Methods for the treatment of Danon disease and other autophagy disorders

Abstract

This provides a treatment for Danon disease and other autophagy disorders. [Solution] This disclosure provides a gene therapy vector comprising an expression cassette containing a polynucleotide encoding one or more isoforms of lysosome-associated membrane protein 2 (LAMP-2), as well as a method of using such a gene therapy vector for the treatment of Danon disease and other autophagy disorders.

Description

[Technical Field]

[0001] This patent application claims priority under 35 U.S.C. § 119(e) based on U.S. Provisional Patent Application No. 62 / 280,269 filed on 19 January 2016, which is incorporated herein by reference in its entirety.

[0002] This research is in part supported by grant numbers PHS 7K23HL107755 and RSA 1268 from the National Institutes of Health. The U.S. Government reserves certain rights in this invention. [Background technology]

[0003] Danon disease is a familial cardiomyopathy associated with impaired autophagy due to mutations in the gene encoding lysosome-associated membrane protein 2 (LAMP-2, also known as CD107b). New evidence highlights the importance of autophagy in regulating the biological function, function, and survival of cardiomyocytes. However, the mechanisms involved in cellular dysfunction and death in cardiomyocytes with impaired autophagy flow remain unclear. In a previous study, to investigate the molecular mechanisms underlying Danon disease, we generated human induced pluripotent stem cells (hiPSCs) from two patients with different LAMP-2 mutations (Non-Patent Literature 1). Danon hiPSC-derived cardiomyocytes (hiPSC-CMs) exhibited impaired autophagy flow and key features of heart failure, such as increased cell size, increased natriuretic peptide expression, and abnormal calcium processing, compared to control hiPSC-CMs. Furthermore, Danon hiPSC-CMs showed excessive mitochondrial oxidative stress and apoptosis. Using the sulfhydryl antioxidant N-acetylcysteine ​​to remove free radicals significantly reduced apoptotic cell death in Danone hiPSC-CMs. We also introduced the coding sequence of the LAMP-2B isoform under the control of a doxycycline-inducible promoter in one of the Danone hiPSC lines using a lentiviral vector. Overexpression of LAMP-2B with the addition of doxycycline also reduced oxidative stress levels and apoptotic cell death in Danone hiPSC-CMs, confirming the importance of LAMP-2B in pathophysiology. In summary, we modeled Danone disease using patient-derived hiPSC-CMs with LAMP-2 mutations, gaining mechanistic insights into the pathogenesis of this disease. We demonstrated that LAMP-2 deficiency leads to impaired autophagy flow, resulting in excessive oxidative stress and subsequent cardiomyocyte apoptosis. Scavenging excess free radicals with antioxidants and overexpressing LAMP-2B improved the disease phenotype in vitro.Prior art does not disclose effective therapeutic strategies for Danon disease or other autophagy-related disorders; therefore, the in vivo studies presented herein were necessary to validate this approach. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Hashem,et al.,Stem Cells.2015 Jul;33(7):2343-50 [Overview of the project] [Means for solving the problem]

[0005] In one embodiment, a gene therapy vector is provided for use in increasing the expression of one or more isoforms of lysosomal-associated membrane protein 2 (LAMP-2) systemically or locally in a subject. The gene therapy vector finds use in the prevention, mitigation, improvement, reduction, inhibition and / or treatment of one or more symptoms of Danon disease or other disorders of insufficient autophagy flux. In various embodiments, the gene therapy vector comprises an expression cassette containing a polynucleotide encoding one or more isoforms of lysosomal-associated membrane protein 2 (LAMP-2). In various embodiments, the vector is a viral vector. In various embodiments, the viral vector is derived from a virus selected from the group consisting of adenoviruses, retroviruses, lentiviruses, herpesviruses and adeno-associated viruses (AAVs). In various forms, the vector is derived from one or more serotypes 1-11 or any subgroup of adeno-associated virus (AAV) serotypes or any subgroup thereof. In various embodiments, the viral vector is encapsulated in an anionic liposome. In various embodiments, the vector is a non-viral vector. In various embodiments, the nonviral vector is selected from the group consisting of naked DNA, cationic liposome complexes, cationic polymer complexes, cationic liposome-polymer complexes, and exosomes. In various forms, the expression cassette includes a first reverse-terminal repeat sequence, an enhancer, a promoter, a polynucleotide encoding one or more isoforms of LAMP-2, a 3' untranslated region, a polyadenylation (poly-A) signal, and a second reverse-terminal repeat, all operably linked in the 5' to 3' direction (from the perspective of the transcribed mRNA). In various embodiments, the promoter is selected from the group consisting of the cytomegalovirus (CMV) promoter and the chicken β-actin (CAG) promoter. In various embodiments, the polynucleotide includes DNA or cDNA. In various embodiments, the polynucleotide encoding one or more isoforms of LAMP-2 includes one or more human LAMP-2 isoforms.In various embodiments, a polynucleotide encoding one or more isoforms of LAMP-2 comprises one or more LAMP-2 isoforms selected from the group consisting of LAMP-2A, LAMP-2B, and LAMP-2C. In various embodiments, a polynucleotide encoding one or more isoforms of LAMP-2 has at least about 90% sequence identity with one or more of SEQ ID NOs: 1, 2, and 3, for example, at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity. In various embodiments, a polynucleotide encoding one or more isoforms of LAMP-2 comprises one or more of SEQ ID NOs: 1, 2, and 3.

[0006] In a further embodiment, a method is provided for preventing, alleviating, improving, reducing, inhibiting, eliminating and / or reversing one or more symptoms of Danon disease or other autophagy disorders in a subject who requires prevention, alleviation, improvement, reduction, inhibition, elimination and / or reversal of such symptoms, comprising administering a gene therapy vector to a subject as described above or herein (see, for example, Figure 2). In a further embodiment, a method is provided for preventing, alleviating, improving, reducing, inhibiting, eliminating and / or reversing one or more symptoms of Danon disease or other autophagy disorders in a subject who requires prevention, alleviation, improvement, reduction, inhibition, elimination and / or reversal of such symptoms, comprising administering an adeno-associated virus (AAV) vector to a subject which comprises an expression cassette containing a polynucleotide encoding one or more isoforms of lysosome-associated membrane protein 2 (LAMP-2). In various embodiments, the vector is administered via a route selected from the group consisting of intravenous, intra-arterial, intracardiac, intracoronary, intramyocardial, intrarenal, intraurethral, ​​epidural, intracranial, subcutaneous and intramuscular. In various embodiments, the vector is delivered or administered via a physical or mechanical method selected from the group consisting of microinjection, jet injection, particle impact, hydrodynamic injection, electroporation, sonoporation, laser irradiation, and magnetofection. In various embodiments, the vector is administered multiple times, with or without immunosuppression or plasmapheresis in the patient. In various embodiments, the autophagy disorder is selected from the group consisting of end-stage heart failure, myocardial infarction, drug toxicity, diabetes, end-stage renal failure, and aging. In various embodiments, the subject is human. In various embodiments, the subject exhibits symptoms of Danon disease or other autophagy disorder. In various embodiments, the subject is identified as having reduced or undetectable LAMP-2 expression. In various embodiments, the subject is identified as having a mutated LAMP-2 gene.

[0007] (definition) The term "Danon disease" refers to an X-linked dominant skeletal and cardiomyopathy with multi-system clinical manifestations. The Danon disease mutation results in a deficiency in lysosomal-associated membrane protein 2 (LAMP-2) protein expression. Key clinical features include skeletal and cardiomyopathy, cardiac conduction abnormalities, cognitive impairment, and retinal disorders. Males are typically affected earlier and more severely than females.

[0008] The terms "Lysosome-Associated Membrane Protein 2" and "LAMP-2" are interchangeable: (1) the amino acid sequence encoded by the LAMP-2 nucleic acid (see, for example, GenBank registration numbers NM_002294.2 (isoform A), NM_013995.2 (isoform B), NM_001122606.1 (isoform C)) or the amino acid sequence of the LAMP-2 polypeptide (see, for example, GenBank registration number accession numbers NP_002285.1 (isoform A), NP_054701) (2) The amino acid sequence of a LAMP-2 polypeptide (e.g., the LAMP-2 polypeptide described herein) having an amino acid sequence having more than 90% amino acid sequence identity, for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more, preferably over a region of at least about 25, 50, 100, 200, 300, 400 or more amino acids or the entire length of LAMP-2 (3) LAMP-2 nucleic acid (e.g., LAMP-2 polynucleotide as described herein, LAMP-2 polynucleotide encoding a LAMP-2 protein) and LAMP-2 polypeptide as described herein, which specifically hybridizes under stringent hybridization conditions to an antisense strand corresponding to the LAMP-2 protein encoding amino acid sequence (e.g., LAMP-2 polynucleotide as described herein) and its conservatively modified variants, and (4) LAMP-2 nucleic acid (e.g., LAMP-2 polynucleotide as described herein, LAMP-2 polynucleotide encoding a LAMP-2 polypeptide as described herein), and nucleic acid and polypeptide polymorphic variants, alleles, mutants and interspecies homologs having nucleic acid sequences having more than about 90%, preferably about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more nucleic acid sequence identity, preferably at least about 25, 50, 100, 200, 500, 1000, 2000, or more nucleic acid regions or the entire length.

[0009] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein and refer to polymers of amino acid residues. These terms apply to amino acid polymers, in which one or more amino acid residues are artificial chemical mimics of corresponding naturally occurring amino acids, as well as to naturally occurring and non-naturally occurring amino acid polymers.

[0010] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimes that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are later modified, such as hydroxyproline, α-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids, namely hydrogen, a carboxyl group, an amino group, and an α-carbon bonded to an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs may have a modified R group (e.g., norleucine) or a modified peptide skeleton, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimes are compounds that have a structure different from the general chemical structure of amino acids, but that function similarly to naturally occurring amino acids.

[0011] Amino acids may be referred to herein by their commonly known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Committee. Similarly, nucleotides may be referred to by their commonly accepted single-letter codes.

[0012] The term "conservatively modified variant" applies to both amino acid sequences and nucleic acid sequences. For a given nucleic acid sequence, a conservatively modified variant refers to a nucleic acid that codes for the same or essentially the same amino acid sequence, or, if the nucleic acid does not code for an amino acid sequence, for an essentially identical sequence. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids code for any given protein. For example, the codons GCA, GCC, GCG, and GCU all code for the amino acid alanine. Therefore, at any position where alanine is identified by the codon, the codon can be changed to one of the corresponding codons listed without altering the coded polypeptide. Such nucleic acid mutations are a type of conservatively modified variant, known as "silent mutations." All nucleic acid sequences in this specification that code for polypeptides also describe all possible silent mutations of the nucleic acid. Those skilled in the art will recognize that modifying each codon in a nucleic acid (except AUG, usually the sole codon for methionine, and TGG, usually the sole codon for tryptophan) can yield a functionally identical molecule. Thus, each silent mutation of a polypeptide-coding nucleic acid is implicit to each described sequence.

[0013] With respect to amino acid sequences, those skilled in the art will know that individual substitutions, deletions, or additions to nucleic acids, peptides, polypeptides, or protein sequences that alter, add, or remove a single amino acid or a small percentage of amino acids in the encoded sequence are “conservatively modified variants” in which the alteration results in the substitution of an amino acid with a chemically similar amino acid. Tables of conservative substitutions that provide functionally similar amino acids are well known in the art. Such conservatively modified variants are added to, and not excluded from, the polymorphic variants, interspecific homologs, and alleles of the present invention.

[0014] The following eight groups each contain amino acids that are conserved substitutions with each other. 1) Alanine (A), Glycine (G), 2) Aspartic acid (D), glutamic acid (E), 3) Asparagine (N), glutamine (Q), 4) Arginine I, Lysine (K), 5) Isoleucine (I), leucine (L), methionine (M), valine (V), 6) Phenylalanine (F), tyrosine (Y), tryptophan (W) and 7) Serine (S), threonine (T).

[0015] A "polynucleotide" is a single- or double-stranded polymer of deoxyribonucleotides or ribonucleotide bases read from the 5' to the 3' end. Polynucleotides include RNA and DNA and may be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules. The size of a polynucleotide is expressed in base pairs (abbreviated as "bp"), nucleotides ("nt"), or kilobases ("kb"). Where the context allows, the latter two terms can describe polynucleotides that are single-stranded or double-stranded. When the term is applied to a double-stranded molecule, it is used to indicate the total length and is understood to be equivalent to the term "base pair." It will be recognized by those skilled in the art that the two strands of a double-stranded polynucleotide may differ slightly in length and their ends may be staggered as a result of enzymatic cleavage. Thus, not all nucleotides in a double-stranded polynucleotide molecule can pair.

[0016] In the context of two or more nucleic acid or polypeptide sequences, the terms “identical” or “percent “identical” refer to two or more sequences or subsequences that, when compared and aligned for maximum correspondence across a comparison window, are identical or have the same percentage of amino acid residues or nucleotides (i.e., sharing at least about 80% identity across a specific region with a reference sequence (e.g., a LAMP-2 polynucleotide or polypeptide sequence described herein), e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity), or refer to a designated region as measured by one of the sequence comparison algorithms described below, or by manual alignment and visual inspection. Such sequences are said to be “substantially identical.” This definition also refers to the complementarity of test sequences. Preferably, the identity exists over a region that is at least about 25 amino acids or nucleotides long, for example, over a region that is 50, 100, 200, 300, or 400 amino acids or nucleotides long, or over the entire length of the reference sequence.

[0017] For sequence comparison, typically one sequence serves as the reference sequence compared to the test sequence. When using a sequence comparison algorithm, the test and reference sequences are entered into a computer, subsequence coordinates are specified as needed, and sequence algorithm program parameters are specified. Either default program parameters can be used, or alternative parameters can be specified. The sequence comparison algorithm then calculates the percentage sequence identity of the test sequence to the reference sequence based on the program parameters. BLAST and BLAST 2.0 algorithms, along with their default parameters, are used for nucleic acid and protein sequence comparison with LAMP-2 nucleic acids and proteins.

[0018] As used herein, a "comparison window" includes reference to any one segment of contiguous positions consisting of a range from 20 to 600, typically about 50 to about 200, more typically about 100 to about 150, which can be compared to a reference sequence at the same number of contiguous positions after the two sequences are optimally aligned. Methods of aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the similarity search method of Pearson & Lipman, Proc. Nat'l Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI) or by manual alignment and visual inspection (see, e.g., Ausubel et al., eds., Current Protocols in Molecular Biology (1995 supplement)). Examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms described in Altschul et al., J. Mol. Biol. 215:403-410 (1990) and Altschul et al., Nucleic Acids Res. 25:3389-34 (1977), respectively. Software for performing BLAST analyses is publicly available from the National Center for Biotechnology Information (world wide web at ncbi.nlm.nih.gov).

[0019] One indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid immunologically cross-reacts with an antibody produced against the polypeptide encoded by the second nucleic acid, as described below. Therefore, the polypeptide is typically substantially identical to the second polypeptide, for example, the two peptides differing only in conserved substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize with each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the sequences can be amplified using the same primers.

[0020] As used herein, “administer” refers to topical and systemic administration, including, for example, intestinal, parenteral, pulmonary, and topical / transdermal administration. Routes of administration of compounds found to be used in the methods described herein (e.g., polynucleotides encoding one or more LAMP-2 isoforms) include, for example, oral (oral (PO) administration), nasal or inhalation administration, administration as a suppository, topical contact, transdermal delivery (e.g., by a transdermal patch), intrathecal (IT) administration, intravenous ("iv") administration, intraperitoneal ("ip") administration, intramuscular ("im") administration, intrafocal administration, or subcutaneous ("sc") administration, or implantation into a subject of a sustained-release device, such as a mini osmotic pump or depot formulation. Administration may be carried out by any route, including parenteral and transmucosal (e.g., oral, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intrarenal, intraurethral, ​​intracardiac, intracoronary, intramyocardial, intradermal, epidural, subcutaneous, intraperitoneal, intraventricular, ionophoretic, and intracranial methods. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, and transdermal patches.

[0021] The terms "systemic administration" and "systemic delivery" refer to a method of administering a compound or composition to a mammal such that the compound or composition is delivered, via the circulatory system, to a site within the body that includes the site targeted for the pharmaceutical action. Systemic administration includes, but is not limited to, oral, intranasal, rectal, and parenteral (e.g., intramuscular, intravenous, intraarterial, transdermal, and subcutaneous, etc., other than those via the gastrointestinal tract) administration.

[0022] The term "co-administer" or "co-administered" when used with respect to, for example, a compound (e.g., a LAMP-2 polynucleotide) and / or an analog thereof and another active agent, refers to the administration of the compound and / or analog and the active agent such that both can achieve a physiological effect simultaneously. However, it is not necessary to administer the two agents together. In certain embodiments, the administration of one agent can precede the administration of the other agent. Simultaneous physiological effects do not necessarily require that both agents be present in the circulation at the same time. However, in certain embodiments, co-administration typically results in both agents being present in the body (e.g., in plasma) at the maximum serum concentration at a significant percentage (e.g., 20% or more, e.g., 30% or 40% or more, e.g., 50% or 60% or more, e.g., 70% or 80% or 90% or more) for any given dose.

[0023] The term "effective amount" or "pharmaceutically effective amount" refers to the amount and / or dosage and / or dosing regimen of one or more compounds (e.g., gene therapy vectors) necessary to provide a desired result (e.g., an increase in the expression of one or more LAMP-2 isoforms sufficient to reduce the ultimate severity of a disease characterized by a disorder or incomplete autophagy (e.g., Danon disease)).

[0024] The term "administer" refers to actions taken by a healthcare professional (e.g., a physician) or person controlling the medical care of a subject to control and / or authorize the administration of a drug / compound in question to a subject. Administering may include diagnosing and / or determining an appropriate treatment or prophylactic regimen, and / or prescribing a specific drug / compound to a subject. Such prescribing may include, for example, writing a prescription and annotating it in a medical record.

[0025] The term "in combination" means, when used in reference to the use of an activator described herein (e.g., one or more isoforms of LAMP-2 polynucleotide) in combination with one or more other drugs described herein (e.g., acetylcholinesterase inhibitors), that the activator and the other drugs are administered such that there is at least some temporal overlap in their physiological activity on the organism. If they are not administered in combination with each other, there is no temporal overlap in their physiological activity on the organism. In certain preferred embodiments, the "other drugs" are not administered to the organism at all (e.g., not co-administered).

[0026] As used herein, the terms “treat” and “cure” mean delaying the onset of any disease or condition, or any one or more symptoms of such disease or condition, slowing or reversing the progression of any of them, reducing the severity of any of them, or mitigating or preventing any of them.

[0027] The term "alleviate" refers to the reduction or elimination of one or more symptoms of the medical condition or disease, and / or the rate or delay of the onset or severity of one or more symptoms of the medical condition or disease, and / or the prevention of the medical condition or disease. In certain embodiments, the reduction or elimination of one or more symptoms of the medical condition or disease may include, for example, a measurable and sustained increase in the expression level of one or more isoforms of LAMP-2.

[0028] As used herein, the phrase "essentially consisting of" refers to a genus or species of active agents listed in a method or composition, and may also include other agents that, by themselves, do not have substantial activity for the listed indication or purpose.

[0029] The terms “subject,” “individual,” and “patient” are interchangeable and refer to mammals, preferably humans or non-human primates, but also to livestock (e.g., dogs or cats), laboratory mammals (e.g., mice, rats, rabbits, hamsters, guinea pigs), and agricultural mammals (e.g., horses, cattle, pigs, sheep). In various embodiments, a subject may be a human being (e.g., adult male, adult female, adolescent male, adolescent female, boy, girl) under the care of a physician or other healthcare professional in an outpatient or other clinical setting, such as a hospital or psychiatric care facility. In certain embodiments, a subject may not be under the care or prescription of a physician or other healthcare professional.

[0030] The terms "gene transfer" or "gene delivery" refer to methods or systems for reliably inserting foreign DNA into a host cell. Such methods may result in transient expression of non-integrated transfer DNA, extrachromosomal replication and expression of transcriptional replicons (e.g., episomes), or integration of the transferred genetic material into the host cell's genomic DNA.

[0031] "AAV vector" means a vector derived from an adeno-associated virus serotype, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, etc. An AAV vector may have one or more AAV wild-type genes (e.g., rep gene and / or cap gene) that are deleted in whole or in part, but retain a functionally adjacent reverse terminal repeat (ITR) sequence. Functional ITR sequences are necessary for the rescue, replication, and packaging of the AAV virus. Therefore, an AAV vector is defined herein as containing at least the sequences required in cis for viral replication and packaging (e.g., functional ITR). The ITR does not have to be a wild-type nucleotide sequence and may be modified, for example, by nucleotide insertion, deletion, or substitution, as long as the sequence provides functional rescue, replication, and packaging. AAV expression vectors are constructed using known techniques to provide regulatory elements, including at least a transcription start region, the DNA of interest (i.e., the LAMP-2 gene), and a transcription termination region, as components operably linked in the direction of transcription. [Brief explanation of the drawing]

[0032] [Figure 1A]Figure 1 shows a schematic diagram of the adeno-associated virus LAMP-2 gene delivery construct. The shown lysosome-associated membrane protein (LAMP) LAMP-2 coding region should generally be understood to include an upstream ribosome-binding sequence and a start codon, although in some embodiments, native elements may be substituted with heterologous elements. The upstream ribosome-binding site is not used in combination with the downstream coding region of the IRES or self-cleaving peptide. The start codon is not necessarily present in the downstream coding region of the self-cleaving peptide. Figures 1A–1F show vector genomes containing one LAMP-2 isoform. Figures 1G–1K show vector genomes containing two LAMP-2 isoforms. Figures 1L–1O show vector genomes containing all three LAMP-2 isoforms. The following abbreviations are used in this figure. ITR: Terminal inversion sequence, LAMP-2: Lysosome-associated membrane protein type 2, UTR: Untranslated region, Poly A: Polyadenylation signal, CAG: Promoter region containing CMV enhancer and CBA promoter sequences, CMV: Cytomegalovirus, CBA: Chicken beta-actin, WPRE: Woodchuck hepatitis virus post-transcriptional regulatory element, RBG: Rabbit β-globin polyadenylation signal, EF-1: Human elongation factor-1, IRES: Internal ribosome entry site, P2A: 2A peptide. Figure 1A shows a schematic diagram of a construct containing protein-coding information for one LAMP-2 isoform-A, B, or C- having common 5' and 3' regulatory regions. [Figure 1B]Figure 1 shows a schematic diagram of the adeno-associated virus LAMP-2 gene delivery construct. The shown lysosome-associated membrane protein (LAMP) LAMP-2 coding region should generally be understood to include an upstream ribosome-binding sequence and a start codon, although in some embodiments, native elements may be substituted with heterologous elements. The upstream ribosome-binding site is not used in combination with the downstream coding region of the IRES or self-cleaving peptide. The start codon is not necessarily present in the downstream coding region of the self-cleaving peptide. Figures 1A–1F show vector genomes containing one LAMP-2 isoform. Figures 1G–1K show vector genomes containing two LAMP-2 isoforms. Figures 1L–1O show vector genomes containing all three LAMP-2 isoforms. The following abbreviations are used in this figure. ITR: terminal inversion sequence, LAMP-2: lysosome-associated membrane protein type 2, UTR: untranslated region, Poly A: polyadenylation signal, CAG: promoter region containing CMV enhancer and CBA promoter sequences, CMV: cytomegalovirus, CBA: chicken beta-actin, WPRE: woodchuck hepatitis virus post-transcriptional regulatory element, RBG: rabbit β-globin polyadenylation signal, EF-1: human elongation factor-1, IRES: internal ribosome entry site, P2A: 2A peptide. Figure 1B shows schematic diagrams of constructs of several embodiments used in the following examples, consisting of 5' and 3' reverse terminal repeat elements, a CAG promoter region containing CMV enhancer and CBA promoter sequences, a CBA intron, a coding sequence for one of the LAMP-2 isoforms containing an upstream ribosome binding sequence and start codon, a WPRE sequence as the 3'UTR, and a rabbit β-globin polyadenylation signal. [Figure 1C]Figure 1 shows a schematic diagram of the adeno-associated virus LAMP-2 gene delivery construct. The shown lysosome-associated membrane protein (LAMP) LAMP-2 coding region should generally be understood to include an upstream ribosome-binding sequence and a start codon, although in some embodiments, native elements may be substituted with heterologous elements. The upstream ribosome-binding site is not used in combination with the downstream coding region of the IRES or self-cleaving peptide. The start codon is not necessarily present in the downstream coding region of the self-cleaving peptide. Figures 1A–1F show vector genomes containing one LAMP-2 isoform. Figures 1G–1K show vector genomes containing two LAMP-2 isoforms. Figures 1L–1O show vector genomes containing all three LAMP-2 isoforms. The following abbreviations are used in this figure. ITR: Terminal inversion sequence, LAMP-2: Lysosome-associated membrane protein type 2, UTR: Untranslated region, Poly A: Polyadenylation signal, CAG: Promoter region containing CMV enhancer and CBA promoter sequences, CMV: Cytomegalovirus, CBA: Chicken beta-actin, WPRE: Woodchuck hepatitis virus post-transcriptional regulatory element, RBG: Rabbit β-globin polyadenylation signal, EF-1: Human elongation factor-1, IRES: Internal ribosome entry site, P2A: 2A peptide. Figure 1C shows a schematic diagram of a construct containing the native human LAMP-2 promoter region. In some embodiments, this construct is used to express a transgene in response to a cellular signal that would typically result in LAMP-2 expression in normal cells. [Figure 1D]Figure 1 shows a schematic diagram of the adeno-associated virus LAMP-2 gene delivery construct. The shown lysosome-associated membrane protein (LAMP) LAMP-2 coding region should generally be understood to include an upstream ribosome-binding sequence and a start codon, although in some embodiments, native elements may be substituted with heterologous elements. The upstream ribosome-binding site is not used in combination with the downstream coding region of the IRES or self-cleaving peptide. The start codon is not necessarily present in the downstream coding region of the self-cleaving peptide. Figures 1A–1F show vector genomes containing one LAMP-2 isoform. Figures 1G–1K show vector genomes containing two LAMP-2 isoforms. Figures 1L–1O show vector genomes containing all three LAMP-2 isoforms. The following abbreviations are used in this figure. ITR: terminal inversion sequence, LAMP-2: lysosome-associated membrane protein type 2, UTR: untranslated region, Poly A: polyadenylation signal, CAG: promoter region containing CMV enhancer and CBA promoter sequences, CMV: cytomegalovirus, CBA: chicken beta-actin, WPRE: woodchuck hepatitis virus post-transcriptional regulatory element, RBG: rabbit β-globin polyadenylation signal, EF-1: human elongation factor-1, IRES: internal ribosome entry site, P2A: 2A peptide. Figure 1D shows a schematic diagram of a construct containing the human elongation factor-1α promoter. In some embodiments, this construct is used to constitutively express a transgene under the control of the human promoter region. Other constitutively active human promoters can also be used instead of EF-1α. [Figure 1E]Figure 1 shows a schematic diagram of the adeno-associated virus LAMP-2 gene delivery construct. The shown lysosome-associated membrane protein (LAMP) LAMP-2 coding region should generally be understood to include an upstream ribosome-binding sequence and a start codon, although in some embodiments, native elements may be substituted with heterologous elements. The upstream ribosome-binding site is not used in combination with the downstream coding region of the IRES or self-cleaving peptide. The start codon is not necessarily present in the downstream coding region of the self-cleaving peptide. Figures 1A–1F show vector genomes containing one LAMP-2 isoform. Figures 1G–1K show vector genomes containing two LAMP-2 isoforms. Figures 1L–1O show vector genomes containing all three LAMP-2 isoforms. The following abbreviations are used in this figure. ITR: terminal inversion sequence, LAMP-2: lysosome-associated membrane protein type 2, UTR: untranslated region, Poly A: polyadenylation signal, CAG: promoter region containing CMV enhancer and CBA promoter sequences, CMV: cytomegalovirus, CBA: chicken beta-actin, WPRE: woodchuck hepatitis virus post-transcriptional regulatory element, RBG: rabbit β-globin polyadenylation signal, EF-1: human elongation factor-1, IRES: internal ribosome entry site, P2A: 2A peptide. Figure 1E shows a schematic diagram of a construct including, but not limited to, a cardiac-specific promoter such as the cardiac troponin T2 promoter. In some embodiments, this construct is used to express the transgene only in cardiac tissue (and, for example, to avoid liver expression). [Figure 1F]Figure 1 shows a schematic diagram of the adeno-associated virus LAMP-2 gene delivery construct. The shown lysosome-associated membrane protein (LAMP) LAMP-2 coding region should generally be understood to include an upstream ribosome-binding sequence and a start codon, although in some embodiments, native elements may be substituted with heterologous elements. The upstream ribosome-binding site is not used in combination with the downstream coding region of the IRES or self-cleaving peptide. The start codon is not necessarily present in the downstream coding region of the self-cleaving peptide. Figures 1A–1F show vector genomes containing one LAMP-2 isoform. Figures 1G–1K show vector genomes containing two LAMP-2 isoforms. Figures 1L–1O show vector genomes containing all three LAMP-2 isoforms. The following abbreviations are used in this figure. ITR: Terminal inversion sequence, LAMP-2: Lysosome-associated membrane protein type 2, UTR: Untranslated region, Poly A: Polyadenylation signal, CAG: Promoter region containing CMV enhancer and CBA promoter sequences, CMV: Cytomegalovirus, CBA: Chicken beta-actin, WPRE: Woodchuck hepatitis virus post-transcriptional regulatory element, RBG: Rabbit β-globin polyadenylation signal, EF-1: Human elongation factor-1, IRES: Internal ribosome entry site, P2A: 2A peptide. Figure 1F shows a schematic diagram of a construct including, but not limited to, a muscle-specific promoter such as a creatinine muscle kinase promoter. In some embodiments, this construct is used to express transgenes in cardiac and skeletal muscle (and to avoid excessive muscle expression). [Figure 1G]Figure 1 shows a schematic diagram of the adeno-associated virus LAMP-2 gene delivery construct. The shown lysosome-associated membrane protein (LAMP) LAMP-2 coding region should generally be understood to include an upstream ribosome-binding sequence and a start codon, although in some embodiments, native elements may be substituted with heterologous elements. The upstream ribosome-binding site is not used in combination with the downstream coding region of the IRES or self-cleaving peptide. The start codon is not necessarily present in the downstream coding region of the self-cleaving peptide. Figures 1A–1F show vector genomes containing one LAMP-2 isoform. Figures 1G–1K show vector genomes containing two LAMP-2 isoforms. Figures 1L–1O show vector genomes containing all three LAMP-2 isoforms. The following abbreviations are used in this figure. ITR: terminal inversion sequence, LAMP-2: lysosome-associated membrane protein type 2, UTR: untranslated region, Poly A: polyadenylation signal, CAG: promoter region containing CMV enhancer and CBA promoter sequences, CMV: cytomegalovirus, CBA: chicken beta-actin, WPRE: woodchuck hepatitis virus post-transcriptional regulatory element, RBG: rabbit β-globin polyadenylation signal, EF-1: human elongation factor-1, IRES: internal ribosome entry site, P2A: 2A peptide. Figure 1G shows a schematic diagram of a construct containing the coding sequences of two LAMP-2 isoforms (using any potential combination) under the control of different promoter regions. In some embodiments, this construct (and those in Figures 1H-1K) is used to express two different LAMP-2 isoforms using the same viral genome. [Figure 1H]Figure 1 shows a schematic diagram of the adeno-associated virus LAMP-2 gene delivery construct. The shown lysosome-associated membrane protein (LAMP) LAMP-2 coding region should generally be understood to include an upstream ribosome-binding sequence and a start codon, although in some embodiments, native elements may be substituted with heterologous elements. The upstream ribosome-binding site is not used in combination with the downstream coding region of the IRES or self-cleaving peptide. The start codon is not necessarily present in the downstream coding region of the self-cleaving peptide. Figures 1A–1F show vector genomes containing one LAMP-2 isoform. Figures 1G–1K show vector genomes containing two LAMP-2 isoforms. Figures 1L–1O show vector genomes containing all three LAMP-2 isoforms. The following abbreviations are used in this figure. ITR: Terminal inversion sequence, LAMP-2: Lysosome-associated membrane protein type 2, UTR: Untranslated region, Poly A: Polyadenylation signal, CAG: Promoter region containing CMV enhancer and CBA promoter sequences, CMV: Cytomegalovirus, CBA: Chicken beta-actin, WPRE: Woodchuck hepatitis virus post-transcriptional regulatory element, RBG: Rabbit β-globin polyadenylation signal, EF-1: Human elongation factor-1, IRES: Internal ribosome entry site, P2A: 2A peptide. Figure 1H shows a schematic diagram of a construct containing sequences of two LAMP-2 isoforms (using any potential combination) under the control of different promoter regions. One isoform is encoded in the (+) direction on a single-stranded AAV genome, and the other isoform is encoded in the (-) direction. In some embodiments, this construct is used to express two different LAMP-2 isoforms on separate DNA strands. [Figure 1I]Figure 1 shows a schematic diagram of the adeno-associated virus LAMP-2 gene delivery construct. The shown lysosome-associated membrane protein (LAMP) LAMP-2 coding region should generally be understood to include an upstream ribosome-binding sequence and a start codon, although in some embodiments, native elements may be substituted with heterologous elements. The upstream ribosome-binding site is not used in combination with the downstream coding region of the IRES or self-cleaving peptide. The start codon is not necessarily present in the downstream coding region of the self-cleaving peptide. Figures 1A–1F show vector genomes containing one LAMP-2 isoform. Figures 1G–1K show vector genomes containing two LAMP-2 isoforms. Figures 1L–1O show vector genomes containing all three LAMP-2 isoforms. The following abbreviations are used in this figure. ITR: terminal inversion sequence, LAMP-2: lysosome-associated membrane protein type 2, UTR: untranslated region, Poly A: polyadenylation signal, CAG: promoter region containing CMV enhancer and CBA promoter sequences, CMV: cytomegalovirus, CBA: chicken beta-actin, WPRE: woodchuck hepatitis virus post-transcriptional regulatory element, RBG: rabbit β-globin polyadenylation signal, EF-1: human elongation factor-1, IRES: internal ribosome entry site, P2A: 2A peptide. Figure 1I shows a schematic diagram of a construct containing the coding sequences of two LAMP-2 isoforms (using any potential combination) under the control of the promoter region and internal ribosome entry site, followed by the 3'UTR and poly A signal. In some embodiments, this construct is used to express two different LAMP-2 isoforms using the same viral genome. [Figure 1J]Figure 1 shows a schematic diagram of the adeno-associated virus LAMP-2 gene delivery construct. The shown lysosome-associated membrane protein (LAMP) LAMP-2 coding region should generally be understood to include an upstream ribosome-binding sequence and a start codon, although in some embodiments, native elements may be substituted with heterologous elements. The upstream ribosome-binding site is not used in combination with the downstream coding region of the IRES or self-cleaving peptide. The start codon is not necessarily present in the downstream coding region of the self-cleaving peptide. Figures 1A–1F show vector genomes containing one LAMP-2 isoform. Figures 1G–1K show vector genomes containing two LAMP-2 isoforms. Figures 1L–1O show vector genomes containing all three LAMP-2 isoforms. The following abbreviations are used in this figure. ITR: Terminal inversion sequence, LAMP-2: Lysosome-associated membrane protein type 2, UTR: Untranslated region, Poly A: Polyadenylation signal, CAG: Promoter region containing CMV enhancer and CBA promoter sequences, CMV: Cytomegalovirus, CBA: Chicken beta-actin, WPRE: Woodchuck hepatitis virus post-transcriptional regulatory element, RBG: Rabbit β-globin polyadenylation signal, EF-1: Human elongation factor-1, IRES: Internal ribosome entry site, P2A: 2A peptide. Figure 1J shows a schematic diagram of a construct containing coding sequences for two LAMP-2 isoforms (using any potential combination) separated by the P2A cleavage site. In some embodiments, this construct is used to express mRNA encoding two different LAMP-2 isoforms in a single polypeptide, which spontaneously cleaves into individual LAMP-2 protein isoforms after translation by 2A peptide autocleavage using the same viral genome. [Figure 1K]Figure 1 shows a schematic diagram of the adeno-associated virus LAMP-2 gene delivery construct. The shown lysosome-associated membrane protein (LAMP) LAMP-2 coding region should generally be understood to include an upstream ribosome-binding sequence and a start codon, although in some embodiments, native elements may be substituted with heterologous elements. The upstream ribosome-binding site is not used in combination with the downstream coding region of the IRES or self-cleaving peptide. The start codon is not necessarily present in the downstream coding region of the self-cleaving peptide. Figures 1A–1F show vector genomes containing one LAMP-2 isoform. Figures 1G–1K show vector genomes containing two LAMP-2 isoforms. Figures 1L–1O show vector genomes containing all three LAMP-2 isoforms. The following abbreviations are used in this figure. ITR: Terminal inversion sequence, LAMP-2: Lysosome-associated membrane protein type 2, UTR: Untranslated region, Poly A: Polyadenylation signal, CAG: Promoter region containing CMV enhancer and CBA promoter sequences, CMV: Cytomegalovirus, CBA: Chicken beta-actin, WPRE: Woodchuck hepatitis virus post-transcriptional regulatory element, RBG: Rabbit β-globin polyadenylation signal, EF-1: Human elongation factor-1, IRES: Internal ribosome entry site, P2A: 2A peptide. Figure 1K is a schematic diagram of a construct containing the coding sequences of LAMP-2 exons 1-8 followed by the intron region (containing all necessary splice signals), the exon 9 coding sequence for one of the LAMP-2 isoforms with 3'UTR and poly A signal, the second intron region (containing all necessary splice signals), and the exon 9 coding sequence for different LAMP-2 isoforms (in any potential combination) with 3'UTR and poly A signal. In some embodiments, this construct is used to express mRNA of two different LAMP-2 isoforms via alternative splicing, a mechanism that generates different LAMP-2 mRNA in normal cells using the same viral genome. [Figure 1L]Figure 1 shows a schematic diagram of the adeno-associated virus LAMP-2 gene delivery construct. The shown lysosome-associated membrane protein (LAMP) LAMP-2 coding region should generally be understood to include an upstream ribosome-binding sequence and a start codon, although in some embodiments, native elements may be substituted with heterologous elements. The upstream ribosome-binding site is not used in combination with the downstream coding region of the IRES or self-cleaving peptide. The start codon is not necessarily present in the downstream coding region of the self-cleaving peptide. Figures 1A–1F show vector genomes containing one LAMP-2 isoform. Figures 1G–1K show vector genomes containing two LAMP-2 isoforms. Figures 1L–1O show vector genomes containing all three LAMP-2 isoforms. The following abbreviations are used in this figure. ITR: terminal inversion sequence, LAMP-2: lysosome-associated membrane protein type 2, UTR: untranslated region, Poly A: polyadenylation signal, CAG: promoter region containing CMV enhancer and CBA promoter sequences, CMV: cytomegalovirus, CBA: chicken beta-actin, WPRE: woodchuck hepatitis virus post-transcriptional regulatory element, RBG: rabbit β-globin polyadenylation signal, EF-1: human elongation factor-1, IRES: internal ribosome entry site, P2A: 2A peptide. Figure 1L shows a schematic diagram of a construct containing coding sequences for all three LAMP-2 isoforms having 3'UTR and polyA signaling under the control of different promoter regions. In some embodiments, this construct is used to express all three different LAMP-2 isoforms using the same viral genome, allowing for the restoration of all potential LAMP-2 functions. [Figure 1M]Figure 1 shows a schematic diagram of the adeno-associated virus LAMP-2 gene delivery construct. The shown lysosome-associated membrane protein (LAMP) LAMP-2 coding region should generally be understood to include an upstream ribosome-binding sequence and a start codon, although in some embodiments, native elements may be substituted with heterologous elements. The upstream ribosome-binding site is not used in combination with the downstream coding region of the IRES or self-cleaving peptide. The start codon is not necessarily present in the downstream coding region of the self-cleaving peptide. Figures 1A–1F show vector genomes containing one LAMP-2 isoform. Figures 1G–1K show vector genomes containing two LAMP-2 isoforms. Figures 1L–1O show vector genomes containing all three LAMP-2 isoforms. The following abbreviations are used in this figure. ITR: terminal inversion sequence, LAMP-2: lysosome-associated membrane protein type 2, UTR: untranslated region, Poly A: polyadenylation signal, CAG: promoter region containing CMV enhancer and CBA promoter sequences, CMV: cytomegalovirus, CBA: chicken beta-actin, WPRE: woodchuck hepatitis virus post-transcriptional regulatory element, RBG: rabbit β-globin polyadenylation signal, EF-1: human elongation factor-1, IRES: internal ribosome entry site, P2A: 2A peptide. Figure 1M shows a schematic diagram of a construct containing the coding sequences for three LAMP-2 isoforms, followed by the 3'UTR and poly A signal, in any order, under the control of the promoter region and two different internal ribosome entry sites. In some embodiments, this construct is used to express all three LAMP-2 isoforms using the same viral genome. [Figure 1N]Figure 1 shows a schematic diagram of the adeno-associated virus LAMP-2 gene delivery construct. The shown lysosome-associated membrane protein (LAMP) LAMP-2 coding region should generally be understood to include an upstream ribosome-binding sequence and a start codon, although in some embodiments, native elements may be substituted with heterologous elements. The upstream ribosome-binding site is not used in combination with the downstream coding region of the IRES or self-cleaving peptide. The start codon is not necessarily present in the downstream coding region of the self-cleaving peptide. Figures 1A–1F show vector genomes containing one LAMP-2 isoform. Figures 1G–1K show vector genomes containing two LAMP-2 isoforms. Figures 1L–1O show vector genomes containing all three LAMP-2 isoforms. The following abbreviations are used in this figure. ITR: Terminal inversion sequence, LAMP-2: Lysosome-associated membrane protein type 2, UTR: Untranslated region, Poly A: Polyadenylation signal, CAG: Promoter region containing CMV enhancer and CBA promoter sequences, CMV: Cytomegalovirus, CBA: Chicken beta-actin, WPRE: Woodchuck hepatitis virus post-transcriptional regulatory element, RBG: Rabbit β-globin polyadenylation signal, EF-1: Human elongation factor-1, IRES: Internal ribosome entry site, P2A: 2A peptide. Figure 1N shows a schematic diagram of a construct containing the coding sequences of all three LAMP-2 isoforms in any potential order, separated by the P2A cleavage site. In some embodiments, this construct is used to express mRNA encoding three different LAMP-2 isoforms in a single polypeptide, which simultaneously cleaves the individual LAMP-2 protein isoforms after translation by 2A peptide autocleavage using the same viral genome. [Figure 10]Figure 1 shows a schematic diagram of the adeno-associated virus LAMP-2 gene delivery construct. The shown lysosome-associated membrane protein (LAMP) LAMP-2 coding region should generally be understood to include an upstream ribosome-binding sequence and a start codon, although in some embodiments, native elements may be substituted with heterologous elements. The upstream ribosome-binding site is not used in combination with the downstream coding region of the IRES or self-cleaving peptide. The start codon is not necessarily present in the downstream coding region of the self-cleaving peptide. Figures 1A–1F show vector genomes containing one LAMP-2 isoform. Figures 1G–1K show vector genomes containing two LAMP-2 isoforms. Figures 1L–1O show vector genomes containing all three LAMP-2 isoforms. The following abbreviations are used in this figure. ITR: Terminal inversion sequence, LAMP-2: Lysosome-associated membrane protein type 2, UTR: Untranslated region, Poly A: Polyadenylation signal, CAG: Promoter region containing CMV enhancer and CBA promoter sequences, CMV: Cytomegalovirus, CBA: Chicken beta-actin, WPRE: Woodchuck hepatitis virus post-transcriptional regulatory element, RBG: Rabbit β-globin polyadenylation signal, EF-1: Human elongation factor-1, IRES: Internal ribosome entry site, P2A: 2A peptide. Figure 1O is a schematic diagram of a construct that includes coding sequences for LAMP-2 exons 1-8, followed by an intron region (containing all necessary splice signals), an exon 9 coding sequence for one of the LAMP-2 isoforms with a 3'UTR and poly-A signal, a second intron region (containing all necessary splice signals), an exon 9 coding sequence for a third LAMP-2 isoform with a 3'UTR and poly-A signal, a third intron region (containing all necessary splice signals), and an exon 9 coding sequence for a third LAMP-2 isoform with a 3'UTR and poly-A signal.In some embodiments, this construct is used to express mRNA of all three LAMP-2 isoforms via alternative splicing, a mechanism that generates different LAMP-2 mRNAs in normal cells using the same viral genome. The exon 9 coding sequence can be arranged in any potential order and combination. [Figure 2] Figure 2 shows a flowchart illustrating a therapeutic approach involving promoting the expression of one or more isoforms of LAMP-2 (e.g., Danon disease or another disease caused at least partially by autophagy deficiency). [Figure 3A] Figures 3A-3F show the coding sequences and protein sequences of the LAMP-2 isoform. Figure 3A: LAMP-2A coding sequence. [Figure 3B] Figures 3A-F show the LAMP-2 isoform coding sequences and protein sequences. Figure 3B: LAMP-2A protein sequence. [Figure 3C] Figures 3A-F show the coding sequences and protein sequences of the LAMP-2 isoforms. Figure 3C: LAMP-2B coding sequence. [Figure 3D] Figures 3A-F show the LAMP-2 isoform coding sequences and protein sequences. Figure 3D: LAMP-2B protein sequence. [Figure 3E] Figures 3A-F show the coding sequences and protein sequences of the LAMP-2 isoforms. Figure 3E: LAMP-2C coding sequence. [Figure 3F] Figures 3A-F show the LAMP-2 isoform coding sequences and protein sequences. Figure 3F: LAMP-2C protein sequence. [Figure 4A] Figures 4A-4C show a comparison of the last 90 amino acids of the human and mouse protein sequences of LAMP-2A (Figure 4A), LAMP-2B (Figure 4B), and LAMP-2C (Figure 4C), demonstrating sequence identity. [Figure 4B]Figures 4A-4C show a comparison of the last 90 amino acids of the human and mouse protein sequences of LAMP-2A (Figure 4A), LAMP-2B (Figure 4B), and LAMP-2C (Figure 4C), demonstrating sequence identity. [Figure 4C] Figures 4A-4C show a comparison of the last 90 amino acids of the human and mouse protein sequences of LAMP-2A (Figure 4A), LAMP-2B (Figure 4B), and LAMP-2C (Figure 4C), demonstrating sequence identity. [Figure 5A] Figures 5A and 5B show the dose-dependent increase in mRNA expression of LAMP-2B (Figure 5A) and LAMP-2A (Figure 5B) after administration of AAV9 vectors containing these genes. [Figure 5B] Figures 5A and 5B show the dose-dependent increase in mRNA expression of LAMP-2B (Figure 5A) and LAMP-2A (Figure 5B) after administration of AAV9 vectors containing these genes. [Figure 6] Figure 6 shows the dose-dependent increase in LAMP-2B and LAMP-2A protein expression after administration of an AAV9 vector containing these genes. [Figure 7A] Figures 7A-7C show fluorescence micrographs of cardiac sections stained with DAPI (blue) and fluorescently labeled anti-LAMP-2 antibody (white). Figure 7A shows cardiac sections from Lamp-2 knockout (KO) mice treated with AAV9.LAMP-2B. [Figure 7B] Figures 7A-C show fluorescence micrographs of cardiac sections stained with DAPI (blue) and fluorescently labeled anti-LAMP-2 antibody (white). Figure 7B shows cardiac sections from Lamp-2 KO mice treated with AAV9.EGFP. [Figure 7C] Figures 7A-7C show fluorescence micrographs of cardiac sections stained with DAPI (blue) and fluorescently labeled anti-LAMP-2 antibody (white). Figure 7C shows a cardiac section from an untreated wild-type (WT) mouse. [Figure 8A]Figures 8A and 8B show fluorescence micrographs of cardiac sections stained with DAPI (blue) and fluorescently labeled anti-LAMP-2 antibody (white; several examples highlighted with arrows). Figure 8A shows cardiac sections from Lamp-2 KO mice treated with AAV9.LAMP-2B. [Figure 8B] Figures 8A and 8B show fluorescence micrographs of cardiac sections stained with DAPI (blue) and fluorescently labeled anti-LAMP-2 antibody (white; several examples highlighted with arrows). Figure 8B shows cardiac sections from Lamp-2 KO mice treated with AAV9.EGFP. [Figure 9A] Figures 9A to 9H show the CAG-RFP-EGFP-LC3B autophagy reporter system. Figure 9A shows a schematic diagram of the system's operation. [Figure 9B-Gprime] Figures 9A–9H show the CAG-RFP-EGFP-LC3B autophagy reporter system. Figures 9B–G' are fluorescence microscopy images, and the X' (X-prime) image is a magnified inset outlined in the image labeled with the same letter without priming. Regarding the grid in Figures 9B–G', the first column is the image of red fluorescence, the second column is the image of green fluorescence, and the third column is a composite image of the first and second columns, where yellow represents red + green fluorescence. The top two columns are images of cardiac sections from WT mice expressing the CAG-RFP-EGFP-LC3B autophagy reporter construct, and the bottom two columns are images of cardiac sections from Lamp-2 KO mice expressing the CAG-RFP-EGFP-LC3B autophagy reporter construct. Immature autolysosomes fluoresce in both green and red (or yellow in fused images). Mature autolysosomes fluoresce in red only. Yellow arrows highlight autophagous vacuoles that fluoresce in both colors, while white arrows highlight autophagous vacuoles that fluoresce only in red. [Figure 9H] Figures 9A–9H show the CAG-RFP-EGFP-LC3B autophagy reporter system. Figure 9H shows the number of autophagy vacuoles, which are autophagy cells or autolysosomes, in WT and Lamp-2 KO mice. [Figure 10A]Figures 10A–E show the effects of gene therapy using the CAG-RFP-EGFP-LC3B autophagy reporter system. Figure 10A is an image of an untreated WT mouse heart section. In images 10A, 10C, and 10D, red arrows are used to highlight some of the autolysosomes present. [Figure 10B] Figures 10A-E show the effects of -2 gene therapy using the CAG-RFP-EGFP-LC3B autophagy reporter system. Figure 10B is an image of a cardiac section showing transduced cells from Lamp-2 KO mice treated with the control vector AAV9.EGFP. [Figure 10C] Figures 10A–E show the effects of the -2 gene therapy using the CAG-RFP-EGFP-LC3B autophagy reporter system. Figure 10C is an image of a cardiac section from a Lamp-2 KO mouse treated with the gene therapy vector AAV9.LAMP-2A. In images 10A, 10C, and 10D, red arrows are used to highlight some of the autolysosomes present. [Figure 10D] Figures 10A–E show the effects of LAMP-2 gene therapy using the CAG-RFP-EGFP-LC3B autophagy reporter system. Figure 10D is an image of a cardiac section from a Lamp-2 KO mouse treated with the gene therapy vector AAV9.LAMP-2B. In images 10A, 10C, and 10D, red arrows are used to highlight some of the autolysosomes present. [Figure 10E] Figures 10A-E show the effects of gene therapy using the CAG-RFP-EGFP-LC3B autophagy reporter system. Figure 10E shows the percentage of vacuoles in total autophagy represented by autophagosomes and autolysosomes under four conditions. [Figure 11A]Figures 11A–C' show electron micrographs of cardiac tissue from WT (Figure 11A, A') and Lamp-2 KO (Figure 11B, B') mice, as well as Lamp-2 KO mice treated with AAV9.LAMP-2B (Figure 11C, C'). White arrows highlight some autophagous vacuoles. Black arrows highlight some damaged mitochondria. [Figure 11B] Figures 11A–C' show electron micrographs of cardiac tissue from WT (Figure 11A, A') and Lamp-2 KO (Figure 11B, B') mice, as well as Lamp-2 KO mice treated with AAV9.LAMP-2B (Figure 11C, C'). White arrows highlight some autophagous vacuoles. Black arrows highlight some damaged mitochondria. [Figure 11C] Figures 11A–C' show electron micrographs of cardiac tissue from WT (Figure 11A, A') and Lamp-2 KO (Figure 11B, B') mice, as well as Lamp-2 KO mice treated with AAV9.LAMP-2B (Figure 11C, C'). White arrows highlight some autophagous vacuoles. Black arrows highlight some damaged mitochondria. [Modes for carrying out the invention]

[0033] 1. Introduction Danon disease is caused by mutations that result in reduced or deleted expression of the lysosomal-associated membrane protein 2 (LAMP-2, also known as CD107b) gene. This method is partly based on introducing one or more polynucleotides (e.g., packaged in an adeno-associated virus (AAV) vector) encoding one or more LAMP-2 isoforms and delivering the LAMP-2 gene / individual isoform to Danon patients. Following delivery of one or more polynucleotides encoding one or more LAMP-2 isoforms, the LAMP-2 transgene is expressed by the patient's own cells. Restoration of LAMP-2 gene expression in Danon patients may result in improvement of the disease phenotype and may serve as a treatment for the disease. Delivery of one or more polynucleotides encoding one or more LAMP-2 isoforms to subjects may also be used to treat other autophagy disorders, including, but are not limited to, end-stage heart failure, myocardial infarction drug toxicity, diabetes, end-stage renal failure, and aging. Abnormalities in autophagy, particularly reduced autophagy flow, are associated with these disorders and many other disorders. Higher levels of LAMP-2 gene expression increase autophagy flow and therefore serve as a therapeutic agent for these disorders.

[0034] Currently, there are no technologies to treat Danon disease by gene therapy. Danon disease is not a traditional lysosomal storage disorder; it is generally defined as a deficiency in lysosomal proteins (e.g., transporters or enzymes) required to process specific cellular substrates, resulting in the toxic accumulation of those specific substrates within lysosomes. Danon disease is understood to be a disorder of autophagy or autophagy vacuolar myopathy, which affects the degradation of all cellular components processed by the autophagy pathway and is not caused by the accumulation of specific substrates. Furthermore, existing technologies do not explicitly describe the delivery of LAMP-2 gene / individual isoforms for the treatment of Danon disease or other autophagy disorders. Therefore, this method provides a unique method for treating Danon disease and improves existing AAV technologies by explicitly including the delivery of the LAMP-2 gene as a means to improve autophagy disorders.

[0035] 2. Patients receiving treatment Subjects / patients susceptible to treatment using the methods described herein include individuals who are at risk of disease or disorder characterized by insufficient autophagy flow (e.g., Danon disease and other known autophagy disorders, but not limited to, systolic and diastolic heart failure, myocardial infarction, drug toxicity (e.g., anthracyclines chloroquine and its derivatives), diabetes mellitus, end-stage renal disease, and aging) but are asymptomatic or currently asymptomatic. Such subjects may be identified as having a mutated LAMP-2 gene or as having reduced or undetectable levels of LAMP-2 expression.

[0036] In some embodiments, subjects present with a disease or disorder characterized by insufficient autophagy flow (e.g., Danon disease and other known autophagy disorders, but not limited to, systolic and diastolic heart failure, myocardial infarction, drug toxicity, diabetes, end-stage renal disease, and aging). Symptoms may be actively present, suppressed or controlled (e.g., by medication), or in remission. Subjects may or may not have been diagnosed with a disorder, for example, by a qualified physician.

[0037] The subject may be any mammal at any stage of development, such as embryonic, fetal, infantile, juvenile, or adult, at the time of delivery. In various embodiments, the subject may be a child, a boy, or an adult. In various embodiments, the subject may be a mammal, such as a human or a domestic mammal (e.g., a dog or a cat).

[0038] 3. Delivery vector for LAMP-2 polynucleotides Generally, the gene therapy vectors described herein comprise an expression cassette containing a polynucleotide encoding one or more isoforms of lysosome-associated membrane protein 2 (LAMP-2), enabling the expression of LAMP-2 in a subject in need (e.g., a subject with Danon disease or another disorder characterized at least partially by a lost autophagy flow due to a loss of LAMP-2 expression) to partially or completely correct the lost LAMP-2 protein expression level and autophagy flow. The gene therapy vectors may be viral or nonviral vectors. Exemplary nonviral vectors include, for example, naked DNA, cationic liposome complexes, cationic polymer complexes, cationic liposome-polymer complexes, and exosomes.

[0039] In some embodiments, the vector has a single isoform, LAMP-2A, LAMP-2B, or LAMP-2C (see Figures 1A-F, for example, schematic diagrams of an AAV vector having the gene for a single LAMP-2 isoform). In other embodiments, the vector has genes for two LAMP-2 isoforms (see Figures 1G-K, for example, schematic diagrams of an AAV vector having genes for two LAMP-2 isoforms, LAMP-2B on one DNA strand and LAMP-2A on the other). Yet another embodiment has all three isoforms (see Figures 1L-O). In addition to the genomic structures described, three-isoform vectors can also be constructed by creating hybrids of the various embodiments shown. For example, there may be two promoters, one driving the expression of a single isoform and the other driving the expression of two isoform cassettes using IRESs, self-cleaving peptides, or alternative splicing. In various embodiments involving multiple isoforms, the isoforms may occur in any order, reflect the natural order of B, A, and C, or modify it. By carrying multiple isoforms in a single vector, co-expression of isoforms in transfected cells can be ensured, and the use of total vector particles in the dose can be reduced.

[0040] Examples of viral vectors include, but are not limited to, adenovirus vectors, retrovirus vectors, lentivirus vectors, herpesvirus vectors, and adeno-associated virus (AAV) vectors. Gene-delivering viral vectors useful for carrying out the present invention can be constructed using methodologies well known in the field of molecular biology. Typically, a viral vector containing a transgene is constructed from a polynucleotide encoding the transgene, appropriate regulatory elements, and elements necessary for the production of a viral protein that mediates cell transduction.

[0041] Such recombinant viruses can be produced by techniques known in the art, such as transfection of packaging cells or transient transfection with helper plasmids or viruses. Typical examples of viral packaging cells include, but are not limited to, PA317 cells, PsiCRIP cells, GPenv+ cells, and 293 cells. Detailed protocols for producing such replication-deficient recombinant viruses can be found, for example, in International Publication No. 95 / 14785, International Publication No. 96 / 22378, U.S. Patent No. 5,882,877, U.S. Patent No. 6,013,516, U.S. Patent No. 4,861,719, U.S. Patent No. 5,278,056 and International Publication No. 94 / 19478, the complete contents of each of these are incorporated herein by reference.

[0042] In some embodiments, the gene viral vector is an adenovirus vector or an adeno-associated virus (AAV) vector. In various embodiments, the AAV vector is selected from AAV serotypes AAV1, AAV2, AAV3, AAV4, AA5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh10, and its subgroups and mixtures including the self-complementary AAV (scAAV) genome, or from any other serotype of AAV that can infect humans, monkeys, or other species. In one embodiment, the AAV vector is AAVrh10. In another embodiment, the AAV vector is AAV9. In yet another embodiment, the AAV vector is AAV8. Recombinant AAV (rAAV) vectors are often used for the delivery of therapeutic genes and have been studied in human clinical trials. rAAV vectors can be designed to deliver transgenes specific to patient cells for expression. Following infection and introduction of the viral genome by an rAAV vector, the viral gene primarily exists as an extrachromosomal structure that is not integrated into the host genome but is expressed by the host cell's translational mechanisms. For successful infection and gene expression in host cells, the rAAV vector requires several components (see Figure 1), including an inverse terminal repeat (ITR), a promoter and / or enhancer region, the transgene and 3' untranslated region, and a polyadenylation signal. After viral infection of host cells, the promoter region initiates the signal for translation of the virally delivered transgene by the host cell's translational mechanisms.

[0043] Generally, regulatory elements are selected to be functional in mammalian cells. The resulting construct, containing operably linked components, is conjugated with a functional AAV ITR sequence (5' and 3'). "Adeno-associated virus inverted terminal repeat" or "AAV ITR" refers to a region recognized in this field, found at each end of the AAV genome, that functions together in cis as a DNA replication origin and as a viral packaging signal. AAV ITRs, along with AAV rep coding regions, result in adequate excision and salvage, as well as integration of nucleotide sequences inserted between two adjacent ITRs in the mammalian cell genome. Nucleotide sequences of AAV ITR regions are publicly known. For example, for the AAV2 sequence, see Kotin, 1994; Berns, KI, "Parvoviridae and their Replication" in Fundamental Virology, 2nd Edition, (BNFields and DMKnipe, eds., the entire content of which is incorporated herein by reference). As used herein, "AAV ITR" does not necessarily have to contain the wild-type nucleotide sequence and may be modified, for example, by nucleotide insertion, deletion, or substitution.

[0044] Furthermore, AAV ITRs may originate from any several AAV serotypes, including but not limited to AAV1, AAV2, AAV3, AAV4, AA5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAVrh10. Additionally, the 5' and 3' ITRs adjacent to selected nucleotide sequences in the AAV vector do not necessarily have to be identical or derived from the same AAV serotype or isolate, as long as they function as intended. That is, they excise and rescue target sequences derived from the host cell genome or vector, enabling the incorporation of heterologous sequences into the recipient cell genome when the AAV Rep gene product is present intracellularly. Furthermore, AAV ITRs may originate from any several AAV serotypes, including but not limited to AAV1, AAV2, AAV3, AAV4, AA5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAVrh10. Furthermore, the 5' and 3' ITRs adjacent to the selected nucleotide sequence in the AAV expression vector do not necessarily have to be identical or derived from the same AAV serotype or isolate, as long as they function as intended. In other words, target sequences derived from the host cell genome or vector can be excised and rescued, allowing for the incorporation of the DNA molecule into the recipient cell genome when the AAV Rep gene product is present in the cell.

[0045] In various embodiments, vectors derived from AAV serotypes that exhibit tropism to mammalian cardiomyocytes, particularly cardiomyocytes and cardiomyocyte progenitor cells, and high transduction efficiency are used. Studies and comparisons of transduction efficiencies of different serotypes are described in Cearley CN et al., Molecular Therapy 16(10);1710-1718, 2008, the full contents of which are incorporated herein by reference. In other non-limiting examples, preferred vectors include vectors derived from any serotype such as AAV1, AAV2, AAV3, AAV4, AA5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAVrh10, which have also been shown to transduce cardiomyocytes.

[0046] In various embodiments, selected nucleotide sequences are operably ligated to regulatory elements that direct their transcription or expression in vivo in a subject. Such regulatory elements may include regulatory sequences that are normally associated with the selected gene.

[0047] Alternatively, heterologous control sequences can be used. Useful heterologous control sequences generally include those derived from sequences encoding mammalian or viral genes. Examples include, but are not limited to, the phosphoglycerate kinase (PKG) promoter, the CAG (CMV enhancer with a chicken β-actin promoter containing the first intron and splice acceptor sequence of the rabbit β-globin gene) promoter, the MCK (muscle creatine kinase) promoter, the SV40 early promoter, the mouse mammary cancer virus LTR promoter, the adenovirus major late promoter (Ad MLP), the herpes simplex virus (HSV) promoter, the cytomegalovirus (CMV) promoter such as the CMV pre-early promoter region (CMVIE), the Rous sarcoma virus (RSV) promoter, synthetic promoters, and hybrid promoters. Promoters may be of human origin or from other species, including mice. Furthermore, sequences derived from non-viral genes, such as the marine metallothionein gene, are also found to be used herein. Such promoter sequences are available, for example, from Stratagene (San Diego, CA). Examples of heterologous promoters include, but are not limited to, the CMV promoter. Examples of inducible promoters include, but are not limited to, DNA response elements to ecdysone, tetracycline, and hypoxia and fin. When multiple isoforms are encoded within a single vector, they are preferably, but not necessarily, operably ligated to different regulatory sequences.

[0048] Similarly, the regulatory elements at the 3' end of the coding region, including the 3' untranslated region (3'UTR) and the polyadenylation signal, may originate from an inserted gene or a heterologous source. Certain embodiments utilize a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) as the 3'UTR or rabbit β-globin polyadenylation signal or both. The 3' regulatory element may also include regulatory elements attached to the coding region. For example, it is possible to express multiple LAMP-2 isoforms under the control of a single promoter by placing an internal ribosome entry site (IRES) (see, e.g., Figures 1I and 1M) or a self-cleaving peptide sequence (e.g., picornavirus 2A peptide, see, e.g., Figures 1J and 1N) between the first and second, and the second and third (if any) isoforms. In the case of an IRES, two or three polypeptides are translated. In the case of self-cleaving peptides, the LAMP-2 isoform, along with the intervening self-cleaving peptide, is present in the vector as a single reading frame that is translated as a single polypeptide that is cleaved post-translation or during translation into the substituted LAMP-2 isoform.

[0049] Transcription under the control of a single promoter is also achievable if the vector is constructed to support alternative splicing. In such constructs, the first eight exons of LAMP-2 are spliced ​​together in cDNA, followed by two or three intron-exon pairs, so as to include all two or three alternative exons that give rise to three LAMP-2 isoforms (see, e.g., Figures 1K and 1O). In various embodiments, the intron-exon pairs arise in any order, reflecting or altering the natural order of B, A, C. Native introns are too long to be included in vectors of the size of AAV. In such cases, the intron is cleaved by removing the central sequence while retaining the required 5' and 3' splice sites and splice signals. Typically, it is sufficient to retain only a few bases at the 5' end of the intron and about 100-200 bases at the 3' end of the intron to ensure splicing. Alternatively, heterologous introns can be substituted for native introns.

[0050] AAV expression vectors containing a target DNA molecule bound by AAV ITRs can be constructed by directly inserting a selected sequence into an AAV genome from which the major AAV open reading frame ("ORF") has been excised. Other parts of the AAV genome can also be excised, as long as a sufficient amount of ITR remains to enable replication and packaging functions. Such constructs can be designed using techniques well known in the art. See, for example, U.S. Patent Nos. 5,173,414 and 5,139,941, International Publication Nos. 92 / 01070 (published January 23, 1992) and International Publication Nos. 93 / 03769 (published March 4, 1993), Lebkowski et al., 1988, Vincent et al., 1990, Carter, 1992, Muzyczka, 1992, Kotin, 1994, Shelling and Smith, 1994, and Zhou et al., 1994 (their entire contents are incorporated herein by reference).

[0051] Alternatively, AAV ITRs can be excised from a viral genome or an AAV vector containing it and fused to the 5' and 3' ends of a selected nucleic acid construct present in another vector using standard ligation techniques. AAV vectors containing ITRs are described, for example, in U.S. Patent No. 5,139,941, the full contents of which are incorporated herein by reference. In particular, several AAV vectors available from the American Type Culture Collection (ATCC) under accession numbers 53222, 53223, 53224, 53225, and 53226 are described. Furthermore, chimeric genes can be synthetically constructed to contain AAV ITR sequences aligned to the 5' and 3' ends of one or more selected nucleic acid sequences. Preferred codons can be used for the expression of chimeric gene sequences in mammalian CNS cells. Complete chimeric sequences are constructed from duplicate oligonucleotides prepared by standard methods. For example, this is described in Edge Nature, vol. 292, 1981, page 756; Nambair et al., Science, vol. 223, 1984, page 1299; and Jay et al., J. Biol. Chem. vol. 259, 1984, page 6311, the full contents of each of which are incorporated herein by reference. To produce the AAV virus, an AAV expression vector is introduced into suitable host cells using known techniques such as transfection. Many transfection techniques are commonly known in the art. See, eg, Graham et al, Virology, 52, 456-467, (1973); Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197.Particularly suitable transfection methods include calcium phosphate coprecipitation (Graham et al., 1973), direct microinjection into cultured cells (Capeechi, 1980), electroporation (Shigekawa et al., 1988), liposome-mediated gene transfer (Mannino et al., 1988), lipid-mediated transduction (Felgner et al., 1987, PNAS USA, 84, 21, 7413-17), and nucleic acid delivery using high-speed microprojectiles (Klein et al., 1987, Endocrinology 120:2339-45). The complete contents of each of the aforementioned references are incorporated herein by reference as a whole.

[0052] In one embodiment, the AAV includes, in addition to the nucleic acid sequence encoding the LAMP-2 isoform, an AAV vector backbone having an ITR derived from AAV2, a promoter such as a cytomegalovirus / chicken β-actin hybrid promoter (CAG) consisting of an enhancer and promoter derived from the mouse PGK (phosphoglycerate kinase) gene or a cytomegalovirus immediate gene, a splice donor and an intron derived from the chicken β-actin gene, a splice acceptor derived from rabbit β-globin, or any promoter such as PGK, CAG, MCK, EF-1, or the natural LAMP-2 promoter. In various embodiments, the viral vector is encapsulated in an anionic liposome, for example, as described in U.S. Patent Application Publication No. 2004 / 0284691.

[0053] In various embodiments, the structure of the expression cassette within the vector includes first and second (e.g., 5' and 3') reverse terminal repeats (ITRs) derived from any known AAV serotype or subgroup including scAAV, any known promoter region (e.g., cytomegalovirus (CMV) promoter or chicken β-actin promoter) in or out of the presence of an enhancer element (e.g., CMV enhancer), lysosome-associated protein 2 genes (all known isoforms, e.g., LAMP-2A, LAMP-2B, and LAMP-2C, and all known polymorphisms of these isoforms), and a polyadenylation signal (including, but not limited to, rabbit β-globin).

[0054] Translation of a LAMP-2 isoform gene or combination of various isoform genes delivered by the virus results in the expression of the LAMP-2 protein for that particular isoform, which is then targeted to the lysosomal membrane of the host cell by the host cell's mechanisms. Subsequently, the restoration of LAMP-2 isoform expression and function restores the flow of autophagy (potentially including, but not necessarily, any known form of autophagy such as macroautophagy, mitophagy, chaperone-mediated autophagy, and DNA / RNA autophagy), which is inadequate and causal in Danon patients, allowing for the removal of toxic cellular components from diseased host cells and damaged organelles, thereby improving cellular function and survival. Ultimately, the restoration of LAMP-2 isoform expression helps treat the underlying genetic defect causing Danon disease and alleviates the phenotype and symptoms of the disease. In other autophagy disorders where LAMP-2 may be expressed but not at levels sufficient to create a sufficient autophagy flow and consequently promote normal cell function and survival, delivery of transgenic LAMP-2 isoforms via rAAV vectors results in overexpression of the LAMP-2 protein. This overexpression restores the autophagy flow to near-normal, normal, or above-normal levels. Restoration of the autophagy flow in diseases that cause disruption of normal autophagy alleviates, mitigates, and / or reverses the disease phenotype and symptoms.

[0055] 4. Vector pharmaceutical compositions For example, a pharmaceutical composition is provided for use in preventing or treating a disorder characterized by a deficient autophagy flow (e.g., Danon disease), comprising a therapeutically effective amount of a vector containing a nucleic acid sequence of polynucleotides encoding one or more LAMP-2 isoforms.

[0056] It will be understood that the single dose or total daily dose of the compounds and compositions of the present invention will be determined by the attending physician within the bounds of best medical judgment. A specific therapeutically effective dose level for any particular patient depends on a variety of factors, including the disorder being treated and its severity; the activity of the specific compound used; the specific composition used, the patient's age, weight, general health, sex, and diet; the timing, route of administration, and excretion rate of the specific compound used; the duration of treatment; drugs used in combination with or simultaneously with the specific nucleic acid or polypeptide used; and similar factors well known in the medical field. For example, it is well within the scope of the art to start with a dose of the compound at a level lower than necessary to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. However, the daily dose of the product can vary over a wide range per adult day. The therapeutically effective dose of the vector according to the present invention to be administered, as well as the dose for treating a pathological condition by the number of viral or nonviral particles and / or pharmaceutical compositions described herein, depends on a number of factors, including the patient's age and condition, the severity of the disturbance or disorder, the method and frequency of administration, and the specific peptide used.

[0057] The pharmaceutical composition containing the vector may be in any form suitable for a selected mode of administration, such as intraventricular, intrathoracic, intracoronary, intravenous, intraarterial, intrarenal, intraurethral, ​​epidural, or intramuscular. Gene therapy vectors containing polynucleotides encoding one or more LAMP-2 isoforms may be administered to animals and humans in unit dose forms as a mixture with conventional pharmaceutical carriers, either as a single activator or in combination with other activators.

[0058] In various embodiments, the pharmaceutical composition contains a pharmaceutically acceptable vehicle for an injectable formulation. These may be, in particular, isotonic sterile saline (such as monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, or mixtures thereof), or dry, and in particular optionally lyophilized, composition that can be prepared into an injectable solution by adding sterile water or saline.

[0059] Pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the immediate preparation of sterile injection solutions or dispersions. In all cases, the form must be sterile and liquid. It must be stable under manufacturing and storage conditions and stored against the contaminants of microorganisms such as bacteria and fungi.

[0060] Solutions containing gene therapy vectors as free bases or pharmacologically acceptable salts can be prepared in water appropriately mixed with a surfactant such as hydroxypropyl cellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, and in oil. Under normal storage and use conditions, these preparations contain preservatives to prevent microbial growth.

[0061] Gene therapy vectors can be formulated into neutral or salty compositions. Pharmacovigilant salts include acid addition salts (formed with free amino groups of proteins), which are formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic salts such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, and procaine.

[0062] The carrier may also be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oil. Appropriate fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by the use of a surfactant. Prevention of microbial action can be provided by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. In many cases, it would be preferable to include an isotonic agent, such as sugar or sodium chloride. Sustained absorption of the injectable composition can be provided by the use of absorption-delaying agents, such as aluminum monostearate and gelatin in the composition.

[0063] A sterile injection solution is prepared by incorporating the required amount of active polypeptide in a suitable solvent along with some other components listed above as needed, and then sterilizing by filtration. Generally, dispersions are prepared by incorporating various sterile active ingredients into a sterile vehicle, which contains a basic dispersion medium and other necessary components from the above. In the case of sterile powders for the preparation of sterile injection solutions, preferred preparation methods are vacuum drying and freeze-drying techniques, which produce a powder of the active ingredient + any additional desired components from a solution containing pre-sterilized filtered powder.

[0064] 5. Methods for treating autophagy-related disorders Further provided is a method for preventing, alleviating, improving, reducing, inhibiting, eliminating and / or reversing one or more symptoms of Danon disease or other autophagy disorders in a subject who requires prevention, alleviation, improvement, reduction, inhibition, elimination and / or reversal of such symptoms, comprising administering to the subject a gene therapy vector as described above and herein, for example, an adeno-associated virus (AAV) vector comprising an expression cassette containing a polynucleotide encoding one or more isoforms of lysosome-associated membrane protein 2 (LAMP-2). The vector is delivered to a subject who requires it, thereby causing the polynucleotide encoding one or more LAMP-2 isoforms to be expressed by transduced cells at therapeutically effective levels.

[0065] In one particular embodiment, the vector is AAV9. In another particular embodiment, the vector is AAV8. In yet another particular embodiment, the vector is AAVrH10. In further aspects of these embodiments, they are used specifically for the treatment of Danon disease.

[0066] In various embodiments, the vector is administered via a route selected from the group consisting of intravenous, intraarterial, intracardiac, intracoronary, intramyocardial, intrarenal, intraurethral, ​​epidural, subcutaneous, and intramuscular. In various embodiments, the vector is delivered directly to the myocardium by epicardial injection via cardiothoracic incision, intracoronary injection, endocardial injection, or another type of injection useful in the heart. Further routes of administration may also include topical application of the vector under direct visualization, such as surface cortical application or other atypical applications.

[0067] Viral vectors typically induce an immune response that may include an antibody response that reduces or completely inhibits the effectiveness of a particular vector in an individual, if repeated dosing is necessary or desirable. Repeated dosing may be appropriate, for example, because cell proliferation, particularly with episomal vectors, can lead to the loss of the vector over time. Tissues that are more difficult to access may require multiple doses of the vector to transfect a sufficient number of cells for effective treatment of the disease. The expression of the introduced electron may be lost over time. The need to administer a gene therapy vector in the face of an inhibitory antibody response can be addressed in various ways. For example, antibody titers can be reduced by apheresis before vector administration, or the patient can be immunosuppressed with an appropriate medical regimen. Alternatively, an empty AAV capsid can be administered to bind to host antibodies and immune cells before injecting the therapeutic AAV containing the LAMP-2 transgene. Depending on the target tissue, direct local administration as an alternative to systemic administration may be useful in overcoming or improving the inhibitory effect of anti-vector antibodies. Another approach involves using vectors based on different serotypes of the virus from which it originates. For example, if an AAV9 vector is initially used, and there are problems with anti-AAV antibody titers, but further gene therapy is needed, an AAV8 vector containing the same (or different) genetic constructs can be administered.

[0068] Once prescribed, the solution can be administered in a therapeutically effective amount in a form suitable for the medication prescription. The prescription is readily administered in various dosage forms, such as the injectable solution type described above, but drug-releasing capsules may also be used. Multiple doses are possible.

[0069] Where necessary, the vectors described herein may be formulated in any suitable vehicle for delivery. For example, they may be placed in pharmaceutically acceptable suspensions, solutions, or emulsions. Suitable media include saline and liposome preparations. More specifically, pharmaceutically acceptable carriers may include sterile aqueous solutions of non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include, but are not limited to, water, alcohol / aqueous solutions, emulsions, or suspensions (including saline and buffer media). Intravenous vehicles include fluid and nutritional supplements, electrolyte supplements (such as those based on Ringer's dextrose), etc.

[0070] Preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents, and inert gases, may also be present.

[0071] Colloidal dispersions can also be used for targeted gene delivery. Colloidal dispersions include oil-in-water emulsions, micelles, mixed micelles, and macromolecular complexes containing liposomes, nanocapsules, microspheres, beads, and lipid-based systems.

[0072] The appropriate regimen can be determined by a physician and depends on the subject's age, sex, weight, and stage of the disease. As an example, for the delivery of nucleic acid sequences encoding LAMP-2 polypeptides using a viral expression vector, each unit dose of the LAMP-2 polypeptide expression vector may contain a composition of 2.5 μl to 100 μl, which is pharmaceutically acceptable in a liquid, for example, 10 11 ~10 16 Contains a viral expression vector at a concentration in the range of viral genome / ml.

[0073] Some regimens use a vector containing the gene for a single LAMP-2 isoform, e.g., LAMP-2B. Other regimens use a vector containing the genes for two LAMP-2 isoforms, e.g., LAMP-2B and LAMP-2A. In some embodiments, the two isoforms are held in different vector preparations. In one embodiment, both vector preparations originate from the same vector, e.g., the AAV9 vector or any other specific vector mentioned above. In other embodiments, both polypeptides are encoded in a single vector. See, for example, Figure 1. Similarly, if a regimen involves the administration of nucleic acid sequences encoding all three isoforms, the isoforms can be carried in individual vectors or in a single vector. In regimens using multiple isoforms carried by individual vectors, the multiple vectors can be administered simultaneously, individually, or mixed together, or sequentially at intervals of several hours, days, or weeks.

[0074] 6. Kit As described above and herein, kits comprising gene therapy vectors comprising polynucleotides encoding one or more LAMP-2 isoforms are further provided. In various embodiments, the kit provides one or more unit dosage forms prepared for administration to a subject, for example, gene therapy vectors prepared in pre-filled syringes or ampoules. In various embodiments, the gene therapy vectors are provided in lyophilized form. [Examples]

[0075] (Example 1) (AAV9 gene therapy) The specific vectors consistent with the above description were constructed as AAV9 vectors derived from AAV2, containing an ITR encoding either LAMP-2A or LAMP-2B under the control of a chicken β-actin promoter with a CMV enhancer (CAG promoter). Furthermore, these vectors incorporated woodchuck hepatitis virus post-transcriptional regulatory elements and rabbit β-globin polyadenylation signals (see Figure 1B). These vectors were obtained from the Gene Therapy Resource Program Preclinical Vector Core facility at the National Heart, Lung, and Blood Institute, University of Pennsylvania. The human LAMP-2 sequence was incorporated with the expectation that it would be functional in mouse due to the high degree of sequence identity among congeners, particularly in the C-terminal region, which forms the transmembrane and cytoplasmic tail components of the protein (see Figure 4).

[0076] The vector is used to deliver LAMP-2 isoforms in vivo. The AAV9 serotype exhibits superior tropism to the heart, skeletal muscle, and nervous tissue, which are the organs most affected by Danon disease. However, Danon is a multisystem disorder, and many different organs may potentially be involved. Therefore, expression of transgenic LAMP-2 isoforms is regulated under a constitutively active chicken β-actin (CBA) promoter with a cytomegalovirus (CMV) enhancer (CAG construct). This promoter is ubiquitously active, enabling LAMP-2 transgene expression in all tissues depending on infection efficiency. An AAV9 vector with an enhanced green fluorescent protein (eGFP) gene was also obtained for use as a control reagent.

[0077] (Example 2) (Administration of AAV9LAMP-2 isoform vector to LAMP-2 knockout mice) Lamp-2 KO mice (Nature. 2000 Aug 24; 406(6798): 902-6; Basic Res Cardiol. 2006 Jul; 101(4): 281-91) develop Danon-like syndrome, but the severity of the disease is milder than in humans, and due to the shorter lifespan of the mice, the time it takes for damage caused by abnormal autophagy flow to accumulate is shorter, possibly at least partially. Therefore, it is desirable to wait until the mice are at least 3 months old, preferably 5-6 months old, before initiating treatment, so that there is sufficient accumulation of pathology for which the reversal can be easily detected.

[0078] In the idealized procedure, 6-month-old Lamp-2 KO mice are used, and 5 × 10⁶ AAV vectors are injected. 11 , 1 x 10 12 , or 2 × 10 12 The genome copy (gc) is administered intravenously into the external jugular vein. In various experiments, WT mice are administered the LAMP-2 isoform vector, and Lamp-2 KO mice are administered the eGFP vector as a control. By using a sufficient number of mice, subpopulations can be evaluated by sacrificing them at various time points, such as 1, 2, and 6 months after administration.

[0079] (Example 3) (Evaluation of LAMP-2 isoform gene transcription after vector administration) Lamp-2 KO mice, approximately 3-4 months old, have a 5x10 11 , 1 x 10 12 , and 2 × 10 12 gc / mice were administered escalating doses of vectors AAV9.LAMP-2B and AAV9.LAMP-2A (see Figure 1B and the explanation above). One mouse was sacrificed per condition, and RT-qPCR was performed three times on digested cardiac tissue to evaluate mRNA expression (gene transcription) of the human transgene. Untreated WT mice were shown not to express human LAMP-2 isoforms. This data indicates a dose-dependent increase in human transgene expression (see Figure 5).

[0080] (Example 4) (Evaluation of LAMP-2 Isoform Protein Expression after Vector Administration) Using the same mice as in Example 3, immunoblotting was performed on digested heart tissues to evaluate protein expression of the human transgene compared to GAPDH. Control Lamp-2 KO mice treated with AAV9.EGFP did not show significant LAMP-2 protein expression. The data show a dose-dependent increase in the expression of the human transgene in mice administered the viral vector (see Figure 6).

[0081] (Example 5) (Intracellular Localization of Transgenic LAMP-2B after Vector Administration) 2×10 12 gc / mouse of AAV9.LAMP-2B or AAV9.EGFP was administered, and heart sections of Lamp-2 KO mice sacrificed 1 month after delivery were stained with DAPI (which binds to AT-rich DNA and makes the cell nucleus visible) and a fluorescent-labeled anti-LAMP-2 antibody (see Figure 7). The pattern of LAMP-2 staining indicates the localization of the human transgenic LAMP-2B protein to intracellular vacuoles and is similar to the staining seen in WT mouse controls for mouse Lamp-2 protein. No human LAMP-2 staining was seen in Lamp-2 KO mice receiving the AAV9.EGFP vector control. These data demonstrate that treatment with the AAV9.LAMP-2B vector leads to the expression of human LAMP-2B protein at physiologically appropriate locations.

[0082] Similarly, experimental human LAMP-2 staining was maintained 2 months after delivery of AAV9.LAMP-2B at a dose of 5×10 11 gc / mouse. Consistent with Example 4, less staining was seen at this lower vector administration dose. In comparison, the AAV9.EGFP vector at 5×10 11 gc / mouse did not show human LAMP-2 staining 3 months after delivery. See Figure 8.

[0083] (Example 6) (CAG-RFP-EGFP-LC3B Autophagy Reporter System) The CAG-RFP-EGFP-LC3B autophagy reporter system enables the evaluation of macroautophagy flow. Microtubule-associated protein 1 light chain 3 (LC3) is expressed on the surface of autophagosomes. A gene fusion is constructed to express LC3 fused to both red fluorescent protein (RFP) and eGFP. When this fusion protein is expressed, the RFP component fluoresces in both autophagosomes and lysosomes, while the eGFP component fluoresces in autophagosomes but is quenched by the acidic environment of lysosomes. As a result, in the merged image, autophagosomes appear yellow and lysosomes appear red (see Figure 9A). When expressed in a WT background, separate red and green images show more red dots than green dots, and the merged image shows a mixture of red and yellow dots (see Figures 9B-D'). Lamp-2 is required for the normal fusion of autophagosomes and lysosomes, forming autolysosomes. Therefore, when this construct is expressed in Lamp-2 KO mice, nearly equal numbers of red and green dots are observed in separate red and green images, while in the merged image, almost entirely yellow dots are observed (see Figures 9E-G'). The accumulation of autophagosomes and the near absence of autolysosomes, along with a greater number of autophagy vacuoles (AVs) overall, reflect a defect in the autophagy flow due to the absence of Lamp-2 (see Figure 9H).

[0084] (Example 7) (Administration of a vector containing either LAMP-2A or LAMP-2B restores the flow of autophagy.) Lamp-2 KO mice expressing the CAG-RFP-EGFP-LC3B construct (Lamp-2 KO / CAG-RFP-EGFP-LC3B reporter mice) were administered either AAV9.LAMP-2B or AAV9.LAMP-2A and compared to Lamp-2 KO mice expressing the CAG-RFP-EGFP-LC3B autophagy reporter system (CAG-RFP-EGFP-LC3B reporter mice). One month after vector delivery, the mice were sacrificed, and autophagy flow was assessed by fluorescence microscopy of cardiac sections. Untreated Lamp-2 KO / reporter mice consistently showed almost exclusively yellow autophagy vacuoles, i.e., autophagosomes (see Figure 10B and Figure 9G'). In contrast, Lamp-2 KO / reporter mice administered with either the gene therapy vector AAV9.LAMP-2B or AAV9.LAMP-2A exhibited a similar proportion of red autophagocytic vacuoles, or autolysosomes, as control WT reporter mice (see Figures 10A, 10C, and 11D, where exemplary points are indicated by arrows). Quantification of AV showed that the proportion of immature autophagy relative to mature autolysosomes was similar in treated Lamp-2 KO mice compared to WT mice (see Figure 10E). Thus, gene therapy with either AAV9.LAMP-2B or AAV9.LAMP-2A restored normal autophagosome-lysosome fusion in cardiomyocytes of Lamp-2 KO mice.

[0085] (Example 9) (Restoration of the ultrastructure of cardiomyocytes as evaluated by electron microscopy) Lamp-2 KO mouse, 5x10 11Gc / mouse AAV9.LAMP-2B was intravenously injected and compared to age-matched Lamp-2 KO mice and untreated WT mice. One month after vector delivery, mice were sacrificed and cardiac sections were analyzed by electron microscopy. Untreated Lamp-2 KO mice showed increased AV accumulation and size (see Figure 11B, B', yellow arrows) and an increased number of abnormal mitochondria (see Figure 11B, red arrows) compared to WT mice (see Figure 11A, A'). In contrast, electron micrographs of Lamp-2 KO mice treated with AAV9.LAMP-2B were more similar to the ultrastructure of untreated WT mice (see Figure 11C, C'). Therefore, treatment with gene therapy vectors restores the ultrastructure of cardiomyocytes.

[0086] In summary, these examples demonstrate that gene therapy vectors based on adeno-associated virus encoding LAMP-2A and LAMP-2B isoforms can be administered intravenously to successfully achieve transgene expression in cardiac tissue. Furthermore, such expression leads to the reversal of defects in autophagy flow and cardiomyocyte ultrastructure, defects also associated with Danon disease. These data support the use of such vectors for gene therapy in the treatment of Danon disease and other disorders associated with autophagy flow defects.

[0087] Finally, while aspects of this specification are emphasized by reference to specific embodiments, it should be understood that those skilled in the art will readily grasp that these disclosed embodiments are merely illustrative of the principles of the subject matter disclosed herein. Therefore, it should be understood that the disclosed subject matter is by no means limited to the specific methodologies, protocols, and / or reagents, etc., described herein. Accordingly, various modifications or alternative configurations of the disclosed subject matter can be made in accordance with the teachings herein without departing from the spirit of this specification. Finally, the terms used herein are for the purpose of describing specific embodiments only and do not limit the scope of the invention as defined solely by the claims. Therefore, the invention is not strictly limited to what is illustrated and described herein.

[0088] Specific embodiments of the Invention, including the best mode known to the inventors for carrying out the Invention, are described herein. Naturally, variations of these described embodiments will be apparent to those skilled in the art by reading the above description. The inventors expect that those skilled in the art will appropriately use such variations, and the inventors intend to carry out the Invention in ways other than those specifically described herein. Accordingly, the Invention includes all modifications and equivalents of the subject matter described in the appended claims, as permitted by applicable law. Furthermore, any combination of the above embodiments in all possible variations is incorporated into the Invention, unless otherwise indicated herein or unless it is clearly inconsistent with the context.

[0089] The grouping of alternative embodiments, elements, or steps of the present invention should not be construed as limiting. Each group member may be referenced and claimed individually or in any combination with other group members disclosed herein. For convenience and / or patentability reasons, it is anticipated that one or more group members may be included in or removed from a group. In the event of such inclusion or omission, the specification shall be deemed to include the modified groups and thus satisfy the description of all Markush groups used in the appended claims.

[0090] Unless otherwise indicated, all numbers representing features, items, quantities, parameters, properties, terms, etc., used herein and in the claims should be understood in all cases as being modified by the term “approximately.” Where used herein, the term “approximately” means that the characteristic, item, quantity, parameter, property, or term thus characterized encompasses ±10% of the stated value of the characteristic, item, quantity, parameter, property, or term. Therefore, unless otherwise indicated, numerical parameters described herein and in the appended claims are variable approximations. At a minimum, each numerical representation should be interpreted using at least the reported number of significant figures and common rounding techniques, not as an attempt to limit the scope of the claims or the application of the doctrine of equivalents. Although the numerical ranges and figures representing the broad scope of the invention are approximations, the numerical ranges and figures shown in specific embodiments are reported as accurately as possible. However, numerical ranges or figures inherently include certain errors that inevitably arise from the standard deviation observed in each test measurement. The enumeration of numerical ranges herein is merely intended to serve as a convenient way to individually refer to each distinct numerical within that range. Unless otherwise indicated herein, individual values ​​within a numerical range are incorporated herein as if they were individually listed herein.

[0091] In the context describing the present invention (particularly in the context of the appended claims), terms such as “a,” “an,” “the,” and similar reference subjects should be interpreted as encompassing both singular and plural forms. Unless otherwise indicated herein, or unless clearly inconsistent with the context, all methods described herein may be performed in any suitable order, unless otherwise indicated herein, or unless clearly inconsistent with the context. Any and all examples or illustrative language provided herein (e.g., “etc.”) are intended merely to better illustrate the present invention and not to limit the scope of the present invention as otherwise claimed. No word herein should be interpreted as indicating an unclaimed element essential to the practice of the present invention.

[0092] The specific embodiments disclosed herein are further limited in claims using terms composed of or essentially consisting of language. Where used in claims, the transitional term "consisting of" excludes elements, steps, or components not described in the claims, whether or not they are described in the claims. The transitional term "essentially consisting of" limits the claims to specific materials or steps and those that do not materially affect the fundamental and novel properties. Embodiments of the invention as thus claimed are essentially or explicitly described and enabled.

[0093] All patents, patent publications, and other publications referenced and identified herein may be used individually, in whole, in connection with the present invention, for example, to describe and disclose compositions and methodologies described in such publications. These publications are provided solely for disclosure prior to the filing date of this application. In this regard, this should not be construed as an acknowledgment that the inventor is not entitled to precede such disclosures, either because of prior inventions or for any other reason. All statements relating to dates or expressions concerning the contents of these documents are based on information available to the applicant and do not constitute an acknowledgment of the accuracy of the dates or contents of these documents.

[0094] (Note) (Note 1) A gene therapy vector comprising an expression cassette containing a polynucleotide encoding one or more isoforms of lysosome-associated membrane protein 2 (LAMP-2).

[0095] (Note 2) The aforementioned vector is a viral vector. A gene therapy vector as described in Appendix 1, characterized by the features described herein.

[0096] (Note 3) The aforementioned viral vector is derived from a virus selected from the group consisting of adenoviruses, retroviruses, lentiviruses, herpesviruses, and adeno-associated viruses (AAVs). A gene therapy vector as described in Appendix 1 or 2, characterized by the above.

[0097] (Note 4) The vector is derived from one or more adeno-associated virus (AAV) serotypes 1-11 or any subgroup thereof. A gene therapy vector as described in Appendix 3, characterized by the features described herein.

[0098] (Note 5) The aforementioned viral vector is encapsulated in an anionic liposome. A gene therapy vector characterized by any one of the appendices 2 to 4.

[0099] (Note 6) The aforementioned vector is a non-viral vector. A gene therapy vector as described in Appendix 1, characterized by the features described herein.

[0100] (Note 7) The non-viral vector is selected from the group consisting of naked DNA, cationic liposome complexes, cationic polymer complexes, cationic liposome-polymer complexes, and exosomes. A gene therapy vector as described in Appendix 6, characterized by the features described herein.

[0101] (Note 8) The expression cassette comprises a first reverse-terminal repeat sequence, an enhancer / promoter region, a polynucleotide encoding one or more isoforms of LAMP-2, a 3' untranslated region containing a polyadenylation signal, and a second reverse-terminal repeat sequence, all operably linked in the 5' to 3' direction. A gene therapy vector characterized by any one of the appendices 1 to 7.

[0102] (Note 9) The promoter is selected from the group consisting of cytomegalovirus (CMV) promoters and CAG promoters. A gene therapy vector as described in Appendix 8, characterized by the features described therein.

[0103] (Note 10) The aforementioned polynucleotide includes DNA or cDNA. A gene therapy vector characterized by any one of the appendices 1 to 9.

[0104] (Note 11) The polynucleotide encoding one or more isoforms of LAMP-2 comprises one or more human LAMP-2 isoforms. A gene therapy vector characterized by any one of the appendices 1 to 10.

[0105] (Note 12) The polynucleotide encoding one or more isoforms of LAMP-2 comprises one or more LAMP-2 isoforms selected from the group consisting of LAMP-2A, LAMP-2B, and LAMP-2C. A gene therapy vector characterized by any one of the appendices 1 to 11.

[0106] (Note 13) The polynucleotides encoding one or more isoforms of LAMP-2 have at least about 90% sequence identity with one or more of SEQ ID NOs: 1, 2, and 3. A gene therapy vector characterized by any one of the appendices 1 to 12.

[0107] (Note 14) The polynucleotide encoding one or more isoforms of LAMP-2 includes one or more of SEQ ID NOs: 1, 2, and 3. A gene therapy vector as described in Appendix 13, characterized by the features described herein.

[0108] (Note 15) This includes administering a gene therapy vector described in any one of the appendices 1 to 14 to a subject. A method for preventing, alleviating, improving, reducing, inhibiting, and / or reversing one or more symptoms of Danon disease or other autophagy disorders in subjects who require prevention, alleviation, improvement, reduction, inhibition, elimination, and / or reversal of such symptoms.

[0109] (Note 16) The procedure involves administering to a subject an adeno-associated virus (AAV) vector containing an expression cassette comprising a polynucleotide encoding one or more isoforms of lysosome-associated membrane protein 2 (LAMP-2), A method for preventing, alleviating, improving, reducing, inhibiting, and / or reversing one or more symptoms of Danon disease or other autophagy disorders in subjects who require prevention, alleviation, improvement, reduction, inhibition, elimination, and / or reversal of such symptoms.

[0110] (Note 17) The vector is administered via a route selected from the group consisting of intravenous, intraarterial, intracardiac, intracoronary, intramyocardial, intrarenal, intraurethral, ​​epidural, and intramuscular. The method described in Appendix 15 or 16, characterized by the features described herein.

[0111] (Note 18) The vector is administered multiple times. A method according to any one of the appendices 15 to 17, characterized by the following:

[0112] (Note 19) The autophagy impairment is selected from the group consisting of end-stage heart failure, myocardial infarction, drug toxicity, diabetes, end-stage renal failure, and aging. The method according to any one of the appendices 15 to 18, characterized by the following:

[0113] (Note 20) The subject is a human being. The method according to any one of the appendices 15 to 19, characterized by the following:

[0114] (Note 21) The aforementioned subjects exhibit symptoms of Danon disease or other autophagy disorders. A method according to any one of the appendices 15 to 20, characterized by the following:

[0115] (Note 22) The aforementioned subjects have been identified as having reduced or undetectable LAMP-2 expression. A method according to any one of the appendices 15 to 21, characterized by the following:

[0116] (Note 23) The aforementioned subject has been identified as having a mutated LAMP-2 gene. A method according to any one of the appendices 15 to 22, characterized by the following:

Claims

[Claim 1] A gene therapy vector comprising an expression cassette containing a polynucleotide encoding one or more isoforms of lysosome-associated membrane protein 2 (LAMP-2).

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