Pharmaceutical Composition

Abstract

To provide methods for the treatment of Danon disease and other autophagy disorders.SOLUTION: This disclosure provides gene therapy vectors that comprise an expression cassette comprising a polynucleotide encoding one or more isoforms of lysosome-associated membrane protein 2 (LAMP-2), and methods of using such gene therapy vectors for the treatment of Danon disease and other autophagy disorders.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

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

[0002] This work was supported in part by Grant Nos. PHS 7K23HL107755 and RSA 1268 from the National Institutes of Health. The U.S. Government has 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). Emerging evidence highlights the importance of autophagy in regulating the biology, function, and survival of cardiomyocytes. However, the mechanisms involved in cellular dysfunction and death in cardiomyocytes with impaired autophagic flux remain unclear. In a previous study, we generated human induced pluripotent stem cells (hiPSCs) from two patients with different LAMP-2 mutations to investigate the molecular mechanisms underlying Danon disease (Non-Patent Document 1). Danon hiPSC-derived cardiomyocytes (hiPSC-CMs) exhibited impaired autophagic flux and key features of heart failure, such as increased cell size, increased expression of natriuretic peptides, and abnormal calcium handling, compared with control hiPSC-CMs. Furthermore, Danon hiPSC-CMs exhibited excessive amounts of mitochondrial oxidative stress and apoptosis. Using the sulfhydryl antioxidant N-acetylcysteine ​​to scavenge free radicals significantly reduced apoptotic cell death in Danone hiPSC-CMs. We also used a lentiviral vector to introduce the coding sequence for the LAMP-2B isoform under the control of a doxycycline-inducible promoter in one of the Danone hiPSC lines. Overexpression of LAMP-2B by 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 hiPSC-CMs derived from patients with LAMP-2 mutations and gained mechanistic insights into the pathogenesis of this disease. We demonstrated that LAMP-2 deficiency leads to impaired autophagic flux, which causes excessive oxidative stress and subsequent cardiomyocyte apoptosis. Scavenging excess free radicals with antioxidants and overexpressing LAMP-2B ameliorated the disease phenotype in vitro.The prior art has not disclosed an effective treatment strategy for Danon disease or other disorders associated with autophagy, and 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 Summary of the Invention [Means for solving the problem]

[0005] In one embodiment, a gene therapy vector is provided for use in, for example, systemically or locally increasing the expression of one or more isoforms of lysosomal-associated membrane protein 2 (LAMP-2) in a subject. The gene therapy vector finds use in preventing, alleviating, ameliorating, reducing, inhibiting, and / or treating one or more symptoms of Danon disease or other disorders of insufficient autophagic flux. In various embodiments, the gene therapy vector comprises an expression cassette comprising 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 adenovirus, retrovirus, lentivirus, herpesvirus, and adeno-associated virus (AAV). In various forms, the vector is derived from one or more of adeno-associated virus (AAV) serotypes 1-11 or any subgroup thereof. In various embodiments, the viral vector is encapsulated in anionic liposomes. In various embodiments, the vector is a non-viral vector. In various embodiments, 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. In various forms, the expression cassette comprises, operably linked in a 5' to 3' direction (from the perspective of the transcribed mRNA), a first inverted terminal repeat, an enhancer, a promoter, a polynucleotide encoding one or more isoforms of LAMP-2, a 3' untranslated region, a polyadenylation (polyA) signal, and a second inverted terminal repeat. In various embodiments, the promoter is selected from the group consisting of a cytomegalovirus (CMV) promoter and a chicken β-actin (CAG) promoter. In various embodiments, the polynucleotide comprises DNA or cDNA. In various embodiments, the polynucleotide encoding one or more isoforms of LAMP-2 comprises one or more human LAMP-2 isoforms.In various embodiments, 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. In various embodiments, the polynucleotide encoding one or more isoforms of LAMP-2 has at least about 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity, to one or more of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. In various embodiments, the polynucleotide encoding one or more isoforms of LAMP-2 comprises one or more of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.

[0006] In a further embodiment, a method is provided for preventing, alleviating, ameliorating, reducing, inhibiting, eliminating, and / or reversing one or more symptoms of Danon disease or other autophagy disorders in a subject in need thereof, comprising administering to the subject a gene therapy vector as described above or herein (see, e.g., Figure 2). In a further embodiment, a method is provided for preventing, alleviating, ameliorating, reducing, inhibiting, eliminating, and / or reversing one or more symptoms of Danon disease or other autophagy disorders in a subject in need thereof, comprising administering to the subject an adeno-associated virus (AAV) vector comprising an expression cassette comprising 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 bombardment, hydrodynamic injection, electroporation, sonoporation, laser irradiation, and magnetofection. In various embodiments, the vector is administered multiple times, with or without patient immunosuppression or plasma exchange. 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 disorders. 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 cardiac disorder with multisystemic clinical manifestations. Danon disease mutations result in the absence of lysosomal-associated membrane protein 2 (LAMP-2) protein expression. Primary clinical features include skeletal and cardiomyopathy, cardiac conduction abnormalities, cognitive impairment, and retinal disease. Males are typically affected earlier and more severely than females.

[0008] The terms "lysosome-associated membrane protein 2" and "LAMP-2" refer interchangeably to (1) the amino acid sequence encoded by a LAMP-2 nucleic acid (see, e.g., GenBank Accession Nos. NM_002294.2 (isoform A), NM_013995.2 (isoform B), NM_001122606.1 (isoform C)) or the amino acid sequence of a LAMP-2 polypeptide (see, e.g., GenBank Accession Nos. NP_002285.1 (isoform A), NP_054701 (2) an amino acid sequence having greater than about 90%, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more amino acid sequence identity with a LAMP-2 polypeptide (e.g., a LAMP-2 polypeptide described herein) or a LAMP-2 polypeptide (e.g., a LAMP-2 polypeptide described herein) over a region or entire length of at least about 25, 50, 100, 200, 300, 400, or more amino acids, respectively, to a LAMP-2 polypeptide (e.g., a LAMP-2 polypeptide described herein) or a LAMP-2 polypeptide (e.g., a LAMP-2 polypeptide described herein) or a LAMP-2 polypeptide (e.g., a LAMP-2 polypeptide described herein) or a LAMP-2 polypeptide (e.g., a LAMP-2 polypeptide described herein) or a LAMP-2 polypeptide (e.g., a LAMP-2 polypeptide described herein) over a region or entire length of at least about 25, 50, 100, 200, 300, 400, or more amino acids, respectively, and / or over a region or entire length of at least about 25, 50, 100, 200, 300, 400, or more amino acids, respectively, to a LAMP-2 polypeptide (e.g., a LAMP-2 polypeptide described herein) or ... (3) specifically hybridize under stringent hybridization conditions to the antisense strand corresponding to the nucleic acid sequence encoding the LAMP-2 protein and conservatively modified variants thereof; and (4) have a nucleic acid and polypeptide polymorphic variants, alleles, mutants, and interspecies homologs that have a nucleic acid sequence that has greater than about 90%, preferably about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more nucleic acid sequence identity with a LAMP-2 nucleic acid (e.g., a LAMP-2 polynucleotide as described herein, a LAMP-2 polynucleotide encoding a LAMP-2 polypeptide as described herein), preferably over a region or entire length of at least about 25, 50, 100, 200, 500, 1000, 2000 or more nucleic acids.

[0009] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics 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 mimetics 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-carboxyglutamate, and O-phosphoserine. An amino acid analog refers to a compound that has the same basic chemical structure as a naturally occurring amino acid, i.e., an α-carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, or methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or altered peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. An amino acid mimetic refers to a compound that has a structure that differs from the general chemical structure of an amino acid but functions similarly to a naturally occurring amino acid.

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

[0012] The term "conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, conservatively modified variants refer to nucleic acids encoding identical or essentially identical amino acid sequences, or, if the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," a type of conservatively modified variation. All nucleic acid sequences herein that encode a polypeptide also describe all possible silent variations of the nucleic acid. Those skilled in the art will recognize that each codon in a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be modified to obtain a functionally identical molecule. Thus, each silent variation of a nucleic acid encoding a polypeptide is implicit in each described sequence.

[0013] With respect to amino acid sequences, those skilled in the art will recognize that individual substitutions, deletions, or additions to nucleic acid, peptide, polypeptide, 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," where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to, and do not exclude, polymorphic variants, interspecies homologs, and alleles of the present invention.

[0014] Each of the following eight groups contains amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G), 2) Aspartic acid (D), glutamic acid (E), 3) Asparagine (N), Glutamine (Q), 4) Arginine 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 deoxyribonucleotide 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 "bp"), nucleotides ("nt"), or kilobases ("kb"). Where the context permits, 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 pairs." Those skilled in the art will recognize that the two strands of a double-stranded polynucleotide may differ slightly in length and that their ends may be staggered as a result of enzymatic cleavage. Therefore, not all nucleotides within a double-stranded polynucleotide molecule may be paired.

[0016] The term "identical" or percent "identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that, when compared and aligned for maximum correspondence over a comparison window, are the same or have the same percentage of amino acid residues or nucleotides (i.e., share at least about 80% identity over a specified region, e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with a reference sequence (e.g., a LAMP-2 polynucleotide or polypeptide sequence described herein), or a specified region, as measured using 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 a test sequence. Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length, for example, over a region that is 50, 100, 200, 300, 400 amino acids or nucleotides in length, or over the entire length of the reference sequence.

[0017] For sequence comparison, typically, one sequence serves as the reference sequence with which the test sequence is compared.When using sequence comparison algorithm, test and reference sequences are input into computer, and partial sequence coordinates are designated as necessary, and sequence algorithm program parameters are designated.Default program parameters can be used, or alternative parameters can be designated.The sequence comparison algorithm then calculates the percent sequence identity of the test sequence to the reference sequence based on program parameters.For the sequence comparison of nucleic acid and protein with LAMP-2 nucleic acid and protein, BLAST and BLAST 2.0 algorithms and default parameters are used.

[0018] As used herein, a "comparison window" refers to any segment of contiguous positions selected from the group consisting of 20 to 600, typically about 50 to about 200, and more typically about 100 to about 150, over which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed, 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 search for similarity 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 suitable algorithms 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. 25:3389-3402 (1977), respectively. Software for performing BLAST analyses is publicly available from the National Center for Biotechnology Information (on the World Wide Web at ncbi.nlm.nih.gov / ).

[0019] An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid cross-reacts immunologically with the antibody produced against the polypeptide encoded by the second nucleic acid, as described below.Therefore, a polypeptide is typically substantially identical to a second polypeptide, for example, the two peptides differ only by conservative 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.Another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequences.

[0020] As used herein, "administering" refers to local and systemic administration, including, for example, enteral, parenteral, pulmonary, and topical / transdermal administration. Routes of administration of compounds (e.g., polynucleotides encoding one or more LAMP-2 isoforms) that find use in the methods described herein include, for example, oral (oral (PO) administration), nasal, or inhalation administration, administration as a suppository, topical contact, transdermal delivery (e.g., via a transdermal patch), intrathecal (IT) administration, intravenous ("iv") administration, intraperitoneal ("ip") administration, intramuscular ("im") administration, intralesional administration, or subcutaneous ("sc") administration, or implantation of a sustained-release device, such as a mini-osmotic pump, depot formulation, or the like, into a subject. Administration can be 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, iontophoretic, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, and the like.

[0021] The terms "systemic administration" and "systemic administration" 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, including a targeted site of pharmaceutical action. Systemic administration includes, but is not limited to, oral, intranasal, rectal, and parenteral (e.g., intramuscular, intravenous, intraarterial, transdermal, and subcutaneous, other than via the digestive tract) administration.

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

[0023] The term "effective amount" or "pharmaceutically effective amount" refers to the amount and / or dosage and / or administration regimen of one or more compounds (e.g., gene therapy vectors) required to produce a desired result (e.g., resulting in increased expression of one or more LAMP-2 isoforms in an amount sufficient to ultimately reduce the severity of a disease characterized by impaired or defective autophagy (e.g., Danon disease)).

[0024] The term "administering" refers to the action taken by a medical professional (e.g., a physician) or someone controlling the medical care of a subject, to control and / or authorize the administration of an agent / compound of interest to a subject. Administering can include diagnosing and / or determining an appropriate therapeutic or prophylactic regimen and / or prescribing a particular agent / compound to a subject. Such prescribing can include, for example, writing the prescription, annotating the medical record, etc.

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

[0026] As used herein, the terms "treat" and "treatment" refer to delaying the onset of, slowing or reversing the progression of, reducing the severity of, or alleviating or preventing the disease or condition to which the term applies, or one or more symptoms of such disease or condition.

[0027] The term "alleviating" refers to the reduction or elimination of one or more symptoms of the condition or disease, and / or the slowing or delaying of the onset or severity of one or more symptoms of the condition or disease, and / or the prevention of the condition or disease. In certain embodiments, the reduction or elimination of one or more symptoms of the condition or disease can 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 "consisting essentially of" refers to the genus or species of active agent(s) recited in the method or composition, and may further include other agents that do not themselves have substantial activity for the recited indication or purpose.

[0029] The terms "subject," "individual," and "patient" refer interchangeably to a mammal, preferably a human or non-human primate, but also to domestic animals (e.g., dogs or cats), laboratory mammals (e.g., mice, rats, rabbits, hamsters, guinea pigs), and agricultural mammals (e.g., horses, cows, pigs, sheep). In various embodiments, a subject may be a human (e.g., an adult male, adult female, adolescent male, adolescent female, boy, girl) under the care of a physician or other medical practitioner in a hospital, psychiatric care facility, outpatient, or other clinical setting. In certain embodiments, a subject may not be under the medical care or prescription of a physician or other practitioner.

[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 transferred DNA, expression of an extrachromosomal replicating and transcribing replicon (e.g., episome), or integration of the transferred genetic material into the genomic DNA of the host cell.

[0031] "AAV vector" refers to a vector derived from an adeno-associated virus serotype, including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, etc. An AAV vector can have one or more AAV wild-type genes (e.g., the rep gene and / or the cap gene) deleted in whole or in part, but retain functional flanking inverted terminal repeat (ITR) sequences. Functional ITR sequences are necessary for AAV viral rescue, replication, and packaging. Thus, an AAV vector is defined herein as containing at least the sequences required in cis for viral replication and packaging (e.g., functional ITRs). The ITRs do not need to be wild-type nucleotide sequences and can be altered, for example, by the insertion, deletion, or substitution of nucleotides, as long as the sequences provide functional rescue, replication, and packaging. AAV expression vectors are constructed using known techniques to provide control elements including at least a transcription initiation region, a 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 drawings]

[0032] [Figure 1A]Figure 1 shows a schematic diagram of an adeno-associated virus LAMP-2 gene delivery construct. The lysosome-associated membrane protein (LAMP) LAMP-2 coding region shown should generally be understood to include an upstream ribosome binding sequence and start codon, although in some embodiments, the native elements can be replaced with heterologous elements. An upstream ribosome binding site is not used in combination with an IRES or the coding region downstream of a self-cleaving peptide. A start codon is not necessarily present in the coding region downstream of a 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: inverted terminal repeat, LAMP-2: lysosome-associated membrane protein type 2, UTR: untranslated region, Poly A: polyadenylation signal, CAG: promoter region including the CMV enhancer and CBA promoter sequences, CMV: cytomegalovirus, CBA: chicken beta-actin, WPRE: woodchuck hepatitis virus post-transcriptional regulatory element, RBG: rabbit beta-globin polyadenylation signal, EF-1: human elongation factor-1, IRES: internal ribosome entry site, P2A: 2A peptide. Figure 1A shows a schematic diagram of the construct containing the protein coding information for one LAMP-2 isoform—A, B, or C—with common 5' and 3' regulatory regions. [Figure 1B]Figure 1 shows a schematic diagram of an adeno-associated virus LAMP-2 gene delivery construct. The lysosome-associated membrane protein (LAMP) LAMP-2 coding region shown should generally be understood to include an upstream ribosome binding sequence and start codon, although in some embodiments, the native elements can be replaced with heterologous elements. An upstream ribosome binding site is not used in combination with an IRES or the coding region downstream of a self-cleaving peptide. A start codon is not necessarily present in the coding region downstream of a 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: inverted terminal repeat, LAMP-2: lysosome-associated membrane protein type 2, UTR: untranslated region, Poly A: polyadenylation signal, CAG: promoter region including the CMV enhancer and CBA promoter sequence, 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 a schematic diagram of the construct of some embodiments, used in the examples below, consisting of 5' and 3' inverted terminal repeat elements, the CAG promoter region including the CMV enhancer and CBA promoter sequence, the CBA intron, the coding sequence for one of the LAMP-2 isoforms including the upstream ribosome binding sequence and start codon, the WPRE sequence as the 3'UTR, and the rabbit β-globin polyadenylation signal. [Figure 1C]Figure 1 shows a schematic diagram of an adeno-associated virus LAMP-2 gene delivery construct. The lysosome-associated membrane protein (LAMP) LAMP-2 coding region shown should generally be understood to include an upstream ribosome binding sequence and start codon, although in some embodiments, the native elements can be replaced with heterologous elements. An upstream ribosome binding site is not used in combination with an IRES or the coding region downstream of a self-cleaving peptide. A start codon is not necessarily present in the coding region downstream of a 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: inverted terminal repeats; LAMP-2: lysosome-associated membrane protein type 2; UTR: untranslated region; Poly A: polyadenylation signal; CAG: promoter region including the CMV enhancer and CBA promoter sequences; CMV: cytomegalovirus; CBA: chicken beta-actin; WPRE: woodchuck hepatitis virus post-transcriptional regulatory element; RBG: rabbit beta-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 cellular signals that would typically result in LAMP-2 expression in normal cells. [Figure 1D]Figure 1 shows a schematic diagram of an adeno-associated virus LAMP-2 gene delivery construct. The lysosome-associated membrane protein (LAMP) LAMP-2 coding region shown should generally be understood to include an upstream ribosome binding sequence and start codon, although in some embodiments, the native elements can be replaced with heterologous elements. An upstream ribosome binding site is not used in combination with an IRES or the coding region downstream of a self-cleaving peptide. A start codon is not necessarily present in the coding region downstream of a 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: inverted terminal repeats; LAMP-2: lysosome-associated membrane protein type 2; UTR: untranslated region; Poly A: polyadenylation signal; CAG: promoter region including the CMV enhancer and CBA promoter sequences; CMV: cytomegalovirus; CBA: chicken beta-actin; WPRE: woodchuck hepatitis virus post-transcriptional regulatory element; RBG: rabbit beta-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 in place of EF-1α. [Figure 1E]Figure 1 shows a schematic diagram of an adeno-associated virus LAMP-2 gene delivery construct. The lysosome-associated membrane protein (LAMP) LAMP-2 coding region shown should generally be understood to include an upstream ribosome binding sequence and start codon, although in some embodiments, the native elements can be replaced with heterologous elements. An upstream ribosome binding site is not used in combination with an IRES or the coding region downstream of a self-cleaving peptide. A start codon is not necessarily present in the coding region downstream of a 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: inverted terminal repeats; LAMP-2: lysosome-associated membrane protein type 2; UTR: untranslated region; Poly A: polyadenylation signal; CAG: promoter region including the CMV enhancer and CBA promoter sequences; CMV: cytomegalovirus; CBA: chicken beta-actin; WPRE: woodchuck hepatitis virus post-transcriptional regulatory element; RBG: rabbit beta-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 containing a cardiac-specific promoter, such as, but not limited to, the cardiac troponin T2 promoter. In some embodiments, this construct is used to express a transgene only in cardiac tissue (and, for example, to avoid liver expression). [Figure 1F]Figure 1 shows a schematic diagram of an adeno-associated virus LAMP-2 gene delivery construct. The lysosome-associated membrane protein (LAMP) LAMP-2 coding region shown should generally be understood to include an upstream ribosome binding sequence and start codon, although in some embodiments, the native elements can be replaced with heterologous elements. An upstream ribosome binding site is not used in combination with an IRES or the coding region downstream of a self-cleaving peptide. A start codon is not necessarily present in the coding region downstream of a 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: inverted terminal repeats; LAMP-2: lysosome-associated membrane protein type 2; UTR: untranslated region; Poly A: polyadenylation signal; CAG: promoter region including the CMV enhancer and CBA promoter sequences; CMV: cytomegalovirus; CBA: chicken beta-actin; WPRE: woodchuck hepatitis virus post-transcriptional regulatory element; RBG: rabbit beta-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 containing a muscle-specific promoter, such as, but not limited to, the creatinine muscle kinase promoter. In some embodiments, this construct is used to express a transgene in cardiac and skeletal muscle (and to avoid expression in excess of muscle). [Figure 1G]Figure 1 shows a schematic diagram of an adeno-associated virus LAMP-2 gene delivery construct. The lysosome-associated membrane protein (LAMP) LAMP-2 coding region shown should generally be understood to include an upstream ribosome binding sequence and start codon, although in some embodiments, the native elements can be replaced with heterologous elements. An upstream ribosome binding site is not used in combination with an IRES or the coding region downstream of a self-cleaving peptide. A start codon is not necessarily present in the coding region downstream of a 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: inverted terminal repeats; LAMP-2: lysosome-associated membrane protein type 2; UTR: untranslated region; Poly A: polyadenylation signal; CAG: promoter region including the CMV enhancer and CBA promoter sequences; CMV: cytomegalovirus; CBA: chicken beta-actin; WPRE: woodchuck hepatitis virus post-transcriptional regulatory element; RBG: rabbit beta-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 under the control of different promoter regions (using any potential combination). 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 an adeno-associated virus LAMP-2 gene delivery construct. The lysosome-associated membrane protein (LAMP) LAMP-2 coding region shown should generally be understood to include an upstream ribosome binding sequence and start codon, although in some embodiments, the native elements can be replaced with heterologous elements. An upstream ribosome binding site is not used in combination with an IRES or the coding region downstream of a self-cleaving peptide. A start codon is not necessarily present in the coding region downstream of a 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: inverted terminal repeat, LAMP-2: lysosome-associated membrane protein type 2, UTR: untranslated region, Poly A: polyadenylation signal, CAG: promoter region including the CMV enhancer and CBA promoter sequences, CMV: cytomegalovirus, CBA: chicken beta-actin, WPRE: woodchuck hepatitis virus post-transcriptional regulatory element, RBG: rabbit beta-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 (+) orientation on a single-stranded AAV genome, and the other isoform is encoded in the (-) orientation. 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 an adeno-associated virus LAMP-2 gene delivery construct. The lysosome-associated membrane protein (LAMP) LAMP-2 coding region shown should generally be understood to include an upstream ribosome binding sequence and start codon, although in some embodiments, the native elements can be replaced with heterologous elements. An upstream ribosome binding site is not used in combination with an IRES or the coding region downstream of a self-cleaving peptide. A start codon is not necessarily present in the coding region downstream of a 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: inverted terminal repeat, LAMP-2: lysosome-associated membrane protein type 2, UTR: untranslated region, Poly A: polyadenylation signal, CAG: promoter region including the CMV enhancer and CBA promoter sequence, CMV: cytomegalovirus, CBA: chicken beta-actin, WPRE: woodchuck hepatitis virus post-transcriptional regulatory element, RBG: rabbit beta-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 their respective promoter regions and internal ribosome entry sites, followed by a 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 an adeno-associated virus LAMP-2 gene delivery construct. The lysosome-associated membrane protein (LAMP) LAMP-2 coding region shown should generally be understood to include an upstream ribosome binding sequence and start codon, although in some embodiments, the native elements can be replaced with heterologous elements. An upstream ribosome binding site is not used in combination with an IRES or the coding region downstream of a self-cleaving peptide. A start codon is not necessarily present in the coding region downstream of a 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: inverted terminal repeat, LAMP-2: lysosome-associated membrane protein type 2, UTR: untranslated region, Poly A: polyadenylation signal, CAG: promoter region including the CMV enhancer and CBA promoter sequences, CMV: cytomegalovirus, CBA: chicken beta-actin, WPRE: woodchuck hepatitis virus post-transcriptional regulatory element, RBG: rabbit beta-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 the coding sequences for two LAMP-2 isoforms (using any potential combination) separated by a P2A cleavage site. In some embodiments, this construct is used to express mRNAs 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 an adeno-associated virus LAMP-2 gene delivery construct. The lysosome-associated membrane protein (LAMP) LAMP-2 coding region shown should generally be understood to include an upstream ribosome binding sequence and start codon, although in some embodiments, the native elements can be replaced with heterologous elements. An upstream ribosome binding site is not used in combination with an IRES or the coding region downstream of a self-cleaving peptide. A start codon is not necessarily present in the coding region downstream of a 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: inverted terminal repeat, LAMP-2: lysosome-associated membrane protein type 2, UTR: untranslated region, Poly A: polyadenylation signal, CAG: promoter region including CMV enhancer and CBA promoter sequence, CMV: cytomegalovirus, CBA: chicken beta-actin, WPRE: woodchuck hepatitis virus post-transcriptional regulatory element, RBG: rabbit beta-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 sequence of LAMP-2 exons 1-8 followed by an intron region (including all necessary splice signals), exon 9 coding sequence for one of the LAMP-2 isoforms with its 3' UTR and poly A signal, a second intron region (including all necessary splice signals), and exon 9 coding sequence for different LAMP-2 isoforms (in any potential combination) with their 3' UTR and poly A signal. In some embodiments, this construct is used to express mRNAs for two different LAMP-2 isoforms using the same viral genome via alternative splicing, a mechanism that generates different LAMP-2 mRNAs in normal cells. [Figure 1L]Figure 1 shows a schematic diagram of an adeno-associated virus LAMP-2 gene delivery construct. The lysosome-associated membrane protein (LAMP) LAMP-2 coding region shown should generally be understood to include an upstream ribosome binding sequence and start codon, although in some embodiments, the native elements can be replaced with heterologous elements. An upstream ribosome binding site is not used in combination with an IRES or the coding region downstream of a self-cleaving peptide. A start codon is not necessarily present in the coding region downstream of a 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: inverted terminal repeat, LAMP-2: lysosome-associated membrane protein type 2, UTR: untranslated region, Poly A: polyadenylation signal, CAG: promoter region including the CMV enhancer and CBA promoter sequences, CMV: cytomegalovirus, CBA: chicken beta-actin, WPRE: woodchuck hepatitis virus post-transcriptional regulatory element, RBG: rabbit beta-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 under the control of different promoter regions, with 3' UTRs and poly A signals. In some embodiments, this construct is used to express all three of the different LAMP-2 isoforms using the same viral genome, allowing for restoration of all potential LAMP-2 functions. [Figure 1M]Figure 1 shows a schematic diagram of an adeno-associated virus LAMP-2 gene delivery construct. The lysosome-associated membrane protein (LAMP) LAMP-2 coding region shown should generally be understood to include an upstream ribosome binding sequence and start codon, although in some embodiments, the native elements can be replaced with heterologous elements. An upstream ribosome binding site is not used in combination with an IRES or the coding region downstream of a self-cleaving peptide. A start codon is not necessarily present in the coding region downstream of a 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: inverted terminal repeat, LAMP-2: lysosome-associated membrane protein type 2, UTR: untranslated region, Poly A: polyadenylation signal, CAG: promoter region including the CMV enhancer and CBA promoter sequence, CMV: cytomegalovirus, CBA: chicken beta-actin, WPRE: woodchuck hepatitis virus post-transcriptional regulatory element, RBG: rabbit beta-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 in any order under the control of a promoter region and two different internal ribosome entry sites, followed by a 3' UTR and poly A signal. 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 an adeno-associated virus LAMP-2 gene delivery construct. The lysosome-associated membrane protein (LAMP) LAMP-2 coding region shown should generally be understood to include an upstream ribosome binding sequence and start codon, although in some embodiments, the native elements can be replaced with heterologous elements. An upstream ribosome binding site is not used in combination with an IRES or the coding region downstream of a self-cleaving peptide. A start codon is not necessarily present in the coding region downstream of a 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: inverted terminal repeat, LAMP-2: lysosome-associated membrane protein type 2, UTR: untranslated region, Poly A: polyadenylation signal, CAG: promoter region including the CMV enhancer and CBA promoter sequences, CMV: cytomegalovirus, CBA: chicken beta-actin, WPRE: woodchuck hepatitis virus post-transcriptional regulatory element, RBG: rabbit beta-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 for all three LAMP-2 isoforms in any potential order, separated by the P2A cleavage site. In some embodiments, this construct is used to express mRNAs 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 1O]Figure 1 shows a schematic diagram of an adeno-associated virus LAMP-2 gene delivery construct. The lysosome-associated membrane protein (LAMP) LAMP-2 coding region shown should generally be understood to include an upstream ribosome binding sequence and start codon, although in some embodiments, the native elements can be replaced with heterologous elements. An upstream ribosome binding site is not used in combination with an IRES or the coding region downstream of a self-cleaving peptide. A start codon is not necessarily present in the coding region downstream of a 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: inverted terminal repeat, LAMP-2: lysosome-associated membrane protein type 2, UTR: untranslated region, Poly A: polyadenylation signal, CAG: promoter region including CMV enhancer and CBA promoter sequence, CMV: cytomegalovirus, CBA: chicken beta-actin, WPRE: woodchuck hepatitis virus post-transcriptional regulatory element, RBG: rabbit beta-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 containing the coding sequence for LAMP-2 exons 1-8, followed by an intron region (including all necessary splice signals), exon 9 coding sequence for one of the LAMP-2 isoforms with a 3'UTR and polyA signal, a second intron region (including all necessary splice signals), exon 9 coding sequence for a second LAMP-2 isoform with a 3'UTR and polyA signal, a third intron region (including all necessary splice signals), and exon 9 coding sequence for a third LAMP-2 isoform with a 3'UTR and polyA signal.In some embodiments, this construct is used to express mRNAs for all three LAMP-2 isoforms using the same viral genome via alternative splicing, a mechanism that generates distinct LAMP-2 mRNAs in normal cells. The exon 9 coding sequence can be arranged in any potential order and combination. [Figure 2] FIG. 2 shows a flowchart of a method for therapeutic treatment (e.g., Danon disease or another disease caused at least in part by autophagy deficiency) by promoting expression of one or more isoforms of LAMP-2. [Figure 3A] Figures 3A-F show the LAMP-2 isoform coding and protein sequences. Figure 3A: LAMP-2A coding sequence. [Figure 3B] Figures 3A-F show the LAMP-2 isoform coding and protein sequences. Figure 3B: LAMP-2A protein sequence. [Figure 3C] Figures 3A-F show the LAMP-2 isoform coding and protein sequences. Figure 3C: LAMP-2B coding sequence. [Figure 3D] Figures 3A-F show the LAMP-2 isoform coding and protein sequences. Figure 3D: LAMP-2B protein sequence. [Figure 3E] Figures 3A-F show the LAMP-2 isoform coding and protein sequences. Figure 3E: LAMP-2C coding sequence. [Figure 3F] Figures 3A-F show the LAMP-2 isoform coding 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-B show a dose-dependent increase in mRNA expression of LAMP-2B (Figure 5A) and LAMP-2A (Figure 5B) after administration of AAV9 vectors carrying these genes. [Figure 5B] Figures 5A-B show a dose-dependent increase in mRNA expression of LAMP-2B (Figure 5A) and LAMP-2A (Figure 5B) after administration of AAV9 vectors carrying these genes. [Figure 6] Figure 6 shows a dose-dependent increase in LAMP-2B and LAMP-2A protein expression after administration of AAV9 vectors carrying these genes. [Figure 7A] Figures 7A-C show fluorescent micrographs of heart sections stained with DAPI (blue) and fluorescently labeled anti-LAMP-2 antibody (white). Figure 7A shows a heart section from a Lamp-2 knockout (KO) mouse treated with AAV9.LAMP-2B. [Figure 7B] Figures 7A-C show fluorescent micrographs of heart sections stained with DAPI (blue) and fluorescently labeled anti-LAMP-2 antibody (white). Figure 7B shows heart sections from Lamp-2 KO mice treated with AAV9.EGFP. [Figure 7C] Figures 7A-C show fluorescent micrographs of heart sections stained with DAPI (blue) and fluorescently labeled anti-LAMP-2 antibody (white). Figure 7C shows a heart section from an untreated wild-type (WT) mouse. [Figure 8A]Figures 8A-B show fluorescent micrographs of heart sections stained with DAPI (blue) and fluorescently labeled anti-LAMP-2 antibody (white; some examples are highlighted by arrows). Figure 8A shows a heart section from a Lamp-2 KO mouse treated with AAV9.LAMP-2B. [Figure 8B] Figure 8A-B show fluorescent micrographs of heart sections stained with DAPI (blue) and fluorescently labeled anti-LAMP-2 antibody (white; some examples are highlighted by arrows). Figure 8B shows heart sections from Lamp-2 KO mice treated with AAV9.EGFP. [Figure 9A] Figures 9A-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) images are enlargements of insets outlined in images labeled with the same letter without prime. Regarding the grid in Figures 9B-G', the first column is an image of red fluorescence, the second column is an image of green fluorescence, and the third column is a merged image of columns 1 and 2. Yellow represents red + green fluorescence. The top two columns are images of heart sections from WT mice expressing the CAG-RFP-EGFP-LC3B autophagy reporter construct, and the bottom two columns are images of heart sections from Lamp-2 KO mice expressing the CAG-RFP-EGFP-LC3B autophagy reporter construct. Immature autolysosomes fluoresce both green and red (or yellow in merged images). Mature autolysosomes fluoresce only red. Yellow arrows highlight autophagic vacuoles that fluoresce in both colors, while white arrows highlight autophagic 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 autophagic vacuoles, which are autophagosomes or autolysosomes, in WT and Lamp-2 KO mice. [Figure 10A]Figures 10A-E show the effects of CAG-RFP-EGFP-LC3B autophagy reporter system-mediated LC3B-mediated autophagy gene therapy. Figure 10A is an image of an untreated WT mouse heart section. In the images in Figures 10A, 10C, and 10D, red arrows are used to highlight some of the autolysosomes present. [Figure 10B] Figures 10A-E show the effects of Lamp-2 gene therapy using the CAG-RFP-EGFP-LC3B autophagy reporter system. Figure 10B is an image of a heart section showing transduced cells from a Lamp-2 KO mouse treated with the control vector AAV9.EGFP. [Figure 10C] Figures 10A-E show the effects of LAMP-2 gene therapy using the CAG-RFP-EGFP-LC3B autophagy reporter system. Figure 10C is an image of a heart section from a Lamp-2 KO mouse treated with the gene therapy vector AAV9.LAMP-2A. In the images in Figures 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 heart section from a Lamp-2 KO mouse treated with the gene therapy vector AAV9.LAMP-2B. In the images of Figures 10A, 10C, and 10D, red arrows are used to highlight some of the autolysosomes present. [Figure 10E] Figures 10A-E show the effect of CAG-RFP-EGFP-LC3B autophagy reporter system-mediated autophagy in vivo. Figure 10E shows the percentage of total autophagic vacuoles represented by autophagosomes and autolysosomes for the 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 from Lamp-2 KO mice treated with AAV9.LAMP-2B (Figure 11C, C'). White arrows highlight several autophagic vacuoles. Black arrows highlight several 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 from Lamp-2 KO mice treated with AAV9.LAMP-2B (Figure 11C, C'). White arrows highlight several autophagic vacuoles. Black arrows highlight several 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 from Lamp-2 KO mice treated with AAV9.LAMP-2B (Figure 11C, C'). White arrows highlight several autophagic vacuoles. Black arrows highlight several damaged mitochondria. DETAILED DESCRIPTION OF THE INVENTION

[0033] 1. Introduction Danon disease is caused by a mutation that results in reduced or absent expression of the lysosome-associated membrane protein 2 (LAMP-2, also known as CD107b) gene. This method is based, in part, on the introduction of one or more polynucleotides (e.g., packaged in an adeno-associated virus (AAV) vector) encoding one or more LAMP-2 isoforms and the delivery of the LAMP-2 gene / individual isoforms to Danon disease patients. After 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 disease patients can result in amelioration of the disease phenotype and serve as a treatment for this disease. Delivery of one or more polynucleotides encoding one or more LAMP-2 isoforms to a subject can also be used to treat other disorders of autophagy, including, but not limited to, end-stage heart failure, myocardial infarction, drug toxicity, diabetes, end-stage renal failure, and aging. Autophagy abnormalities, particularly reduced autophagic flux, have been implicated in these and many other disorders. Higher levels of expression of the LAMP-2 gene increase autophagic flux and thus serve as a treatment for these disorders.

[0034] Currently, no technology exists for treating Danon disease via gene therapy. Danon disease is not a traditional lysosomal storage disorder; it is generally defined as a defect in a lysosomal protein (e.g., a transporter or enzyme) required to process a specific cellular substrate, resulting in the toxic accumulation of that specific substrate within the lysosome. Danon disease is understood to be a disorder of autophagy or autophagic vacuolar myopathy, which affects the degradation of all cellular components processed by the autophagic pathway and is not caused by the accumulation of a specific substrate. Furthermore, existing technology does not explicitly describe the delivery of LAMP-2 genes or individual isoforms for the treatment of Danon disease or other autophagy disorders. Therefore, the present method provides a unique method for treating Danon disease and improves upon existing AAV technology by explicitly including delivery of the LAMP-2 gene as a means to ameliorate impaired autophagy.

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

[0036] In some embodiments, the subject exhibits a disease or disorder characterized by insufficient autophagic flux (e.g., Danon disease and other known disorders of autophagy, including, but not limited to, systolic and diastolic heart failure, myocardial infarction, drug toxicity, diabetes, end-stage renal disease, and aging). Symptoms may be active, suppressed or controlled (e.g., with medication), or in remission. The subject may or may not have been diagnosed with the 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 is a child, juvenile, or adult. In various aspects, the subject is 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 contain an expression cassette containing a polynucleotide encoding one or more isoforms of lysosome-associated membrane protein 2 (LAMP-2) and allow expression of LAMP-2 in a subject in need thereof (e.g., a subject with Danon disease or another disorder characterized by defective autophagic flux due, at least in part, to the loss of LAMP-2 expression), thereby enabling partial or complete correction of defective LAMP-2 protein expression levels and autophagic flux. Gene therapy vectors can be viral or non-viral. Exemplary non-viral vectors include, for example, naked DNA, cationic liposome complexes, cationic polymer complexes, cationic liposome-polymer complexes, and exosomes.

[0039] In some embodiments, the vector carries a single isoform, LAMP-2A, LAMP-2B, or LAMP-2C (see, e.g., Figures 1A-F, which show schematic diagrams of AAV vectors carrying genes for a single LAMP-2 isoform). In other embodiments, the vector carries genes for two LAMP-2 isoforms (see, e.g., Figures 1G-K, which show schematic diagrams of AAV vectors carrying genes for two LAMP-2 isoforms, LAMP-2B on one DNA strand and LAMP-2A on the other). Still other embodiments carry all three isoforms (see Figures 1L-O). In addition to the depicted genomic structures, three-isoform vectors can also be constructed by creating hybrids of the various embodiments shown. For example, there can be two promoters, one driving expression of a single isoform and another driving expression of a two-isoform cassette using an IRES, a self-cleaving peptide, or alternative splicing. In various embodiments involving multiple isoforms, the isoforms may occur in any order, reflecting or altering the natural order of B, A, C. Carrying multiple isoforms in a single vector can ensure co-expression of isoforms in transfected cells and can allow for the use of fewer whole vector particles in a dose.

[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.The gene delivery viral vectors useful for carrying out the present invention can be constructed using well-known methodologies in the field of molecular biology.Typically, viral vectors carrying transgenes are constructed from the polynucleotides encoding transgenes, appropriate regulatory elements and the elements required for the production of viral proteins that mediate 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, 293 cells, etc. Detailed protocols for producing such replication-defective recombinant viruses can be found, for example, in International Publication No. WO 95 / 14785, International Publication No. WO 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. WO 94 / 19478, the complete contents of each of which 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 aspects, the AAV vector is selected from the AAV serotypes AAV1, AAV2, AAV3, AAV4, AA5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh10, and subgroups and mixtures thereof, including self-complementary AAV (scAAV) genomes, or 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 to deliver therapeutic genes and are being studied in human clinical trials.The rAAV vector can be designed to deliver specific transgenes to patient cells for expression. After infection and transfer of the viral genome by the rAAV vector, the viral genes are primarily present as extrachromosomal structures that are not integrated into the host genome but are expressed by the host cell's translational machinery. For successful host cell infection and gene expression, the rAAV vector requires several components (see Figure 1): inverted terminal repeats (ITRs), a promoter and / or enhancer region, a transgene and 3' untranslated region, and a polyadenylation signal. After viral infection of the host cell, the promoter region initiates a signal for translation of the virally delivered transgene by the host cell's translational machinery.

[0043] Generally, the control elements are selected to be functional in mammalian cells. The resulting construct containing the operably linked components is linked to functional AAV ITR sequences (5' and 3'). "Adeno-associated virus inverted terminal repeat" or "AAV ITR" refers to the art-recognized regions found at each end of the AAV genome that function together in cis as an origin of DNA replication and a viral packaging signal. The AAV ITRs, together with the AAV rep coding region, result in sufficient excision and rescue, as well as integration of the nucleotide sequence inserted between the two adjacent ITRs in the mammalian cell genome. The nucleotide sequences of the AAV ITR regions are known. For example, for the AAV2 sequence, see Kotin, 1994; Berns, K.I. "Parvoviridae and Their Replication" in Fundamental Virology, 2nd Edition, (B.N. Fields and D.M.K. Knipe, eds., the entire contents of which are incorporated herein by reference). As used herein, an "AAV ITR" need not necessarily comprise the wild-type nucleotide sequence, but may be altered, for example, by the insertion, deletion or substitution of nucleotides.

[0044] Furthermore, the AAV ITRs may be derived from any of several AAV serotypes, including, but not limited to, AAV1, AAV2, AAV3, AAV4, AA5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAVrhlO. Furthermore, the 5' and 3' ITRs flanking a selected nucleotide sequence in an AAV vector do not necessarily need 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 the sequence of interest derived from the host cell genome or vector, allowing integration of the heterologous sequence into the recipient cell genome when the AAV Rep gene product is present in the cell. Furthermore, the AAV ITRs may be derived from any of several AAV serotypes, including, but not limited to, AAV1, AAV2, AAV3, AAV4, AA5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAVrhlO. Furthermore, the 5' and 3' ITRs flanking a selected nucleotide sequence in an AAV expression vector do not necessarily need to be identical or derived from the same AAV serotype or isolate, so long as they function as intended, i.e., excising and rescuing sequences of interest derived from the host cell genome or vector, and allowing integration 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 have tropism and high transduction efficiency for mammalian cardiac muscle cells, particularly cardiomyocytes and cardiac progenitor cells, are used. A review and comparison of the transduction efficiency of different serotypes is described in Cearley CN et al., Molecular Therapy 16(10);1710-1718, 2008, the entire 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 cardiac muscle cells.

[0046] In various embodiments, the selected nucleotide sequence is operably linked to control elements that direct its transcription or expression in a subject in vivo. Such control elements can include control sequences normally associated with the selected gene.

[0047] Alternatively, heterologous regulatory sequences can be used. Useful heterologous regulatory 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 (chicken β-actin promoter with CMV enhancer 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 tumor virus LTR promoter, the adenovirus major late promoter (Ad MLP), the herpes simplex virus (HSV) promoter, the cytomegalovirus (CMV) promoter, such as the CMV immediate-early promoter region (CMVIE), the Rous sarcoma virus (RSV) promoter, synthetic promoters, hybrid promoters, and the like. Promoters may be of human origin or from other species, including mice. Additionally, sequences derived from non-viral genes, such as the marine metallothionein gene, also find use herein. Such promoter sequences are available, for example, from Stratagene (San Diego, CA). Examples of heterologous promoters include, but are not limited to, CMV promoters. Examples of inducible promoters include, but are not limited to, DNA response elements for ecdysone, tetracycline, and hypoxia androgenicity. When multiple isoforms are encoded in a single vector, they are preferably, but not necessarily, operably linked 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 polyadenylation signal, can be derived from an inserted gene or a heterologous source. Certain embodiments utilize the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) as the 3' UTR or rabbit β-globin polyadenylation signal, or both. The 3' regulatory element can also include regulatory elements connected to the coding region. For example, multiple LAMP-2 isoforms can be expressed 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 second and third (if present) isoforms. In the case of an IRES, two or three polypeptides are translated. In the case of a self-cleaving peptide, 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- or during translation into the replacement LAMP-2 isoform.

[0049] Transcription under the control of a single promoter can also be achieved when the vector is constructed to support alternative splicing. In such constructs, the first eight exons of LAMP-2 are present in the cDNA, followed by two or three intron-exon pairs spliced ​​together to contain two or all three of the alternative exons that give rise to the three LAMP-2 isoforms (see, for example, Figures 1K and 1O). In various embodiments, the intron-exon pairs occur in any order, reflecting or altering the natural order of B, A, and C. Natural introns are too long to be included in vectors as large as AAV. In such cases, the intron is truncated by removal of the central sequence while retaining the necessary 5' and 3' splice sites and splice signals. Typically, retaining only a few bases at the 5' end of the intron and approximately 100-200 bases at the 3' end of the intron is sufficient to ensure splicing. Alternatively, a heterologous intron can replace the natural intron.

[0050] AAV expression vectors with the target DNA molecule bound by AAV ITRs can be constructed by directly inserting the selected sequence into the AAV genome from which the main AAV open reading frame ("ORF") has been excised.Other parts of the AAV genome can also be deleted, as long as a sufficient amount of ITR remains to allow replication and packaging functions.Such constructs can be designed using techniques well known in the art. See, for example, U.S. Pat. Nos. 5,173,414 and 5,139,941, WO 92 / 01070 (published January 23, 1992) and WO 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, the entire contents of which are incorporated herein by reference.

[0051] Alternatively, AAV ITRs can be excised from the viral genome or an AAV vector containing them 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 entire 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 produced to contain AAV ITR sequences aligned 5' and 3' to one or more selected nucleic acid sequences. Preferred codons for expression of the chimeric gene sequence in mammalian CNS cells can be used. The complete chimeric sequence is assembled from overlapping oligonucleotides prepared by standard methods. For example, Edge Nature, vol. 292, 1981, page 756; Nambair et al., Science, vol. 223, 1984, page 1299; Jay et al., J. Biol. Chem. vol. 259, 1984, page 6311, the entire contents of each of which are incorporated herein by reference. To produce AAV virus, AAV expression vectors are 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-velocity microprojectiles (Klein et al., 1987, Endocrinology 120:2339-45). The complete contents of each of the foregoing references are incorporated herein by reference in their entirety.

[0052] In one embodiment, the AAV comprises, in addition to a nucleic acid sequence encoding a LAMP-2 isoform, an AAV vector backbone with ITRs derived from AAV2, a promoter such as the cytomegalovirus / chicken β-actin hybrid promoter (CAG), which consists of an enhancer and promoter from the mouse PGK (phosphoglycerate kinase) gene or the cytomegalovirus immediate-start gene, a splice donor and intron from the chicken β-actin gene, a splice acceptor from rabbit β-globin, or any promoter such as the PGK, CAG, MCK, EF-1, or native LAMP-2 promoter. In various embodiments, the viral vector is encapsulated in anionic liposomes, as described, for example, 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') inverted terminal repeats (ITRs) from any known AAV serotype or subgroup, including scAAV, any known promoter region (e.g., the cytomegalovirus (CMV) promoter or chicken β-actin promoter) with or without an enhancer element (e.g., a CMV enhancer), the lysosome-associated protein 2 gene (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 the virally delivered LAMP-2 isoform gene or a combination of various isoform genes 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 machinery. Restoring LAMP-2 isoform expression and function then restores autophagy (potentially including, but not necessarily limited to, any known form of autophagy, such as macroautophagy, mitophagy, chaperone-mediated autophagy, and DNA / RNA autophagy), which is deficient and causally impaired in Danon disease patients, allowing the removal of toxic cellular components from disease-affected host cells and damaged organelles, improving cellular function and survival. Ultimately, restoring LAMP-2 isoform expression can help treat the underlying genetic defect causing Danon disease and alleviate the disease phenotype and symptoms. In other autophagy disorders in which LAMP-2 is expressed but not at levels sufficient to generate sufficient autophagic flux and thereby promote normal cell function and survival, delivery of transgenic LAMP-2 isoforms via rAAV vectors results in overexpression of LAMP-2 protein. This overexpression restores autophagic flux to near-normal, normal, or even supranormal levels. Restoring autophagic flux in disorders that cause a disruption of normal autophagy can alleviate, mitigate, and / or reverse disease phenotypes and symptoms.

[0055] 4. Vector Pharmaceutical Composition For example, provided is a pharmaceutical composition for use in preventing or treating a disorder characterized by defective autophagic flux (e.g., Danon disease), comprising a therapeutically effective amount of a vector comprising a nucleic acid sequence of a polynucleotide encoding one or more isoforms of LAMP-2.

[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 scope of best medical judgment. The specific therapeutically effective dose level for any particular patient will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific compound used; the specific composition used, the patient's age, weight, general health, sex, and diet; the time of administration, route of administration, and rate of excretion of the specific compound used; the duration of treatment; drugs used in combination with or concomitantly with the specific nucleic acid or polypeptide used; and similar factors well known in the medical field. For example, it is well within the skill of one in the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. However, the daily dosage of the product may vary over a wide range per adult per day. The therapeutically effective amount of the vectors according to the present invention to be administered, as well as the dosage for treating a pathological condition, depending on the number of viral or non-viral particles and / or pharmaceutical compositions described herein, will depend on numerous factors, including the age and condition of the patient, the severity of the disorder or disorder, the method and frequency of administration, and the specific peptide used.

[0057] Pharmaceutical compositions containing the vectors may be in any form suitable for the selected mode of administration, such as intracerebroventricular, intrathoracic, intracoronary, intravenous, intraarterial, intrarenal, intraurethral, ​​epidural, or intramuscular, etc. Gene therapy vectors containing polynucleotides encoding one or more LAMP-2 isoforms can be administered to animals and humans in unit dosage forms in admixture with conventional pharmaceutical carriers as the sole active agent or in combination with other active agents.

[0058] In various embodiments, the pharmaceutical composition contains a pharmaceutically acceptable vehicle for an injectable formulation, which may in particular be isotonic sterile saline (monosodium phosphate or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, etc. or a mixture of their salts), or a dried, in particular lyophilized, composition that can be prepared into an injectable solution by optionally adding sterile water or saline.

[0059] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and fluid. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0060] Solutions containing the gene therapy vector as a free base or pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0061] Gene therapy vectors can be formulated into compositions in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein) and are formed with inorganic acids such as hydrochloric or phosphoric acids, or organic salts such as acetic, oxalic, tartaric, mandelic, and the like. 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, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Proper 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 dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of injectable compositions can be achieved by the use of agents delaying absorption, for example, aluminum monostearate and gelatin in the compositions.

[0063] Sterile injection solution is prepared by incorporating the required amount of active polypeptide in suitable solvent together with some other components as listed above as needed, and then sterilizing by filtration.Generally, dispersion is prepared by incorporating various sterilized active components into a sterile vehicle, which contains basic dispersion medium and other necessary components from above.For the preparation of sterile powder for sterile injection solution, the preferred preparation method is vacuum drying and freeze-drying technology, which produces powder of active component plus any additional desired component from the solution containing the powder that has been previously sterilized and filtered.

[0064] 5. How to treat autophagy disorders Also provided is a method for preventing, alleviating, ameliorating, reducing, inhibiting, eliminating, and / or reversing one or more symptoms of Danon disease or other autophagy disorders in a subject in need thereof, comprising administering to the subject a gene therapy vector as described above and herein, e.g., an adeno-associated virus (AAV) vector comprising an expression cassette comprising a polynucleotide encoding one or more isoforms of lysosomal-associated membrane protein 2 (LAMP-2). The vector is delivered to a subject in need thereof, such that the polynucleotide encoding one or more LAMP-2 isoforms is expressed by the transduced cells at therapeutically effective levels.

[0065] In one specific embodiment, the vector is AAV9. In another specific embodiment, the vector is AAV8. In another specific embodiment, the vector is AAVrH10. In further aspects of these embodiments, they are specifically used to treat Danon disease.

[0066] In various aspects, 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 a cardiothoracic incision, intracoronary injection, endocardial injection, or another type of injection useful in the heart. Additional administration routes may also include local application of the vector under direct visualization, such as, for example, superficial cortical or other atypical application.

[0067] Viral vectors typically elicit immune responses, including antibody responses, that can reduce or completely inhibit the effectiveness of a particular vector in an individual, making repeated dosing necessary or desirable. Repeated dosing may be appropriate, for example, because vectors can be lost over time due to cell proliferation, particularly episomal vectors. Tissues that are more difficult to access may require multiple administrations of the vector to transfect a sufficient number of cells for effective treatment of the disease. Expression of the transgene 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, empty AAV capsids can be administered to bind to host antibodies and immune cells before injecting a therapeutic AAV containing a LAMP-2 transgene. Depending on the target tissue, direct local administration instead of systemic administration may be useful to overcome or ameliorate the inhibitory effects of anti-vector antibodies. Yet another approach could be to use a vector based on a different serotype of the virus from which it is derived. For example, if an AAV9 vector is used initially and there is a problem with anti-AAV antibody titers, but there is a need for further gene therapy, an AAV8 vector carrying the same (or a different) gene construct could be administered.

[0068] Once formulated, solutions can be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the injectable solution types described above, although drug-release capsules and the like can also be used. Multiple doses are also possible.

[0069] If necessary, the vectors described herein can be formulated in any suitable vehicle for delivery. For example, they can be placed in a pharmaceutically acceptable suspension, solution, or emulsion. Suitable vehicles include saline and liposomal preparations. More specifically, pharmaceutically acceptable carriers can 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, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like.

[0070] Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.

[0071] Colloidal dispersion systems can also be used for targeted gene delivery, including macromolecule complexes including oil-in-water emulsions, micelles, mixed micelles, and 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 disease. As an example, for delivery of a nucleic acid sequence encoding a LAMP-2 polypeptide using a viral expression vector, each unit dose of a LAMP-2 polypeptide expression vector may contain 2.5 μl to 100 μl of the composition, which may be, for example, 10 μl in a pharmaceutically acceptable liquid. 11 ~10 16 Contains viral expression vectors at concentrations ranging from 1000 to 1000 viral genomes / ml.

[0073] Some regimens use a vector carrying a gene for a single LAMP-2 isoform, e.g., LAMP-2B. Other regimens use a vector carrying genes for two LAMP-2 isoforms, e.g., LAMP-2B and LAMP-2A. In some embodiments, the two isoforms are carried in different vector preparations. In one aspect, both vector preparations are derived from the same vector, e.g., an AAV9 vector or any other specific vector described above. In other embodiments, both polypeptides are encoded in a single vector. See, e.g., FIG. 1. Similarly, when a regimen includes 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, with intervals of several hours, days, or weeks between administrations.

[0074] 6. Kit Further provided is a kit comprising a gene therapy vector comprising a polynucleotide encoding one or more LAMP-2 isoforms as described above and herein.In various embodiments, the kit provides the gene therapy vector in one or more unit dosage forms, such as pre-filled syringes or ampoules, ready for administration to a subject.In various aspects, the gene therapy vector is provided in a lyophilized form. [Example]

[0075] Example 1 (AAV9 gene therapy) Specific vectors consistent with the above description were constructed as AAV9 vectors containing ITRs derived from AAV2 and encoding either LAMP-2A or LAMP-2B under the control of the chicken β-actin promoter with a CMV enhancer (CAG promoter). Additionally, these vectors incorporated woodchuck hepatitis virus posttranscriptional regulatory elements and a rabbit β-globin polyadenylation signal (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 integrated because it is predicted to be functional in mice due to the high degree of sequence identity between homologs, 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 has excellent tropism for heart and skeletal muscle, as well as nervous tissue, which are the organs most affected by Danon disease. However, Danon disease is a multisystem disorder, potentially involving many different organs. Therefore, expression of the transgenic LAMP-2 isoforms is under the control of a constitutively active chicken β-actin (CBA) promoter with a cytomegalovirus (CMV) enhancer (CAG construct). This promoter is ubiquitously active, allowing LAMP-2 transgene expression in all tissues, depending on infection efficiency. An AAV9 vector carrying the 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 a Danon-like syndrome, but the severity of the disease is milder than the human disease, likely due to the shorter lifespan of the mice, which allows less time for damage to accumulate, likely due at least in part to abnormal autophagic flux. Therefore, it is advisable to wait until the mice are at least 3 months old, preferably 5-6 months old, before initiating treatment so that there has been sufficient accumulation of pathology for its reversal to be easily detectable.

[0078] In an idealized procedure, 6-month-old Lamp-2 KO mice were transfected with 5 × 10 of an AAV vector. 11 , 1×10 12 , or 2 × 10 12 Genome copies (gc) are administered intravenously into the external jugular vein. In various experiments, WT mice receive a LAMP-2 isoform vector, while Lamp-2 KO mice receive an eGFP vector as a control. By using a sufficient number of mice, subpopulations can be sacrificed and evaluated 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 were treated with 5 × 10 11 , 1×10 12 , and 2 × 10 12 gc / mice received increasing doses of the vectors AAV9.LAMP-2B and AAV9.LAMP-2A (see Figure 1B and above). One mouse per condition was sacrificed, and RT-qPCR was performed in triplicate on digested heart tissue to assess mRNA expression (gene transcription) of the human transgene. Untreated WT mice were used to demonstrate no expression of the human LAMP-2 isoform. The data show 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 tissue to assess human transgene protein expression relative to GAPDH. Control Lamp-2 KO mice treated with AAV9.EGFP showed no significant LAMP-2 protein expression. The data show a dose-dependent increase in human transgene expression in mice administered the viral vector (see Figure 6).

[0081] Example 5 (Intracellular localization of transgenic LAMP-2B after vector administration) 2×10 12 Heart sections from Lamp-2 KO mice administered AAV9.LAMP-2B or AAV9.EGFP in gc / mice and sacrificed 1 month after delivery were stained with DAPI (which binds to AT-rich DNA, making cell nuclei visible) and a fluorescently labeled anti-LAMP-2 antibody (see Figure 7). The pattern of LAMP-2 staining indicates localization of the human transgenic LAMP-2B protein to intracellular vacuoles, similar to the staining seen in WT mouse controls for mouse Lamp-2 protein. No human LAMP-2 staining is observed 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 in a physiologically relevant location.

[0082] Similarly, experimental human LAMP-2 staining was performed at 5 × 10 in test mice. 11 The staining was maintained 2 months after delivery of AAV9.LAMP-2B at a dose of 10 ... 11 The AAV9.EGFP vector in gc / mice showed no 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 allows for evaluation of macroautophagy flux. Microtubule-associated protein 1 light chain 3 (LC3) is expressed on the surface of autophagosomes. A gene fusion was constructed to express LC3 fused to both red fluorescent protein (RFP) and eGFP. When this fusion protein was expressed, the RFP component fluoresced in both autophagosomes and lysosomes, while the eGFP component fluoresced in autophagosomes but was quenched by the acidic environment of the lysosomes. As a result, in the merged image, autophagosomes appeared yellow and lysosomes appeared red (see Figure 9A). When expressed in a WT background, more red dots than green dots were observed in the separate red and green images, whereas mixed red and yellow dots were observed in the merged image (see Figure 9B-D'). Lamp-2 is required for successful fusion of autophagosomes with lysosomes to form autolysosomes. Thus, when this construct was expressed in Lamp-2 KO mice, approximately equal numbers of red and green dots were seen in the separate red and green images, and almost entirely yellow dots were seen in the merged images (see Figure 9E-G'). The accumulation of autophagosomes and the near absence of autolysosomes, along with an overall higher number of autophagic vacuoles (AVs), reflects a defect in autophagic flux due to the absence of Lamp-2 (see Figure 9H).

[0084] Example 7 Administration of vectors carrying either LAMP-2A or LAMP-2B restores autophagic flux. 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 with Lamp-2 KO mice expressing the CAG-RFP-EGFP-LC3B autophagy reporter system (CAG-RFP-EGFP-LC3B reporter mice). One month after vector delivery, mice were sacrificed, and cardiac sections were evaluated by fluorescence microscopy to assess autophagy flux. Untreated Lamp-2 KO / reporter mice continued to exhibit mostly only yellow autophagic vacuoles, i.e., autophagosomes (see Figure 10B and Figure 9G'). In contrast, Lamp-2 KO / reporter mice administered either the gene therapy vector AAV9.LAMP-2B or AAV9.LAMP-2A exhibited many red autophagic vacuoles, or autolysosomes, at a similar rate to control WT reporter mice (see Figures 10A, 10C, and 11D, where arrows indicate representative points within). Quantification of AVs showed that the ratio of immature to mature autolysosomes was similar in treated Lamp-2 KO mice compared with 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 (Recovery of cardiomyocyte ultrastructure assessed by electron microscopy) Lamp-2 KO mice, 5 × 10 11gc / mice were intravenously injected with AAV9.LAMP-2B and compared with 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 accumulation and size of AVs (see yellow arrows in Figure 11B, B') and an increased number of abnormal mitochondria (see red arrows in Figure 11B) compared with WT mice (see Figure 11A, A'). In contrast, electron micrographs of Lamp-2 KO mice treated with AAV9.LAMP-2B more closely resembled the ultrastructure of untreated WT mice (see Figure 11C, C'). Thus, treatment with the gene therapy vector restores the ultrastructure of cardiomyocytes.

[0086] In summary, these examples demonstrate that adeno-associated virus-based gene therapy vectors encoding the LAMP-2A and LAMP-2B isoforms can be administered intravenously to successfully achieve transgene expression in cardiac tissue. In addition, such expression leads to the reversal of defects in autophagic flux 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 defects in autophagic flux.

[0087] Finally, while aspects of the present specification are emphasized by reference to specific embodiments, it should be understood that those skilled in the art will readily appreciate that these disclosed embodiments are merely illustrative of the principles of the subject matter disclosed herein. Accordingly, it should be understood that the disclosed subject matter is in no way limited to the particular methodology, 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 the specification. Finally, the terminology used herein is for the purpose of describing particular embodiments only and does not limit the scope of the present invention, which is defined solely by the claims. Accordingly, the present invention is not limited to that precisely as shown and described.

[0088] Certain embodiments of the present invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those skilled in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as they see fit, and the inventors intend to practice the invention otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Furthermore, this invention includes any combination of the above-described embodiments in all possible variations thereof unless otherwise indicated herein or clearly contradicted by context.

[0089] Groupings of alternative embodiments, elements, or steps of the invention are not to be construed as limiting. Each group member may be referenced and claimed individually or in any combination with other group members disclosed herein. It is anticipated that one or more group members may be included in, or deleted from, a group for reasons of convenience and / or patentability. In the event of such inclusion or deletion, the specification is deemed to include the modified group and, therefore, to satisfy all Markush group descriptions used in the appended claims.

[0090] Unless otherwise indicated, all numbers expressing features, items, quantities, parameters, characteristics, terms, etc. used in the specification and claims should be understood to be modified in all instances by the term "about." As used herein, the term "about" means that the characteristic, item, quantity, parameter, characteristic, or term so characterized encompasses ±10% of the value of the stated characteristic, item, quantity, parameter, property, or term. Thus, unless indicated to the contrary, the numerical parameters set forth in this specification and the appended claims are approximations that may vary. At the very least, and not as an attempt to limit the scope of the claims and the application of the doctrine of equivalents, each numerical designation should be construed in light of at least the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and values ​​setting forth the broad scope of the invention are approximations, the numerical ranges and values ​​set forth in the specific examples are reported as precisely as possible. However, numerical ranges or values ​​inherently contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value within that range. Unless otherwise stated herein, each value of a numerical range is incorporated herein as if each individual value were individually recited herein.

[0091] The terms "a," "an," "the," and similar referents as used in the context of describing the present invention (particularly in the context of the appended claims) should be construed to encompass both the singular and the plural. Unless otherwise indicated herein or clearly contradicted by context, all methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "etc.") provided herein is intended merely to better illustrate the invention and does not limit the scope of the invention, as otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0092] Certain embodiments disclosed herein are further limited in the claims using language consisting of or consisting essentially of language. When used in a claim, the transitional term "consisting of" excludes any element, step, or ingredient not recited in the claim, whether or not recited in the claim. The transitional term "consisting essentially of" limits the claim to specific materials or steps and those that do not materially affect the basic and novel characteristics. Embodiments of the invention so claimed are essentially or explicitly described and enabled.

[0093] All patents, patent publications, and other publications referenced and identified herein may be individually incorporated by reference in their entireties in connection with the present invention, for example, for the purpose of describing and disclosing the compositions and methodologies described in such publications. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or presentation of the contents of these documents are based on the information available to the applicants and do not constitute any admission as to the accuracy of the dates or contents of these documents.

[0094] (Addendum) (Appendix 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] (Appendix 2) The vector is a viral vector. 2. A gene therapy vector according to claim 1.

[0096] (Appendix 3) The viral vector is derived from a virus selected from the group consisting of adenovirus, retrovirus, lentivirus, herpesvirus, and adeno-associated virus (AAV). 3. A gene therapy vector according to claim 1 or 2.

[0097] (Appendix 4) The vector is derived from one or more adeno-associated virus (AAV) serotypes 1-11 or any subgroup thereof, 4. A gene therapy vector according to claim 3.

[0098] (Appendix 5) The viral vector is encapsulated in an anionic liposome. 5. A gene therapy vector according to any one of claims 2 to 4.

[0099] (Appendix 6) The vector is a non-viral vector. 2. A gene therapy vector according to claim 1.

[0100] (Appendix 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. 7. A gene therapy vector according to claim 6.

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

[0102] (Appendix 9) The promoter is selected from the group consisting of a cytomegalovirus (CMV) promoter and a CAG promoter. 9. A gene therapy vector according to claim 8.

[0103] (Appendix 10) The polynucleotide comprises DNA or cDNA. 10. A gene therapy vector according to any one of claims 1 to 9.

[0104] (Appendix 11) The polynucleotide encoding one or more isoforms of LAMP-2 includes one or more human LAMP-2 isoforms. 11. A gene therapy vector according to any one of claims 1 to 10.

[0105] (Appendix 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; 12. A gene therapy vector according to any one of claims 1 to 11.

[0106] (Appendix 13) The polynucleotide encoding one or more isoforms of LAMP-2 has at least about 90% sequence identity with one or more of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3; 13. A gene therapy vector according to any one of claims 1 to 12.

[0107] (Appendix 14) The polynucleotide encoding one or more isoforms of LAMP-2 comprises one or more of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; 14. A gene therapy vector according to claim 13.

[0108] (Appendix 15) administering to a subject a gene therapy vector of any one of claims 1 to 14. A method for preventing, alleviating, ameliorating, reducing, inhibiting, eliminating, and / or reversing one or more symptoms of Danon disease or other autophagy disorders in a subject in need thereof.

[0109] (Appendix 16) administering to a subject an adeno-associated virus (AAV) vector containing an expression cassette comprising a polynucleotide encoding one or more isoforms of lysosomal-associated membrane protein 2 (LAMP-2). A method for preventing, alleviating, ameliorating, reducing, inhibiting, eliminating, and / or reversing one or more symptoms of Danon disease or other autophagy disorders in a subject in need thereof.

[0110] (Appendix 17) The vector is administered via a route selected from the group consisting of intravenous, intra-arterial, intracardiac, intracoronary, intramyocardial, intrarenal, intraurethral, ​​epidural, and intramuscular; 17. The method according to claim 15 or 16,

[0111] (Appendix 18) The vector is administered multiple times. 18. The method of any one of appendices 15 to 17.

[0112] (Appendix 19) 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. 19. The method of any one of claims 15 to 18.

[0113] (Appendix 20) the subject is a human; 20. The method of any one of appendices 15 to 19.

[0114] (Appendix 21) The subject exhibits symptoms of Danon disease or other autophagy disorders. 21. The method of any one of claims 15 to 20.

[0115] (Appendix 22) The subject is identified as having reduced or undetectable LAMP-2 expression. 22. The method of any one of claims 15 to 21.

[0116] (Appendix 23) The subject has been identified as having a mutant LAMP-2 gene. 23. The method of any one of claims 15 to 22.

Claims

1. 1. A pharmaceutical composition for use in a method for treating impaired or insufficient autophagic flux in the myocardium of a subject with Danon disease, comprising: an adeno-associated virus (AAV) vector comprising an expression cassette comprising a polynucleotide encoding lysosomal-associated membrane protein 2B (LAMP-2B) operably linked to a promoter, wherein the AAV vector has tropism for cells of mammalian myocardium.

2. 2. The pharmaceutical composition of claim 1, wherein the LAMP-2B has at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO:

11.

3. The pharmaceutical composition according to claim 1 or 2, wherein the cells are cardiomyocytes and cardiac progenitor cells.

4. The pharmaceutical composition according to claim 1 , wherein the AAV vector is encapsulated in an anionic liposome.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the expression cassette comprises, operably linked in a 5' to 3' direction, a first inverted terminal repeat sequence, an enhancer / promoter region, a polynucleotide encoding LAMP-2B, a 3' untranslated region comprising a polyadenylation signal, and a second inverted terminal repeat sequence.

6. 6. The pharmaceutical composition according to claim 1, wherein the promoter is selected from the group consisting of a cytomegalovirus (CMV) promoter, a hybrid promoter comprising a chicken β-actin promoter and a CMV enhancer (CAG promoter), a human elongation factor-1α promoter, and a phosphoglycerate kinase (PGK) promoter.

7. The pharmaceutical composition of claim 6 , wherein the promoter comprises a cytomegalovirus (CMV) promoter.

8. The pharmaceutical composition of claim 6, wherein the promoter comprises a hybrid promoter (CAG promoter) comprising a chicken β-actin promoter and a CMV enhancer.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the polynucleotide encodes the LAMP-2B having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO:

2.

10. 10. The pharmaceutical composition of any one of claims 1 to 9, wherein the method comprises administering the AAV vector to the subject via intravenous, intra-arterial, intracardiac, intracoronary, intramyocardial, intrarenal, intraurethral, ​​epidural, or intramuscular administration.

11. The pharmaceutical composition of claim 10, wherein the AAV vector is administered multiple times.

12. The pharmaceutical composition of any one of claims 1 to 11, wherein the subject is identified as having reduced or undetectable LAMP-2 expression.

13. The pharmaceutical composition of any one of claims 1 to 12, wherein the subject is identified as having a mutated LAMP-2 gene.

14. 14. The pharmaceutical composition of claim 1, wherein the use ameliorates impaired autophagic flux in the subject.

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