Treatment of neuromuscular diseases by gene therapy expressing KLOTHO proteins

Abstract

The present disclosure provides: a gene construct comprising a nucleic acid sequence encoding a mammalian s-KL, which nucleic acid sequence is operatively linked to a muscle cell specific promoter, the present invention relates to gene constructs for use in the treatment of dyskinesia, which may be expressed, for example, in neuromuscular disorders or diseases, using viral and non-viral vectors having myoblast and motoneuron tropies, containing gene constructs, which are useful in the treatment of dyskinesia, which may be expressed, for example, in neuromuscular disorders or diseases, the gene construct contains a nucleic acid sequence for coding mammalian s-KL, and the nucleic acid sequence is operatively connected with a promoter such as a muscle cell specific promoter and is delivered in a pharmaceutical composition containing an expression vector; an isolated cell containing the expression vector; and methods of treating dyskinesia and motor neuron diseases.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63 / 330,684, filed on April 13, 2022, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure relates to the field of medicine, and more particularly, to medical methods for treating neuromuscular disorders or diseases characterized by movement disorders and motor neuron diseases such as amyotrophic lateral sclerosis.

[0004] Sequence Listing

[0005] This application contains a sequence listing that has been submitted electronically in ASCHII format and is hereby incorporated in its entirety. The XML copy created on April 11, 2023 is named 58578-001PCT.xml and is 162KB in size. Background Art

[0006] According to the International Neuromodulation Society, movement disorders involve partial or complete loss of function of a body part (usually one or more limbs). Diseases that cause movement disorders may cause muscle weakness, poor endurance (i.e., fatigue), loss of muscle control, or complete paralysis. Movement disorders are the leading cause of physical disability. They are primarily caused by peripheral problems affecting muscles, problems in the central nervous system that affect muscle output, and sensory problems that affect muscles, movement, and balance. Movement disorders are often evident in neuromuscular diseases, neurological conditions, and motor neuron diseases, but can also be caused by cancer or even traumatic injuries to the central and peripheral nervous systems.

[0007] Neuromuscular disease is any disease that affects the motor neurons in 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), ultimately affecting the subject's ability to move. Damage to motor neurons in the spinal cord, CNS, PNS, neuromuscular junction or skeletal muscle can cause muscle atrophy and weakness. Sensory problems may also occur. Neuromuscular diseases can be acquired or hereditary. Mutations in more than 500 genes have been shown to be the cause of neuromuscular diseases. Other causes include nerve or muscle degeneration, autoimmunity, toxins, drugs, malnutrition, metabolic disorders, hormone imbalances, infections, nerve compression / entrapment, including blood supply and trauma.

[0008] 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-Eatondisease, 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 (MNDs), a group of rare neurodegenerative disorders that selectively affect motor neurons, the cells that control the body's voluntary muscles. Examples of MNDs 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 muscular atrophy (MMA), as well as some rare variants that resemble ALS.

[0009] Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder of the motor system and the most common form of motor neuron disease (MND), with an incidence of 1-5 cases per 100,000 people per year. Patients with ALS suffer from progressive muscle weakness and paralysis, and usually die within 2-5 years after diagnosis, mainly due to respiratory failure. Any skeletal muscle may be affected, which can cause patients to have a variety of different clinical symptoms. These symptoms range from spasticity to fasciculations, obvious weakness and muscle atrophy, hyperreflexia and spasticity, and in some cases, mood and memory function will also change. Sporadic ALS (sALS) accounts for the majority of cases, with a peak age of onset of about 60 years old. About 5-10% of cases are familial (fALS) caused by genetic mutations (mostly autosomal dominant), and the age of onset is ten years earlier than sporadic cases. Cases of patients under 25 years old are designated as juvenile-onset ALS (jALS), and are usually autosomal recessive. Some of the most common genetic causes of fALS are mutations in Cu / Zn superoxide dismutase 1 (SOD1), TAR-DNA binding protein (TDP-43), fusions in sarcomas (FUS), and hexanucleotide repeat expansions in chromosome 9 open reading frame 72 (C9orf72). The neuropathological hallmark of ALS is that it affects both upper and lower motor neurons (MNs), distinguishing it from other MNDs. Neurodegeneration occurs in the corticospinal and corticobulbar tracts, with loss of large pyramidal neurons in the primary motor cortex as well as the anterior horns of the brainstem and spinal cord.

[0010] Denervation of the endplate and axonal retraction are thought to lead to MN death and subsequent muscle atrophy in a ‘death backwards’ mode. A ‘death forwards’ process is also under debate, in which the primary injury occurs in either the superior or inferior MNs and degeneration extends anterogradely to descending axonal projections.

[0011] Unfortunately, as with other neuromuscular diseases, especially motor neuron disease, there is no effective treatment for ALS to date. Despite decades of intensive research and clinical trials, most promising preclinical therapies have failed to translate into successful human trials. ) and Edaravone ) are the only drugs approved for the treatment of ALS. Both extend survival by only a few months and slightly improve motor function, so symptomatic and palliative measures (including feeding and respiratory support) are the mainstay of patient management. Interestingly, most of the compounds studied interfere with a single mechanism involved in MN death. Therefore, one of the main difficulties in developing effective treatments for ALS lies in the multiple events that lead to MN death. Given the multifactorial mechanisms of ALS pathogenesis, the use of combination or multi-target therapies that act on several mechanisms simultaneously and target different cell types may enhance therapeutic efficacy and maximize translational effects.

[0012] Therefore, despite the efforts made to date, there remains a need for effective and safe treatments for movement disorders of different etiologies. Summary of the invention

[0013] The present disclosure relates to a novel therapy for preventing and / or treating motor impairment and other movement disorders in neuromuscular diseases based on the administration of certain variants of mammalian klotho protein, in particular alternative RNA splice variants of Klotho (s-KL). To date, there is no evidence that certain klotho variants can be safely administered to muscle cells (in protein form or as gene therapy) to treat movement disorders.

[0014] On the one hand, 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 a 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 neuronal 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 a zinc driven metallothionein promoter.

[0015] In another aspect, the present disclosure provides a plasmid comprising a gene construct comprising (e.g., integrated or cloned therein) 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 myocyte-specific promoter, and a start sequence operably linked to the first promoter. Although the plasmid itself can function as a vector, it can be used to prepare more complex expression vectors, such as AAV and lentiviral vectors. The plasmid can also be used to obtain mRNA after linearization of the plasmid and appropriate in vitro transcription, which can be further encapsulated or complexed with a cationic lipid if desired.

[0016] On the other hand, the present disclosure provides an expression vector comprising (e.g., having integrated or cloned therein) a gene construct or plasmid containing (a) a first myocyte-specific 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 myocyte tropism; or (b) a first promoter that functions in myocytes, neuronal cells, or induced pluripotent stem cells (iPSCs), the first promoter being operably linked to a nucleic acid sequence encoding mammalian s-KL or a functional variant thereof, wherein the expression vector has myocyte tropism. In some embodiments, the promoter is a myocyte-specific promoter, and the expression vector has a neuronal cell tropism for preferentially targeting the vector to neuronal 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 an adeno-associated virus (AAV) vector of a serotype having myocyte tropism; or the expression vector is an AAV vector of a serotype having neuronal cell tropism.

[0017] In some embodiments, the s-KL polypeptide comprises or consists of SEQ ID NO: 1, which is the 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 to SEQ ID NO: 1. In some embodiments, the polypeptide has a sequence at least 90% identical to SEQ ID NO: 1. In some embodiments, the polypeptide has a sequence at least 95% identical to SEQ ID NO: 1. In some embodiments, the polypeptide has a sequence at least 98% identical to SEQ ID NO: 1. In some embodiments, the polypeptide has a sequence at least 99% identical to SEQ ID NO: 1. In other embodiments, the polypeptide has a sequence of SEQ ID NO: 2, which is murine s-KL.

[0018] In some embodiments, the expression vector is a viral vector. In some embodiments, the viral vector is an adeno-associated virus of a serotype, and in some embodiments, the serotype is any one of the following: AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9p31, AAVrh10, PHPeB, 9P31, AAVrh74, and AAV Moyo.

[0019] In some embodiments, the AAV vector contains an adeno-associated virus capsid polypeptide having an amino acid sequence of any one of SEQ ID NOs: 17-29. In some embodiments, the AAV vector contains an adeno-associated virus capsid polypeptide having an amino acid sequence of SEQ ID NOs: 17 or 26.

[0020] In some embodiments, the viral vector is an AAV vector of a serotype having neuronal cell tropism, and in some embodiments, the AAV vector is any one of AAV1, AAV8, or AAV9.

[0021] In some embodiments, the carrier is a lipid-based carrier. In some embodiments, the lipid-based carrier is a lipid nanoparticle (LNP) or a liposome.

[0022] 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.

[0023] On the other hand, the present disclosure provides a kind of cell of separation, it contains the gene construct or plasmid as disclosed herein, wherein the cell of separation is human muscle cell, human neuron cell or human induced pluripotent stem cell (iPSC) (it can be induced in vitro to differentiate into muscle cell or neuron cell, or can be differentiated in vivo).In some embodiments, the cell of separation is neuron cell, for example, motor neuron.In some embodiments, the cell of separation is striated muscle cell or skeletal muscle cell.

[0024] Another aspect of the present disclosure is a method for treating a disease or condition characterized by movement disorders 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 spinal cord injury, peripheral nerve injury or traumatic nerve injury. In some embodiments, the method may require administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of the plasmid or the expression vector containing the gene construct. In some embodiments, the method may require continuous administration (e.g., infusion) of the composition to the subject. In some embodiments, the continuous infusion can be performed over a period of about 1 hour to about 24 hours / day for one or more days. In some embodiments, the subject does not exhibit movement disorder symptoms.

[0025] Another aspect of the present invention is a cell therapy, which includes applying the isolated cells disclosed herein to a subject in need. This method may require an in vitro method, wherein myocytes or neuronal cells are separated from the subject and transformed or transfected with the gene construct, the plasmid or the expression vector, and then applied to the subject. In some embodiments, the myocytes or neuronal cells are enriched before or after conversion. In other embodiments, some pluripotent or stem cell-like cells are transformed or transfected with the expression vector, and induced to differentiate into specific cell types (e.g., myocytes or neurons) in vitro. Once integrated with the expression vector (which may occur before or after differentiation), the cell is applied to the subject. In some embodiments, the method is autologous, in this regard, it may be necessary to separate terminally differentiated cells from the subject, reprogram (i.e., induce) pluripotency in these cells to produce iPSC, integrate the expression vector into the iPSC, differentiate the iPSC into neurons or myocytes, and apply differentiated cells to the subject. In some embodiments, the cells are differentiated into myocytes. In some embodiments, the cells are differentiated into neuronal cells. In some embodiments, iPSC cells are allowed to differentiate after administration to a subject.

[0026] Without intending to be bound by theory, the constructs, methods, etc. disclosed herein can increase mammalian s-KL protein levels in skeletal muscle cells, which can protect the cells from toxic insults and preserve myocyte function and / or motor neuron function, or improve movement disorders associated with diseases and conditions characterized by movement disorders, such as ALS.

[0027] As shown in the working examples herein, the inventors have found that administration of a mammalian secreted RNA splice variant of Klotho (s-KL) protein significantly delayed disease progression in a mouse model of ALS disease. In addition, administration of a mammalian s-KL protein in gene therapy using an AAVmyo myocyte-tropic 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.

[0028] The present disclosure may overcome one or more disadvantages associated with known therapies, such as treatment with Klotho protein. The half-life of Klotho protein in vivo is relatively short, about 7.5 hours, resulting in a one-log reduction approximately every 24 hours. In contrast, treatment according to the present disclosure may have an effect similar to that of administering mammalian s-Klotho protein by continuous infusion. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1Shown is s-KL mRNA expression (fold change in ALS mice compared to WT animals) in several tissues studied, displayed as mean ± SEM (n = 7SOD1, 3-10 WT mice per group, ***p<0.001, **p<0.01, *p<0.05).

[0030] Figure 2A and Figure 2B Shown is the preserved neuronal survival at the ventral horn (VH) of the spinal cord under glutamate-induced excitotoxicity (GLUT+) triggered by induced overexpression of s-KL compared to samples that received GLUT+ excitotoxicity and were transduced with a blank vector (or not transduced). Figure 2A Fluorescence microscopy images of Figure 2B The bar graph in is the second bar of glutamate-induced excitotoxicity (GLUT+) in each group. All data are mean ± SEM (n = 6 per group, ***p<0.001, **p<0.01, *p<0.05). SMI 32 is the motor neuron cell recognized by SMI32 antibody.

[0031] Figure 3 Schematic representation of the gene therapy strategy for systematic treatment of mice with a myocyte-tropic vector and a gene construct containing a promoter specific for striated (skeletal) muscle cells (SOD1 model).

[0032] Figure 4A and Figure 4B is a double line graph showing the plantar muscles of wild type (WT), SOD1 mimetic (AAV-blank), SOD1s-KL low dose (AAV-s-KL), SOD1 s-KL high dose (AAV-s-KL) ( Figure 4A ) or tibialis anterior ( Figure 4B ) of the compound muscle action potential (CMAP) value (amplitude, in mV).

[0033] Figure 5 Is a bar graph showing motor evoked potentials (MEP) recorded from the tibialis anterior (TA). The first bar in the group represents the assay in SOD1 mimics, the second represents SOD1 s-KL low dose, and the third is SOD1 s-KL high dose. (***p<0.001, **p<0.01, *p<0.05, relative to SOD1 mimics, mean ± SEM).

[0034] Fig. 6A and Figure 6B As shown previously FIG. 4A to FIG. 5 The rotarod fall observed in animals was reported in Fig. 6A ) time (time, seconds) and grip strength ( Figure 6B Line graph of force (g) in (***p<0.001, **p<0.01, *p<0.05, SOD1 high dose vs. SOD1 mimetic, mean ± SEM).

[0035] Figure 7 Shown as before FIG. 4A to FIG. 5 Clinical disease onset in animals as disclosed in .

[0036] FIG. 8A to FIG. 8C The neuromuscular junction (NMJ) is shown to visualize the occupancy of the presynaptic terminals. Fig. 8A In Figure 2, a fluorescence microscopy image of a longitudinal section of gastrocnemius muscle labeled with neurofilament 200 (NF200, dark grey in the image), anti-synaptophysin, and α-bungarotoxin (BTX, light grey in the image). Figure 8B In the Figure, the percentage of endplates occupied in WT mice as well as mock (SOD1 mimetic) and treated (SOD1s-KL) is indicated. Figure 8C In the figure, muscle mass / body weight (mg / g) of WT mice as well as mock (SOD1 mimetic) and treated (SOD1s-KL) are indicated. (***p<0.001, **p<0.01, *p<0.05, SOD1 mimetic vs. SOD1 high dose, data presented as mean ± SEM).

[0037] FIG. 9A to FIG. 9B Shown is preserved motor neuron survival at the lumbar spinal cord in SOD1 mice triggered by induced overexpression of s-KL compared to samples from wild-type and SOD1 mice transduced with empty vector. Fig.9A is a white light microscopic image, and Fig. 9B Is to show or quantify Fig.9A Bar graph showing the number of motor neurons shown in (all data are mean ± SEM (n = 8 per group, ***p < 0.001)).

[0038] FIG. 10A to FIG. 10D Shown is reduced microglial activity in the lumbar spinal cord in SOD1 mice triggered by induced overexpression of s-KL compared to samples from wild-type and SOD1 mice transduced with empty vector. Fig. 10A is a fluorescence microscopy image, and FIG. 10B to FIG. 10D is a set of bar graphs showing or quantifying the number of integrated densities (arbitrary units) per image in images stained with Iba1, GFAP, and vimentin (all data are mean ± SEM (n = 8 per group, **p<0.01, *p<0.05)).

[0039] Fig.11A gene therapy strategy that systematically treats mice with an AAV vector with myocyte tropism (AAVmyo) and a gene construct containing a promoter specific for striated (skeletal) muscle cells (SOD1 G93A Schematic view of the model).

[0040] FIG. 12A to FIG. 12B is a set of line graphs showing the plantar muscles ( Fig. 12A ) and tibialis anterior ( Fig. 12B ) of the compound muscle action potential (CMAP) value (amplitude, in mV).

[0041] Fig.13 is a bar graph showing motor evoked potentials (MEPs) recorded from the tibialis anterior (TA). The first bar in the group represents the assay in SOD1 mimetic, the second bar represents SOD1 AAVmyo-s-KL low dose, and the third bar is SOD1 AAVmyo-s-KL high dose (***p<0.001, **p<0.01, *p<0.05, vs. SOD1 mimetic, mean±SEM).

[0042] FIG. 14A to FIG. 14B It is shown as FIG. 12A to FIG. 13 The rotarod falls observed in animals are shown in Fig.14A ) time (time, seconds) and grip strength ( Fig. 14B Line graph of the force (g) in .

[0043] Fig.15 Shows FIG. 12A to FIG. 14B Clinical disease onset in the animals shown. DETAILED DESCRIPTION

[0044] definition

[0045] Unless otherwise stated, all terms used herein in this application should be understood with their common meanings known in the art. Other more specific definitions of certain terms used in this application are described below and are intended to be uniformly applied throughout the entire specification and claims, unless otherwise explicitly stated definitions provide a broader definition.

[0046] As used herein, the indefinite articles "a" and "an" are synonymous with "at least one" or "one or more." Definite articles such as "the" as used herein also include the plural form of the noun unless otherwise stated.

[0047] Throughout the specification and claims, the word "comprise" and its variants are not intended to exclude other technical features, additives, components, elements or steps. In addition, the word "comprise" covers the situation of "consisting of..."

[0048] Gene constructs

[0049] 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 synonymously herein and refer to a nucleic acid sequence encoding a mammalian secreted RNA splice variant of mammalian Klotho (s-KL) or a functional variant thereof, the nucleic acid sequence being operably linked to a muscle cell-specific promoter.

[0050] As used herein, the term "nucleic acid" is also referred to as "nucleic acid sequence" in this article, and refers to the polymer of nucleotides, each of which is an organic molecule composed of nucleosides (nucleobases and pentose) and phosphate. Unless otherwise specified or apparent from the context, the term nucleotide includes nucleosides with ribose sugar (i.e., ribonucleotides forming ribonucleic acid RNA) or 2'-deoxyribose sugar (ie, deoxyribonucleotides forming deoxyribonucleic acid DNA). Nucleotide is used as a monomer unit of nucleic acid polymers or polynucleotides. The four kinds of nucleobases in DNA are guanine (G), adenine (A), cytosine (C) and thymine (T). The four kinds of nucleobases in RNA are guanine (G), adenine (A), cytosine (C) and uracil (U). Nucleic acid is a linear chain of nucleotides (e.g., at least 3 nucleotides), which is chemically bound by a series of ester bonds between the phosphate group of a nucleotide and the hydroxyl group of the sugar in the adjacent nucleotide (i.e., ribose or 2'-deoxyribose).

[0051] As used herein, the term "promoter" refers to a nucleic acid sequence that directly or indirectly regulates the transcription of a corresponding nucleic acid sequence to which it is operably linked, which in the context of the present disclosure is a mammalian s-KL or a functional variant thereof. A promoter may act alone to regulate transcription, or it 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 of the DNA (towards the 5' region of the sense strand). Promoters generally range in length from about 100 to 1000 base pairs. A "constitutive promoter" is a promoter that is active under all conditions in a cell, in contrast to other regulated promoters that are only active in a cell in response to a specific stimulus, such as an "inducible promoter".

[0052] As used herein, the term "muscle" encompasses skeletal muscle, which refers to a volitionally controlled striated muscle type attached to bones, representative examples of which include the diaphragm, biceps, triceps, quadriceps, tibialis medialis, and gastrocnemius.

[0053] As used herein in the context of a promoter, the terms "muscle cell specificity" and "muscle specificity" refer to preferential, selective or predominant expression of mammalian s-KL (or its functional variant) in muscle cells or muscle tissues compared to other (i.e., non-muscle) cells and tissues. In some embodiments, at least 50%, 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% of the expression occurs in muscle cells or tissues. In some embodiments, "muscle cell specificity" means that the expressed mammalian s-KL (or its functional variant) does not substantially leak into other organs or tissues outside of muscle, such as the lungs, liver, brain, kidneys and / or spleen.

[0054] As used herein in the context of a promoter, the term "functions in muscle cells" refers to a promoter that directly or indirectly regulates the transcription of a mammalian s-KL nucleic acid to which it is operably linked; however, it is not necessarily muscle cell specific.

[0055] As used herein, the term "operably linked" is understood to mean that the nucleic acid sequence encoding s-KL or its functional variant is spatially positioned or arranged relative to the promoter in the gene construct of the expression vector to drive the expression of the nucleic acid coding sequence.

[0056] Klotho (KL) is a protein expressed primarily in the kidney and brain, and to a lesser extent in skeletal muscle, lung, bladder, testis, and ovary. Klotho has two different isoforms, one encoding a full-length transmembrane protein of approximately 140 KDa (mKL), which can be processed into a soluble form of approximately 130 KDa (p-KL). The other isoform encodes a secreted but shorter isoform of approximately 63 KDa (s-KL). mKL contains a KL1 domain and a KL2 domain, which can be released by α / β / γ-secretase to produce p-KL or further cleaved to release the p-KL1 domain and the p-KL2 domain (each approximately 70 KDa). s-KL contains a KL1-like domain plus an additional tail (15 amino acids at its C-terminus in mouse s-KL and 16 amino acids at its C-terminus in human s-KL). The two isoforms, m-KL and s-KL, have different expression levels and spatial-temporal expression profiles, suggesting that they have different roles.

[0057] Soluble and secreted klotho are forms that are present in the circulation (e.g., blood) and cerebrospinal fluid (CSF) and function as circulating hormones that exert biological effects on distant organs and multiple systems by binding to cell surface receptors and inhibiting signals such as insulin and insulin-like growth factor 1 (IGF1) signaling. It must be noted that although similar abbreviations (s-KL) are sometimes used in the prior art to designate soluble Klotho, which is a processed version of transmembrane Klotho, and secreted Klotho, which is an RNA splice variant, these two variants exhibit different structures.

[0058] The term "secreted RNA splice variant of mammalian Klotho", abbreviated herein as "s-KL", refers to a protein produced by alternative RNA splicing of a transcript encoding the full-length transmembrane form of the Klotho protein (m-KL), which produces a truncated form of the protein (i.e., s-KL) formed by a KL1-like domain, with an approximate weight of 63 kDa, and a specific secretion signal consisting of a 15 amino acid tail in mice and a 16 amino acid tail in humans, which is not found in the m-KL transcript, and is therefore also referred to as a secreted isoform of klotho, s-KL, or a secreted RNA splice variant of the klotho protein. S-KL is distinct from other forms of soluble klotho, i.e., p-KL, p-KL1, and p-KL2. In the present disclosure, m-KL refers to the full-length transmembrane form; p-KL represents soluble proteolytic klotho (i.e., KL1-KL2), which is produced by the cleavage of m-KL and has a molecular weight of approximately 130 kDa; and p-KL1 and p-KL2 refer to soluble klotho forms composed of the KL1 domain and the KL2 domain of p-KL, respectively. m-KL refers to the full-length transcript (m-KL) encoding a single-pass transmembrane protein with a molecular weight of approximately 140 kDa. The protein contains three domains, including a short transmembrane domain located at the C-terminus, an extracellular domain composed of two internal repeats of approximately 550 amino acids, respectively referred to as KL1 and KL2, 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, producing another form of soluble Klotho of about 130 kDa (the proteolytic membrane subisoform is abbreviated as p-KL). In addition, the proteases ADAM10 and 17 located between the KL1 domain and the KL2 domain have a second recognition site, which produces 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 encompass full-length m-KL or p-KL (containing both the KL1 domain and the KL2 domain). The mammalian s-KL polypeptides encoded by the nucleic acids described herein and their functional variants exclude the full-length m-KL and p-KL (containing both the KL1-KL2 domains) forms of Klotho, each of which has a molecular weight of approximately 130 kDa.

[0059] SEQ ID NO: 1 is the amino acid sequence of human s-KL, which, as described above, is an alternatively spliced ​​transcript from the α-klotho human gene, including the KL1 domain, which has an approximate weight of about 63 kDa, and a specific secretion signal consisting of a 16 amino acid tail not found in m-KL or KL1 transcripts. The α-klotho human gene is located on chromosome 13NC_000013.11 (33016063..33066145) of the assembly GRCh38 of the human genome maintained by the Genome Reference Consortium (December 24, 2013). 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 the mRNA having GenBank database accession number NM_004795 (version 3 of May 3, 2014, the sequence of which is hereby incorporated by reference) of 5012 base pairs.

[0060] The amino acid sequence of human s-KL is as follows (SEQ ID NO: 1):

[0061]

[0062] The nucleic acid sequence of a representative complementary DNA (cDNA) encoding human s-KL (SEQ ID NO: 1) is shown below and identified as SEQ ID NO: 3 (s-KL-specific secretory sequence boxed below):

[0063]

[0064]

[0065] Rodent s-KL may also be useful in the present disclosure. SEQ ID NO: 2 is the amino acid sequence of an alternatively spliced ​​transcript from the α-klotho mouse gene, including a KL1 domain sequence, which is approximately 70 kDa in weight and has a specific secretion signal consisting of a 15 amino acid tail not found in the m-KL transcript. The α-klotho mouse gene is located on chromosome 5 (150,952,607-150,993,809) of the July 2007 mouse UCSC Genome Browser (NCBI37 / mm9) assembly of the mouse genome. SEQ ID NO: 2 is derived from the corresponding cDNA of SEQ ID NO: 4, which in turn is derived from an alternatively spliced ​​transcript of the mRNA sequence of GenBank database accession number NM_013823 (version 2 of February 15, 2015 (the sequence of which is hereby incorporated by reference)) having 5124 base pairs.

[0066] The amino acid sequence of mouse s-KL (SEQ ID NO: 2) is shown below:

[0067]

[0068]

[0069] The nucleic acid sequence of a representative complementary DNA (cDNA) nucleic acid sequence of mouse s-KL is shown below (SEQ ID NO: 4):

[0070]

[0071]

[0072] Additional mammalian s-KL may be suitable for use in the present disclosure. Mammalian klotho genes containing a KL1 domain and a KL2 domain are known in the art and / or can be readily identified according to standard techniques. If an s-KL isoform has not yet been identified for a given mammalian species, an s-KL isoform may be readily derived from an identified mammalian klotho gene, particularly from a KL1 domain and / or a KL2 domain with an appropriate C-terminal tail, according to standard techniques.

[0073] Native to mammal species: bonobo (Pan paniscus, pygmy chimpanzee, accession number: XP_034792458.1), chimpanzee (Pan troglodytes, chimpanzee, accession number: XP_522655.2), gorilla (Gorilla gorilla, western lowland gorilla, accession number: XP_030857339.1), crab-eating macaque (Macaca fascicularis, crab-eating macaque, accession numbers: AAC77917.1, Q8WP17.1, XP_005586019.2, XP_005586019.3), Sumatran orangutan (Pongo abelii, Sumatranorangutan, accession numbers: XP_024086695.2, PNJ48590.1), Bornean orangutan (Pongo pygmaeus, Bornean orangutan, accession number: XP_054302967.1), Ugandan red Colobus (Piliocolobus tephrosceles, Ugandan red Colobus, accession number: XP_023064573.2), northernwhite-cheekedgibbon (Nomascus leucogenys, northernwhite-cheekedgibbon, accession number: XP_030674267.1), Tibetan macaque (Macaca thibetana, Tibetan macaque, accession number: XP_050621876.1), pig-tailed macaque (Macaca nemestrina, pig-tailed macaque, accession number: XP_011746941.1), macaque (Macaca mulatta, Rhesus monkey, accession number: EHH28941.1, XP_001101127.2), silvery gibbon (Hylobates moloch, silvery gibbon, accession number: XP_032005041.1), gelada (Theropithecus gelada, gelada, accession number: XP_025220255.1), golden snub-nosed monkey (Rhinopithecus roxellana, golden snub-nosed monkey, accession number: XP_030778249.1), Francois's langur (Trachypithecus francoisi, Francois's langur, accession number: XP_033091442.The amino acid sequences of additional mammalian klotho genes of 1), green monkey (Chlorocebus sabaeus, green monkey, accession number: XP_007958284.1) and East African baboon (Papioanubis, olive baboon, accession number: XP_031511616.1) are disclosed in the attached sequence listing as SEQ ID No: 42-63 (the sequences of which are hereby incorporated by reference). Each mammalian klotho has at least 85% amino acid sequence identity with SEQ ID NO: 1 and SEQ ID NO: 2. .

[0074] The present disclosure encompasses functional variants of mammalian s-KL and their use in the disclosed compositions and methods.The functional variant may be non-naturally occurring.

[0075] Protein variants are well understood by those skilled in the art and may involve amino acid modifications that generally fall into one or more of three categories: substitution variants, insertion variants, or deletion variants. In the present disclosure, a variant is "functional" in that it is therapeutically effective in treating movement disorders that may occur, for example, in various diseases or disorders, such as neuromuscular diseases and disorders.

[0076] When referring to a polypeptide, as used herein, the term "substitution variant" refers to a polypeptide in which at least one amino acid in the native or starting sequence is removed and a different amino acid is inserted in its place. The substitution may be single, in which only one amino acid is substituted in the polypeptide molecule, or multiple, in which two or more amino acids are substituted in the same polypeptide molecule.

[0077] When referring to polypeptides, as used herein, the term "conservative substitution" refers to that at least one amino acid in the native or starting sequence of a polypeptide is replaced by a different amino acid of similar size, charge or polarity. The example of conservative substitution includes replacing another non-polar residue with a non-polar (hydrophobic) residue, such as exchanging isoleucine, valine or leucine. Similarly, the example of conservative substitution includes replacing another polar residue with a polar (hydrophilic) residue, such as the exchange between arginine and lysine, between glutamine and asparagine, and between glycine and serine. Other examples of conservative substitution include replacing another basic amino acid with a basic amino acid (such as lysine, arginine or histidine), or replacing another acidic residue with an acidic residue (such as aspartic acid or glutamic acid).

[0078] When referring to polypeptides, as used herein, the term "insertion variant" refers to a variant in which one or more amino acids are inserted adjacent to an amino acid at a particular position in a native or starting sequence. As used herein, the term "adjacent to" an amino acid refers to attachment of the amino acid via the α-carboxyl or α-amino functional group of the amino acid.

[0079] When referring to polypeptides, as used herein, the term "deletion variant" refers to a variant in which one or more of the natural or starting amino acids are removed. Typically, a deletion variant will lack one or more amino acids in a specific region of the molecule.

[0080] In the present disclosure, the term "identity" refers to the percentage of identical residues in two sequences when the sequences are optimally aligned. If, in the optimal alignment, a position in the first sequence is occupied by the same amino acid as the corresponding position in the second sequence, the sequence exhibits identity relative to the position. The percentage of identity determines the number of identical residues within the length defined in a given alignment. Therefore, the level of identity between two sequences or ("percentage of sequence identity") is measured as the ratio of the number of identical positions shared by the sequences to the number of positions compared (i.e., percentage of sequence identity = (number of identical positions / total number of positions compared) × 100). Gaps, i.e. positions in the alignment where residues are present in one sequence but not in the other, are considered to be positions with different residues and are counted as positions compared.

[0081] As an illustration, a polypeptide having an amino acid sequence that is at least, for example, 95% identical to the reference amino acid sequence of SEQ ID NO: 1 means that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may contain up to five amino acid changes per 100 amino acids in the reference amino acid sequence of SEQ ID NO: 1. 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 one amino acid, or up to 5% of the amino acid residues of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These changes to the reference sequence may occur at the amino or carboxyl terminal positions of the reference amino acid sequence or at any position between these terminal positions, interspersed between residues in the reference sequence or in one or more contiguous groups within the reference sequence, respectively.

[0082] A variety of mathematical algorithms for quickly obtaining the best comparison and calculating the identity between two or more sequences are known, and are incorporated into a plurality of available software programs. For purposes of the present disclosure, preferably an algorithm based on a global comparison is used to determine the sequence identity between two amino acid sequences, such as Needleman-Wunsch algorithm (Needleman and Wunsch, " Journal of Molecular Biology (J.Mol.Biol.) " 48:443-453 (1970), preferably implemented in the Needle program of the EMBOSS package (Rice et al., " Genetics Trend (Trends Genet) " .16 (6): 276-277 (2000)); or using the BLAST global comparison tool (Altschul et al., " Journal of Molecular Biology " 215 (3): 403-410 (1990)) of default settings. When the sequence lengths compared are substantially the same, local comparisons can also be used.

[0083] Representative functional variant mammalian s-KL (which may be naturally occurring or non-naturally occurring) 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 may 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. A representative functional variant of a mammalian s-KL having at least 85% amino acid sequence identity to SEQ ID NO: 1 is the KL1 domain (eg, the cleavage product of p-KL, p-KL1).

[0084] In some embodiments, the polypeptide has an amino acid sequence having 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 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 can be removed and replaced with SQLTASVSSPPTRALSLASSAFLLGWRSWRILCPEPTR (SEQ ID NO: 17).

[0085] Polypeptides having a percent identity of at least 88% to either of SEQ ID NO: 1 or SEQ ID NO: 2 encompass mammalian s-KL other than human and mouse s-KL.

[0086] In another embodiment, optionally in combination with any of the embodiments provided below, the length of the mammalian s-KL polypeptide or its functional variant is equal to or less than 645 amino acids, 600 amino acids or 550 amino acids. 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 the length ... identity with SEQ ID NO: 1, and wherein the length of the variant is 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, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 1, 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 is 545 to 600 amino acids in length.

[0087] 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 the heterologous moiety via a peptide bond.

[0088] As used herein, a "heterologous moiety" refers to any molecule coupled to a polypeptide via a covalent peptide bond. In specific embodiments, the heterologous moiety is located at the N-terminus or C-terminus of the polypeptide. In specific embodiments, the heterologous moiety is located at both the N-terminus and the C-terminus of the polypeptide.

[0089] The heterologous moiety can be, for example, a molecule that facilitates the purification of a polypeptide. In a specific embodiment, the heterologous moiety is a peptide. In an even more specific embodiment, the heterologous moiety is a polyhistidine track. It will be appreciated by those skilled in the art that small peptides that aid in the purification of a protein can be maintained in the final compound without affecting its function.

[0090] In some embodiments, the heterologous moiety is a vehiculization agent. As known in the art, these agents promote the absorption, transport and delivery of polypeptides. Representative examples of vehiculization agents include dipalmitoyl-phosphatidyl-choline (DPCC) liposomes, micellar emulsions, dimethylformamide (DMF) and halogenated phenothiazines.

[0091] The promoter operably linked to the nucleic acid encoding mammalian s-KL or a functional variant thereof is a myocyte-specific promoter. Representative examples of myocyte-specific promoters that may be suitable for use in the present disclosure include mammalian desmin (DES, also known as CSM1 or CSM2) promoter; α2 actinin (ACTN2, also known as CMD1AA) promoter; filamin-C (FLNC, also known as actin binding-like protein (ABLP), filamin-2 (FLN2), ABP-280, ABP280A, ABPA, ABPL, MFM5 or MPD4) promoter; sarcoplasmic / endoplasmic reticulum calcium ATPase 1 (ATP2A1, also known as AT P2A or SERCA1) promoter; troponin I type 1 (TNNI1, also known as SSTNI or 25TTNI) promoter; myosin-1 (MYH1) promoter, i.e., the phosphorylated fast skeletal muscle myosin light chain (MYLPF) promoter; myosin 1 (MYH1, also known as MYHSA1, MYHa, MyC-2X / D, or MyHC-2x) promoter; alpha-3 chain tropomyosin (TPM3, also known as CFTD, NEM1, OK / Scl.5, TM-5, TM3, TM30, TM30 nm, TM5, TPMsk3, TRK, h TM5 or hscp30) promoter; ankyrin repeat domain-containing protein 2 (ANKRD2, also known as ARPP) promoter; myosin heavy chain (MHC) promoter; myosin light chain (MLC) promoter; muscle creatine kinase (MCK) promoter, i.e., synthetic muscle promoter, as described in Li et al., Nature Biotechnology (Nat. Biotechnol.) 17(3):241-245 (1999), such as SPc5-12 promoter, muscle creatine kinase (MCK) promoter, dMCK promoter, tMCK promoter, which are respectively composed of a double-stranded or triple-stranded tandem of the MCK enhancer and the MCK basal promoter, as described in Wang et al., Gene Ther. 15(22):1489-1499 (2008).

[0092] Hybrid (synthetic) myocyte-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 from viral or human sequences (e.g., CMV, CAG, or PGK). Representative examples of hybrid promoters include hybrid α-myosin heavy chain enhancer / MCK enhancer (MHCK7; 770 bp); MCK-C5-12 promoter (as described in Wang et al., Gene Therapy 15(22): 1489-1499 (2008)) and cardiac and skeletal muscle-specific myosin chaperone Unc45b (195 bp) promoter (as described in Rudeck et al., Genesis 54(8): 431-438 (2016)). In some embodiments, the promoter is a mammalian muscle cell promoter, such as a human or murine desmin promoter, examples of which are described in, e.g., U.S. Patent Application Publication No. 2020 / 00407746.

[0093] In some embodiments, the promoter is a promoter naturally associated with a gene encoding human desmin, whose promoter sequence is shown below in SEQ ID NO:5:

[0094]

[0095]

[0096] Therefore, in some embodiments, the gene construct of the present invention may comprise a human desmin promoter of SEQ ID NO: 5 operably linked to a mouse s-KL of SEQ ID NO: 2, and have the following nucleic acid sequence (SEQ ID NO: 6):

[0097]

[0098]

[0099] In some embodiments, the gene construct comprises a human desmin gene promoter operably linked to a nucleic acid sequence encoding human s-KL (eg, a nucleic acid sequence of SEQ ID NO: 3).

[0100] In some embodiments, the nucleic acid encoding mammalian s-KL or a functional variant thereof is operably linked to more than one, 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 myocyte-specific promoter. In some embodiments, the additional promoter is a neuronal cell-specific promoter. In some embodiments, the additional promoter is a ubiquitous promoter. A "ubiquitous promoter" is a promoter that drives expression in almost all tissues. 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 neuronal cell-specific promoter. In some embodiments, the genetic construct contains three promoters: a first myocyte-specific promoter, a neuronal cell-specific promoter, and an inducible promoter, all of which are operably linked to the nucleic acid encoding s-KL.

[0101] In certain embodiments, other promoters are hybrid (synthetic) neuron-specific promoters and muscle-specific promoters and may also be useful. The type of such promoters 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 certain embodiments, neuron-specific promoters are hybrid promoters, wherein a promoter in the promoter described above is fused with a CMV enhancer. Representative examples of hybrid promoters include 5' cytomegalovirus enhancers (CMV E-380bp) fused to PDGF-β promoters (Liu et al., Gene Therapy. 11 (1): 52-60 (2004) described) and CMV enhancers fused to SYN promoters (such as Hioki et al., Gene Therapy. 14 (11): 872-882 (2007) described). For example, other hybrid neuron-specific promoters are described in U.S. Patent Application Publication 2009 / 0055941. Yet other examples of muscle cell-specific promoters include synthetic neuronal cell-specific promoters, which have greater activity than naturally occurring promoters.

[0102] In other embodiments, the other promoter is not a tissue-specific promoter, and its representative type includes a constitutive promoter and an inducible promoter. The constitutive promoter is independent of the regulatory influence to initiate RNA synthesis. Inducible promoters allow regulatory gene expression, and can be regulated by the presence of exogenously supplied compounds, environmental factors (such as temperature) or specific physiological states. Representative examples of specific promoters include CMV promoters (e.g., cytomegalovirus intermediate early (CMVIE) promoters), β-actin promoters (e.g., chicken β-actin (CAG) promoters), CASI promoters (e.g., described as CMV enhancers, chicken β-actin promoters and the synthetic promoters of the combination of splicing donors and splicing acceptors flanking ubiquitin (UBC) enhancers, as described in U.S. Patent No. 8,865,881), human phosphoglycerate kinase-1 (PGK) promoters, TBG promoters, retrovirus Rous sarcoma (Rous sarcoma) virus LTR promoter, SV40 promoter, dihydrofolate reductase promoter, phosphoglycerol kinase (PGK) promoter, EF1a promoter, zinc-inducible sheep metallothionein (MT) promoter, dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, T7 polymerase promoter system, ecdysone insect promoter, tetracycline repressible system, tetracycline inducible system, RU486 inducible system and rapamycin inducible system.

[0103] In some embodiments, the additional promoter is a zinc-driven inducible promoter. Representative examples of zinc-driven promoters include zinc-driven metallothionein promoters, promoter / operator region of the zntA gene of Escherichia coli (Brocklehurst et al., Mol. Microbiol. 31(3): 893-902 (1999) described) and zinc-regulated promoters (described in U.S. Pat. No. 8,354,272). In some embodiments, the additional promoter is a zinc-driven metallothionein promoter.

[0104] In some embodiments, the additional promoter operably linked to the nucleic acid encoding mammalian s-KL or its functional variant is a neuronal cell-specific promoter. As used herein, the term "neuronal cell" encompasses cells of the CNS, PNS, and spinal cord, representative cell types of which include neurons (including motor neurons, sensory neurons, and interneurons), glial cells, and astrocytes. Motor neurons send signals from the brain and spinal cord to control everything from muscle contraction to glandular output. The terms "neuronal cell specificity" and "neuronal specificity" are used interchangeably herein and refer to the preferential, selective, or dominant expression of mammalian s-KL (or its functional variant) in neuronal cells or neuronal tissues compared to other (i.e., non-neuronal) cells and tissues. In some embodiments, at least 50%, 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% of the expression occurs in neuronal cells or tissues. In some embodiments, "neuronal cell specificity" means that the expressed mammalian s-KL (or its functional variant) does not leak into other cells, organs or tissues other than neurons, such as lung, liver, brain, kidney and / or spleen.

[0105] Representative examples of neuronal cell-specific promoters that may be suitable for use in the present disclosure include synapsin 1 (SYN) promoter, calcium / calmodulin-dependent protein kinase II promoter, tubulin αI promoter, neuron-specific enolase (NSE) promoter, platelet-derived growth factor β chain (PDGF-β) promoter, microtubule-associated protein 1B (MAP1B), dopaminergic receptor 1 (Drd1a) promoter, 67 kDa glutamate decarboxylase (GAD67) promoter, homeobox Dlx5 / 6, glutamate receptor 1 (GluR1) promoter, glial fibrillary acidic protein (GFAP) promoter, and pretachykinin 1 (Tac1) promoter.

[0106] In addition to one or more promoters, the gene construct / expression cassette may further comprise one or more other non-coding regulatory elements, also referred to as expression control sequences, as long as they are not contained in the expression vector. Representative examples of regulatory elements include appropriate transcription initiation sequences, termination sequences, promoter sequences, and enhancer sequences; effective RNA processing signals, such as, for example, splicing and polyadenylation (poly A) tail sequences; poly A consensus sequences, tetracycline-regulatable systems, post-transcriptional regulatory elements, sequences that stabilize cytoplasmic mRNA; sequences that improve 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 a viral vector of the present disclosure. Representative examples of post-transcriptional regulatory elements include hepatitis B virus post-transcriptional regulatory elements (HPRE) and woodchuck hepatitis post-transcriptional regulatory elements (WPRE). WPRE is a tripartite cis-acting element that has been shown to enhance transgene expression driven by certain, but not all, promoters.

[0107] In some embodiments, the genetic construct comprises a cytomegalovirus intermediate early (CMV IE) promoter operably linked to nucleic acids encoding mammalian s-KL, post-transcriptional regulatory elements of woodchuck hepatitis virus (WPRE), and poly A.

[0108] In some embodiments, the genetic construct comprises a CAG promoter operably linked to a nucleic acid encoding s-KL (mouse cDNA or human s-KL) and a poly A tail.

[0109] The genetic construct can be DNA (e.g., for plasmids and various viral vectors (e.g., adenoviral vectors and adeno-associated viral (AAV) vectors) or RNA (e.g., for linearized mRNA constructs or for other viral vectors, such as retroviral vectors and lentiviral vectors). In some embodiments where the genetic construct is in the form of RNA of a linearized mRNA construct, the genetic construct is incorporated into a plasmid, which is treated with an enzyme (e.g., a restriction endonuclease) to produce linear DNA, which is transcribed in vitro in a bioreactor to produce mRNA. The linearized mRNA is complexed with a positively charged polymer, cationic lipid, or other complex to form extruded nanoparticles, extruded micron-sized particles, or micellar emulsions.

[0110] In some embodiments in which the gene construct is in the form of RNA for retroviral and lentiviral delivery, the gene construct contains group antigen (gag), reverse transcriptase (pol) and envelope (env) genes, as well as 5' and 3' long terminal repeats (LTR). In some embodiments, the 5'LTR is a chimeric 5'LTR containing a heterologous promoter that is not dependent on transactivation by the lentiviral tat protein. The lentiviral LTR can be divided into three elements, named 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, a portion of the LTR is modified, substituted or deleted, for example, a portion of the 3'U3 is deleted. In one embodiment, the 3'U5 element is replaced by poly A. In one embodiment, 5'U3 is replaced by a truncated CMV immediate early (IE) enhancer / TATA promoter. Additional lentiviral non-coding regulatory elements are known in the art, see, for example, 2021 / 181108A1 and WO 2021 / 160993A1 and U.S. Patents 6,669,936, 6,924,123 and 10,544,429.

[0111] In some embodiments, the gene construct (or nucleic acid construct) further comprises one or more of the following:

[0112] (a) poly A signal, for example, SV40 poly A tail;

[0113] (b) protein translation start site consensus nucleic acid;

[0114] (c) post-transcriptional regulatory element nucleic acid;

[0115] (d) 5' and 3' inverted terminal repeats; and

[0116] (e)Intron.

[0117] As known in the art, the term "intron" encompasses any portion of the entire intron that is large enough to be recognized and spliced ​​by the nuclear spliceosome. Generally, short sequences, functional sequences, intron sequences are preferred to keep the size of the expression cassette as small as possible, which helps the construction and operation of the expression cassette. In certain embodiments, introns are obtained from genes encoding proteins encoded by the coding sequence in the expression cassette. Introns can be located at 5' of the coding sequence, 3' of the coding sequence, or within the coding sequence. The advantage of locating the intron 5' to the coding sequence is to minimize the chance that the intron interferes with the function of the polyadenylation signal. Representative examples of suitable introns include minute virus of mice (MVM) introns, β-globin introns (βIVS-1), factor IX (FIX) intron A, simian virus 40 (SV40) small t introns, and β-actin introns.

[0118] All these elements (a) to (e) are sequences that allow the correct expression of the gene operably linked to the promoter. A person skilled in the art will understand the sequences referred to and their functions.

[0119] In some embodiments, the gene construct may include muscle-specific regulatory elements, for example, in addition to a myocyte-specific promoter, which may enhance muscle-specific expression of a mammalian s-KL coding sequence. Representative examples of such sequences include CSk-SH1, CSk-SH2, CSk-SH3, CSk-SH4, CSk-SH51, and CSk-SH6 regulatory elements. See, for example, U.S. Patent Application Publication 2020 / 00407746. In some embodiments, such regulatory elements may be located upstream of the promoter.

[0120] The nucleic acid sequence of the gene construct comprising the nucleic acid encoding SEQ ID NO:6, WPRE and poly A is shown below (SEQ ID NO:7):

[0121]

[0122]

[0123]

[0124]

[0125]

[0126] Methods for cloning nucleic acids and gene constructs into vectors are known in the art. In certain embodiments, one or more sequences or the entire gene construct can be codon optimized, for example, depletion 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 gene construct synthesis or by any suitable molecular biology technique.

[0127] Expression vector

[0128] Expression vectors, also called expression constructs, are typically viruses or plasmids designed for protein expression in cells (e.g., may contain a viral genome or part thereof). Expression vectors have the characteristics that any vector may have, such as an origin of replication, a selectable marker, and a suitable site for inserting a gene construct, such as a multiple cloning site (MCS).

[0129] In one aspect, the present disclosure provides an expression vector comprising: (a) a nucleic acid construct 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) a nucleic acid construct comprising a first promoter functionally operable in muscle cells, the first promoter operably linked to a nucleic acid encoding mammalian s-KL or a functional variant thereof, wherein the expression vector has muscle cell tropism.

[0130] Viral expression vector

[0131] In some embodiments, the expression vector is a viral vector, e.g., a retroviral vector, a lentiviral vector, an adenoviral vector, a herpes virus vector, an adenovirus, or an adeno-associated virus (AAV) vector.

[0132] As used herein, the term "adeno-associated virus" refers to a viral vector that infects dividing and resting primate (and human) cells. Due to the lack of any pathogenicity and the fact that they usually integrate at the same location in the genome (the AAVS1 site in chromosome 19), this viral vector can be safely used to transduce foreign DNA into human cells in gene therapy applications.

[0133] In some embodiments, the expression vector is an adeno-associated virus (AAV). The AAV vector can be derived from any suitable host species, including humans (h), baboons, chimpanzees and rhesus monkeys (rh), pig-tailed monkeys (pi) and cynomolgus monkeys. The genomic organization of all known AAV serotypes is similar. The genome of AAV is a linear single-stranded DNA molecule with a length of less than about 5,000 nucleotides (nt). The reverse terminal repeat sequence (ITR) is located on the flank of the unique coding nucleotide sequence of non-structural replication (Rep) protein and structural (VP) protein. VP protein (VP1, VP2 and VP3) forms capsid and contributes to the tropism of the virus. The terminal 145nt ITR is self-complementary and is organized so that the energetically stable intramolecular duplex formed by forming a T-shaped hairpin can be formed. These hairpin structures are used as the starting point for viral DNA replication, thereby being used as primers for cellular DNA polymerase complexes. After wild-type (wt) AAV infection in mammalian cells, the Rep gene is expressed and plays a role in the replication of the viral genome.

[0134] 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. It is known that AAV serotype 9 (AAV9) achieves efficient transduction in heart and skeletal muscle. Therefore, in some embodiments, the expression vector is an AAV9 vector, for example, a self-complementary AAV9 vector (scAAV9).

[0135] In some embodiments, the expression vector is an AAV with a serotype having a myocyte tropism. As used herein, the term "myocyte tropism" refers to a vector that preferentially affects myocytes, for example, a viral vector that preferentially infects myocytes rather than other cell types in the body. Representative examples of such AAVs with a serotype having a myocyte tropism include AAV1, AAV5, AAV6, AAV8, AAV9 (BBB), AAVrh.74, and AAVmyo. Other AAVs with myocyte tropism are known in the art. See U.S. Patent Application Publication 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 with a serotype having a myocyte tropism, the serotype being selected from AAV8 and AAVmyo. In these embodiments, the promoter does not need to be a myocyte-specific promoter.

[0136] In some embodiments, the expression vector is an adeno-associated virus containing an AAV capsid polypeptide, the AAV capsid polypeptide comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 18-29, or encoded by a nucleic acid having a sequence of any one of SEQ ID NOs: 30-41. See Grimm et al., U.S. Patent Application Publication 2021 / 0363193.

[0137] In some embodiments, the AAV capsid polypeptide comprises or is 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 U.S. Patent Application Publication 2021 / 0363193. In some embodiments, the AAV capsid polypeptide comprises or is 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 U.S. Patent Application Publication 2021 / 0363193. In some embodiments, the AAV capsid polypeptide comprises or is 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 U.S. Patent Application Publication 2021 / 0363193.

[0138] In some embodiments, the expression vector is an adeno-associated virus of the AAV8 serotype, which includes or is the nucleic acid sequence of SEQ ID NO:7, which is a genetic construct including a human desmin promoter, a nucleic acid encoding mouse s-KL, a WPRE sequence, and a SV40 poly A tail.

[0139] In some embodiments, the expression vector is an adeno-associated virus of the AAV8 serotype, which includes or is the nucleic acid sequence of SEQ ID NO:7, but wherein the gene encodes human s-KL, such as the nucleic acid sequence of SEQ ID NO:3.

[0140] The positions of the different sequences in SEQ ID NO:7 are shown in Table A.

[0141] Table A. Sequence legend for SEQ ID NO:7.

[0142]

[0143] In some embodiments, the expression vector is an AAV with a serotype having neuronal tropism. As used herein, the term "neuronal cell tropism" refers to a vector that preferentially affects neuronal cells, for example, a viral vector that preferentially infects motor neuron cells rather than other cell types in the body. Representative examples of such AAVs with serotypes having neuronal cell tropism include AAV1, AAV6, and AAV7. In some embodiments, the expression vector is an adeno-associated virus with a serotype having a neuronal cell tropism specific to astrocytes, for example, AAV5. In some embodiments, the expression vector is a vector with neuronal cell tropism, such as described in, for example, U.S. Patent Application Publications 2019 / 0030138 and 2020 / 0339960. In these embodiments, the promoter does not need to be a neuron-specific promoter.

[0144] In some embodiments, the expression vector is an intraparenchymal injection of AAV delivery targeted to the spinal cord and is a serotype with neuronal cell 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 neuronal cell tropism. AAV vector serotypes with neuronal cell tropism are well suited for intravenous injection and include AAV9, AAVhr.10, AAVrh.8, and AAVrh43.

[0145] In some embodiments, the expression vector is configured to undergo axonal retrograde 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 the muscle or sciatic nerve and preferentially targets motor neurons.

[0146] In some embodiments, the viral vector is a recombinant AAV (rAAV), which includes at least one capsid protein from an AAV serotype. The AAV capsid protein can provide tissue-specific targeting so that the AAV will infect the gene construct and deliver the gene construct to the desired tissue or organ. In some embodiments, AAV is a pseudotype AAV (pAAV), which includes a virus or a viral vector having a viral envelope protein from more than one virus. pAAV may have changed the host or tissue tropism, or increased or reduced particle stability. In some embodiments, pAAV includes nucleic acids from two or more different AAVs, for example, nucleic acids from an AAV source encode capsid proteins, and nucleic acids in at least one other AAV source encode other viral proteins and / or viral genomes. In some embodiments, pAAV refers to an AAV including an inverted terminal repeat sequence (ITR) of an AAV serotype and capsid proteins of different AAV serotypes. For example, a pAAV vector containing an ITR of a serotype X encapsulated with a Y protein will be designated as AAVX / Y (e.g., AAV2 / 1 has an ITR of AAV2 and a capsid of AAV1). In some embodiments, pAAV can be used to combine the tissue-specific targeting capabilities of the capsid protein from one AAV serotype with the viral DNA (e.g., viral ITRs) from another AAV serotype, thereby allowing targeted delivery of a transgene to a target tissue.

[0147] AAV vector production is most commonly achieved by double plasmid transfection, followed by in vitro auxiliary adenovirus infection. The two plasmids in the double plasmid transfection are a vector plasmid and a packaging plasmid. The vector plasmid contains a gene construct described herein, which is flanked by an AAV reverse terminal repeat (ITR), and the packaging plasmid provides an AAV replicase (Rep) and capsid (Cap) gene for vector DNA replication and packaging. Once a suitable cell line (e.g., HeLa) is transfected with two plasmids, then infected with an auxiliary adenovirus, and the AAV gene is expressed, the gene from the two plasmids is expressed and packaged into the AAV particles. The protein encoded on the packaging plasmid forms the AAV particles, and the vector plasmid (containing the gene construct) is packaged into the AAV particles. AAV particles are either purified from auxiliary adenovirus particles, or adenovirus particles are selectively heat-inactivated. This procedure produces a replication-defective AAV containing a gene construct, which can be further deployed and delivered to a subject.

[0148] Alternatively, instead of in vitro helper adenovirus infection, a third adenovirus-derived plasmid containing essential replication genes (e.g., E1A, E1B, E2A, E4, and VARNA) from adenovirus can also be transfected with the vector plasmid and packaging plasmid. In some embodiments, the packaging plasmid and adenovirus-derived plasmid can be stably integrated into the cell line.

[0149] Additional methods for producing and delivering AAV, rAAV, and pAAV are known in the art. See, for example, U.S. Patent Publications 2015 / 0065560, 2013 / 0090374, and 2012 / 0309050, and U.S. Patents 11,041,171, 11,020,443, and 7,858,367.

[0150] 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 has been described, for example, in the following: U.S. Patents 5,665,577, 5,981,276, 6,013,516, 7,090,837, 8,119,119 and 10,954,530. The lentiviral vector comprises a defective lentiviral genome, i.e., at least one of the lentiviral genes gag, pol and env has been inactivated or deleted. Additional lentiviral genes and sequences include a Rev response element (RRE) and a DNA flap at the center of the viral cDNA between the central polypurine segment (cPPT) and the central termination sequence (CTS), usually abbreviated as cPPT CTS.

[0151] In some embodiments, the expression vector is a recombinant lentivirus comprising a recombinant genome comprising a lentiviral encapsulation psi sequence, an RNA nuclear export element, a transgene and a promoter and / or a sequence that facilitates nuclear import of RNA between the LTR5' and 3' lentiviral sequences, and a mutant integrase that prevents its genome from integrating into the genome of an isolated cell. The lentiviral vector may comprise, for example, the sequence 5'LTR-psi-RRE-cPPT CTS-transgene-LTR3'.

[0152] In some embodiments, a viral vector encoding a nucleic acid sequence of the present disclosure is administered with an empty vector comprising a capsid protein and excluding any nucleic acid sequence of the present disclosure. In some embodiments, an empty vector is administered at a ratio of 5:1 to 3:1 of an empty vector to a viral vector to immunomodulate the immune response of a subject to viral rector.

[0153] In some embodiments, the viral vector is immunomodulatory. Immunomodulation can 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 2021 / 0139860 and U.S. Patents 6,399,385 and 10,022,457. Pegylation allows the circulation time of viral vector particles to be longer and the immune response to be reduced (i.e., reduced immunogenicity).

[0154] 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 2019 / 0030138 can be combined with the viral vectors described herein.

[0155] Non-viral expression vectors

[0156] In other embodiments, the expression vector is a non-viral vector, representative examples of which include plasmids, minicircles, and transposon-based vectors, such as Sleeping Beauty (SB)-based vectors and piggyBac (PB)-based vectors. In yet other embodiments, the vector may include both viral and non-viral elements.

[0157] 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), a start sequence, a poly-A tail sequence, optional regulatory elements, and other optional sequences (e.g., a multiple cloning site). In some embodiments, mRNA is produced by a circular plasmid. The plasmid may be linearized with a restriction endonuclease, transcribed in vitro to produce mRNA, and modified with a 5' cap and a 3' poly-A tail.

[0158] In some embodiments, the carrier encapsulates a gene construct or plasmid. The carrier can be lipid-based, for example, a lipid nanoparticle (LNP), a liposome, a lipid vesicle or a lipid complex. In some embodiments, the carrier is an LNP. In certain embodiments, the LNP comprises two or more concentric bilayers separated by an aqueous compartment. The lipid bilayers can be functionalized and / or cross-linked to each other. The lipid bilayer can comprise one or more ligands, proteins or channels.

[0159] Lipid carriers, for example, LNPs can include one or more cationic / ionizable lipids, one or more polymer-conjugated lipids, one or more structural lipids and / or one or more phospholipids. "Cationic lipids" refer to lipids with a positive charge or a lipid that can maintain a positive charge. Cationic lipids include one or more amine groups that carry a positive charge, depending on pH. "Polymer-conjugated lipids" refer to lipids with conjugated polymer moieties. Polymer-conjugated lipids include PEGylated lipids, which are lipids conjugated to polyethylene glycol. "Structural lipids" refer to non-cationic lipids that do not have a net charge at physiological pH. Exemplary structural lipids include cholesterol, coprosterol, sitosterol, ergosterol, campesterol, etc. "Phospholipids" refer to lipids with triglycerides, two fatty acids, and a phosphate ion. The phospholipids in LNPs assemble lipids into one or more lipid bilayers. LNPs, methods of preparation, formulation, and delivery thereof are disclosed, for example, in U.S. Patents 9,364,435, 9,518,272, 10,022,435, and 11,191,849, and U.S. Patent Application Publications 2004 / 0142025, 2007 / 0042031, and 2020 / 0237679.

[0160] Lipoplexes, liposomes and lipid nanoparticles can comprise combinations of lipid molecules, e.g., cationic lipids, neutral lipids, anionic lipids, polypeptide lipid conjugates and other stabilizing components. Representative stabilizing components include antioxidants, surfactants and salts. Compositions and methods for preparing lipid complexes, liposomes and lipid nanoparticles are known in the art. See, e.g., U.S. Pat. Nos. 8,058,069, 8,969,353, 9,682,139, 10,238,754, U.S. Patent Application Publications 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).

[0161] Pharmaceutical composition

[0162] In some embodiments, pharmaceutical compositions containing expression vectors of the present disclosure can be formulated with pharmaceutically acceptable carriers and optionally pharmaceutically acceptable excipients (collectively referred to as pharmaceutically acceptable "vehicles") for administration to subjects by any suitable and medically acceptable mode of administration. In some embodiments, administration is delivered parenterally (e.g., intravenously).

[0163] The expressions "pharmaceutically acceptable carrier" and "pharmaceutically acceptable excipient" refer to ingredients that are physiologically inert (e.g., non-immunogenic) and non-toxic, and that are compatible with all the other ingredients of the pharmaceutical composition and suitable for contact with tissues or organs of subjects (e.g., mammals, including both humans and non-human animals), commensurate with a reasonable benefit / risk ratio.

[0164] In some embodiments, the expression vector is formulated for systemic (e.g., parenteral) administration. In some embodiments, the carrier is aqueous, and its representative examples include water, saline solutions (e.g., physiological saline, antibacterial water, and phosphate buffered saline (PBS)) and dextrose and glycerol aqueous solutions. The aqueous carrier may also include polyols, such as glycerol, propylene glycol, and liquid polyethylene glycol. The water-based composition may include additional excipients, such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives. Such liquid-based preparations may be in the form of solutions, suspensions, or dispersions. In some embodiments, the carrier is non-aqueous.

[0165] The pharmaceutical composition may further contain an excipient. As with the case of a carrier, the selection of an excipient will depend in part on the specific carrier and the specific method for administering the composition. Therefore, it will be readily appreciated by those skilled in the art that the pharmaceutical composition of the present disclosure has a variety of suitable formulations. Representative examples of excipients include coating agents, surfactants and emulsifiers, antimicrobial agents and other preservatives, solubilizers, isotonic agents, absorption blockers, and suspending agents. Lecithin is an exemplary coating agent that establishes appropriate fluidity and maintains the desired particle size in the case of dispersion. Surfactants may also contribute to particle size maintenance. Exemplary antimicrobial agents include antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. Exemplary isotonic agents include sugars and polyols (for example, mannitol, sorbitol, and sodium chloride). Exemplary absorption blockers include aluminum monostearate and gelatin, which prolong the absorption of the gene construct in the composition.

[0166] Additional pharmaceutically acceptable excipients for preparing pharmaceutical compositions, formulations thereof, and delivery expression vectors (such as viral vectors) are known in the art. See, for example, U.S. Patent Application Publications 2013 / 0090374 and 2012 / 0309050, and U.S. Patents 7,858,367, 11,020,443, and 11,041,171.

[0167] Pharmaceutical compositions containing the expression vectors can also be formulated for several other routes or modes of administration, including, for example, oral (including both liquid and solid dosage forms), inhalation (via pressurized propellants), topical (e.g., lotions, creams, gels, ointments, medicated sticks, sprays, and pastes), and mucosal (e.g., vaginal).

[0168] The compositions may be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials.

[0169] Treatment

[0170] The method of the present invention needs to treat a subject suffering from dyskinesia due to loss of muscle function or due to neuron-muscle dysfunction at the motor neuron level. As understood in the art, "treatment" is a method for obtaining a beneficial or desired result, comprising clinical results. Such results can include the alleviation or improvement of one or more symptoms of dyskinesia, the alleviation of the degree of disease or illness, the stabilization of the state of disease or illness, the delay or slowing down of disease or illness, the improvement or alleviation of disease or illness, and the alleviation (whether in part or in whole) of disease or illness, whether detectable or undetectable. As used herein, the term "subject" includes humans and non-human animals (e.g., non-human primates, livestock, domestic pets and laboratory animals (such as rodents)). As used herein, the term "dyskinesia" refers to the loss of mobility caused by loss of muscle function. Functional loss is mainly due to muscle weakness, muscle atrophy, loss of motor neuron function, muscle contractility disorders and other muscle cell diseases (e.g., metabolism), spinal cord injury, motor neuron disorders or motor neuron diseases. Examples of movement disorders include complete or partial paralysis (e.g., inability to walk or stand, speak, swallow, and ultimately breathe (ie, respiratory insufficiency), the latter leading to death), lack of muscle control, poor endurance, muscle spasms, spasticity, muscle weakness, and muscle atrophy.

[0171] According to the International Society of Neuromodulation, movement disorders involve partial or complete loss of function of a body part, usually one or more limbs. Diseases that cause movement disorders may cause muscle weakness, poor endurance (i.e., fatigue), loss of muscle control, or complete paralysis. Movement disorders are a leading cause of physical disability. They are primarily caused by peripheral problems affecting muscles, problems in the central nervous system affecting muscle output, and sensory problems affecting muscles, movement, and balance. Movement disorders are often evident in neuromuscular diseases and neurological conditions, but can also be caused by cancer or even traumatic injuries to the central and peripheral nervous systems.

[0172] Many neuromuscular and muscle diseases and conditions, including motor neuron diseases, such as some neurodegenerative diseases can be manifested as one or more symptoms of movement disorders. Diseases and conditions characterized by one or more symptoms of movement disorders or manifested as one or more symptoms of movement disorders include neuromuscular diseases. As used herein, the term "neuromuscular disease" includes motor neurons (all of which are components of motor units) in the spinal cord or central nervous system (CNS), peripheral nervous system (PNS), neuromuscular junctions or skeletal muscles, thereby ultimately affecting any disease of the subject's ability to move. The damage of motor neurons in the spinal cord, CNS, PNS, neuromuscular junctions or skeletal muscles can cause muscle atrophy and weakness. Sensory problems may also occur. Neuromuscular diseases can be acquired or hereditary. Mutations exceeding 500 genes have been shown to be the cause of neuromuscular diseases. Other causes include nerve or muscle degeneration, autoimmunity, toxins, drugs, malnutrition, metabolic disorders, hormone imbalance, infection, nerve compression / entrapment, including blood supply and trauma.

[0173] Representative examples of diseases or disorders characterized by movement disorders include amyotrophic lateral sclerosis (ALS), including the more common or sporadic form (sALS) and the familial form (fALS), Charcot-Marie-Tooth disease, multiple sclerosis, muscular dystrophy, Duchenne and Becker muscular dystrophy, myasthenia gravis, myopathies, myositis, including polymyositis and dermatomyositis, peripheral neuropathy, neuromyotonia, Lambert-Eaton disease, Friedrich's ataxia, traumatic nerve injury, diabetic neuropathy, motor skill disorders and spinal muscular atrophy (SMA), spinal cord injury, peripheral nerve injury or traumatic nerve injury, and muscle metabolic diseases. However, still other diseases and disorders that may be amenable to treatment with the disclosed expression vectors include hereditary myopathies, toxic neuropathies, autoimmune peripheral polyneuropathies, acute inflammatory demyelinating polyradiculoneuropathy (AIDP), chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), vascular polyneuritis, polyneuropathy, idiopathic ganglionitis, amyotrophic lateral sclerosis, multifocal motor block neuropathy or lower motor neuron syndrome, neuromuscular diseases, muscular atrophy, drug-induced myopathy, sarcopenia, cachexia, type II myofasciitis, age-related muscular atrophy, and acquired autoimmune primary muscle disorders.

[0174] Some diseases and conditions that may be suitable for treatment with the disclosed gene constructs and expression vectors include those classified as motor neuron diseases (MNDs), which are a group of rare neurodegenerative disorders that selectively affect motor neurons (cells that control the body's voluntary muscles). Examples of MNDs include ALS, progressive bulbar palsy (PBP), pseudobulbar palsy, progressive muscular atrophy (PMA), primary lateral sclerosis (PLS), spinal muscular atrophy (SMA), and monomelar muscular atrophy (MMA), as well as some rare variants similar to ALS.

[0175] In some embodiments, the methods entail treating a subject suffering from dyskinesia in ALS, including dyskinesia in sALS, including dyskinesia in fALS, and including dyskinesia in jALS.

[0176] In some embodiments, the methods may be "preventative" or "prophylactic" because the expression vector (or transformed cells according to the ex vivo embodiments) is administered to the subject before the movement disorder occurs. These embodiments may be particularly advantageous in cases where the subject is suspected of having an early stage neuromuscular disorder and / or has received a preliminary positive diagnosis, has been tested or is believed (e.g., through family history) to have a susceptibility to a disease or condition (e.g., ALS). Preventative or prophylactic treatment may delay the onset of the movement disorder or reduce its severity once the movement disorder becomes apparent. Tests for determining susceptibility to movement disorders may include, for example, genetic tests, muscle biopsies, and electromyography as are known in the art. For example, fALS can be diagnosed by genetic testing.

[0177] Mode of administration

[0178] In some embodiments, the pharmaceutical composition is administered parenterally to a subject (e.g., by intrathecal, subcutaneous, intravenous, intraventricular, intramuscular, or intraarterial injection, bolus injection, or infusion, which may be continuous or discontinuous). Other routes / modes of administration include any medically acceptable route, representative examples of which may include oral, inhalation, topical, and mucosal.

[0179] In some embodiments, the method of treatment may require the administration of an expression vector to a cell. 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 having a gene construct encoding mammalian s-KL, and administering the isolated cells back into the subject. In some embodiments, the method comprises optionally differentiating the isolated cells into cell types with a differentiation 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, neuronal cells, or iPSCs. In some embodiments, the neuronal cells are motor neurons.

[0180] In some embodiments, the isolated cell is a pluripotent stem cell, which is induced or has been induced to pluripotency (e.g., iPSC). PSC can be obtained from a subject (e.g., from adipose tissue), in which case it can be reprogrammed to a pluripotent state (autologous). Alternatively, iPSC can be obtained from a suitable cell bank (allogeneic), for example, an umbilical cord cell bank. Pluripotency induction can be carried out by any suitable means known in the art. Generally, pluripotency induction involves directly or indirectly regulating a specific cell pathway in an isolated cell with a pluripotency factor, which can be a nucleic acid sequence, a polypeptide, a small molecule, or a combination thereof. In one embodiment, the pluripotency factor is a polypeptide transcription factor or a polynucleotide encoding a transcription factor. 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, YY 1, 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 factor comprises a nucleic acid sequence encoding the transcription factors Oct4, Sox2, Klf4, c-Myc and Nanog.

[0181] As known in the art, iPSC can be differentiated into myocytes or neuronal cells with differentiation agents. Representative examples of differentiation agents for iPSC differentiation into myocytes 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 iPSC differentiation into neuronal cells include retinoic acid, bone morphogenetic protein 4 (BMP4) nerve growth factor (NGF), retinoic acid receptor (RAR) agonists (e.g., TTNPB), glycogen synthase kinase 3 inhibitors (e.g., CHIR99021), neurotrophin-3 (NT-3) and combinations thereof. Additional enrichment or selection can be performed to cells processed and / or differentiated. For example, in some embodiments, CD34+ cells are selectively enriched to separate smooth muscle cells. In certain embodiments, iPSC is differentiated and selectively enriched to separate striated muscle cells. In some embodiments, neuronal cells are enriched by selecting forkhead box A2 (FOXA2, also known as HNF3β and TCF-3B) positive cells. In some embodiments, neuronal cells are enriched by selecting CD133 positive cells.

[0182] In other embodiments, muscle cells and / or neuronal cells are isolated from a subject. In some embodiments, myoblasts and satellite cells (in a quiescent state and an activated state) are isolated from a subject by surgical methods (e.g., muscle biopsy) and separate fluorescence activated cell sorting (separation of, e.g., Pax7 positive cells). In some embodiments, induced pluripotent stem cells, central angioblasts, immortalized muscle precursor cells, or other pluripotent cell lines are obtained and administered locally or systemically to a subject.

[0183] The method of introducing the expression vector into the cell is well known in the art. Those skilled in the art will easily understand the specific method depending on the nature of the vector. In some embodiments, delivery or integration may include transfection, infection or transduction of cells with the expression vector. In some embodiments, the expression vector including the expression vector is delivered to the muscle cell by lipofection. Lipofection is described in, for example, U.S. Patents 5,049,386, 4,946,787 and 4,897,355. Electroporation may be beneficial to plasmid vectors. In some embodiments, factor H is increased during treatment, for example, by collecting adipose tissue from the subject, purifying stem cells from adipose tissue, treating stem cells to increase the secretion of factor H (e.g., small molecule compounds, optionally selegiline (Selegiline)), and the treated stem cells are applied to the subject. For example, the collection and treatment of adipose tissue are described in U.S. Patent Publication No. 2016 / 0193251.

[0184] After the cells are transformed with the gene construct and any optional differentiation and / or enrichment is complete, the cells are administered to a subject. Administration of these cells can be accomplished by any suitable means known in the art and disclosed elsewhere herein.

[0185] Therapeutic effective dose

[0186] In the context of the present disclosure, a dose administered to a subject, particularly a human, is "therapeutically effective" in the sense that it should be sufficient to achieve beneficial or desired results, including clinical results as described above, in the subject within a reasonable time frame. The dose will depend on a variety of factors, including the strength of the particular expression vector employed, the subject's condition and the subject's weight, and the severity of the movement disorder. The size of the dose will also be determined by the presence, nature, and extent of any adverse side effects that may accompany the administration of the expression vector.

[0187] The unit dosage form of the pharmaceutical composition containing the expression vector can be formulated. As used herein, the term "unit dose" refers to a physically discrete unit suitable as a unit dose for human and animal subjects, each unit containing a predetermined amount of expression vectors calculated in an amount sufficient to produce the desired effect. The unit dosage form can be administered by suitable infusion or injection required for the embodiment with any suitable volume of liquid. Dosage can be determined by unit body weight (e.g., kg of dosage / subject), by body surface area (BSA) (usually expressed as square meter BSA) or any suitable measurement result of the subject.

[0188] In some embodiments utilizing viral expression vectors, the viral vector is expressed at 1×10 6 Up to 5×10 14 The term "vector genome" (or "vg") as used herein refers to the nucleic acid sequence that constitutes the viral vector genome plus any transgene (e.g., mammalian s-KL) encoded therein.

[0189] In some embodiments, the viral vector is a lentivirus and each dose (e.g., a single intravenous infusion) is administered with 1×10 8 Up to 5×10 12 Transduction units / kg (TU / kg).

[0190] In some embodiments, at least about 1×10 5 viral genomes / mL to at least about 100×10 16The expression vector is administered to a subject at a titer of 10 viral genomes / mL. The term "viral genome" (vg) (also referred to as "genome equivalent," "genome copy" (gc), or "genomic particle" (gp)) used to refer to viral titer refers to the number of viral particles containing an expression vector (e.g., recombinant AAV), regardless of their infectivity or function.

[0191] In some embodiments, the viral vector delivering the s-KL gene construct is expressed as 1×10 5 Viral genomes to 5×10 14 The viral vector is administered in a suitable volume to achieve blood and tissue levels of an effective amount of viral genomes, which is an s-KL genome dose that achieves 200-1300 pg / ml of s-KL protein or polypeptide in the blood, CSF or desired target tissues such as muscle. In some embodiments, the viral vector is administered in an appropriate volume of 1×10 6 vg or more in a single vial in a deliverable volume. In some embodiments, administration of 1×10 9 An initial dose of 1 × 10 viral genomes was administered, and subsequently 1 × 10 11 The dosage of vg.

[0192] In some embodiments, the effective amount is a dose that achieves 1-2,000 pg / ml of polypeptide in the blood, CSF, or desired target tissue. In some embodiments, the effective amount is a dose that achieves 200-1300 pg / ml of polypeptide in the blood, CSF, or desired target tissue. In some embodiments, the effective amount is a dose that achieves an average of about 550 pg / ml in the circulation of adult subjects or within a range of 200 above or below the average. In some embodiments, the effective amount is a dose that achieves an average of about 950 pg / ml in the circulation of pediatric subjects or within a range of 300 above or below the average.

[0193] In some embodiments, the therapeutically effective dose doubles the gene expression of the nucleic acid sequence encoding mammalian s-KL or a functional variant thereof. In some embodiments, the effective dose increases expression by at least two times. In some embodiments, the effective dose increases gene expression by at least three times, or at least four times, or at least five times, or at least six times, or at least eight times, or at least ten times.

[0194] The number of times the composition is administered to a subject in need may depend on any one or more of many factors and is determined by a medical professional, including a disease or condition and its severity, and the subject's response to the formulation. The administration of a therapeutically effective amount of the expression vector occurs at least once. In other embodiments, multiple administrations occur within a given period, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more, to achieve a desired therapeutic effect. The dosage administered each time and / or the frequency of administration can be adjusted as needed based on the subject's condition and physiological response. In the case where the subject's condition is not improved, at the doctor's discretion, the composition can be administered for a long time, i.e., within an extended period, included in the duration of the subject's entire life, to mitigate or otherwise control or limit the subject's disease or symptoms of condition. In some embodiments, for example, where the subject's state does improve, at the doctor's discretion, the composition can be administered continuously.

[0195] In some embodiments, treatment can be applied in the time period of about one hour. In some embodiments, treatment can be applied in the time period of about one hour to about 24 hours in a day. In some embodiments, treatment can be applied 24 hours a day, for many days, including only 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 15 days and 20 days by way of example. In some other embodiments, treatment can temporarily reduce or temporarily suspend the time of a certain length (that is, "intermittent period"). The length of the intermittent period varies greatly between such as 2 days and 1 year, including only 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days and 365 days by way of example. In some other embodiments, treatment can temporarily reduce the time of a certain length. The dose reduction during the reduction period can be 10% to less than 100%, including by way of example only 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% and 95%.

[0196] Combination therapy

[0197] The treatment method may include co-administration (i.e., administration within the same treatment period) of another active agent known to be effective in treating movement disorders (e.g., associated with neuromuscular diseases and disorders). Representative additional active agents include riluzole and edaravone and combinations thereof. In some embodiments, the treatment method is used in combination with direct infusion of recombinant proteins or gene therapy expressing 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.

[0198] Although the present invention is described in conjunction with the above aspects and embodiments, the foregoing description and the following examples are intended to illustrate rather than 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 belongs.

[0199] Examples

[0200] Materials and methods

[0201] Animal housing. The mouse model used in this study was based on (SOD1 G93A , also abbreviated as SOD1) ALS mouse model was generated. The animals had free access to food and water and were maintained under standard temperature conditions (22±2°C) and a 12-h light / dark cycle (300lux / 0lux).

[0202] Transgenic mice. Transgenic mice with the G93A human SOD1 mutation (C57bl6-Tg[SOD1-G93A]1Gur) were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). Hemizygous C57bl6 SOD1 G93A Male. Offspring were identified by PCR of DNA extracted from tail tissue. Experimental procedures were approved by the Ethics Committee of the Universitat Autònoma de Barcelona. The following experimental groups of mice were used: SOD1 G93AWT littermates (n=15-19 / condition, 7-10 per sex) were injected with AAV8-hDes-s-KL (SEQ ID NO: 7) or AAV8-mock (SEQ ID NO: 8). To compensate for the experimental groups, animals were assigned based on body weight and litter origin. Animals were weighed every 4 weeks to monitor their overall health.

[0203] Table B. Sequence legend for SEQ ID NO: 8:

[0204]

[0205] Virus production and injection. Mouse s-KL cDNA was cloned between AAV2 ITRs under the control of the human desmin promoter. AAV8 virus stock was produced by triple transfection of expression plasmids, Rep8Cap2 plasmids containing AAV genes, and pXX6 plasmids containing adenoviral genes required for helper viruses into HEK293-AAV cells. Plasmids encoding AAV2 ITRs are described in Piedra et al., Hum. Gene Ther. Methods 26(1):35-42 (2015). Plasmids encoding pXX6 are described in Xiao et al., J. Virol. 72(3):2224-2232 (1998). Plasmids encoding Rep8Cap2 are described in Gao et al., Proc. Natl. Acad. Sci. USA 99(18): 11854-11859 (2002).

[0206] AAV particles were purified by iodixanol gradient. Titration was assessed by picogreen (Invitrogen) quantification and calculated as viral genomes / ml (vg / ml). A control serotype-matched AAV empty vector (mock) was used as a control.

[0207] For intravenous administration, 1.8 × 10 14 and 3×10 14 vg / kg of AAV8-s-KL or AAV8-mock was injected into the tail vein of 6-week-old mice in a total volume of 250 μl of suspension.

[0208] 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).

[0209] One μg of RNA was reverse transcribed using 10 μmol / l DTT, 200U M-MuLV reverse transcriptase (New England BioLabs), 10u RNase Out ribonuclease inhibitor (Invitrogen), 1 μmol / l oligo(dT) and 1 μmol / l random hexamer (BioLabs). The reverse transcription cycle conditions were 25°C for 10 minutes, 42°C for 1 hour and 72°C for 10 minutes. The mRNA expression of s-KL was analyzed by a specific primer set (s-KL-Fw: 5'-TCATAATGGAAACCTTAAAAGCAA -3' (SEQ ID NO: 9) and s-KL-Rv: 5'-CACTGGGTTTTGTCAAAGGA -3' (SEQ ID NO: 10)) using the Taqman probe 5'-6FAM-3'BHQ-1s-KL-Pr: 5'-AGAAGAGTCCTCGCCGGATGCTGTA-3' (SEQ ID NO: 11). The expression level of s-KL was normalized using mouse 36B4 (m36b4-Fw (Rplp0): 5'-ATGGGTACAAGCGCGTCCTG-3' (SEQ ID NO: 12); m36b4-Rv (Rplp0): 5'-AGCCGCAAATGCAGATGGATC-3' (SEQ ID NO: 13); probe 5-HEX-3'BHQ-1 m36B4-Pr (Rplp0): 5'-TGTGGAGACTGAGTACACCTTCCCA-3' (SEQ ID NO: 14)).

[0210] Thermal cycling conditions included 5 min polymerase activation at 95° C., 45 15 sec cycles at 95° C., 30 sec at 60° C., 30 sec at 72° C., and 5 sec at 65° C. to 95° C. (increments of 0.5° C. every 5 sec.) Fluorescence detection was performed at the end of PCR extension, and melting curves were analyzed by monitoring the fluorescence of the Taqman probe.

[0211] Spinal cord organotypic culture. Sprague-Dawley rats on postnatal day 8 were euthanized and the spinal cords were aseptically harvested and placed in ice-cold high-glucose Gey's Balanced Salt Solution, from which the meninges were removed. The spinal cords were cut transversely into 350 mm thick slices using a chopper. The L4-L5 lumbar vertebrae slices were transferred to Millicell-CM porous membranes in plates containing incubation medium.

[0212] After axotomy during the culture procedure, a large number of neurons die naturally, and glial cells show strong reactivity, so the culture needs to be left for a week to stabilize. Then, add one microliter of 10 8 IU of Ad5-CMV-s-KL or Ad5-CMV-blank droplets or culture medium as a control. In alternative assays, adeno-associated viruses, i.e., AAV9-CMV-s-KL, AAV9-CMV-blank vector or culture medium were used as controls. The slices were maintained for another week to allow transgene expression. 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, which was then replaced with culture medium. At 19 DIV, slices were collected and fixed with paraformaldehyde 4% for histological staining.

[0213] Electrophysiological testing. Motor nerve conduction testing was performed every 4 weeks from 8 to 16 weeks of age. The sciatic nerve was stimulated by single pulses (Grass S88 stimulator) delivered by needle electrodes placed at the sciatic notch. Evoked compound muscle action potentials (CMAPs) from the gastrocnemius (GM) and plantar interosseous muscles (PL) were recorded with microneedle electrodes. The electromyographic signals were amplified and displayed on a digital oscilloscope (Tektronix 450S) for measurement of the amplitude and latency of the CMAP.

[0214] Motor evoked potentials were assessed to evaluate central motor pathways. Supramaximal electrical stimulation was delivered by subcutaneous placement of needle electrodes above the skull overlying the sensorimotor cortex, and MEPs from the GM muscle were recorded using microneedle electrodes.

[0215] Motor test. The rotarod test was used to test the activity of treated and untreated SOD1 G93AAnimals were evaluated for motor coordination, strength, and balance. Each mouse was placed in a rotarod at a constant speed of 14 rpm three times, and the maximum time until falling was recorded. A maximum time of 180 seconds was set. Testing was performed every other week from 8 weeks of age to 20 weeks of age. The onset of clinical disease in each mouse was determined as the first week in which the duration was less than 180 seconds.

[0216] 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.

[0217] For NMJ labeling, GM muscle was cryopreserved in 30% sucrose in PBS, 60 μm longitudinal sections were cut continuously with a cryostat, and collected in a continuous series of 10. 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 secondary antibodies conjugated with Alexa 594 (1:200; Life Science) and Alexa 488 conjugated α-bungarotoxin (1:200, B-13422, Life Technologies). The proportion of endplates innervated was determined by classifying each endplate as occupied (when presynaptic terminals overran the endplate) or vacant (no presynaptic markers contacting the endplate). At least four fields were analyzed per muscle, totaling >100 endplates. For collateral sprouting, the number of sprouts per endplate was counted as neurofilament-positive projections from presynaptic terminals or pre-terminal nodes on confocal z-projections. For analysis, the total number of sprouts and the proportion of occupied endplates innervated by collateral sprouts were considered.

[0218] Data analysis.All experiments were performed by researchers who blinded the different treatments of each mouse group and randomly assigned animals to groups taking into account body weight and litter.Data are expressed as mean ± SEM.Statistical analysis of electrophysiological and motor test results was performed using one-way or repeated measures ANOVA and Tukey post-hoc test.For MEP electrophysiological results, Student's ttest was applied.For clinical disease onset, log-rank (Mantel-Cox) test was applied.Histological and molecular biological data were analyzed using t-Student, one-way or 2-way ANOVA and Tukey or Holm-Sídák post-hoc test.

[0219] result

[0220] 1-Klotho in SOD1 G93 Down-regulated in mouse models

[0221] Once mRNA was extracted from several tissues of the mouse model, it was observed that SOD1 G93A The mRNA expression of secreted Klotho (s-KL) was decreased in the motor cortex, spinal cord, gastrocnemius and soleus muscles of the 16th week of the disease end stage, relative to the wild type (WT) s-KL expression ratio (n = 7SOD1, 3-10 WT mice per group, ***p<0.001, **p<0.01, *p<0.05). Data are in Figure 1 Shown in Figure , where s-KL mRNA expression (fold change) is presented as mean ± SEM in several investigated tissues.

[0222] 2- In vitro assay. Klotho protects spinal motor neurons from excitotoxicity in organotypic culture

[0223] Rat spinal cord organotypic slices were treated with AAV9 encoding s-KL (SEQ ID NO: 15) or blank sequence (SEQ ID NO: 16) and exposed to glutamate. Figure 2A Fluorescence microscopy images and Figure 2B Shown in the bar graph, where the second bar of each group corresponds to glutamate-induced excitotoxicity (GLUT+). Overexpression of s-KL protected neurons located in the ventral horn (VH) of the spinal cord from glutamate-induced excitotoxicity compared to untreated controls or AAV9-blank. All data are mean ± SEM (n = 6 per group, ***p < 0.001, **p < 0.01, *p < 0.05).

[0224] Tables C and D list specific fragment sequences in each of SEQ ID NOs: 15 and 16, respectively.

[0225] Table C. Sequence Legend for SEQ ID NO: 15

[0226]

[0227] Table D. Sequence Legend for SEQ ID NO: 16

[0228]

[0229] An equivalent assay with equivalent results was performed with rat spinal cord organotypic slices treated with adenoviral vectors encoding sKL or blank sequences and exposed to glutamate (GLUT+) that induces cytotoxic effects. Data from this alternative assay are not shown, but overexpression of s-KL protected neurons located in the ventral horn (VH) of the spinal cord from glutamate-induced excitotoxicity and survival compared to untreated controls (GLUT-) or Ad-blank (which showed high mortality).

[0230] 3-Gene therapy strategy

[0231] At 6 weeks of age, SOD1 female mice were treated with 1.8 × 10 14 vg / kg or 3×10 14 vg / kg of AAV-hDesmin-s-KL-WPRE vector (n = 15) were treated intravenously. WT (n = 10) and SOD1 female mice (n = 15) were treated with AAV-hDesmin-blank-WPRE at the same dose as a control. The human desmin promoter was used to restrict expression to striated muscle. The WPRE sequence was used to stabilize mRNA. Animals were subjected to rotarod and grip strength tests weekly, and nerve conduction tests were performed at 8, 12 and 16 weeks. Tissues were collected at the terminal stage of the disease and processed for histological and expression analysis.

[0232] Figure 3 A schematic diagram of an example gene therapy strategy is depicted in FIG.

[0233] 4- Improved Compound Muscle Action Potential (CMAP)

[0234] After gene therapy according to the previous section, motor nerve conduction testing was performed using two needle electrodes placed at the sciatic notch, and the sciatic nerve was stimulated with a single pulse of 20 μs duration. At 8, 12, and 16 weeks of age, compound muscle action potentials (CMAPs) from the tibialis anterior (TA) and plantar interosseous muscles (PL) were recorded with microneedle electrodes.

[0235] Treatment promoted significant preservation of CMAP amplitude in the tibialis anterior and plantar interosseous muscles, indicating that s-KL overexpression promoted SOD1 G93A Motor function improved in female mice.

[0236] These data suggest that motor function is maintained due to good communication between motor neurons and muscle cells.

[0237] CMAP values ​​(amplitude, in mV) of wild type (WT), SOD1 mimetic, SOD1 s-KL low dose, and SOD1 s-KL high dose were Figure 4A (plantar muscles, PL) and Figure 4B (tibialis anterior, TA). (***p<0.001, **p<0.01, *p<0.05, SOD1 mimetic vs. SOD1 high dose, mean±SEM).

[0238] 5- Increased motor evoked potential amplitude (MEP)

[0239] To evaluate the central corticospinal descending pathway, motor evoked potentials (MEPs) from TA muscles were recorded. The motor cortex was electrically stimulated with pulses of 0.1 millisecond duration and ultra-high intensity, delivered with needle electrodes placed subcutaneously above the skull. Gene therapy increased the amplitude of MEPs, indicating enhanced connectivity between upper motor neurons and lower motor neurons (***p<0.001, **p<0.01, *p<0.05, relative to SOD1 mimics, mean ± SEM). Figure 5 The data are depicted in , where the first bar in the group represents the assay in SOD1 mimetic, the second bar represents SOD1 s-KL low dose, and the third bar is SOD1 s-KL high dose.

[0240] 6-Klotho enhances SOD1 G93A Motor function in mice

[0241] The rotarod test was performed to assess the motor coordination and balance of the animals. The mice were placed on a rotating rod at a constant speed of 14 rpm; 180 seconds was chosen as the cutoff time. To test the grip strength of the forelimbs and hindlimbs, the mice were placed above a metal grid and allowed to grasp the bars with all four paws while the tail was pulled apart. During the test, the instrument recorded the peak pulling force before release.

[0242] Compared with untreated controls, overexpression and secretion of s-KL in muscle improved SOD1 G93A Motor performance of mice on the rotarod. As assessed by the grip strength test, high-dose treated mice also had improved strength at the terminal stage (***p<0.001, **p<0.01, *p<0.05, SOD1 high dose vs. SOD1 mimetic, mean ± SEM). Data are available at FIG. 6A to FIG. 6BAs depicted in FIG. 1 , for a rotarod apparatus, Fig. 6A The time (seconds) in , and for grip strength, Figure 6B The force in (g).

[0243] 7- Delayed onset of clinical illness

[0244] The onset of clinical disease for each mouse in the previous section was determined as the first week in which the animal could not maintain 180 seconds on the rotarod. Treatment was able to significantly delay the onset of disease, and at 16 weeks, only 35% of the high-dose treated mice (treated with AAV9-s-KL vector SEQ ID NO: 15) showed motor deficits compared to the mock-treated mice (treated with AAV9-empty vector SEQ ID NO: 16). Figure 7 The data depicted in show the probability of seizures for each mouse type.

[0245] 8-s-KL maintains neuromuscular junction (NMJ) and muscle mass

[0246] Gastrocnemius longitudinal sections were labeled with neurofilament 200 (NF200), anti-synaptophysin, and alpha-bungarotoxin (BTX). Endplates were classified as occupied (when presynaptic terminals covered or contacted the endplate) or vacant (no presynaptic markers contacting the endplate). Secretion of s-KL from skeletal muscle significantly increased the proportion of NMJs occupied by presynaptic terminals and attenuated muscle wasting, as evidenced by greater muscle mass (***p<0.001, **p<0.01, *p<0.05, SOD1 mimetic vs. SOD1 high dose, data presented as mean ± SEM). FIG. 8A to FIG. 8C The data are depicted in . Fig. 8A are fluorescence microscopy images showing sections labeled with NF200 (dark grey) and sections labeled with BTX (light grey). Figure 8B The percentage of endplates occupied in WT mice as well as mock (SOD1 mimetic) and treated (SOD1s-KL) are indicated. Figure 8C Muscle mass / body weight (mg / g) of WT mice as well as mock (SOD1 mimetic) and treated (SOD1s-KL) are indicated.

[0247] 9-Muscle s-KL secretion protects spinal motor neurons

[0248] At 16 weeks of age, mice were transcardially perfused with 4% paraformaldehyde and lumbar spinal cords were harvested. For spinal MN assessment, the spinal cords were postfixed over a 4-hour period, cryopreserved in 30% sucrose in PBS, and 20 μm transverse sections were serially cut using a cryostat. One of every two slides from each animal covering the lumbar region from L4 to L6 was stained with cresyl violet. Motor neurons were identified by their localization in the ventral horn and by strict size and morphological criteria.

[0249] s-KL secreted by skeletal muscle protected motor neurons in the lumbar spinal cord, a 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). Figure 9A-9B Courage is depicted in it. Fig.9A is a white light microscopy image showing a section labeled with cresyl violet (dark grey), which stains Nissl substance in neurons and nuclei, thereby revealing neuronal structure. Fig. 9B Neuronal structures in WT mice (WT mimic), mimic (SOD1 mimic), and treated (SOD1sKL) mice were quantified.

[0250] 10-Klotho protects the spinal cord from neuroinflammatory damage in ALS

[0251] Intramuscular delivery of s-KL reduced processed SOD1 as assessed by Iba1 fluorescence G93A Microglial reactivity in the lumbar spinal cord of mice. Astrocyte proliferation was analyzed by GFAP and vimentin labeling and corrected to near WT levels (n = 8 / group, two-way ANOVA test, **p<0.01, *p<0.05). Data are expressed as mean ± SEM. FIG. 10A to FIG. 10D The data are depicted in . Fig. 10A are fluorescence microscopy images showing sections labeled for lba1 (top row), sections labeled for GFAP (middle row), and sections labeled for vimentin (bottom row). FIG. 10B to FIG. 10D Shows Fig. 10A Quantification of staining in WT mice (WT mock), mock (SOD1 mock), and treated (SOD1 sKL) in , demonstrating reduced staining of Iba1, GFAP, and vimentin after s-KL treatment. Fig. 10B The integrated density (in arbitrary units) of the images labeled with Lba1 is indicated. Fig. 10C The integrated density (in arbitrary units) of the images for GFAP labeling is indicated. Fig. 10D The integrated density (in arbitrary units) of the images for vimentin labeling is indicated.

[0252] 11-Gene therapy strategies using AAVmyo vectors

[0253] AAVmyo is an AAV serotype (Tabebordbar et al., Cell 184(19):4919-4938 (2021)) that has a tropism for striated muscle with higher transduction efficiency in mice and non-human primates. In order to reduce the dose of AAV vector administered and avoid possible side effects, the AAVmyo serotype was tested for s-KL gene therapy. Fig.11 A schematic view of an embodiment of a gene therapy strategy is depicted in FIG.

[0254] Six-week-old SOD1 mice were treated with 2×10 12 vg / kg (n=6; AAVmyo-s-KL low dose) or 8×10 12 vg / kg (n=8; AAVmyo-s-KL high dose) of AAVmyo-hDesmin-sKL-WPRE expression vector was intravenously treated. Figure 3 Compared to the doses used in gene expression experiments using AAV8 expression vectors, AAVmyo-s-KL high dose and AAVmyo-sKL low dose were 1 / 150 and 1 / 37.5, respectively. WT (n=14) and SOD1 female mice (n=18) were treated with AAVmyo-hDesmin-blank-WPRE at the same dose as controls. As in previous studies, animals were followed up and euthanized at 16 weeks of age for tissue collection.

[0255] 12-AAVmyo-Des-sKL improves compound muscle action potential (CMAP)

[0256] When compared with control SOD1 G93A When compared to mice treated with higher doses of AAVmyo-Des-sKL, the expression of SOD1 G93A The plantar (PL) and GM compound muscle action potential (CMAP) amplitudes of mice were significantly preserved from week 12 and week 8, respectively. Using this novel serotype, a similar level of neuromuscular connection preservation as AAV8-Des-sKL was achieved while reducing the administered virus titer by 37.5-fold (two-way ANOVA test, *p<0.05; *SOD1 mimetic vs. SOD1 sKL high dose; data are expressed as mean ± SEM).

[0257] Fig.11 A to Fig.11 Data are depicted in B. CMAP values ​​(amplitude, in mV) of wild-type WT mimic, SOD1 mimic, SOD1 Myo-AAV-s-KL low dose, and SOD1 Myo-AAV-s-KL high dose were Fig.11 A (plantar muscles, PL) and Fig.11 The results are presented in B (tibialis anterior muscle, TA). *p<0.05, SOD1 mimetic versus SOD1Myo-AAV-s-KL, mean ± SEM).

[0258] 13-AAVmyo-Des-sKL increases motor evoked potential amplitude (MEP)

[0259] Compared with SOD1-mimetic mice, treated SOD1 G93A The motor evoked potential amplitude of mice was higher, which also indicates a higher preservation of central motor pathways. AAVmyo-Des-sKL treatment preserved MEP amplitude to a greater extent than AAV8-DessKL treatment (two-way ANOVA test, **p<0.01, *p<0.05; *SOD1 mimics vs. SOD1 sKL high dose; data are expressed as mean ± SEM). The data are depicted in Figure 12, where the first bar in the group represents the assay in the SOD1 mimic, the second bar represents the SOD1 AAVmyo-s-KL low dose, and the third bar is the SOD1 AAVmyo-s-KL high dose.

[0260] 14-Klotho enhances SOD1 G93A Motor function in mice

[0261] A slower progression of motor decline was observed in SOD1 mice treated with AAVmyo-Des-sKL. The animals' strength was also preserved, especially in the high-dose treated group, where SOD1 mice performed better than animals injected with AAV8-DessKL (two-way ANOVA, ***p<0.001, **p<0.01, *p<0.05; *SOD1 mimetic vs. SOD1 sKL high dose, #SOD1 mimetic vs. WT mimetic; mean ± SEM). Data are in Fig.13 A to Fig.13 As depicted in B, for the rotarod apparatus, Fig.13 A in seconds, and for grip strength, Fig.13 The force in B (g).

[0262] 15-Delayed onset of clinical illness

[0263] The onset of clinical disease in both groups of AAVmyo-Des-sKL treated SOD1 mice was significantly delayed, similar to that of mice treated with a high dose of AAV8-Des-sKL (log-rank Mantel-Cox test). The data are depicted in Figure 14, which shows the probability of onset for each mouse type.

[0264] Other publications incorporated herein by reference

[0265] Zeldich et al., “Klotho Is Neuroprotective in the Superoxide Dismutase (SOD1G93A) Mouse Model of ALS” J. Mol. Neurosci. 69(2): 264-285 (2019)

[0266] Minamizaki et al., “Soluble Klotho causes hypomineralization in Klotho-deficient mice.” J. Endocrinol. 237(3): 285-300 (2018)

[0267] WO2017085317 (Autonomous University of Barcelona et al.)

[0268] All patent publications and non-patent publications are indicative of the level of skill of those skilled in the art to which the present disclosure pertains. All of these publications, including any specific portions thereof cited, are incorporated herein by reference to the same extent as if each individual publication was expressly and individually indicated to be incorporated by reference.

[0269] Although the present invention has been described herein with reference to specific embodiments, it should be understood that these embodiments are merely illustrative of the principles and applications of the present invention. Therefore, it should be understood that many modifications may be made to the illustrative embodiments, and other arrangements may be designed without departing from the spirit and scope of the present invention as defined by the appended claims.

Claims

1. A gene construct comprising a nucleic acid 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. 2 . The gene construct according to claim 1 , wherein the mammalian s-KL encoded by the nucleic acid sequence is human s-KL having the amino acid sequence of SED ID NO:

1. 3 .

3. The gene construct according to claim 2, wherein the nucleic acid sequence is SEQ ID NO:

3.

4. The gene construct of claim 1, wherein the mammalian s-KL encoded by the nucleic acid sequence is murine s-KL having the amino acid sequence of SEQ ID NO:

2. The gene construct according to claim 4 , wherein the nucleic acid sequence is SEQ ID NO:

4. 6 . The gene construct according to claim 1 , wherein the functional variant has at least 85% amino acid sequence identity with SEQ ID NO:

1.

7. The gene construct according to claim 1, wherein the functional variant has at least 88% amino acid sequence identity with SEQ ID NO:

1.

8. The gene construct according to claim 1, wherein the functional variant has at least 95% amino acid sequence identity with SEQ ID NO:

1.

9. The gene construct according to claim 1, wherein the functional variant has at least 98% amino acid sequence identity with SEQ ID NO:

1.

10. The genetic construct according to any one of claims 1 to 9, wherein the first promoter is a mammalian desmin promoter.

11. The gene construct according to claim 10, wherein the mammalian desmin promoter is the human desmin promoter. 12 . The gene construct according to claim 11 , wherein the human desmin promoter has a nucleic acid sequence of SEQ ID NO:

5.

13. The genetic construct according to any one of claims 1 to 12, further comprising a second promoter operably linked to the nucleic acid sequence encoding the mammalian s-KL or a functional variant thereof; and wherein the first promoter and the second promoter are different.

14. The genetic construct of claim 13, wherein the second promoter is a muscle cell-specific promoter; or wherein the second promoter is a neuronal cell-specific promoter.

15. The genetic construct of claim 13, wherein the second promoter is a constitutive promoter.

16. The genetic construct of claim 15, wherein the constitutive promoter is a cytomegalovirus (CMV) promoter.

17. The genetic construct of claim 13, wherein the second promoter is an inducible promoter.

18. The genetic construct of claim 17, wherein the inducible promoter is a zinc-driven metallothionein promoter.

19. A plasmid comprising the gene construct according to any one of claims 1 to 18 and an initiation sequence operably linked to the first promoter.

20. An expression vector comprising (b) the gene construct according to any one of claims 1 to 18 or the plasmid according to claim 19, wherein the expression vector may or may not have myocyte tropism; or (b) a nucleic acid construct comprising a first promoter that functions in muscle cells, neuronal cells or induced pluripotent stem cells (iPSCs), the first promoter being operably linked to a nucleic acid sequence encoding mammalian s-KL or a functional variant thereof, wherein the expression vector has muscle cell tropism. The expression vector according to claim 20 , which is a viral expression vector.

22. The expression vector according to claim 21, which is an adeno-associated virus (AAV) vector having a serotype with muscle cell tropism; or it is an AAV vector having a serotype with neuronal cell tropism.

23. The expression vector of claim 22, wherein the serotype of the AAV vector has myocyte tropism.

24. The expression vector of claim 23, wherein the AAV vector is an AAV1, AAV8, AAV9 or AAVmyo vector; or wherein the AAV vector comprises an AAV capsid polypeptide comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 17-29.

25. The expression vector of claim 24, wherein the AAV vector is an AAVmyo vector.

26. The expression vector of claim 24, wherein the AAV vector comprises an AAV capsid polypeptide comprising or containing the amino acid sequence of any one of SEQ ID Nos: 17-29.

27. The expression vector of claim 26, wherein the AAV vector comprises an AAV capsid polypeptide comprising or containing the amino acid sequence of SEQ ID NO: 17 or 26.

28. The expression vector of claim 24, wherein the AAV vector is an AAV9 vector.

29. The expression vector of claim 22, wherein the serotype of the AAV vector has neuronal cell tropism.

30. The expression vector of claim 29, wherein the AAV vector is an AAV1 vector.

31. The expression vector of claim 29, wherein the AAV vector is an AAV8 vector.

32. The expression vector of claim 29, wherein the AAV vector is an AAV9 vector.

33. The expression vector of claim 20, which is a lipid-based vector.

34. The expression vector of claim 33, which is a lipid nanoparticle (LNP) or a liposome.

35. The expression vector according to any one of claims 20 to 34, further comprising at least one non-coding regulatory element.

36. The expression vector of claim 35, wherein the regulatory element is any one or more of the following: a poly A sequence, a protein translation start site consensus nucleic acid sequence, a post-transcriptional regulatory element nucleic acid sequence, a 5' inverted terminal repeat nucleic acid sequence, a 3' inverted terminal repeat nucleic acid sequence, or an intron.

37. The expression vector of claim 36, wherein the poly A sequence is an SV40 Poly A sequence.

38. The expression vector of claim 37, wherein the post-transcriptional regulatory element nucleic acid sequence is a hepatitis B virus post-transcriptional regulatory element (HPRE) or a woodchuck hepatitis post-transcriptional regulatory element (WPRE).

39. A pharmaceutical composition comprising the plasmid according to claim 19 or the expression vector according to any one of claims 20 to 38 and a pharmaceutically acceptable carrier.

40. An isolated cell comprising a genetic construct according to any one of claims 1 to 18 or a plasmid according to claim 19; wherein the isolated cell is a human muscle cell, a human neuronal cell, or a human induced pluripotent stem cell (iPSC) capable of differentiating into a muscle cell or a neuronal cell.

41. The isolated cell of claim 40, which is a muscle cell.

42. The isolated cell of claim 41 which is a skeletal muscle cell.

43. The isolated cell of claim 41 which is a striated muscle cell.

44. The isolated cell of claim 40, which is a neuronal cell.

45. The isolated cell of claim 44, wherein the neuronal cell is a motor neuron.

46. ​​The isolated cell of claim 40, which is an iPSC capable of differentiating into a muscle cell or a neuronal cell.

47. A cell therapy comprising administering to a subject in need thereof the isolated cells of any one of claims 40 to 46.

48. A method of treating a movement disorder, the method comprising administering the pharmaceutical composition of claim 39 to a subject in need thereof.

49. The method of claim 48, wherein the subject suffers from a disease or condition characterized by or manifested by symptoms of movement disorders.

50. The method of claim 48, wherein the disease or disorder is a neuromuscular disease or disorder.

51. The method of claim 50, wherein the neuromuscular disease is amyotrophic lateral sclerosis (ALS).

52. The method of claim 51, wherein the ALS is sporadic ALS (sALS).

53. The method of claim 51, wherein the ALS is familial ALS (fALS).

54. The method of claim 35, wherein the subject in need thereof does not exhibit movement disorders but is susceptible to movement disorders.

55. The method of any one of claims 35 to 41, wherein the pharmaceutical composition is administered parenterally.

56. The method of claim 42, wherein the pharmaceutical composition is administered intravenously, intramuscularly, intracranially, or intrathecally.

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