dermatomyositis, peripheral neuropathy, neuromyotonia, Lambert-Eaton myasthenic syndrome, Friedreich's ataxia, traumatic nerve injury, diabetic neuropathy, motor disability, and spinal muscular atrophy (SMA), spinal cord injury, peripheral nerve injury or traumatic nerve injury, as well as muscle metabolic diseases. Further other diseases and disorders that may be suitable for treatment using the disclosed expression vectors include hereditary myopathy, toxic neuropathy, autoimmune peripheral polyneuropathy, acute inflammatory demyelinating polyneuropathy (AIDP), chronic inflammatory demyelinating polyneuropathy (CIDP), vasculitic polyneuropathy, paran neuropathy, idiopathic ganglionitis, amyotrophic lateral sclerosis, multifocal motor conduction block neuropathy, or lower motor neuron syndrome, neuromuscular diseases, muscular atrophy, drug-induced myopathy, sarcopenia, cachexia, type II muscle fiber atrophy, age-related muscular atrophy, and acquired autoimmune primary myopathy.

[0129] Some diseases and disorders that may be suitable for treatment using the gene constructs and expression vectors of the present disclosure include diseases and disorders classified as motor neuron diseases (MND), which are a group of rare neurodegenerative disorders that selectively affect motor neurons, the cells that control the voluntary muscles of the body. Examples of MND include ALS, progressive bulbar palsy (PBP), pseudobulbar palsy, progressive muscular atrophy (PMA), primary lateral sclerosis (PLS), spinal muscular atrophy (SMA), and monomelic amyotrophy (MMA), as well as some rarer variants similar to ALS.

[0130] In some embodiments, the method involves treating a subject having a motor impairment in ALS, including motor impairment in sALS, motor impairment in fALS, and motor impairment in jALS.

[0131] In some embodiments, the method may be "preventative" or "prophylactic" in that the expression vector (or transformed cells according to an ex vivo embodiment) is administered to the subject prior to the onset of signs of the movement disorder. These embodiments may be particularly advantageous in situations where the subject is suspected of having an early stage of a neuromuscular disorder and / or has received a preliminary positive diagnosis, has a predisposition to a disease or disorder (e.g., ALS) (e.g., by family history), or is otherwise so considered. Prophylactic or preventative treatment may result in a delay in the onset of the movement disorder or a reduction in the severity of the movement disorder when symptoms appear. Tests for determining the cause of the movement disorder may include, for example, genetic tests, muscle biopsies, and electromyography tests known in the art. For example, fALS may be diagnosed by genetic testing.

[0132] Route of Administration In some embodiments, the pharmaceutical composition is administered to the subject parenterally (e.g., via intrathecal, subcutaneous, intravenous, intraventricular, intramuscular, or intraarterial injection, either as a bolus or infusion, which may be continuous or discontinuous). Other routes / methods of administration include any medically acceptable route, representative examples of which may include oral, inhalation, topical, and mucosal.

[0133] In some embodiments, the treatment method may involve the administration of an expression vector to cells. In some embodiments, the method comprises isolating cells from a subject in need of treatment, also referred to herein as “isolated cells,” placing the isolated cells in a suitable ex vivo culture system, exposing the isolated cells to an expression vector with a gene construct encoding mammalian s-KL, and administering the isolated cells back to the subject. In some embodiments, the method optionally comprises differentiating the isolated cells into a cell type with a differentiating agent. In some embodiments, the method comprises optionally enriching the isolated cells before or after exposing the isolated cells to the expression vector. The cells isolated from the subject may be muscle cells, nerve cells, or iPSCs. In some embodiments, the nerve cells are motor neurons.

[0134] In some embodiments, the isolated cells are pluripotent (e.g., iPSCs) or pluripotent stem cells induced to pluripotency. The PSCs may be obtained from a subject (e.g., adipose tissue), in which case they may be reprogrammed to a pluripotent state (self). Alternatively, the iPSCs can be obtained from a suitable cell bank (allogeneic), e.g., a cord blood cell bank. Pluripotency induction can be carried out by any suitable means known in the art. Generally, pluripotency induction involves the regulation of specific cell pathways, either directly or indirectly, in isolated cells having pluripotency factors, which can be nucleic acid sequences, polypeptides, small molecules, or combinations thereof. In one embodiment, the pluripotency factors are polypeptide transcription factors or polynucleotides encoding transcription factors. Representative examples of pluripotency factor transcription factors include Oct-3 / 4, Cdx-2, Gbx2, Gsh1, HesX1, HoxA10, HoxA11, HoxB1, Irx2, Isl1, Meis1, Meox2, Nanog, Nkx2.2, Onecut, Otx1, Oxt2, Pax5, Pax6, Pdx1, Tcf1, Tcf2, Zfhx1b, Klf-4, Atbf1, Esrrb, Gcnf, Jarid2, Jmjd1a, Jmjd2c, Klf-3, Klf-5, Mel-18, Myst3, Nac1, REST, Rex-1, Rybp, Sall4, Sall1, Tif1, YY1, Zeb2, Zfp281, Zfp57, Zic3, Coup-Tf1, Coup-Tf2, Bmi1, Rnf2, Mta1, Pias1, Pias2, Pias3, Piasy, Sox2, Lef1, Sox15, Sox6, Tcf-7, Tcf711, c-Myc, L-Myc, N-Myc, Hand1, Mad1, Mad3, Mad4, Mxi1, Myf5, Neurog2, Ngn3, Olig2, Tcf3, Tcf4, Foxc1, Foxd3, BAF155, C / EBPβ, mafa, Eomes, Tbx-3, Rfx4, Stat3, Stella, and UTF-1. In one embodiment, the pluripotency factors include nucleic acid sequences encoding the transcription factors Oct4, Sox2, Klf4, c-Myc, and Nanog.

[0135] As is known in the art, iPSCs can be differentiated into muscle cells or nerve cells using differentiation agents. Representative examples of differentiation agents for the differentiation of iPSCs into muscle cells include TGF-β, all-trans retinoic acid, dibutyryl cyclic adenosine monophosphate (cAMP), platelet-derived growth factor-BB (PDGF-BB), and combinations thereof. Representative examples of differentiation agents for the differentiation of iPSCs into nerve cells include retinoic acid, bone morphogenetic protein 4 (BMP4), nerve growth factor (NGF), retinoic acid receptor (RAR) agonist (e.g., TTNPB), glycogen synthase kinase 3 inhibitor (e.g., CHIR99021), neurotrophin-3 (NT-3), and combinations thereof. Additional enrichment or selection can be performed on the treated cells and / or differentiated cells. For example, in some embodiments, CD34+ cells are selectively enriched to isolate smooth muscle cells. In some embodiments, iPSCs are differentiated and selectively enriched to isolate striated muscle cells. In some embodiments, nerve cells are enriched by selecting forkhead box A2 (FOXA2, also known as HNF3β, and TCF-3B) positive cells. In some embodiments, nerve cells are enriched by selecting CD133 positive cells.

[0136] In other embodiments, muscle cells and / or nerve cells are isolated from a subject. In some embodiments, myoblasts, and satellite cells (either in a quiescent or activated state) are isolated from a subject by a surgical method (e.g., muscle biopsy), and fluorescence-activated cell sorting (e.g., isolating Pax7 positive cells) is performed. In some embodiments, induced pluripotent stem cells, mesodermal angioblasts, immortalized muscle progenitor cells, or other pluripotent cell lines are obtained and administered to the subject locally or systemically.

[0137] Methods for introducing an expression vector into a cell are known in the art. One of ordinary skill in the art will readily appreciate a particular method depending on the nature of the vector. In some embodiments, delivery or integration may include transfecting, infecting, or transducing the cell with the expression vector. In some embodiments, an expression vector containing the expression vector is delivered to muscle cells by lipofection. Lipofection is described, for example, in U.S. Patent Nos. 5,049,386, 4,946,787, and 4,897,355. Electroporation may be advantageous for plasmid vectors. In some embodiments, Factor H is increased during treatment, for example, by harvesting adipose tissue from a subject, purifying stem cells from the adipose tissue, treating the stem cells to increase the secretion of Factor H (e.g., a small molecule compound, optionally serglycin), and administering the treated stem cells to the subject. Harvesting and treatment of adipose tissue are described, for example, in U.S. Patent Publication No. 2016 / 0193251.

[0138] The cells are administered to a subject after the cells have been transformed with the genetic construct and any optional differentiation and / or enrichment has been completed. Administration of these cells is known in the art and can be accomplished by any suitable means disclosed elsewhere in this specification.

[0139] Therapeutically effective dose In the context of the present disclosure, a dose administered to a subject, particularly a human, should be "therapeutically effective" in the sense that it is sufficient to achieve beneficial or desired results in the subject over a reasonable time frame, including the clinical results as described above. The dose depends on various factors including the strength of the particular expression vector used, the condition of the subject, and the weight of the subject, as well as the severity of the movement disorder. The size of the dose is also determined by the presence, nature, and extent of any adverse side effects that may be associated with the administration of the expression vector.

[0140] The unit dosage form of the pharmaceutical composition containing the expression vector may be formulated. As used herein, the term "unit dosage form" refers to physically discrete units suitable as unit doses for human and animal subjects, and each unit contains a predetermined amount of the expression vector calculated to be in an amount sufficient to produce the desired effect. The unit dosage form may be administered in any suitable volume of liquid over the appropriate infusion or injection required by the embodiment. The dosage may be determined by unit body weight (e.g., dosage per kg of subject), body surface area (BSA) often expressed in square meters BSA, or any suitable measurement of the subject.

[0141] In some embodiments utilizing viral expression vectors, the viral vector is at 1×10 6 ~5×10 14 vector genomes per kg of subject body weight (vg / kg). As used herein, the term "vector genome" (or "vg") refers to the nucleic acid sequence that constitutes the gene of the viral vector and any transgene encoded therein (e.g., mammalian s-KL).

[0142] In some embodiments, the viral vector is a lentivirus and is administered at 1×10 8 ~5×10 12 transducing units (TU / kg) per kg per dose (e.g., by a single intravenous infusion).

[0143] In some embodiments, the expression vector is administered to the subject at a titer of at least about 1×10 5 viral genomes / mL to at least about 100×10 16 viral genomes / mL. When used with respect to viral titer, the term "viral genome" (vg) (also known as "genomic equivalent", "genomic copy" (gc), or "genomic particle" (gp)) refers to the number of virions containing the expression vector, e.g., recombinant AAV, whether infectious or functional.

[0144] In some embodiments, the viral vector delivering the s-KL gene construct is administered to a human in a suitable volume from 1×10 5 to 5×10 14 viral genomes in order to achieve blood and tissue levels of the viral genome in an effective amount, which is an s-KL genomic dosage that achieves 200 - 1300 pg / ml of s-KL protein or polypeptide in a desired target tissue such as blood, CSF, or muscle. In some embodiments, the viral vector is administered at 1×10 6 vg or more in a deliverable volume within a single vial. In some embodiments, a first dose of 1×10 9 viral genomes is administered, followed by a subsequent dose of 1×10 11 vg.

[0145] In some embodiments, the effective amount is a dosage that achieves 1 - 2,000 pg / ml of polypeptide in blood, CSF, or a desired target tissue. In some embodiments, the effective amount is a dosage that achieves 200 - 1300 pg / ml of polypeptide in blood, CSF, or a desired target tissue. In some embodiments, the effective amount is a dosage that achieves an average of about 550 pg / ml within a range of 200 above or below, or above or below the average, in the circulation of an adult subject. In some embodiments, the effective amount is a dosage that achieves an average of about 950 pg / ml within a range of 300 above or below, or above or below the average, in the circulation of a pediatric subject.

[0146] In some embodiments, the therapeutically effective dosage results in a multiple increase in gene expression of a nucleic acid sequence encoding mammalian s-KL or a functional variant thereof. In some embodiments, the effective dosage results in an increase in expression of at least 2-fold. In some embodiments, the effective dosage results in an increase in gene expression of at least 3-fold, or at least 4-fold, or at least 5-fold, or at least 6-fold, or at least 8-fold, or at least 10-fold.

[0147] The number of times the composition is administered to a subject in need thereof may depend on any one of a number of factors and may be at the discretion of a medical professional, including the disease or disorder and its severity, as well as the subject's response to the formulation. Administration of a therapeutically effective amount of the expression vector is performed at least once. In other embodiments, administration is performed multiple times, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times, over a given period to achieve the desired therapeutic effect. The dosage and / or frequency of each administration can be adjusted as needed based on the subject's condition and physiological response. If the subject's condition does not improve, the composition may be administered chronically, i.e., over a long period including the entire lifespan of the subject, at the discretion of the physician, to improve or otherwise control or limit the symptoms of the subject's disease or condition. In some embodiments, e.g., if the subject's condition improves, the composition may be administered continuously, at the discretion of the physician.

[0148] In some embodiments, treatment can be administered over a period of about 1 hour. In some embodiments, treatment may be administered over a period of about 1 hour to about 24 hours per day. In some embodiments, treatment can be administered 24 hours a day over a plurality of days including, by way of example only, 1, 2, 3, 4, 5, 6, 7, 10, 15, and 20 days. In some other embodiments, treatment can be temporarily reduced or temporarily interrupted for a specific period (i.e., an "off period"). The length of the off period can vary widely, e.g., between 2 days and 1 year, including, by way of example only, 2, 3, 4, 5, 6, 7, 10, 12, 15, 20, 28, 35, 50, 70, 100, 120, 150, 180, 200, 250, 280, 300, 320, 350, and 365 days. In some other embodiments, treatment can be temporarily reduced for a period of time of a specific length. The dosage reduction during the reduction period can be from 10% to less than 100% and can include, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95%.

[0149] Combination therapy The treatment method may include co - administration (i.e., administration within the same treatment period) of another active agent known to be effective in the treatment of movement disorders (e.g., related to neuromuscular diseases and disorders). Representative additional active agents include riluzole and edavarone, and combinations thereof. In some embodiments, the treatment method is used in combination with direct injection of recombinant proteins or gene therapy that express IGF - 1, TDP - 43 (TAR DNA - binding protein 43), EEAT2 (excitatory amino acid transporter 2), GDNF (glial - derived neurotrophic factor), cardiotrophin - 1, brain - derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), follistatin 344 (FSTN - 344), and factor H.

[0150] The present invention has been described in conjunction with the above - mentioned aspects and embodiments. However, the foregoing description and the following examples are intended to illustrate, not to limit, the scope of the present invention. Other aspects, advantages, and modifications within the scope of the present invention will be apparent to those skilled in the art to which the present invention pertains.

Examples

[0151] Materials and Methods Animal Breeding. The mouse model for this study was generated according to the mouse model of ALS (SOD1 G93A , also abbreviated as SOD1). Animals had free access to food and water and were maintained under standard temperature conditions (22 ± 2°C) and a 12 - hour light / dark cycle (300 lux / 0 lux).

[0152] Transgenic Mice. Transgenic mice with the G93A human SOD1 mutation (C57bl6 - Tg[SOD1 - G93A]1Gur) were obtained from the Jackson Laboratory (Bar Harbor, Maine, USA). Hemizygous C57bl6 SOD1 G93AThe males were obtained by mating with C57bl6 females. The offspring were identified by PCR of DNA extracted from tail tissue. The experimental procedures were approved by the Ethics Committee of the Universitat Autonoma de Barcelona. The following experimental groups of mice were used: SOD1 injected with AAV8-hDes-s-KL (SEQ ID NO: 7) or AAV8-Mock (SEQ ID NO: 8). G93A WT littermates treated with AAV8-hDes-s-KL or AAV8-Mock (n = 15 - 19 / condition, 7 - 10 per sex). To correct for experimental groups, animals were assigned according to body weight and original littermates. Animal body weight was measured every 4 weeks to monitor overall health status.

[0153] Table B. Sequence legend of SEQ ID NO: 8

Table 2

[0154] Virus production and injection. Mouse s-KL cDNA was cloned between AAV2 ITRs under the control of the human desmin promoter. AAV8 virus stocks were produced by triple transfection of HEK293-AAV cells with an expression plasmid, a Rep8Cap2 plasmid containing the AAV gene, and a pXX6 plasmid containing the adenovirus genes required as helper virus. The AAV2 ITR coding plasmid is described in Piedra et al., Hum. Gene Ther. Methods 26(1):35 - 42(2015). The pXX6 coding plasmid is as described in Xiao et al., J. Virol. 72(3):2224 - 2232(1998). The Rep8Cap2 coding plasmid is described in Gao et al., Proc. Natl. Acad. Sci. U.S.A. 99(18):11854 - 11859(2002).

[0155] AAV particles were purified by iodixanol gradient. Titration was evaluated by PicoGreen (Invitrogen) quantification and calculated as viral genomes per milliliter (vg / ml). A control serotype-matched AAV empty vector (mock) was used as a control.

[0156] For intravenous administration, 1.8×10 14 and 3×10 14 vg / kg of AAV8-s-KL or AAV8-Mock in a total volume of 250 μl suspension was injected into the tail vein of 6-week-old mice.

[0157] Real-time PCR. For RNA extraction, 1000 μl of Qiazol (Qiagen) was added and the tissue was homogenized twice at 50 Hz for 6 minutes using a Tyssue Lyser LT (Qiagen). The sample was then purified with chloroform, precipitated with isopropanol, washed with 70% ethanol, and resuspended in 20 μl of RNase-free water. RNA concentration was measured using a NanoDrop ND-1000 (Thermo Scientific).

[0158] 1 μg of RNA was reverse-transcribed using 10 μmol / l DTT, 200 U of M-MuLV reverse transcriptase (New England BioLabs), 10 u of RNase Out ribonuclease inhibitor (Invitrogen), 1 μmol / l oligo(dT), and 1 μmol / l random hexamer (BioLabs). The reverse transcription cycle conditions were 10 minutes at 25°C, 1 hour at 42°C, and 10 minutes at 72°C. The mRNA expression of s-KL was analyzed using the Taqman probe 5’-6FAM-3’BHQ-1 s-KL-Pr:5’- AGAAGAGTCCTCGCCGGATGCTGTA -3’ (SEQ ID NO: 11) and the specific primer set (s-KL-Fw:5’- TCATAATGGAAACCTTAAAAGCAA -3’ (SEQ ID NO: 9) and s-KL-Rv:5’- CACTGGGTTTTGTCAAAGGA -3’ (SEQ ID NO: 10)). The expression level of mouse 36B4 was normalized using s-KL(m36b4-Fw(Rplp0):5’-ATGGGTACAAGCGCGTCCTG-3’ (SEQ ID NO: 12), m36b4-Rv(Rplp0):5’-AGCCGCAAATGCAGATGGATC-3’ (SEQ ID NO: 13), and the probe 5-HEX-3’BHQ-1 m36B4-Pr(Rplp0):5’-TGTGGAGACTGAGTACACCTTCCCA-3’ (SEQ ID NO: 14).

[0159] The thermal cycle conditions were polymerase activation at 95°C for 5 minutes, 45 cycles of 15 seconds at 95°C, 30 seconds at 60°C, 30 seconds at 72°C, and 5 seconds at 65°C to 95°C (increasing 0.5°C every 5 seconds). Fluorescence detection was performed at the end of PCR elongation, and the melting curve was analyzed by monitoring the fluorescence of the Taqman probe.

[0160] Spinal cord organotypic culture. On the 8th day after birth, Sprague-Dawley rats were euthanized, the spinal cord was aseptically collected, and placed in ice-cold high-glucose Gey's balanced salt solution from which the meninges had been removed. The spinal cord was transversely cut into 350-mm thick sections using a chopper. The L4-L5 lumbar sections were transferred to a Millicell-CM porous membrane in a plate containing incubation medium.

[0161] After axotomy performed during the culture treatment, many neurons die naturally and glial cells show strong reactivity, so the culture needs to be left for one week to stabilize. Then, 10 8 IU of Ad5-CMV-s-KL or Ad5-CMV-Null, or 1 microliter drops containing medium as a control were added onto each slice. In an alternative assay, adeno-associated virus, namely, AAV9-CMV-s-KL, AAV9-CMV-Null vectors, or medium were used as a control. To allow expression of the transgene, the slices were maintained for an additional week. At 14 days in vitro (DIV), glutamate (50 μM) in Locke's solution was added to the plates for 30 minutes to induce acute excitotoxicity and then this was replaced with medium. At 19 DIV, the slices were harvested and fixed with 4% paraformaldehyde for histological staining.

[0162] Electrophysiological examination. Motor nerve conduction velocity tests were performed every 4 weeks from 8 to 16 weeks of age. The sciatic nerve was stimulated by a single pulse (Grass S88 stimulator) delivered through a needle electrode placed at the sciatic notch. The evoked compound muscle action potential (CMAP) was recorded from the gastrocnemius muscle (GM) and the plantaris lateralis muscle (PL) using a micro-needle electrode. To measure the amplitude and latency of the CMAP, the electromyogram signal was amplified and displayed on a digital oscilloscope (Tektronix 450S).

[0163] Motor evoked potentials were evaluated to assess the central motor pathway. Electrical stimulation of maximum supramaximal intensity was delivered with a needle electrode placed subcutaneously on the skull covering the sensorimotor cortex, and MEP was recorded from the GM muscle using a micro-needle electrode.

[0164] Spontaneous movement test. Motor coordination, intensity, and balance were evaluated in treated and untreated SOD1 G93AEvaluated by the rotarod test in animals. Each mouse was placed three times into a rotarod that rotated at a constant speed of 14 rpm for the longest time until a fall was recorded. The maximum time was set at 180 seconds. The tests were performed bi-weekly on mice aged 8 - 20 weeks. The onset of clinical disease for each mouse was determined as the first week when the maintenance time was lower than 180 seconds.

[0165] Histology. At 16 weeks of age, mice were transcardially perfused with 4% paraformaldehyde in PBS, and the lumbar spinal cord, tibial nerve, and gastrocnemius muscle were harvested.

[0166] For NMJ labeling, GM muscle was cryopreserved with 30% sucrose in PBS, and longitudinal sections of 60 μm were serially cut with a cryotome and 10 consecutive sections were collected. The sections were blocked with PBS - Triton - FBS and incubated with primary antibodies, anti - synaptophysin (1:500, AB130436, Abcam), anti - neurofilament 200 (NF200, 1:1000, AB5539, Millipore), and anti - S100β (1:1, 22520, Immunostar) for 48 hours at 4°C. After washing, the sections were incubated overnight with Alexa 594 - conjugated secondary antibody (1:200; Life Science) and Alexa 488 - conjugated α - bungarotoxin (1:200, B - 13422, Life Technologies). The percentage of innervated endplates was determined by classifying each endplate as either occupied (when a presynaptic terminal is over the endplate) or empty (no presynaptic label in contact with the endplate). At least 4 fields with a total of >100 endplates per muscle were analyzed. For sprouting, the number of sprouts per endplate was counted as the neurofilament - positive projections from presynaptic terminals or terminal nodes on confocal z - projections. For analysis, we considered both the total number of sprouts and the percentage of occupied endplates innervated by sprouting.

[0167] Data analysis. All experiments were performed by blinded researchers with regard to different treatments of each mouse group and random allocation of animals in groups taking into account body weight and littermates. Data are presented as mean ± SEM. Results of electrophysiological and spontaneous locomotion tests were statistically analyzed using one-way or repeated measures analysis of variance (ANOVA) with Tukey's post hoc test. For MEP electrophysiological results, Student's t-test was applied. For clinical disease onset, log-rank (Mantel-Cox) test was applied. Histological and molecular biology data were analyzed using one-way or two-way ANOVA with t-Student, Tukey or Holm-Sidak's post hoc test.

[0168] Results 1 - Crotau downregulates SOD1 G93 in a mouse model When mRNA extraction of several tissues of the mouse model was performed, mRNA expression of secreted crotau (s-KL) decreased in the motor cortex, spinal cord, gastrocnemius, and soleus muscles of SOD1 G93A and was observed to be significant in the analyzed muscles. Expression ratio of s-KL to wild type (WT) at 16 weeks, the end stage of the disease (n = 7 SOD1, 3 - 10 WT mice per group, ***p < 0.001, **p < 0.01, *p < 0.05). Data are shown in Fig. 1 and s-KL mRNA expression (fold change) is shown as mean ± SEM of several studied tissues.

[0169] 2 - In vitro assay. Crotau protects spinal cord motor neurons from excitotoxicity in organotypic cultures Rat spinal cord organotypic slices were treated with AAV9 encoding s-KL (SEQ ID NO: 15) or a null sequence (SEQ ID NO: 16) and exposed to glutamate. Data are shown as fluorescence microscopy images in Figure 2A and bar graphs in Figure 2B, with the second bar in each set corresponding to glutamate-induced excitotoxicity (GLUT+). Overexpression of s-KL preserves neurons located in the ventral horn (VH) of the spinal cord from glutamate-induced excitotoxicity compared to untreated controls or AAV9-Null. All data are mean ± SEM (n = 6 per group, ***p < 0.001, **p < 0.01, *p < 0.05).

[0170] Tables C and D list the specific fragment sequences in each of SEQ ID NO: 15 and SEQ ID NO: 16, respectively.

[0171] Table C. Sequence legend for SEQ ID NO: 15

Table 3

[0172] Table D. Sequence legend for SEQ ID NO: 16

Table 4

[0173] Equivalent assays with equivalent results were performed on rat spinal cord organotypic slices, which were treated with an adenoviral vector encoding sKL or a null sequence and exposed to glutamate (GLUT +) that induces a cytotoxic effect. Data from this alternative assay are not shown, but overexpression of s-KL-mediated neuron survival in the ventral horn (VH) of the spinal cord from glutamate-induced excitotoxicity is shown compared to untreated controls (GLUT-) or Ad-Null that showed high mortality.

[0174] 3 - Gene therapy strategy SOD1 female mice were at 6 weeks of age at 1.8×10 14 vg / kg or 3×10 14The animals were intravenously treated with the AAV-hDesmin-s-KL-WPRE vector (n = 15) at vg / kg. WT (n = 10) and SOD1 female mice (n = 15) were treated at the same dose with AAV-hDesmin-Null-WPRE as a control. The human desmin promoter was used to restrict expression to skeletal muscle. The WPRE sequence was used to stabilize the mRNA. The animals were followed weekly at 8, 12, and 16 weeks by rotarod and grip strength tests, as well as nerve conduction tests. Tissues were harvested at the end stage of the disease and processed for histological and expression analysis.

[0175] A schematic diagram of an embodiment of the gene therapy strategy is shown in Figure 3.

[0176] 4 - Improvement of compound muscle action potential (CMAP) After gene therapy according to the previous section, motor nerve conduction velocity tests were performed using two needle electrodes placed at the sciatic notch and stimulating the sciatic nerve with single pulses of 20 μs duration. Compound muscle action potentials (CMAPs) were recorded from the tibialis anterior (TA) and plantaris lateralis (PL) muscles using micro-needle electrodes at 8, 12, and 16 weeks of age.

[0177] Treatment promoted significant preservation of CMAP amplitudes in the tibialis anterior and plantaris lateralis muscles, indicating that s-KL overexpression promotes improvement of motor function in SOD1 G93A female mice.

[0178] These data indicate that motor function is maintained because motor neurons and muscle cells communicate well.

[0179] For CMAP values (amplitude in mV), the plantaris muscle, PL, is shown in Figure 4A, the tibialis anterior muscle, TA, is shown in Figure 4B, for wild type (WT), SOD1 mock, SOD1 s-KL low dose, and SOD1 s-KL high dose (***p < 0.001, **p < 0.01, *p < 0.05 SOD1 mock vs SOD1 high dose, mean ± SEM).

[0180] 5 - Increase in motor evoked potential amplitude (MEP) To assess the central corticospinal descending pathway, motor evoked potentials (MEPs) were recorded from the TA muscle. The motor cortex was electrically stimulated with pulses of 0.1 ms duration and supramaximal intensity, delivered with needle electrodes placed subcutaneously above the skull. Gene therapy increased the amplitude of MEPs, indicating enhanced connectivity between upper and lower motor neurons (***p<0.001, **p<0.01, *p<0.05, mean±SEM when compared to SOD1 mock). Data are shown in Figure 5, where the first bar of the set is for the assay in SOD1 mock, the second bar is for SOD1 s-KL low dose, and the third bar is for SOD1 s-KL high dose.

[0181] 6-Klotho is SOD1 G93A Enhance mouse motor function The rotarod test was performed to evaluate the motor coordination and balance of the animals. Mice were placed on the rotating rod at a constant speed of 14 rpm, and 180 seconds was selected as the cut-off time. To test the grip strength of the forelimbs and hindlimbs, the mice were placed on a metal grid, allowing the four paws to grasp the bar while being pulled away by the tail. During the test, the instrument recorded the peak pulling force before release.

[0182] Overexpression and secretion of s-KL by muscle enhances SOD1 activity on the rotarod compared to untreated controls G93A Improved the motor skills of the mice. The strength of mice treated with the high dose was also improved at the end stage, as assessed by the grip strength test (***p<0.001, **p<0.01, *p<0.05, SOD1 high dose compared to SOD1 mock, mean ± SEM). Data are shown in Figure 6A-6B for the rotarod as time (s) in Figure 6A and for grip strength as force (g) in Figure 6B.

[0183] 7- Delayed onset of clinical disease The onset of clinical disease in each mouse of the previous section was determined as the first week in which the animal could not be maintained on the rotarod for 180 seconds. Treatment was able to significantly delay the onset of disease, and at the 16-week time point, only 35% of the mice treated at high dose (treated with the AAV9-s-KL vector, SEQ ID NO: 15) showed motor impairment compared to the mock-treated mice (treated with the AAV9-null vector, SEQ ID NO: 16). The data shown in Figure 7 indicate the probability of onset in each mouse type.

[0184] 8-s-KL preserves the neuromuscular junction (NMJ) and muscle mass Longitudinal sections of the gastrocnemius muscle were labeled for neurofilament 200 (NF200), anti-synaptophysin, and α-bungarotoxin (BTX). Endplates were classified as occupied (when the presynaptic terminal overlapped or contacted the endplate) or empty (not pre-synaptically labeled while in contact with the endplate). Secretion of s-KL by skeletal muscle significantly increased the proportion of NMJs occupied by presynaptic terminals and reduced muscle wasting as evidenced by a larger muscle mass (***p < 0.001, **p < 0.01, *p < 0.05, SOD1 mock vs. SOD1 high dose, data are shown as mean ± SEM). The data are shown in Figures 8A-8C. Figure 8A, a fluorescence microscopy image, shows sections labeled for NF200 (dark gray) and sections labeled for BTX (light gray). Figure 8B shows the proportion of occupied endplates in WT mice, as well as in mock (SOD1 mock) and treated (SOD1 s-KL). Figure 8C shows the muscle mass (mg / g) per body weight in WT mice, as well as in mock (SOD1 mock) and treated (SOD1 s-KL).

[0185] 9-Secretion of s-KL by muscle protects spinal motor neurons At 16 weeks of age, mice were transcardially perfused with 4% paraformaldehyde and the lumbar spinal cord was harvested. For spinal cord MN evaluation, the spinal cord was fixed for 4 hours, cryopreserved with 30% sucrose in PBS, and serially sectioned transversely at 20 μm using a cryotome. One out of every two slides from the lumbar spinal cord region containing L4-L6 of each animal was stained with cresyl violet. Motor neurons were identified by their localization in the anterior horn and according to strict size and morphological criteria.

[0186] Skeletal muscle-secreted s-KL protected motor neurons in the lumbar spinal cord, which is the region that undergoes more motor neuron death in the SOD1 mouse model (n = 8 / group, two-way ANOVA test, ***p < 0.001, data are presented as mean ± SEM). The data are shown in Figures 9A-9B. Figure 9A, which is a white light microscopy image, shows a section labeled with cresyl violet (dark gray), which stains neurons and Nissl substance in the cell nucleus, thereby revealing the neuronal structure. Figure 9B quantifies the neuronal structure in WT mice (WT mock), as well as mock (SOD1 mock) and treated (SOD1 sKL) mice.

[0187] 10-Croto protects the spinal cord from neuroinflammatory damage in ALS Delivery of s-KL by muscle, as evaluated by Iba1 fluorescence, in treated SOD1 G93AMicroglial reactivity in the lumbar spinal cord of mice was reduced. Astrocytosis was analyzed by GFAP and vimentin labeling and corrected to be near WT levels (n = 8 / group, two-way ANOVA test, **p < 0.01, *p < 0.05). Data are presented as mean ± SEM. The data are shown in Figures 10A - 10D. Figure 10A, a fluorescence microscopy image, shows sections labeled for lba1 (upper panel), GFAP (middle panel), and vimentin (lower panel). Figures 10B - 10D show the quantification of the staining in Figure 10A for WT mice (WT mock) and mock (SOD1 mock) and treatment (SOD1 sKL), showing a decrease in the staining of Iba1, GFAP, and vimentin after s-KL treatment. Figure 10B shows the integrated density (arbitrary units) of the lba1-labeled image. Figure 10C shows the integrated density (arbitrary units) of the GFAP-labeled image. Figure 10D shows the integrated density (arbitrary units) of the vimentin-labeled image.

[0188] Gene therapy strategy using the 11-AAVmyo vector AAVmyo is an AAV serotype with striated muscle tropism that has excellent transduction efficiency in mice and non-human primates (Tabebordbar et al., Cell 184(19):4919 - 4938(2021)). The AAVmyo serotype was tested for s-KL gene therapy to reduce the dose of the administered AAV vector and avoid possible side effects. A schematic diagram of one embodiment of the gene therapy strategy is shown in Figure 11.

[0189] Six-week-old SOD1 mice were given 2×10 12 vg / kg (n = 6, AAVmyo-s-KL low dose) or 8×10 12They were intravenously treated with the AAVmyo-hDesmin-sKL-WPRE expression vector at vg / kg (n = 8, high dose of AAVmyo-s-KL). The high dose of AAVmyo-s-KL and the low dose of AAVmyo-sKL were 150-fold and 37.5-fold lower, respectively, compared to the doses used in the gene expression experiment using the AAV8 expression vector in Figure 3. WT (n = 14) and SOD1 female mice (n = 18) were treated at the same dose with AAVmyo-hDesmin-Null-WPRE as a control. The animals were followed as in previous studies and euthanized at 16 weeks of age for tissue collection.

[0190] 12 - AAVmyo-Des-sKL improves compound muscle action potential (CMAP) Treatment with higher doses of AAVmyo-Des-sKL G93A In treated SOD1 G93A mice showed significant preservation of the amplitude of compound muscle action potential (CMAP) of the plantaris (PL) and gastrocnemius (GM) muscles from week 12 and week 8, respectively, compared to control SOD1

[0191] The data are shown in Figures 11A - 11B. For CMAP values (amplitude in mV), the plantaris muscle, PL is shown in Figure 11A, the tibialis anterior muscle, TA is shown in Figure 11B, for wild WT mock, SOD1 mock, SOD1 Myo-AAV-s-KL low dose, SOD1 Myo-AAV-s-KL high dose (*p < 0.05 SOD1 mock vs SOD1 Myo-AAV-s-KL, mean ± SEM).

[0192] 13 - AAVmyo-Des-sKL increases the amplitude of motor-evoked potential (MEP) The amplitude of the motor-evoked potential G93AIt was also higher in mice and showed higher preservation of the central motor pathway. Compared with the AAV8-DessKL treatment, the AAVmyo-Des-sKL treatment maintained the MEP amplitude more significantly (two-way analysis of variance test, **p < 0.01, *p < 0.05; SOD1 mock vs. SOD1 sKL high dose, data are presented as mean ± SEM). The data are shown in Figure 12, where the first bar of the set is for the assay in SOD1 mock, the second bar is for the low dose of SOD1 AAVmyo-s-KL, and the third bar is for the high dose of SOD1 AAVmyo-s-KL.

[0193] 14-Croto is SOD1 G93A Enhance the motor function of mice In SOD1 mice treated with AAVmyo-Des-sKL, a slower progression of spontaneous motor decline was observed. The strength of the animals was also preserved, especially in the high-dose treatment group, and the SOD1 mice performed better than those injected with AAV8-DessKL (two-way analysis of variance test, ***p < 0.001, **p < 0.01, *p < 0.05, *SOD1 mock vs. SOD1 sKL high dose, #SOD1 mock vs. WT mock, mean ± SEM). The data for the rotarod as time (s) are shown in Figure 13A, and the data for the grip strength as force (g) are shown in Figure 13B for Figures 13A - 13B.

[0194] 15 - Delay in clinical disease onset Clinical disease onset was significantly delayed in both groups of SOD1 mice treated with AAVmyo-Des-sKL, similar to those treated with high-dose AAV8-Des-sKL (log-rank Mantel-Cox test). The data are shown in Figure 14, which shows the probability of onset for each mouse type.

[0195] Other publications incorporated herein by reference Zeldich et al., "Klotho Is Neuroprotective in the Superoxide Dismutase (SOD1G93A) Mouse Model of ALS" J. Mol. Neurosci. 69(2):264-285 (2019) Minamizaki et al., "Soluble Klotho causes hypomineralization in Klotho-deficient mice" J. Endocrinol. 237(3):285-300(2018) WO2017085317 (Universitat Autonoma de Barcelona et al.)

[0196] All patent publications and non-patent publications indicate the state of the art relevant to the present disclosure. All of these publications (including any specific portions thereof that are referenced) are hereby incorporated by reference into this specification to the same extent as if each individual publication had been specifically and individually indicated to be incorporated by reference.

[0197] Although the invention herein has been described with reference to specific embodiments, it should be understood that these embodiments are merely illustrative of the principles and applications of the invention. Accordingly, it is to be understood that numerous modifications may be made to the exemplary embodiments, and other arrangements may be devised without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. A pharmaceutical composition for the treatment of a motor disorder in a subject requiring the same, wherein the pharmaceutical composition is any one of (i) to (iii): (i) A plasmid comprising a gene construct comprising a nucleic acid comprising a first promoter operably ligated to a nucleic acid sequence encoding mammalian s-KL or a functional variant thereof, wherein the first promoter is a muscle cell-specific promoter, and an initiation sequence operably ligated to the first promoter; (ii) Expression vectors containing the following: (a) The gene construct or plasmid, wherein the expression vector may or may not be muscle cell-targeting; or (b) A nucleic acid construct comprising a first promoter that functions in muscle cells, nerve cells, or induced pluripotent stem cells (iPSCs) operably linked to a nucleic acid sequence encoding mammalian s-KL or a functional variant thereof, wherein the expression vector is muscle cell-targeted; or (iii) Isolated cells comprising the gene construct or plasmid, wherein the isolated cells are human muscle cells, human nerve cells, or human induced pluripotent stem cells (iPSCs) that can differentiate into muscle cells or nerve cells; A pharmaceutical composition comprising and a pharmaceutically acceptable carrier.

2. The mammalian s-KL encoded by the nucleic acid sequence is a human s-KL having the amino acid sequence of SEQ ID NO: 1, and optionally the mammalian s-KL nucleic acid sequence is SEQ ID NO: 3; or The pharmaceutical composition according to claim 1, wherein the mammalian s-KL encoded by the nucleic acid sequence is a mouse s-KL having the amino acid sequence of SEQ ID NO: 2, and optionally the mammalian s-KL nucleic acid sequence is SEQ ID NO:

4.

3. The functional variant has at least 85% amino acid sequence identity with SEQ ID NO: 1; or The functional variant has at least 88% amino acid sequence identity with SEQ ID NO: 1; or The functional variant has at least 95% amino acid sequence identity with SEQ ID NO: 1; or The pharmaceutical composition according to claim 2, wherein the functional variant has at least 98% amino acid sequence identity with SEQ ID NO:

1.

4. The pharmaceutical composition according to claim 1, wherein the first promoter is a mammalian desmin promoter, optionally the mammalian desmin promoter is a human desmin promoter, and optionally the human desmin promoter has the nucleic acid sequence of SEQ ID NO:

5.

5. The pharmaceutical composition according to claim 1, further comprising a second promoter operably ligated to the nucleic acid sequence encoding the mammalian s-KL or a functional variant thereof, wherein the first and second promoters are different.

6. The second promoter is a muscle cell-specific promoter, or the second promoter is a neuron cell-specific promoter; or The second promoter is a constitutive promoter; optionally, the constitutive promoter is a cytomegalovirus (CMV) promoter; or The pharmaceutical composition according to claim 5, wherein the second promoter is an inductive promoter; optionally, the inductive promoter is a zinc-driven metallothionein promoter.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the expression vector is a viral expression vector; optionally, the expression vector is a myocyte-targeting serotype adeno-associated virus (AAV) vector, or the AAV vector is a neuron-targeting serotype.

8. The AAV vector is a serotype having myocyte-targeting properties, and the AAV vector is an AAV1, AAV8, AAV9, or AAVmyo vector, or the AAV vector comprises an AAV capsid polypeptide comprising one of the amino acid sequences of SEQ ID NOs. 17 to 29, or any of those amino acid sequences; or The pharmaceutical composition according to claim 7, wherein the AAV vector is an AAVmyo vector.

9. The AAV vector comprises an AAV capsid polypeptide comprising any one amino acid sequence from SEQ ID NOs. 17 to 29, or consisting of any of those amino acid sequences; optionally, the AAV vector comprises an AAV capsid polypeptide comprising the amino acid sequence of SEQ ID NOs. 17 or 26, or consisting of any of those amino acid sequences; or The pharmaceutical composition according to claim 8, wherein the AAV vector is an AAV9 vector.

10. The pharmaceutical composition according to claim 7, wherein the AAV vector is a serotype having neuronal tropism, and the AAV vector is an AAV1 vector, an AAV8 vector, or an AAV9 vector.

11. The expression vector further comprises at least one non-coding regulatory element; Optionally, the regulatory element is one or more of the following: a polyA sequence, a protein translation initiation site consensus nucleic acid sequence, a post-transcriptional regulatory element nucleic acid sequence, a 5' and 3' terminal inverted repeat nucleic acid sequence, or an intron; Optionally, the polyA sequence is an SV40 polyA sequence; The pharmaceutical composition according to claim 1, wherein the post-transcriptional regulatory element nucleic acid sequence is optionally a hepatitis B virus post-transcriptional regulatory element (HPRE) or a woodchuck hepatitis post-transcriptional regulatory element (WPRE).

12. The cell is a nerve cell; optionally, the cell is a motor neuron; or The pharmaceutical composition according to claim 1, wherein the cells are muscle cells; optionally, the cells are skeletal muscle cells or striated muscle cells.

13. The pharmaceutical composition according to claim 1, wherein the subject has a disease or disorder characterized by or exhibiting symptoms of motor impairment; optionally, the disease or disorder is a neuromuscular disease or disorder; optionally, the disease or disorder is a motor neuron disease (MND).

14. The neuromuscular disease is amyotrophic lateral sclerosis (ALS), Charcot-Marie-Tooth disease, multiple sclerosis, muscular dystrophy, Duchenne and Becker muscular dystrophy, myasthenia gravis, muscle disease, myositis, peripheral neuropathy, neurogenic myotonia, Lambert-Eaton myasthenic syndrome, Friedreich ataxia, traumatic nerve injury, diabetic neuropathy, motor impairment, spinal muscular atrophy (SMA), spinal cord injury, peripheral nerve injury, traumatic nerve injury, muscle metabolic disease, hereditary myopia Chi, toxic neuropathy, autoimmune peripheral polyneuropathy, acute inflammatory demyelinating polyneuropathy (AIDP), chronic inflammatory demyelinating polyneuropathy (CIDP), mononeuropathy with vasculitis, paraneuropathy, idiopathic ganglionitis, amyotrophic lateral sclerosis, multifocal motor conduction Locke neuropathy, lower motor neuron syndrome, muscular atrophy, drug-induced myopathy, sarcopenia, cachexia, type II fibrous atrophy, age-related muscular atrophy, or acquired autoimmune primary myopathy; or The pharmaceutical composition according to claim 13, wherein the MND is ALS, progressive bulbar palsy (PBP), pseudobulbar palsy, progressive muscular atrophy (PMA), primary lateral sclerosis (PLS), smaorrhea (SMA), or monolimb muscular atrophy (MMA).

15. The pharmaceutical composition according to claim 14, wherein the neuromuscular disease is amyotrophic lateral sclerosis (ALS); and optionally, the ALS is idiopathic ALS (sALS) or familial ALS (fALS).

16. The pharmaceutical composition according to claim 1, wherein the subject requiring it does not exhibit motor impairment but has a predisposition to motor impairment.

17. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is administered parenterally; or the pharmaceutical composition is administered intravenously, intramuscularly, intracranially, or subarachnoidally.

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