RU2777571C2 - Methods for treatment of danon's disease and other autophagy disorders - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology, in particular to a vector based on adeno-associated virus (AAV) of serotype 9, containing an expression cassette containing polynucleotide encoding lysosome-associated membrane protein 2B (LAMP-2B), functionally connected to a hybrid promotor containing chicken β-actin promotor and CMV enhancer (CAG promotor).

EFFECT: invention is effective for the treatment of Danon’s disease.

14 cl, 11 dwg, 8 ex

Description

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

[0002] This work was supported in part by National Institutes of Health Grant Nos. PHS 7K23HL107755 and RSA 1268. The US government has certain rights to this invention.

BACKGROUND

[0003] Danon's 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). Evidence has emerged highlighting the importance of autophagy in regulating cardiomyocyte bioenergetics, function, and survival. However, the mechanisms responsible for the disruption of cellular functions and the death of cardiomyocytes in a weakened autophagic flow remain unclear. In a previous study, in order to study the molecular mechanisms responsible for Danon's disease, the inventors generated induced human pluripotent stem cells (hiPSC) from two patients with different LAMP-2 mutations (Hashem, et al., Stem Cells. 2015 Jul;33(7 ):2343-50). Danon hiPSC-derived cardiomyocytes (hiPSC-CM) showed reduced autophagic flux and key signs of heart failure such as increased cell size, increased expression of natriuretic peptides, and abnormal calcium metabolism compared to control hiPSC-CM. Additionally, Danon hiPSC-CM exhibited excessive amounts of mitochondrial oxidative stress and apoptosis. The use of the sulfhydryl antioxidant N-acetylcysteine to scavenge free radicals resulted in a significant reduction in apoptotic cell death among Danon hiPSC-CMs. The inventors also used a lentiviral vector to introduce the LAMP-2B isoform coding sequence under the control of a doxycycline-inducible promoter into one of the Danon hiPSC lines. Overexpression of LAMP-2B with the addition of doxycycline also reduced levels of oxidative stress and apoptotic cell death among Danon hiPSC-CM, confirming the importance of LAMP-2B in the pathophysiology of the disease. Briefly, the inventors modeled Danon's disease using hiPSC-CM from patients with mutations in LAMP-2, which allowed them to make a mechanistic hypothesis about the pathogenesis of this disease. The inventors demonstrated that LAMP-2 deficiency led to a decrease in autophagic flux, leading to excessive oxidative stress and subsequent apoptosis of cardiomyocytes. Removal of excessive free radicals using antioxidants and overexpression of LAMP-2B improved the disease phenotype in vitro. The prior art does not disclose effective strategies for the treatment of Danon's disease or other diseases associated with autophagy, and thus the in vivo studies described herein are necessary to validate the approach.

SUMMARY

[0004] In one aspect, gene therapy vectors are provided, for example, for use in systemically or locally upregulating one or more isoforms of lysosome associated membrane protein 2 (LAMP-2) in a subject. Gene therapy vectors find use in the prevention, amelioration, amelioration, reduction, inhibition and/or treatment of one or more symptoms of Danon's disease or other disorder of insufficient autophagic flow. In various embodiments, gene therapy vectors comprise an expression cassette containing a polynucleotide encoding one or more isoforms of lysosome associated membrane protein 2 (LAMP-2). In various embodiments, the implementation of the vector is a viral vector. In various embodiments, the viral vector is from a virus selected from the group consisting of adenovirus, retrovirus, lentivirus, herpesvirus, and adeno-associated virus (AAV). In various embodiments, a vector from one or more of adeno-associated virus (AAV) serotypes 1-11, or any subgroups thereof. In various embodiments, the viral vector is encapsulated in an anionic liposome. In various embodiments, the implementation of the vector is a non-viral vector. In various embodiments, the non-viral vector is selected from the group consisting of deproteinized DNA, a cationic liposomal complex, a cationic polymer complex, a cationic liposomal polymer complex, and an exosome. In various embodiments, the expression cassette comprises a 5' to 3' (in terms of mRNA to be transcribed) operably linked first inverted terminal repeat, enhancer, promoter, polynucleotide encoding one or more isoforms of LAMP-2, 3'- an untranslated region, a polyadenylation signal (polyA), and a second inverted terminal repeat. In various embodiments, the promoter is selected from the group consisting of the cytomegalovirus (CMV) promoter and the chicken β-actin (CAG) promoter. In various embodiments, the implementation of the polynucleotide contains DNA or cDNA. In various embodiments, the polynucleotide encoding one or more isoforms of LAMP-2 comprises one or more isoforms of human LAMP-2. In various embodiments, the polynucleotide encoding one or more LAMP-2 isoforms comprises one or more LAMP-2 isoforms selected from the group consisting of LAMP-2A, LAMP-2B, and LAMP-2C. In various embodiments, a polynucleotide encoding one or more isoforms of LAMP-2 has at least about 90% sequence identity, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, 99% sequence identity, with one or more of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. In various embodiments, a polynucleotide encoding one or more isoforms of LAMP-2 contains one or more of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.

[0005] In a further aspect, methods are provided for preventing, alleviating, ameliorating, reducing, inhibiting, eliminating and/or reversing one or more symptoms of Danon's disease or other autophagy disorder in a subject in need thereof, comprising administering to the subject a gene therapy vector as previously described. and in the present description (see, for example, Fig. 2). In a further aspect, methods are provided for preventing, alleviating, improving, reducing, inhibiting, eliminating and/or reversing one or more symptoms of Danon's disease or other autophagy disorder in a subject in need thereof, comprising administering to the subject an adeno-associated virus (AAV) vector containing an expression cassette, containing a polynucleotide encoding one or more isoforms of lysosome-associated membrane protein 2 (LAMP-2). In various embodiments, the vector is administered via a route selected from the group consisting of intravenous, intra-arterial, intracardial, intracoronary, intramyocardial, intrarenal, intraurethral, epidural, intracranial, subcutaneous, and intramuscular. In various embodiments, the vector is delivered or administered by a physical or mechanical means selected from the group consisting of microinjection, jet injection, particle bombardment, hydrodynamic infusion, electroporation, sonoporation, laser irradiation, magnetofection. In various embodiments, the implementation of the vector is administered several times with or without the use of immunosuppression or plasmapheresis in the patient. In various embodiments, the autophagy disorder is selected from the group consisting of end-stage heart failure, myocardial infarction, drug toxicities, diabetes, end-stage renal disease, and aging. In various embodiments, the subject is a human. In various embodiments, the subject exhibits symptoms of Danon's disease or other autophagy disorder. In various embodiments, the subject is identified as having reduced or undetectable LAMP-2 expression. In various embodiments, the subject is identified as having a mutant LAMP-2 gene.

DEFINITIONS

[0006] The term "Danon's disease" refers to an X-linked dominant skeletal and cardiac muscle disorder with multisystem clinical manifestations. Mutations in Danon's disease lead to a lack of protein expression of lysosome-associated membrane protein 2 (LAMP-2). Major clinical features include skeletal and cardiomyopathy, cardiac conduction abnormalities, cognitive impairment, and retinal disease. Men are usually affected earlier and more severely than women.

[0007] The terms "lysosome-associated membrane protein 2" and "LAMP-2" interchangeably refer to nucleic acids and polymorphic polypeptide variants, alleles, mutants, and cross-species homologues that: (1) have an amino acid sequence that has more than about 90 % amino acid sequence identity, e.g. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher amino acid sequence identity, preferably over a region of at least about 25, 50, 100, 200, 300, 400 or more amino acids, or over the entire length, with the amino acid sequence encoded by the LAMP-2 nucleic acid (see, for example, GenBank accession numbers NM_002294.2 (isoform A), NM_013995.2 ( isoform B), NM_001122606.1 (isoform C)), or with the amino acid sequence of the LAMP-2 polypeptide (see, for example, GenBank accession numbers NP_002285.1 (isoform A), NP_054701.1 (isoform B), NP_001116078.1 ( iso form C)); (2) bind to antibodies, eg, polyclonal antibodies raised against an immunogen containing the amino acid sequence of a LAMP-2 polypeptide (eg, the LAMP-2 polypeptides described herein); or an amino acid sequence encoded by a LAMP-2 nucleic acid (eg, the LAMP-2 polynucleotides described herein) and conservatively modified variants thereof; (3) hybridize specifically under stringent hybridization conditions with an antisense strand corresponding to the nucleic acid sequence encoding the LAMP-2 protein and conservatively modified variants thereof; (4) have a nucleic acid sequence that has greater than about 90%, preferably greater than about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater identity nucleotide sequences, preferably over a region of at least about 25, 50, 100, 200, 500, 1000, 2000 or more nucleotides, or over the entire length, with a LAMP-2 nucleic acid (e.g., LAMP-2 polynucleotides as disclosed herein, and LAMP-2 polynucleotides that encode LAMP-2 polypeptides as disclosed herein).

[0008] The terms "polypeptide", "peptide", and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to polymers of amino acids in which one or more amino acid residues is an artificial chemical mimetic of the corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0009] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics, that function in a manner that is similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are subsequently modified, such as hydroxyproline, α-carboxyglutamate and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e. an α-carbon that is bonded to hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide , methionine methylsulfonium. Such analogs have modified R groups (eg, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that differs from the basic chemical structure of an amino acid, but that functions in a manner that is similar to a naturally occurring amino acid.

[0010] Amino acids may be referred to herein by their commonly known three-letter designations or by the one-letter designations recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their common single letter codes.

[0011] "Conservatively modified variants" are applicable to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, conservatively modified variants refer to those nucleic acids that encode identical or substantially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, substantially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids code for any given protein. For example, all codons GCA, GCC, GCG and GCU code for the amino acid alanine. Thus, at each position where alanine is defined by a codon, the codon can be changed to any of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid variations are "silent variations", which are one kind of conservatively modified variations. Each nucleic acid sequence in the present description that encodes a polypeptide also describes every possible silent variation of the nucleic acid. One skilled in the art will appreciate that every codon in a nucleic acid (with the exception of AUG, which is typically the only codon for methionine, and TGG, which is typically the only codon for tryptophan) can be modified to produce a functionally identical molecule. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is latent in each described sequence.

[0012] With regard to amino acid sequences, one skilled in the art will appreciate that individual substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or protein sequence that alter, add, or delete one amino acid or a small percentage of amino acids in the encoded sequence are "conservatively modified variant", where the change leads to the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables listing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to, and do not exclude, the polymorphic variants, interspecies homologues and alleles of the invention.

[0013] Each of the following eight groups contains amino acids that are conservative substitutions for each other:

1) alanine (A), glycine (G);

2) aspartic acid (D), glutamic acid (E);

3) asparagine (N), glutamine (Q);

4) arginine I, lysine (K);

5) isoleucine (I), leucine (L), methionine (M), valine (V);

6) phenylalanine (F), tyrosine (Y), tryptophan (W); and

7) serine (S), threonine (T)

[0014] 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 can be isolated from natural sources, synthesized in vitro, or made from a combination of natural and synthetic molecules. The sizes of polynucleotides are expressed in base pairs (abbreviated "bp"), nucleotides ("n.") or thousand bases ("t. O."). When the context allows, the last two terms can describe polynucleotides that are single-stranded or double-stranded. When the term is applied to double-stranded molecules, it is used to denote length and should be understood as equivalent to the term "base pairs". Those skilled in the art will appreciate that the two strands of a double-stranded polynucleotide may differ slightly in length and that their ends may be misaligned as a result of enzymatic cleavage; thus, all nucleotides in a double-stranded polynucleotide molecule may not be paired.

[0015] The terms "identical" or percent "identity" in the context of two or more nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a particular percentage of amino acid residues or nucleotides that are the same (i.e., e. have at least about 80% identity, e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity over a precisely defined region of a reference sequence, e.g., a LAMP-2 polynucleotide or a polypeptide sequence as disclosed herein, when compared and aligned for maximum match over a comparison window, or designated region, as measured using one of the following sequence comparison algorithms or through manual alignment and visual inspection, then such sequences are so-called "substantially identical". This definition also applies to the completion of a test sequence. Preferably, the identity occurs 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 sequences.

[0016] When comparing sequences, usually one sequence functions as a reference sequence against which test sequences are compared. When using the sequence comparison algorithm, the test and reference sequences are entered into the computer, the coordinates of the subsequences are indicated, if necessary, and the parameters of the program with the sequence algorithm are set. You can use the program's default settings, or you can specify alternative settings. The sequence comparison algorithm then calculates a percent sequence identity for the test sequences relative to the reference sequence, based on the program parameters. To compare nucleic acid and protein sequences with LAMP-2 nucleic acids and proteins, the BLAST and BLAST 2.0 algorithms and default parameters are used.

[0017] A "comparison window" as used herein includes an indication of a segment of any one of a plurality of contiguous positions selected from the group consisting of 20 to 600, typically about 50 to about 200, more typically about 100 to about up to 150, where the sequence can be compared to a reference sequence with the same number of contiguous positions after optimal alignment of the two sequences. Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed, for example, using the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), using the homologous alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), using the similarity search method of Pearson and Lipman, Proc. Nat'l. Acad. sci. USA 85:2444 (1988), using computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI) or using manual alignment and visual inspection ( see, for example, Ausubel et al., Ed., Current Protocols in Molecular Biology (1995, Supplement)). Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., J. Mol. Biol. 215:403-410 (1990) and Altschul et al., Nucleic Acids Res. 25:3389-3402 (1977), respectively. BLAST analysis software is publicly available through the National Center for Biotechnology Information (on the web at ncbi.nlm.nih.gov/).

[0018] An indication that two nucleic acid or polypeptide sequences are substantially identical is that the polypeptide encoded by the first nucleic acid has immunological cross-reactivity with antibodies formed against the polypeptide encoded by the second nucleic acid, as described below. Thus, the polypeptide is usually substantially identical to the second polypeptide, for example, when the two peptides differ only in conservative substitutions. Another indication that two nucleic acid sequences are essentially identical is that the two molecules, or their complements, hybridize with each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.

[0019] As used herein, "administration" refers to local and systemic administration, for example, including enteral, parenteral, pulmonary, and topical/transdermal administration. Routes of administration for compounds (e.g., a polynucleotide encoding one or more isoforms of LAMP-2) that find use in the methods described herein include, for example, oral (per os (P.O.)) administration, nasal or inhalation administration, administration suppository, topical contact, transdermal delivery (eg, via a transdermal patch), intrathecal (IT) administration, intravenous (“iv”) administration, intraperitoneal (“ip”) administration, intramuscular (“im”) administration, administration within a lesion or subcutaneous ("sc") administration or implantation of a slow release device, such as a mini-osmotic pump, a depot formulation, etc., to a subject. Administration can be via any route, including parenteral and transmucosal (eg, oral, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intrarenal, intraurethral, intracardial, intracoronary, intramyocardial, intradermal, epidural, subcutaneous, intraperitoneal, intraventricular, iontophoretic, and intracranial. Other delivery methods include, but are not limited to, liposomal formulations, intravenous infusion, transdermal patches, and the like.

[0020] The terms "systemic administration" and "systemically administered" refer to a method of administering a compound or composition to a mammal so as to deliver the compound or composition to specific locations in the body, including pharmaceutical target sites, via the circulatory system. Systemic administration includes, but is not limited to, oral, intranasal, rectal, and parenteral (eg, other than through the digestive tract, such as intramuscular, intravenous, intra-arterial, transdermal, and subcutaneous) administration.

[0021] The term "co-administration" or "parallel administration", when used, for example, in relation to compounds (for example, LAMP-2 polynucleotides) and/or their analogs and another active agent, refers to the administration of the compound and/or analogs and the active means so that both can simultaneously achieve a physiological effect. However, the two agents are not necessarily administered together. In certain embodiments, the administration of one agent may precede the administration of another. Simultaneous physiological effect does not necessarily require the presence of both agents in the circulation at the same time. However, in certain embodiments, co-administration will typically result in a significant proportion (e.g., 20% or more, such as 30% or 40% or more, such as 50% or 60% or more, such as 70% or 80% or 90% or more) of both agents are simultaneously present in the body (eg, in plasma) of the maximum serum concentration for any given dose.

[0022] The term "effective amount" or "pharmaceutically effective amount" refers to the amount and/or dose and/or dosing schedule of one or more compounds (e.g., gene therapy vectors) necessary to obtain the desired result, for example, increased expression of one or several isoforms of LAMP-2 in an amount sufficient to reduce the brunt of a disease characterized by impaired or deficient autophagy (eg, Danon's disease).

[0023] The phrase "cause administration" refers to an action taken by a medical professional (e.g., physician) or person supervising the medical care of a subject, who controls and/or authorizes administration of the agent(s)/compound(s) in question to the subject. The administration may be prompted by diagnosing and/or determining an appropriate therapeutic or prophylactic regimen and/or prescribing a particular agent(s)/compounds to a subject. Such a prescription may include, for example, writing a prescription, annotating a medical record, and the like.

[0024] The phrase "in combination with" when used in relation to the use of the active agent(s) described herein (e.g., one or more isoforms of a LAMP-2 polynucleotide) in combination with one or more other drugs described as used herein (eg, an acetylcholinesterase inhibitor), the active agent(s) and other drug(s) are administered so that there is at least some chronological overlap of their physiological activities in the body. When they are not administered in combination with each other, there is no chronological overlay of physiological activity in the body. In certain preferred embodiments, the "other drug(s)" is not administered at all (eg, not co-administered) to the body.

[0025] As used herein, the terms "treat" and "treatment" refer to delaying the onset, slowing or reversing progress, lessening or alleviating or preventing the disease or condition to which the term is applied, or one or more symptoms of such disease or states.

[0026] The term "mitigation" refers to the reduction or elimination of one or more symptoms of this pathology or disease and / or to reduce the speed or delay the onset or severity of one or more symptoms of this pathology or disease and / or the prevention of this pathology or disease. In certain embodiments, the reduction or elimination of one or more symptoms of a pathology or disease may include, for example, a measurable and lasting increase in expression levels of one or more LAMP-2 isoforms.

[0027] As used herein, the phrase "consists essentially of" refers to the families or types of active pharmaceutical agents listed in the method or composition, and may additionally include other agents that, by themselves, do not have significant activity in relation to the stated indication. or goals.

[0028] The terms "subject", "individual", and "patient" interchangeably refer to a mammal, preferably a human or non-human primate, as well as domesticated mammals (e.g., canines or felines), laboratory mammals (e.g., mouse, rat, rabbit, hamster, guinea pig) and farm mammals (e.g. horse, cow, pig, sheep). In various embodiments, the subject may be a human (e.g., adult male, adult female, male adolescent, female adolescent, boy, girl) being cared for by a physician or other healthcare professional in a hospital, mental health facility, as an outpatient or in another clinical context. In certain embodiments, the subject may not have the care or prescription of a physician or other healthcare professional.

[0029] The terms "gene transfer" or "gene delivery" refer to methods or systems for securely inserting foreign DNA into host cells. Such methods can lead to transient expression of non-integrated transferred DNA, extrachromosomal replication and expression of transferred replicons (eg, episomes), or integration of the transferred genetic material into the genomic DNA of host cells.

[0030] By "AAV vector" is meant a vector derived from an adeno-associated virus of a particular serotype, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, etc. AAV vectors may have one or more wild-type AAV genes , which are deleted in whole or in part, for example, the rep and/or cap genes, but retain the sequences of functional flanking inverted terminal repeats (ITRs). Functional ITR sequences are required for rescue, replication, and packaging of the AAV virion. Thus, an AAV vector is defined herein as containing at least those sequences that are required in cis for replication and packaging (eg, functional ITRs) of the virus. ITRs are not necessarily wild-type nucleotide sequences, and can be altered, for example, by insertion, deletion, or substitution of nucleotides, as long as the sequences provide for functional rescue, replication, and packaging. AAV expression vectors are constructed using known methods to at least provide, as operably linked components in the direction of transcription, control elements including a transcription initiation region, a DNA of interest (i.e., the LAMP-2 gene), and a termination region. transcription.

BRIEF DESCRIPTION OF THE FIGURES

[0031] In FIG. 1 illustrates a scheme of adeno-associated viral constructs for the delivery of LAMP-2 genes. In general, it should be understood that said lysosome-associated membrane protein (LAMP) LAMP-2 coding region includes a ribosome binding sequence and an upstream start codon, but in some embodiments, the native element can be replaced with a heterologous one. An upstream ribosome binding site is not used in combination with coding regions downstream of an IRES or self-cleaving peptide. The start codon is not necessarily present in coding regions downstream of the self-cleaving peptide. In FIG. 1A-1F are vector genomes containing a single isoform of LAMP-2. In FIG. 1G-1K are vector genomes containing two isoforms of LAMP-2. In FIG. 1L-1O are vector genomes containing all three LAMP-2 isoforms. In FIG. the following abbreviations are used: ITR: inverted terminal repeat; LAMP-2: lysosome-associated membrane protein type 2; UTR: untranslated region; Poly A: polyadenylation signal; CAG: promoter region containing the CMV enhancer and CBA promoter sequences; CMV: cytomegalovirus; CBA: chicken β-actin; WPRE: woodchuck hepatitis virus post-transcriptional regulatory element; RBG: rabbit β-globin polyadenylation signal; EF-1: human elongation factor-1; IRES: internal ribosome binding site; P2A: 2A peptide.

[0032] FIG. 1A is a diagram of a construct containing protein coding information for one LAMP-2 isoform - A, B, or C - with conventional 5' and 3' control regions.

[0033] FIG. 1B is a schematic of a construct in some embodiments and used in the examples below, which consists of 5' and 3' inverted terminal repeat units, a CAG promoter region containing CMV enhancer and CBA promoter sequences, a CBA intron, a coding sequence for one of the LAMP- 2, including the ribosome binding sequence and upstream start codon, the WPRE sequence as the 3' UTR, and the rabbit beta globin polyadenylation signal.

[0034] FIG. 1C is a schematic 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 normally lead to LAMP-2 expression in normal cells.

[0035] FIG. 1D is a 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 a human promoter region. Other constitutively active human promoters can also be used in place of EF-1α.

[0036] FIG. 1E is a 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 the transgene only in cardiac tissue (and avoid expression in the liver, for example).

[0037] FIG. 1F is a diagram of a construct containing a muscle specific promoter such as, but not limited to, the muscle creatinine kinase promoter. In some embodiments, this construct is used to express the transgene in cardiac and skeletal muscle (and avoid expression outside of muscle).

[0038] FIG. 1G is a schematic of a construct containing the coding sequences for two LAMP-2 isoforms (using any possible combination) driven by different promoter regions. In some embodiments, this construct (as well as those in FIGS. 1H to 1K) is used to express two different isoforms of LAMP-2 using the same viral genome.

[0039] FIG. 1H is a schematic of a construct containing sequences for two LAMP-2 isoforms (using any possible combination) driven by different promoter regions. One isoform is encoded in the (+) direction on the single stranded AAV genome and the other isoform is encoded in the (-) direction. In some embodiments, this construct is used to express two different isoforms of LAMP-2 on separate strands of DNA.

[0040] FIG. 1I is a schematic of a construct containing the coding sequences for two LAMP-2 isoforms (using any possible combination) driven by the promoter region and internal ribosome binding site, respectively, followed by the 3' UTR and Poly A signals. In some embodiments, this construct used to express two different isoforms of LAMP-2 using the same viral genome.

[0041] In FIG. 1J is a schematic of a construct containing the coding sequences for two LAMP-2 isoforms (using any possible combination) separated by a P2A cleavage site. In some embodiments, this construct is used to express mRNA encoding two different isoforms of LAMP-2 in a single polypeptide that will spontaneously cleave into individual isoforms of the LAMP-2 protein after translation via self-cleavage of the 2A peptide using the same viral genome.

[0042] FIG. 1K shows a schematic of a construct containing the coding sequence for LAMP-2 exons 1-8, followed by an intron region (containing all necessary splicing signals) encoding the exon 9 sequence for one of the LAMP-2 isoforms with a 3' UTR and a Poly A signal, a second intron region (containing all necessary splicing signals), and the exon 9 coding sequence for another LAMP-2 isoform (in any possible combination) with a 3' UTR and a Poly A signal. In some embodiments, this construct is used to express mRNA for two different LAMP-2 isoforms through alternative splicing, which is the mechanism that creates different LAMP-2 mRNAs in normal cells using the same viral genome.

[0043] FIG. 1L is a schematic of a construct containing coding sequences for all three LAMP-2 isoforms, with 3' UTR and Poly A signals, driven by different promoter regions. In some embodiments, this construct is used to express all three different LAMP-2 isoforms using the same viral genome, allowing all possible functions of LAMP-2 to be restored.

[0044] FIG. 1M is a diagram of a construct containing the coding sequences for all three LAMP-2 isoforms, in any order, under the control of a promoter region and two different internal ribosome binding sites, followed by 3' UTR and Poly A signals. In some embodiments, this construct is used to express all three LAMP-2 isoforms using the same viral genome.

[0045] FIG. 1N is a diagram of a construct containing the coding sequences for all three LAMP-2 isoforms, in any possible order, separated by P2A cleavage sites. In some embodiments, this construct is used to express an mRNA encoding three different LAMP-2 isoforms in a single polypeptide that spontaneously cleaves into individual LAMP-2 isoform proteins after translation via self-cleavage of the 2A peptide using the same viral genome.

[0046] FIG. 1O is a schematic of a construct containing the coding sequence for exons 1-8 of LAMP-2, followed by an intron region (containing all the necessary splicing signals) encoding an exon 9 sequence for one of the LAMP-2 isoforms with a 3' UTR and a Poly A signal, second intron region (containing all necessary splicing signals) and exon 9 coding sequence for second LAMP-2 isoform with 3' UTR and Poly A signal, third intron region (containing all necessary splicing signals) and exon 9 coding sequence for third isoform LAMP- 2 with a 3' UTR and a Poly A signal. In some embodiments, this construct is used to express mRNA for all three LAMP-2 isoforms via alternative splicing, which is the mechanism that creates different LAMP-2 mRNAs in normal cells using the same the viral genome. Exon 9 coding sequences can be changed in any possible order and combination.

[0047] FIG. 2 illustrates a flow diagram of a method for therapeutic treatment (eg, Danon's disease or other disease caused at least in part by autophagy deficiency) by promoting the expression of one or more LAMP-2 isoforms.

[0048] FIG. 3A-F show LAMP-2 coding isoforms and protein sequences: FIG. 3A: LAMP-2A coding sequence; fig. 3B: LAMP-2A protein sequence; fig. 3C: LAMP-2B coding sequence; fig. 3D: LAMP-2B protein sequence; fig. 3E: LAMP-2C coding sequence; and fig. 3F: LAMP-2C protein sequence.

[0049] FIG. 4A-C compare the last 90 amino acid sequences of human and mouse LAMP-2A (FIG. 4A), LAMP-2B (FIG. 4B) and LAMP-2C (FIG. 4C) proteins, which show sequence identity.

[0050] FIG. 5A-B illustrate the dose-dependent increase in LAMP-2B (FIG. 5A) and LAMP-2A (FIG. 5B) mRNA expression following administration of AAV9 vectors carrying these genes.

[0051] FIG. 6 illustrates the dose-dependent increase in LAMP-2B and LAMP-2A protein expression following administration of AAV9 vectors carrying these genes.

[0052] FIG. 7A-C are fluorescence micrographs of heart sections stained with DAPI (blue) and fluorescently labeled with anti-LAMP-2 antibody (white). In FIG. 7A shows a heart section of a Lamp-2 knockout (KO) mouse treated with AAV9.LAMP-2B. In FIG. 7B shows a heart section of a KO Lamp-2 mouse treated with AAV9.EGFP. In FIG. 7C is a heart section of a wild-type (WT) mouse that has not been treated.

[0053] FIG. 8A-B show fluorescence micrographs of heart sections stained with DAPI (blue) and fluorescently labeled with anti-LAMP-2 antibody (white; some examples are marked with arrows). In FIG. 7A is a heart section of a KO Lamp-2 mouse treated with AAV9.LAMP-2B. In FIG. 7B is a heart section of a KO Lamp-2 mouse treated with AAV9.EGFP.

[0054] FIG. 9A-H illustrate the CAG-RFP-EGFP-LC3B autophagic reporter system. In FIG. 9A is a schematic representation of the operation of the system. In FIG. 9B-G' are fluorescence microscopy images; images X' (X stroke) represent an increase in the inserts indicated in the images marked with the same letter without a stroke. With respect to the mesh of FIG. 9B-G'; the first column is the red fluorescence images; the second column is the green fluorescence images; the third column is the fusion images from the first and second columns, yellow represents red+green fluorescence; the top two rows are images of heart sections from WT mice expressing the CAG-RFP-EGFP-LC3B autophagic reporter construct; the bottom two rows are heart sections from KO Lamp-2 mice expressing the CAG-RFP-EGFP-LC3B autophagic reporter construct. immature autophagosomes fluoresce in both green and red (or yellow in fused images). Mature autolysosomes fluoresce only in red. Yellow arrows indicate autophagic vacuoles fluorescing in both colors, while white arrows indicate autophagic vacuoles fluorescing in red only. In FIG. 9H illustrates the number of autophagic vacuoles, which are autophagosomes or autolysosomes in WT and KO Lamp-2 mice.

[0055] FIG. 10A-E illustrate the effect of -2 gene therapy using the CAG-RFP-EGFP-LC3B autophagic reporter system. In FIG. 10A is a sectional image of a WT mouse heart without treatment. In FIG. 10B is a sectional image of the heart showing transduced cells from a KO Lamp-2 mouse treated with the AAV9.EGFP control vector. In FIG. 1°C is a sectional image of the heart of a KO Lamp-2 mouse treated with the AAV9.LAMP-2A gene therapy vector. In FIG. 10D is a sectional view of the heart of a KO Lamp-2 mouse treated with the AAV9.LAMP-2B gene therapy vector. In the images of Fig. 10A, 1°C and 10D red arrows are used to indicate some of the autolysosomes present. In FIG. 10E illustrates the proportion of all autophagic vacuoles represented by autophagosomes and autolysosomes for four conditions.

[0056] FIG. 11A-C' are electron micrographs of heart tissue from WT (FIGS. 11A, A') and KO Lamp-2 (FIGS. 11B, B') and KO Lamp-2 mice treated with AAV9.LAMP- 2B (FIGS. 11C, C'). White arrows indicate some of the autophagic vacuoles. Black arrows indicate some of the damaged mitochondria.

DETAILED DESCRIPTION

1. Introduction

[0057] Danon's disease is caused by mutations leading to reduced or null expression of the lysosome-associated membrane protein 2 (LAMP-2, also known as CD107b) gene. These methods are based in part on the introduction of one or more polynucleotides encoding one or more isoforms of LAMP-2, eg packaged in an adeno-associated virus (AAV) vector, in order to deliver the LAMP-2 gene/isoforms to patients with Danon's disease. Following delivery of one or more polynucleotides encoding one or more LAMP-2 isoforms, the LAMP-2 transgene is expressed in the patient's own cells. Restoration of LAMP-2 gene expression in patients with Danon's disease can lead to an improvement in the disease phenotype, which serves as a therapy 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 toxicities, diabetes, end-stage renal disease, and aging. Autophagy abnormalities, in particular reduced autophagic flux, are implicated in these disorders, as well as many others. Expression of higher levels of the LAMP-2 gene increases autophagic flux and therefore serves as a treatment for these disorders.

[0058] At the present level of technology there is no treatment of Danon's disease by methods of gene therapy. Danon's disease is not a traditional lysosomal storage disease, which is generally defined as a deficiency of a lysosomal protein (eg, transporter or enzyme) that is required to process a particular cellular substrate, resulting in toxic accumulation of that particular substrate in lysosomes. Danon's disease is understood as a disorder of autophagy or autophagic vacuolar myopathy, which causes the destruction of all cellular components subject to processing by the autophagic pathway and which is not due to the accumulation of a specific substrate. Additionally, the current state of the art does not explicitly describe the delivery of the LAMP-2 gene/individual isoforms for the treatment of Danon's disease or other disorders of autophagy. Therefore, these methods provide a unique way to treat Danon's disease and improve existing AAV technologies by explicitly incorporating LAMP-2 gene delivery as a means to improve autophagy disorders.

2. Patients to be treated

[0059] Subjects/patients treatable using the methods described herein include individuals at risk for a disease or disorder that is characterized by deficient autophagic flow (e.g., Danon's disease, as well as other known disorders of autophagy, including, but not limited to, systolic and diastolic heart failure, myocardial infarction, drug toxicities (eg, anthracyclines, chloroquine and its derivatives), diabetes, end-stage renal disease, and aging) but not showing symptoms, as well as subjects currently showing symptoms. Such a subject can be identified as having a mutant LAMP-2 gene or as having reduced or undetectable levels of LAMP-2 expression.

[0060] In some embodiments, the subject exhibits symptoms of a disease or disorder that is characterized by insufficient autophagic flux (e.g., Danon's disease, as well as other known disorders of autophagy, including, but not limited to, systolic and diastolic heart failure, myocardial infarction, drug toxicities, diabetes , end stage renal disease and aging). Symptoms may be active or may be suppressed or controlled (eg, with medication) or in remission. The subject may or may not have been diagnosed with a disorder, for example, by a qualified physician.

[0061] The subject may be any mammal at any stage of development at the time of delivery, such as fetal, fetal, childhood, adolescent, or adult. In various embodiments, the subject is a child, adolescent, or adult. In various embodiments, the subject is a mammal, such as a human or a domesticated mammal (eg, canine or feline).

3. LAMP-2 polynucleotide delivery vectors

[0062] In general, the gene therapy vectors described herein comprise an expression cassette containing a polynucleotide encoding one or more isoforms of lysosome-associated membrane protein 2 (LAMP-2) that allows expression of LAMP-2 for partial or completely correcting deficient LAMP-2 protein expression levels and autophagic flux in a subject in need thereof (e.g., a subject having Danon's disease or another disorder characterized by deficient autophagic flux due at least in part to deficient LAMP-2 expression). Vectors for gene therapy can be viral or non-viral vectors. Illustrative non-viral vectors include, for example, deproteinized DNA, cationic liposomal complexes, cationic polymer complexes, cationic liposomal polymer complexes, and exosomes.

[0063] In some embodiments, the implementation of the vector carries one isoform, LAMP-2A, LAMP-2B or LAMP-2C; see, for example, FIG. 1A-F, which is a diagram of an AAV vector carrying a gene for one isoform of LAMP-2. In other embodiments, the implementation of the vector carries genes for two isoforms of LAMP-2; see, for example, FIG. 1G-K, which is a schematic of AAV vectors carrying the gene for two LAMP-2 isoforms, LAMP-2B on one DNA strand and LAMP-2A on the other DNA strand. Other embodiments carry all three isoforms (see FIG. 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 may be two promoters, one driving the expression of one isoform and the other driving the expression of a dual isoform cassette using an IRES, a self-cleaving peptide, or alternative splicing. In various embodiments containing multiple isoforms, the isoforms occur in any order, either reflecting the natural order of B, A, C, or changing it. With a single vector carrying multiple isoforms, it is possible to co-express isoforms in transfected cells and use fewer full vector particles per dose.

[0064] Examples of a viral vector include, but are not limited to, adenoviral, retroviral, lentiviral, herpesvirus vectors, and adeno-associated virus (AAV) vectors. Viral gene delivery vectors that can be used in the practice of the present invention can be constructed using methods well known in the field of molecular biology. Typically, viral vectors carrying transgenes are assembled from transgene-encoding polynucleotides, appropriate regulatory elements, and elements necessary for the production of viral proteins that mediate cell transduction,

[0065] Such recombinant viruses can be obtained by methods known in the art, for example, by transfection of packaging cells or by transient transfection using helper plasmids or viruses. Typical examples of virus packaging cells include, but are not limited to, PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc. Detailed protocols for the production of such replication-defective recombinant viruses can be found, for example, in WO95/14785, WO96/22378 , US Pat. No. 5,882,877, US Pat. No. 6,013,516, US Pat. No. 4,861,719, US Pat. No. 5,278,056, and WO94/19478, the entire contents of each of which are hereby incorporated by reference.

[0066] In some embodiments, the gene viral vector is an adenoviral vector or an adeno-associated virus (AAV) vector. In various embodiments, 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 genomes (scAAV) or any other serotypes AAVs 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 another embodiment, the AAV vector is AAV8. Often, recombinant AAV (rAAV) vectors, which have been studied in human clinical trials, are used to deliver therapeutic genes. rAAV vectors can be designed to deliver specific transgenes to patient cells for expression. After infection and introduction of the viral genome with the rAAV vector, the viral genes exist primarily as extrachromosomal structures that do not integrate into the host genome, but are expressed by the host cell's translation apparatus. Several components are required for successful host cell infection and rAAV gene expression (see Figure 1): inverted terminal repeat (ITR) elements, a promoter and/or enhancer region, a transgene and a 3' untranslated region, and a polyadenylation signal. Upon viral infection of host cells, the promoter region initiates signals for translation of the virus-delivered transgene by the host cell's translation machinery.

[0067] In general, controls are selected that are functional in the mammalian cell. The resulting construct, which contains operably linked components, is linked (5' and 3') to functional AAV ITR sequences. By "adeno-associated viral inverted terminal repeats" or "AAV ITR" is meant the regions accepted in the art that occur at each end of the AAV genome that function together in cis as origins of replication for DNA replication and as packaging signals for the virus. The AAV ITR, together with the AAV rep coding region, allows efficient excision and rescue as well as insertion of the nucleotide sequence located between the two flanking ITRs into the mammalian cell genome. The nucleotide sequences of the AAV ITR regions are known. For AAV2 sequences see, for example, Kotin, 1994; Berns, K I "Parvoviridae and their Replication" in Fundamental Virology, 2nd ed., (Eds. B. N. Fields and D. M. Knipe, the entire contents of which are hereby incorporated by reference). As used herein, "AAV ITR" does not necessarily contain a wild-type nucleotide sequence, and it can be changed, for example, by insertion, deletion or substitution of nucleotides.

[0068] Additionally, AAV ITR can be derived from any of several AAV serotypes, including but not limited to AAV1, AAV2, AAV3, AAV4, AA5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh10. In addition, the 5' and 3' ITRs that flank a selected nucleotide sequence in an AAV vector need not be identical or derived from the same AAV serotype or isolate, provided they perform their intended function, i.e., do allow expression and rescue of the sequence of interest from the genome of the host cell or vector and allow the insertion of the heterologous sequence into the genome of the recipient cell when AAV Rep gene products are present in the cell. Additionally, AAV ITR can be derived from any of several AAV serotypes, including but not limited to AAV1, AAV2, AAV3, AAV4, AA5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh10. In addition, the 5' and 3' ITRs that flank the selected nucleotide sequence in the AAV expression vector do not have to be identical or derived from the same AAV serotype or isolate, provided they serve their intended function, i.e. allow the excision and rescue of a sequence of interest from the genome of the host cell or vector; and allow the insertion of the DNA molecule into the genome of the recipient cell when AAV Rep gene products are present in the cell.

[0069] In various embodiments, vectors derived from AAV serotypes having tropism for, and high transduction efficiencies in, mammalian myocardial cells, in particular cardiomyocytes and cardiomyocyte progenitors, are used. For a review and comparison of transduction efficiencies in various serotypes, see Cearley C N et al., Molecular Therapy 16(10); 1710-1718, 2008, the entire contents of which are hereby incorporated 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 also shown to transduce cardiomyocyte cells .

[0070] 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 may contain control sequences typically associated with the selected gene.

[0071] Alternatively, heterologous escape sequences can be used. Effective heterologous control sequences generally include those derived from mammalian or viral coding sequences. Examples include, but are not limited to, phosphoglycerate kinase (PKG) promoter, CAG promoter (CMV enhancer with chicken β-actin promoter up to the 1st intron and rabbit beta-globin gene splicing acceptor), MCK (muscle creatine kinase) promoter ), SV40 early promoter, mouse mammary tumor virus LTR promoter; adenovirus major late promoter (Ad MLP); herpes simplex virus (HSV) promoter, cytomegalovirus (CMV) promoter such as the CMV immediate early promoter (CMVIE) region, Rous sarcoma virus (RSV) promoter, synthetic promoters, hybrid promoters, and the like. Promoters can be derived from humans or other species , including from mice. In addition, sequences derived from non-viral genes, such as the marine animal metallothionein gene, will also find use in the present disclosure. Such promoter sequences are commercially available, for example, from Stratagene (San Diego, Calif.). Examples of heterologous promoters include, but are not limited to, the CMV promoter. Examples of inducible promoters include, but are not limited to, DNA responsive elements for ecdysone, tetracycline, and hypoxiandauphin. When a single vector encodes multiple isoforms, they are preferably, but not necessarily, operably linked to different control sequences.

[0072] Similarly, control elements at the 3'end of the coding region, including the 3'untranslated region (3'UTR) and the polyadenylation signal, may be from an inserted gene or from a heterologous source. In specific embodiments, woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) is used as the 3' UTR or rabbit beta-globin polyadenylation signal, or both. 3' controls may also include controls connecting to coding regions. For example, it is possible to express multiple LAMP-2 isoforms under the control of a single promoter by placing an internal ribosome binding site (IRES) (see, for example, FIGS. 1I and 1M) or a self-cleaving peptide sequence (such as picornavirus peptide 2A; see, for example, Fig. 1J and 1N) between the 1st and 2nd and 2nd and 3rd (if present) isoforms. In the case of IRES, 2 or 3 polypeptides are translated. In the case of a self-cleaving peptide, the LAMP-2 isoforms along with the intermediate self-cleaving peptide are present in the vector as a single reading frame, which is translated as a single polypeptide, which is cleaved into its replacement LAMP-2 isoforms after or during translation.

[0073] Transcription under the control of a single promoter can also be achieved when the vector is designed to support alternative splicing. In such constructs, the first eight exons of LAMP-2 are present spliced together in the cDNA, followed by two or three intron-exon pairs to include two or all three alternative exons that give rise to the three LAMP-2 isoforms (see e.g. , Fig. 1K and 1O). In various embodiments, the intron-exon pairs occur in any order, either reflecting the natural order B, A, C, or changing it. Native introns are too long to be included in vectors that are size limited such as AAV. In such cases, the introns are truncated by removing the central sequences while retaining the necessary 5' and 3' splicing sites and splicing signals. Typically, only a few bases at the 5' end of the intron and approximately 100-200 bases at the 3' end of the intron are sufficient to allow for splicing. Alternatively, heterologous introns can be replaced with native introns.

[0074] An AAV expression vector that carries the DNA molecule of interest restricted by the AAV ITR can be constructed by directly inserting the selected sequence(s) into the AAV genome from which the AAV basic open reading frames (“ORFs”) have been excised. Other portions of the AAV genome may also be removed, provided that a sufficient portion of the ITR remains to allow for replication and packaging functions. Such designs can be developed using methods well known in the art. See, for example, US Pat. Nos. 5,173,414 and 5,139,941; international publications 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 full contents of each of which are hereby incorporated by reference.

[0075] Alternatively, the AAV ITR can be excised from the viral genome or from an AAV vector containing the same and 5' and 3' fusion of the selected nucleic acid construct that is present in another vector using standard ligation techniques. AAV vectors that contain ITRs are described, for example, in US Pat. No. 5,139,941, the entire contents of which are hereby incorporated by reference. In particular, it describes several AAV vectors that are available from the American Type Culture Collection ("ATCC") under accession numbers 53222, 53223, 53224, 53225, and 53226. Additionally, chimeric genes can be synthetically generated that contain AAV ITR sequences located 5 ' and 3' from one or more selected nucleic acid sequences. Preferred codons may be used to express the chimeric gene sequence in mammalian CNS cells. The complete chimeric sequence is assembled from overlapping oligonucleotides prepared by standard methods. See, for example, Edge Nature, vol. 292, 1981, p. 756; Nambair et al., Science, vol. 223, 1984, p. 1299; Jay et al., J. Biol. Chem. vol. 259, 1984, p. 6311, the entire contents of each of which are hereby incorporated by reference. In order to produce AAV virions, an AAV expression vector is introduced into a suitable host cell using known methods, for example, by transfection. In general, a variety of transfection methods are known in the art. See, for example, 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. In particular, 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 entire contents of each of the above sources are hereby incorporated by reference in their entirety.

[0076] In one embodiment, the AAV comprises, in addition to the nucleic acid sequence(s) encoding the LAMP-2 isoform, an AAV vector backbone with an ITR derived from AAV2, a promoter such as the mouse PGK (phosphoglycerate kinase) gene promoter or a fusion promoter cytomegalovirus/chicken β-actin (CAG) that contains an enhancer from the immediate cytomegalovirus gene and a promoter, a splice donor and intron from the chicken β-actin gene, a splicing acceptor site from rabbit β-globin, or any promoter such as the PGK promoter, CAG , MCK, EF-1 or native LAMP-2 promoter. In various embodiments, the viral vector is encapsulated in an anionic liposome, for example, as described in US Patent Publication No. 2015/0284691.

[0077] In various embodiments, the expression cassette structure in the vector contains 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., promoter cytomegalovirus (CMV) or chicken β-actin promoter) with or without any known enhancer element (e.g., CMV enhancer), lysosome-associated protein 2 gene (including 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 or ).

[0078] Translation of a virus-delivered LAMP-2 isoform gene or a combination of different isoform genes results in the expression of that particular isoform of the LAMP-2 protein, which is then targeted to the host cell's lysosomal membranes by the host cell machinery. Restoration of LAMP-2 isoform(s) expression and function then restores the autophagic flux (potentially including, but not necessarily limited to, all known forms of autophagy such as macroautophagy, mitophagy, chaperone-mediated autophagy, and DNA/RNA autophagy), which is deficient and causes patients with Danon's disease, which allows the affected host cells to eliminate toxic cellular components and damaged organelles and thus increase cell function and survival. Ultimately, restoring the expression of LAMP-2 isoform(s) serves to treat the underlying genetic deficiency that causes Danon's disease and attenuates the phenotype and symptoms of the disease. In other autophagy disorders where LAMP-2 can be expressed but not at a sufficient level to generate sufficient autophagic flux and thus promote normal cell function and survival, delivery of LAMP-2 transgenic isoform(s) via the rAAV vector leads to overexpression of the resulting proteins (protein) LAMP-2. This overexpression restores autophagic flux to nearly normal, normal, or above normal levels. Restoration of autophagic flux in a disease that causes disruption of normal autophagy then alleviates, alleviates, and/or reverses the phenotype and symptoms of the disease.

4. Vector pharmaceutical compositions

[0079] Pharmaceutical compositions are provided, e.g., for use in the prevention or treatment of a disorder that is characterized by insufficient autophagic flux (e.g., Danon's disease), which contain a therapeutically effective amount of a vector that contains a nucleic acid sequence of a polynucleotide that encodes one or more isoforms of LAMP -2.

[0080] It is understood that the decision on a single dose or total daily dose of the compounds and compositions of the present invention will be made by the attending physician within the framework of sound medical judgment. The specific level of therapeutically effective dose for any particular patient depends on various factors, including the disorder being treated and the severity of the disorder; the activity of the particular compound used; the particular composition used, the age, body weight, general health, sex, and diet of the patient; the time of administration, route of administration, and rate of excretion of the particular compound used; duration of treatment; drugs used in combination or incidentally with the specific nucleic acid or polypeptide used; and similar factors well known in the medical field. For example, skill in the art would well permit starting doses of a compound at levels below those needed to achieve the desired therapeutic effect and gradually increasing the dose until the desired effect is achieved. However, the daily dose of products may vary over a wide range per adult per day. The therapeutically effective amount of the vector according to the invention to be administered, as well as the dosage for treating a disease state using a variety of viral or non-viral particles and/or pharmaceutical compositions described herein, will depend on a variety of factors, including age and condition. the patient, the severity of the disorder or disorder, the route and frequency of administration, and the particular peptide to be used.

[0081] Pharmaceutical compositions that contain the vector may be in any form that is suitable for the chosen mode of administration, for example, for intraventricular, intramyocardial, intracoronary, intravenous, intraarterial, intrarenal, intraurethral, epidural, or intramuscular administration. A gene therapy vector containing a polynucleotide encoding one or more isoforms of LAMP-2 can be administered, as the sole active agent or in combination with other active agents, in a standard form for administration, as a mixture with standard pharmaceutical carriers, animal and human .

[0082] In various embodiments, the implementation of the pharmaceutical compositions contain carriers that are pharmaceutically acceptable for the composition, which can be injected. In particular, they can be isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride, etc., or mixtures of such salts) or dry, in particular lyophilized compositions which, when added, depending on the case, sterilized water or saline, allow the preparation of injectable solutions.

[0083] 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 protected from the contaminating action of microorganisms such as bacteria and fungi.

[0084] Solutions containing gene therapy vectors as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant such as hydroxypropyl cellulose. Dispersions can also be obtained in glycerol, liquid polyethylene glycols and mixtures thereof, and in oils. Under normal conditions of storage and use, these preparations contain a preservative in order to prevent the growth of microorganisms.

[0085] Vectors for gene therapy can be formulated in the composition in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein) and those formed with inorganic acids such as, for example, hydrochloric or phosphoric acid, or organic acids such as acetic, oxalic, tartaric, mandelic, etc. n. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium or iron hydroxides, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc.

[0086] The carrier may also be as a solvent or dispersion medium containing, for example, water, ethanol, polyol (eg, glycerol, propylene glycol and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the desired particle size in the case of a dispersion, and by using surfactants. Prevention of the action of microorganisms can be achieved by using various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. 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 through the use of agents in the compositions that delay absorption, such as aluminum monostearate and gelatin.

[0087] Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in a suitable solvent with several other ingredients listed above, if necessary, followed by filtered sterilization. In general, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and other necessary ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and lyophilization methods which yield a powder of the active ingredient plus any additional desired ingredient from its pre-sterile filtered solution.

5. Ways to treat autophagy diseases

[0088] Additionally provided are methods for preventing, mitigating, improving, reducing, inhibiting, eliminating and/or reversing one or more symptoms of Danon's disease or other autophagy disorder in a subject in need thereof, comprising administering to the subject a gene therapy vector as described previously and in the present description, for example, an adeno-associated virus (AAV) vector containing an expression cassette containing a polynucleotide encoding one or more isoforms of lysosome-associated membrane protein 2 (LAMP-2). The vector is delivered to a subject in need thereof, whereby the transduced cells express a polynucleotide encoding one or more LAMP-2 isoforms at a therapeutically effective level.

[0089] 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 a further aspect of these embodiments, they are used in particular in the treatment of Danon's disease.

[0090] 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, subcutaneous, and intramuscular. In various embodiments, the vector is delivered directly to the myocardium via epicardial injection via minithoracotomy, via intracoronary injection, via endomyocardial injection, or via another type of injection that can be used in the heart. Additional routes of administration may also include local application of the vector in direct imaging, such as superficial cortical application or other non-stereotactic application.

[0091] Viral vectors typically elicit an immune response, which may include the formation of antibodies that may reduce or completely inhibit the efficacy of a particular vector in that individual when repeated dosing becomes necessary or desirable. Repeated dosing may become appropriate, for example, since the vector may be lost over time due to cell proliferation, in particular episomal vectors. Tissue that is more difficult to access may require multiple injections of the vector in order to transfect enough cells to effectively treat the disease. Transgene expression can also be lost over time. The need to introduce a gene therapy vector due to inhibition of antibody formation can be met in various ways. For example, antibody titers can be reduced by apheresis prior to vector administration, or the patient can be immunosuppressed using an appropriate medical regimen. Alternatively, an empty AAV capsid can be administered to bind host antibodies and immune system cells prior to injection of a therapeutic AAV containing the LAMP-2 transgene(s). Depending on the target tissue, direct topical administration instead of systemic administration may be effective in overcoming or improving the inhibitory effect of antibodies against the vector. In yet another approach, a vector based on a different serotype of virus from which it was derived can be used. For example, if an AAV9 vector was originally used and there is a problem with anti-AAV antibody titer, but there is a need for additional gene therapy, an AAV8 vector carrying the same (or different) gene construct can be introduced.

[0092] Once formulated, solutions may be administered in a manner that is compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are readily administered in various dosage forms such as the injectable type described above, but drug-release capsules and the like may also be used. Multiple doses may also be administered.

[0093] As appropriate, the vectors described herein may be formulated in any suitable delivery vehicle. For example, they can be placed in a pharmaceutically acceptable suspension, solution or emulsion. Suitable media include saline and liposome preparations. More specifically, pharmaceutically acceptable carriers may include sterile aqueous or 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 buffer media. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like.

[0094] Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases, and the like.

[0095] A colloidal dispersion system can also be used for targeted gene delivery. Colloidal dispersion systems include macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes.

[0096] The physician may determine an appropriate schedule, which depends on the subject's age, sex, weight, and disease stage. For example, to deliver 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 a composition comprising the viral expression vector in a pharmaceutically acceptable fluid, for example, at a concentration in the range of 10 ¹¹ to 10 ¹⁶ viral genomes per ml.

[0097] Some schemes use a vector carrying a gene for one isoform of LAMP-2, such as LAMP-2B. Other schemes use a vector carrying a gene for two LAMP-2 isoforms, eg LAMP-2B and LAMP-2A. In some embodiments, different preparations of vectors carry two isoforms. In one aspect, both vector preparations are derived from the same vector, such as the AAV9 vector or any other specific vector described above. In other embodiments, both polypeptides are encoded in the same vector; see, for example, FIG. 1. Similarly, if the scheme includes the introduction of nucleic acid sequences encoding all three isoforms, the isoforms may carry separate vectors or a single vector. In schemes using multiple isoforms that carry separate vectors, multiple vectors can be entered simultaneously, individually or mixed together, or they can be entered sequentially at intervals of hours, days or weeks.

6. Sets

[0098] Additionally provided are kits containing a gene therapy vector containing a polynucleotide encoding one or more isoforms of LAMP-2, as previously described and herein. In various embodiments, the kits provide gene therapy vectors provided in one or more unit dosage forms ready for administration to a subject, eg, in a pre-filled syringe or ampoule. In various embodiments, the gene therapy vector is provided in lyophilized form.

EXAMPLES

Example 1 AAV9 Gene Therapy

[0099] Specific vectors as described above were constructed as an AAV9 vector with ITRs derived from AAV2 that encode LAMP-2A or LAMP-2B under the control of the chicken β-actin promoter with CMV enhancer (CAG promoter). Additionally, these vectors contained the woodchuck hepatitis virus post-transcriptional regulatory element and the rabbit β-globin polyadenylation signal (see Figure 1B). These vectors were obtained from the National Heart, Lung, and Blood Institute Gene Therapy Resource Program Preclinical Vector Core Facility at the University of Pennsylvania. Human LAMP-2 sequences were inserted with the expectation that they would be functional in the mouse due to the high degree of sequence identity between homologues, particularly in the C-terminal region that forms the components of the protein's transmembrane and cytoplasmic tail (see Figure 4).

[0100] The vector is used to deliver LAMP-2 isoforms in vivo. The AAV9 serotype has excellent tropism for cardiac and skeletal muscle, as well as nervous tissue, the organs most affected by Danon's disease. However, Danon's disease is a multisystem disorder in which many different organs can be involved; therefore, the expression of transgenic LAMP-2 isoforms is driven by a constitutively active chicken β-actin (CBA) promoter with a cytomegalovirus (CMV) enhancer (CAG construct). This promoter is ubiquitously active and allows the expression of the LAMP-2 transgene in all tissues, depending on the effectiveness of the infection. An AAV9 vector carrying an enhanced green fluorescent protein (eGFP) gene was also prepared for use as a control reagent.

Example 2 Administration of AAV9 vectors with LAMP-2 isoforms to LAMP-2 knockout mice.

[0101] mice with KO Lamp-2 (Nature. 2000 Aug 24;406(6798):902-6; Basic Res Cardiol. 2006 Jul;101(4):281-91) develop a syndrome similar to Danon's disease, although disease severity is more moderate than in human disease, probably due at least in part to the shorter lifespan of mice, allowing less time for damage to accumulate due to reduced autophagic flux. Therefore, it is desirable to wait until the mice are at least 3 months old, and preferably 5-6 months old, before initiating treatment so that there is sufficient accumulation of pathology so that its reversal is easily detectable.

[0102] In an ideal procedure, KO Lamp-2 mice at 6 months of age receive 5×10 ¹¹ , 1×10 ¹² , or 2×10 ¹² genomic copies (gc) of the AAV vector via intravenous injection into the external jugular vein. In various experiments, WT mice receive LAMP-2 isoform vectors and KO Lamp-2 mice receive an eGFP vector as controls. Sufficient mice are used so that subpopulations can be sacrificed and assessed at various time points, eg 1, 2 and 6 months after administration.

Example 3 Transcription Evaluation of LAMP-2 Gene Isoform After Vector Introduction

[0103] KO Lamp-2 mice, approximately 3-4 months of age, received escalating doses of AAV9.LAMP-2B and AAV9.LAMP-2A vectors (see FIG. 1B and descriptions above) at 5×10 ¹¹ , 1 ×10 ¹² and 2×10 ¹² g.k./mouse. One mouse was sacrificed for each condition and RT-qPCR was performed in triplicate on split heart tissue in order to assess mRNA expression (gene transcription) of human transgenes. an untreated WT mouse was used to demonstrate the lack of expression of human LAMP-2 isoforms. The data suggest a dose-dependent increase in the expression of human transgenes (see Fig. 5).

Example 4 Evaluation of LAMP-2 protein isoform expression after vector injection

[0104] Using the same mice as in Example 3, immunoblotting was performed on split heart tissue in order to evaluate human transgene protein expression for GAPDH. The KO Lamp-2 control mouse treated with AAV9.EGFP did not show significant LAMP-2 protein expression. The data suggest a dose-dependent increase in human transgene expression in mice receiving viral vectors (see FIG. 6).

Example 5 Subcellular Location of Transgenic LAMP-2Bs after Vector Introduction

[0105] Sections of the heart of a KO Lamp-2 mouse treated with 2×10 ¹² g. k./mouse of either AAV9.LAMP-2B or AAV9.EGFP, which was sacrificed 1 month after delivery, were stained using DAPI (which binds to AT-rich DNA and thus makes cell nuclei visible) and a fluorescently labeled anti-LAMP-2 antibody (see Fig. 7). The LAMP-2 staining pattern indicates the localization of the transgenic human LAMP-2B protein in intracellular vacuoles and is similar to the staining observed in WT control mice for the mouse Lamp-2 protein. No human LAMP-2 staining was observed in a KO Lamp-2 mouse receiving a control AAV9.EGFP vector. These data demonstrate that treatment with the AAV9.LAMP-2B vector leads to expression of the human LAMP-2B protein at a physiologically appropriate location.

[0106] In a similar experiment, human LAMP-2 staining was maintained 2 months after delivery of AAV9.LAMP-2B at 5×10 ¹¹ g. k./mouse to the test mouse. Although in accordance with example 4, less staining was observed using this lower dose of vector. As a comparison, no human LAMP-2 staining was detected 3 months after delivery of 5×10 ¹¹ g.k./mouse AAV9.EGFP vector. See fig. eight.

Example 6 CAG-RFP-EGFP-LC3B Autophagic Reporter System

[0107] The CAG-RFP-EGFP-LC3B autophagic reporter system allows assessment of macroautophagic flux. Light chain 3 (LC3) of microtubule associated protein 1 is expressed on the surface of autophagosomes. Fusion genes are constructed to express LC3 fused to both red fluorescent protein (RFP) and eGFP. This fusion protein is expressed, the RFP component fluoresces in both autophagosomes and lysosomes, while the eGFP component fluoresces in autophagosomes but is quenched by the acidic environment of lysosomes. As a result, in the fused image, autophagosomes are yellow and lysosomes are red (see Fig. 9A). When expressed in WT context, more red dots than green dots are seen in the individual red and green images and a mixture of red and yellow dots in the fused images (see FIGS. 9B-D'). Lamp-2 is required for normal fusion of autophagosomes with lysosomes to form autolysosomes. Thus, when this construct is expressed in a KO Lamp-2 mouse, approximately equal numbers of red and green dots are observed in separate red and green images, with nearly all yellow dots in fused images (see FIGS. 9E-G'). Accumulation of autophagosomes and almost no autolysosomes, along with an overall higher number of autophagic vacuoles (AV), reflects a defect in autophagic flow due to the absence of Lamp-2 (see Fig. 9H).

Example 7 Introduction of a Vector Carrying either LAMP-2A or LAMP-2B Restores Autophagic Flux

[0108] KO Lamp-2 mice expressing the CAG-RFP-EGFP-LC3B construct (KO Lamp-2/CAG-RFP-EGFP-LC3B reporter mice) were treated with AAV9.LAMP-2B or AAV9.LAMP-2A and compared with KO Lamp-2 mice expressing the CAG-RFP-EGFP-LC3B autophagic reporter system (CAG-RFP-EGFP-LC3B reporter mice). One month after vector delivery, mice were sacrificed and heart sections were assessed by fluorescence microscopy in order to assess autophagic flux. mice with KO Lamp-2/reporter without treatment continued to show almost only yellow autophagic vacuoles, ie autophagosomes (see Fig. 10B and Fig. 9G'). In contrast, KO Lamp-2/reporter mice receiving either gene therapy vector, AAV9.LAMP-2B or AAV9.LAMP-2A, showed many red autophagic vacuoles, which are autolysosomes, in a similar proportion to the control reporter. mouse WT (see Fig. 10 A, C and D, exemplary points on them are indicated by arrows). AV quantification demonstrated that the ratio of immature autophagosomes to mature autolysosomes is similar in KO Lamp-2 treated mice when compared to WT mice (see FIG. 10E). Thus, gene therapy using AAV9.LAMP-2B or AAV9.LAMP-2A restored normal autophagosome-lysosome fusion in KO Lamp-2 mouse cardiomyocytes.

Example 8 Restoration of Cardiomyocyte Ultrastructure as Assessed by Electron Microscopy

[0109] KO Lamp-2 mice were injected intravenously with 5×10 ¹¹ g. k./mouse AAV9.LAMP-2B, and compared with a retrospective mouse of the same age with KO Lamp-2 and WT mouse, which were not treated. Mice were sacrificed 1 month after vector delivery and heart sections were analyzed by electron microscopy. Compared to WT (see Fig. 11A, A'), the untreated KO Lamp-2 mouse showed AV accumulation and enlargement (see Fig. 11B, B', yellow arrows) and an increase in abnormal mitochondria (see Fig. 11B, B', yellow arrows). Fig. 11B, red arrows). In contrast, electron micrographs of a KO Lamp-2 mouse treated with AAV9.LAMP-2B more closely resembled the ultrastructure of an untreated WT mouse (see FIG. 11C, C'). Thus, treatment with a gene therapy vector restores the ultrastructure of cardiomyocytes.

[0110] Briefly, these examples show that adeno-associated virus gene therapy vectors encoding LAMP-2A and LAMP-2B isoforms can be administered intravenously to successfully achieve transgene expression in heart tissue. Additionally, such expression leads to the reversal of defects in autophagic flux and cardiomyocyte ultrastructure, defects that are also associated with Danon's disease. These data support the use of such gene therapy vectors in the treatment of Danon's disease and other disorders associated with defects in autophagic flow.

[0111] In conclusion, it should be understood that while aspects of this disclosure are illuminated through reference to specific embodiments, one skilled in the art will readily appreciate that these disclosed embodiments are only illustrative of the principles of the subject matter disclosed in the present description. Therefore, it should be understood that the disclosed subject matter is in no way limited to the specific method, protocol and/or reagents, etc., which are described in the present description. As such, various modifications or changes or alternative configurations to the disclosed object of the invention can be made in accordance with the provisions of the present description, without departing from the essence of this description. Finally, the terminology used in the present description is only for the purpose of describing specific embodiments and is not intended to limit the scope of the present invention, which is defined only by the claims. Accordingly, the present invention is not limited to this, exactly as shown and described.

[0112] Certain embodiments of the present invention are described in the present description, including the best option known to the inventors for carrying out the invention. Of course, variations in these described embodiments will be apparent to those skilled in the art upon reading the above description. The inventor expects those skilled in the art to use such variations as the case may be, and the inventors contemplate practicing the present invention in a manner other than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended herein, as applicable law permits. In addition, any combination of the embodiments described above, in all their possible variations, is included in the invention, unless otherwise indicated in the present description or otherwise contrary to the context.

[0113] The groupings of alternative embodiments, elements or steps of the present invention should not be construed as limitations. Each element of the group can be specified and declared individually or in any combination with other elements of the group disclosed in the present description. It is contemplated that one or more members of the group may be included in or removed from the group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the description is deemed to include the group as modified, thus fulfilling the written description of all Markush groups used in the appended claims.

[0114] Unless otherwise indicated, all numbers expressing characteristic, element, quantity, parameter, property, term, and so on, used in the present description and claims, should be understood as modified in all cases by the term "approximately". As used herein, the term "approximately" means that a characteristic, element, quantity, parameter, property, or member thus defined spans a range of ±10% above and below the value of the specified characteristic, element, quantity, parameter, property, or member. . Accordingly, unless otherwise indicated, the numerical parameters set forth in the description and the appended claims are approximations that may vary. As a last resort, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical designation should be read at least in the light of the number of significant digits given and using the usual methods of rounding. While these numerical ranges and values defining the broad scope of the invention are approximations, the numerical ranges and values set forth in the specific examples are given as accurately as possible. However, any numerical range or value inherently contains certain errors that are an inherent result of the standard deviation encountered in their respective test measurements. The presentation of numerical value ranges in this specification is only intended to serve as a shorthand way of indicating individually each individual numerical value falling within the range. Unless otherwise indicated in the present description, each individual value of the numerical range is included in this description, as if they were individually listed in the present description.

[0115] Singular forms used in the context of the description of the present invention (in particular, in the context of the following claims) should be interpreted as covering the singular and plural, unless otherwise indicated in the present description or does not conflict with the context explicitly. All of the methods described herein can be performed in any suitable order, unless otherwise indicated in the present description or otherwise expressly contradicted by the context. The use of any and all examples or approximate language (eg, "such as") given in the present description is intended only to more fully illuminate the present invention and does not impose a limitation on the scope of the invention, as claimed otherwise. Nothing in this specification should be construed as an indication of any unclaimed element required for the practice of the invention.

[0116] Specific embodiments disclosed herein may further be limited in the claims using the wording consists of or essentially consists of. When used in a claim, as given or as added in an amendment, the transitional term "consisting of" excludes any element, step, or ingredient not listed in the claims. The transitional term "consisting essentially of" limits the scope of the claims to precisely defined materials or steps and that does not significantly affect the essential and novel feature(s). Embodiments of the present invention, as claimed, are inherently or explicitly described and used in the present description.

[0117] All patents, patent publications and other publications mentioned and identified in the present description are individually and expressly incorporated herein by reference in their entirety for the purpose of describing and disclosing, for example, the compositions and methods described in such publications, which can be used in connection with the present invention. These publications are provided for disclosure only prior to the filing date of this application. Nothing in this connection should be construed as an assumption that the inventors are not entitled to predate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the content of these documents are based on the information available to the applicants and do not constitute any admission as to the correctness of the dates or the content of these documents.

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SEQUENCE LIST

<110> The Regents of the University of California

<120> TREATMENTS FOR DANON DISEASE AND OTHER AUTOPHAGY DISORDERS

<130> 1959169-00006

<150> US62/280,269

<151> 2016-01-19

<160> 12

<170> PatentIn version 3.5

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Thr Thr Thr Pro Thr Pro Lys Glu Lys Pro Glu Ala Gly Thr Tyr Ser

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Val Asn Asn Gly Asn Asp Thr Cys Leu Leu Ala Thr Met Gly Leu Gln

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Leu Asn Ile Thr Gln Asp Lys Val Ala Ser Val Ile Asn Ile Asn Pro

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Asn Thr Thr His Ser Thr Gly Ser Cys Arg Ser His Thr Ala Leu Leu

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Arg Leu Asn Ser Ser Thr Ile Lys Tyr Leu Asp Phe Val Phe Ala Val

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Lys Asn Glu Asn Arg Phe Tyr Leu Lys Glu Val Asn Ile Ser Met Tyr

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Leu Val Asn Gly Ser Val Phe Ser Ile Ala Asn Asn Asn Leu Ser Tyr

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Trp Asp Ala Pro Leu Gly Ser Ser Tyr Met Cys Asn Lys Glu Gln Thr

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Val Ser Val Ser Gly Ala Phe Gln Ile Asn Thr Phe Asp Leu Arg Val

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Gln Pro Phe Asn Val Thr Gln Gly Lys Tyr Ser Thr Ala Gln Asp Cys

355 360 365

Ser Ala Asp Asp Asp Asn Phe Leu Val Pro Ile Ala Val Gly Ala Ala

370 375 380

Leu Ala Gly Val Leu Ile Leu Val Leu Leu Ala Tyr Phe Ile Gly Leu

385 390 395 400

Lys His His His Ala Gly Tyr Glu Gln Phe

405 410

<210> 11

<211> 410

<212> Protein

<213> Homo sapiens

<400> 11

Met Val Cys Phe Arg Leu Phe Pro Val Pro Gly Ser Gly Leu Val Leu

1 5 10 15

Val Cys Leu Val Leu Gly Ala Val Arg Ser Tyr Ala Leu Glu Leu Asn

20 25 30

Leu Thr Asp Ser Glu Asn Ala Thr Cys Leu Tyr Ala Lys Trp Gln Met

35 40 45

Asn Phe Thr Val Arg Tyr Glu Thr Thr Asn Lys Thr Tyr Lys Thr Val

50 55 60

Thr Ile Ser Asp His Gly Thr Val Thr Tyr Asn Gly Ser Ile Cys Gly

65 70 75 80

Asp Asp Gln Asn Gly Pro Lys Ile Ala Val Gln Phe Gly Pro Gly Phe

85 90 95

Ser Trp Ile Ala Asn Phe Thr Lys Ala Ala Ser Thr Tyr Ser Ile Asp

100 105 110

Ser Val Ser Phe Ser Tyr Asn Thr Gly Asp Asn Thr Thr Phe Pro Asp

115 120 125

Ala Glu Asp Lys Gly Ile Leu Thr Val Asp Glu Leu Leu Ala Ile Arg

130 135 140

Ile Pro Leu Asn Asp Leu Phe Arg Cys Asn Ser Leu Ser Thr Leu Glu

145 150 155 160

Lys Asn Asp Val Val Gln His Tyr Trp Asp Val Leu Val Gln Ala Phe

165 170 175

Val Gln Asn Gly Thr Val Ser Thr Asn Glu Phe Leu Cys Asp Lys Asp

180 185 190

Lys Thr Ser Thr Val Ala Pro Thr Ile His Thr Thr Val Pro Ser Pro

195 200 205

Thr Thr Thr Pro Thr Pro Lys Glu Lys Pro Glu Ala Gly Thr Tyr Ser

210 215 220

Val Asn Asn Gly Asn Asp Thr Cys Leu Leu Ala Thr Met Gly Leu Gln

225 230 235 240

Leu Asn Ile Thr Gln Asp Lys Val Ala Ser Val Ile Asn Ile Asn Pro

245 250 255

Asn Thr Thr His Ser Thr Gly Ser Cys Arg Ser His Thr Ala Leu Leu

260 265 270

Arg Leu Asn Ser Ser Thr Ile Lys Tyr Leu Asp Phe Val Phe Ala Val

275 280 285

Lys Asn Glu Asn Arg Phe Tyr Leu Lys Glu Val Asn Ile Ser Met Tyr

290 295 300

Leu Val Asn Gly Ser Val Phe Ser Ile Ala Asn Asn Asn Leu Ser Tyr

305 310 315 320

Trp Asp Ala Pro Leu Gly Ser Ser Tyr Met Cys Asn Lys Glu Gln Thr

325 330 335

Val Ser Val Ser Gly Ala Phe Gln Ile Asn Thr Phe Asp Leu Arg Val

340 345 350

Gln Pro Phe Asn Val Thr Gln Gly Lys Tyr Ser Thr Ala Gln Glu Cys

355 360 365

Ser Leu Asp Asp Asp Thr Ile Leu Ile Pro Ile Ile Val Gly Ala Gly

370 375 380

Leu Ser Gly Leu Ile Ile Val Ile Val Ile Ala Tyr Val Ile Gly Arg

385 390 395 400

Arg Lys Ser Tyr Ala Gly Tyr Gln Thr Leu

405 410

<210> 12

<211> 411

<212> Protein

<213> Homo sapiens

<400> 12

Met Val Cys Phe Arg Leu Phe Pro Val Pro Gly Ser Gly Leu Val Leu

1 5 10 15

Val Cys Leu Val Leu Gly Ala Val Arg Ser Tyr Ala Leu Glu Leu Asn

20 25 30

Leu Thr Asp Ser Glu Asn Ala Thr Cys Leu Tyr Ala Lys Trp Gln Met

35 40 45

Asn Phe Thr Val Arg Tyr Glu Thr Thr Asn Lys Thr Tyr Lys Thr Val

50 55 60

Thr Ile Ser Asp His Gly Thr Val Thr Tyr Asn Gly Ser Ile Cys Gly

65 70 75 80

Asp Asp Gln Asn Gly Pro Lys Ile Ala Val Gln Phe Gly Pro Gly Phe

85 90 95

Ser Trp Ile Ala Asn Phe Thr Lys Ala Ala Ser Thr Tyr Ser Ile Asp

100 105 110

Ser Val Ser Phe Ser Tyr Asn Thr Gly Asp Asn Thr Thr Phe Pro Asp

115 120 125

Ala Glu Asp Lys Gly Ile Leu Thr Val Asp Glu Leu Leu Ala Ile Arg

130 135 140

Ile Pro Leu Asn Asp Leu Phe Arg Cys Asn Ser Leu Ser Thr Leu Glu

145 150 155 160

Lys Asn Asp Val Val Gln His Tyr Trp Asp Val Leu Val Gln Ala Phe

165 170 175

Val Gln Asn Gly Thr Val Ser Thr Asn Glu Phe Leu Cys Asp Lys Asp

180 185 190

Lys Thr Ser Thr Val Ala Pro Thr Ile His Thr Thr Val Pro Ser Pro

195 200 205

Thr Thr Thr Pro Thr Pro Lys Glu Lys Pro Glu Ala Gly Thr Tyr Ser

210 215 220

Val Asn Asn Gly Asn Asp Thr Cys Leu Leu Ala Thr Met Gly Leu Gln

225 230 235 240

Leu Asn Ile Thr Gln Asp Lys Val Ala Ser Val Ile Asn Ile Asn Pro

245 250 255

Asn Thr Thr His Ser Thr Gly Ser Cys Arg Ser His Thr Ala Leu Leu

260 265 270

Arg Leu Asn Ser Ser Thr Ile Lys Tyr Leu Asp Phe Val Phe Ala Val

275 280 285

Lys Asn Glu Asn Arg Phe Tyr Leu Lys Glu Val Asn Ile Ser Met Tyr

290 295 300

Leu Val Asn Gly Ser Val Phe Ser Ile Ala Asn Asn Asn Leu Ser Tyr

305 310 315 320

Trp Asp Ala Pro Leu Gly Ser Ser Tyr Met Cys Asn Lys Glu Gln Thr

325 330 335

Val Ser Val Ser Gly Ala Phe Gln Ile Asn Thr Phe Asp Leu Arg Val

340 345 350

Gln Pro Phe Asn Val Thr Gln Gly Lys Tyr Ser Thr Ala Glu Glu Cys

355 360 365

Ser Ala Asp Ser Asp Leu Asn Phe Leu Ile Pro Val Ala Val Gly Val

370 375 380

Ala Leu Gly Phe Leu Ile Ile Val Val Phe Ile Ser Tyr Met Ile Gly

385 390 395 400

Arg Arg Lys Ser Arg Thr Gly Tyr Gln Ser Val

405 410

<---

Claims (14)

1. A vector based on adeno-associated virus (AAV) serotype 9 for the treatment of Danon's disease, containing an expression cassette containing a polynucleotide encoding lysosome-associated membrane protein 2B (LAMP-2B) operably linked to a hybrid promoter containing the chicken β-actin promoter and CMV enhancer (CAG promoter). 2. The vector according to claim 1, wherein the vector is encapsulated in an anionic liposome. 3. The vector according to claim 1 or 2, in which the expression cassette contains functionally linked in the direction from 5' to 3' the first inverted terminal repeat, the enhancer/promoter region, the polynucleotide encoding LAMP-2B, the 3'-untranslated region containing the signal polyadenylation, and a second inverted terminal repeat. 4. Vector according to any one of paragraphs. 1-3, in which the polynucleotide contains DNA or cDNA. 5. Vector according to any one of paragraphs. 1-4, wherein the polynucleotide encoding LAMP-2B contains human LAMP-2. 6. Vector for gene therapy according to any one of paragraphs. 1-5, wherein the polynucleotide encoding LAMP-2B 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. 7. The vector of claim 6, wherein the polynucleotide encoding LAMP-2B comprises one or more of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. 8. A method of treating Danon's disease in a subject in need thereof, comprising administering to the subject a vector according to any one of paragraphs. 1-7, where the subject is identified as having a mutant LAMP-2 gene. 9. A method of treating Danon's disease in a subject in need, comprising administering to the subject an adeno-associated virus 9 (AAV9) vector containing an expression cassette containing a polynucleotide encoding lysosome-associated membrane protein 2B (LAMP-2B) operably linked to a fusion promoter containing the chicken β-actin promoter and the CMV enhancer (CAG promoter), wherein the subject is identified as having a mutant LAMP-2B gene. 10. The method according to any one of paragraphs. 8, 9, wherein the vector is administered via a route selected from the group consisting of intravenous, intra-arterial, intracardiac, intracoronary, intramyocardial, intrarenal, intraurethral, epidural, and intramuscular. 11. The method according to any one of paragraphs. 8-10, in which the vector is injected several times. 12. The method according to any one of paragraphs. 8-11, in which the subject is a human. 13. The method according to any one of paragraphs. 8-12, in which the subject is identified as having reduced or undetectable LAMP-2B expression. 14. The use of a vector according to any one of paragraphs. 1-7 for the treatment of Danon's disease in a subject in need thereof, wherein the subject is identified as having a mutant LAMP-2 gene.

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