Gene therapy vector for treating dainong disease

Abstract

The present disclosure provides gene therapy vectors comprising a polynucleotide sequence encoding a LAMP-2 polypeptide, methods of use thereof, pharmaceutical compositions, and the like. In particular, the present disclosure provides recombinant AAV vectors having an AAVrh74 serotype that expresses LAMP-2A, LAMP-2B, or LAMP-2C, for use as, for example, a therapeutic agent for Darnong disease.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 202080011825.7, with an application date of February 12, 2020 and an invention title of "Gene Therapy Vector for the Treatment of Danon Disease".

[0002] Related Applications

[0003] This application claims priority to U.S. Provisional Patent Application No. 62 / 934,928, filed November 13, 2019, and U.S. Provisional Patent Application No. 62 / 804,521, filed February 12, 2019, each of which is incorporated herein by reference in its entirety.

[0004] Sequence Listing

[0005] This application is being filed electronically via EFS-Web and includes a sequence listing in.txt format that is being filed electronically. The.txt file contains a sequence listing entitled "ROPA_013_02WO_ST25.txt" created on February 11, 2020, and is approximately 61 kilobytes in size. The sequence listing contained in the.txt file is part of the specification and is incorporated herein by reference in its entirety. Technical Field

[0006] The present invention generally relates to gene therapy for diseases associated with mutations in lysosome-associated membrane protein 2 (LAMP-2, also known as CD107b). Background Art

[0007] Lysosome-associated membrane protein 2 (LAMP-2, also known as CD107b) is a gene that encodes a lysosome-associated membrane glycoprotein. Alternative splicing of the gene results in three isoforms: LAMP-2A, LAMP-2B, and LAMP-2C. Loss-of-function mutations in LAMP-2 are associated with human diseases, including Danon disease (a familial cardiomyopathy associated with impaired autophagy function). Danon disease is a rare but severe cardiac and skeletal muscle disease that results in significant morbidity and early mortality due to arrhythmias and cardiomyopathy. The inherited X-linked nature explains the reported differences in phenotypic severity between males and females. Boucek et al., Genetics in Medicine 13:563-568 (2011). It is now understood that the disease is caused by a primary deficiency of lysosome-associated membrane protein-2 (LAMP-2), which serves as a lysosomal membrane receptor in chaperone-mediated autophagy. Nishino et al., Nature 406:906-910 (2000). Summary of the Invention

[0008] The present disclosure provides such gene therapy vectors related to LAMP2, methods of using the same, pharmaceutical compositions, etc. Although the clinical use of adeno-associated virus (AAV) vectors is known, the selection of the preferred serotype of AAV for gene therapy remains challenging and unpredictable.

[0009] The present disclosure provides improved gene therapy vectors comprising polynucleotide sequences encoding LAMP-2 polypeptides, methods of using the same, pharmaceutical compositions, etc. In particular, the present disclosure provides recombinant AAV vectors having the AAVrh74 serotype expressing LAMP-2A, LAMP-2B, or LAMP-2C for use as, for example, therapeutic agents for Danon disease.

[0010] Other features and advantages of the invention will be apparent from and are encompassed by the following detailed description and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 An embodiment of an AAV vector having the AAVrh74 serotype is depicted.

[0012] Figure 2 A bar graph showing vector DNA quantification by qPCR in the organs most affected by Danon disease is shown.

[0013] Figure 3A A bar graph showing vector DNA quantification by qPCR in the cardiac region is shown.

[0014] Figure 3B A bar graph showing vector DNA quantification by qPCR in muscle is shown.

[0015] Figure 4 A bar graph showing mRNA quantification by RT-qPCR in the organs most affected by Danon disease is shown.

[0016] Figure 5A A bar graph showing mRNA quantification by RT-qPCR in the cardiac region is shown.

[0017] Figure 5B A bar graph showing mRNA quantification by RT-qPCR in muscle is shown.

[0018] Figure 6A Micrographs showing semi-quantitative mRNA analysis by RNAscope in untreated left ventricles are shown.

[0019] Figure 6B Micrographs showing semi-quantitative mRNA analysis by RNAscope in treated left ventricles are shown.

[0020] Figure 7AMicrographs showing semi - quantitative mRNA analysis by RNAscope in untreated quadriceps muscles.

[0021] Figure 7B Micrographs showing semi - quantitative mRNA analysis by RNAscope in treated quadriceps muscles.

[0022] Figure 8 Micrographs showing semi - quantitative mRNA analysis by RNAscope in treated gastrocnemius muscles.

[0023] Figure 9 Bar graphs showing protein quantification by ELISA in the organs most affected by Danon disease.

[0024] Figure 10A Bar graphs showing protein quantification by ELISA in the cardiac region.

[0025] Figure 10B Bar graphs showing protein quantification by ELISA in muscles.

[0026] Figure 11A-11D Line graphs showing clinicopathological measurements in NHP serum during the study. Clinicopathological levels were evaluated as changes in the following during the study: ( Figure 11A ) alanine aminotransferase, ALT; ( Figure 11B ) aspartate aminotransferase, AST; ( Figure 11C ) white blood cells, WBC; ( Figure 11D ) neutrophils. Detailed Description

[0027] The present disclosure provides an AAVrh74 - based gene therapy vector that employs an optimized expression cassette to deliver a polynucleotide encoding one of the lysosome - associated membrane protein 2 (LAMP2) proteins (also known as CD107b). Generally, LAMP2 is human LAMP2, but expression of any mammalian LAMP - 2 is contemplated. The native LAMP2 gene encodes through alternative splicing three variants: LAMP - 2A, LAMP - 2B, and LAMP - 2C. LAMP - 2B is associated with Danon disease. Although the present disclosure mainly relates to Danon disease, LAMP2 is involved in various other diseases, including cancer. The disclosed vectors can be used to treat any of these diseases.

[0028] The present disclosure also relates to an AAVrh74 capsid or a capsid that has substantial homology with the AAVrh74 capsid and retains the functions of the AAVrh74 capsid. The present disclosure provides the sequences listed in Table 1. Table 1 also provides polynucleotide sequences for various embodiments. These sequences are not intended to limit the invention, as it is contemplated to replace or modify these sequences with different promoters, enhancers or other genetic elements.

[0029] Table 1: Sequences

[0030]

[0031]

[0032] The present disclosure provides a recombinant adeno-associated virus (rAAV) gene therapy vector. As used herein, an "rAAV gene therapy vector" refers to a complete virus comprising nucleic acid and protein components, including a capsid protein. In some embodiments, the capsid protein is encoded by a polynucleotide provided on a plasmid that is trans-linked to the transfer plasmid. The polynucleotide sequence of wild-type AAVrh74cap is as follows:

[0033] AAVrh74 capsid coding sequence (SEQ ID NO:1)

[0034]

[0035] The present disclosure also provides the protein sequences of AAVrh74 VP1, VP2 and VP3, including SEQ ID NO:2-4, as well as their homologs or functional variants.

[0036] AAVrh74 VP1 (SEQ ID NO:2)

[0037] MAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYNFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTAGTQQLLFSQAGPNNMSAQAKNWLPGPCYRQQRVSTTLSQNNNSNFAWTGATKYHLNGRDSLVNPGVAMATHKDDEERFFPSSGVLMFGKQGAGKDNVDYSSVMLTSEEEIKTTNPVATEQYGVVADNLQQQNAAPIVGAVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQAKLASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGTYSEPRPIGTRYLTRNL

[0038] AAVrh74 VP2 (SEQ ID NO:3)

[0039] STIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYNFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTAGTQQLLFSQAGPNNMSAQAKNWLPGPCYRQQRVSTTLSQNNNSNFAWTGATKYHLNGRDSLVNPGVAMATHKDDEERFFPSSGVLMFGKQGAGKDNVDYSSVMLTSEEEIKTTNPVATEQYGVVADNLQQQNAAPIVGAVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQAKLASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGTYSEPRPIGTRYLTRNL

[0040] AAVrh74 VP3 (SEQ ID NO:4)

[0041] RTGNNFEFSYNFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTAGTQQLLFSQAGPNNMSAQAKNWLPGPCYRQQRVSTTLSQNNNSNFAWTGATKYHLNGRDSLVNPGVAMATHKDDEERFFPSSGVLMFGKQGAGKDNVDYSSVMLTSEEEIKTTNPVATEQYGVVADNLQQQNAAPIVGAVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQAKLASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGTYSEPRPIGTRYLTRNL

[0042] In some cases, the AAVrh74 capsid contains the amino acid sequence shown in SEQ ID NO:2. In some embodiments, the rAAV vector contains a polypeptide that comprises, consists essentially of, or consists of: a sequence that is, for example, at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, more typically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of AAVrh74 VP1 shown in SEQ ID NO:2. In some embodiments, the rAAV vector contains a polypeptide that comprises, consists essentially of, or consists of: a sequence that is, for example, at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, more typically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of AAVrh74 VP2 shown in SEQ ID NO:3. In some embodiments, the rAAV vector contains a polypeptide that comprises, consists essentially of, or consists of: a sequence that is, for example, at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, more typically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of AAVrh74 VP3 shown in SEQ ID NO:4.

[0043] The wild-type polypeptide sequence of LAMP-2B (SEQ ID NO:5) and the wild-type polynucleotide sequence of LAMP-2B (SEQ ID NO:6) are respectively:

[0044] MVCFRLFPVPGSGLVLVCLVLGAVRSYALELNLTDSENATCLYAKWQMNFTVRYETTNKTYKTVTISDHGTVTYNGSICGDDQNGPKIAVQFGPGFSWIANFTKAASTYSIDSVSFSYNTGDNTTFPDAEDKGILTVDELLAIRIPLNDLFRCNSLSTLEKNDVVQHYWDVLVQAFVQNGTVSTNEFLCDKDKTSTVAPTIHTTVPSPTTTPTPKEKPEAGTYSVNNGNDTCLLATMGLQLNITQDKVASVININPNTTHSTGSCRSHTALLRLNSSTIKYLDFVFAVKNENRFYLKEVNISMYLVNGSVFSIANNNLSYWDAPLGSSYMCNKEQTVSVSGAFQINTFDLRVQPFNVTQGKYSTAQECSLDDDTILIPIIVGAGLSGLIIVIVIAYVIGRRKSYAGYQT(SEQ ID NO:5); and

[0045]

[0046] In one embodiment, the transgene has at least 95% identity with the polynucleotide sequence of SEQ ID NO:5. In one embodiment, the transgene has at least 99% identity with the polynucleotide sequence of SEQ ID NO:5. In one embodiment, the transgene comprises the polynucleotide sequence of SEQ ID NO:5. In embodiments, the transgene has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or complete identity with SEQ ID NO:5.

[0047] In one embodiment, the transgene encodes a polypeptide having at least 95% identity with the amino acid sequence of SEQ ID NO:6. In one embodiment, the transgene encodes a polypeptide having at least 99% identity with the amino acid sequence of SEQ ID NO:6. In one embodiment, the polypeptide encoded by the transgene comprises the amino acid sequence of SEQ ID NO:6. In embodiments, the polypeptide encoded by the transgene has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or complete identity with SEQ ID NO:6.

[0048] Modifications to the gene sequence of LAMP-2B are disclosed herein, including: codon optimization, CpG depletion, removal of cryptic splice sites, and reduction of alternative open reading frames (ORFs). In embodiments, the present disclosure provides a transgene encoding an isoform of lysosome-associated membrane protein 2 (LAMP-2) or a functional variant thereof. In embodiments, the transgene has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or complete identity with a sequence selected from SEQ ID NOs:7-9. The present disclosure provides at least three variant gene sequences of LAMP-2B (SEQ ID NOs:7-9):

[0049]

[0050]

[0051]

[0052] In one embodiment, the transgene has at least 95% identity with a sequence selected from SEQ ID NO: 7 - 9. In one embodiment, the transgene has at least 99% identity with a sequence selected from SEQ ID NO: 7 - 9. In one embodiment, the transgene comprises a sequence selected from SEQ ID NO: 7 - 9. In an embodiment, the transgene has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or complete identity with SEQ ID NO: 7. In an embodiment, the transgene has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or complete identity with SEQ ID NO: 8. In an embodiment, the transgene has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or complete identity with SEQ ID NO: 9.

[0053] In some cases, the transgene has a polynucleotide sequence different from the polynucleotide sequence of a reference sequence, e.g., the "native" or "wild - type" LAMP - 2B sequence. In some embodiments, the transgene has at most 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% or 95% identity with the reference sequence. In some embodiments, the reference sequence is SEQ ID NO: 6. For example, SEQ ID NO: 7 has 78.5% identity with SEQ ID NO: 6.

[0054] In some embodiments, the transgene is similar or identical to a subsequence of any one of SEQ ID NO:5 or 7-9. In some embodiments, the transgene comprises a subsequence of any one of SEQ ID NO:5 or 7-9. In various embodiments, the subsequence may comprise any set of consecutive nucleotides (nt) in the full sequence that is at least about 50 nt, at least about 100 nt, at least about 150 nt, at least about 250 nt, at least about 200 nt, at least about 350 nt, at least about 450 nt, at least about 400 nt, at least about 450 nt, at least about 550 nt, at least about 600 nt, at least about 650 nt, at least about 600 nt, at least about 650 nt, at least about 700 nt, at least about 750 nt, at least about 800 nt, at least about 850 nt, at least about 900 nt, at least about 950 nt, at least about 1000 nt, at least about 1050 nt, at least about 1100 nt, at least about 1150 nt, or at least about 1200 nt in length.

[0055] In some embodiments, the transgene is similar or identical to a subsequence of any one of SEQ ID NO:6 or 16-18. In some embodiments, the transgene encodes a polypeptide comprising a subsequence of any one of SEQ ID NO:6 or 16-18. In various embodiments, the subsequence may comprise any set of consecutive amino acids (aa) in the full sequence that is at least about 20 aa, at least about 30 aa, at least about 50 aa, at least about 70 aa, at least about 80 aa, at least about 100 aa, at least about 120 aa, at least about 130 aa, at least about 150 aa, at least about 170 aa, at least about 180 aa, at least about 200 aa, at least about 220 aa, at least about 230 aa, at least about 250 aa, at least about 270 aa, at least about 280 aa, at least about 300 aa, at least about 320 aa, at least about 330 aa, at least about 350 aa, at least about 370 aa, at least about 380 aa, or at least about 400 aa in length.

[0056] In some embodiments, the transgenic encodes a LAMP-2 polypeptide that comprises an N-terminal truncation of 1 to 10 amino acids (aa), 1 to 20 aa, 1 to 30 aa, 1 to 40 aa, or 1 to 50 aa, or an N-terminal truncation of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 48, 50 or more aa; and / or a C-terminal truncation of 1 to 10 amino acids (aa), 1 to 20 aa, 1 to 30 aa, 1 to 40 aa, or 1 to 50 aa, or a C-terminal truncation of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 48, 50 or more aa.

[0057] In some embodiments, a subsequence of the LAMP2 polypeptide comprises a functional variant of LAMP-2A, LAMP-2B, or LAMP-2C. As used herein, "functional variant" refers to a polypeptide that shares sequence similarity with a reference LAMP-2A, LAMP-2B, or LAMP-2C and has at least one biological property of LAMP-2A, LAMP-2B, or LAMP-2C. Biological properties can include the ability to specifically interact with one or more binding partners, the ability to bind anti-LAMP2 antibodies, and / or the ability to complement a defect in LAMP2 activity in a cell, tissue, and / or organism.

[0058] In some embodiments, a subsequence of the LAMP2 polypeptide comprises a functional fragment of LAMP-2A, LAMP-2B, or LAMP-2C. As used herein, "functional fragment" refers to a polypeptide that shares sequence similarity with a reference LAMP-2A, LAMP-2B, or LAMP-2C and has at least one biological property of LAMP-2A, LAMP-2B, or LAMP-2C. Biological properties can include the ability to specifically interact with one or more binding partners, the ability to bind anti-LAMP2 antibodies, and / or the ability to complement a defect in LAMP2 activity in a cell, tissue, and / or organism.

[0059] In one embodiment, the transgene is codon-optimized for expression in human host cells. In one embodiment, the transgene coding sequence is modified or "codon-optimized" to enhance expression by replacing infrequently represented codons with more frequently represented codons. The coding sequence is the portion of the mRNA sequence that encodes amino acids for translation. During translation, each of the 61 trinucleotide codons is translated into one of 20 amino acids, resulting in the degeneracy or redundancy of the genetic code. However, different cell types and different animal species utilize tRNAs (each carrying an anticodon) that encode the same amino acid at different frequencies. When a gene sequence contains codons that are infrequently represented by the corresponding tRNAs, the ribosomal translation machinery may slow down, thereby hindering efficient translation. Expression can be improved via "codon optimization" for a particular species, in which the coding sequence is altered to encode the same protein sequence but utilizes highly represented codons, and / or codons utilized by highly expressed human proteins (Cid-Arregui et al., 2003; J. Virol. 77:4928).

[0060] In some embodiments, the coding sequence of the transgene is modified to replace codons that are infrequently expressed in mammals or in primates with codons that are frequently expressed in primates. For example, in some embodiments, the transgene encodes a polypeptide having at least 85% sequence identity with a reference polypeptide (e.g., wild-type LAMP-2B; SEQ ID NO:16), such as at least 90% sequence identity, at least 95% sequence identity, at least 98% identity, or at least 99% identity with the reference polypeptide, wherein at least one codon of the coding sequence has a higher tRNA frequency in humans than the corresponding codon in the sequences disclosed above or herein.

[0061] In one embodiment, the transgene contains alternative open reading frames that are fewer than SEQ ID:6. In one embodiment, the transgene is modified to enhance expression by terminating or removing open reading frames (ORFs) that do not encode the desired transgene. An open reading frame (ORF) is a nucleic acid sequence that follows a start codon and contains no stop codons. The ORF may be in a forward or reverse orientation and may be "in frame" or "out of frame" compared to the gene of interest. Such open reading frames have the potential to be expressed together with the gene of interest in an expression cassette and can have unwanted adverse effects. In some embodiments, the transgene has been modified to remove open reading frames by further altering codon usage. This is accomplished by eliminating one or more start codons (ATG, TTG, CTG) and / or introducing one or more stop codons (TAG, TAA or TGA) in a reverse orientation or out of frame into the desired ORF while preserving the encoded amino acid sequence and optionally maintaining highly utilized codons (i.e., avoiding codons with a frequency <20%) in the gene of interest.

[0062] In variants of the present disclosure, the transgene coding sequence can be optimized by codon optimization and removal of non-transgenic ORFs or by using either of these two techniques. In some cases, after codon optimization, non-transgenic ORFs are removed or minimized in order to remove ORFs introduced during codon optimization.

[0063] In one embodiment, the transgene contains fewer CpG sites than SEQ ID:6. Without being bound by theory, it is believed that the presence of CpG sites in a polynucleotide sequence is associated with an unwanted immune response of the host to a viral vector containing the polynucleotide sequence. In some embodiments, the transgene is designed to reduce the number of CpG sites. Exemplary methods are provided in U.S. Patent Application Publication No. US20020065236A1.

[0064] In one embodiment, the transgene contains fewer cryptic splicing sites than SEQ ID:6. For optimization, software can be used, for example, to increase the GC content and / or remove cryptic splicing sites in order to avoid transcriptional silencing and thus increase transgene expression. Alternatively, any optimization method known in the art can be used. The removal of cryptic splicing sites is described, for example, in International Patent Application Publication No. WO2004015106A1. software, such as to increase the GC content and / or remove cryptic splicing sites in order to avoid transcriptional silencing and thus increase transgene expression. Alternatively, any optimization method known in the art can be used. The removal of cryptic splicing sites is described, for example, in International Patent Application Publication No. WO2004015106A1.

[0065] The present disclosure also provides expression cassettes encoding LAMP-2B and gene therapy vectors, such as the codon-optimized LAMP-2B sequences disclosed herein, which comprise: a consensus optimal Kozak sequence, a full-length polyadenylation (polyA) sequence (or replacing a truncated polyA with a full-length polyA), and minimal or no upstream (i.e., 5') start codon (i.e., ATG site).

[0066] In some cases, the expression cassette contains two or more of a first inverted terminal repeat, an enhancer / promoter region, a consensus optimal Kozak sequence, a transgene (e.g., a transgene encoding LAMP-2B disclosed herein), a 3' untranslated region including a full-length polyA sequence, and a second inverted terminal repeat.

[0067] In one embodiment, the expression cassette comprises a Kozak sequence operably linked to a transgene. In one embodiment, the Kozak sequence is a consensus optimal Kozak sequence that comprises or consists of SEQ ID NO: 20.

[0068] GCCGCCACCATGG(SEQ ID NO:20)

[0069] In one embodiment, the expression cassette comprises an alternative Kozak sequence operably linked to a transgene. In one embodiment, the Kozak sequence is an alternative Kozak sequence that comprises or consists of SEQ ID NO. 21-25.

[0070] (gcc)gccRccAUGG(SEQ ID NO:21)

[0071] AGNNAUGN(SEQ ID NO:22)

[0072] ANNAUGG(SEQ ID NO:23)

[0073] ACCAUGG(SEQ ID NO:24)

[0074] GACACCAUGG(SEQ ID NO:25)

[0075] In SEQ ID NO: 21, lowercase letters represent the most common bases at positions where the bases can still vary; uppercase letters represent highly conserved bases; 'R' indicates adenine or guanine. In SEQ ID NO: 21, the sequence (gcc) in parentheses is optional. In SEQ ID NO: 22-23, "N" represents any base.

[0076] A variety of sequences can be used in place of this consensus optimal Kozak sequence as a translation start site, and the identification and testing of other sequences is within the skill of one of ordinary skill in the art. See Kozak M. An analysis of vertebrate mRNA sequences: intimations of translational control. J. Cell Biol. 115(4):887–903 (1991).

[0077] In one embodiment, the expression cassette comprises a full-length polyA sequence operably linked to a transgene. In one embodiment, the full-length polyA sequence comprises SEQ ID NO:26.

[0078] TGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATC(SEQ ID NO:26)

[0079] A variety of alternative polyA sequences can be used in the expression cassettes of the present disclosure, including but not limited to the bovine growth hormone polyadenylation signal (bGHpA) (SEQ ID NO:27), the SV40 early / late polyadenylation signal (SEQ ID NO:28), and the human growth hormone (HGH) polyadenylation signal (SEQ ID NO:29).

[0080] TCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAGGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTG(SEQ ID NO:27)

[0081] CAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTA(SEQ ID NO:28)

[0082] CTGCCCGGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTTGTCCTAATAAAATTAAGTTGCATCATTTTGTCTGACTAGGTGTCCTTCTATAATATTATGGGGTGGAGGGGGGTGGTATGGAGCAAGGGGCCCAAGTTGGGAAGAAACCTGTAGGGCCTGC(SEQ ID NO:29)

[0083] In some embodiments, the expression cassette comprises an active fragment of the polyA sequence. In certain embodiments, the active fragment of the polyA sequence comprises, consists of, for example, less than 20 base pairs (bp), less than 50 bp, less than 100 bp, or less than 150 bp of any of the polyA sequences disclosed herein.

[0084] In some cases, transgene expression is increased by ensuring that the expression cassette does not contain competing ORFs. In one embodiment, the expression cassette does not contain a start codon within 20, 30, 40, 50, 60, 70, 80, 90, 100, 200 or 300 base pairs 5' of the start codon of the transgene. In one embodiment, the expression cassette does not contain a start codon 5' of the start codon of the transgene. In some embodiments, the expression vector does not contain alternative transcripts. In some embodiments, the expression cassette does not contain alternative transcripts except for small transcripts, such as transcripts of 300 base pairs or fewer.

[0085] In one embodiment, the expression cassette comprises a first inverted terminal repeat, an enhancer / promoter region, an intron, a consensus optimal Kozak sequence, a transgene, a 3' untranslated region comprising a full-length polyA sequence, and a second inverted terminal repeat, operably linked in the 5' to 3' direction, wherein the expression cassette does not contain a start codon 5' of the start codon of the transgene.

[0086] In one embodiment, the enhancer / promoter region comprises a CMV IE enhancer and a chicken β-actin promoter in the 5' to 3' direction. In one embodiment, the enhancer / promoter region comprises a CAG promoter. As used herein, "CAG promoter" refers to a polynucleotide sequence comprising a CMV early enhancer element, a chicken β-actin promoter, the first exon and the first intron of the chicken β-actin gene, and a splice acceptor from the rabbit β-myoglobin gene.

[0087] In one embodiment, the expression cassette has at least 95% identity to a sequence selected from SEQ ID NOs: 10-12. In one embodiment, the expression cassette has complete identity to a sequence selected from SEQ ID NOs: 10-12, or has at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to a sequence selected from SEQ ID NOs: 10-12. In certain embodiments, the expression cassette comprises one or more modifications compared to a sequence selected from SEQ ID NOs: 10-12. In a particular embodiment, one or more of the modifications comprise one or more of the following: removal of one or more (e.g., all) upstream ATG sequences, replacement of the Kozak sequence with an optimized consensus Kozak sequence or another Kozak sequence (including but not limited to any sequence disclosed herein), and / or replacement of the polyadenylation sequence with a full-length polyadenylation sequence or another polyadenylation sequence (including but not limited to any sequence disclosed herein). An illustrative configuration of the genetic elements within these exemplary expression cassettes is depicted in Figure 1 in.

[0088]

[0089]

[0090]

[0091] In one embodiment, the vector is an adeno-associated virus (AAV) vector. In one embodiment, the expression cassette comprises ITR sequences selected from SEQ ID NO: 13 and 14.

[0092] CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT(SEQ ID NO:13)

[0093] AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAG(SEQ ID NO:14)

[0094] In related embodiments, the present disclosure provides a gene therapy vector comprising the expression cassette disclosed herein. Generally, the gene therapy vectors described herein comprise an expression cassette that comprises a polynucleotide that encodes one or more isoforms of lysosomal-associated membrane protein 2 (LAMP-2), which permits expression of LAMP-2 to partially or fully correct the LAMP-2 protein expression level and / or autophagy flux deficiency in a subject in need thereof (e.g., a subject having Danon disease or another disease characterized at least in part by insufficient autophagy flux due to insufficient LAMP-2 expression).

[0095] LAMP-2A protein sequence

[0096] MVCFRLFPVPGSGLVLVCLVLGAVRSYALELNLTDSENATCLYAKWQMNFTVRYETTNKTYKTVTISDHGTVTYNGSICGDDQNGPKIAVQFGPGFSWIANFTKAASTYSIDSVSFSYNTGDNTTFPDAEDKGILTVDELLAIRIPLNDLFRCNSLSTLEKNDVVQHYWDVLVQAFVQNGTVSTNEFLCDKDKTSTVAPTIHTTVPSPTTTPTPKEKPEAGTYSVNNGNDTCLLATMGLQLNITQDKVASVININPNTTHSTGSCRSHTALLRLNSSTIKYLDFVFAVKNENRFYLKEVNISMYLVNGSVFSIANNNLSYWDAPLGSSYMCNKEQTVSVSGAFQINTFDLRVQPFNVTQGKYSTAQDCSADDDNFLVPIAVGAALAGVLILVLLAYFIGLKHHHAGYEQF(SEQ ID NO:15)

[0097] LAMP-2B protein sequence

[0098] MVCFRLFPVPGSGLVLVCLVLGAVRSYALELNLTDSENATCLYAKWQMNFTVRYETTNKTYKTVTISDHGTVTYNGSICGDDQNGPKIAVQFGPGFSWIANFTKAASTYSIDSVSFSYNTGDNTTFPDAEDKGILTVDELLAIRIPLNDLFRCNSLSTLEKNDVVQHYWDVLVQAFVQNGTVSTNEFLCDKDKTSTVAPTIHTTVPSPTTTPTPKEKPEAGTYSVNNGNDTCLLATMGLQLNITQDKVASVININPNTTHSTGSCRSHTALLRLNSSTIKYLDFVFAVKNENRFYLKEVNISMYLVNGSVFSIANNNLSYWDAPLGSSYMCNKEQTVSVSGAFQINTFDLRVQPFNVTQGKYSTAQECSLDDDTILIPIIVGAGLSGLIIVIVIAYVIGRRKSYAGYQTL(SEQ ID NO:16)

[0099] LAMP-2C protein sequence

[0100] MVCFRLFPVPGSGLVLVCLVLGAVRSYALELNLTDSENATCLYAKWQMNFTVRYETTNKTYKTVTISDHGTVTYNGSICGDDQNGPKIAVQFGPGFSWIANFTKAASTYSIDSVSFSYNTGDNTTFPDAEDKGILTVDELLAIRIPLNDLFRCNSLSTLEKNDVVQHYWDVLVQAFVQNGTVSTNEFLCDKDKTSTVAPTIHTTVPSPTTTPTPKEKPEAGTYSVNNGNDTCLLATMGLQLNITQDKVASVININPNTTHSTGSCRSHTALLRLNSSTIKYLDFVFAVKNENRFYLKEVNISMYLVNGSVFSIANNNLSYWDAPLGSSYMCNKEQTVSVSGAFQINTFDLRVQPFNVTQGKYSTAEECSADSDLNFLIPVAVGVALGFLIIVVFISYMIGRRKSRTGYQSV(SEQ ID NO:17)

[0101] In one embodiment, the expression cassette comprises a polynucleotide sequence encoding LAMP-2 disclosed herein, e.g., SEQ ID NO: 15-17 or a sequence having at least 90%, at least 95%, at least 98% or at least 99% identity to any one of SEQ ID NO: 15-17. The gene therapy vector can be a viral or non-viral vector. Exemplary non-viral vectors include, for example, naked DNA, cationic liposome complexes, cationic polymer complexes, cationic liposome-polymer complexes, and exosomes. Examples of viral vectors include, but are not limited to, adenovirus, retrovirus, lentivirus, herpesvirus, and adeno-associated virus (AAV) vectors.

[0102] In some embodiments, the expression cassette comprises a polynucleotide sequence encoding one or more, two or more, or all three of SEQ ID NO: 15-17. In some embodiments, a polynucleotide sequence comprising the native introns of the LAMP-2 gene enables the expression of more than one isoform in the same cell using a single vector. In some embodiments, artificial introns, splice acceptors, and / or splice donors are used to optimize the length of the polynucleotide and / or to optimize the ratio of isoforms expressed by a polynucleotide encoding two or more or all three of SEQ ID NO: 15-17.

[0103] In some embodiments, the expression cassette, AAV capsid gene, and / or helper gene are delivered to cells using transduction, transfection, electroporation, liposomes, and any other methods known in the art. In some embodiments, the expression cassette, AAV capsid gene, and / or helper gene are delivered in liposomes or lipid nanoparticles (LNPs). The expression cassette, AAV capsid gene, and / or helper gene can be provided as DNA, such as on one or more plasmids, bacmids, or other DNA molecules. In some embodiments, the expression cassette, AAV capsid gene, and / or helper gene are delivered as RNA molecules. In some embodiments, the RNA molecules comprise one or more mRNA molecules, such as one or more in vitro transcribed mRNA molecules. In some embodiments, the mRNA molecules are modified mRNA molecules. Illustrative modifications include locked nucleic acids, phosphorothioate bonds, and modified nucleosides (e.g., pseudouridine, 5-methylcytosine, or 5-methylcytidine). In some embodiments, the modified mRNA comprises a cap, such as an ARCA cap. The expression cassette, AAV capsid gene, and / or helper gene can be delivered in vitro or in vivo. In some embodiments, the AAV capsid gene comprises one or more of the AAV9 capsid gene and the AAVrh74 capsid gene.

[0104] The gene delivery viral vectors useful in the practice of the present invention can be constructed using methods well known in the field of molecular biology. Generally, a viral vector carrying a transgene is assembled from a polynucleotide encoding the transgene, appropriate regulatory elements, and elements necessary to produce viral proteins that mediate cell transduction. Such recombinant viruses can be produced by techniques known in the art, such as by transfection of packaging cells or by transient transfection with a helper plasmid or virus. Typical examples of viral packaging cells include, but are not limited to, HeLa cells, SF9 cells (optionally with a baculovirus helper vector), 293 cells, etc. Herpesvirus-based systems can be used to produce AAV vectors, as described in US20170218395A1. Detailed protocols for producing such replication-defective recombinant viruses can be found, for example, in W095 / 14785, W096 / 22378, U.S. Patent No. 5,882,877, U.S. Patent No. 6,013,516, U.S. Patent No. 4,861,719, U.S. Patent No. 5,278,056, and W094 / 19478, the entire contents of each of which are incorporated herein by reference.

[0105] AAV is a 4.7 kb single-stranded DNA virus. AAV-based recombinant vectors are associated with excellent clinical safety because wild-type AAV is non-pathogenic and has no etiological association with any known disease. Additionally, AAV provides the ability to achieve efficient gene delivery and sustained transgene expression in many tissues. "AAV vector" means a vector derived from an adeno-associated virus serotype, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.10, AAVrh74, etc. The AAV vector may have one or more AAV wild-type genes that are entirely or partially deleted, such as the rep and / or cap genes, but retain functional flanking inverted terminal repeat (ITR) sequences. The functional ITR sequences are necessary for rescuing, replicating, and packaging AAV virions. Thus, an AAV vector is defined herein as including at least those sequences necessary for cis-replication and packaging of the virus (e.g., functional ITRs). The ITRs do not have to be wild-type nucleotide sequences and can be altered, for example, by insertion, deletion, or substitution of nucleotides, as long as the sequences provide functional rescue, replication, and packaging. The AAV vector may contain other modifications, including but not limited to one or more modified capsid proteins (e.g., VP1, VP2, and / or VP3). For example, the capsid proteins can be modified to alter tropism and / or reduce immunogenicity. AAV expression vectors are constructed using known techniques to provide at least as control elements components operably linked in the transcriptional direction, the control elements including a transcription initiation region, the DNA of interest (i.e., the LAMP-2 gene), and a transcription termination region.

[0106] Adeno-associated virus (AAV) is a single-stranded DNA virus. The AAV genome is constructed from either sense or antisense single-stranded deoxyribonucleic acid (ssDNA), which is approximately 4.7 kilobases in length. The genome contains inverted terminal repeats (ITRs) at both ends of the DNA strand, as well as two open reading frames (ORFs): rep and cap. The first, rep, consists of four overlapping genes that encode the Rep proteins required for the AAV life cycle, and the second, cap, encodes three capsid proteins: VP1, VP2, and VP3. The cap genes are expressed as messenger RNA (mRNA) from the p40 promoter of AAV. Alternatively, the mRNA is spliced into 2.3 kb and 2.6 kb transcripts, with the 2.3 kb transcript being more abundant. VP1 is expressed only from the 2.6 kb transcript, and the molecular weight of the VP1 protein is 87 kilodaltons (kDa). Due to the presence of an optimal Kozak sequence for translation initiation, VP2 is expressed from an open reading frame that starts at the ACG codon rather than the canonical AUG codon. VP2 is 72 kDa. Only 62 kDa VP3 is expressed by the presence of the ATG sequence in both the 2.3 kb transcript and the 2.6 kb transcript. The relative abundance of VP1:VP2:VP3 is 1:1:10. VP1, VP2, and VP3 interact to form an icosahedrally symmetric capsid.

[0107] AAV-based recombinant vectors are associated with excellent clinical safety because wild-type AAV is non-pathogenic and has no etiological association with any known disease. Additionally, AAV provides the ability to achieve efficient gene delivery and sustained transgene expression in many tissues. A variety of AAV serotypes are known, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.10, AAVrh74, etc. An AAV vector may have one or more AAV wild-type genes that are entirely or partially deleted, e.g., the rep and / or cap genes, but retains functional flanking inverted terminal repeat (ITR) sequences. The serotype of a recombinant AAV vector is determined by its capsid. International Patent Publication No. WO2003042397A2 discloses various capsid sequences, including those of AAV1, AAV2, AAV3, AAV8, AAV9, and AAVrh10. International Patent Publication No. WO2013078316A1 discloses the polypeptide sequence of VP1 from AAVrh74. Numerous different naturally occurring or genetically modified AAV capsid sequences are known in the art.

[0108] The present disclosure also provides a pharmaceutical composition comprising an expression cassette or vector (e.g., a gene therapy vector) disclosed herein and one or more pharmaceutically acceptable carriers, diluents, or excipients. In certain embodiments, the pharmaceutical composition comprises an AAV vector comprising an expression cassette disclosed herein, e.g., wherein the expression cassette comprises a codon-optimized transgene encoding LAMP-2B, e.g., any one of SEQ ID NOs: 7-9. The present invention provides, for example, a pharmaceutical composition for preventing or treating a disorder characterized by insufficient autophagic flux (e.g., Danon disease), comprising a therapeutically effective amount of a vector comprising a nucleic acid sequence of a polynucleotide encoding one or more isoforms of LAMP-2.

[0109] The pharmaceutical composition containing the expression cassette or vector can be in any form suitable for the selected mode of administration, such as for intraventricular, intramyocardial, intracoronary, intravenous, intraarterial, intrarenal, intraurethral, epidural, or intramuscular administration. The gene therapy vector comprising a polynucleotide encoding one or more LAMP-2 isoforms can be administered as the sole active agent, or in combination with other active agents, in unit dosage form, as a mixture with a conventional pharmaceutical carrier, to animals and humans. In some embodiments, the pharmaceutical composition comprises cells transduced ex vivo with any gene therapy vector of the present disclosure.

[0110] In various embodiments, the pharmaceutical composition contains a pharmaceutically acceptable vehicle (e.g., carrier, diluent, and excipient) for a formulation that can be injected. These solutions can in particular be isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium chloride, or magnesium chloride, etc., or mixtures of these salts). Illustrative pharmaceutical forms suitable for injectable use include, for example, sterile aqueous solutions or dispersions; formulations comprising sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.

[0111] In another aspect, the present disclosure provides a method for preventing, alleviating, ameliorating, reducing, inhibiting, eliminating, and / or reversing one or more symptoms of Danon disease or another autophagic disorder in a subject in need thereof, comprising administering to the subject a gene therapy vector of the present disclosure. The term "Danon disease" refers to an X-linked dominant skeletal and cardiac disorder with multisystem clinical manifestations. Danon disease mutations result in the absence of lysosome-associated membrane protein 2 (LAMP-2) protein expression. The main clinical features include skeletal and cardiac myopathy, cardiac conduction abnormalities, cognitive difficulties, and retinal disease. Males are generally affected earlier and more severely than females.

[0112] In one embodiment, the vector is administered via a route selected from the group consisting of: parenteral, intravenous, intra-arterial, intracardiac, intracoronary, intramuscular, intra-renal, intra-urethral, epidural, and intramuscular. In one embodiment, the vector is administered multiple times. In one embodiment, the vector is administered by intramuscular injection of the vector. In one embodiment, the vector is administered by injecting the vector into skeletal muscle. In one embodiment, the expression cassette comprises a muscle-specific promoter, optionally the muscle creatine kinase (MCK) promoter or the MCK / SV40 hybrid promoter, as described in Takeshita et al. Muscle creatine kinase / SV40 hybrid promoter for muscle-targeted long-term transgene expression. Int J Mol Med. February 2007; 19(2):309-15. In one embodiment, the vector is administered by intracardiac injection.

[0113] In one embodiment, the present disclosure provides a method of treating a disease or disorder, optionally Danon disease, in a subject in need thereof, comprising contacting cells with a gene therapy vector according to the present disclosure and administering the cells to the subject. In one embodiment, the cells are stem cells, optionally pluripotent stem cells. In one embodiment, the stem cells are capable of differentiating into cardiac tissue. In one embodiment, the stem cells are capable of differentiating into muscle tissue, such as cardiac muscle tissue and / or skeletal muscle tissue. In one embodiment, the stem cells are autologous. In one embodiment, the stem cells are induced pluripotent stem cells (iPSCs).

[0114] In one embodiment, the autophagy disorder is selected from the group consisting of: end-stage heart failure, myocardial infarction, drug toxicity, diabetes, end-stage renal failure, and aging. In one embodiment, the subject is a mammal, such as a human. In one embodiment, the subject is exhibiting symptoms of Danon disease or another autophagy disorder. In one embodiment, the subject has been identified as having reduced or undetectable LAMP-2 expression. In one embodiment, the subject has been identified as having a mutated LAMP-2 gene.

[0115] Subjects / patients suitable for treatment using the methods described herein include individuals at risk of a disease or disorder characterized by insufficient autophagic flux (e.g., Danon disease and other known autophagy disorders, including but not limited to systolic and diastolic heart failure, myocardial infarction, drug toxicity (e.g., anthracyclines, chloroquine and its derivatives), diabetes, end-stage kidney disease, and aging) but not showing symptoms, as well as subjects currently showing symptoms. Such subjects may have been identified as having a mutated LAMP-2 gene or having a reduced or undetectable level of LAMP-2 expression.

[0116] In some embodiments, a subject is exhibiting symptoms of a disease or disorder characterized by insufficient autophagic flux (e.g., Danon disease and other known autophagy disorders including, but not limited to, systolic and diastolic heart failure, myocardial infarction, drug toxicity, diabetes, end-stage renal disease, and aging). The symptoms may be manifesting, or may be suppressed or controlled (e.g., by pharmacological treatment) or alleviated. The subject may or may not have been diagnosed with the disorder, for example, by a qualified physician.

[0117] Definitions

[0118] The terms “lysosome-associated membrane protein 2” and “LAMP-2” are used interchangeably to refer to nucleic acid and polypeptide polymorphic variants, alleles, mutants, and interspecies homologs: (1) that preferably have greater than about 90% amino acid sequence identity, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or greater amino acid sequence identity, in a region of at least about 25, 50, 100, 200, 300, 400 or more amino acids or over the full length, with an amino acid sequence encoded by a LAMP-2 nucleic acid (see, e.g., GenBank accession numbers NM_002294.2 (isoform A), NM_013995.2 (isoform B), NM_001122606.1 (isoform C)) or with an amino acid sequence of a LAMP-2 polypeptide (see, e.g., GenBank accession numbers NP_002285.1 (isoform A), NP_054701.1 (isoform B), NP_001116078.1 (isoform C)); (2) that bind to an antibody, such as a polyclonal antibody, raised against an immunogen comprising the amino acid sequence of a LAMP-2 polypeptide (e.g., a LAMP-2 polypeptide as described herein); or an amino acid sequence encoded by a LAMP-2 nucleic acid (e.g., a LAMP-2 polynucleotide as described herein) and its conservatively modified variants; (3) that specifically hybridize under stringent hybridization conditions to an antisense strand corresponding to the nucleic acid sequence encoding the LAMP-2 protein and its conservatively modified variants; (4) that preferably have greater than about 90%, preferably greater than about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher nucleotide sequence identity in a region of at least about 25, 50, 100, 200, 500, 1000, 2000 or more amino acids or over the full length, with LAMP-2 (e.g., a LAMP-2 polynucleotide as described herein, and a LAMP-2 polynucleotide encoding a LAMP-2 polypeptide as described herein).

[0119] When, for example, using one of the following sequence comparison algorithms or by manual alignment and visual inspection to measure the greatest correspondence when comparing and aligning over a comparison window or designated region, in the case of two or more nucleic acid or polypeptide sequences, the term "identical" or "identity" percentage refers to two or more sequences or subsequences that are the same or have a specified percentage of the same amino acid residues or nucleotides (i.e., having at least about 80% identity with a reference sequence, such as the LAMP-2 polynucleotide or polypeptide sequences described herein, for example at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity over a designated region. Such sequences are then referred to as "substantially identical". This definition also refers to the complement of the test sequence. Preferably, the identity exists over a region of at least about 25 amino acids or nucleotides, such as a region of 50, 100, 200, 300, 400 amino acids or nucleotides, or over the full length of the reference sequence.

[0120] For sequence comparison, typically one sequence acts as a reference sequence, and the test sequence is compared to the reference sequence. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, and if necessary, subsequence coordinates are specified, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be specified. Subsequently, the sequence comparison algorithm calculates the percentage of sequence identity of the test sequence relative to the reference sequence based on the program parameters. For sequence comparison of nucleic acids and proteins to LAMP-2 nucleic acids and proteins, the BLAST and BLAST 2.0 algorithms and default parameters are used.

[0121] As used herein, a "comparison window" includes a segment that refers to any one of a number of consecutive positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150, where after the two sequences are optimally aligned, the sequence can be compared to a reference sequence of the same number of consecutive positions. Methods of aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm, Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm, Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method, Pearson & Lipman, Proc. Nat’l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA, Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wl) or by manual alignment and visual inspection (see, e.g., Ausubel et al., eds., Current Protocols in Molecular Biology (1995 Supplement)). Examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, 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. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (on the World Wide Web at ncbi.nlm.nih.gov / ).

[0122] An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with an antibody raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, the polypeptide is generally substantially identical to the second polypeptide, e.g., where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules, or their complements, hybridize to each other under stringent conditions. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequences.

[0123] As used herein, "administer" refers to local and systemic administration, including, for example, enteral, parenteral, pulmonary, and topical / transdermal administration. Routes of administration of compounds (e.g., polynucleotides encoding one or more LAMP-2 isoforms) useful in the methods described herein include, for example, oral (per os (P.O.)) administration, nasal or inhaled administration, administration as a suppository, topical contact, transdermal delivery (e.g., via a transdermal patch), intrathecal (IT) administration, intravenous ("iv") administration, intraperitoneal ("ip") administration, intramuscular ("im") administration, intralesional administration, or subcutaneous ("sc") administration, or implantation of a slow-release device, such as a microosmotic pump, depot formulation, etc., into a subject. Administration can be by any route, including parenteral and transmucosal (e.g., oral, intranasal, vaginal, rectal, or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intrarenal, intraurethral, intracardiac, intracoronary, intramyocardial, intradermal, epidural, subcutaneous, intraperitoneal, intraventricular, iontophoretic, and intracranial. Other delivery modes include, but are not limited to, the use of liposomal formulations, intravenous infusions, transdermal patches, etc. In some embodiments, the dose of the administered rAAV gene therapy vector is from about 1E+11 vector genomes (vg) / kg to about 1E+12 vg / kg, from about 1E+12 vg / kg to about 2E+12 vg / kg, from about 2E+12 vg / kg to about 3E+12 vg / kg, from about 3E+12 vg / kg to about 3E+13 vg / kg, or from about 3E+13 vg / kg to about 3E+14 vg / kg. In some embodiments, the dose of the administered rAAV gene therapy vector is from about 3E+12 vg / kg to about 3E+14 vg / kg.

[0124] The terms "systemic administration" and "systemically administered" refer to methods of administering a compound or composition to a mammal such that the compound or composition is delivered via the circulatory system to a site in the body, including the site targeted for drug action. Systemic administration includes, but is not limited to, oral, intranasal, rectal, and parenteral (e.g., other than via the digestive tract, such as intramuscular, intravenous, intraarterial, transdermal, and subcutaneous) administration.

[0125] When used, for example, with respect to a compound (e.g., a LAMP-2 polynucleotide) and / or an analogue thereof and another active agent, the terms “co-administer” or “simultaneously administer” mean administering the compound and / or analogue and the active agent such that both can achieve a physiological effect simultaneously. However, the two agents need not be administered together. In certain embodiments, the administration of one agent can precede the administration of the other agent. Simultaneous physiological effects do not necessarily require the presence of both agents in the circulation simultaneously. However, in certain embodiments, co-administration generally results in the presence of both agents in the body (e.g., in the plasma) simultaneously at a significant fraction (e.g., 20% or greater, e.g., 30% or 40% or greater, e.g., 50% or 60% or greater, e.g., 70% or 80% or 90% or greater) of their maximum serum concentration for any given dose.

[0126] The term “effective amount” or “pharmaceutically effective amount” refers to the amount and / or dose and / or dosing regimen of one or more compounds (e.g., a gene therapy vector) required to produce a desired result, such as an increase in the expression of one or more LAMP-2 isoforms, in an amount sufficient to reduce the ultimate severity of a disease (e.g., Danon disease) characterized by impaired or insufficient autophagy. In some embodiments, the effective amount is an rAAV gene therapy vector of from about 1E+11 vg / kg to about 1E+12 vg / kg, from about 1E+12 vg / kg to about 2E+12 vg / kg, from about 2E+12 vg / kg to about 3E+12 vg / kg, from about 3E+12 vg / kg to about 3E+13 vg / kg, or from about 3E+13 vg / kg to about 3E+14 vg / kg. In some embodiments, the effective amount is an rAAV gene therapy vector of from about 3E+12 vg / kg to about 3E+14 vg / kg.

[0127] The phrase “causing to be administered” refers to an action taken by a medical professional (e.g., a doctor) or a person controlling the medical care of a subject, which controls and / or permits the administration of a controversial agent / compound to the subject. Causing to be administered can involve diagnosing and / or determining an appropriate treatment or prevention regimen, and / or prescribing a specific agent / compound for the subject. Such prescribing can include, for example, drafting a prescription form, annotating medical records, etc.

[0128] As used herein, the terms “treat” and “treatment” mean delaying the onset of a disease or condition or one or more symptoms of the disease or condition to which the term applies, delaying or reversing its progression, reducing its severity, or alleviating or preventing the disease or condition. The terms “treat” and “treatment” also include preventing, alleviating, improving, reducing, inhibiting, eliminating, and / or reversing one or more symptoms of a disease or condition.

[0129] The term "alleviate" refers to reducing or eliminating one or more symptoms of the pathology or disease, and / or reducing or delaying the rate of onset or severity of one or more symptoms of the pathology or disease, and / or preventing the pathology or disease. In certain embodiments, the reduction or elimination of one or more symptoms of a pathology or disease can include, for example, a measurable and sustained increase in the expression level of one or more isoforms of LAMP-2.

[0130] As used herein, the phrase "consisting essentially of" refers to the genus or species of active agents recited in a method or composition, and also includes other agents that are not themselves substantially active for the recited indication or purpose.

[0131] The terms "subject", "individual", and "patient" are used interchangeably to refer to a mammal, preferably a human or non-human primate, but also to domesticated mammals (e.g., canine or feline), laboratory mammals (e.g., mouse, rat, rabbit, hamster, guinea pig), and agricultural mammals (e.g., horse, cow, pig, sheep). In various embodiments, the subject can be a human (e.g., adult male, adult female, adolescent male, adolescent female, male child, female child).

[0132] The term "gene transfer" or "gene delivery" refers to methods or systems for reliably inserting exogenous DNA into a host cell. Such methods can result in transient expression of non-integrated transferred DNA, extrachromosomal replication, and expression of transferred replicons (e.g., episomes), or integration of the transferred genetic material into the genomic DNA of the host cell.

[0133] The term "vector" is used herein to refer to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. The transferred nucleic acid is typically ligated (e.g., inserted) within the vector nucleic acid molecule. A vector can include sequences that direct autonomous replication or reverse transcription in a cell, or can include sequences sufficient to allow integration into the host cell DNA. "Vector" includes gene therapy vectors. As used herein, the term "gene therapy vector" refers to a vector capable of being used to perform gene therapy, such as delivering a polynucleotide sequence encoding a therapeutic polypeptide to a subject. A gene therapy vector can contain a nucleic acid molecule encoding a therapeutically active polypeptide ("transgene"), e.g., LAMP-2B or other genes that can be used for gene therapy when introduced into a subject. Useful vectors include, but are not limited to, viral vectors.

[0134] As used herein, the term "expression cassette" refers to a DNA segment that is capable of driving the expression of a polynucleotide ("transgene") encoding a therapeutically active polypeptide (e.g., LAMP-2B) incorporated into the expression cassette in an appropriate context. When introduced into a host cell, the expression cassette is particularly capable of directing the cell's machinery to transcribe the transgene into RNA, which is then typically further processed and ultimately translated into a therapeutically active polypeptide. The expression cassette may be contained within a gene therapy vector. Generally, the term expression cassette does not include the polynucleotide sequences of the 5' of the 5' ITR and the 3' of the 3' ITR.

[0135] All patents, patent publications, and other publications cited and identified in this specification are hereby incorporated by reference in their entirety for all purposes.

[0136] Examples

[0137] Example 1: Preclinical and Clinical Evaluation of AAVrh74-LAMP-2B

[0138] Generate a plasmid vector comprising a gene expression cassette as depicted in Figure 1 . The transgene was modified to encode LAM2B-HA-FLAG such that the protein could be detected using anti-HA or anti-FLAG antibodies. The AAVrh74-LAMP2B viral vector was generated using a three-plasmid, helper virus-free system to produce recombinant AAV particles containing the serotype rh74 capsid protein and a viral genome with AAV2 ITRs flanking the human LAMP-2B expression cassette. The viral vector was tested in non-human primates.

[0139] Pharmacological and toxicological studies were performed in LAMP-2B - / - and wild-type mice. Based on the preclinical safety and efficacy data observed in the mouse and non-human primate studies, a clinical study of patients with Danon disease was conducted.

[0140] Example 2: DNA, RNA, and Protein Expression in Non-Human Primates after Intravenous Administration of 1×10 13 vg / kg AAV9.LAMP2B and AAVrh74.LAMP2B

[0141] A non-human primate study of AAV9 versus AAVrh74 vectors was conducted in paired male and female African green monkeys (AGMs). The subjects received AAV9.LAMP2B-HA-Flag or AAVrh74.LAMP2B-HA-Flag. "AAV9.LAMP2B-HA-Flag" is an adeno-associated virus vector of AAV9 serotype encoding LAMP2B fused to the C-terminus of the HA-Flag tag. "AAVrh74.LAMP2B-HA-Flag" is an adeno-associated virus vector of AAVrh74 serotype encoding LAMP2B fused to the C-terminus of the HA-Flag tag. One subject was given a vehicle control. The vectors were administered by intravenous injection of 2 mL of 1.85×10 13 vector genomes (vg) / mL (as determined by quantitative polymerase chain reaction (qPCR)) using a plasmid containing the WPRE sequence to generate a reference curve. This injection achieved the target dose of the vector, which was approximately 1.0×10 13 vg / kg. Due to their lower body weight, female rats received a corresponding vector dose of approximately 1.2×10 13 vg / kg. This experiment is summarized in Table 2.

[0142] Table 2

[0143]

[0144] The subjects were humanely euthanized two months after injection, and tissues were collected for DNA, RNA, and protein analysis. The following tissues were examined: heart (left atrium, right atrium, left ventricle, and right ventricle); skeletal muscle (quadriceps and gastrocnemius); liver (left lobe, right lobe, middle lobe, and quadrate lobe); brain (frontal lobe, parietal lobe, temporal lobe, occipital lobe, cortex, hippocampus, medulla, and cerebellum); and gonads.

[0145] Vector DNA – Quantitative PCR

[0146] DNA was extracted from frozen tissues using a Qiagen kit. DNA purity (A260 / A280) and concentration were evaluated on a NanoDrop One TM spectrophotometer (Thermo). On a real-time PCR system (QuantStudio5, Thermo), quantitative PCR (qPCR) of 20 ng of DNA was performed using the following primers / probes and TaqMan Universal Master Mix II (Thermo, 4440038):

[0147] WPRE (box):

[0148] Forward primer: 5`-ATCATGCTATTGCTTCCCGTA-3`(SEQ ID NO:30)

[0149] Reverse primer: 5`-GGGCCACAACTCCTCATAAA-3`(SEQ ID NO:31)

[0150] Probe: 5`-CCTCCTTGTATAAATCCTGGTTGCTGTCT-3`(SEQ ID NO:32)

[0151] RNaseP (housekeeping gene, Thermo)

[0152] A standard curve was generated using plasmid DNA containing the WPRE sequence. Figure 2 Bar graph showing vector DNA quantification by qPCR in the organs most affected by Danon disease.

[0153] LAMP2B mRNA – Quantitative RT-PCR

[0154] RNA was extracted from heart and muscle tissues using the RNeasy Fibrous Tissue Kit (Qiagen), and from liver and brain using the RNeasy Lipid Tissue Kit (Qiagen). Purity (A260 / A280) and concentration were evaluated on a NanoDrop One spectrophotometer (Thermo). RNA was reverse transcribed into cDNA using the Superscript IV VILO Master Mix (Thermo). On a real-time PCR system (QuantStudio5, Thermo), qPCR was performed on 10 ng of RNA using the following primers / probes and TaqMan Universal Master Mix II (Thermo):

[0155] WPRE (box):

[0156] Forward primer: 5`-ATCATGCTATTGCTTCCCGTA-3`(SEQ ID NO:33)

[0157] Reverse primer: 5`-GGGCCACAACTCCTCATAAA-3`(SEQ ID NO:34)

[0158] Probe: 5`-CCTCCTTGTATAAATCCTGGTTGCTGTCT-3`(SEQ ID NO:35)

[0159] HPRT-1 (housekeeping gene, Thermo)

[0160] A standard curve was generated using plasmid DNA containing the WPRE sequence.Figure 3A A bar graph showing the quantification of vector DNA in the heart region by qPCR. Figure 3B A bar graph showing the quantification of vector DNA in muscle by qPCR. Figure 4 A bar graph showing the quantification of mRNA in the organs most affected by Danon disease by RT-qPCR. Figure 5A A bar graph showing the quantification of mRNA in the heart region by RT-qPCR. Figure 5B A bar graph showing the quantification of mRNA in muscle by RT-qPCR.

[0161] LAMP2B mRNA-RNAscope

[0162] Fix 5 mm tissue cubes in 10% neutral buffered formalin, embed in paraffin and section. Transgenic mRNA was detected using the WPRE-O3 ZZ probe (ACD) with RNAscope 2.5LS RED. Semi-quantitative visual assessment was performed on one section from each tissue, and cells with ≥ 1 dot per cell were considered positive. The percentage of positive cells was grouped into five categories: 0%, 1 - 25%, 26 - 50%, 51 - 75% or 100%.

[0163] Figure 6A Micrographs showing semi-quantitative mRNA analysis by RNAscope in untreated left ventricles. Figure 6B Micrographs showing semi-quantitative mRNA analysis by RNAscope in treated left ventricles. Figure 7A Micrographs showing semi-quantitative mRNA analysis by RNAscope in untreated quadriceps. Figure 7B Micrographs showing semi-quantitative mRNA analysis by RNAscope in treated quadriceps. Figure 8 Micrographs showing semi-quantitative mRNA analysis by RNAscope in treated gastrocnemius. Results for vehicle control (Table 3A), AAV9 (Table 3A) and AAVrh74 (Table 3C) were pooled.

[0164] Table 3A

[0165] Animal Tissue Location Treatment % of expressing cells B059 Heart Left ventricle Untreated, vehicle 0％ B059 Muscle Quadriceps Untreated, vehicle 0％ B059 Liver Left lobe Untreated, vehicle 0％

[0166] Table 3B

[0167] Animal Tissue Location Treatment % of expressing cells A991 Heart Left ventricle Treated, AAV9 26-50％ A991 Heart Right ventricle Treated, AAV9 26-50％ A991 Heart Left atrium Treated, AAV9 1-25％ A991 Heart Right atrium Treated, AAV9 26-50％ A991 Muscle Quadriceps Treated, AAV9 0％ A991 Muscle Gastrocnemius Treated, AAV9 0％ A991 Liver Left lobe Treated, AAV9 26-50％ A991 Liver Right lobe Treated, AAV9 51-75％ A602 Heart Left ventricle Treated, AAV9 1-25％ A602 Heart Right ventricle Treated, AAV9 1-25％ A602 Heart Left atrium Treated, AAV9 26-50％ A602 Heart Right atrium Treated, AAV9 1-25％ A602 Muscle Quadriceps Treated, AAV9 1-25％ A602 Muscle Gastrocnemius Treated, AAV9 1-25％ A602 Liver Left lobe Treated, AAV9 51-75％ A602 Liver Right lobe Treated, AAV9 51-75％

[0168] Table 3C

[0169]

[0170]

[0171] LAMP2B Protein - ELISA

[0172] Homogenize approximately 125 mg of tissue in 500 μL of lysis buffer using 0.9 - 2.00 mm stainless steel beads (Next Advance) and a Next Advance Bullet Blender 24. The lysis buffer contains 300 mM NaCl, 20 mM EDTA, 100 mM Tris pH 8.0, 2% NP - 40, 0.2% SDS, and Complete TM EDTA - free protease inhibitor and PhosSTOP TM phosphatase inhibitor. Total protein was evaluated by BCA (Thermo). 100 μg of total protein was loaded per well. A standard curve was constructed using purified human LAMP2 protein (Origene). ELISA was performed using a mouse monoclonal antibody (H4B4, Novus Biologicals) as the capture antibody, a goat polyclonal antibody (R&D Systems) as the detection antibody, and an HRP - conjugated antibody: donkey anti - goat (Millipore) as the secondary antibody. The plate was developed with TMB (Thermo) and quantified on a spectrophotometer (Spectramax M5c).

[0173] Figure 9 A bar graph showing protein quantification by ELISA in the organs most affected by Danon disease is presented. Figure 10A A bar graph showing protein quantification by ELISA in the cardiac region is presented. Figure 10B A bar graph showing protein quantification by ELISA in muscle is presented.

[0174] Clinical Pathology

[0175] Evaluate the pathological effects of the vector. Figure 11A-11D A line graph showing clinical pathology measurements in NHP serum during the study is presented. Clinical pathology levels were evaluated as changes in the following during the study:( Figure 11A ) alanine aminotransferase, ALT;( Figure 11B ) aspartate aminotransferase, AST;( Figure 11C ) white blood cells, WBC;( Figure 11D ) neutrophils. B059 is the vector control. A991 and A602 are animals treated with AAV9. A710 and A981 are animals treated with AAVrh74.

[0176] Conclusion

[0177] At a vector dose of 1.0×10 13 vg / kg, AAV-based gene therapy using the LAMP2B transgene was well tolerated in non-human primates. This result is an important and unexpected result because experiments on AAV-based gene therapy for some other transgenes have demonstrated pathological effects at doses equal to or lower than 1.0×10 13 vg / kg. In addition, both AAV9 and AAVrh74 were well tolerated. At day 21, elevated levels of certain markers were observed in A602 and A710 animals, but these outliers may be due to experimental error or self-resolving pathology.

[0178] Both vectors localized to the target tissues and transduced the target tissues for the treatment of Danon disease (heart and muscle), but had little effect in the brain or gonads. For safety reasons, expression in the gonads is not desired. As expected, a significant amount of the vector accumulated in the liver, which is desired because the liver is the tissue affected by Danon disease. The vector was present in each quadrant of the heart and in both the quadriceps and gastrocnemius muscles. This is the desired result for the treatment of Danon disease.

[0179] The tropism of the AAV vector serotype (e.g., AAV9) does not predict the tropism of other serotypes (e.g., AAVrh74). This experiment confirmed that AAVrh74 achieved the desired tropism for the treatment of Danon disease or other diseases with an etiology related to heart and muscle tissues. LAMP2B transgene mRNA and protein were both expressed in the same group of tissues in the AAV9 and AAVrh74 groups. The expression was comparable between the vector serotypes. The number of animals in the study was too small to discern a statistically significant trend in the expression levels between AAV9 and AAVrh74. RNAscope indicated that a similar fraction of cells were infected in the heart, muscle, and liver tissues of the AAV9 and AAVrh74 groups.

[0180] These data confirm that AAVrh74 can be used as a vector to deliver LAMP2B to tissues relevant to the treatment of Danon disease. In these experiments, AAVrh74 was not inferior to AAV9.

[0181] The present invention relates to the following embodiments:

[0182] 1. A recombinant adeno-associated virus (rAAV) gene therapy vector comprising a polynucleotide and a capsid protein, wherein the polynucleotide comprises a 5' ITR, an expression cassette, and a 3' ITR,

[0183] wherein the expression cassette comprises a transgene encoding lysosome-associated membrane protein 2 (LAMP-2) or a functional variant thereof,

[0184] wherein the expression cassette is flanked by the 5' ITR and the 3' ITR, and

[0185] wherein the capsid protein comprises an AAVrh.74 capsid protein or a functional variant thereof.

[0186] 2. The rAAV gene therapy vector according to embodiment 1, wherein the LAMP-2 is selected from LAMP-2A, LAMP-2B, and LAMP-2C.

[0187] 3. The rAAV gene therapy vector according to embodiment 1, wherein the capsid protein has at least 95% sequence identity with an amino acid sequence selected from SEQ ID NO: 2-4.

[0188] 4. The rAAV gene therapy vector according to embodiment 3, wherein the capsid protein has at least 95% sequence identity with SEQ ID NO: 2.

[0189] 5. The rAAV gene therapy vector according to embodiment 4, wherein the capsid protein has at least 97% sequence identity with SEQ ID NO: 2.

[0190] 6. The rAAV gene therapy vector according to embodiment 5, wherein the capsid protein has at least 99% sequence identity with SEQ ID NO: 2.

[0191] 7. The rAAV gene therapy vector according to any one of embodiments 1 to 6, wherein the capsid protein is an AAVrh.74 capsid protein.

[0192] 8. The rAAV gene therapy vector according to any one of embodiments 1 to 7, wherein the 5' ITR and the 3' ITR are respectively the 5' ITR of AAV2 and the 3' ITR of AAV2, or variants thereof.

[0193] 9. The rAAV gene therapy vector according to embodiment 8, wherein the 5' ITR has at least 98% identity with SEQ ID NO: 13, and the 3' ITR has at least 98% identity with SEQ ID NO: 14.

[0194] 10. The rAAV gene therapy vector according to any one of embodiments 1 to 9, wherein the transgene is codon-optimized for expression in human host cells.

[0195] 11. The rAAV gene therapy vector according to any one of embodiments 1 to 10, wherein the expression cassette contains fewer CpG sites than SEQ ID: 6.

[0196] 12. The rAAV gene therapy vector according to any one of Embodiments 1 to 11, wherein the expression cassette contains fewer cryptic splicing sites than SEQ ID: 6.

[0197] 13. The rAAV gene therapy vector according to any one of Embodiments 1 to 12, wherein the expression cassette encodes fewer alternative open reading frames than SEQ ID: 6.

[0198] 14. The rAAV gene therapy vector according to any one of Embodiments 1 to 13, wherein the transgene shares at least 95% identity with a sequence selected from SEQ ID NO: 7-9.

[0199] 15. The rAAV gene therapy vector according to Embodiment 14, wherein the transgene shares at least 99% identity with a sequence selected from SEQ ID NO: 7-9.

[0200] 16. The rAAV gene therapy vector according to Embodiment 15, wherein the transgene contains a sequence selected from SEQ ID NO: 7-9.

[0201] 17. The rAAV gene therapy vector according to any one of Embodiments 1 to 16, wherein the expression cassette contains a consensus optimal Kozak sequence operably linked to the transgene, wherein the consensus optimal Kozak sequence contains SEQ ID NO: 20.

[0202] 18. The rAAV gene therapy vector according to any one of Embodiments 1 to 17, wherein the expression cassette contains a full-length polyA sequence operably linked to the transgene, wherein the full-length polyA sequence contains SEQ ID NO: 26.

[0203] 19. The rAAV gene therapy vector according to any one of Embodiments 1 to 18, wherein the expression cassette does not contain a start codon on the 5' side of the start codon of the transgene.

[0204] 20. The rAAV gene therapy vector according to any one of Embodiments 1 to 19, wherein the expression cassette contains a first inverted terminal repeat sequence, an enhancer / promoter region, an intron, a consensus optimal Kozak sequence, the transgene, a 3' untranslated region including a full-length polyA sequence, and a second inverted terminal repeat sequence operably linked in the 5' to 3' direction, wherein the expression cassette does not contain a start codon on the 5' side of the start codon of the transgene.

[0205] 21. The rAAV gene therapy vector as described in Embodiment 20, wherein the enhancer / promoter region contains the CMV IE enhancer and the chicken β-actin promoter in the 5' to 3' direction, and optionally wherein the enhancer / promoter region further contains the first exon and the first intron of the chicken β-actin gene and the splice acceptor of the rabbit β-globin gene.

[0206] 22. The rAAV gene therapy vector as described in Embodiment 20, wherein the enhancer / promoter region contains a tissue-specific promoter capable of mediating increased expression in cardiac tissue and / or skeletal muscle tissue compared to liver tissue.

[0207] 23. The rAAV gene therapy vector as described in any one of Embodiments 1 to 21, wherein the expression cassette has at least 95% identity with a sequence selected from SEQ ID NOs: 10 - 12.

[0208] 24. The rAAV gene therapy vector as described in Embodiment 23, wherein the expression cassette contains a sequence selected from SEQ ID NOs: 10 - 12.

[0209] 25. A pharmaceutical composition comprising the rAAV gene therapy vector as described in any one of Embodiments 1 to 24.

[0210] 26. A method of treating or preventing Danon disease or another autophagy disorder in a subject in need thereof, comprising administering to the subject the rAAV gene therapy vector as described in any one of Embodiments 1 to 24 or the pharmaceutical composition as described in Embodiment 25.

[0211] 27. The method as described in Embodiment 26, wherein the rAAV gene therapy vector or the pharmaceutical composition is administered via a route selected from the group consisting of intravenous, intra-arterial, intracardiac, intracoronary, intramyocardial, intrarenal, intraurethral, epidural, and intramuscular.

[0212] 28. The method as described in Embodiment 26 or Embodiment 27, wherein the autophagy disorder is selected from the group consisting of end-stage heart failure, myocardial infarction, drug toxicity, diabetes, end-stage renal failure, and aging.

[0213] 29. The method as described in any one of Embodiments 26 to 28, wherein the subject is a human.

[0214] 30. The method as described in any one of Embodiments 26 to 29, wherein the subject exhibits symptoms of Danon disease or another autophagy disorder.

[0215] 31. The method according to any one of embodiments 26 to 30, wherein the subject has been identified as having reduced or undetectable endogenous LAMP-2 expression.

[0216] 32. The method according to any one of embodiments 26 to 31, wherein the subject has been identified as having a mutated LAMP-2 gene.

[0217] 33. The method according to any one of embodiments 26 to 32, wherein the rAAV gene therapy vector is administered at a dose of about 3×10 12 vg / kg to about 3×10 14 vg / kg.

[0218] 34. The method according to any one of embodiments 26 to 33, wherein the rAAV gene therapy vector is administered at a dose of about 3×10 12 vg / kg to about 1.2×10 13 vg / kg.

[0219] 35. The method according to any one of embodiments 26 to 33, wherein the rAAV gene therapy vector is administered at a dose of about 1.0×10 13 vg / kg.

[0220] 36. The method according to any one of embodiments 26 to 35, wherein the dose of the rAAV gene therapy vector, optionally when administered at a dose of about 1.0×10 13 vg / kg, does not cause clinical pathology.

[0221] 37. The method according to any one of embodiments 26 to 36, wherein the administration of the rAAV gene therapy vector transduces one or more of the heart, muscle, and liver.

[0222] 38. The method according to any one of embodiments 26 to 37, wherein the administration of the rAAV gene therapy vector results in LAMP2B mRNA expression in one or more of the heart, muscle, and liver.

[0223] 39. The method according to any one of embodiments 26 to 38, wherein the administration of the rAAV gene therapy vector results in LAMP2B protein expression in one or more of the heart, muscle, and liver.

[0224] 40. The method according to any one of embodiments 26 to 39, wherein the administration of the rAAV gene therapy vector results in at least about 10%, at least about 20%, or at least about 30% of the cells in one or more of the heart, muscle, and liver being infected with the rAAV gene therapy vector.

[0225] 41. The method according to any one of embodiments 26 to 40, wherein administration of the rAAV gene therapy vector results in transduction of the rAAV gene therapy vector in the gonad at less than 0.1 vector genomes (vg) per diploid genome.

[0226] 42. The method according to any one of embodiments 26 to 41, wherein administration of the rAAV gene therapy vector causes LAMP2B mRNA expression in the gonad at less than 2×10 4 mRNA copies per microgram of total RNA.

[0227] 43. The method according to any one of embodiments 26 to 42, wherein administration of the rAAV gene therapy vector does not cause LAMP2B protein expression in the brain and / or gonad.

[0228] 44. The method according to any one of embodiments 26 to 43, wherein administration of the rAAV gene therapy vector transduces and / or causes transgene expression at a level substantially the same as that of an AAV9 gene therapy vector having the same expression cassette.

[0229] 45. A method of delivering a LAMP-2 polynucleotide encoding a LAMP-2 protein to a cell, comprising contacting the cell with an rAAV gene therapy vector according to any one of embodiments 1 to 24 or a pharmaceutical composition according to embodiment 25, wherein the cell is optionally selected from cardiac cells, lung cells, and / or muscle cells.

[0230] 46. A method of transducing a cell, comprising contacting the cell with an rAAV gene therapy vector according to any one of embodiments 1 to 24 or a pharmaceutical composition according to embodiment 25, wherein the cell is optionally selected from cardiac cells, lung cells, and / or muscle cells.

[0231] 47. A method of delivering a LAMP-2 polynucleotide encoding a LAMP-2 protein to a tissue and / or expressing a LAMP-2 protein in a tissue, comprising contacting the tissue with an rAAV gene therapy vector according to any one of embodiments 1 to 24 or a pharmaceutical composition according to embodiment 25, wherein the tissue is optionally selected from cardiac tissue, lung tissue, and / or muscle tissue.

[0232] 48. A method of delivering a LAMP-2 polynucleotide encoding a LAMP-2 protein to a subject and / or expressing a LAMP-2 protein in a subject, comprising administering to the subject an rAAV gene therapy vector according to any one of embodiments 1 to 24 or a pharmaceutical composition according to embodiment 25.

[0233] 49. The method according to embodiment 48, wherein the rAAV gene therapy vector or pharmaceutical composition is administered via a route selected from the group consisting of intravenous, intra-arterial, intracardiac, intracoronary, intramyocardial, intrarenal, intraurethral, epidural, and intramuscular.

[0234] 50. The method according to embodiment 48 or embodiment 49, wherein the subject has an autophagy disorder or is at risk of an autophagy disorder, and the autophagy disorder is selected from the group consisting of Danon disease, end-stage heart failure, myocardial infarction, drug toxicity, diabetes, end-stage renal failure, and aging.

[0235] 51. The method according to any one of embodiments 48 to 50, wherein the subject is a human.

[0236] 52. The method according to any one of embodiments 48 to 51, wherein the subject exhibits symptoms of an autophagy disorder.

[0237] 53. The method according to any one of embodiments 48 to 52, wherein the subject has been identified as having reduced or undetectable endogenous LAMP-2 expression.

[0238] 54. The method according to any one of embodiments 48 to 53, wherein the subject has been identified as having a mutated LAMP-2 gene.

[0239] 55. The method according to any one of embodiments 48 to 54, wherein the rAAV gene therapy vector is administered at a dose of about 3×10 12 vg / kg to about 3×10 14 vg / kg.

[0240] 56. The method according to any one of embodiments 48 to 55, wherein the rAAV gene therapy vector is administered at a dose of about 3×10 12 vg / kg to about 1.2×10 13 vg / kg.

[0241] 57. The method according to any one of embodiments 48 to 56, wherein the rAAV gene therapy vector is administered at a dose of about 1.0×10 13 vg / kg.

[0242] 58. The method according to any one of embodiments 48 to 57, wherein the dose of the rAAV gene therapy vector, optionally when administered at a dose of about 1.0×10 13 vg / kg, does not cause clinical pathology.

[0243] 59. The method according to any one of embodiments 48 to 58, wherein administration of the rAAV gene therapy vector transduces one or more of the heart, muscle, and liver.

[0244] 60. The method according to any one of embodiments 48 to 59, wherein administration of the rAAV gene therapy vector causes LAMP2B mRNA expression in one or more of the heart, muscle, and liver.

[0245] 61. The method according to any one of embodiments 48 to 60, wherein administration of the rAAV gene therapy vector causes LAMP2B protein expression in one or more of the heart, muscle, and liver.

[0246] 62. The method according to any one of embodiments 48 to 61, wherein administration of the rAAV gene therapy vector results in at least about 10%, at least about 20%, or at least about 30% of the cells in one or more of the heart, muscle, and liver being infected with the rAAV gene therapy vector.

[0247] 63. The method according to any one of embodiments 48 to 62, wherein administration of the rAAV gene therapy vector results in transduction of the rAAV gene therapy vector in the gonad at less than 0.1 vector genomes (vg) per diploid genome.

[0248] 64. The method according to any one of embodiments 48 to 63, wherein administration of the rAAV gene therapy vector causes LAMP2B mRNA expression in the gonad at less than 2×10 4 mRNA copies per microgram of total RNA.

[0249] 65. The method according to any one of embodiments 48 to 64, wherein administration of the rAAV gene therapy vector does not cause LAMP2B protein expression in the brain and / or gonad.

[0250] 66. The method according to any one of embodiments 48 to 65, wherein administration of the rAAV gene therapy vector transduces and / or causes transgene expression at a level substantially the same as that of an AAV9 gene therapy vector having the same expression cassette.

Claims

1. A recombinant adeno-associated virus (rAAV) gene therapy vector, comprising a polynucleotide and a capsid protein, wherein the polynucleotide comprises a 5' ITR, an expression cassette, and a 3' ITR, wherein the expression cassette comprises a transgene encoding lysosome-associated membrane protein 2 (LAMP-2) or a functional variant thereof, wherein the expression cassette is flanked by the 5’ ITR and the 3’ ITR, and wherein the capsid protein comprises an AAVrh.74 capsid protein or a functional variant thereof.

2. The rAAV gene therapy vector according to claim 1, wherein the LAMP-2 is selected from LAMP-2A, LAMP-2B, and LAMP-2C.

3. The rAAV gene therapy vector according to claim 1, wherein the capsid protein has at least 95% sequence identity with an amino acid sequence selected from SEQ ID NO: 2-4.

4. The rAAV gene therapy vector according to any one of claims 1 to 3, wherein the expression cassette comprises a first inverted terminal repeat, an enhancer / promoter region, an intron, a consensus optimal Kozak sequence, the transgene, a 3' untranslated region including a full-length polyA sequence, and a second inverted terminal repeat, operably linked in the 5’ to 3’ direction, and wherein the expression cassette does not contain a start codon 5' to the start codon of the transgene.

5. The rAAV gene therapy vector according to any one of claims 1 to 4, wherein the expression cassette has at least 95% identity with a sequence selected from SEQ ID NO: 10-12.

6. A pharmaceutical composition comprising the rAAV gene therapy vector according to any one of claims 1 to 5.

7. A method of treating or preventing Danon disease or another autophagy disorder in a subject in need thereof, comprising administering to the subject the rAAV gene therapy vector according to any one of claims 1 to 5 or the pharmaceutical composition according to claim 6.

8. The method according to claim 7, wherein the rAAV gene therapy vector or pharmaceutical composition is administered via a route selected from the group consisting of intravenous, intraarterial, intracardiac, intracoronary, intramyocardial, intrarenal, intraurethral, epidural, and intramuscular.

9. The method according to claim 7 or claim 8, wherein the autophagy disorder is selected from the group consisting of end-stage heart failure, myocardial infarction, drug toxicity, diabetes, end-stage renal failure, and aging.

10. A method of delivering a LAMP-2 polynucleotide encoding a LAMP-2 protein to a cell, comprising contacting the cell with the rAAV gene therapy vector according to any one of claims 1 to 5 or the pharmaceutical composition according to claim 6, wherein the cell is optionally selected from cardiac cells, lung cells, and / or muscle cells.

11. A method of transducing a cell, comprising contacting the cell with the rAAV gene therapy vector according to any one of claims 1 to 5 or the pharmaceutical composition according to claim 6, wherein the cell is optionally selected from cardiac cells, lung cells, and / or muscle cells.

12. A method of delivering a LAMP-2 polynucleotide encoding a LAMP-2 protein to a tissue and / or expressing the LAMP-2 protein in a tissue, comprising contacting the tissue with the rAAV gene therapy vector according to any one of claims 1 to 5 or the pharmaceutical composition according to claim 6, wherein the tissue is optionally selected from cardiac tissue, lung tissue, and / or muscle tissue.

13. A method of delivering a LAMP-2 polynucleotide encoding a LAMP-2 protein to a subject and / or expressing the LAMP-2 protein in a subject, comprising administering to the subject the rAAV gene therapy vector according to any one of claims 1 to 5 or the pharmaceutical composition according to claim 6.

14. The method according to claim 13, wherein the rAAV gene therapy vector or the pharmaceutical composition is administered via a route selected from the group consisting of intravenous, intra-arterial, intracardiac, intracoronary, intramyocardial, intrarenal, intraurethral, epidural, and intramuscular.

15. The method according to claim 13 or claim 14, wherein the subject has an autophagy disorder or is at risk of an autophagy disorder, the autophagy disorder being selected from Danon disease, end-stage heart failure, myocardial infarction, drug toxicity, diabetes, end-stage renal failure, and aging.

16. The method according to any one of claims 13-15, wherein the subject is human.

17. The method according to any one of claims 13-16, wherein the subject exhibits symptoms of an autophagy disorder.

18. The method according to any one of claims 13-17, wherein the subject has been identified as having reduced or undetectable endogenous LAMP-2 expression.

19. The method according to any one of claims 13-18, wherein the subject has been identified as having a mutated LAMP-2 gene.

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