Gene therapy to treat protein misfolding disorders

Abstract

The disclosed gene therapy vectors, compositions, and methods, such as adeno-associated viruses (AAVs), for treating diseases caused by misfolded proteins are provided. The disclosed rAAVs include nucleotide sequences encoding the BCL2-associated atanogen 3 (BAG-3) protein, and methods for administering these rAAVs to subjects of interest to treat diseases and disorders associated with protein misfolding and / or aggregation, thereby resulting in increased targeting of agglutinating proteins for degradation by the BAG3-mediated selective macroautophagy pathway, thereby restoring protein homeostasis. The disclosed gene therapy vectors, such as rAAV constructs, are also used in the treatment of inclusion body myositis (IBM) associated with Paget's disease of bone and frontotemporal dementia (IBMPFD), as well as multisystem proteinosis.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 499,712, filed on 2 May 2023, which is incorporated herein by reference in its entirety.

[0002] Integration by referencing electronically submitted documents The computer-readable nucleotide / amino acid sequence listing submitted concurrently with this specification and identified as follows is incorporated in its entirety by reference: 58564_SeqListing.xml; size: 54,030 bytes; created: April 30, 2024.

[0003] This disclosure provides gene therapy vectors, such as recombinant adeno-associated virus (rAAV) constructs, designed for the treatment of diseases and disorders associated with protein misfolding and / or aggregation, such as neurodegenerative protein misfolding disorders, e.g., amyotrophic lateral sclerosis (ALS) and protein aggregation myopathy (PAM), e.g., limb-girdle muscular dystrophy type 1A (LGMD1A). This disclosure also provides gene therapy vectors, such as rAAV constructs, designed for the treatment of inclusion body myositis (IBM) associated with Paget's disease of bone and frontotemporal dementia (IBMPFD), and / or multisystem protein disorders. The disclosed rAAVs include nucleotide sequences encoding the BCL2-associated atanogen 3 (BAG-3) protein, and methods for administering these rAAVs to subjects requiring treatment for diseases and disorders associated with protein misfolding and / or aggregation. [Background technology]

[0004] Most proteins must fold properly into a clearly defined three-dimensional structure in order to function. Furthermore, cells must maintain protein homeostasis (proteostasis) and balance the synthesis, folding, transport, and timely degradation of proteins that are no longer needed, or proteins that are damaged or misfolded beyond repair or refolding. Therefore, cells have evolved a vast and tightly connected proteostatic network (PN) that encompasses translation mechanisms, molecular chaperones and co-chaperones, the ubiquitin-proteasome system (UPS), and autophagy mechanisms to regulate proteostasis. These multiple quality control mechanisms ensure that nascent polypeptides fold properly, mature proteins maintain their functional conformation, and misfolded proteins are correctly refolded or degraded.

[0005] However, despite the presence of numerous effective regulatory systems, the accumulation of misfolded proteins, leading to proteotoxicity and disruption of protein homeostasis, is not uncommon. Factors such as mistranslation by defective ribosomes, cellular senescence, physiological stressors such as oxidative stress and pH changes, and protein mutations can all shift the equilibrium from its native conformational state to an unfolded or partially folded state, causing misfolding. Compared to unfolded proteins, partially folded protein intermediates are more susceptible to degradation by intracellular quality control systems. However, these folding intermediates are also more prone to self-assembly, forming higher-order aggregates, due to the presence of large, continuous patches of surface hydrophobicity (Non-Patent Literature 1). These higher-order aggregates are highly resistant to degradation and tend to mistarget the wrong cellular compartments. Protein misfolding or alterations in the stability and aggregation of proteins are underlying mechanisms for many human disorders collectively known as proteinopathy, including, but not limited to, those affecting the nervous system (neuroproteinopathies), such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), and Huntington's disease (HD); striated muscle (protein aggregate myopathy (PAM)), such as limb-girdle muscular dystrophy type 2Q (LGMD2Q / LGMDR17) and limb-girdle muscular dystrophy type 1A (LGMD1A); and the cardiovascular system (protein-toxic heart disease, such as desmin-related myopathy (DRM)).

[0006] There are no known effective therapies for treating diseases associated with misfolded proteins or protein aggregates, and available treatments remain temporary relief. Therefore, there is a need for therapies that reverse or eliminate protein misfolding and aggregation, thereby restoring proteostasis. Clearance of misfolded proteins by upregulating autophagy represents a promising therapeutic strategy in these diseases. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Fink AL.1998.Fold Des.3(1):R9-R23 [Overview of the Initiative]

[0008] Provided herein are polynucleotide sequences comprising a transcriptional regulatory element and a nucleotide sequence encoding human Bcl-associated atanogen 3 protein (hBAG3). In some embodiments, the nucleotide sequence encoding hBAG3 is at least 90% identical to nucleotides 1112-2839 of SEQ ID NO: 1, or at least 90% identical to nucleotides 981-2708 of SEQ ID NO: 2, which encodes a protein that retains BAG3 activity. In some embodiments, the nucleotide sequence encoding hBAG3 comprises nucleotides 1112-2839 of SEQ ID NO: 1 or nucleotides 981-2708 of SEQ ID NO: 2. In some embodiments, the nucleotide sequence encoding hBAG3 comprises the nucleotide sequence shown in SEQ ID NO: 8.

[0009] In one embodiment, the disclosure includes using the BAG-3 protein to increase the recognition and degradation of misfolded or aggregated proteins or protein inclusions. In certain embodiments, the recognition and degradation of misfolded or aggregated proteins via the BAG3 protein described herein can treat or prevent diseases or disorders associated with misfolded or aggregated proteins.

[0010] For example, in one embodiment, the degradation of mutant copper-zinc superoxide dismutase enzyme (SOD1) via BAG3 as described herein can treat or prevent amyotrophic lateral sclerosis (ALS). In another exemplary embodiment, the degradation of mutant myotilin via BAG3 as described herein can treat or prevent myotilinopathy, such as limb-girdle muscular dystrophy 1A (LGMD1A), and other diseases associated with the degradation of misfolded or aggregated proteins.

[0011] In one embodiment, the present disclosure provides compositions and methods for robust and long-term increase in BAG3 expression and / or activity in muscle or CNS tissue in the treatment of protein misfolding disorders or disabilities. In certain embodiments, the compositions include nucleic acid molecules, expression vectors, proteins, peptides, small molecules, etc., that increase the expression, activity, or both of the BAG3 protein.

[0012] This disclosure provides methods and compositions for treating or preventing diseases or disorders associated with misfolded proteins or protein aggregates in mammals. This disclosure describes gene therapy vectors expressing the human Bcl-associated atanogen 3 (BAG3) gene, such as AAV, and methods for delivering BAG3 to muscles and CNS to reduce the accumulation of misfolded or aggregated proteins and / or prevent or treat diseases or disorders associated with misfolded proteins or protein aggregates. In addition, this disclosure provides combination therapies and approaches for promoting nerve regeneration and further improving muscle function using gene therapy vectors for delivering BAG3 to address protein misfolding and aggregation, and gene therapy vectors for delivering the neurotrophic factor neurotrophin-3 (NT-3).

[0013] This disclosure is at least in part based on the finding that increasing BAG3 expression (e.g., using a vector) in animal models of myofibrild myopathy (MFM) with protein aggregation, e.g., LGMD1A, reduced the accumulation of intracellular inclusions of disease-associated cytotoxic protein aggregates, reduced motor neuron cell death, and improved muscle function. Similarly, in a mouse model of ALS, a neurodegenerative disease associated with protein misfolding, BAG3 gene therapy reduced neuronal cell death, improved muscle function, extended overall survival, and amplified the synergistic effects observed when BAG3 therapy was combined with NT3.

[0014] Accordingly, in one embodiment, provided herein is a polynucleotide sequence comprising a transcriptional regulatory element and a nucleotide sequence encoding human Bcl-related atanogen 3 protein (hBAG3). In some embodiments, the nucleotide sequence encoding hBAG3 is at least 90% identical to nucleotides 1112-2839 of SEQ ID NO: 1, or at least 90% identical to nucleotides 981-2708 of SEQ ID NO: 2, which encodes a protein that retains BAG3 activity. In some embodiments, the nucleotide sequence encoding hBAG3 comprises nucleotides 1112-2839 of SEQ ID NO: 1 or nucleotides 981-2708 of SEQ ID NO: 2. In some embodiments, the nucleotide sequence encoding hBAG3 consists of the nucleotide sequence shown in SEQ ID NO: 8.

[0015] Transcriptional regulatory elements include, but are not limited to, promoters, enhancers, and / or polyadenylation signal sequences. Examples of transcriptional regulatory elements include the cytomegalovirus (CMV promoter), CMV enhancer, miniCMV promoter, MHCK7, CK8 promoter, chicken β-actin promoter, P546 promoter, monkey virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long-terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus very early promoter, Rous sarcoma virus promoter, and human gene promoters, such as, but are not limited to, actin promoters, myosin promoters, elongation factor-1a promoters, hemoglobin promoters, and creatine kinase promoters.

[0016] In exemplary embodiments, the transcriptional regulatory element is a muscle-specific regulatory element. The term “muscle-specific regulatory element” refers to a nucleotide sequence that regulates the expression of a coding sequence that is specific to expression in muscle tissue. These regulatory elements include enhancers and promoters.

[0017] Examples of muscle-specific promoters include one or more of the following: human skeletal actin gene element, cardiac actin gene element, desmin promoter, skeletal alpha-actin (ASKA) promoter, troponin I (TNNI2) promoter, muscle cell-specific enhancer-binding factor (MEF) binding element, muscle creatine kinase (MCK) promoter, truncated MCK (tMCK) promoter, myosin heavy chain (MHC) promoter, hybrid α-myosin heavy chain enhancer / MHC enhancer-promoter (MHCK7) promoter, C5-12 promoter, mouse creatine kinase enhancer element, skeletal fast-twitch muscle troponin C gene element, slow-twitch muscle cardiac troponin c gene element, slow-twitch muscle troponin I gene element, hypoxia-inducible nuclear factor (HIF)-response element (HRE), steroid-inducible element, and glucocorticoid-response element (GRE).

[0018] This disclosure provides constructs comprising a muscle-specific regulatory element shortening MCK (tMCK) promoter. For example, the tMCK promoter nucleotide sequence comprises nucleotides 164-884 of SEQ ID NO: 1. In some embodiments, the tMCK comprises the nucleotide sequence shown in SEQ ID NO: 11.

[0019] In certain embodiments, the transcriptional regulatory element includes a chicken beta-actin (CBA) promoter. For example, the CBA promoter sequence includes nucleotides 495-749 of SEQ ID NO: 2. In certain embodiments, the transcriptional regulatory element includes a CMV enhancer. For example, the CMV enhancer sequence includes nucleotides 209-463 of SEQ ID NO: 2. In certain embodiments, the transcriptional regulatory element is a hybrid CMV enhancer / chicken β-actin (CBA) promoter.

[0020] In exemplary embodiments, the transcriptional regulatory elements are neuron-specific regulatory elements. The term “neuron-specific regulatory element” refers to a nucleotide sequence that regulates the expression of a coding sequence that is specific to expression in neuronal tissue. These regulatory elements include neuron-specific enhancers and promoters.

[0021] Exemplary neuron-specific promoters include one or more of the following: platelet-derived growth factor B chain (PDGFβ) promoter, synapsin-1 (Syn) promoter, synapsin-2 promoter, tyrosine hydroxylase promoter, dopamine β-hydroxylase (DBH) promoter, hypoxanthine-guanine phosphoribosyltransferase (HPRT) promoter, low affinity nerve growth factor receptor (LNGFR) promoter, calcitonin gene-related peptide promoter (CGRP promoter), choline acetyltransferase (ChAT) promoter, neuron-specific enolase (NSE) promoter, calcium / calmodulin-dependent protein kinase II (CaMKII) promoter, methyl CpG-binding protein 2 (MeCP2) promoter, glial fibrillary acidic protein (GFAP) promoter, calbindin 2 promoter, motor neuron and pancreatic homeobox 1 (MNX1) promoter (also known as the Hb9 promoter), nestin promoter, parvalbumin (PVALB) promoter, and somatostation (SST) promoter.

[0022] In some embodiments, any of the polynucleotides disclosed herein further comprises an SV40 enhancer and / or an intron such as an SV40 intron or a chimeric intron. For example, the SV40 intron comprises nucleotides 830-926 of SEQ ID NO: 2. In some embodiments, the chimeric intron comprises nucleotides 937-1069 of SEQ ID NO: 1. In addition, any of the polynucleotides disclosed herein further comprises a polyadenylation signal sequence, which is optionally a synthetic polyadenylation signal sequence. The polynucleotide sequences disclosed herein include inverted terminal repeats (ITRs), such as mutant ITRs or wild-type ITRs.

[0023] This disclosure also provides polynucleotide sequences that constitute an AAV genome. For example, this disclosure provides an AAV genome or polynucleotide sequence comprising a nucleotide sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to nucleotides 1-3245 of SEQ ID NO: 1. In certain embodiments, the polynucleotide sequence that constitutes an AAV genome comprises nucleotides 1-3245 of SEQ ID NO: 1. In some embodiments, the polynucleotide sequence that constitutes an AAV genome comprises the nucleotide sequence described in SEQ ID NO: 6. In addition, the rAAV genomes provided herein hybridize under stringent conditions to the polynucleotide sequence of nucleotides 1-3245 of SEQ ID NO: 1, the nucleotide sequence of SEQ ID NO: 6, or its complement.

[0024] This disclosure also provides polynucleotide sequences that constitute an AAV genome. For example, this disclosure provides an AAV genome or polynucleotide sequence comprising a nucleotide sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to nucleotides 1-3114 of SEQ ID NO: 2. In certain embodiments, the polynucleotide sequence that constitutes an AAV genome comprises nucleotides 1-3114 of SEQ ID NO: 2. In some embodiments, the polynucleotide sequence that constitutes an AAV genome comprises the nucleotide sequence described in SEQ ID NO: 7. In addition, the rAAV genomes provided herein hybridize under stringent conditions to the polynucleotide sequence of nucleotides 1-3114 of SEQ ID NO: 2, the nucleotide sequence of SEQ ID NO: 7, or its complement.

[0025] In the context of nucleic acid sequences or amino acid sequences, the terms “sequence identity,” “percent sequence identity,” or “percent identity” refer to residues in two sequences that are identical when aligned to the greatest extent possible. The length of the sequence identity comparison can span the entire genome, the entire gene coding sequence, or preferably a fragment of at least about 500–5000 nucleotides. However, identity between smaller fragments, e.g., at least about 9 nucleotides, typically at least about 20–24 nucleotides, at least about 28–32 nucleotides, or at least about 36 or more nucleotides, may also be desired. The percentage of sequence identity can be determined by techniques known in the art. For example, homology can be determined by directly comparing the sequence information of two polypeptide molecules by aligning the sequence information and using readily available computer programs such as ALIGN, ClustalW2, and BLAST. In one embodiment, when BLAST is used as an alignment tool, the following default parameters are used: Genetic code = standard; filter = none; strand = both; cutoff = 60; prediction = 10; matrix = BLOSUM62; description = 50 sequences; sort = high score; database = non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swiss protein+Spupdate+PIR.

[0026] This disclosure also provides recombinant adeno-associated viruses (rAAVs) comprising any of the polynucleotide sequences described herein. For example, rAAVs include AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, AAVrh.74, AAVrh.10 capsid proteins, or variants thereof. In some embodiments, rAAV is the AAVrh.74 serotype. In some embodiments, rAAV is the AAV1 serotype. In some embodiments, rAAV is the AAV9 serotype.

[0027] This disclosure also provides recombinant AAV particles comprising any of the polynucleotide sequences disclosed herein or any of the rAAVs disclosed herein.

[0028] In another embodiment, the Disclosure provides a method for producing rAAV vector particles, the method comprising culturing cells transfected with any rAAV vector of the Disclosure and recovering rAAV particles from the supernatant of the transfected cells. The Disclosure also provides viral particles comprising any of the recombinant AAV vectors of the Disclosure.

[0029] This disclosure also provides compositions comprising any of the rAAVs disclosed herein or any of the rAAV particles described herein. In some embodiments, the compositions further comprise a pharmaceutically acceptable carrier. The compositions may also comprise other components such as diluents and adjuvants. The acceptable carriers, diluents and adjuvants are nontoxic to the recipient, preferably inert at the dosage and concentration used, and comprise buffers and surfactants such as Pluronic®.

[0030] This disclosure also provides compositions for treating muscular dystrophy in subjects requiring such treatment, the compositions comprising any of the rAAVs disclosed herein, or any rAAV particles disclosed herein, or any of the compositions disclosed herein.

[0031] This disclosure also provides a method for reducing misfolded proteins or protein aggregates in subjects suffering from myofibril myopathy associated with protein misfolding or aggregation, comprising administering to a subject in need of such reduction any of the rAAVs disclosed herein, any rAAV particles disclosed herein, or any of the compositions disclosed herein. For example, a subject suffering from limb-girdle muscular dystrophy type 1A (LGMD1A) caused by aggregation of mutant myotirin (MYOT).

[0032] This disclosure also provides a method for reducing misfolded proteins or protein aggregates in subjects suffering from neurodegenerative diseases or disorders associated with protein misfolding, comprising administering to subjects in need of such reduction any of the rAAVs disclosed herein, any rAAV particles disclosed herein, or any of the compositions disclosed herein. For example, a subject suffering from amyotrophic lateral sclerosis (ALS) caused by misfolded superoxide dismutase 1 (SOD1).

[0033] Exemplary proteins associated with protein misfolding disorders include desmin, alpha-crystallin B chain, myotilin, filamin C, BAG family molecular chaperone regulator 3 (BAG-3), Z-band alternative splicing PDZ motif-containing proteins, HSPB8, 4.5LIM domain protein 1, titin, plectin, α-actin, or DNAJ heat shock protein family (Hsp40) member B6, alpha-synuclein, amyloid-beta, mutant huntingtin, tau protein, prion protein, misfolded superoxide dismutase 1 (SOD1), islet amyloid polypeptide (IAPP), Musashi protein, p53, fusion sarcoma (FUS), progranulin, and TAR. Examples include, but are not limited to, DNA-binding protein 43 (TDP-43), misfolded transthyretin protein (TTR), valosin-containing protein (VCP), NOTCH3 receptor, mutant cystatin C, polyglutamine repeats, serum amyloid A (SAA), mutant gelzolin, misfolded rhodopsin, mezin, dipeptide repeat proteins, or atrial natriuretic peptides.

[0034] This disclosure also provides a method for preventing or treating a disease or disorder associated with misfolded proteins or protein aggregates in a subject who requires prevention or treatment of such a disease or disorder, comprising administering to the subject requiring such treatment any of the rAAVs disclosed herein, any rAAV particles disclosed herein, or any of the compositions disclosed herein. For example, the subject suffers from protein aggregate myopathy (PAM) or a neurodegenerative disease associated with misfolded proteins or protein aggregates.

[0035] This disclosure also provides a method for treating or preventing inclusion body myositis (IBM) or multiple system proteinopathy in subjects requiring treatment or prevention of IBM or multiple system proteinopathy, the method comprising administering to a subject requiring treatment or prevention of IBM or multiple system proteinopathy any of the rAAVs disclosed herein, or any rAAV particles disclosed herein, or any of the compositions disclosed herein. For example, IBM is associated with Paget's disease of bone and / or frontotemporal dementia (IBMPFD). In some embodiments, the subject has a mutation in the balossin-containing protein (VCP) gene. Furthermore, in any of the disclosed methods, the subject suffers from amyotrophic lateral sclerosis or Charcot-Marie-Tooth type 2.

[0036] This disclosure also provides the use of any of the rAAVs disclosed herein, any rAAV particles disclosed herein, or any of the compositions disclosed herein, for the preparation of a medicament for the treatment or prevention of inclusion body myositis (IBM) or multiple system proteinopathy in subjects requiring treatment or prevention of inclusion body myositis (IBM) or multiple system proteinopathy. For example, IBM is associated with Paget's disease of bone and / or frontotemporal dementia (IBMPFD). In some embodiments, the subject has a mutation in the balossin-containing protein (VCP) gene. Furthermore, in any of the disclosed uses, the subject suffers from amyotrophic lateral sclerosis or Charcot-Marie-Tooth type 2.

[0037] This disclosure also provides compositions for treating or preventing inclusion body myositis (IBM) or multiple system proteinopathy in subjects requiring treatment or prevention of IBM or multiple system proteinopathy, wherein the composition comprises any of the rAAVs disclosed herein, or any rAAV particles disclosed herein, or any of the compositions disclosed herein. For example, IBM is associated with Paget's disease of bone and / or frontotemporal dementia (IBMPFD). In some embodiments, the subject has a mutation in the balossin-containing protein (VCP) gene. Furthermore, in any of the disclosed uses, the subject suffers from amyotrophic lateral sclerosis or Charcot-Marie-Tooth type 2.

[0038] Combination therapy is also intended. In this regard, any of the aforementioned methods described herein may further include administering a second nucleic acid encoding the NT-3 polypeptide. In some embodiments, the second nucleic acid encoding the NT-3 polypeptide comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 9. In some embodiments, the second nucleic acid encoding the NT-3 polypeptide comprises the nucleotide sequence shown in SEQ ID NO: 9. In some embodiments, the second nucleic acid encoding the NT-3 polypeptide comprises a nucleotide sequence encoding an amino acid sequence that is at least 90% identical to SEQ ID NO: 10. In some embodiments, the second nucleic acid encoding the NT-3 polypeptide comprises a nucleotide sequence encoding an amino acid sequence shown in SEQ ID NO: 10. In some embodiments, the second nucleic acid encoding the NT-3 polypeptide comprises a nucleotide sequence that is at least 90% identical to nucleotides 1077-1850 of SEQ ID NO: 12. In some embodiments, or 100% identical to nucleotides 1077-1850 of SEQ ID NO: 12.

[0039] In some embodiments, a second nucleic acid encoding the NT-3 polypeptide is operably linked to a muscle-specific regulatory element. For example, the muscle-specific regulatory element is a human skeletal actin gene element, a cardiac actin gene element, a muscle cell-specific enhancer-binding factor (MEF) element, a muscle creatine kinase (MCK) promoter, a tMCK (shortened MCK) promoter, a myosin heavy chain (MHC) promoter, an MHCK7 promoter (a hybrid of MHC and MCK), a C5-12 (synthetic promoter), a mouse creatine kinase enhancer element, a skeletal fast-twitch muscle troponin C gene element, a slow-twitch muscle cardiac troponin C gene element, a slow-twitch muscle troponin I gene element, a hypoxia-inducible factor-binding element, a steroid-inducible element, or a glucocorticoid-response element (GRE). In some embodiments, a second nucleic acid encoding the NT-3 polypeptide is operably linked to a muscle creatine kinase promoter / enhancer sequence. For example, the enhancer / promoter includes the sequence shown at nucleotides 147-860 of SEQ ID NO: 12.

[0040] In some embodiments, a second nucleic acid encoding the NT-3 polypeptide is administered using a second viral vector. The second AAV can be any serotype, e.g., AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV12, AAV13, Anc80, AAV-B1, AAVrh.10, AAVrh.74, or variants thereof. In some embodiments, the serotype of the second recombinant AAV capsid is AAV-1. In some embodiments, the second recombinant AAV vector contains the scAAV1.tMCK.NTF3 rAAV genome which is at least 90% identical to SEQ ID NO: 12, as described in U.S. Patent Publication No. US-2020-0339960 (the disclosure of which is incorporated herein by reference in its entirety). In some embodiments, the scAAV1.tMCK.NTF3 rAAV genome contained the nucleotide sequence shown in Sequence ID No. 12.

[0041] In some embodiments, the second nucleic acid encoding the NT-3 polypeptide further comprises an intron, such as an SV40-intron or a chimeric intron. In some embodiments, the second nucleic acid encoding the NT-3 polypeptide comprises the chimeric intron sequence shown in nucleotides 892-1024 of SEQ ID NO: 12. In addition, the second nucleic acid encoding the NT-3 polypeptide disclosed herein further comprises a polyadenylation signal sequence, which is optionally a synthetic polyadenylation signal sequence. For example, the polyadenylation signal sequence comprises the sequence shown in nucleotides 1860-2059 of SEQ ID NO: 12. The second nucleic acid encoding the NT-3 polypeptide disclosed herein further comprises an inverted-end repeat sequence (ITR), such as a mutant ITR or a wild-type ITR.

[0042] In any of the disclosed methods, rAAV, rAAV particles, or compositions are administered by systemic administration, intramuscular injection, or intravenous injection. In addition, in any of the methods of the present invention, rAAV is administered systemically, such as by parenteral administration by injection, infusion, or implantation.

[0043] The compositions of this disclosure are formulated for intramuscular or intravenous injection. In addition, the compositions of this invention are formulated for systemic administration, such as parenteral administration by injection, infusion, or transplantation. In addition, any of the compositions of this disclosure are formulated for administration to subjects suffering from myofibrillary myopathy associated with protein misfolding or aggregation (e.g., limb-girdle muscular dystrophy type 1A (LGMD1A) or neurodegenerative diseases associated with misfolded proteins or protein aggregates, such as ALS), or inclusion body myositis, such as IBMPFD or multiple system proteinopathy.

[0044] In any use of the present disclosure, the pharmaceutically acceptable drug is formulated for intramuscular or intravenous injection. In addition, in any use of the present invention, the pharmaceutically acceptable drug is formulated for systemic administration, such as parenteral administration by injection, infusion, or implantation. In addition, any of the pharmaceutically acceptable drugs may be prepared for administration to subjects suffering from myofibrillary myopathy associated with protein misfolding or aggregation (e.g., limb-girdle muscular dystrophy type 1A (LGMD1A) or neurodegenerative diseases associated with misfolded proteins or protein aggregates, such as ALS), or inclusion body myositis, such as IBMPFD or multiple system proteinopathy.

[0045] As used herein, “subject” may be any animal and may be referred to as a patient. Preferably, the subject is a vertebrate, and more preferably, the subject is a mammal such as livestock (e.g., cattle, horses, pigs) or pets (e.g., dogs, cats). In some embodiments, the subject is a human. In some embodiments, the subject contains one or more mutations in the BAG3 gene, such as the BAG3 P209L mutation or the GGGGCC hexanucleotide repeat in C9orf72. In some embodiments, the subject contains a mutation in any one of the following genes: VCP, DES, FLNC, MYOT, CRYAB, ZASP, BAG3, FHL1, TTN, PLEC, ACTA1, HSPB8, SOD1, or DNAJB6.

[0046] The preceding paragraphs are not intended to define all aspects of the Disclosure, and additional aspects are described in other sections, such as the Detailed Description. The entire document is intended to be related as a unified disclosure, and it should be understood that all combinations of features described herein are contemplated, even if combinations of features are not found together in the same sentences, paragraphs, or sections in this document. The Disclosure includes, as additional aspects, all embodiments of the Invention that are somewhat narrower in scope than the variations defined in the particular paragraphs above. For example, if a particular aspect of the Disclosure is described as a genus, each member of the genus should be understood to be an individual aspect of the Disclosure. [Brief explanation of the drawing]

[0047] [Figure 1] Figure 1A provides a schematic diagram of a gene therapy cassette containing a tMCK enhancer / promoter, full-length BAG3 cDNA, SV40 poly-A tail, and 5' and 3' ITRs. Figure 1B provides a schematic diagram of a gene therapy cassette containing a CBA promoter / CMV enhancer, SV-40 intron, full-length BAG3 cDNA, SV40 poly-A tail, and 5' and 3' ITRs. [Figure 2] Figures 2A and 2B provide plasmid maps and ORF analyses of ss.pAAV.tMCK.BAG3 and ss.pAAV.CMV.CBA.BAG3, respectively. [Figure 3] A schematic diagram of a full-length NTF3 cDNA cassette having a tMCK enhancer / promoter, gene expression-enhancing introns (I), ITRs, and an SV40 poly-A tail is provided. [Figure 4] The presence of the 74kDA hBAG3 protein is observed in WP-4 lysates (left panel) 8 weeks after injection of ss.pAAV.CMV.hBAG3 (2.5 × 10¹¹ vg in 50 μl of PBS) (right panel), as well as in the left GAS muscle of TgT57I transgenic (MYOT) mice (No. 97) and wild-type (WT) mice (No. 98). Muscle tissue that was not injected does not show an hBAG3 band. [Figure 5A-C]Figures 5A and 5B provide representative images of hematoxylin and eosin (H&E) stained sections of untreated control right GAS muscle (B) and TgT57I transgenic (MYOT) mice (A) and BAG3-treated left GAS muscle (B) 12 weeks after BAG3 gene therapy by IM injection of 2.5 × 10¹¹ vg of ssAAV9.CMV.hBAG3 vector, showing a significant reduction in dark purple intracytoplasmic aggregates compared to untreated control right GAS, which has more mutant aggregates. Figure 5C provides fluorescence micrographs of tissue sections from untreated right GAS muscle control tissue and AAV-treated left GAS muscle tissue of TgT57I transgenic (MYOT) mice, fixed 8 weeks post-injection and immunostained with anti-myotilin antibody. Left GAS muscle injected with AAV9.BAG3 (left panel) showed fewer myotylin-positive areas, while untreated control right GAS muscle (right panel) revealed numerous clearly defined aggregates and showed strong immunopositivity for myotylin. [Figure 5D] Figure 5C provides a graph showing the quantification of myotilin inclusion density in immunostained sections. The left panel provides quantification by pixel count, and the right panel provides quantification by mean pixel count. These results show a significant reduction (p<0.0001) in both the pixel count and mean pixel count of treated left GAS muscle (calculated as a percentage of untreated right GAS in each mouse). [Figure 6] Figures 6A–6C provide representative images of H&E-stained sections of tibialis anterior muscle from C57BL / 6 wild-type (WT) mice 8 weeks after systemic administration of the ssrAAVrh74.tMCK.hBAG3 vector at a dose of 6 × 10¹²vg. Figures 6B and 6C provide representative images of H&E-stained sections of GAS muscle from male MYOT mice 8 weeks after systemic delivery of ssrAAVrh74.tMCK.hBAG3 in 3 × 10¹²vg (B) or untreated male mice (C). Tissues from treated mice showed an overall reduction in particle size compared to tissues from untreated mice. [Figure 7]Figures 7A–7D provide representative images (A and C, respectively) of H&E-stained sections of GAS and Quad muscle from male MYOT mice 8 weeks after systemic delivery of ssrAAVrh74.tMCK.hBAG3 at 3 × 10¹²vg, as well as age- and sex-matched untreated control muscle (B and D, respectively). Treated muscle showed smaller aggregate sizes and lower basophilia compared to untreated muscle. [Figure 8] Figures 8A–8J provide representative images of H&E stained sections from the right GAS muscle of a male-only cohort of MYOT mice 8 weeks after intramuscular injection of the ssrAAVrh74.tMCK.hBAG3 vector at low dose (LD, 3 × 10¹⁰ vg), intermediate dose (ID, 1 × 10¹¹ vg), and high dose (HD, 2 × 10¹¹ vg), with the left GAS muscle being an untreated control. Aggregate size was smaller and basophilic was lower in the muscle treated with all three doses. [Figure 9] Figures 9–9C provide bar graphs showing the myotilin aggregate size distribution of untreated left GAS muscle and right GAS mouse muscle treated with LD, ID, and HD ssrAAVrh74.tMCK.hBAG3 vectors, with H&E images provided in Figure 8 (two-way ANOVA, Bonferroni multiple comparison test, ****p<0.0001, ***p<0.001). The treated right muscle showed a significant shift to smaller aggregate sizes at all three doses. [Figure 10] Figures 10A-10C provide bar graphs showing the average aggregate fluorescence intensity, average aggregate area (μm2), and percentage of area occupied by myotirin aggregates in LD, ID, and HD treated muscles, as well as untreated control muscles (t-test, *p<0.05). [Figure 11] Figures 11A–11D provide bar graphs showing significant improvements in rotor rod treadmill, grip strength test, and maximal tetanus response in MYOT mice 8 months after systemic delivery of AAVrh74.tMCK.hBAG3 at 3 × 10¹²vg. Figure 11E provides a bar graph showing no significant difference in maximal seizure response 8 months after systemic delivery of AAVrh74.tMCK.hBAG3 at 3 × 10¹²vg. [Figure 12] Figure 12A provides fluorescence micrographs of quadriceps femoris muscle sections from untreated (left panel) and BAG3-treated (right panel) MYOT mice, immunostained with myotilin antibody 8 months after injection. Figure 12B provides a bar graph showing the quantification of significantly reduced myotilin aggregate density (number of aggregates / mm2) in treated muscle (t-test, *p<0.05). Figure 12C provides a bar graph of myotilin aggregate size distribution / mm2 in treated and untreated cohorts (two-way ANOVA, *p<0.05, ****p<0.0001). [Figure 13] Figure 13A provides Kaplan-Meier survival curves showing the overall survival of SOD1-G93A mouse cohorts treated with ss.AAV9.CBA.hBAG3 (4 × 10¹²vg) and sc.AAV1.tMCK.NT-3 (Neurotrophin-3, 1 × 10¹¹vg) as monotherapy or combination therapy at preclinical, early, and paralysis stages (40, 70, and 100 days, respectively). Figure 13B provides bar graphs showing the significant increase in survival rates between different treatment cohorts compared to untreated mice, and the differences in survival rates between treatment cohorts. [Figure 14] Figures 14A–14C provide line graphs of rotarod (A) and grip strength (B) test performance over time for the mouse cohort described in Figure 13. Asterisks indicate significant changes (p<0.05) in the treated cohort compared to the untreated cohort at a given time point (t-test). The untreated cohort showed a steady decline in rotarod, grip strength, and electrophysiological tests. In the rotarod test, both the BAG3-100, Combo-70, and Combo-40 cohorts showed significant improvement compared to untreated mice, starting one week after injection at 128–135 days of age. Similar improvements were observed in the grip strength test, mainly in the Combo-70 and Combo-40 cohorts. [Figure 15A] Figure 15A provides a micrograph of a cresyl violet-stained spinal cord section showing the anterior horn region used for neuron counting. [Figure 15B-C]Figures 15B and 15C provide bar graphs showing the quantification of large (≥15 μm in diameter) and total neuronal populations in the anterior horn of the lumbar spinal cord from endpoint WT and SOD1.G93A mice, and from 70-day-old untreated mice (RL-70), respectively. [Figure 16] Representative micrographs of cresyl violet-stained paraffin-embedded spinal cord tissue sections are provided, showing the distribution of anterior horn cells from the SOD1.G93A mouse cohort of BAG3-100(A), RL(B), BAG3-40(E), NT-3-40(F), Combo(G), and BAG3-70(H). The framed areas in Figures 14A and B are shown at higher magnification (20x objective lens) in Figures 14C and D, respectively. [Figure 17-1] Provides the sequence ss.pAAV.tMCK.BAG3 (sequence number 1). [Figure 17-2] Same as above. [Figure 17-3] Same as above. [Figure 17-4] Same as above. [Figure 17-5] Same as above. [Figure 17-6] Same as above. [Figure 17-7] Same as above. [Figure 17-8] Same as above. [Figure 18-1] Provides the sequence for ss.pAAV.CMV.CBA.BAG3 (sequence number 2). [Figure 18-2] Same as above. [Figure 18-3] Same as above. [Figure 18-4] Same as above. [Figure 18-5] Same as above. [Figure 18-6] Same as above. [Figure 18-7] Same as above. [Figure 18-8] Same as above. [Figure 19] Figures 19A–19C provide graphs of functional outcomes 9 months after hBAG3 gene delivery, showing (A) treadmill, (B) rotord, and (C) grip strength data. [Figure 20]Figures 20A–20E provide representative (A) H&E, (B, C) Gomori trichrome, (D) COX, and (E) SDH stained images of the gastrocnemius muscle of 4-month-old VCP-A232E mice, showing myopathic changes (basophilic regenerating fibers, increased internal nuclei in A), excessive submembrane mitochondrial accumulation (arrows, CE), and COX-deficient fibers (D). [Figure 21] Figures 21A and 21B provide representative SDH-stained sections from the gastrocnemius muscle of BAG3-treated mice at the endpoint, showing (A) untreated fibers with altered fiber size variability and SDH content (abnormal mitochondria), and (B) improved mitochondrial content and distribution after gene therapy. [Figure 22] Figures 22A–22D show the effects of BAG3 gene therapy on the gastrocnemius muscle. Graphs show the changes observed in (A) fiber size and (B) distribution percentage for different fiber types in treated and untreated cohorts. Histograms show the fiber size distribution based on (C) number of fibers and (D) percentage of fibers. [Figure 23] Figures 23A–23C provide Western blots (A) of the gastrocnemius muscle of VCP-A232E mice, as well as graphs showing the protein levels of (B) p62 and (C) LC3 proteins. [Figure 24] Figures 24A-24E provide graphs showing the relative expression levels of (A) Norad, (B) Pum2, (C) Pgc1α, (D) Cox1, and (E) Cox3 in the gastrocnemius muscle of treated and untreated VCP-A232E mice. [Modes for carrying out the invention]

[0048] This disclosure demonstrates that upregulation of the molecular cochaperone BCL2-associated atanogen 3 (BAG3) protein selectively increases the degradation of misfolded proteins and proteins prone to aggregation. Since misfolded proteins and proteins prone to aggregation play a role in the pathology of various disorders and diseases associated with the accumulation of misfolded proteins and / or aggregates, upregulation of BAG3 protein is an effective therapy for these disorders and diseases.

[0049] The therapies disclosed are based on the premise that manipulating autophagy effectively treats protein aggregate-related diseases. The pathogenesis of neurodegenerative phenotypes of protein aggregate myopathy (PAM), such as myofibrillar myopathy, or central nervous system (CNS) disorders associated with agglutinating proteins, is linked to the successive presence of mutant proteins. Studies have shown that neurodegenerative symptoms in a mouse model of Huntington's disease are linked to direct RNA interference targeting mutant huntingtin. 1 It has been shown that this can be alleviated by stopping the expression of mutant aggregate-prone proteins in symptomatic mice, or by discontinuing the expression of mutant aggregate-prone proteins. 2、3 A second approach to increasing the toxicity of aggregate-prone proteins can be achieved by enhancing their degradation. The highly conserved BAG family co-chaperone BAG3 (Bcl-2-associated atanogen 3) is uniquely positioned in a degradation pathway that has the ability to induce autophagy to remove misfolded proteins. 4、5、6~8Under acute stress and during cellular senescence, BAG3, in coordination with the molecular chaperone HSP70 (heat shock protein 70) and the smaller heat shock protein HSPB8, as well as the ubiquitin-binding protein or receptor p62 / SQSTM1, specifically targets proteins prone to aggregation for autophagy-mediated degradation. Therefore, BAG3-mediated selective macroautophagy represents the ultimate adaptive safeguard and emergency system of protein quality control, which is activated under pathophysiological conditions to ensure cellular proteostasis. In addition, BAG3-mediated selective macroautophagy is also involved in the clearance of aggregated proteins associated with neurodegenerative disorders and protein aggregate myopathy. 9、10 .

[0050] Another BAG family protein, BAG1, which possesses HSP70 and STUB1 (STIP1 homology and U-Box-containing protein 1), primarily mediates the degradation of polyubiquitinated proteins by the proteasome under physiological conditions. 11、12 BAG3 induces the conversion of polyubiquitinated proteins by the autophagy-lysosomal system under pathophysiological conditions. 13~15 The expression levels of BAG1 and BAG3 are mutually regulated during cellular senescence and under acute stress. Under physiological conditions, high BAG1 expression and low BAG3 expression may be detected, while under pathophysiological conditions, BAG3 levels increase and BAG1 levels decrease. The BAG1 to BAG3 expression switch involves a functional switch from HSP70-BAG1-mediated proteasomal degradation to HSP70-BAG3-mediated selective macroautophagy. 16Therefore, the present disclosure provides BAG3 gene therapy as a treatment option investigated in rodent models of myopathic and neurodegenerative diseases associated with protein aggregates. These results described herein show for the first time that wild-type (WT) BAG3 gene therapy using an adeno-associated virus (AAV) reduces mutant protein aggregates via BAG3-mediated clearance and that these gene therapy vectors are potential treatments for human disorders resulting from misfolded protein aggregates involved in muscle or CNS tissue.

[0051] BAG3 and Myopathy with Abnormal Protein Aggregates BAG3 in Muscle BAG3 is highly expressed in muscle and heart tissues and co-localizes with Z-disk proteins such as α-actinin and desmin. 17 Patients characterized by BAG3 mutations feature disrupted Z-disks, myofibril degeneration, and disarray. 18 Muscle biopsies from patients with severe infantile myopathy and restrictive cardiomyopathy due to a mutation (P209L) in the BAG3 gene showed accumulation of abnormal in-fiber substances with strong immunoreactivity to BAG3, αB-crystallin, desmin, gelsolin, myotilin, ubiquitin, and dystrophin. In normal muscle, BAG3 was immunolocalized to the Z-disk (co-localized with myotilin). Studies suggest a mechanism whereby P209L BAG3 tends to aggregate with the available BAG3 pool, causing BAG3 insufficiency, which leads to impairment of autophagy activity. 19 .

[0052] Myofibrillar Myopathy (MFM) Myofibrillar muscle myopathy (MFM) is a growing group of protein aggregate disorders with significant clinical and genetic heterogeneity. Disorders in this group are characterized by a distinctive histopathology of abnormal protein aggregation and myofibril breakdown. All genes causing myofibrillar myopathy encode proteins that reside in or associate with the Z-disk. In addition to the known disease genes DES, FLNC, MYOT, CRYAB, ZASP, BAG3, FHL1, and TTN, mutations in PLEC, ACTA1, HSPB8, and DNAJB6 have also been associated with the proximal MFM clinical phenotype. Subgroups of MFM, desmynopathies, distal myotirinopathy, ZASPopathy, and alpha-B crystallin mutation distal myopathy present with distal dominant muscle weakness but show similar pathological changes on muscle biopsy. A common feature of distal MFM myopathy is that the symptoms are dominant and adult-onset, starting in the legs and slowly progressing to include other muscle groups. Cardiomyopathy is not a common feature in distal MFM myopathy. 20 .

[0053] Neurodegenerative diseases with BAG3 and abnormal protein aggregates Neurodegenerative diseases arising from different mutant / misfolded protein aggregates share a common unifying feature: the inability of protein clearance mechanisms to efficiently degrade misfolded proteins and maintain proteostasis. Disposal of abnormal, misfolded proteins occurs through either the ubiquitin-proteasome mechanism or the autophagy-lysosome system, but aggregated proteins are primarily degraded by a process called selective macroautophagy. The selective macrophagy pathway is mediated by the multifunctional HSP70 co-chaperone BAG3 (BCL-2-associated atanogen 3). A well-recognized feature of many neurodegenerative disorders is the aggregation and accumulation of misfolded proteins associated with cytotoxic effects in neurons. Examples of misfolded proteins characteristic of these disorders include tau protein found in neurofibrillary tangles (in AD), mutant huntingtin with enlarged polyQ tubules (in HD), mutant androgen receptor (SBMA) with enlarged polyQ tubules, and mutant superoxide dismutase 1 (SOD1, in familial amyotrophic lateral sclerosis, fALS). In this context, BAG3 is tau 5 , α-synuclein 21 , mutant SOD1 4 , and mutant huntingtin 13 It has been shown to promote the clearance of disease-related aggregation-prone proteins, including BAG3. These observations suggest that BAG3 is a key player in maintaining neuronal proteostasis, and therefore, therapies that upregulate autophagy may improve neurodegenerative diseases. In various experimental systems, autophagy activation has been shown to reduce inclusion body accumulation and further mitigate neurodegenerative phenotypes (see, for example, the data provided in Example 7).

[0054] BAG3 and LGMD1A (Myochirinopathy) Myotirin is primarily expressed in skeletal and cardiac muscle and localizes to the sarcomere Z disk, a highly electron-density structure that contributes to sarcomere assembly, actin filament stabilization, and muscle force transmission. 22In the Z disk, myotirin interacts with α-actinin-2 (ACTN2). 23 This suggests that actin filaments can be bundled together, which plays an important role in the fixation and stabilization of F-actin. 24 Several myotirin point mutations have been observed in patients with limb-girdle muscular dystrophy type 1A (LGMD1A), an autosomal dominant muscle disorder with adult-onset, characterized by early vulnerability of the proximal girdle muscles. 25 Muscle histology reveals myopathic changes including extensive autophagy vacuoles and Z-band streaming. 26 Recent combined proteomic and immunolocalization analyses from patient biopsies offer new insights into the complex regulation of proteolysis in myotilinopathy, which signifies a combination of the acquisition of toxic function leading to myotirin-positive protein aggregates and the loss of function caused by a shift in subcellular distribution accompanied by myotirin deficiency in Z-disks, impairing the integrity of myofibrils. 27 .

[0055] Amyotrophic lateral sclerosis (ALS) ALS is a rapidly progressive neurodegenerative disease caused by the selective loss of upper and lower motor neurons in the brain and spinal cord. Most ALS cases are sporadic and lack apparent genetic linkage, but 10% of cases are familial ALS (fALS) with dominant inheritance. The most common genotype of ALS is due to elongated GGGGCC repeats in the non-coding region of C9ORF72. The mechanism by which hexonucleotide repeat elongation (HRE) causes toxicity is unknown, but it has been proposed that the gain-of-function disease mechanism of toxic HNE (repetitive RNA toxicity and dipeptide repeat protein production), in conjunction with C90rf72-mediated autophagy impairment, leads to the aggregation of dipeptide repeat proteins and TAR DNA-binding proteins, TARBP / TDP-43.

[0056] The second most common cause of these familial cases (approximately 20%) is mutations in the gene encoding ubiquitous cytoplasmic Cu / Zn superoxide dismutase (SOD1), which leads to the accumulation of misfolded SOD1 protein and motor neuron death. 32 The accumulation of misfolded proteins in spinal motor neurons is characteristic of both familial and sporadic ALS. The cellular mechanisms involved in maintaining protein homeostasis and preventing protein aggregation were impaired in both familial and sporadic ALS cases that were SOD1 immunoreactive, supported by the presence of ubiquitinated insoluble inclusion bodies, not only in familial ALS patients but also in sporadic ALS patients.

[0057] In one embodiment, the present disclosure provides compositions and methods for treating or preventing diseases or disorders associated with misfolded proteins or protein aggregates. This specification shows that BAG3 plays a role in targeting misfolded proteins or aggregated proteins for degradation by autophagy. Therefore, in certain embodiments, the compositions and methods of the present disclosure may be used to selectively remove intracellular or extracellular misfolded proteins, protein aggregates, or protein inclusions.

[0058] For example, in certain embodiments, the Disclosure provides compositions and methods for treating or preventing protein aggregate myopathy (PAM) in subjects requiring treatment or prevention of PAM. PAM is a group of hereditary or acquired muscle disorders morphologically characterized by the abnormal accumulation of proteins within muscle fibers, including, but not limited to, myofibril myopathy (MFM), actin-related myopathy (actinopathy), and myosin storage myopathy (MSM) (Olive et al. 2015 Hum Mol Genet. 24(21):6264). For example, in a particular embodiment, the composition and method include α-actin (ACTA1), nebulin / nebulet (NEBL), myosin (MYO), titin (TTN), filamin C (FLNC), myotirin (MYOT), Z-band alternative splicing PDZ motif-containing protein (ZASP), 4.5LIM domain protein 1 (FHL1), desmin (DES), plectin (PLEC), valosin-containing protein (VCP), and DNAJ heat shock protein family (Hsp40) member B6 (DNAJB6). It is used for the treatment or prevention of PAM associated with misfolded proteins and / or protein aggregates of BAG family molecular chaperone regulator 3 (BAG-3), αB-crystallin, synemine, alpha-crystallin B chain (CRYAB), bifunctional UDP-N-acetylglucosamine 2-epimerase / N-acetylmannosamine kinase (GNE), selenoprotein N (SELENON), LIM domain-binding protein (LDB), synemine (SYNM), and poly(A)-binding protein nucleus 1 (PABPN1).

[0059] For example, in certain embodiments, the Disclosure provides compositions and methods for treating or preventing myofibril myopathy (MFM) in subjects requiring treatment or prevention of MFM. Exemplary MFMs include, but are not limited to, desminopathy (DES gene), alpha-B crystallinopathy (CRYAB gene), myotilinopathy (MYOT gene), filaminopathy (FLNC gene), BAG3-associated myofibril myopathy (BAG3 gene), ZASPopathy (ZASP gene), or HSPB8 myopathy (HSPB8 gene).

[0060] In some embodiments, the disclosure encompasses the treatment or prevention of neuromuscular conditions having diseases that cause mutations characteristic of myofibrilding myopathy. Exemplary neuromuscular conditions characterized by MFM include, but are not limited to, reductol myopathy (FHL1 gene), hereditary myopathy with early respiratory failure (HMERF gene), epidermolysis bullosa simplex with muscular dystrophy (PLEC gene), MFM-actinopathy (ACTA1 gene), and limb-girdle muscular dystrophy type 1D (DNAJB6 gene).

[0061] In some embodiments, the present disclosure provides compositions and methods for the treatment of neurodegenerative diseases or disorders associated with misfolded proteins or protein aggregates. For example, in certain embodiments, the compositions and methods are used to treat or prevent diseases and disorders associated with misfolded proteins and / or protein aggregates of polyglutamine repeat-related proteins such as amyloid-beta, alpha-synuclein, tau, prions, SOD1, TDP-43, FUS, p53, p53 variants, or huntingtin and ataxin.

[0062] Exemplary neurodegenerative diseases associated with misfolded proteins or protein aggregates include amyotrophic lateral sclerosis (ALS), transmissible spongiform encephalopathy (prion disease), synucleinopathies, Lewy body dementia (DLB), multiple system atrophy (MSA), tauopathy, frontotemporal lobar degeneration (FTLD), frontotemporal dementia (FTD), sporadic or familial motor neuron disease (MND) with or without, corticobasal degeneration, frontotemporal lobar degeneration with ubiquitin-positive inclusions, argyrophilic granule disease, This includes, but is not limited to, Pick's disease, amyotrophic lateral sclerosis (ALS), sporadic ALS, Alzheimer's disease (AD, sporadic and familial), Down syndrome, familial dementia, polyglutamine (polyQ) diseases (Huntington's disease (HD), dentatorubral-pallidoluysian atrophy (DRPLA), spinal-bulbar muscular atrophy (SBMA), and six spinocerebellar degenerations (SCA1, SCA2, SCA3, SCA6, SCA7, and SCA17)), hippocampal sclerosis dementia, or Parkinson's disease (PD).

[0063] However, the present invention is not limited to the treatment or prevention of neurodegenerative disorders and myofibrild myopathy. Rather, this disclosure encompasses the treatment or prevention of any disease or disorder associated with misfolded proteins or protein aggregates. Other such diseases and disorders include, but are not limited to, AL amyloidosis, AA amyloidosis, familial Mediterranean fever, senile systemic amyloidosis, familial amyloid polyneuropathy, hemodialysis-associated amyloidosis, ApoAI amyloidosis, ApoAII amyloidosis, ApoAIV amyloidosis, Finnish hereditary amyloidosis, lysozyme amyloidosis, fibrinogen amyloidosis, Icelandic hereditary cerebral amyloid angiopathy, type II diabetes mellitus, medullary thyroid carcinoma, atrial amyloidosis, hereditary cerebral hemorrhage with amyloidosis, pituitary prolactinoma, injection-induced amyloidosis, aortic medial amyloidosis, hereditary lattice keratopathy, corneal amyloidosis associated with trichiasis, cataract, odontogenic calcifying epithelioma, alveolar proteinosis, inclusion body myositis, and lichenoid amyloidosis.

[0064] In one embodiment, the disclosure includes using the BAG-3 protein to increase the recognition and degradation of misfolded or aggregated proteins or protein inclusions. In certain embodiments, the recognition and degradation of misfolded or aggregated proteins via the BAG3 protein described herein can treat or prevent diseases or disorders associated with misfolded or aggregated proteins.

[0065] For example, in one embodiment, the degradation of mutant copper-zinc superoxide dismutase enzyme (SOD1) via BAG3 as described herein can treat or prevent amyotrophic lateral sclerosis (ALS). In another exemplary embodiment, the degradation of mutant myotilin via BAG3 as described herein can treat or prevent myotilinopathy, such as limb-girdle muscular dystrophy 1A (LGMD1A), and other diseases associated with the degradation of misfolded or aggregated proteins.

[0066] In one embodiment, the present disclosure provides compositions and methods for robust and long-term increase in BAG3 expression and / or activity in muscle or CNS tissue in the treatment of protein misfolding disorders or disabilities. In certain embodiments, the compositions include nucleic acid molecules, expression vectors, proteins, peptides, small molecules, etc., that increase the expression, activity, or both of the BAG3 protein.

[0067] BAG3, as well as inclusion body myositis and multisystem proteinosis Inclusion body myositis (IBM), associated with Paget's disease of bone and frontotemporal dementia (IBMPFD), is considered one of the genetic forms of IBM. It is an adult-onset, progressive, dominant disorder characterized by proximal and distal muscle weakness and mild to severe myopathy. Myopathy is present in 90% of cases, and 42% of Paget's disease and 30% of Paget's disease cases are associated with frontotemporal dementia. 45 Myopathy, like bone disease, begins at an average age of 43, but dementia usually begins about 10 years later. 45IBMPFD is caused by mutations in the balossin-containing protein (VCP) gene. There is no correlation between the type of mutation and the development of clinical features associated with the balossin-containing protein (VCP) gene. The spectrum of other diseases associated with VCP mutations includes amyotrophic lateral sclerosis (ALS). 462 and Charcot-Marie-Tooth2 type 47 This explains why this condition has come to be known by the preferred name: "Multisystem Proteinosis". 47、48 Serum CK levels may be normal, but they fluctuate and are generally elevated in IBMPFD. The VCP gene has 17 exons, with mutations reported in 11 exons. All mutations associated with IBMPFD and / or familial ALS are exon missense mutations. The A232E mutation is associated with a more severe clinical phenotype, characterized by earlier onset and more invasive myopathy, compared to cases with other VCP mutations. 49 .

[0068] definition Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention pertains. Any methods and materials similar to or equivalent to those described herein may be used in carrying out the tests of the present invention, but certain materials and methods are described herein. The following terms are used in the description and claims of the present invention:

[0069] As used herein, “BAG3,” “BAG3 molecule,” “BCL2-associated atanogen 3 (BAG3) gene,” and “BCL2-associated atanogen 3 (BAG3) molecule” include all family members, variants, cDNA sequences, alleles, fragments, species, coding and non-coding sequences, sense and antisense polynucleotide chains, etc. (HGNC(939)Entrez Gene(9531)Ensembl(ENSG00000151929)OMIM(603883)UniProtKB(095817)). For example, the BAG3 gene or cDNA nucleotide sequence is indicated as SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 8, or as nucleotides 1112-2839 of SEQ ID NO: 1 or nucleotides 981-2708 of SEQ ID NO: 2. Similarly, “BAG3,” “BAG3 molecule,” and “BCL2-associated atanogen 3 (BAG3) protein” also refer to the BAG3 polypeptide or its fragments, proteins, variants, derivatives, etc. For example, the BAG3 protein contains the amino acid sequence of SEQ ID NO: 5. Therefore, the term "molecule" encompasses both the nucleic acid sequence and the amino acid sequence of BAG3.

[0070] As used herein, the term "AAV" is a common abbreviation for adeno-associated virus. Adeno-associated viruses are single-stranded DNA parvoviruses that grow only in cells where certain functions are provided by co-infecting helper viruses. Currently, there are 13 serotypes of AAV that have been characterized. General information and an overview of AAV can be found, for example, in Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169-228, and Berns, 1990, Virology, pp. 1743-1764, Raven Press, (New York). However, since it is well known that various serotypes are very closely related both structurally and functionally, even at the genetic level, it is quite expected that these same principles may apply to additional AAV serotypes. (See, for example, Blacklowe, 1988, pp. 165-174 of Parvoviruses and Human Disease, JR Pattison, ed., and Rose, Comprehensive Virology 3:1-61 (1974)). For example, all AAV serotypes clearly exhibit very similar replication characteristics mediated by homologous rep genes, all possessing three related capsid proteins, including the one expressed in AAV2. The degree of relatedness is further suggested by heteroduplex analysis, which reveals extensive cross-hybridization between serotypes along genome length and the presence of similar self-annealing segments at the terminals corresponding to "reverse terminal repeats" (ITRs). Similar infectivity patterns also suggest that replication function in each serotype is under similar regulatory control.

[0071] As used herein, recombinant “vector” or “AAV vector” refers to a vector derived from the wild-type genome of a virus, such as AAV, by using molecular methods to remove the wild-type genome from the virus (e.g., AAV) and replace it with a non-natural nucleic acid, such as a heterologous polynucleotide sequence or transgene (e.g., a therapeutic gene expression cassette). Typically, for AAV, one or both inverted terminal repeat (ITR) sequences of the wild-type AAV genome are retained in the AAV vector if they are adjacent to the inserted transgene. Recombinant viral vectors are distinguished from viral genomes because all or part of the viral genome is replaced with a non-natural sequence with respect to viral genomic nucleic acids, such as a heterologous polynucleotide sequence. Therefore, the incorporation of a non-natural sequence defines a viral vector (e.g., AAV) as a “recombinant” vector, and in the case of AAV, it may be referred to as an “rAAV vector.” Such an AAV vector can be replicated and packaged into infectious viral particles when present in a host cell transfected with a vector encoding and expressing rep and cap gene products.

[0072] As used herein, the term “self-complementary AAV vector” (scAAV) refers to a vector containing a double-stranded vector genome generated by the deletion of a terminal degradation site (dTR) from one of the ITRs of AAV. The absence of the TR prevents replication initiation at the vector ends where the TR is absent. Generally, scAAV vectors generate a single-stranded inverted repeat genome with wild-type (wt) AAV TRs at each end and a mutant TR (mTR) in the middle.

[0073] An "AAV virion," "AAV virus particle," or "AAV vector particle" refers to a viral particle consisting of at least one AAV capsid protein and a capsid-forming polynucleotide AAV vector. If the particle contains heterologous polynucleotides (i.e., polynucleotides other than the wild-type AAV genome, such as transgenes delivered to mammalian cells), it is typically referred to as an "AAV vector particle" or simply an "AAV vector." Therefore, since such vectors are contained within AAV vector particles, the production of AAV vector particles necessarily involves the production of AAV vectors.

[0074] As used herein, the term “operably linked” refers to the juxtaposition of polynucleotide (or polypeptide) elements that are in a functional relationship that enables them to operate in the expected manner. For example, a promoter is operably linked to a coding sequence if it assists in the initiation of transcription of that sequence. Intervening residues may exist between the promoter and the coding region as long as this functional relationship is maintained. Generally, though not always, operably linked means that the linked nucleic acid sequences are contiguous and, if it is necessary to link two protein coding regions, they are contiguous and within the same reading frame.

[0075] A “transcriptional regulatory element” refers to an element or nucleic acid sequence of an element that regulates the expression of a nucleotide sequence to which it is operably ligated. A transcriptional regulatory element is “operably ligated” to a nucleotide sequence if the transcriptional regulatory element controls and regulates the transcription and / or translation of the nucleotide sequence. Thus, transcriptional regulatory elements may include promoters, enhancers, internal ribosome entry sites (IRESs), transcriptional terminators, start codons before protein-coding genes, intron splicing signals, and stop codons. The term “transcriptional regulatory element” is intended to include, at a minimum, elements or sequences of elements designed so that their presence affects expression, and may also include additional beneficial components. The term may also include nucleic acid sequence designs such that undesirable, potential start codons inside or outside the frame are removed from the sequence. It may also include nucleic acid sequence designs such that undesirable, potential splice sites are removed. It may include poly-A tails, i.e., sequences that direct the addition of a chain of adenine residues at the 3' end of mRNA, or polyadenylation sequences (PAs), sequences referred to as poly-A sequences. It may also be designed to enhance mRNA stability. Transcriptional regulatory elements that affect transcriptional and translational stability, such as promoters, and sequences that affect translation, such as Kozak sequences, are known in the art. Transcriptional regulatory elements can be configured to modulate the nucleotide sequence to which they are operably linked, so that lower or higher expression levels are achieved.

[0076] As used herein, the term “promoter” typically refers to a nucleotide sequence located upstream (5') of its coding sequence that controls the expression of the coding sequence by providing recognition for RNA polymerase and other factors necessary for proper transcription. “Promoters” include minimal promoters, which are short DNA sequences consisting of a TATA box and other sequences that help identify the transcription start site, to which regulatory elements are added for control of expression. “Promoter” also refers to a nucleotide sequence containing a minimal promoter + regulatory elements that can control the expression of a coding sequence or functional RNA. This type of promoter sequence consists of proximal and more distal upstream elements, the latter of which are often referred to as enhancers. Thus, an “enhancer” is a DNA sequence that can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the promoter's level or tissue specificity. It can act in both directions (normal or reverse) and can function even when moved upstream or downstream from the promoter. Both enhancers and other upstream promoter elements bind to sequence-specific DNA-binding proteins that mediate their effects. A promoter may be entirely derived from a native gene, or it may be composed of different elements derived from different naturally occurring promoters, or it may be further composed of synthetic DNA segments. A promoter may also include DNA sequences involved in the binding of protein factors that control the effectiveness of transcription initiation in response to physiological or developmental conditions. A “constitutive” promoter is one that is active in most tissues under most physiological and developmental conditions. An “inducible” promoter is one that is physiologically or developmentally regulated, for example, by the application of a chemical inducer. A “tissue-specific” promoter is active only in specific types of tissues or cells.

[0077] As used herein, the terms “nucleic acid construct,” “expression construct,” or “expression cassette” are intended to mean a nucleic acid molecule (typically composed of DNA) capable of directing the expression of a particular nucleotide sequence in a suitable host cell, comprising a promoter operably ligated to a nucleotide sequence of interest operably ligated to a termination signal. It also typically contains sequences necessary for the proper translation of the nucleotide sequence. The coding region usually codes for the protein of interest, but may also code for a functional RNA of interest, e.g., antisense RNA or uncoding RNA, in sense or antisense directions. An expression cassette containing the nucleotide sequence of interest may be a chimeric, meaning that at least one of its components is heterogeneous with respect to at least one of the other components. An expression cassette may also be naturally occurring but obtained in a recombinant form useful for heterogeneous expression. Such an expression cassette includes a transcription start region ligated to the nucleotide sequence of interest. Such an expression cassette may have multiple restriction sites for insertion of the gene of interest under the transcriptional regulation of a regulatory region. The expression cassette may further include a selection marker gene.

[0078] As used herein, the term “intron” refers to a sequence encoded in a DNA sequence that is transcribed into an RNA molecule by RNA polymerase but is removed by splicing to form mature messenger RNA. “Synthetic intron” refers to a sequence that is not initially replicated from a naturally occurring intron sequence and generally does not have a naturally occurring sequence, but is removed from the RNA transcript during normal post-transcriptional processing. Such synthetic introns can be designed to have a variety of different properties, in particular, they can be designed to have a splice site of a desired strength and a desired length. In preferred embodiments of the present invention, both molecular switch expression cassettes and therapeutic gene expression cassettes include synthetic introns. The synthetic introns include consensus sequences for a 5' splice site, a 3' splice site, and a branching point. When incorporated into a eukaryotic vector designed to express a therapeutic gene, the synthetic introns direct the splicing of the RNA transcript in a highly efficient and precise manner, thereby minimizing potential splicing and maximizing the production of the desired gene product.

[0079] As used herein, the terms “treat,” “treat” (and its grammatical variations) mean obtaining a desired pharmacological and / or physiological effect, including reversing, alleviating, inhibiting, or preventing the progression of one or more symptoms of a disease, disorder, or condition to which such terms apply.

[0080] The terms “individual,” “subject,” and “patient” are used interchangeably herein and refer to mammals including, but not limited to, humans and non-human primates, including monkeys and humans; mammalian sports animals (e.g., horses); mammalian farm animals (e.g., sheep, goats, etc.); mammalian pets (e.g., dogs, cats, etc.); and rodents (e.g., mice, rats, etc.). In any of the therapeutic methods described herein, the subject may include mutations associated with diseases or disorders related to misfolded proteins or protein aggregates. Exemplary mutations include the BAG3 P209L mutation or the GGGGCC hexanucleotide repeat in C9orf72. In some embodiments, the subject includes mutations in any one of the following genes: DES, FLNC, MYOT, CRYAB, ZASP, BAG3, FHL1, TTN, PLEC, ACTA1, HSPB8, SOD1, or DNAJB6.

[0081] As used herein, the term “unit dosage form” refers to a physically distinct unit of the formulation of the present invention appropriate for the subject being treated, each unit containing a predetermined amount in optional association with a pharmaceutical carrier (excipient, diluent, vehicle, or filler) that, when administered in one or more doses, produces the desired effect (e.g., prophylactic or therapeutic effect). However, it will be understood that the total daily dose of the compositions of this disclosure is to be determined by the attending physician within the bounds of sound medical judgment. A specific effective dose level for any particular subject will depend on a variety of factors, including the disorder being treated and its severity, the specific composition used, the subject’s age, weight, overall health, sex, and diet, the time of administration, the duration of treatment, any drugs and / or additional therapies used in combination with or concurrently with the compositions of the present invention, and similar factors well known in the medical field. In some embodiments, unit dosage forms may be, for example, in ampoules and vials containing liquid compositions or compositions in a freeze-dried or lyophilized state, and for example, sterile liquid carriers may be added before in vivo administration or delivery. Individual unit dosage forms can be included in multi-dose kits or containers. AAV vectors or AAV virions, and their pharmaceutical compositions, can be packaged in single-dose or multi-dose units to facilitate administration and ensure uniformity of dosage.

[0082] The "therapeutic effective dose" falls within a relatively broad range that can be determined through experiments and / or clinical trials. For example, in the case of in vivo injection, e.g., direct injection into the target tissue (e.g., muscle tissue), the therapeutic effective dose is approximately 10 per kilogram of the subject's body weight. 6 ~about 10 15 This is approximately 10 AAV virions. In some embodiments, the therapeutically effective dose is about 10 per kilogram of the subject's body weight. 8 ~10 12 This is approximately 100 AAV virions. Other effective dosages can be easily determined by those skilled in the art through routine trials to establish dose-response curves.

[0083] As used herein, “effective dose” means an amount of compound, drug, substance, formulation, or composition sufficient to result in a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or prevention of disability or defect due to the distress of the disease. The dose may be a single dose or, according to a multi-dose regimen, alone or in combination with other compounds, drugs, or substances. A person skilled in the art will be able to determine such a dose based on factors such as the size of the subject, the severity of the symptoms of the subject, and the particular composition or route of administration selected.

[0084] When referring to measurable values ​​such as the amount, dose, time, or temperature of a polynucleotide or polypeptide sequence, the terms “about” or “approximately” as used herein mean to include variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% from the specified value, where such variations are appropriate for performing the disclosed method.

[0085] Furthermore, as used herein, “and / or” means any and all possible combinations of one or more of the related enumerated items, as well as the absence of any combination when interpreted as an alternative ("or").

[0086] This disclosure provides BCL2-associated atanogen 3 (BAG3) gene therapy as a feasible therapeutic strategy for treating human disorders (proteinopathy) caused by the accumulation of misfolded protein aggregates. Proteinopathy includes, but is not limited to, neurodegenerative diseases and disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), and Huntington's disease (HD), as well as neuromuscular diseases and disorders also known as protein aggregate myopathy (PAM), such as limb-girdle muscular dystrophy type 2Q (LGMD2Q / LGMDR17) and limb-girdle muscular dystrophy type 1A (LGMD1A). The disclosed compositions and methods enable robust and long-term expression of the BAG-3 gene in muscle and central nervous system (CNS) in the treatment of protein misfolding diseases or disorders, and BAG3 selectively increases the autophagy flux and clearance of misfolded proteins or proteins prone to aggregation. For example, the disclosed compositions and methods increase the recognition and elimination of misfolded proteins. Therefore, the present invention can be used to treat or prevent misfolded proteins, protein aggregates, or protein inclusions both intracellularly and extracellularly. This disclosure relates to the discovery of the role of BAG3 as a molecular cochaperone for refolding or degrading misfolded proteins or proteins prone to aggregation, which play a role in the pathology of various muscle and neurodegenerative disorders.

[0087] polypeptides and nucleic acids that encode polypeptides In certain embodiments, the disclosure relates to polypeptides comprising BAG3 or a functional isoform of BAG3, or fragments thereof.

[0088] The human BAG3 gene, located on the long arm of chromosome 10, encodes a ubiquitously expressed 575-amino acid multifunctional protein primarily expressed in skeletal muscle, cardiac muscle, and the central nervous system (CNS). The NCBI reference nucleic acid sequence of BAG3 can be found in Genbank under accession number NM_004281.4, referred herein as SEQ ID NO: 8. The NCBI reference amino acid sequence of BAG3 can be found in Genbank under accession number NP_004272.2, referred herein as SEQ ID NO: 5.

[0089] During acute proteotoxic stress, BAG3 mediates a non-standard macroautophagy pathway called chaperone-assisted selective autophagy (CASA). Functioning as a scaffold protein, BAG3 interacts and coordinates with the chaperones HSP70 (heat shock protein 70) and HSPB8 (heat shock protein beta-8), as well as the classical selective autophagy receptor p62 / SQSTM1, to selectively identify, refold, or target misfolded and aggregation-prone proteins for autophagy-lysosomal degradation (Arndt et al. 2010. Curr. Biol. 20, 143-148). Therefore, the BAG3-induced selective macroautophagy pathway is a crucial cellular safeguard for protein quality control in response to stress, including pathophysiological conditions. This pathway significantly contributes to protein homeostasis in postmittal cells such as differentiated neurons and striated muscle cells. As a result, BAG3 dysfunction and / or mutations that confer conformational instability to aggregation-prone proteins cause a wide range of diseases, including skeletal muscle myopathy and cardiomyopathy, as well as numerous neurodegenerative diseases.

[0090] In summary, these diseases affect millions of lives worldwide and have devastating economic consequences. However, despite the attention of the scientific community, these disorders remain far from being resolved, as treatments remain palliative. This disclosure focuses on providing a BAG3 transgene for upregulating the BAG3 protein as a therapeutic option for skeletal muscle myopathy and neurodegenerative diseases associated with protein aggregates by activating BAG3-mediated selective autophagy.

[0091] To achieve this, a polynucleotide sequence is provided comprising a transcriptional regulatory element and a nucleotide sequence encoding the human Bcl-related atanogen 3 protein. In a particular embodiment, the nucleotide sequence encoding the BAG3 polypeptide encodes the amino acid sequence of SEQ ID NO: 5.

[0092] In various embodiments, the BAG3 polypeptide includes an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, or 70% identity with the amino acid sequence shown in SEQ ID NO: 5, or the amino acid sequence encoded by nucleotides 1112-2839 of SEQ ID NO: 1, nucleotides 981-2708 of SEQ ID NO: 2, or the nucleotide sequence of SEQ ID NO: 8.

[0093] Amino acid sequence modifications can, for example, be substitutions, deletions, or insertions of one or more amino acids, preferably conservative substitutions. The BAG3 polypeptide may have any combination of amino acid substitutions, deletions, or insertions that preserve the polypeptide's activity. In one embodiment, the BAG3 polypeptide may have numerous amino acid changes such that its amino acid sequence shares at least 60, 70, 80, 85, 90, 95, 97, 98, 99, or 99.5% identity with the amino acid sequence encoded by the BAG3 cDNA (SEQ ID NO: 5), which is shown as nucleotides 1112-2839 of SEQ ID NO: 1, nucleotides 981-2708 of SEQ ID NO: 2, or the nucleotide sequence of SEQ ID NO: 8.

[0094] nucleic acids that encode polypeptides In certain embodiments, the present invention relates to isolated nucleic acids and / or recombinant nucleic acids encoding BAG3 polypeptides. The nucleic acids of the subject may be single-stranded or double-stranded, DNA, or RNA molecules. These nucleic acids are useful as therapeutic agents. For example, these nucleic acids are useful in the production of recombinant polypeptides that are administered to cells or subjects as therapeutic agents. Alternatively, these nucleic acids can be administered directly to cells or organisms as therapeutic agents, such as in gene therapy.

[0095] In some embodiments, the BAG3 nucleic acid comprises a nucleotide sequence containing nucleotides 1112-2839 of SEQ ID NO: 1 and nucleotides 981-2708 of SEQ ID NO: 2, or the nucleotide sequence of SEQ ID NO: 8. In various embodiments, the BAG3 nucleic acid comprises a nucleotide sequence having 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, or 70% identity with the nucleotide sequence of SEQ ID NO: 1 and 2839 of SEQ ID NO: 2, or the nucleotide sequence of SEQ ID NO: 8. Those skilled in the art will understand that nucleic acid sequences complementary to the subject nucleic acid, and variants of the subject nucleic acid, are also within the scope of this disclosure. In further embodiments, the nucleic acid sequences of the present invention may be isolated, recombinant, and / or fused with heterologous nucleotide sequences, or may be present in a DNA library.

[0096] In other embodiments, the nucleic acids of the present invention also include nucleotide sequences that hybridize under highly stringent conditions to nucleotides 1112-2839 of SEQ ID NO: 1, nucleotides 981-2708 of SEQ ID NO: 2, or the nucleotide sequence of SEQ ID NO: 8, or their complementary sequences. As discussed above, those skilled in the art will readily understand that the appropriate stringency conditions for promoting DNA hybridization can be varied. For example, hybridization can be performed by washing with 6.0 × sodium chloride / sodium citrate (SSC) at about 45°C, followed by 2.0 × SSC at 50°C. For example, the salt concentration in the washing step can be selected from low stringency of about 2.0 × SSC at 50°C to high stringency of about 0.2 × SSC at 50°C. In addition, the temperature in the washing step can be increased from low stringency conditions at room temperature or about 22°C to high stringency conditions at about 65°C. Both temperature and salt concentration may be varied, or the temperature or salt concentration may be kept constant while other variables are varied. In one embodiment, the present invention provides nucleic acids that are hybridized with 6×SSC under low stringency conditions at room temperature and subsequently washed with 2×SSC at room temperature.

[0097] Isolated nucleic acids different from the target nucleic acid due to the degeneracy of the genetic code are also within the scope of this invention. For example, some amino acids are specified by two or more triplets. Codons that specify the same amino acid or synonym (e.g., CAU and CAC are synonyms for histidine) may result in "silent" mutations that do not affect the amino acid sequence of the protein. However, DNA sequence polymorphisms that result in changes in the amino acid sequence of the target protein are expected to exist between mammalian cells. Those skilled in the art will understand that these mutations in one or more nucleotides (up to about 3-5% of nucleotides) of the nucleic acid encoding a particular protein may exist between individuals of a given species due to innate allelic variation. Any and all such nucleotide mutations and resulting amino acid polymorphisms are within the scope of this disclosure.

[0098] In some embodiments, a polynucleotide sequence is operably ligated to one or more transcriptional regulatory elements within an expression construct that is functional in the target cell (including, but not limited to, promoters, enhancer or activator sequences, polyadenylation sequences, reader or signal sequences, ribosome binding sites, transcription start and termination sequences, and translation start and termination sequences). Constitutive or inducible promoters known in the art are contemplated in this disclosure. The promoter may be a naturally occurring promoter or a hybrid promoter combining elements of two or more promoters. The expression construct may reside in the cell on an episome, such as a plasmid, or the expression construct may be inserted into a chromosome. In some embodiments, the expression vector includes a selectable marker gene to allow selection of transformed host cells. The selectable marker gene is well known in the art and will vary depending on the host cell used.

[0099] In certain embodiments of this disclosure, the nucleic acid of subject matter is provided in an expression vector comprising a nucleotide sequence encoding a BAG3 polypeptide and operably ligated to at least one transcriptional regulatory element. The transcriptional regulatory element is recognized in the art and selected to direct the expression of the polypeptide. Thus, the term transcriptional regulatory element includes promoters, enhancers, and other expression regulatory elements. Exemplary transcriptional regulatory elements are described in Goeddel; Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, Calif. (1990). For example, any of the broad range of transcriptional regulatory elements that control the expression of a DNA sequence when operably ligated to a DNA sequence may be used in these vectors to express the DNA sequence encoding the polypeptide.Such useful transcriptional regulatory elements include, for example, the H1 promoter, EF1-alpha promoter, minimal EF1-alpha promoter, unc45b promoter, CK1 promoter, CK6 promoter, CK7 promoter, CK8e promoter, cardiac troponin C (cTnC) promoter, miniCMV promoter, CMV promoter, cytomegalovirus / chicken β-actin promoter (also known as CBA or CAG promoter), short CMV early enhancer / chicken β-actin / short β-globin intron (sCAG), muscle creatine kinase (MCK) promoter, hybrid alpha-myosin heavy chain enhancer / MCK enhancer promoter (MHCK7), truncated MCK (tMCK) promoter, minimal MCK promoter, desmin promoter, P546 promoter, Simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV) promoter, and human immunodeficiency virus Examples include the HIV long-terminal repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the Epstein-Barr virus early promoter, the Roussarcoma virus promoter, and, but not limited to, human gene promoters such as the actin promoter, myosin promoter, elongation factor-1a promoter, hemoglobin promoter, and creatine kinase promoter; mouse creatine kinase enhancer elements; skeletal fast-twitch muscle troponin C gene elements; slow-twitch muscle cardiac troponin C gene elements; slow-twitch muscle troponin I gene elements; hypoxia-inducible nuclear factor response elements; steroid-inducible elements or glucocorticoid response elements (GREs); woodchuck hepatitis virus post-transcriptional regulatory elements (WPREs); bovine growth hormone polyadenylation sequences; and other elements known to regulate the expression of genes in prokaryotic or eukaryotic cells, or their viruses, as well as various combinations thereof.

[0100] This also includes inductive promoters. Non-exclusive examples of inductive promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline regulatory promoters.

[0101] Expression cassettes containing the BAG3 transgene also contain intron sequences in some embodiments. As is generally known in the art, introns are DNA polynucleotides that are transcribed into RNA and removed during mRNA processing through intron splicing. Polynucleotide cassettes containing introns generally have higher expression than cassettes without introns, i.e., intron-mediated enhancement (IME). Introns can stimulate expression 2 to 500 times (Buchman and Berg, 1988. Mol Cel Bio, 8(10):4395). Efficiently spliced ​​introns contain pre-splicing donors, branching points, and Py-rich regions (Senapathy et al, 1990; Meth. Enzymol. 183, 252-78, Wu and Krainer, 1999, Mol Cell Biol 19(5):3225-36). 5' introns are generally more efficient than 3' terminal introns (Huang and Gorman, 1990; Mol Cell Bio, 10:1805). Examples of such introns include, but are not limited to, small t-introns, SV40 introns, rabbit β-globin introns, chimeric introns (human β-globin donor and immunoglobulin heavy chain acceptor), chicken β-actin introns, hybrid introns (adenovirus / mouse immunoglobulin), and mouse microvirus (MVM) VP introns. Any intron can be used in the expression cassette so that it can be spliced ​​out from the obtained mRNA product, as long as it contains a splice donor / acceptor region recognized in mammalian cells. In one embodiment, the intron contains, essentially consists of, or comprises an SV40 intron, e.g., nucleotides 830-927 of SEQ ID NO: 2. In another embodiment, the intron includes, essentially consists of, or comprises a chimeric intron, for example, nucleotides 937-1069 of SEQ ID NO: 1.

[0102] In some embodiments, the promoter is the CMV promoter, tMCK promoter, MHCK7 promoter, or CBA promoter. The CMV promoter consists of two elements: a CMV enhancer and the CMV promoter itself, and is selected as a stable, constitutive, and ubiquitous promoter for transgene expression in all cell types. The MHCK7 promoter is highly specific for expression in skeletal muscle, including diaphragm and cardiac tissue, and includes the following elements, an αMHC enhancer, a del63MCK enhancer, and an MCK promoter, to minimize off-target effects in other tissues. The tMCK promoter drives expression in skeletal muscle and consists of a triple tandem of an MCK enhancer to a basal MCK promoter, as described by Wang et al. (Gene Ther. 2008;15(22):1489-1499). The CBA promoter drives high neuronal gene expression.

[0103] In some embodiments, the CBA promoter contains nucleotides 495-749 of SEQ ID NO: 2. In some embodiments, the tMCK promoter contains nucleotides 165-884 of SEQ ID NO: 1. In some embodiments, the CMV enhancer contains nucleotides 209-463 of SEQ ID NO: 2.

[0104] Gene therapy vectors In some embodiments, the Disclosure includes nanoparticles, extracellular vesicles, exosomes, or vectors containing any of the nucleic acids of the Disclosure, or a combination of any one or more thereof, for providing a BAG3 transgene. In some embodiments, one or more copies of these sequences are combined into a single nanoparticle, extracellular vesicle, exosome, or vector.

[0105] Accordingly, the Disclosure includes vectors, which include the nucleic acids of the Disclosure or combinations of the nucleic acids of the Disclosure. Embodiments of the Disclosure deliver the nucleic acids disclosed herein using vectors (e.g., viral vectors, e.g., adeno-associated virus (AAV), adenovirus, retrovirus, lentivirus, equine-associated virus, alphavirus, poxvirus, herpesvirus, herpes simplex virus, poliovirus, Sindbisvirus, vaccinia virus, or synthetic viruses, e.g., chimeric viruses, mosaic viruses, or pseudotyped viruses, and / or viruses containing foreign proteins, synthetic polymers, nanoparticles).

[0106] This disclosure provides a recombinant(r) AAV gene therapy vector containing a nucleic acid comprising a polynucleotide encoding the BAG3 protein for use in subjects requiring treatment for neuroproteinopathy, such as amyotrophic lateral sclerosis (ALS), and myoproteinopathy, such as LGMD1A (myotilinopathy). AAV, as a viral genome, is unique in its safety profile, and once transduced into its carrier cells, remains stably expressed as episomal DNA and is incorporated into the host genome only very rarely.

[0107] Therefore, in some embodiments, the present disclosure utilizes AAV to deliver a BAG3 transgene, such as a cDNA encoding the BAG3 protein. As used herein, the term "AAV" is a common abbreviation for adeno-associated virus. AAV is a replication-deficient parvovirus whose single-stranded DNA genome is approximately 4.7 kb long and contains structural elements known as inverted terminal repeats (ITRs) that form dsDNA hairpin structures at each end. ITRs can form hairpin structures by self-annealing, and they contain Rep-binding elements (RBEs) and terminal degradation sites (TRS) that together constitute the AAV origin of replication. ITRs are also required as packaging signals for post-replication genome capsidation. Construction of an rAAV vector genome requires only that the polynucleotide (or transgene) of interest is adjacent to an AAV terminal ITR and that the total length of the vector genome does not exceed a packaging limit of approximately 5 kb. In any of the AAV genomes described herein, the genome includes at least one inverted terminal repeat (ITR), such as a mutant ITR or a wild-type ITR.

[0108] A key limitation of recombinant adeno-associated virus (rAAV) vector efficiency is the need for host cell-mediated synthesis of double-stranded DNA from a single-stranded genome. The ability to package double-stranded transgene DNA into AAV vectors, referred to as self-complementary AAV (scAAV) vectors, offers an opportunity to bypass the requirement of viral double-stranded DNA synthesis, thus overcoming this limitation. In scAAV vectors, deletion of a terminal degradation site from one of the ITRs prevents replication initiation by the Rep protein from the mutated end. The genome then replicates as an inverse dimer, which will self-anneal after viral decoating (McCarty et al. 2001. Gene Ther 8:1248-1254, McCarty et al. 2003. Gene Ther 10:2112-2118, Wang et al. 2003 Gene Ther. 10(26):2105-2111). Compared to single-stranded AAV (ssAAV) vectors, scAAV vectors have been shown to improve transduction efficiency in vivo, but they reduce the cloning capacity of AAV by half, or to 2.2 kB.

[0109] Exemplary ITR sequences may be 130 base pairs or 141 base pairs long, such as an exemplary 5' ITR containing nucleotides 1-128 or nucleotides 1-128 of SEQ ID NO: 1 or SEQ ID NO: 2, and a deletion of a terminal degradation site, or an exemplary 3' ITR containing nucleotides 3118-3245 of SEQ ID NO: 1 or nucleotides 2987-3114 of SEQ ID NO: 2. In any of the AAV genomes described herein, the genome contains at least one inverted terminal repeat sequence (ITR), such as a mutant ITR or a wild-type ITR.

[0110] Such AAV vectors can replicate and package into infectious viral particles when present in host cells transfected with vectors encoding and expressing rep and cap gene products. AAV is a single-strand replication-deficient DNA parvovirus that grows only in cells where certain functions are provided by co-infecting helper viruses. The AAV genome is approximately 4.7 kb long and contains a 145-nucleotide inverted terminal repeat (ITR). Several serotypes of AAV have been characterized. General information and an overview of AAV can be found, for example, in Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169-228, and Berns, 1990, Virology, pp. 1743-1764, Raven Press, (New York). However, since it is well known that various serotypes are very closely related both structurally and functionally, even at the genetic level, it is quite expected that these same principles may apply to additional AAV serotypes. (See, for example, Blacklowe, 1988, pp. 165-174 of Parvoviruses and Human Disease, JR Pattison, ed., and Rose, Comprehensive Virology 3:1-61 (1974)). For example, all AAV serotypes clearly exhibit very similar replication characteristics mediated by homologous rep genes, all possessing three related capsid proteins, including the one expressed in AAV2. The degree of relatedness is further suggested by heteroduplex analysis, which reveals extensive cross-hybridization between serotypes along genome length and the presence of similar self-annealing segments at the terminals corresponding to "reverse terminal repeats" (ITRs). Similar infectivity patterns also suggest that replication function in each serotype is under similar regulatory control.

[0111] Multiple serotypes of AAV exist. The nucleotide sequences of the genomes of AAV serotypes are known. For example, the nucleotide sequence of the AAV serotype 2 (AAV2) genome is presented in Srivastava et al., J Virol, 45:555-564 (1983), modified by Ruffing et al., J Gen Virol, 75:3385-3392 (1994). As other examples, the complete genome of AAV-1 is available under GenBank acceptance number NC_002077, the complete genome of AAV-3 is available under GenBank acceptance number NC_1829, the complete genome of AAV-4 is available under GenBank acceptance number NC_001829, the genome of AAV-5 is available under GenBank acceptance number AF085716, the complete genome of AAV-6 is available under GenBank acceptance number NC_001862, at least portions of the genomes of AAV-7 and AAV-8 are available under GenBank acceptance numbers AX753246 and AX753249, respectively (see also U.S. Patent Nos. 7,282,199 and 7,790,449 relating to AAV-8), and the genome of AAV-9 is available under GenBank acceptance number NC_002077, the complete genome of AAV-3 is available under GenBank acceptance number NC_1829, the complete genome of AAV-4 is available under GenBank acceptance number NC_001829, the complete The AAV-10 genome is provided in al., J. Virol., 78:6381-6388 (2004), the AAV-11 genome is provided in Mol. Ther., 13(1):67-76 (2006), and the AAV-11 genome is provided in Virology, 330(2):375-383 (2004). Cloning of AAVrh.74 serotypes is described in Rodino-Klapac., et al. Journal of Translational Medicine 5, 45 (2007). The Cis action sequence that directs viral DNA replication (rep), capsid formation / packaging, and host cell chromosome integration is contained within the ITR. Three AAV promoters (named p5, p19, and p40 relative to their relative map locations) drive the expression of two AAV internal open reading frames encoding the rep and cap genes.Two rep promoters (p5 and p19), coupled with differential splicing of a single AAV intron (at AAV2 nucleotides 2107 and 2227), result in the production of four rep proteins (rep78, rep68, rep52, and rep40) from the rep gene. The rep proteins possess multiple enzymatic properties that ultimately contribute to the replication of the viral genome. The cap gene, expressed from the p40 promoter, encodes three capsid proteins: VP1, VP2, and VP3. Alternative splicing and non-consensus translation initiation sites are involved in the production of the three related capsid proteins. A single consensus polyadenylation site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are outlined in Muzyczka, Current Topics in Microbiology and Immunology, 158:97-129 (1992).

[0112] The Cis action sequence, which directs viral DNA replication (rep), capsid formation / packaging, and host cell chromosome integration, is contained within the AAV ITR. Three AAV promoters (named p5, p19, and p40 relative to their relative map locations) drive the expression of two AAV internal open reading frames encoding the rep and cap genes. Coupled with differential splicing of a single AAV intron (at nucleotides 2107 and 2227), the two rep promoters (p5 and p19) result in the production of four rep proteins (rep78, rep68, rep52, and rep40) from the rep gene. The rep proteins possess multiple enzymatic properties that ultimately contribute to the replication of the viral genome. The cap gene is expressed from the p40 promoter and encodes three capsid proteins: VP1, VP2, and VP3. Alternative splicing and non-consensus translation initiation sites are involved in the production of the three related capsid proteins. A single consensus polyadenylation site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are outlined in Muzyczka, Current Topics in Microbiology and Immunology, 158:97-129 (1992).

[0113] AAV possesses unique characteristics that make it attractive as a vector for delivering foreign DNA to cells, for example, in gene therapy. AAV infection of cells in culture is non-cellular, and natural infection in humans and other animals is silent and asymptomatic. Furthermore, AAV infects many mammalian cells and allows for the potential to target many different tissues in vivo. Additionally, AAV can transduce slow-dividing and non-dividing cells and, as a transcriptionally active nuclear episome (extrachromosomal element), can essentially persist for the lifetime of those cells. The AAV proviral genome is infectious as DNA cloned into a plasmid, enabling the construction of recombinant genomes. Furthermore, because the signals directing AAV replication, genomic capsid formation, and integration are contained within the ITR of the AAV genome, part or all of the internal approximately 4.3 kb of genome (rep-cap encoding replication and structural capsid proteins) can be replaced with foreign DNA, such as a gene expression cassette containing a promoter, the DNA of interest, and polyadenylation signals. The rep and cap proteins can be supplied trans. Another important characteristic of AAV is that it is an extremely stable virus. This means it easily withstands the conditions used to inactivate adenoviruses (56°C to 65°C for several hours), reducing the importance of chilling AAV. AAV can even be freeze-dried. Finally, cells infected with AAV do not show resistance to co-infection.

[0114] In some embodiments, AAV lacks rep and cap genes. In some embodiments, AAV is recombinant linear AAV (rAAV), single-stranded AAV (ssAAV), or recombinant self-complementary AAV (scAAV). The self-complementary (sc) technique allows single-stranded viral DNA genomes to bind to themselves, thereby priming double-stranded DNA synthesis. This sc element both accelerates and enhances gene expression compared to constructs lacking the sc element.

[0115] Advances in AAV vectors have resulted in safer and more efficient viral vehicles that deliver therapeutic transgenes with a single injection, making gene therapy a preferred therapeutic intervention for monogenic diseases. AAV vectors can provide long-term expression of gene products in postmittal target tissues. Therefore, current AAV-based strategies may require only a single vector administration.

[0116] The recombinant AAV genomes of this disclosure include, for example, one or more AAV ITRs flanked by polynucleotides encoding one or more MPZ inhibitory RNAs or MPZ miRNAs. The rAAV genomes provided herein either further include RNAi-resistant substituted MPZ genes, or the RNAi-resistant substituted MPZ genes reside in separate rAAVs. The miRNAs and polynucleotides encoding the substituted MPZs are operably ligated to transcriptional regulatory DNA, such as promoter DNA, which is functional in the target cell. Commercial suppliers such as Ambion Inc. (Austin, TX), Darmacon Inc. (Lafayette, CO), InvivoGen (San Diego, CA), and Molecular Research Laboratories, LLC (Herndon, VA) produce custom inhibitory RNA molecules. In addition, commercially available kits such as the SILENCER® siRNA construction kit (Ambion Inc., Austin, TX) or the psiRNA system (InvivoGen, San Diego, CA) are available for generating custom siRNA molecules.

[0117] In some embodiments, AAV is AAV1, AAV9, or AAVrh.74. AAV9 has become the most widely used vector for muscular and / or neurological indications, with a clinically established safety profile. Intrathecal administration of AAV9 allows for the seeding of the transgene throughout the nervous system and is currently FDA approved for spinal muscular atrophy (SMA, NCT03381729), and is in clinical trials for the treatment of neuronal ceroid lipofuscinosis 3 (CLN3, NCT03770572), CLN6 (NCT02725580), giant axonal neuropathy (GAN, NCT02362438), mucopolysaccharidosis type 3A (NCT02716246) and type 3B (NCT03315182), as well as exon 2 duplication in the DMD gene (NCT04240314). These characteristics make AAV9 an ideal gene delivery method for treating disorders in which muscle and heart are the most affected organs. AAV9 has also been shown to target Schwann cells and other peripheral neuropathy. More importantly, AAV9 has been reported to transduce Schwann cells in macro animals and non-human primates, which indicates that it is a desirable viral vector for clinical applications requiring the delivery of therapeutic genes to human Schwann cells. Finally, data from studies in other models of muscle disease show that the AAV9 vector efficiently transfects skeletal muscle, heart, and diaphragm in mice and non-human primates.

[0118] In various forms, AAV is AAVrh.74. Cloning of the AAVrh.74 serotype was described in Rodino-Klapac., et al. Journal of Translational Medicine 5, 45 (2007). AAVrh.74 isolated from rhesus monkeys has good transduction properties with a high tendency to skeletal and cardiac muscle, and it also has lower pre-existing human herd immunity compared to AAV2. AAVrh.74 is used for muscular indications and is in clinical trials for the treatment of limb-girdle muscular dystrophy, type 2E (LGMD2E, NCT03652259), dyspharinopathy, e.g., limb-girdle muscular dystrophy type 2B (LGMD2B) (NCT02710500) by intramuscular injection, and Duchenne muscular dystrophy (NCT03375164) by systemic delivery via peripheral limb venous injection.

[0119] In various forms, AAV is AAV1. AAV1 is commonly used to treat neuromuscular disorders. GLYBERA®, the world's first approved viral gene therapy, uses AAV1 as a vector to deliver an intact copy of the human lipoprotein lipase gene via intramuscular administration in patients with familial lipoprotein lipase deficiency (LPLD). AAV1 is also in clinical trials for the treatment of frontotemporal dementia (NCT04747431) via direct administration into the CSF by intracisional cisterna magna (ICM) injection, Charcot-Marie-tooth neuropathy type 1A (NCT03520751) via intramuscular injection, and congestive heart failure (NCT04703842) via intracoronary delivery to the heart.

[0120] In summary, this highlights the relative safety of gene therapy in diseases requiring local or systemic delivery.

[0121] The DNA plasmid of this disclosure comprises the rAAV genome of this disclosure. In some embodiments, the DNA plasmid is transferred to cells to which infection with an AAV helper virus (e.g., adenovirus, E1-deficient adenovirus, or herpesvirus) is tolerated in order to assemble the rAAV genome into infectious viral particles. Thus, in some embodiments, this disclosure comprises an AAV vector for delivering a therapeutic agent into cells. In some embodiments, the cells are neuronal cells. In some embodiments, the neuronal cells are Schwann cells.

[0122] An "AAV virion," "AAV virus particle," "AAV particle," or "AAV vector particle" refers to a viral particle consisting of at least one AAV capsid protein and a capsid-forming polynucleotide AAV vector. If the particle contains heterologous polynucleotides (i.e., polynucleotides other than the wild-type AAV genome, such as transgenes delivered to mammalian cells), it is typically referred to as an "AAV vector particle" or simply an "AAV vector." Therefore, since such vectors are contained within the AAV vector particle, the production of an AAV vector particle necessarily involves the production of an AAV vector. Techniques for producing rAAV particles, which provide the packaged AAV genome, rep and cap genes, and helper virus function to a cell, are standard in the art. The production of rAAV requires the presence of the following components within a single cell (indicated herein as a packaging cell): the rAAV genome, the AAV rep and cap genes separate from (i.e., not present in) the rAAV genome, and the helper virus function. The AAV rep gene may originate from any AAV serotype capable of inducing recombinant virus, but is not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.74, AAVrh.8, or AAVrh.10, AAVAnc80, AAV7m8, AAV2 / 1, AAV2 / 8, AAV2 / 9, or AAVMYO, and their derivatives, and may originate from an AAV serotype different from the rAAV genome ITR. In some embodiments, the AAV DNA in the rAAV genome is derived from any AAV serotype capable of inducing recombinant viruses, including but not limited to the following: AAV serotypes are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.74, AAVrh.8, or AAVrh.10, AAVAnc80, AAV7m8, AAV2 / 1, AAV2 / 8, AAV2 / 9, or AAVMYO, and their derivatives.For example, other types of rAAV variants, including those with capsid mutations, are also included in this disclosure. Such variants include MyoAAV or AAVMYO, as well as other variants described, for example, in Marsic et al., Molecular Therapy 22(11):1900-1909 (2014), Weismann, J., et al., Nat Commun 11(1):5432 (2020), and Tabebordbar, M. et al., Cell 184(19):4919-4938 e22 (2021), which are incorporated herein by reference in their entirety. As described above, nucleotide sequences of various AAV serotype genomes are known in the art. The use of cognitive components is specifically intended. The production of pseudotype rAAV is disclosed, for example, in WO01 / 83692, which is incorporated herein by reference in its entirety.

[0123] In some embodiments, the viral vector is a pseudotype AAV containing an ITR from one AAV serotype and a capsid protein from a different AAV serotype. In some embodiments, the pseudotype AAV is AAV2 / 9 (i.e., an AAV containing an AAV2 ITR and an AAV9 capsid protein). In some embodiments, the pseudotype AAV is AAV2 / 8 (i.e., an AAV containing an AAV2 ITR and an AAV8 capsid protein). In some embodiments, the pseudotype AAV is AAV2 / 1 (i.e., an AAV containing an AAV2 ITR and an AAV1 capsid protein).

[0124] In some embodiments, AAV contains recombinant capsid proteins such as capsid proteins containing one or more chimeras from capsid proteins of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.74, AAVrh.8, or AAVrh.10, AAVAnc80, AAV7m8, AAV2 / 1, AAV2 / 8, AAV2 / 9, or AAVMYO, and their derivatives. Other types of rAAV variants, e.g., rAAV with capsid mutations, are also intended. See, for example, Marsic et al., Molecular Therapy, 22(11):1900-1909 (2014). Nucleotide sequences of genomes of various AAV serotypes are known in the art.

[0125] Multiple studies have demonstrated long-term (over 1.5 years) recombinant AAV-mediated protein expression in muscle. See Clark et al., Hum Gene Ther, 8:659-669 (1997), Kessler et al., Proc Nat. Acad Sc. USA, 93:14082-14087 (1996), and Xiao et al., J Virol, 70:8098-8108 (1996). Also see Chao et al., Mol Ther, 2:619-623 (2000) and Chao et al., Mol Ther, 4:217-222 (2001). Furthermore, because muscles are highly vascularized, recombinant AAV transduction resulted in the appearance of the transgene product in systemic circulation after intramuscular injection, as described by Herzog et al., Proc Natl Acad Sci USA, 94:5804-5809 (1997) and Murphy et al., Proc Natl Acad Sci USA, 94:13921-13926 (1997). In addition, Lewis et al., J Virol, 76:8769-8775 (2002) demonstrated that skeletal muscle fibers possess the necessary cellular factors for correct antibody glycosylation, folding, and secretion, showing that muscles are capable of stable expression of secreted protein therapeutics.

[0126] A recombinant AAV genome, in various embodiments, comprises the nucleic acid molecule of this disclosure and one or more AAV ITRs adjacent to the nucleic acid molecule. The AAV DNA of the rAAV genome may be derived from any AAV serotype capable of inducing recombinant viruses, including but not limited to AAV serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVRH10, AAVRH74, AAV11, AAV12, AAV13, or Anc80, AAV7m8, and their derivatives). The production of pseudotyped rAAV is disclosed, for example, in WO01 / 83692. Other types of rAAV variants, e.g., rAAV with capsid mutations, are also intended. See, for example, Marsic et al., Molecular Therapy, 22(11):1900-1909 (2014). As described in the background technology section above, the nucleotide sequences of various AAV serotype genomes are known in the art.

[0127] The recombinant AAV provided (i.e., infectious capsid-forming rAAV particles) contains an rAAV genome. The term “rAAV genome” refers to a polynucleotide sequence derived from a modified native AAV genome. In some embodiments, the rAAV genome is modified to remove native cap and rep genes. In some embodiments, the rAAV genome contains endogenous 5' and 3' reverse end repeats (ITRs). In some embodiments, the rAAV genome contains ITRs from an AAV serotype different from the AAV serotype from which the AAV genome is derived. In some embodiments, the rAAV genome contains a transgene of interest flanked at the 5' and 3' ends by reverse end repeats (ITRs). In some embodiments, the rAAV genome contains a “gene cassette”. In exemplary embodiments, both rAAV genomes lack AAV rep and cap DNA, i.e., there is no AAV rep or cap DNA between the ITRs of the rAAV genome.

[0128] The DNA plasmids of this disclosure include the rAAV genome of this disclosure. The DNA plasmids are transferred to cells that are tolerant of infection with an AAV helper virus (e.g., adenovirus, E1 deletion adenovirus, or herpesvirus) in order to assemble the rAAV genome into infectious viral particles. Techniques for producing rAAV particles that provide the cell with the packaged AAV genome, rep and cap genes, and helper virus function are standard in the art. The production of rAAV requires that the following components be present in a single cell (indicated herein as the packaging cell): the rAAV genome, the AAV rep and cap genes separate from (i.e., not present in) the rAAV genome, and the helper virus function. The AAV rep and cap genes may be derived from any AAV serotype capable of inducing recombinant virus, including but not limited to AAV serotypes AAV9, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAVrh.74, AAV8, AAV10, AAV11, AAV12, and AAV13, and may be derived from an AAV serotype different from the rAAV genome ITR. The production of pseudotyped rAAV is disclosed, for example, in WO01 / 83692, which is incorporated in its entirety herein by reference.

[0129] The method for generating packaging cells involves creating a cell line that stably expresses all the components necessary for AAV particle production. For example, a plasmid (or multiple plasmids) containing an rAAV genome lacking AAV rep and cap genes, AAV rep and cap genes isolated from the rAAV genome, and selectable markers such as neomycin resistance genes, is incorporated into the cell genome. The AAV genome is introduced into bacterial plasmids by procedures such as GC tailing (Samulski et al. 1982. Proc Natl Acad Sci USA. 79, 2077-2081), addition of a synthetic linker containing restriction endonuclease cleavage sites (Laughlin et al. 1983. Gene. 23, 65-73), or direct blunt-end ligation (Senapathy et al. 1984. J Biol Chem. 259, 4661-4666). Subsequently, the packaging cell line is infected with a helper virus such as adenovirus. The advantages of this method are that the cells are selectable and it is suitable for large-scale production of rAAV. Another example of a suitable method is to use adenovirus or baculovirus instead of plasmid to introduce the rAAV genome and / or rep and cap genes into packaging cells.

[0130] The general principles of rAAV production are outlined, for example, in Carter, 1992, Current Opinions in Biotechnology, 1533-539, and Muzyczka, 1992, Curr. Topics in Microbial. and Immunol., 158:97-129. Various approaches have been proposed by Ratschin et al., Mol. Cell. Biol. 4:2072 (1984), Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984), Tratschin et al., Mol. Cell. al., J.Virol.,62:1963(1988), and Lebkowski et al.,Mol.Cell.Biol.,Oct;8(10):3988-96(1988), Samulski et al. al., J. Virol., 63:3822-3828 (1989), U.S. Patent No. 5,173,414, WO95 / 13365 and corresponding U.S. Patent No. 5,658,776, WO95 / 13392, WO96 / 17947, PCT / US98 / 18600, WO97 / 09441 (PCT / US96 / 14423), WO97 / 08298 (PCT / US96 / 13872), WO97 / 21825 (PCT / US96 / 20777), WO97 / 06243 (PCT / FR96 / 01064), WO99 / 11764, Perrin et al. Vaccine 13:1244-1250 (1995), Paul et al. This is described in al. Human Gene Therapy 4:609-615 (1993), Clark et al. Gene Therapy 3:1124-1132 (1996) 51, U.S. Patent Nos. 5,786,211, 5,871,982, and 6,258,595. The above documents are incorporated herein by reference in their entirety, with particular emphasis on the portions relating to rAAV production. The production and use of self-complementary (sc)rAAV are particularly intended and illustrated.

[0131] Accordingly, this disclosure provides packaging cells that produce infectious rAAV. In one embodiment, the packaging cells are stably transformed cancer cells such as HeLa cells, 293 cells, and PerC.6 cells (allogeneic 293 strain). In another embodiment, the packaging cells are non-transformed cancer cells such as low-passage 293 cells (human fetal kidney cells transformed with adenovirus E1), MRC-5 cells (human fetal fibroblasts), WI-38 cells (human fetal fibroblasts), Vero cells (monkey kidney cells), and FRhL-2 cells (rhesus monkey fetal lung cells).

[0132] In some embodiments, rAAV is purified by methods standard in the art, for example, by column chromatography or a cesium chloride gradient. Methods for purifying rAAV vectors from helper viruses are known in the art and include, for example, those disclosed in Clark et al., Hum. Gene Ther., 10(6):1031-1039 (1999), Schenpp and Clark, Methods Mol. Med., 69 427-443 (2002), U.S. Patent No. 6,566,118 and WO98 / 09657.

[0133] Composition and method of use Compositions comprising nucleic acids and viral vectors as disclosed herein are provided. Compositions comprising delivery vehicles (such as rAAV) as described herein are provided. In various embodiments, such compositions also comprise a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is a diluent, excipient, or buffer. The compositions may also comprise other components, such as adjuvants.

[0134] Acceptable carriers, diluents, excipients, and adjuvants are nontoxic to the recipient and preferably inert at the dose and concentration used, and include buffers such as phosphates, citrates, or other organic acids; antioxidants such as ascorbic acid; low molecular weight polypeptides; proteins, e.g., serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as Tween®, Pluronic®, or polyethylene glycol (PEG).

[0135] Sterile injectable solutions are prepared by incorporating the required amount of rAAV into a suitable solvent, along with various other components listed above as needed, and then sterilizing by filtration. Generally, dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle containing a basic dispersion medium and other required components from those listed above. For sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient and any additional desired components from its previously sterile filtered solution.

[0136] The dose of rAAV administered by the method of this disclosure will vary depending, for example, the specific rAAV, the mode of administration, the time of administration, the therapeutic target, the individual, and the targeted cell type, and may be determined by methods standard in the art. The dose may be expressed in units of viral genome (vg). The dose intended herein is approximately 1 × 10⁻⁶ 7 , 1 x 10 8 , 1 x 10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , 9×10 9 , 1 x 10 10, 2×10 10 , 3 x 10 10 , 4×10 10 , 5×10 10 , 1 x 10 11 , about 1×10 12 , about 1×10 13 , about 1.1×10 13 , about 1.2×10 13 , about 1.3×10 13 , about 1.5×10 13 , about 2×10 13 , about 2.5×10 13 , about 3×10 13 , about 3.5×10 13 , about 4×10 13 , about 4.5×10 13 , about 5×10 13 , about 6×10 13 , about 7×10 13 , about 8×10 13 , about 9×10 13 , about 1×10 14 , about 2×10 14 , about 3×10 14 , about 4×10 14 , about 5×10 14 , about 1×10 15 , about 1×10 16 This includes the entire viral genome up to or beyond.

[0137] Approximately 1×10 9 ~Approx. 1×10 10 , about 5×10 9 ~Approx. 5×10 10 , about 1×10 10 ~Approx. 1×10 11 , about 1×10 11 ~Approx. 1×10 15 vg, approx. 1×10 12 ~Approx. 1×10 15 vg, approx. 1×10 12 ~Approx. 1×10 14 vg, approx. 1×10 13 ~about 6×10 14 vg, approx. 1×10 13 ~Approx. 1×10 15 vg, and approximately 6 × 10 13 ~Approx. 1.0×10 14The dosage of vg is also contemplated. The single dosages exemplified herein are 1×10 13 vg administered via intravenous or intraperitoneal delivery.

[0138] The dosage can also be expressed in units of vg / kg. The dosages contemplated herein are approximately 1×10 7 vg / kg, 1×10 8 vg / kg, 1×10 9 vg / kg, 5×10 9 vg / kg, 6×10 9 vg / kg, 7×10 9 vg / kg, 8×10 9 vg / kg, 9×10 9 vg / kg, 1×10 10 vg / kg, 2×10 10 vg / kg, 3×10 10 vg / kg, 4×10 10 vg / kg, 5×10 10 vg / kg, 1×10 11 vg / kg, approximately 1×10 12 vg / kg, approximately 1×10 13 vg / kg, approximately 1.1×10 13 vg / kg, approximately 1.2×10 13 vg / kg, approximately 1.3×10 13 vg / kg, approximately 1.5×10 13 vg / kg, approximately 2×10 13 vg / kg, approximately 2.5×10 13 vg / kg, approximately 3×10 13 vg / kg, approximately 3.5×10 13 vg / kg, approximately 4×10 13 vg / kg, approximately 4.5×10 13 vg / kg, approximately 5×10 13 vg / kg, approximately 6×10 13 vg / kg, approximately 7×10 13 , approximately 8×10 13 , approximately 9×10 13 , approximately 1×10 14 vg / kg, approximately 2×10 14 vg / kg, approximately 3×10 14 vg / kg, approximately 4×10 14 vg / kg, approximately 5×10 14vg / kg, approx. 1×10 15 vg / kg, approx. 1×10 16 Includes up to vg / kg.

[0139] Approximately 1×10 9 vg / kg ~ approx. 1×10 10 vg / kg, approx. 5×10 9 vg / kg ~ approx. 5×10 10 vg / kg, approx. 1×10 10 vg / kg ~ approx. 1×10 11 vg / kg, approx. 1×10 11 vg / kg ~ approx. 1×10 15 vg / kg, approx. 1×10 12 vg / kg ~ approx. 1×10 15 vg / kg, approx. 1×10 12 vg / kg ~ approx. 1×10 14 vg / kg, approx. 1×10 13 vg / kg ~ approx. 2×10 14 vg / kg, approx. 1×10 13 vg / kg ~ approx. 1×10 15 vg / kg, and approximately 6 × 10 13 vg / kg ~ approx. 1.0×10 14 Dosages of vg / kg are also intended. The single dose exemplified herein is 1 × 10⁶ when administered via intravenous or intraperitoneal delivery. 13 It is vg / g.

[0140] Transduction or transfection of cells with rAAV as described herein results in sustained expression of the BAG3 gene / protein. As used herein, the terms “transduction” and “transfection” are interchangeable. The terms “transduction” or “transfection” are used, for example, to refer to the administration / delivery of the BAG3 gene to target cells, either in vivo or in vitro, via replication-deficient rAAV as described herein, resulting in the expression of the BAG3 gene / protein by the target cells. Therefore, this disclosure provides methods for administering / delivering rAAV expressing the BAG3 gene to cells or subjects. In some embodiments, the subjects are mammals. In some embodiments, the mammals are humans. These methods include transduction of cells and tissues (including, but not limited to, peripheral motor neurons, sensorimotor neurons, neurons, Schwann cells, and other tissues or organs such as muscle, heart, liver, and brain) using one or more rAAVs as described herein. Transduction may be performed with a gene cassette containing cell-specific regulatory elements.

[0141] A method is provided for transducing target cells in vivo or in vitro using a delivery vehicle (e.g., nanoparticles, extracellular vesicles, exosomes, or a vector (e.g., rAAV)). The in vivo method includes the step of administering an effective dose or effective multiple doses of a composition comprising a delivery vehicle (such as rAAV) to an animal (including a human subject or patient) that requires it. If the dose is administered before the onset of the disorder / disease, the administration is prophylactic. If the dose is administered after the onset of the disorder / disease, the administration is therapeutic. "Effective dose" is a dose that alleviates (eliminates or reduces) at least one symptom associated with the disorder / disease condition being treated, a dose that slows or prevents progression to the disorder / disease condition, a dose that slows or prevents progression to the disorder / disease condition, a dose that reduces the severity of the disease, a dose that results in remission (partial or complete) of the disease, and / or a dose that prolongs survival. Therefore, this specification provides a method for administering an effective dose of rAAV (or a dose that is essentially administered simultaneously or at intervals) to a subject who requires it.

[0142] Provided herein are pharmaceuticals and methods for treating, improving, or preventing diseases or disorders associated with misfolded proteins or protein aggregates, or diseases or disorders associated with mutant BAG3 genes or abnormal BAG3 gene expression. Molecular, biochemical, histological, and functional outcome measures demonstrate the therapeutic efficacy of the methods. Levels of human BAG3 transcripts in animals can be confirmed by RT-PCR and / or RNA-seq. BAG3 protein expression levels in skeletal muscle, including the heart and diaphragm, can be assessed using Western blotting. Localization of BAG3 in Z-disks and proper co-localization with its binding partner, as well as the pathological features of muscle and inflammation, can be confirmed by immunohistochemistry. To evaluate the potential therapeutic efficacy in mice, muscle contraction function can be measured using the Aurora Whole Animal Muscle Test System. In patients, various functional outcome measures may be used to assess successful treatment, including a 100-meter timed test, a 10-meter walking / running test, the North Star gait scale (NSAD) for limb girdle muscular dystrophy, upper limb performance (PUL) 2.0, and force myometry assessments (including measures of shoulder abduction, elbow flexion / extension, and knee flexion / extension force).

[0143] In the method of the present disclosure, BAG3 protein expression is increased by at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 95, at least 98 percent, at least 99 percent, or 100 percent.

[0144] Combination therapy Combination therapies are also contemplated in this disclosure. The combinations used herein include both concurrent and sequential therapies. Combinations of the methods disclosed with standard medical procedures, along with combinations with novel therapies, are particularly contemplated.

[0145] Combination therapy with immunosuppressants In some embodiments, combination therapy includes administering an immunosuppressant in combination with a gene therapy disclosed herein.

[0146] Immunosuppressants may be administered after gene therapy, before or after the onset of the immune response to rAAV in the subject. In addition, immunosuppressants may be administered concurrently with gene therapy or protein replacement therapy. The immune response in the subject includes adverse immune responses or inflammatory reactions that follow or are triggered by the administration of rAAV to the subject. The immune response may be the production of antibodies in the subject in response to the administered rAAV.

[0147] Examples of immunosuppressants include cell quiescent agents such as glucocorticosteroids, Janus kinase inhibitors, calcineurin inhibitors, mTOR inhibitors, purine analogs, methotrexate, and cyclophosphamide; biologics such as inosine monophosphate dehydrogenase (IMDH) inhibitors, monoclonal antibodies or fusion proteins and polypeptides; and dipeptide boronic acid molecules such as bortezomib.

[0148] Immunosuppressants may be anti-inflammatory steroids, which are steroids that reduce inflammation and suppress or modulate the target immune system. Exemplary anti-inflammatory steroids are glucocorticoids such as prednisolone, betamethasone, dexamethasone, methotrexate, hydrocortisone, methylprednisolone, deflazacort, budesonide, or prednisone.

[0149] Janus kinase inhibitors are inhibitors of the JAK / STAT signaling pathway that target one or more enzymes in the Janus kinase family. Exemplary Janus kinase inhibitors include tofacitinib, baricitinib, upadacitinib, peficitinib, and oclacitinib.

[0150] Calcineurin inhibitors bind to cyclophylline and inhibit the activity of calcineurin. Examples of calcineurin inhibitors include cyclosporine, tacrolimus, and piceclorimus.

[0151] mTOR inhibitors reduce or inhibit the serine / threonine-specific protein kinase mTOR. Exemplary mTOR inhibitors include rapamycin (also known as sirolimus), everolimus, and temsirolimus.

[0152] Immunosuppressants include immunosuppressive macrolides. The term "immunosuppressive macrolide" refers to macrolide agents that suppress or modulate the target immune system. Macrolides are a class of drugs that contain a large macrocyclic lactone ring to which one or more deoxy sugars, such as cladinose or desamine, are attached. The lactone ring is usually 14, 15, or 16 members. Macrolides belong to the polyketide class of drugs and can be natural products. Examples of immunosuppressive macrolides include tacrolimus, pimecrolimus, and rapamycin (also known as sirolimus).

[0153] Purine analogs inhibit nucleotide synthesis and include IMDH inhibitors. Exemplary purine analogs include azathioprine, mycophenolates such as mycophenolate acids or mycophenolate mofetil, and refnomide.

[0154] Examples of immunosuppressive biologics include abatacept, adalimumab, anakinra, certolizumab, etanercept, golimumab, infliximab, ixekizumab, natalizumab, rituximab, secukinumab, tocilizumab, ustekinenumab, vedolizumab, basiliximab, belatacept, and daclizumab.

[0155] In particular, immunosuppressants are anti-CD20 antibodies. The term anti-CD20-specific antibody refers to an antibody that specifically binds to CD20 or inhibits or reduces the expression or activity of CD20. Examples of anti-CD20 antibodies include rituximab, ocrelizumab, or ofatumumab.

[0156] Examples of additional immunosuppressive antibodies include anti-CD25 antibodies (or anti-IL2 antibodies or anti-TAC antibodies) such as basiliximab and daclizumab, as well as anti-CD3 antibodies such as muromonab-CD3, otelixizumab, teprizumab, and vizilizumab, and anti-CD52 antibodies such as alemtuzumab.

[0157] One exemplary combination therapy is the delivery of rapamycin and rituximab before or concurrently with the delivery of the AAV vector. Another exemplary combination therapy is the delivery of rapamycin, rituximab, and a corticosteroid, such as prednisone.

[0158] Combination therapy with neurotrophic factors (NTFs) In some embodiments, combination therapy involves administering neurotrophic factors (NTFs) in combination with gene therapies disclosed herein. NTFs are necessary to modulate several physiological processes in the nervous system, including neurogenic differentiation and survival of stem cells, axonal growth and synapse maintenance, proliferation and differentiation (Chao MV.2003.Nat Rev Neurosci.4(4):299-309). Furthermore, reduced levels of NTF have been reported in several neurodegenerative disorders, including ALS (Anand et al. 1995. Nat Med. 1(2):168-72.9-11, Lee et al. 1996 J Neuropathol Exp Neurol. 55(8):915-23, Ono et al. 1999. Eur Neurol. 42(3):163-8.8092) (Connor B, Dragunow M. 1998. Brain Res Brain Res Rev. 27(1):1-39), suggesting that loss of nutritional support may be important in the pathophysiology of the disease. In the context of amyotrophic lateral sclerosis (ALS), it has long been hypothesized that a lack of neurotrophic growth factor is one of the neurotoxic contributing factors to the disease, leading to motor neuron death. Furthermore, in ALS mouse models, for example, SOD1 G93A Previous studies in mice have demonstrated preclinical efficacy using BDNF, CNTF, and GDNF (reviewed in Gouel et al. 2019. Front Neurol. 10:835). Therefore, the combination of NFT and the BAG3 substitution therapy of this disclosure represents a promising synergistic therapeutic strategy for treating neurodegenerative diseases caused by protein aggregation, such as ALS.

[0159] In some embodiments, NTFs are selected from the group consisting of brain-derived growth factor (BDNF), neurotrophin-3 (NT-3), nerve growth factor (NGF), fibroblast growth factor (FGF), and glial cell-derived growth factor (GDNF). In some embodiments, the NFT is NT-3. NT-3 belongs to a family of growth factors called neurotrophins and has been shown to be a major mediator of neurodevelopment not only throughout adulthood but also during the early neurogenic period. In humans, NT-3 is encoded by the NTF3 gene, which encodes a 119-amino acid polypeptide that localizes to chromosome 12p13 and forms a homodimer (Maisonpierre et al. 1991. Genomics. 10:558-568). NT3 exerts its effects by binding to high-affinity tropomyosin receptor kinase (e.g., tropomyosin-related kinase C receptor (TrkC)) receptors and low-affinity p75 neurotrophin receptor (p75NTR). NTF3 binding to TrkC induces PI3K / Akt and RAS / ERK signaling pathways that regulate cell growth, survival, and differentiation (Chao MV.2003. Nat Rev Neurosci 4(4):299-309, Liot et al. Exp Neurol.187(1):38-46). Functional deficiency of NTF3 has been shown to cause severe neuronal deficits and early postnatal death in mice (Conover JC, Yancopoulos GD.1997. Rev Neurosci.8(1):13-27). Furthermore, motor neurons derived from conditionally Ntf3 knockout embryos show increased apoptosis and abnormal projections in central branch nerve-stimulated motor neurons, suggesting that NTF3 is crucial for neuronal survival and axonal projection (Usin et al.2012. Development 139(6):1125-1132).

[0160] In some embodiments, the NT-3 polynucleotide in the rAAV genome is the NT-3 cDNA shown in SEQ ID NO: 9. In some embodiments, the NT-3 polynucleotide in the rAAV genome is the NT-3 cDNA shown in Genbank accession number NM_001102654 or the NT-3 cDNA sequence shown in SEQ ID NO: 9, or a polynucleotide having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the NT-3 cDNA. In some embodiments, the NT-3 polynucleotide encodes the same NT-3 polypeptide as encoded by the NT-3 cDNA of SEQ ID NO: 9. The amino acid sequence of the NT-3 polypeptide encoded by the NT-3 cDNA is shown as SEQ ID NO: 9. Amino acid sequence modifications can, for example, be substitutions, deletions, or insertions of one or more amino acids, preferably conservative substitutions. The NT-3 polypeptide may have any combination of amino acid substitutions, deletions, or insertions in which polypeptide activity is preserved. In one embodiment, the NT-3 polypeptide may have numerous amino acid changes such that its amino acid sequence shares at least 60, 70, 80, 85, 90, 95, 97, 98, 99, or 99.5% identity with the amino acid sequence (SEQ ID NO: 10) encoded by the NT-3 cDNA shown as SEQ ID NO: 9 or provided as Genbank accession number NM_001102654.

[0161] In some embodiments, the rAAV genome is the sc.AAV1.tMCK.NTF3 genome. An exemplary scAAV1.tMCK.NT-3 is disclosed in International Publication No. WO2019 / 079755, which is incorporated herein by reference in its entirety.

[0162] In yet another embodiment, an isolated nucleic acid is provided comprising the nucleotide sequence shown in SEQ ID NO: 12. Also provided is an isolated nucleic acid comprising, in 5' to 3' order, (i) a first AAV2 inverted terminal repeat (ITR); (ii) a muscle creatine kinase promoter sequence; (iii) a nucleotide sequence encoding a human NT-3 polypeptide (SEQ ID NO: 9); and (iv) a second AAV2 ITR sequence, wherein the human NT-3 polypeptide has an amino acid sequence that is at least 90% identical and 100% identical to SEQ ID NO: 10.

[0163] The intended recombinant AAVs include the nucleic acids mentioned above, and rAAVs include nucleotide sequences that are identical to the nucleotide sequence shown in Sequence ID No. 12 by at least 60, 70, 80, 85, 90, 95, 97, 98, 99, or 99.5%.

[0164] The administration of an effective dose of the nucleic acids, nanoparticles, extracellular vesicles, exosomes, viral vectors, or compositions of this disclosure may be by routes standard in the art, including but not limited to intramuscular, parenteral, intravascular, intravenous, oral, buccal, nasal, transpulmonary, intracranial, intraventricular, intrathecal, intraosseous, intraocular, intrarectal, or vaginal. In various embodiments, the effective dose is delivered by a combination of routes. For example, in various embodiments, the effective dose is delivered intravenously and / or intramuscularly, or intravenously and intraventricularly, etc. In some embodiments, the effective dose is delivered sequentially or consecutively. In some embodiments, the effective dose is delivered simultaneously. The routes of administration and serotypes of the AAV components of rAAV of this disclosure (in particular, AAV ITR and capsid protein) may be selected and / or adapted by those skilled in the art, taking into account the infection and / or disease state being treated, as well as the target cells / tissues expressing miRNA.

[0165] In particular, the actual administration of a delivery vehicle (such as rAAV) can be achieved by using any physical method to transport the delivery vehicle (such as rAAV) to target cells of interest. Administration methods include, but are not limited to, intramuscular injection, bloodstream injection, and / or direct injection into the nervous system or liver. It has been demonstrated that simply resuspending rAAV in phosphate-buffered saline is sufficient to provide a vehicle useful for muscle tissue expression, and there are no known limitations on carriers or other components that may be co-administered with rAAV (however, compositions that degrade DNA should be avoided in the usual manner with rAAV). The capsid protein of rAAV may be modified so that rAAV targets specific target tissues of interest, such as neurons. See, for example, WO02 / 053703, the disclosure of which is incorporated herein by reference. Pharmaceutical compositions can be prepared as injectable formulations or as topical formulations delivered to muscle by transdermal transport. Numerous formulations for both intramuscular injection and transdermal delivery have been previously developed and can be used in carrying out the methods of this disclosure. The delivery vehicle (such as rAAV) can be used with any pharmaceutically acceptable carrier to facilitate administration and handling.

[0166] Dispersions of delivery vehicles (such as rAAV) can also be prepared in glycerol, sorbitol, liquid polyethylene glycol, and mixtures thereof, as well as in oil. Under normal storage and use conditions, these formulations contain preservatives to prevent microbial growth. In this regard, all sterile aqueous media used are readily available by standard techniques well known to those skilled in the art.

[0167] Pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. In all cases, these forms must be sterile and fluid enough to allow for easy syringe injection. They must be stable under manufacturing and storage conditions and protected against microbial contamination such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, sorbitol, etc.), suitable mixtures thereof, and vegetable oils. Adequate fluidity can be maintained, for example, by the use of coating agents such as lecithin, by maintaining the required particle size in the case of dispersants, and by the use of surfactants. Prevention of microbial action can be provided by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it would be preferable to include an isotonic agent, such as suMPZ or sodium chloride. The prolonged absorption of injectable compositions can be achieved by using absorption-delaying agents, such as aluminum monostearate and gelatin.

[0168] Sterile injectable solutions are prepared by incorporating the required amount of rAAV into a suitable solvent, along with various other components listed above as needed, and then sterilizing by filtration. Generally, dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle containing a basic dispersion medium and other required components from those listed above. For sterile powders for the preparation of sterile injectable solutions, preferred methods of preparation are vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient plus any additional desired components from those solutions that have been previously sterilized and filtered.

[0169] This disclosure also provides kits for use in the treatment of diseases or disorders described herein. Such kits include at least a first sterile composition comprising, in a pharmaceutically acceptable carrier, one of the nucleic acids described herein or one of the viral vectors described herein. Another component is optionally a second therapeutic agent for the treatment of the disorder, together with a suitable container and vehicle for administering the therapeutic composition. The kit optionally includes a solution or buffer for suspending, diluting, or affecting the delivery of the first and second compositions.

[0170] In one embodiment, such a kit comprises a nucleic acid or vector in a diluent packaged in a container such as a sealed bottle or vessel, with a label affixed to the container or included in a package describing the use of the nucleic acid or vector. In one embodiment, the diluent is contained in the container such that the amount of headspace within the container (e.g., the amount of air between the liquid formulation and the top of the container) is very small. Preferably, the amount of headspace is negligible (i.e., almost none).

[0171] In some embodiments, the formulation includes a stabilizer. The term “stabilizer” refers to a substance or excipient that protects the formulation from harmful conditions, such as those that occur during heating or freezing, and / or extends the stability or shelf life of the formulation in a stable state. Examples of stabilizers include, but are not limited to, stabilizers such as sucrose, lactose, and mannose; sugar alcohols such as mannitol; amino acids such as glycine or glutamic acid; and proteins such as human serum albumin or gelatin.

[0172] In some embodiments, the formulation includes an antimicrobial preservative. The term “antimicrobial preservative” refers to any substance added to the composition that inhibits the growth of microorganisms that may be introduced during repeated punctures of the vial or container used. Examples of antimicrobial preservatives include, but are not limited to, thimerosal, 2-phenoxyethanol, benzethonium chloride, and phenol.

[0173] In some embodiments, the kit includes a label and / or instructions describing the use of the reagents provided in the kit. The kit also optionally includes a catheter, syringe, or other delivery device for delivering one or more of the compositions used in the methods described herein.

[0174] The entire document is intended to be related as a unified disclosure, and it should be understood that all combinations of features described herein are contemplated even if combinations of features do not appear together in the same sentence, paragraph, or section as in this document. The disclosure also includes all embodiments of the disclosure, which are, for example, somewhat narrower in scope than the variations specifically mentioned above. With respect to aspects of the disclosure described as a genus, all individual species are considered distinct aspects of the disclosure. With respect to aspects of the disclosure described or claimed as "a" or "an," these terms should be understood to mean "one or more" unless the context explicitly requires a more limited meaning. Where an aspect of the disclosure is described as "containing" a feature, the embodiment is also contemplated to "consist of" or "essentially consist of" that feature.

[0175] All publications, patents, and patent applications cited herein are incorporated herein by reference in such a manner as each individual publication or patent application is specifically and individually indicated to be incorporated in whole by reference, to the extent that it does not conflict with this disclosure.

[0176] The examples and embodiments described herein are for illustrative purposes only, and various modifications or changes will be suggested to those skilled in the art from their perspective and should be understood to be within the scope of the spirit and scope of this application and the appended claims. The following examples are provided as examples and not as limitations.

[0177] The numerical ranges described include each integer value within each range, as well as the minimum and maximum integers described. [Examples]

[0178] Example 1 Construction of a BAG3 expression construct The AAV genome constructs encoding BAG3 used in the following examples are non-replicating recombinant adeno-associated viruses called ssAAVrh74.tMCK.BAG3 (Figure 1A) and ssAAV9.CMV.CBA.BAG3 (Figure 1B).

[0179] First, the ss.pAAV.CMV.BAG3 plasmid was cloned, and an AAV serotype 9 vector containing human BAG3 cDNA was produced under the CMV promoter using standard methodology. The ss.AAV.CMV.BAG3 cassette for gene transfer, containing the CMV enhancer / promoter, full-length BAG3 cDNA, and SV40 poly-A tail, is provided as Sequence ID No. 13.

[0180] Generation of novel plasmids suitable for systemic treatment of protein aggregate myopathy and neurodegenerative disorders. We generated additional plasmids suitable for systemic treatment of protein aggregate myopathy and neurodegenerative disorders. To avoid potential off-target effects, the CMV promoter was replaced with a muscle-specific promoter, the tMCK enhancer / promoter, and the serotype was switched to the AAVrh74 serotype. The construct also included an SV40 poly-A tail. For the treatment of an animal model of myofibrild myopathy, we generated the AAVrh74.tMCK.hBAG3 vector (Figure 1B). The sequence of AAVrh74.tMCK.hBAG3 is provided in Figure 17 (SEQ ID NO: 1).

[0181] In addition, plasmids were generated in which the CMV promoter in the initial cassette was replaced with a chicken beta-actin (CBA) promoter / CMV enhancer, an SV40 tail was included, and the serotype was switched to the AAV9 serotype. The AAV9.CBA.hBAG3 vector was generated for systemic treatment of a rodent model of misfolded protein-associated neurodegenerative disease (Figure 1C). The sequence of AAV9.CBA.hBAG3 is provided in Figure 18 (SEQ ID NO: 2).

[0182] rAAV vectors were produced by a modified cross-packaging approach, which involved packaging the vector genome adjacent to the AAV2 ITR onto a non-AAV2 capsid, in this case AAVrh74, AAV1, or AAV9 capsid, using Rep proteins from the AAV2 serotype (Rabinowitz et al., J Virol. 76(2):791-801 (2002)). Production was achieved using a standard 3 plasmid DNA / CaPO4 precipitation method with HEK293 cells. HEK293 cells were maintained in 10% fetal bovine serum (FBS) and DMEM supplemented with penicillin and streptomycin. The produced plasmids were (i) plasmids encoding BAG3 therapeutic protein or NT-3 therapeutic protein, (ii) rep2-capX modified AAV helper plasmid encoding cap serotype rh.74 isolate, and (iii) adenovirus type 5 helper plasmid (pAdhelper) expressing adenovirus E2A, E4 ORF6, and VA I / II RNA genes. Capsid-formed vector genome (vg) titers were determined using a quantitative PCR-based titration method with a Prism 7500 Taqman detector system (PE Applied Biosystems). [Clark et al., Hum Gene Ther. 10(6):1031-1039 (1999)]. Final titer (vg ml) -1 The virus was identified by quantitative reverse transcriptase PCR using specific primers and probes with a Prism 7500 real-time detector system (PE Applied Biosystems, Grand Island, NY, USA). The divided viruses were stored at 80°C.

[0183] All plasmids used to construct the packaged AAV genome also contain kanamycin (KanR) or ampicillin (AmpR) resistance genes outside the ITR sequence used for genome packaging. This allows the DNA encoding the AAV genome to be transformed into bacteria, which will produce large amounts of DNA in the presence of either antibiotic, killing all untransformed bacteria. KanR and AmpR are not packaged in the AAV capsid in the AAV genome used to treat patients, but their presence enables DNA production in bacteria.

[0184] The description of ss.pAAV.tMCK.BAG3.Kan is shown as Sequence ID No. 1 and is presented in Table 1 below. The plasmid map of this vector is shown in Figure 2A. [Table 1]

[0185] The description of ss.pAAV.CMV.CBA.BAG3 is given as Sequence ID No. 2 and is presented in Table 2 below. The plasmid map of this vector is shown in Figure 2B. [Table 2]

[0186] The description of ss.pAAV.CMV.hBAG3 is given as sequence number 13 and is presented in Table 3 below. [Table 3]

[0187] Purification of AAV particles by density gradient centrifugation with iodixanol AAV particles were purified by iodixanol gradient centrifugation. Virus-containing cell lysates were purified by centrifugation (3700 g, 4°C, 20 min), and the clarified lysates were transferred to a Quick Seal ultracentrifugation tube (26 × 77 mm, Beckman). Different concentrations of iodixanol solution (Sigma) were layered beneath the virus-containing lysates. This created an iodixanol gradient consisting of 6.0 ml of 60% iodixanol, 5.0 ml of 40% iodixanol, 6.0 ml of 25% iodixanol, and 9.0 ml of 15% iodixanol, with the virus solution at the top. The gradient was centrifuged in an ultracentrifuge at 416,000 g at 18°C ​​for 1 hour. Next, the 40% phase containing AAV particles was extracted using a cannula by penetrating the tube at the bottom of the 40% phase and dropping the solution into a collection tube until the 25% phase was reached. The 40% phase AAV capsid titer was determined using a commercially available ELISA (AAV titer ELISA, Progen).

[0188] Example 2 Construction of an NT-3 expressing AAV construct The design of a self-complementary rAAV virus vector having serotype 1 containing NTF3 cDNA under tMCK was previously described in Sahenk et al., Mol Ther, 22(3):511-521 (2014), which is incorporated herein by reference in its entirety. Ali-coated viruses were kept at -80°C until use. Blood samples were collected from treated and untreated mice by ocular hemorrhage under anesthesia at 6 and 16 weeks post-injection, and serum was assayed for NT-3 levels using capture ELISA. The construct is referred herein to as scAAV1.tMCK.NTF3.

[0189] The tMCK promoter / enhancer sequence is used to drive muscle-specific gene expression and consists of a muscle creatine kinase promoter to which a fused enhancer element (enh358MCK, 584bp) is added. A triple tandem of MCK enhancers (206bp) was ligated to the 87bp basal promoter in the tMCK promoter / enhancer.

[0190] The scAAV1.tMCK.NTF3 drug product was produced by three plasmid DNA transfections of human HEK293 master cell bank cells with (i) the pAAV.tMCK.NTF3 vector plasmid (see Figure 3), (ii) an AAV1 helper plasmid called R88 / C1 containing AAV rep2 and Cap1 wild-type genes, and (iii) a helper adenovirus plasmid.

[0191] A schematic diagram of the plasmid having molecular characteristics and an open reading frame is shown in FIG. 3. The rAAV genome derived from the pAAV.tMCK.NTF3 plasmid is a self-complementary DNA genome containing a human NTF3 cDNA expression cassette adjacent to the AAV2 inverted terminal repeat sequence (ITR). This sequence is encapsulated in the AAV1 virion. The plasmid pAAV.tMCK.NTF3 was constructed by inserting a tMCK expression cassette driving the NTF3 gene sequence into the AAV cloning vector psub201. The human NTF3 gene is expressed from the mouse triple tandem MCK promoter, which is a modification of the aforementioned CK6 promoter and contains a triple E box sequence. The SV40 polyadenylation signal is used for efficient transcription termination. The cassette also contains a chimeric intron for increased gene expression, consisting of the 5' donor site from the first intron of the human β-globin gene, the branch point, and the 3' splice acceptor site from an intron between the leader and the body of the immunoglobulin gene heavy chain variable region. The NTF3 expression cassette has a consensus Kozak immediately before the ATG start and a 200 bp SV40 polyA signal for efficient mRNA termination. The entire NTF3 cDNA is included (NCBI reference sequence: NM_001102654). The only viral sequence contained in this vector is the inverted terminal sequence of AAV2, which is required for both viral DNA replication and packaging. The AAV ITR is a sequence that is nearly identical at both ends but in opposite orientations. The "left" (mutated) ITR has a terminal resolution site deleted to allow hairpin formation of the genome. The identity of all DNA plasmid elements was confirmed by DNA plasmid sequencing on the plasmid source stock.

[0192] The base pair positions of the relevant molecular characteristics within the rAAV vector DNA plasmid of SEQ ID NO: 12 are shown in Table 4. [Table 4]

[0193] Example 3 Mouse model Rodent model expressing the myotilin T57I mutation The myotilin T57I transgenic mice used in the studies of the present inventors modeled autosomal dominant LGMD1A by co-expressing a mutant human myotilin transgene in the presence of normal levels of endogenous WT mouse myotilin28. The TgT57I levels are 2.6-fold higher than the endogenous levels. Immunostaining of cross-sections of frozen muscle shows that the expression of the transgene is uniform across muscle fibers within a given muscle group and is similar in both slow type I and fast type II fibers. Muscle pathology is progressive, with both the size and number of aggregates increasing with age starting as small foci at 2 weeks of age and reaching up to 40 μm in older mice. Immunostaining shows that the aggregates in TgT57I muscle have some Z-disk proteins (ACTN2, FLNC, and desmin), and the sarcomere proteins titin and myosin are similarly localized to the aggregates. In addition, the aggregates contain ubiquitinated proteins.

[0194] TgT57I mice show contractile dysfunction of the extensor digitorum longus muscle tested at 8 months of age, and muscle mass and muscle fiber cross-sectional area are similarly decreased. However, the soleus muscle and diaphragm muscle are completely spared from any pathological or physiological defects 28 。

[0195] SOD1-G93A mouse model for ALS Amyotrophic lateral sclerosis (ALS or Lou Gehrig's disease) is a neurodegenerative disease characterized by the degeneration of upper and lower motor neurons, leading to progressive muscle atrophy and weakness, and ultimately paralysis. ALS typically develops between the ages of 55 and 75, and death, often due to respiratory failure, usually occurs within three years of disease onset in 50% of patients. While most ALS cases (90%) have no clear genetic basis (sporadic ALS), approximately 10% of ALS cases are familial (familial ALS), usually inherited in an autosomal dominant pattern and caused by mutations in multiple genes. However, both forms of the disease are molecularly and clinically indistinguishable (Bosco and Landers, 2010; Gros-Louis et al., 2006). Currently, more than 20 genes are associated with fALS, four of which account for the majority of familial cases: the chromosome 9 open reading frame 72 gene (C9ORF72, 40%), superoxide dismutase 1 (SOD1, 20%), sarcoma fusion (FUS, 1-5%), and TAR DNA-binding protein (TARDBP, 1-5%).

[0196] Although the etiology of ALS remains poorly understood, abnormal protein aggregation and altered proteostasis are common features of sporadic and familial forms of ALS. In familial ALS, several mutated genes lead to the formation of abnormally folded proteins, which aggregate to form inclusion bodies and actively impair proteostatic mechanisms (Soo et al. 2015. Acta Neuropathol. 130, 679-97). These are mainly SOD1, TAR DNA-binding protein 43 (TDP-43), FUS, and C9ORF72 (Andersen PM, Al-Chalabi A., 2011. Nat Rev Neurol. 2011;7(11):603-615; DeJesus-Hernandez et al., 2011 Neuron. 72(2):245-256; Guo et al. 2011. Nat Struct Mol Biol. 18:822-830; Guo et al. 2010. Brain Res. 1353,234-44). Furthermore, dominant mutations in genes encoding components of protein quality control mechanisms related to vesicular transport, autophagy, ER homeostasis, and UPS, such as tubulin 4A (TUB4A), dynein, dynactin, sequestosome-1 (p62), optinulin, valosin-containing protein (VCP or p97), vesicle-associated membrane protein-associated protein B (VAPB), TANK1-binding kinase 1 (TBK1), and ubiquilin-2, are all associated with ALS (Deng et al., 2011; Fecto et al. 2011; Arch Neurol. 68, 1440-6; Freischmidt et al. 2015; Nat Neurosci. 18, 631-6; Hipp et al. 2014 Trends in Cell Biology. 24, 506-514; Johnson et al. 2010; Neuron. 68, 857-64; Rademakers and van Blitterswijk,2014, Smith et al.2014.Neuron.84,324-31, Synofzik et al.,2012, Williams et al.2012.Neurobiol Aging.33,2527 e3-10).Furthermore, intracellular protein inclusions are a prominent neuropathological feature of ALS. The majority of ALS patients exhibit inclusions not only in the brainstem and spinal cord, but also in the cerebellum, hippocampus, and frontal and temporal lobes (as outlined in Al-Chalabi et al. 2012. Acta Neuropathol. 124(3):339-352), in both degenerated neurons and surrounding glial cells (Wood et al., 2003 Neuropathology and Applied Neurobiology. Vol.29, ed., pp.529-545; Piao et al. 2003. Brain Pathol. 13, 10-22; Nishihira et al., 2008. Acta Neuropathol. 116, 169-182; Zhang et al. 2008. Acta Neuropathol. 115, 115-122). The most common inclusion bodies are ubiquitinated proteins found in both upper and lower motor neurons (Neumann et al. 2006. Science 314, 130-133.), suggesting defects in protein turnover (Blokhuis et al. 2013. Acta Neuropathol. 125 777-794).

[0197] Preclinical ALS research is based on the same genetic mutations observed in patients and requires, in parallel, animal models that closely mimic the pathogenesis of the disease. The association of the aforementioned genes with ALS has primarily driven the creation of transgenic animal models expressing ALS-binding mutations to understand the pathological mechanisms leading to neuronal cell death and to develop therapeutic strategies. In 1993, SOD1 became the first gene associated with ALS (Rosen et al. Nature. 1993;362(6415):59-62, Dend et al. Science. 1993;261(5124):1047-1051). SOD1 encodes cytoplasmic Cu / Zn superoxide dismutase, which catalyzes the disproportionation of toxic superoxide anions to oxygen and hydrogen peroxide. Mutations in SOD1 account for approximately 20% of fALS cases. The first mouse models of ALS were developed after the identification of mutations in the SOD1 gene. Transgenic mice overexpressing mutant (G93A) SOD1 were created by randomly inserting multiple copies of mutant human SOD1 into chromosome 12 of the mouse genome (Gurney et al. 1994. Science 264, 1772-1775, Achilli et al. 2005 Amyotroph. Lateral Scler. Other Motor Neuron Disord. 6, 111-114). SOD1-G93A mice develop phenotypes and pathological symptoms that reproduce the characteristic signs of ALS in human ALS patients, including the progressive onset of muscle weakness leading to paralysis (Kong, J and Xu, Z. 1998. J. Neurosci. 18, 3241-3250) (Gurney et al. 1994. Science 264, 1772-1775). The moment of disease onset and the lifespan of these mice are related to the level of overexpression of mutant SOD1, while overexpression of non-mutant SOD1 does not result in a specific phenotype.SOD1-G93A mice develop neurodegeneration of spinal motor neurons with adult onset (approximately 90 days), leading to progressive motor impairment resulting in paralysis of one or more limbs within a few weeks of age, which closely resembles the pathology of human ALS (Synofzik et al., 2010. J Neurol Neurosurg Psychiatry. 81(7):764-767). Furthermore, intracellular protein aggregates, a clear and important feature of motor neurons in ALS patients, are also evident in mutant SOD1 mice (Bruijn et al. Science. 1998; 281(5384): 1851-1854, Watanabe et al Neurobiol Dis. 2001; 8(6): 933-941), representing a key feature of ALS. The accumulation of misfolded proteins and proteins prone to aggregation suggests an imbalance in protein homeostasis (proteostasis). However, the rate of disease progression and death in SOD1-G93A mice can be slower or faster depending on the genetic background in which mutant SOD1 is expressed. The average lifespan of the B6 / SJL mixed line is 129 days, while the B6 background line survives for approximately 144–161 days, the SJL line for approximately 119 days, and the 129 line for approximately 125 days (10, 11). In addition, copy number variations in the mutant SOD1 transgene can alter the severity of the phenotype (Acevedo-Arozena et al. 2011. Dis Model Mech. 4(5):686-700).

[0198] Since its development in 1994, the SOD1G93A mouse remains the most commonly used animal model of ALS, and therefore, much of the inventors' knowledge regarding the etiology and pathogenesis of this disease stems from research conducted using this animal model. This model is also freely available and is gaining popularity.

[0199] At present, it remains unclear how SOD1 mutations selectively induce motor neuron death. Studies using SOD1G93A transgenic mice and SOD1 knockout mice suggest that motor neuron degeneration is not due to a loss of SOD1 enzyme activity, but rather to a toxic effect ("gain of toxic function") induced by the presence of the mutant SOD1 itself (Gurney et al. 1994, Reaume et al. 1996, Wong et al. 2002). However, the accumulation of misfolded and aggregate-prone proteins suggests an imbalance in protein homeostasis (proteostasis). Molecular chaperones are crucial factors in maintaining proteostasis by promoting protein folding and quality control. Indeed, in the mutant SOD1 (mutSOD1) ALS model, aggregation correlates with dysfunction of both proteasomes and / or autophagy, which are essential for intracellular chaperone-mediated protein quality control (PQC), as well as decreased mutSOD1 clearance from motor neurons. In fact, alimoclomol, a heat shock response co-inducer that upregulates molecular chaperone expression, can protect against mutant SOD1 toxicity in vivo.

[0200] The studies described herein investigated whether overexpression of the cochaperone, BAG3, has beneficial effects in a SOD1-G93A mouse model of ALS, such as reducing mutant SOD1 aggregation, improving cell viability, and / or mitigating motor neuron degeneration.

[0201] Mouse supply source MYOT (TgT57I), C57BL / 6 wild-type (WT), and SOD1-G93A (B6SJL-Tg(SOD1*G93A)1Gur / J, strain number: 002726) mice were obtained from Jackson Laboratory (Bar Harbor, ME). Hemizygous SOD1-G93A mice were bred with C57BL / 6 wild-type mice. Hemizygous SOD1-G93A female mice were included in the experiments to minimize sex-dependent variability in the animals. All animal experiments were conducted in accordance with guidelines approved by the Research Institute at Nationwide Children's Hospital Animal Care and Use Committee.

[0202] Example 4 Materials and methods cell line The HEK293 cell line was derived from immortalized human fetal kidney cells and purchased from ATCC. The WP-4 mouse tumor cell line, a clone of the methylcholanthrene-induced mouse fibrosarcoma cell line MCA205, was obtained from Agonox (Wexler et al., J. Natl. Cane. Inst., Vol. 63, pg. 1393, 1979; Asher et al., J. Inmunol, Vol. 146, pg. 3227, 1991; Mule et al, Hum Gene Ther. 7(13): 1545-53, 1996).

[0203] cell culture HEK293 cells were maintained as a monolayer in Dulbecco's Modified Eagle (DMEM) medium (Corning no. 10013CV or alternative) supplemented with 10% thermo-inactivated fetal bovine serum (FBS) (Life Technologies no. 10437-028 or alternative) and 1% penicillin / streptomycin antibiotic solution (10000 U / mL; Life Technologies no. 15140-122). WP-4 tumor cells were cultured in C10 culture medium (Roswell Park Memorial Institute (RPMI) 1640 Growth Medium (Gibco Life Technologies)) containing 2 mM L-glutamine (MilliporeSigma no. TMS-002-C), 1% penicillin / streptomycin (10000 U / mL; Life Technologies), 1 × non-essential amino acids (Gibco no. 11140050), 1 mM sodium pyruvate (Gibco no. 11360070), 10 mM HEPES buffer (ThermoFisher no. 15630-080), 1 × β-mercaptoethanol (Sigma-Aldrich no. ES-007-E), and 10% FBS (Life Technologies no. 10437-028 or alternative)) (Roswell Park Memorial Institute (RPMI) 1640 Growth Medium (Gibco Life Technologies)). Cells were cultured in (no. 11875-093). The cell lines were genetically authenticated using STR-based DNA profiling and multiplex PCR, and confirmed to be free of mycoplasma and endotoxins using the MycoAlert detection kit (Lonza) and the Endosafe-PTS system (Charles River Laboratories). The cells were cultured in a humidified incubator at 37°C and 5% CO2.

[0204] Electroporation and transfection of WP-4 cells In vitro rAAV vector infection WP-4 cells are placed in a 6cm diameter dish in a 5x10 4Cells were seeded at a density of [density]. Cells were allowed to adhere for 24 hours. After 24 hours, normal complete medium was replaced with RPMI1640 and FBS was reduced by 2%. The rAAV vector was moved from -80°C to room temperature and subjected to gentle vortexing and short spins. The rAAV suspension was prepared using buffer (PBS) only and then added directly to cells at infection multiplicity (MOI) of 40,000 genome copies or 0 genome copies (non-transduction control group). After incubation with the virus for 2 hours, the medium was replaced to remove vectors that had not transduced. Subsequently, the used medium was replaced every 48 hours. After 7 days, the efficiency of cell transduction was evaluated using several methods. Cells stably expressing the gene of interest were selected by treatment with 1-3 μg / ml puromycin over 2 days (InvivoGen).

[0205] The plates were examined for GFP-positive cells using a fluorescence microscope, and the number of positive cells was calculated.

[0206] AAV vector delivery to mice Intramuscular (IM) injection of TA in mice 3-4 week old mdx mice and age-matched C57 / BL10 mice were used for IM injection. Mice were anesthetized and maintained on 1-4% isoflurane (in oxygen). Both hind limbs were shaved, and 3 × 10¹⁰ vg of rAAV8, microdystrophin, FLAG, or saline (30 μl volume) was injected into the TA muscle using a 30-gauge insulin syringe.

[0207] Intramuscular (IM) AAV delivery IM injection was administered to the tibialis anterior muscle of wild-type (C57BL / 6J) or TgT571 transgenic (MYOT) mice by direct injection of 50 μL of PBS, while control mice were administered PBS alone in the same volume. The total dose of ssAAV9.CMV.hBAG3 administered was 2.5 × 10⁻⁶. 11It was vg. Alternatively, the same MYOT mice were used, the left GAS muscle was injected, and the right GAS muscle was used as a control. The titer for IM injection was measured using a linear DNA standard. The injected mice showed no abnormal clinical signs after any of the injections.

[0208] In the IM safety experiment using asAAVrh74.tMCK.hBAG3, 1.0×10 11 vg, 4.0×10 11 vg, 8.0×10 11 vg doses were injected intramuscularly into wild-type (C57BL / 6J) mice. For the IM ssAAVrh74.tMCK.hBAG3 dose titration, only the male cohort of MYOT mice was injected with 3.0×10 11 vg, 1.0×10 11 vg, 2.0×10 11 vg.

[0209] For the ALS mouse model, SOD-G93A mice were injected with ssAAV9.CBA.hBAG3 at a dose of 3.0×10 11 vg and / or ssAAV1.tMCK.NT-3 at a dose of 1.0×10 11 vg.

[0210] Systemic AAV delivery The mice were placed in a mouse tube strainer (Braintree Scientific; Braintree, MA, USA) with the tail outside the tube. The injection area was prepared with povidone-iodine and 70% ethanol, and then the AAV vector or AAV buffer was usually injected into the tail vein in a volume of 150 μL, not exceeding a volume of 200 μL. For the systemic dose titration, 3×10 12 vg or 6×10 1212-16 week old MYOT mice were injected with a 1g dose of ssAAVrh74.tMCK.hBAG3. The titer for IV injection was measured using a linear DNA standard. Mice were sacrificed 8 weeks after injection, and GAS, Quad, TA, and Tri muscles were collected. The diaphragm was subjected to force measurement (see below), FDB was tested for membrane repair capacity, and muscles and organs were collected for expression and histopathology.

[0211] For effectiveness evaluation, 3 × 10 12 vg ssAAVrh74.tMCK.hBAG3 was injected into the tail vein of 12-16 week old MYOT mice (n=3). The titer for IV injection was measured using a linear DNA standard.

[0212] Immunofluorescence The mouse groups were euthanized by an overdose of xylazine / ketamine anesthetic 8 weeks (n=2 in each cohort) and 12 weeks (5MYOT and 3WT mice) after gene injection. GAS muscle tissue (i.e., 2-3 months post-injection) collected from 4-month and 5-month-old male mice was rapidly frozen in liquid nitrogen-frozen isopentane. The tissue was frozen into 12 μm thick sections and mounted on Superfrost Plus microscope slides (Fisherbrand Superfrost Plus). The mounted tissue sections were fixed with 4% paraformaldehyde for 10 minutes at room temperature. The sections were washed with phosphate-buffered saline (PBS) containing 0.01% Triton X-100, permeabilized with 0.3% Triton X-100 for 5 minutes, and then incubated in a humidified chamber at room temperature for 1 hour in a barrier buffer (1×PBS, 10% goat serum). Tissue sections were incubated overnight at 4°C with polyclonal rabbit anti-human MYOT primary antibody (1:400, LSBio catalog no. LS-C33471 or MyBioSource catalog no. MBS9127412). After washing three times for 5 minutes with wash buffer (1×PBS, 1% goat serum), the tissue sections were incubated at room temperature for 1 hour with AlexaFluor-594 conjugate goat anti-rabbit secondary antibody (1:500; Life Technologies, Grand Island, NY, USA; catalog no. A11032). The sections were washed three times for 5 minutes with wash buffer, then counterstained with 0.5 mg / ml 4',6-diamidino-2-phenylindole (DAPI, Sigma catalog no. D-9542), and covered with ProLong Gold Antifade Mountant (Molecular Probes catalog no. P36961). Images were acquired at 10x magnification using a Nikon Ti2-E microscope (Nikon Instruments, Tokyo, Japan), and analyzed using NIS-Elements (v5.3) software to determine aggregate size, fluorescence density of individual aggregates, and average aggregate fluorescence density. The signal threshold was set using tissue sections stained with secondary antibody alone to exclude nonspecific signals.Representative 20x magnified images were obtained from randomly selected mice using a Zeiss Axioskop microscope and Axiovision Rel software. The number of red pixels in each image was determined using Bioquant software (Bioquant Image Analysis Software, version 2016, R&M Biometrics Inc., Nashville, TN) and expressed as a percentage of untreated control muscle in each mouse.

[0213] H&E staining As described in the immunofluorescence section above, muscle tissue was rapidly frozen in liquid nitrogen-cooled isopentane. 12 μm sections were cut using a cryostat (Leica Biosystems, Wetzlar, Germany) and subjected to hematoxylin and eosin (H&E) as follows. Sections were fixed for 5 minutes, placed in 70% ethanol, and then rehydrated in deionized water. Slides were stained in Harris modified hematoxylin solution (Sigma-Aldrich HHS32) for 4.5 minutes, then washed in deionized water, immersed in eosin Y solution (Sigma-Aldrich HT110116) for 1.5 minutes, and then washed again in deionized water until clear. Slides were dehydrated in an ascending alcohol series, removed in xylene, and then mounted with Cytoseal (Richard-Allan Scientific® Cytoseal 60® Thermo Scientific, 8310-16).

[0214] Histological analysis Aggregate particle size and density were compared with untreated, age-matched littermates. 12 μm thick cryostat sections were stained with H&E for conventional histopathology or immunostained with antibodies against myotilin using standard protocols. Representative 20x magnification images were obtained from each biopsy using a Zeiss Axioskop microscope and Axiovision Rel software. 10x magnification images covering the entire surface of the immunostained sections were acquired using a Nikon Ti2-E microscope and analyzed using NIS-Elements (v5.3) software to determine aggregate size, individual aggregate fluorescence density, and mean aggregate fluorescence density. Myotilin aggregate load was quantified using immunofluorescence techniques for four randomly selected representative images at 20x magnification from the quadriceps femoris muscle of the BAG3-treated (n=10) and UT (n=8) cohorts. Aggregate count / mm³ was determined in both the treated and untreated cohorts. 2 and size distribution / mm 2 The aggregate density was calculated.

[0215] Western blot WP-4 cell lysates were prepared from 80% confluent tissue culture plates three days after transduction with ssAAV9.CMV.hBAG3 in radioimmunoprecipitation (RIPA) buffer (Sigma) supplemented with a protease inhibitor cocktail (Roche Diagnostics). Serial frozen sections of gastrocnemius muscle from treated and untreated mice, or treated and untreated muscle from the same mice, were collected for protein preparation. Samples were collected in a buffer containing a protease inhibitor (Roche Diagnostics) (125 mM Tris-HCl buffer [pH 6.8], 4% SDS, 5% glycerol, and 4 M urea). Insoluble material in the lysates was discarded after rapid centrifugation at 4°C. Protein concentrations were determined using the Lowry assay (RC DC, Bio-Rad). Equal amounts of protein were packed onto 3-8% TRIS acetate gradient gels (NuPAGE, Invitrogen) and transferred to polyvinylidene fluoride (PVDF) membranes (Amersham Biosciences). The membranes were blocked at room temperature for 30 minutes with 5% skim milk powder in 1× Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBST). The membranes were then incubated overnight at 4°C with rabbit anti-BAG3 monoclonal primary antibody (1:500, Abcam, catalog no. ab92309) in the blocking solution, followed by incubation at room temperature for 1 hour with horseradish peroxidase conjugate secondary antibody (0.02 μg / mL goat anti-rabbit IgG-HRP, Vector Lab). After washing with TBST, the blot was developed using enhanced chemiluminescence (ECL) reagent (Amersham Biosciences), and images were acquired by ChemiDoc (Bio-Rad Laboratories, Hercules, CA) or X-ray film exposure.

[0216] Cell counting by cresyl violet staining and stereochemistry. The mice were euthanized, and their calf muscles were harvested. The animals were then perfused transcardially with saline solution, followed by perfusion with 10% formaldehyde for fixation. The spinal cord was removed, and the animals were post-fixed overnight in 4% PFA at 4°C.

[0217] Cervical, thoracic, and lumbar segments of the spinal cord were paraffin-embedded, and cross-sections were made for motor neuron counting. For each segment, a total of seven 7 μm sections were cut at 49 μm intervals. For cresyl violet staining of paraffin-embedded spinal cord sections, the tissue was deparaffinized by two or three changes of xylene for 3 minutes each, followed by rehydration in 100% ethanol, twice for 3 minutes each. The tissue was then stained with 0.1% cresyl violet for 4 minutes and promptly washed with tap water to remove excess staining. The tissue was washed with 70% ethanol and then dehydrated by two changes of 100% ethanol for 3 minutes each. The tissue was then removed in xylene, mounted in Depex, and air-dried in a fume hood for 15–20 minutes.

[0218] Alternatively, spinal cords were prepared from NaCl-perfused mice, separated into cervical, thoracic, and lumbar segments, and cryopreserved. For motor neuron counting, 12 μm frozen sections cut at 84 μm intervals were fixed with acetone and subsequently stained with cresyl violet. For cresyl violet staining of frozen sections, the tissue was rehydrated in 95% ethanol, 75% ethanol, and 70% ethanol, and the solution was stained in cresyl violet for 30–45 seconds while protecting it from light. The tissue was then dehydrated in 75% ethanol, 95% ethanol, 100% ethanol, and 100% ethanol for 30 seconds each. 0.1% cresyl violet was prepared in water, filtered, and stored at 4°C for up to one month. After dehydration, the sections were air-dried under a fume hood for 15–20 minutes and immediately imaged, or stored in a box with desiccant at -80°C for up to several weeks.

[0219] Using a Nikon Ti2-E microscope, 20x magnified images of spinal cord sections were obtained. Anterior horn cells in lumbar spinal cord sections with identifiable nucleoli were counted, and their diameter was measured as the short axis passing through the center of the nucleus. All counted neurons, including interneurons with a diameter of less than 15 μm (estimated motor neurons with a diameter greater than 15 μm), were located ventrally on a drawn straight line that was in contact with the ventral boundary of the central canal and perpendicular to the semi-axis of the spinal cord. The total number of neurons originating from both the right and left anterior horn regions included those representing a screening of the lumbar spinal cord thickness of approximately 350 μm.

[0220] Neurological function assessment All measures of disease progression in animals were conducted blindly by independent investigators to avoid subjective bias. Body weight was assessed weekly throughout the study. Nerve conduction studies, rotarod tests, and grip strength tests were initiated at 8 weeks and repeated weekly until 17 weeks of age.

[0221] Motor function test / Grip strength: Mice were tested for baseline motor function within one week prior to receiving an im injection of ssAAV1.CMV.NT-3 or PBS. Motor function tests included bilateral simultaneous hindlimb grip strength and left hindlimb grip strength using a grip strength meter (Chatillon Digital Meter; Model DFIS-2; Columbus Instruments, Columbus, OH). Bilateral or unilateral grip strength was assessed by having the animal grip a platform and then gently pull it with its tail, while the sensor recorded the force (Newtons, N) resisting the pull before the mouse released the platform as a measure of muscle strength. Force measurements were recorded in four separate tests. Measurements were performed on the same day and at the same time each week. Bilateral and ipsilateral grip strength measurements for the endpoint were performed in two sessions (morning and afternoon), three tests per day for three consecutive days, before obtaining nerve conduction studies. The mean of these measurements was used to correlate with conduction studies.

[0222] Rotor rod test Mouse motor function and balance were tested at baseline and endpoints using an accelerating rotorod (Columbus Instruments, OH, USA). Mice were trained with the rotorod device for two weeks to adapt to the test protocol before data collection. The protocol was performed at 5 rpm with a constant acceleration of 0.5 rpm / s, and the average of the two best trials out of three was included in the analysis.

[0223] Nerve conduction research Animals were anesthetized under 2% isoflurane, and body temperature was maintained using a heating pad set to 37°C. Right sciatic nerve conduction studies were performed using the Nicolet Viasys Viking Select EMG EP System (Nicolet Biomedical, Wisconsin, USA) and 27G disposable subcutaneous needle electrodes for both stimulation and recording, as previously described (Pollari et al. 2018 J Vis Exp.(136):57741). The stimulating electrodes were placed subcutaneously on the proximal (stimulation 1) and distal (stimulation 2) sides of the ischial notch, with a distance of approximately 2 cm between electrodes. The recording electrode was placed subcutaneously aligned with the long axis of the gastrocnemius muscle, and the reference electrode was inserted subcutaneously at a 30-degree angle next to the Achilles tendon, leaving a needle 2–5 mm below the skin. Measured parameters included compound muscle action potential (CMAP) amplitude, distal latency, area, duration, and nerve conduction velocity. The nerve conduction velocity was determined using the distance between the two stimulation sites.

[0224] Sciatic motor nerve conduction studies were performed bilaterally in each cohort of mice at baseline and 16 weeks after the start of treatment, using an electrodiagnostic system (Synergy N2 electromyograph; Natus, Middletown, WI), as previously reported (Yalvac et al., 2015, 2014). Briefly, sciatic motor nerve conduction responses were recorded using two thin ring electrodes (Alpine Biomed, Skovlunde, Denmark) used as activity (E1) and reference (E2) electrodes. The activity recording electrode was placed on the proximal portion of the gastrocnemius muscle, and the reference electrode was placed on the mid-metatarsal region of the foot. Skin impedance was reduced by precisely coating the ring electrodes with electrode gel (Spectra 360 by Parker Laboratories, Fairfield, NJ) using an irrigation syringe. A pair of 28-gauge unipolar needle electromyography electrodes (Teca, Oxford Instruments Medical, New York, NY) were used to deliver supramaximal stimulation to the sciatic nerve in the distal femur and ischial notch. Measured parameters included compound muscle action potential (CMAP) amplitude, distal latency, and conduction velocity.

[0225] Example 5 BAG3 transduction efficiency in vitro and in vivo To test the transduction efficiency of rAAV-mediated expression of BAG3 in vitro, WP-4 tumor cells were transduced with ssAAV9.CMV.hBAG3, and the expression of the transgene hBAG3 was detected in the lysate using a monoclonal antibody against hBAG3. The expression of the hBAG3 transgene was detected in WP-4 cell lysates using a monoclonal antibody against hBAG3 (Figure 4, left panel).

[0226] The viability of ssAAV9.CMV.hBAG3 was tested in vivo using TgT57I transgenic (MYOT) mice (No. 97) and wild-type (WT) mice. TgT57I mice reproduce the progressive MYOT protein aggregation deficiency that characterizes LGMD1A. In 3-month-old TgT57I mice, aggregates are associated with further systemic muscle pathology, including deficits in myofibril size and gastrocnemius muscle weight, as well as a slight but significant increase in myofibrils with centrally located nuclei, which are histological indicators of muscle degeneration followed by repair. Importantly, these phenotypes are useful outcome measures for gene therapy. Therefore, the effects of ssAAV9.CMV.hBAG3-mediated MYOT degradation were investigated against LGMD1A-related aggregate formation, myofibril diameter, muscle weight, and central nucleus deficits in TgT57I mice.

[0227] To test hBAG3 expression in vivo, ssAAV9.CMV.hBAG3 was used in 2.5 × 10⁶ units. 11 The 1 / 2 dose was intramuscularly injected (IM) into the left gastrocnemius muscle (GAS) of TgT57I transgenic (MYOT) and WT mice. hBAG3 expression was detected by Western blotting in the injected left myolysate 8 weeks after injection, but BAG3 expression was not detected in the right muscle of untreated controls (Figure 4, right panel).

[0228] Protein aggregation in myofibrils is a histological feature of myofibrils and appears to play a crucial role in their pathogenesis. To confirm the potential of the hBAG3 transgene to reduce the accumulation of protein aggregates in myofibrils in vivo, 2.5 × 10⁶ units were introduced into the left GAS of MYOT mice. 11 ssAAV9.CMV.hBAG3 was administered via immunosuppression (IM) injection, and the target muscle of the right gastrointestinal stenosis (GAS) was used as an untreated control. Aggregate accumulation was examined 12 weeks after AAV delivery by staining frozen sections of ssAAV9.CMV.hBAG3-treated and untreated GAS muscle with hematoxylin and eosin (H&E), as well as MYOT immunoreactive antibody.

[0229] H&E sections from untreated muscle of MYOT mice stained a dark purple, indicating the abundance of intracytoplasmic protein aggregates (Figure 5A). Compared to GAS muscle from untreated muscle, H&E sections from treated muscle stained a pale purple, indicating a significant decrease in the amount of intracytoplasmic aggregates (Figure 5B). These results were confirmed by immunohistochemistry.

[0230] Frozen sections of untreated control muscle, immunostained with anti-myotirin antibody, showed a high presence of immunoreactive MYOT-seeded protein aggregates, while sections from treated left GAS muscle showed a significant decrease in MYOT-seeded protein aggregates (Figure 5C). Quantification of myotirin inclusion density on fluorescence imaging from immunostained sections confirmed that ssAAV9.CMV.hBAG3 treatment significantly reduced the abundance of protein aggregates in TgT57I gastrocnemius muscle 12 weeks after injection (Figure 5D). In summary, these data demonstrate that hBAG3 gene therapy significantly reduced the abundance of intracytoplasmic protein aggregates in treated left GAS muscle compared to untreated right muscle.

[0231] Example 6 Toxicity evaluation and dose escalation study of AAVrh74.tMCK.hBAG3 Switching from the ssAAV9.CMV.hBAG3 vector to the AAVrh74.tMCK.hBAG3 vector resulted in sustained high expression of hBAG3 over the long term in disease-affected muscles, while exhibiting limited activity in other tissues. The potent constitutive CMV promoter in the ssAAV9.CMV.hBAG3 vector is known to drive high levels of gene expression, but it is also a non-discriminatory promoter, which is not necessarily ideal for use in restricted muscle transduction. In contrast, the tMCK promoter exhibits high muscle specificity and provides high in vivo expression levels in skeletal muscle and cardiac muscle, both intramuscularly and systemically. The tMCK promoter is also attractive to AAV vectors due to its compact size and small packaging volume of approximately 4.7 Kb. The muscle specificity of the tMCK promoter can be further enhanced by pairing it with the AAVrh74 serotype, considering its orientation towards muscle (skeletal and cardiac) tissue, making this pairing ideal for muscle gene therapy. Furthermore, AAVrh.74, isolated from rhesus monkeys, exhibits superior transduction characteristics and lower existing human herd immunity to it compared to AAV2 (Mendell et al. JAMA Neurol. 2020;77(9):1122-1131).

[0232] A crucial factor for the success of gene therapy is the demonstration of efficacy at doses of a vector that is safe and easily manufactured. The toxicity of the ssrAAVrh74.tMCK.hBAG3 vector is shown at three different doses: 1 × 10⁻⁶ 11 , 4×10 11 , or 8×10 11 After injecting IM into the right GAS muscle at vg / kg, the first evaluation was performed in C57BL / 6WT mice (n=4). 1×10 in WT mice 11 Injection of ssrAAVrh74.tMCK.hBAG3 in VG did not cause muscle inflammation or necrosis 8 weeks after injection. 4×10 11 At the VG dose, myofibrils necrotized and micro-inflammation were observed in several areas of muscle in one of three mice. However, at 8 × 10 11The vg dose resulted in widespread inflammation and a marked increase in internal nuclei in all mice, and progressive muscle fiber necrosis in 2 out of 3 mice. These histological observations suggested dose-dependent toxicity after IM injection. 3 × 10 in WT 12 VG and 6×10 12 Systemic administration of the vector in vg did not cause muscle inflammation or necrosis 8 weeks after injection. However, there was an increase in fibers with internal nuclei in several regions of the GAS, Quad, and TA muscles, at 6 × 10⁻⁶. 12 This was observed at the VG dose, and this observation is consistent with regeneration after previous necrosis (Figure 6A). No ongoing necrosis or inflammation was observed.

[0233] Based on these results in WT mice, for systemic delivery of ssrAAVrh74.tMCK.hBAG3, 3 × 10¹⁶ doses were administered to the tail vein of 12-16 week old TgT57I transgenic mice (n=3, Table 2). 12 The starting dose of vg was injected. There was significant variability in the degree of aggregate formation among female mice (likely due to non-random X chromosome inactivation), and therefore, only males were used in subsequent experiments to evaluate the efficacy of BAG3 gene therapy in this mouse model. ssrAAVrh74.tMCK.hBAG3(3×10 12 Eight weeks after systemic delivery of vg), an overall decrease in the abundance, size, and staining intensity of aggregates in GAS and Quad muscles from male MYOT mice was observed compared to control muscles from untreated, age-matched male MYOT mice (Figures B, 6C, and 7). The aggregates were more basophilic in the untreated muscles.

[0234] These represent low dose (LD), intermediate dose (ID), and high dose (HD), respectively, in a 3x10⁻¹⁰ 10 vg, 1×10 11 vg, and 2×10 11Intramuscular dose titration experiments were performed using three doses of the vg ssrAAVrh74.tMCK.hBAG3 vector in a cohort of male MYOT mice only. In H&E sections from treated GAS muscle, a significant reduction in eosinophil aggregate size and number was observed for all three doses compared to uninjected contralateral GAS muscle (Figure 8). [Table 5]

[0235] Myotylin aggregate quantification: Quantitative histology was performed on treated and untreated GAS muscle sections collected 8 weeks after vector injection to quantify myotylin-positive aggregates and determine the effective vector dose. Immunostaining of the sections identified myotylin-positive aggregates. In the treated group, a significant shift to smaller aggregate sizes was observed at all test doses (Figure 9). Average aggregate fluorescence intensity, average aggregate area (μm²) 2 ), and the percentage of area occupied by aggregates also decreased with BAG3 treatment. The reduction in average aggregate strength and average aggregate area was significant in the low-dose (LD) cohort, while the percentage of area occupied by aggregates was significantly lower in the intermediate-dose (ID) cohort (Table 6 and Figure 10). [Table 6]

[0236] Systemic AAVrh74.tMCK.hBAG3 in TgT57I transgenic mice Based on these observations, 3 × 10 12 The VG dose was selected as the systemic dose. Ten male mice were given 3 × 10 at 4-8 weeks of age. 12The mice were administered a vg dose of AAVrh74.tMCK.hBAG3. Eight sex- and age-matched TgT57I mice served as untreated (UT) controls. Functional studies performed on these mice included rotarods, treadmill tests, grip strength tests, and assays including in vivo muscle contraction assays to test maximal spasmodic and tetanic responses. At the endpoint (8 months post-injection), the BAG3-treated cohort (n=10) showed a 43.2% increase in rotarod time [BAG3, 39.54±1.33 sec; UT (n=8), 27.6±1.77 sec; p=0.0001] and a 70.5% increase in treadmill distance (BAG3, 156.03±13.93 m; UT, 91.53±13.35 m; p=0.0048) compared to the UT cohort. Grip strength improved by 38.7% (BAG3, 0.102±0.008 kg; UT, 0.074±0.004 kg; p=0.006) in the treated cohort. In vivo muscle contraction assays showed a significant increase in muscle strength for the maximal tetanic response (BAG3, 6.26±0.44 mN*m; UT, 5.19±0.23 mN*m; p=0.047), but the increase for the maximal spasmodic response was not significant (Figures 11A-E).

[0237] Myotylin aggregate load in muscle after systemic delivery of AAVrh74.tMCK.hBAG3 was quantified 8 months post-injection using immunofluorescence technology. Four randomly selected representative images at 20x magnification from quadriceps femoris muscles of BAG3-treated (n=10) and UT (n=8) cohorts were analyzed (Figure 12A). The mean number of myotylin-positive aggregates per unit area (aggregate density) was significantly reduced in the treated group compared to the untreated counterpart (BAG3, 466.1±31.3 / mm2 vs. UT, 585.6±47.9 / mm2; per mouse analysis, p=0.0351; per image analysis, p=0.0002, Figure 12B). A decrease in mean aggregate fluorescence intensity was also observed with treatment, but it did not reach statistical significance. Aggregate size distribution analysis revealed a reduction in the numerical values ​​of all aggregate sizes due to the treatment, and showed a significant decrease for aggregate sizes up to 10 μm in diameter (Figure 12C).

[0238] Example 7 BAG3 gene therapy in SOD1-G93A ALS mouse model The following studies support the hypothesis that overexpression of BAG3 accelerates autophagy, promoting the degradation of misfolded proteins in neurodegeneration and improving survival and function in the SOD1G93A ALS model: 1) Overexpression of SOD1G93A in immortalized motor neurons (NSC34 cells) induced compensatory increases in the expression of BAG3, HspB8, and Hsp70, resulting in increased SOD1G93A removal, but SOD1G93A was not degraded when autophagy was blocked; 2) SOD1G93A was found to be immunoprecipitated by BAG3, HspB8, and Hsc70, demonstrating the importance of BAG3-mediated autophagy in the removal of aggregated misfolded proteins in familial ALS43. In the spinal cord of SODG93A and SODG85R transgenic mouse models, BAG3 and HspB8 were upregulated compared to control mice, and BAG3 colocalized with mutant SOD1 in perinuclear inclusions, indicating that BAG3, in cooperation with Hsp70, directs the mutant protein towards aggresomes for degradation.

[0239] Efficacy of AAVrh74.CBA.hBAG3 in SOD1-G93A mice SOD1-G93A mice were treated with monotherapy or combination therapy with ss.AAV9.CBA.hBAG3(4×10 12 Systemic administration of vg), or sc.AAV1.tMCK.NT-3 (1×10 11 I received an intramuscular injection of vg) (Table 7). [Table 7]

[0240] To investigate whether the onset time of intraneuronal aggregate formation affects the efficacy of BAG3, systemic BAG3 gene delivery was performed in P40 [preclinical stage without intraneuronal aggregates (BAG3-40 cohort)], P70 [early stage of intraneuronal aggregate formation correlated with the first significant decline in functional tests (BAG3-70 cohort)], and P100, stage with signs of paralysis and more widespread intraneuronal inclusions (BAG3-100 cohort). Systemic delivery of AAV9.BAG3 at day 100 (BAG3-100), AAV1.NT-3 IM injection at day 40 (NT-3-40), and combination therapy at day 70 significantly extended the mean lifespan of SOD1-G93A mice. The mean age to death was 142 days in the untreated cohort (Figure 13), but this increased to 156 days in the BAG3-100 cohort, 161 days in the NT-3-40 cohort, and 153 days in the Combo-70 cohort, suggesting rates of 9.8% (p=0.0073), 13.3% (p=0.0019), and 8.17% (p=0.0099), respectively. The combination group, which received NT-3 / BAG3 combination therapy at 40 days of age, also showed a nearly statistically significant improvement in survival (p=0.055), with one of the four mice in this cohort surviving for 164 days compared to the untreated RL group, corresponding to a 15.6% increase in survival [141.8±2.7 (mean ± SE), n=7].

[0241] For functional assays, steady declines in rotarod, grip strength, and electrophysiological test performance were observed in the untreated cohort. In the rotarod test, both the BAG3-100, Combo-70, and Combo-40 cohorts showed significant improvement compared to untreated mice, starting 1–2 weeks after injection at 128–135 days of age (Figure 14A). The NT-3-40, BAG3-70, and BAG3-40 cohorts also showed significantly better performance for 5 weeks (77–107 days), 4 weeks (92–114 days), and 3 weeks (77–92 days), respectively. Overall, the performance of the NT-3-40 and BAG3-40 cohorts was equally better than that of the RL cohort. The Combo-40 cohort outperformed only the BAG3-40 and NT-3-40 cohorts, and the Combo-70 cohort surpassed the BAG3-70 cohort, which showed significantly better performance than the RL cohort throughout the study, strongly suggesting that NT-3 / BAG3 combination therapy is more beneficial than any monotherapy. While the survival time of the Combo-70 cohort was not different from that of the Combo-40 cohort, the rotarod performance of the Combo-70 cohort was significantly better than that of the Combo-40 cohort for four consecutive weeks (100-121 days), indicating that the duration of combination therapy may influence functional improvement. In the BAG3-only cohort, an overall trend toward better performance was observed in BAG3-70 and BAG3-100 compared to BAG3-40, but this was not statistically significant. The duration of BAG3 treatment was important for survival, and early treatment did not increase survival, but improvements in functional performance were observed regardless of the BAG3 injection time.

[0242] Similar improvements were detected in grip strength tests, primarily in the Combo-70 and Combo-40 cohorts. These mice exhibited significantly better grip strength for 6 weeks, starting at 4 weeks post-injection (Figure 14B). The BAG3-70 cohort showed lower grip strength at days 70 and 77, but significant improvement was observed at 3 and 4 weeks post-injection. Overall, the performance of the NT-3-40, BAG3-100, and BAG3-40 cohorts was better than that of the RL cohort, particularly at 84-92 days, 128 days, and 92 days, respectively. Compound muscle action potentials (CMAPs) showed a rapid decline in all cohorts at the time points tested (every 14-16 days), without statistically significant differences between groups.

[0243] Quantitative histopathology: In each treatment cohort, anterior horn cells were counted from the lumbar segment, and their short-axis diameter was measured on paraffin-embedded spinal sections stained cresyl violet at the endpoint. Both large motor neurons and small interneurons were included according to the parameters detailed in the Methods section (Figure 15).

[0244] Quantitative histopathology, which assessed the number of neurons in the anterior horn region of the lumbar segment from each cohort, revealed that large neurons (diameter / motor neuron ≥ 15 μm) were well-conserved in the Combo-40, Combo70, BAG3-100, and NT-3-40 cohorts. A mean total of 147.3 (±18.6) motor neurons were counted at 70 days of age, decreasing to 63.0 (±10.5) at 142 days in untreated mice. The number of motor neurons counted for the Combo-40, Combo70, BAG3-100, and NT-3-40 cohorts were 87.0 (±14.8), 50.2 (±6.5), 48.0 (±9.6), and 44.3 (±3.7), respectively (Figure 14A). These figures indicate that these cohorts did not experience further motor neuron loss compared to UTs who died two weeks prior, suggesting that the treatment reduced the natural rate of decline in many large neurons (Figures 15B and 16).

[0245] The total number of neurons counted in the anterior horn region of the lumbar segment showed a similar decline over time in the untreated cohort. SOD1.G93A mice had a mean total of 779.3 (±30.3) neurons at 70 days of age, but this number decreased to 534.4 (±43.0) at 142 days. The total number of neurons counted for the Combo-40, Combo70, BAG3-100, NT-3-40, and WT cohorts was 526.0 (±25.1), 525.8 (±26.8), 445.4 (±13.4), 750.0 (±56.7), and 821.7 (±36.1), respectively (Figure 14B). This data suggests that the conservation observed for large neurons is also valid for total neuron counts in the Combo-40, Combo-70, BAG3-100, and NT-3-40 cohorts, and is particularly pronounced in the NT-3-40 cohort, which has a number of neuron counts close to that of the WT and RL-70 data.

[0246] Example 8 BAG3 gene therapy in the VCP-A232E mouse model IBM PFD VCP-A232E mouse model The VCP-A232E mouse is an inclusion body myositis (IBM) model associated with Paget's disease of bone and frontotemporal dementia (IBMPFD), previously generated and characterized by Custer et al. (Hum.Mol.Genet.19:1741-1755, 2010). This model possesses a human VCP transgene with the A232E mutation, associated with a more severe clinical phenotype characterized by earlier onset and more invasive myopathy compared to cases with the most commonly observed R155H mutation. The VCP-A232E mouse model, with its more severe phenotype than the VCP-R155H mouse model, accurately reproduces the complete IBMPFD phenotype, as the pathology is observed in the muscles, brain, and bone. Early symptoms of muscle weakness are reported around 3–6 months of age and are observed as hindlimb clenching.

[0247] Histological analysis of quadriceps and gastrocnemius muscles isolated from 9-month-old VCP-A232E mice revealed myopathic changes (including irregular fiber size, central nuclei, and inflammatory infiltration) and rimmed vacuoles. TDP-43 accumulation was also observed in the muscles of this model at 9 months. Mice begin to show weight loss compared to wild-type mice at approximately 9 months of age.

[0248] Experimental Design In this model, the transgene is integrated into the X chromosome. Therefore, to avoid the variability that may be observed in females due to X inactivation, male mice were used throughout this study. Seven 3-month-old VCP-A232E male mice (BAG3 cohort) were subjected to 3 × 10⁻¹⁰ 12 Eleven mice were administered a vg dose of AAVrh74.tMCK.hBAG3 systemically via injection and used as an untreated control (UT cohort). Mice were euthanized at 12 months of age, 9 months after gene delivery. Mice were tested for rotord strength and grip strength at baseline (3 months of age) and endpoint (12 months of age). Treadmill performance testing was performed at the endpoint. Gastrocnemius, quadriceps femoris, and tibialis anterior muscles were collected from the mice at the endpoint.

[0249] result The treadmill performance test showed that the BAG3 treatment cohort had a 27.8% longer treadmill distance compared to the UT cohort at the endpoint (BAG3, 70.00±4.36m, n=7; UT, 54.78±3.60m, n=11; p=0.017) (Figure 19A). Similarly, the rotarod test performed at the endpoint revealed that the BAG3 cohort showed a 46.2% increase in rotarod duration (BAG3, 32.33±2.47 seconds, n=7; UT, 22.12±2.09 seconds, n=11, p=0.0197), although no significant decrease was observed between the two cohorts at baseline (Figure 19B). The UT cohort showed a decline in rotarod that matched disease progression when comparing baseline and endpoint data (UT; baseline, 45.27±1.17 seconds, n=5; endpoint, 22.12±2.09 seconds, n=11; p<0.0001), while BAG3 gene therapy maintained rotarod function (BAG3, baseline, 40.71±4.18 seconds, n=7; endpoint, 32.33±2.47 seconds, n=7; p=0.095). The treatment did not alter grip strength.

[0250] H&E, Gomori trichrome, COX, and SDH staining were performed on muscle tissue from VCP-A232E mice exhibiting mitochondrial abnormalities, including muscle fibers with central nuclei showing degenerative / regenerative processes, as well as mitochondrial abnormalities, as indicated by mitochondrial staining and submembrane accumulation of COX-negative fibers (irregular red, brown, and blue fibers in trichrome, COX, and SDH staining, respectively) (Figure 20). In addition, BAG3 gene therapy improved mitochondrial abnormalities observed in SDH-stained sections (Figure 21).

[0251] The effects of BAG3 gene therapy on muscle fiber size and fiber type composition at the endpoint were quantified in VCP-A232E mice. Increased fiber size was observed in all fiber types, with a significant increase in fast-twitch glycolytic (FTG) fibers in the gastrocnemius muscle (BAG3, 38.04±1.23, n=6; UT, 33.81±1.20 μm, p=0.049) (Figure 22A). The distribution percentages of different fiber types did not show significant changes after BAG3 treatment (Figure 22B). The treatment effect was reflected in the muscle fiber size distribution histogram with a shift to larger diameter subgroups on the right (Figures 22C, 22D).

[0252] Since BAG3 is one of the key factors in the macroautophagy pathway, we analyzed the protein levels of two important autophagy markers, p62 and LC3 proteins. Significant increases in both proteins were observed in the treated group compared to the untreated counterpart (Figure 23).

[0253] Previous preliminary studies have suggested that NORAD (non-coding RNA activated by DNA damage), a long non-coding RNA, may be involved in the evolution of myopathic diseases, playing a hypothetical protective role against disease progression. To understand how BAG3 gene therapy may affect NORAD expression, qRT-PCR was performed on the calf muscles of treated and untreated VCP-A232E mice. Compared to untreated mice, a tendency toward higher levels of NORAD was observed in the treated cohort, but the increase observed in this cohort was more pronounced compared to age-matched WT mice (Figure 24A), indicating that BAG3 treatment may be beneficial for increasing NORAD levels.

[0254] NORAD is a preferred target of the RNA-binding protein PUM2 in mouse tissues, and upon loss of NORAD, PUM2 hyperrepresses key genes necessary for mitosis and mitochondrial function. Therefore, Pum2 levels were analyzed, and it was observed that Pum2 expression levels decreased to WT levels in treated mice (Figure 24B). PGC1α is a mitochondrial biosynthesis marker, and COX1 and COX3 are important mtDNA coding subunits of COX in respiratory complex IV. The clear trend of increase observed in Pgc1α levels, as well as the significant increase in the expression of Cox1 and Cox3 subunits, compared to the untreated cohort, suggests a positive effect of BAG3 gene therapy on mitochondrial biosynthesis and function (Figures 24C-24E).

[0255] These results are from AAVrh74.tMCK.hBAG3(3×10 12 vg) This study demonstrates that systemic delivery of VCP-A232E to male mice resulted in improved functional and histopathological outcomes compared to untreated counterparts.

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[0257] Sequence SEQ ID NO:1 - FIG. 17 ssAAVrh74.tMCK.BAG3

Chem.

Chem.

change

change

change

change

change

change

change

change

Claims

1. A polynucleotide sequence comprising a transcriptional regulatory element and a nucleotide sequence encoding human Bcl-associated atanogen 3 protein.

2. The nucleotide sequence encoding the human Bcl-related atanogen 3 protein is, (a) A nucleotide sequence having at least 90% sequence identity with nucleotides 1112-2839 of Sequence ID No. 1, or (b) A nucleotide sequence containing nucleotides 1112-2839 of SEQ ID NO: 1, (c) A nucleotide sequence having at least 90% sequence identity with nucleotides 981-2708 of Sequence ID No. 2, (d) A nucleotide sequence containing nucleotides 981 to 2708 of SEQ ID NO: 2, or (e) The polynucleotide sequence according to claim 1, comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:

8.

3. The polynucleotide sequence according to claim 1 or 2, wherein the transcriptional regulatory element is a muscle-specific regulatory element.

4. The polynucleotide sequence according to claim 3, wherein the muscle-specific control comprises one or more of the following: human skeletal actin gene element, cardiac actin gene element, desmin promoter, skeletal alpha-actin (ASKA) promoter, troponin I (TNNI2) promoter, muscle cell-specific enhancer-binding factor MEF-binding element, muscle creatine kinase (MCK) promoter, truncated MCK (tMCK) promoter, myosin heavy chain (MHC) promoter, hybrid α-myosin heavy chain enhancer / MHC enhancer-promoter (MHCK7) promoter, C5-12 promoter, mouse creatine kinase enhancer element, skeletal fast-twitch muscle troponin C gene element, slow-twitch muscle cardiac troponin c gene element, slow-twitch muscle troponin I gene element, hypoxia-inducible nuclear factor (HIF)-response element (HRE), steroid-inducible element, and glucocorticoid-response element (GRE).

5. The nucleic acid molecule according to claim 1 or 2, wherein the transcriptional regulatory element is a neuron-specific regulatory element.

6. The neuron-specific regulatory elements include the platelet-derived growth factor B chain (PDGFβ) promoter, synapsin-1 (Syn) promoter, synapsin-2 promoter, tyrosine hydroxylase promoter, dopamine β-hydroxylase (DBH) promoter, hypoxanthine-guanine phosphoribosyltransferase (HPRT) promoter, low affinity nerve growth factor receptor (LNGFR) promoter, calcitonin gene-related peptide promoter (CGRP promoter), choline acetyltransferase (ChAT) promoter, and neuron-specific enolase. The nucleic acid molecule according to claim 5, comprising one or more of the following: (NSE) promoter, calcium / calmodulin-dependent protein kinase II (CaMKII) promoter, methyl CpG-binding protein 2 (MeCP2) promoter, glial fibrillary acidic protein (GFAP) promoter, calbindin 2 promoter, motor neuron and pancreatic homeobox 1 (MNX1) promoter, also known as the Hb9 promoter, nestin promoter, parvalbumin (PVALB) promoter, and somatostation (SST) promoter.

7. The polynucleotide sequence according to claim 1 or 2, wherein the transcriptional regulatory element comprises a tMCK promoter containing nucleotides 165 to 884 of SEQ ID NO:

1.

8. The polynucleotide sequence according to any one of claims 1 to 7, further comprising a CMV enhancer containing nucleotides 209 to 463 of SEQ ID NO:

2.

9. The polynucleotide sequence according to any one of claims 1 to 8, further comprising a CBA promoter sequence containing nucleotides 495 to 749 of SEQ ID NO:

2.

10. The polynucleotide sequence according to any one of claims 1 to 9, further comprising a chimeric intron containing nucleotides 937 to 1069 or an SV40 intron sequence containing nucleotides 830 to 926 of SEQ ID NO:

2.

11. The polynucleotide sequence according to any one of claims 1 to 10, further comprising a polyadenylated sequence containing nucleotides 2849 to 3048 of SEQ ID NO: 1 or nucleotides 2718 to 2917 of SEQ ID NO:

2.

12. The polynucleotide sequence according to any one of claims 1 to 11, further comprising one or more inverted terminal repeat sequences (ITRs).

13. A polynucleotide sequence comprising a polynucleotide sequence having 90% identity with nucleotides 1-3245 of SEQ ID NO: 1 or 90% identity with nucleotides 1-3114 of SEQ ID NO:

2.

14. A polynucleotide sequence comprising a polynucleotide sequence containing nucleotides 1-3245 of SEQ ID NO: 1 or nucleotides 1-3114 of SEQ ID NO:

2.

15. A viral vector comprising the polynucleotide sequence described in any one of claims 1 to 14.

16. The viral vector according to claim 15, wherein the viral vector is an adeno-associated virus (AAV), an adenovirus, a lentivirus, a retrovirus, a poxvirus, a baculovirus, a herpes simplex virus, a vaccinia virus, or a synthetic virus.

17. The viral vector according to claim 15 or 16, wherein the viral vector is AAV.

18. The viral vector according to any one of claims 12 to 14, wherein the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVanc80, AAVrh. 74, AAVrh. 8, AAVrh. 10, AAV2 / 1, AAV2 / 8, AAV2 / 9, AAVMYO, or a variant thereof.

19. rAAV particles comprising the AAV described in claim 17 or 18.

20. A composition comprising a polynucleotide according to any one of claims 1 to 14, a viral vector according to any one of claims 15 to 18, or AAV particles according to claim 19, and a pharmaceutically acceptable carrier.

21. The composition according to claim 20, wherein the composition is formulated for intravenous or intramuscular delivery.

22. A method for treating or preventing a disease or disorder associated with misfolded proteins or protein aggregates in a subject requiring treatment or prevention of such a disease or disorder, the method comprising administering to the subject an effective amount of a polynucleotide according to any one of claims 1 to 14, a viral vector according to any one of claims 15 to 18, AAV particles according to claim 19, or a composition according to claim 20 or 21.

23. A method for reducing the accumulation of misfolded proteins or aggregated proteins in a subject suffering from a disease or disorder related to misfolded proteins or protein aggregates, the method comprising administering to the subject an effective amount of a polynucleotide according to any one of claims 1 to 14, a viral vector according to any one of claims 15 to 18, AAV particles according to claim 19, or a composition according to claim 20 or 21.

24. Use of the polynucleotide according to any one of claims 1 to 14, the viral vector according to any one of claims 15 to 18, the AAV particle according to claim 19, or the composition according to claim 20 or 21, for the preparation of a pharmaceutical for treating or preventing a disease or disorder associated with a misfolded protein or protein aggregate, in a subject requiring treatment or prevention of such a disease or disorder.

25. Use of a polynucleotide according to any one of claims 1 to 14, a viral vector according to any one of claims 15 to 18, AAV particles according to claim 19, or a composition according to claim 20 or 21A for the preparation of a pharmaceutical for reducing the accumulation of misfolded proteins or aggregated proteins in subjects suffering from a disease or disorder related to misfolded proteins or protein aggregates.

26. A composition for treating or preventing a disease or disorder associated with misfolded proteins or protein aggregates in a subject requiring treatment or prevention of such a disease or disorder, wherein the composition comprises a polynucleotide according to any one of claims 1 to 14, a viral vector according to any one of claims 15 to 18, AAV particles according to claim 19, or the composition according to claim 20 or 21.

27. A composition for reducing the accumulation of misfolded proteins or aggregated proteins in subjects suffering from diseases or disorders related to misfolded proteins or protein aggregates, wherein the composition comprises a polynucleotide according to any one of claims 1 to 14, a viral vector according to any one of claims 15 to 18, AAV particles according to claim 19, or the composition according to claim 20 or 21.

28. The method, use, or composition according to any one of claims 22 to 27, wherein the disease or disorder associated with the misfolded protein or protein aggregate is protein aggregate myopathy (PAM).

29. The method, use, or composition according to claim 28, wherein the PAM is myofibrild myopathy (MFM), desminopathy, alpha-B crystallinopathy, myotirinopathy (limbrild muscular dystrophy type 1A (LGMD1A)), filaminopathy, BAG3-associated myofibrild myopathy, ZASPopathy, HSPB8 myopathy, reduced myopathy (RBM), hereditary myopathy with early respiratory failure, simple epidermolysis bullosa with muscular dystrophy, MFM-actinopathy, or limbbrild muscular dystrophy type 1D (LGMD1D).

30. The method, use, or composition according to claim 29, wherein the misfolded or aggregated protein associated with myofibril myopathy is desmin, alpha-crystallin B chain, myotilin, filamin C, BAG family molecular chaperone regulator 3 (BAG-3), Z-band alternative splicing PDZ motif-containing protein, HSPB8, 4.5LIM domain protein 1, titin, plectin, α-actin, or DNAJ heat shock protein family (Hsp40) member B6.

31. The method, use, or composition according to claim 29 or 30, wherein the myofibril myopathy is LGMD1A, and the misfolded protein or aggregated protein associated with LGMD1A is myotirin.

32. The method, use, or composition according to any one of claims 22 to 27, wherein the disease or disorder related to misfolded proteins or protein aggregates is a neurodegenerative disease.

33. The neurodegenerative diseases associated with misfolded proteins or protein aggregates include amyotrophic lateral sclerosis (ALS), transmissible spongiform encephalopathy (prion disease), synucleinopathies, Lewy body dementia (DLB), multiple system atrophy (MSA), tauopathy, frontotemporal lobar degeneration (FTLD), frontotemporal dementia (FTD), sporadic or familial motor neuron disease (MND) with or without, corticobasal degeneration, frontotemporal lobar degeneration with ubiquitin-positive inclusions, argyrophilic granule disease, Pick's disease, and muscle The method, use, or composition according to claim 32, wherein the condition is atrophic lateral sclerosis (ALS), sporadic ALS, Alzheimer's disease (AD, sporadic and familial), Down syndrome, familial British dementia, polyglutamine (polyQ) disease (Huntington's disease (HD), dentatorubral-pallidoluysian atrophy (DRPLA), spinal-bulbar muscular atrophy (SBMA), and six spinocerebellar degenerations (SCA1, SCA2, SCA3, SCA6, SCA7, and SCA17)), hippocampal sclerosis dementia, or Parkinson's disease (PD).

34. The method, use, or composition according to claim 32 or 33, wherein the misfolded protein or protein aggregate is alpha-synuclein, amyloid-beta, mutant huntingtin, tau protein, prion protein, misfolded superoxide dismutase 1 (SOD1), islet amyloid polypeptide (IAPP), Musashi protein, p53, fusion sarcoma (FUS), progranulin, TAR DNA-binding protein 43 (TDP-43), misfolded transthyretin protein (TTR), valosin-containing protein (VCP), NOTCH3 receptor, mutant cystatin C, polyglutamine repeat, serum amyloid A (SAA), mutant gelzolin, misfolded rhodopsin, mezin, dipeptide repeat protein, or atrial natriuretic peptide.

35. The method, use, or composition according to any one of claims 33 to 35, wherein the neurodegenerative disease is ALS, and the misfolded protein or protein aggregate associated with ALS is SOD1.

36. A method for treating or preventing inclusion body myositis or multiple system proteinopathy in a subject requiring treatment or prevention of such condition, the method comprising administering to the subject an effective amount of a polynucleotide according to any one of claims 1 to 14, a viral vector according to any one of claims 15 to 18, AAV particles according to claim 19, or a composition according to claim 20 or 21.

37. Use of the polynucleotide according to any one of claims 1 to 14, the viral vector according to any one of claims 15 to 18, the AAV particle according to claim 19, or the composition according to claim 20 or 21 for the preparation of a pharmaceutical for treating or preventing inclusion body myositis or multiple system proteinopathy in subjects requiring treatment or prevention of inclusion body myositis or multiple system proteinopathy.

38. A composition for treating or preventing inclusion body myositis or multiple system proteinopathy in subjects requiring treatment or prevention of inclusion body myositis or multiple system proteinopathy, wherein the composition comprises a polynucleotide according to any one of claims 1 to 14, a viral vector according to any one of claims 15 to 18, AAV particles according to claim 19, or the composition according to claim 20 or 21.

39. The method, use, or composition according to any one of claims 36 to 38, wherein the inclusion body myositis (IBM) is associated with Paget's disease of bone and / or frontotemporal dementia (IBMMPFD).

40. The method, use, or composition according to any one of claims 36 to 39, wherein the subject has a mutation in the balossin-containing protein (VCP) gene.

41. The method, use, or composition according to any one of claims 36 to 39, wherein the subject is suffering from amyotrophic lateral sclerosis or Charcot-Marie-Tooth type 2.

42. The method further comprises administering a second nucleic acid encoding the NT-3 polypeptide, a) The nucleic acid contains a nucleotide sequence that is at least 90% identical to the nucleotide sequence of Sequence ID No. 9, b) The nucleic acid contains the nucleotide sequence of SEQ ID NO: 9, c) The nucleic acid contains a nucleotide sequence that encodes an amino acid sequence that is at least 90% identical to SEQ ID NO: 10, or d) The method, use, or composition according to any one of claims 22 to 241, wherein the nucleic acid comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:

10.

43. The method, use, or composition according to claim 36, wherein the nucleic acid encoding the NT-3 polypeptide is operably linked to a muscle-specific promoter.

44. The method, use, or composition according to claim 43, wherein the muscle-specific promoter is a muscle-specific creatine kinase promoter (MCK).

45. The method, use, or composition according to claim 44, wherein the muscle creatine kinase promoter has the nucleotide sequence shown in SEQ ID NO:

11.

46. The method, use, or composition according to any one of claims 42 to 45, wherein the second nucleic acid encoding NT-3 is administered using a viral vector.

47. The method, use, or composition according to claim 40, wherein the viral vector is recombinant adeno-associated virus (rAAV).

48. The method, use, or composition according to claim 47, wherein the capsid serotype of the rAAV is AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV12, AAV13, Anc80, AAV-B1, AAVrh. 10, AAVrh. 74, or a variant thereof.

49. The method, use, or composition according to claim 48, wherein the capsid serotype of the rAAV is AAV-1.

50. The second nucleotide sequence is in the order from 5' to 3'. (i) The first AAV2 inverted terminal repeat sequence (ITR), (ii) Muscle creatine kinase promoter / enhancer sequence shown in nucleotides 147-860 of Sequence ID No. 12, (iii) Nucleotide sequences encoding human NT-3 polypeptides, and (iv) Contains an rAAV genome sequence containing a second AAV2 ITR sequence, The method, use, or composition according to any one of claims 42 to 49, wherein the human NT-3 polypeptide has an amino acid sequence that is at least 90% identical to SEQ ID NO: 10, or 100% identical to SEQ ID NO: 10, or is encoded by a nucleotide sequence that is at least 90% identical to nucleotides 1077 to 1850 of SEQ ID NO: 12, or 100% identical to nucleotides 1077 to 1850 of SEQ ID NO:

12.

51. The method, use, or composition according to claim 50, wherein the nucleic acid sequence further comprises a chimeric intron shown in nucleotides 892-1024 of SEQ ID NO: 12 on the 3' side of the promoter / enhancer.

52. The method, use, or composition according to claim 50 or 51, wherein the nucleic acid sequence further comprises an SV40 polyadenylation signal, shown in nucleotides 1860-2059 of SEQ ID NO: 12, on the 3' side of the nucleotide sequence encoding a human NT-3 polypeptide.

53. The method, use, or composition according to any one of claims 42 to 52, wherein the second nucleic acid comprises an scAAV1.tMCK.NTF3 rAAV genome which is at least 90% identical to SEQ ID NO:

12.

54. The method, use, or composition according to any one of claims 42 to 53, wherein the nucleic acid comprising the scAAV1.tMCK.NTF3 genome is shown in Sequence ID No. 12.

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