GENE THERAPY VECTORS FOR TREATMENT OF DANON DISEASE

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Appl. No. 62/697,302, filed July 12, 2018, which is incorporated herein by reference in its entirety. SEQUENCE LISTING

This application is being filed electronically via EFS-Web and includes an electronically submitted sequence listing in .txt format. The .txt file contains a sequence listing entitled “ROPA 01 l_OlWO_SeqList_ST25.txf’ created on July 11, 2019 and having a size of ~62 kilobytes. The sequence listing contained in this .txt file is part of the specification and is incorporated herein by reference in its entirety.

FIELD OF INVENTION

The invention relates generally to gene therapy for diseases associated with mutations in lysosome-associated membrane protein 2 (LAMP-2, also known as CD 107b).

BACKGROUND

Lysosome-associated membrane protein 2 (LAMP-2, also known as CD 107b) is a gene that encodes a lysosome-associated membrane glycoprotein. Alternative splicing of the gene produces 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.

International Patent Application Publication No. WO2017127565A1 discloses that overexpression of LAMP-2 in human induced pluripotent stem cells (hiPSCs) derived from patients with LAMP-2 mutations, as described in Hashem, et ah, Stem Cells. 2015

Jul;33(7):2343-50, results in reduced oxidative stress levels and apoptotic cell death, confirming the importance of LAMP-2B in disease pathophysiology.

There remains a need in the art for gene therapy vectors for LAMP-2. The present disclosure provides such gene therapy vectors, methods of use thereof, pharmaceutical compositions, and more. SUMMARY OF THE INVENTION

The present disclosure provides improved gene therapy vectors comprising a polynucleotide sequence encoding a LAMP-2 polypeptide, methods of use thereof, pharmaceutical compositions, and more.

In one aspect, the disclosure provides a gene therapy vector comprising an expression cassette comprising a transgene encoding an isoform of lysosome-associated membrane protein 2 (LAMP-2) or a functional variant thereof, wherein the transgene is codon-optimized for expression in a human host cell.

In an embodiment, the expression cassette contains fewer CpG sites than SEQ ID: 2. In an embodiment, the expression cassette contains fewer cryptic splice sites than

SEQ ID: 2.

In an embodiment, the expression cassette encodes fewer alternative open reading frames than SEQ ID: 2.

In an embodiment, the transgene shares at least 95% identity to a sequence selected from SEQ ID NOs: 3-5.

In an embodiment, the transgene shares at least 99% identity to a sequence selected from SEQ ID NOs: 3-5.

In an embodiment, the transgene comprises a sequence selected from SEQ ID NOs: 3- 5.

In an embodiment, the transgene shares at least 95% identity to SEQ ID NO: 3.

In an embodiment, the transgene shares at least 99% identity to SEQ ID NO: 3.

In an embodiment, the transgene comprises a sequence identical to SEQ ID NO: 3.

In an embodiment, the expression cassette comprises a consensus optimal Kozak sequence operatively linked to the transgene, wherein optionally the consensus optimal Kozak sequence comprises SEQ ID NO: 6.

In an embodiment, the expression cassette comprises a full-length polyA sequence operatively linked to the transgene, wherein optionally the full-length polyA sequence comprises SEQ ID NO: 7. In an embodiment, the expression cassette comprises no start site 5' to the transgene capable of generating alternative mRNAs.

In an embodiment, the expression cassette comprises operatively linked, in the 5' to 3' direction, a first inverse terminal repeat, an enhancer/promoter region, a consensus optimal Kozak sequence, the transgene, a 3' untranslated region including a full-length polyA sequence, and a second inverse terminal repeat.

In an embodiment, the enhancer/promoter region comprises in the 5' to 3' direction a CMV IE enhancer and a chicken beta-actin promoter.

In an embodiment, the expression cassette shares at least 95% identity to a sequence selected from SEQ ID NOs: 8-10.

In an embodiment, the expression cassette shares complete identity to a sequence selected from SEQ ID NOs: 8-10.

In a second aspect, the disclosure provides a method of preventing, mitigating, ameliorating, reducing, inhibiting, eliminating and/or reversing one or more symptoms of Danon disease or another autophagy disorder in a subject in need thereof, comprising administering to the subject any gene therapy vector of the disclosure.

In an embodiment, the vector is administered via a route selected from the group consisting of intravenous, intra-arterial, intracardiac, intracoronary, intramyocardial, intrarenal, intraurethral, epidural, and intramuscular.

In an embodiment, the autophagy disorder is selected from the group consisting of end-stage heart failure, myocardial infarction, drug toxicities, diabetes, end-stage renal failure, and aging.

In an embodiment, the subject is a human.

In an embodiment, the subject is exhibiting symptoms of Danon disease or another autophagy disorder.

In an embodiment, the subject has been identified as having reduced or non-detectable LAMP-2 expression.

In an embodiment, the subject has been identified as having a mutated LAMP -2 gene. In a third aspect, the disclosure provides a pharmaceutical composition for use in preventing, mitigating, ameliorating, reducing, inhibiting, eliminating and/or reversing one or more symptoms of Danon disease or another autophagy disorder, comprising any gene therapy vector of the disclosure.

Other features and advantages of the invention will be apparent from and

encompassed by the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A provides a diagram of an illustrative embodiment of a viral vector of the disclosure.

FIG. IB provides a diagram of an illustrative embodiment of an expression cassette of an adeno-associated virus (AAV) gene therapy vector.

FIG. 2 shows the expression cassette of plasmid-based green fluorescence protein (GFP) reporter system used to test and compare wildtype verses codon-optimized LAMP-2B constructs.

FIG. 3 is a graph showing transfection expression efficiency of LAMP-2B constructs tested using transfection of a plasmid-based GFP reporter system and measured as GFP+ cells per well. A wild-type LAMP-2B construct (WT) is compared to three codon-optimized (“CO”) constructs, CO 1, CO 2, and CO 3, and a no-vector control (labeled“ViaFect Only”).

FIG. 4 is a graph showing the gene expression level in cells transfected with plasmid- based GFP reporting systems used to test LAMP-2B constructs, which is measured as mean GFP intensity of GFP+ cells in absorbance units (A.U.). GFP expression by cells transfected with a wild-type LAMP-2B construct (WT) is compared to GFP expression by three codon- optimized (“CO”) constructs, CO 1, CO 2, and CO 3, or a no-vector control (labeled “ViaFect Only”).

FIG. 5 shows immunofluorescence images of induced pluripotent stem cell (iPSC)- derived cardiomyocytes two days after transfection with a plasmid-based GFP reporter system. Cells were transfected with no DNA, or LAMP-2B constructs expressing wild-type LAMP-2B, the CO 1 variant, the CO 2 variant, or the CO 3 variant.

FIG. 6 shows immunofluorescence images of iPSC-derived cardiomyocytes seven days after transfection with a plasmid-based GFP reporter system. Cells were transfected with no DNA, or LAMP-2B constructs expressing wild-type LAMP-2B, the CO 1 variant, the CO 2 variant, or the CO 3 variant.

FIG. 7A shows an immunoblot of human LAMP-2B protein in CHO-Lec2 cells transduced with AAV9 viral vectors comprising the wild-type LAMP-2B vl.O transgene (AAV9 1.0), the optimized variant LAMP-2B vl.2 transgene (AAV9 1.2) or a GFP transgene (AAV9 GFP). Molecular weight markers (MW) and a control LAMP-2B recombinant protein sample (LAMP2B (+ve control)) were also included.

FIG. 7B shows quantification of LAMP-2B protein by ELISA in CHO-Lec2 cells transduced with AAV9-wild-type LAMP-2B (vl.O), AAV9-optimized LAMP-2B (vl.2) or AAV9-GFP (GFP) vectors.

FIG. 8A shows immunofluorescence images of Danon patient iPSC-derived cardiomyocytes transduced with the indicated amounts of AAV9-Luc (Luc), AAV9-wild- type LAMP-2B (LAMP2B vl .O) or AAV9-optimized LAMP-2B (LAMP2B vl .2) vectors.

FIG. 8B shows quantification of immunofluorescence of human LAMP-2B protein in Danon patient iPSC-derived cardiomyocytes transduced with AAV9-Luc, AAV9-wild-type LAMP-2B (vl.O) or AAV9-optimized LAMP-2B (vl.2) vectors.

FIG. 8C shows an immunoblot of human LAMP-2B protein in Danon patient iPSC- derived cardiomyocytes transduced with AAV9-Luc, AAV9-wild-type LAMP-2B (vl.O) or AAV9-optimized LAMP-2B (vl.2) vectors.

FIG. 9A shows PCR quantification of viral vector DNA in cardiac tissue isolated from LAMP -2-deficient mice treated with AAV9-wild-type LAMP-2B (vl.O), AAV9- optimized LAMP-2B (vl.2) or an AAV9 vehicle control. Vector copy number was quantified as VCN/Diploid Nucleus in the cardiac tissue. Control wild-type mice not injected with vector were included as controls (WT).

FIG. 9B shows quantitative RT-PCR analyses of transgene mRNA, measured by RT- PCR using probes specific for the WPRE element, in cardiac tissue isolated from LAMP -2- deficient mice treated with AAV9-wild-type LAMP-2B (vl.O), AAV9-optimized LAMP-2B (vl.2) or an AAV9 vehicle control (Vehicle). Expression of mRNA was quantified as vector genomes (vg) per pg total cellular RNA using a standard curve to convert copy number to vector genomes. FIG. 9C shows an immunoblot of LAMP-2B protein in cardiac tissue isolated from LAMP-2-deficient mice treated with AA9-wild-type LAMP-2B (vl.0), AAV9-optimized LAMP-2B (vl.2) or the AAV9 vehicle control (Vehicle) compared to untreated wild-type mice (Untreated).

FIG. 9D shows immunofluorescence images of human LAMP-2B protein in cardiac tissue isolated from LAMP-2-deficient mice treated with AAV9-wild-type LAMP-2B (vl.0), AAV9-optimized LAMP-2B (vl.2) or the AAV9 vehicle control.

FIG. 10A shows PCR quantification of viral vector DNA in heart, muscle, liver and brain tissue isolated from primates treated with the AAV9-optimized human LAMP-2B (treated) vector or no vector vehicle control (untreated). Individuals are denoted as black or white squares.

FIG. 10B shows PCR quantification of viral vector DNA in cardiac chambers isolated from primates treated with AAV9-optimized human LAMP-2B vector (treated) or no vector vehicle control (untreated). Individuals are denoted as B059 (male, M), A991 (female, F), and A602 (male, M).

FIG. 10C shows quantitative RT-PCR analyses of transgene mRNA, measured by RT-PCR using probes specific for the WPRE element, in heart, muscle, liver and brain tissue isolated from primates treated with the AAV9-optimized human LAMP-2B vector (treated) or no vector vehicle control (untreated).

FIG. 10D shows quantitative RT-PCR analyses of transgene mRNA in cardiac chambers isolated from primates injected with the AAV9-optimized human LAMP-2B vector (treated) or no vector vehicle control (untreated).

FIG. 10E shows percentage of cells expressing transgene mRNA in situ in heart, muscle and liver tissue isolated from primates injected with AAV9-optimized human LAMP- 2B vector (treated) or no vector vehicle control (untreated). Individuals are denoted as B059 (male, M), A991 (female, F), and A602 (male, M).

FIG. 10F shows transgene mRNA staining in situ in heart tissue isolated from primates injectioned with the AAV9-optimized human LAMP-2B vector or no vector vehicle control (untreated). Individuals are denoted as B059 (male, M), A991 (female, F), and A602 (male, M). FIG. 10G shows fold change of LAMP-2B protein assessed by western blot in heart, muscle and liver tissue isolated from primates treated with the AAV9-optimized human LAMP-2B vector relative to no vector (untreated).

FIG. 10H shows fold change of LAMP-2B protein assessed by western blot in cardiac chambers isolated from primates treated with the AAV9-optimized human LAMP-2B vector relative to no vector (untreated).

FIG. 101 shows quantification of LAMP-2B protein by ELISA in heart, muscle and liver tissue isolated from primates treated with the AAV9-optimized human LAMP-2B vector relative to no vector (untreated).

FIG. 10 J shows quantification of LAMP-2B protein by ELISA in cardiac chambers isolated from primates treated with the AAV9-optimized human LAMP-2B vector relative to no vector (untreated).

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure provides improved polynucleotide sequences, expression cassettes, and vectors encoding an isoform of LAMP-2 (e.g., LAMP-2B), as well as related pharmaceutical compositions, and their use to treat diseases and disorders associated with LAMP-2 deficiency or mutation. The present inventors have discovered that modifications to the gene sequence of LAMP-2B result in increased transgene expression. In addition, the presence of specific sequence elements in the expression cassettes of gene therapy vectors encoding LAMP-2B result in an improvement in transgene expression. Accordingly, the LAMP-2 polynucleotide sequences, expression cassettes, and vectors disclosed herein offer advantages for gene therapy as compared to previous gene therapy vectors, including the ability to achieve higher levels of LAMP-2 expression in therapeutically relevant tissues.

The wild-type polypeptide sequence of human LAMP-2B (SEQ ID NO: 1) and the wild-type polynucleotide sequence encoding human LAMP-2B (SEQ ID NO: 2) are, respectively:

1 MVCFRLFPVP GSGLVLVCLV LGAVRSYALE LNLTDSENAT CLYAKWQMNF TVRYETTNKT

61 YKTVTISDHG TVTYNGSICG DDQNGPKIAV QFGPGFSWIA NFTKAASTYS IDSVSFSYNT

121 GDNTTFPDAE DKGILTVDEL LAIRIPLNDL FRCNSLSTLE KNDWQHYWD VLVQAFVQNG

181 TVSTNEFLCD KDKTSTVAPT IHTTVPSPTT TPTPKEKPEA GTYSWNGND TCLLATMGLQ

241 LNITQDKVAS VININPNTTH STGSCRSHTA LLRLNSSTIK YLDFVFAVKN ENRFYLKEW

301 ISMYLWGSV FSIANNNLSY WDAPLGSSYM CNKEQTVSVS GAFQINTFDL RVQPFNVTQG

361 KYSTAQECSL DDDTILIPII VGAGLSGLII VIVIAYVIGR RKSYAGYQT (SEQ ID NO: 1); and

1 ATGGTGTGCT TCCGCCTCTT CCCGGTTCCG GGCTCAGGGC TCGTTCTGGT CTGCCTAGTC

61 CTGGGAGCTG TGCGGTCTTA TGCATTGGAA CTTAATTTGA CAGATTCAGA AAATGCCACT

121 TGCCTTTATG CAAAATGGCA GATGAATTTC ACAGTTCGCT ATGAAACTAC AAATAAAACT

181 TATAAAACTG TAACCATTTC AGACCATGGC ACTGTGACAT ATAATGGAAG CATTTGTGGG

241 GATGATCAGA ATGGTCCCAA AATAGCAGTG CAGTTCGGAC CTGGCTTTTC CTGGATTGCG

301 AATTTTACCA AGGCAGCATC TACTTATTCA ATTGACAGCG TCTCATTTTC CTACAACACT

361 GGTGATAACA CAACATTTCC TGATGCTGAA GATAAAGGAA TTCTTACTGT TGATGAACTT

421 TTGGCCATCA GAATTCCATT GAATGACCTT TTTAGATGCA ATAGTTTATC AACTTTGGAA

481 AAGAATGATG TTGTCCAACA CTACTGGGAT GTTCTTGTAC AAGCTTTTGT CCAAAATGGC

541 ACAGTGAGCA CAAATGAGTT CCTGTGTGAT AAAGACAAAA CTTCAACAGT GGCACCCACC

601 ATACACACCA CTGTGCCATC TCCTACTACA ACACCTACTC CAAAGGAAAA ACCAGAAGCT

661 GGAACCTATT CAGTTAATAA TGGCAATGAT ACTTGTCTGC TGGCTACCAT GGGGCTGCAG

721 CTGAACATCA CTCAGGATAA GGTTGCTTCA GTTATTAACA TCAACCCCAA TACAACTCAC

781 TCCACAGGCA GCTGCCGTTC TCACACTGCT CTACTTAGAC TCAATAGCAG CACCATTAAG

841 TATCTAGACT TTGTCTTTGC TGTGAAAAAT GAAAACCGAT TTTATCTGAA GGAAGTGAAC

901 ATCAGCATGT ATTTGGTTAA TGGCTCCGTT TTCAGCATTG CAAATAACAA TCTCAGCTAC

961 TGGGATGCCC CCCTGGGAAG TTCTTATATG TGCAACAAAG AGCAGACTGT TTCAGTGTCT

1021 GGAGCATTTC AGATAAATAC CTTTGATCTA AGGGTTCAGC CTTTCAATGT GACACAAGGA

1081 AAGTATTCTA CAGCCCAAGA GTGTTCGCTG GATGATGACA CCATTCTAAT CCCAATTATA

1141 GTTGGTGCTG GTCTTTCAGG CTTGATTATC GTTATAGTGA TTGCTTACGT AATTGGCAGA

1201 AGAAAAAGTT ATGCTGGATA TCAGACTCTG TAA

(SEQ ID NO: 2).

Disclosed herein are modified polynucleotide sequences encoding an isoform of lysosome-associated membrane protein 2 (LAMP-2) or a functional variant thereof. In certain embodiments, the modified polynucleotide sequences comprise one or more of the following modifications as compared to the wild-type polynucleotide encoding the isoform of LAMP-2: codon-optimization, CpG depletion, removal of cryptic splice sites, or a reduced number of alternative open-reading frames (ORFs). In some embodiments, the modified polynucleotide encodes LAMP-2A, LAMP-2B, LAMP-2C or a functional variant of any of these isoforms.

In embodiments, the disclosure provides a polynucleotide sequence or transgene encoding LAMP-2B or a functional variant thereof comprising one or more nucleotide substitutions as compared to SEQ ID NO:2. In embodiments, the transgene shares at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or complete identity to a sequence selected from SEQ ID NOs: 3-5. The disclosure provides at least three illustrative variant transgene sequences encoding LAMP-2B (SEQ ID NOs: 3-5):

1 ATGGTCTGCT TCAGACTGTT CCCTGTCCCT GGATCTGGTC TGGTGCTTGT GTGCTTGGTG 61 CTGGGTGCTG TGAGATCCTA TGCCCTTGAG CTGAACCTGA CTGACTCAGA AAATGCCACT 121 TGCCTGTATG CCAAGTGGCA GATGAACTTC ACTGTGAGAT ATGAGACTAC CAACAAGACC 181 TACAAGACTG TGACCATCTC AGACCATGGC ACTGTCACCT ACAATGGATC AATCTGTGGT 241 GATGATCAGA ATGGCCCAAA GATAGCAGTG CAGTTTGGGC CCGGTTTTTC CTGGATTGCT 301 AACTTCACCA AGGCAGCCTC CACCTACAGC ATTGACTCAG TCAGCTTCAG CTACAACACT 361 GGGGATAACA CCACCTTCCC TGACGCAGAG GACAAGGGAA TCCTTACTGT GGACGAACTC 421 CTGGCAATCA GAATCCCCCT TAACGACCTG TTCAGATGCA ACTCCCTTTC AACCCTTGAA 481 AAGAATGATG TGGTGCAACA CTATTGGGAC GTCCTGGTGC AAGCCTTTGT GCAGAATGGG 541 ACAGTGAGTA CCAACGAGTT CCTCTGTGAC AAGGACAAGA CCAGCACTGT GGCCCCCACT 601 ATCCACACCA CTGTGCCCAG CCCTACCACT ACCCCCACCC CTAAAGAGAA GCCAGAAGCT 661 GGAACCTACT CAGTCAACAA TGGAAATGAC ACATGCCTCC TTGCCACCAT GGGACTGCAG 721 CTGAACATCA CTCAGGACAA GGTGGCCTCA GTGATTAACA TCAACCCTAA CACCACTCAT 781 AGCACTGGGA GCTGCAGATC ACATACAGCT CTGCTGAGGC TCAACTCCTC CACCATCAAG 841 TACCTGGACT TTGTGTTTGC TGTGAAGAAT GAGAACAGGT TCTACCTCAA GGAAGTGAAC 901 ATTTCCATGT ACCTGGTCAA TGGTTCAGTG TTCTCTATTG CCAACAACAA TCTGAGCTAC 961 TGGGATGCAC CCCTGGGATC CTCCTACATG TGCAACAAGG AGCAGACTGT GAGTGTGTCA 1021 GGTGCTTTTC AGATCAACAC TTTTGACCTG AGGGTGCAGC CCTTCAATGT GACTCAGGGA 1081 AAGTACTCCA CTGCACAAGA GTGTTCCTTG GATGATGACA CTATCCTCAT CCCCATTATT 1141 GTGGGAGCTG GACTGTCAGG ATTGATTATA GTGATTGTGA TTGCTTATGT GATTGGAAGG 1201 AGAAAGAGCT ATGCTGGCTA CCAGACCCTG TAA

(SEQ ID NO: 3);

1 ATGGTGTGCT TTAGACTGTT TCCTGTGCCT GGTTCAGGGC TGGTCCTGGT CTGTCTGGTG 61 CTGGGGGCTG TCAGAAGCTA TGCCTTGGAG CTGAACCTCA CTGATAGTGA AAATGCCACT 121 TGTCTGTATG CTAAGTGGCA GATGAACTTC ACTGTGAGAT ATGAAACCAC CAACAAGACT 181 TACAAAACAG TGACCATCTC AGATCATGGA ACTGTGACCT ACAACGGCAG CATTTGTGGA 241 GACGACCAGA ACGGACCAAA AATCGCTGTC CAATTTGGGC CTGGATTCTC CTGGATTGCC 301 AATTTCACTA AAGCTGCCTC CACATATTCA ATTGACTCAG TGTCCTTCTC CTACAACACT 361 GGGGACAACA CTACTTTCCC TGATGCTGAA GATAAGGGAA TCTTGACAGT GGATGAGCTG 421 CTGGCTATCA GGATCCCTTT GAATGACCTG TTTAGGTGTA ATTCACTGAG CACTCTGGAG 481 AAGAACGACG TGGTGCAGCA CTACTGGGAC GTGCTGGTGC AGGCCTTTGT GCAGAACGGC 541 ACTGTGTCCA CCAACGAATT CCTGTGTGAT AAGGACAAAA CTTCCACTGT GGCACCTACA 601 ATTCACACTA CTGTGCCTTC ACCTACCACC ACTCCAACTC CAAAGGAAAA GCCTGAAGCA 661 GGAACCTACT CTGTGAACAA TGGCAATGAT ACCTGTCTGT TGGCCACCAT GGGCCTCCAA 721 CTGAACATTA CTCAGGACAA GGTGGCCTCA GTGATTAACA TTAACCCCAA CACTACCCAC 781 TCCACTGGCA GCTGTAGATC ACACACAGCC TTGCTCAGAC TGAATAGCAG CACCATCAAG 841 TATTTGGATT TTGTGTTTGC AGTGAAGAAT GAAAACAGGT TCTACCTGAA GGAAGTCAAC 901 ATCTCAATGT ACCTGGTGAA CGGCTCAGTG TTCAGCATTG CCAACAACAA CCTCTCCTAT 961 TGGGACGCTC CACTGGGGAG CAGCTACATG TGTAACAAGG AACAGACTGT GTCAGTGTCA 1021 GGAGCCTTCC AGATTAACAC CTTTGATCTG AGGGTCCAAC CCTTTAATGT CACTCAAGGA 1081 AAGTATAGCA CTGCCCAGGA GTGCTCCCTG GATGATGACA CCATTCTGAT TCCAATCATT 1141 GTGGGTGCAG GACTTTCTGG GCTTATTATT GTGATTGTGA TTGCCTATGT GATTGGCAGA 1201 AGGAAATCCT ATGCAGGGTA CCAAACTCTG TAA

(SEQ ID NO: 4); and

1 ATGGTCTGTT TTAGGCTGTT CCCTGTCCCT GGTTCAGGAC TGGTCTTAGT GTGTCTGGTG 61 CTTGGAGCTG TCAGAAGCTA TGCCCTGGAG CTGAACCTGA CTGACTCAGA AAATGCCACT 121 TGCCTGTATG CCAAGTGGCA GATGAACTTC ACTGTCAGAT ATGAAACCAC CAACAAGACC 181 TATAAGACTG TGACCATCTC AGACCATGGC ACTGTGACTT ACAATGGGTC AATTTGTGGA 241 GATGACCAGA ATGGCCCTAA GATAGCTGTC CAGTTTGGTC CAGGATTCAG CTGGATTGCC 301 AACTTCACCA AGGCAGCCAG CACCTACAGC ATTGACTCTG TGTCCTTCTC CTACAACACA 361 GGAGACAACA CCACTTTCCC TGATGCAGAG GACAAAGGTA TCCTGACTGT GGATGAGTTG 421 CTGGCAATCA GGATCCCACT GAACGATCTG TTCAGGTGCA ACTCACTGTC CACTCTGGAA 481 AAGAATGATG TGGTGCAGCA CTATTGGGAT GTGCTAGTCC AGGCCTTTGT CCAGAATGGG 541 ACTGTGTCAA CTAATGAGTT CCTGTGTGAC AAGGACAAGA CAAGCACTGT AGCCCCCACT 601 ATCCATACCA CAGTACCTAG CCCCACCACT ACTCCAACCC CCAAGGAGAA GCCTGAGGCT 661 GGCACCTACT CAGTGAACAA TGGGAATGAC ACCTGTTTGC TGGCCACTAT GGGACTCCAA 721 CTGAACATCA CCCAGGACAA AGTGGCCTCT GTGATCAATA TCAATCCCAA CACCACCCAC 781 AGCACTGGGT CCTGCAGAAG CCACACTGCC CTCCTGAGGC TCAACTCATC AACTATCAAG 841 TACTTGGATT TTGTGTTTGC AGTGAAGAAT GAGAACAGAT TCTACCTCAA AGAGGTCAAC 901 ATTTCAATGT ACCTGGTGAA TGGGAGTGTG TTCTCCATTG CTAACAACAA CCTGAGCTAC 961 TGGGATGCCC CTCTGGGCTC CTCATACATG TGCAACAAGG AACAGACTGT GAGTGTGTCA 1021 GGGGCCTTCC AGATCAACAC TTTTGACCTG AGAGTGCAGC CCTTTAATGT GACACAGGGA 1081 AAGTACAGCA CTGCTCAGGA GTGCAGCCTG GATGATGACA CTATCCTGAT CCCTATCATT 1141 GTGGGGGCAG GCCTGTCTGG ACTCATTATT GTGATTGTGA TTGCCTATGT GATAGGGAGA 1201 AGGAAGTCTT ATGCTGGATA CCAGACCCTG TAA

(SEQ ID NO: 5).

In an embodiment, the transgene shares at least 95% identity to a sequence selected from SEQ ID NOs: 3-5. In an embodiment, the transgene shares at least 99% identity to a sequence selected from SEQ ID NOs: 3-5. In an embodiment, the transgene comprises a sequence selected from SEQ ID NOs: 3-5. In an embodiment, the transgene shares at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or complete identity to SEQ ID NO: 3. In an embodiment, the transgene shares at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or complete identity to SEQ ID NO: 4. In an embodiment, the transgene shares at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or complete identity to SEQ ID NO: 5.

In some embodiments, the transgene is similar to or identical to a subsequence of any one of SEQ ID NOs: 3-5. In some embodiments, the transgene comprises a subsequence of any one of SEQ ID NOs: 3-5. In various embodiments, the subsequence may comprise any set of consecutive nucleotides (nt) in the full sequence having a length of 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, or at least abou 1000 nt.

In some embodiments, the transgene shares at least 95% identity to a subsequence that comprises nucleotides 1-500, 250-750, 500-1000, or 750-1240 of any one of SEQ ID NO: 3- 5. In an embodiment, the transgene shares at least 99% identity to a subsequence that comprises nucleotides 1-500, 250-750, 500-1000, or 750-1240 of any one of SEQ ID NO: 3- 5. In an embodiment, the transgene comprises a sequence that comprises nucleotides 1-500, 250-750, 500-1000, or 750-1240 of any one of SEQ ID NOs: 3-5. In embodiment, the transgene shares at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,

96%, 97%, 98%, 99%, or complete identity to a subsequence that comprises nucleotides 1- 500, 250-750, 500-1000, or 750-1240 of any one of SEQ ID NOs: 3-5. In embodiments, the transgene shares at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,

96%, 97%, 98%, 99%, or complete identity to a subsequence that comprises nucleotides 1- 500, 250-750, 500-1000, or 750-1240 of SEQ ID NO: 3. In embodiment, the transgene shares at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,

99%, or complete identity to a subsequence that comprises nucleotides 1-500, 250-750, 500- 1000, or 750-1240 of SEQ ID NO: 3.

In certain embodiments, the transgene encodes any of the various isoforms of LAMP- 2, including any of LAMP-2A, LAMP-2B, or LAMP-2C, or a functional fragment or variant of any of these isoforms. Thus, in particular embodiments, the expression cassette is an optimized polynucleotide sequence encoding any of LAMP-2A, LAMP-2B, or LAMP-2C, or a functional fragment or variant thereof, which comprises one or more modifications as compared to the corresponding wild-type polynucleotide sequence, including one or more modification selected from: codon-optimization of the transgene sequence encoding LAMP- 2A, LAMP-2B, or LAMP-2C; the expression cassette or transgene sequence contains fewer CpG sites than its corresponding wild-type sequence; the expression cassette or transgene sequence contains fewer CpG sites than its corresponding wild-type sequence; the expression cassette or transgene sequence contains fewer cryptic splice sites than its corresponding wild- type sequence; and/or the expression cassette or transgene sequence contains fewer open reading frames than its corresponding wild-type sequence. In particular embodiments, the optimized sequence is optimized for increased expression in human cells. The wild-type human polynucleotide sequences encoding the LAMP-2A and LAMP-2C isoforms are set forth in SEQ ID NOs: 29 and 30, respectively. The wild-type sequences of human LAMP- 2A and LAMP-2C proteins are set forth in SEQ ID NOs: 34 and 35, respectively. The sequences of the wild-type LAMP-2 isoforms and coding sequences are also publicly available. While the specification describes specific embodiments with respect to LAMP-2B, it is understood that LAMP-2A or LAMP-2C could alternatively be used in each

embodiment.

The coding sequences of wild-type LAMP-2A (SEQ ID NO: 29) and wild-type LAMP-2C (SEQ ID NO: 30) are 100% identical to the coding sequence of wild-type LAMP- 2B (SEQ ID NO: 2) across at least nucleotides 1 - 1080. Accordingly, it will be readily recognized by those in the art that that transgenes, expression cassettes, and vectors disclosed herein can be adapted for expression of these isoforms of LAMP-2 by substituting the 3' end (nucleotides 1081 - end) of either of LAMP -2 A (SEQ ID NO: 29) or wild-type LAMP-2C (SEQ ID NO: 30) in place of nucleotides 1081 - 1233 of LAMP-2B (e.g., an optimized LAMP-2B of any of SEQ ID NO: 3-5). For example, embodiments of the invention utilize nucleotides 1-1080 of the optimized LAMP-2B gene sequences, SEQ ID NOs: 3-5, which are, respectively, SEQ ID NOs: 31-33.

In an embodiment, the transgene shares at least 95% identity to a sequence selected from SEQ ID NOs: 31-33. In an embodiment, the transgene shares at least 99% identity to a sequence selected from SEQ ID NOs: 31-33. In an

embodiment, the transgene comprises a sequence selected from SEQ ID NOs: 31-33.

In an embodiment, the transgene shares at least 85%, 86%, 87%, 88%, 89%, 90%,

91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or complete identity to SEQ ID NO: 31. In an embodiment, the transgene shares at least 85%, 86%, 87%, 88%, 89%,

90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or complete identity to SEQ ID NO: 32. In an embodiment, the transgene shares at least 85%, 86%, 87%, 88%,

89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or complete identity to SEQ ID NO: 33. In some cases, the transgene has a polynucleotide sequence that is different from the polynucleotide sequence of a reference sequence, e.g., a“native” or “wild-type” LAMP-2B sequence. In some embodiments, the transgene shares 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 a reference sequence. In some embodiments, the reference sequence is SEQ ID NO: 2.

For example, SEQ ID NO: 3 shares 78.5% identity to SEQ ID NO: 2.

In some cases, the transgene has a polynucleotide sequence that is different from the polynucleotide sequence of a reference sequence, e.g, a“native” or“wild- type” LAMP-2A sequence. In some embodiments, the transgene shares 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 a reference sequence. In some embodiments, the reference sequence is the wild-type human LAMP-2A sequence set forth in SEQ ID NO: 29.

In some cases, the transgene has a polynucleotide sequence that is different from the polynucleotide sequence of a reference sequence, e.g, a“native” or“wild- type” LAMP-2C sequence. In some embodiments, the transgene shares 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 a reference sequence. In some embodiments, the reference sequence is the wild-type human LAMP-2A sequence set forth in SEQ ID NO: 30.

In an embodiment, the transgene is codon-optimized for expression in a human host cell. In an 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 the amino acids for translation. During

translation, each of 61 trinucleotide codons are translated to one of 20 amino acids, leading to a degeneracy, or redundancy, in the genetic code. However, different cell types, and different animal species, utilize tRNAs (each bearing an anticodon) coding for the same amino acids at different frequencies. When a gene sequence contains codons that are infrequently represented by the corresponding tRNA, the ribosome translation machinery may slow, impeding efficient translation. Expression can be improved via“codon optimization” for a particular species, where the coding

sequence is altered to encode the same protein sequence, but utilizing codons that are highly represented, and/or utilized by highly expressed human proteins (Cid-Arregui et al., 2003; J. Virol. 77: 4928).

In some embodiments, the coding sequence of the transgene is modified to replace codons infrequently expressed in mammal or in primates with codons

frequently expressed in primates. For example, in some embodiments, the transgene encodes a polypeptide having at least 85% sequence identity to a reference

polypeptide ( e.g . wild-type LAMP-2B; SEQ ID NO: 3)— for example, at least 90% sequence identity, at least 95% sequence identity, at least 98% identity, or at least 99% identity to 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 sequence disclosed above or herein.

In an embodiment, the transgene comprises fewer alternative open reading frames than SEQ ID: 2. In an embodiment, the transgene is modified to enhance expression by termination or removal of open reading frames (ORFs) that do not encode the desired transgene. An open reading frame (ORF) is the nucleic acid sequence that follows a start codon and does not contain a stop codon. ORFs may be in the forward or reverse orientation, and may be“in frame” or“out of frame” compared with the gene of interest. Such open reading frames have the potential to be expressed in an expression cassette alongside the gene of interest, and could lead to undesired adverse effects. In some embodiments the transgene has been modified to remove open reading frames by further altering codon usage. This may be done by eliminating one or more start codons (ATG) and/or introducing one or more stop codons (TAG, TAA, or TGA) in reverse orientation or out-of-frame to the desired ORF, while preserving the encoded amino acid sequence and, optionally, maintaining highly utilized codons in the gene of interest (i.e., avoiding codons with frequency < 20%).

In some embodiments, the expression cassette comprises at most one, at most two, at most three, at most four, or at most five start codons 5' to the start codon of the transgene. In some embodiments, the expression cassette comprises no start codon 5' to the start codon of the transgene. In some embodiments, one or more ATG codons in the 5' UTR, the promoter, the enhance, the promoter/enhancer element, or other sequences 5' to the start codon of the transgene remain after one or more cryptic start sites are removed. In some embodiments, the expression cassette comprises no cryptic starts sites upstream of transgene to generate erroneous mRNAs.

In variations of the present disclosure, the transgene coding sequence may be optimized by either codon optimization or removal of non-transgene ORFs or using both techniques. In some cases, one removes or minimizes non-transgene ORFs after codon optimization in order to remove ORFs introduced during codon optimization.

In an embodiment, the transgene contains fewer CpG sites than SEQ ID: 2. Without being bound by theory, it is believed that the presence of CpG sites in a polynucleotide sequence is associated with the undesirable immunological responses of the host against a viral vector comprising the polynucleotide sequence. In some embodiments, the transgene is designed to reduce the number of CpG sites. Exemplary methods are provides in ET.S. Patent Application Publication No. ETS20020065236A1.

In an embodiment, the transgene contains fewer cryptic splice sites than SEQ ID: 2. For the optimization, GeneArt® software may be used, e.g, to increase the GC content and/or remove cryptic splice sites in order to avoid transcriptional silencing and, therefore, increase transgene expression. Alternatively, any optimization method known in the art may be used. Removal of cryptic splice sites is described, for example, in International Patent Application Publication No. W02004015106A1.

Also disclosed herein are expression cassettes and gene therapy vectors encoding LAMP-2B. In certain embodiments, the expression cassettes and gene therapy vectors comprise a codon-optimized or variant LAMP-2B polynucleotide sequence or transgene sequence disclosed herein.

In particular embodiments, an expression cassette or gene therapy vector encoding LAMP-2B comprises: a consensus optimal Kozak sequence, a full-length polyadenylation (poly A) sequence (or substitution of full-length polyA by a truncated poly A), and minimal or no upstream ( i.e . 5') or cryptic start codons (i.e. ATG sites). In some embodiments, the expression cassette comprises no start site 5' to the transgene capable of generating alternative mRNAs. In certain embodiments, the expression cassette or gene therapy vector comprises a sequence encoding LMAP-2B, e.g ., a codon-optimized or variant LAMP-2B polynucleotide sequence or transgene sequence disclosed herein.

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 a LAMP-2B disclosed herein), a 3' untranslated region including a full-length polyA sequence, and a second inverted terminal repeat. In some embodiments, one or both of the inverted terminal repeats (ITRs) are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, or AAV9 ITRs, or any one ITR known in the art. In some embodiments, the expression cassette comprises exactly two ITRs. In some embodiments, both ITRs are AAV2, AAV5, or AAV9 ITRs. In some embodiments, both ITRs are AAV2 ITRs.

In an embodiment, the expression cassette comprises a Kozak sequence operatively linked to the transgene. In an embodiment, the Kozak sequence is a consensus optimal Kozak sequence comprising or consisting of SEQ ID NO: 6:

GCCGCCACCATGG (SEQ ID NO: 6).

In various embodiments, the expression cassette comprises an alternative

Kozak sequence operatively linked to the transgene. In an embodiment, the Kozak sequence is an alternative Kozak sequence comprising or consisting of any one of

SEQ ID NOs. 14-18:

(gcc)gccRccAUGG (SEQ ID NO: 14);

AGNNAUGN (SEQ ID NO: 15);

ANNAUGG (SEQ ID NO: 16);

ACCAUGG (SEQ ID NO: 17);

GACACCAUGG (SEQ ID NO: 18).

In some embodiments, the expression cassette comprises no Kozak sequence.

In SEQ ID NO: 14, a lower-case letter denotes the most common base at a position where the base can nevertheless vary; an upper-case letter indicate a highly conserved base; indicates adenine or guanine. In SEQ ID NO: 14, the sequence in parentheses (gcc) is optional. In SEQ ID NOs: 15-17,‘N’ denotes any base.

A variety of sequences can be used in place of this consensus optimal Kozak sequence as the translation-initiation site and it is within the skill of those in the art to identify and test other sequences. See Kozak M. An analysis of vertebrate mRNA sequences: intimations of translational control. J Cell Biol. 115 (4): 887-903 (1991).

In an embodiment, the expression cassette comprises a full-length polyA sequence operatively linked to the transgene. In an embodiment, the full-length polyA sequence comprises SEQ ID NO: 7:

1 TGGCTAATAA AGGAAATTTA TTTTCATTGC AATAGTGTGT TGGAATTTTT TGTGTCTCTC

61 ACTCGGAAGG ACATATGGGA GGGCAAATCA TTTAAAACAT CAGAATGAGT ATTTGGTTTA 121 GAGTTTGGCA ACATATGCCC ATATGCTGGC TGCCATGAAC AAAGGTTGGC TATAAAGAGG 181 TCATCAGTAT ATGAAACAGC CCCCTGCTGT CCATTCCTTA TTCCATAGAA AAGCCTTGAC 241 TTGAGGTTAG ATTTTTTTTA TATTTTGTTT TGTGTTATTT TTTTCTTTAA CATCCCTAAA 301 ATTTTCCTTA CATGTTTTAC TAGCCAGATT TTTCCTCCTC TCCTGACTAC TCCCAGTCAT 361 AGCTGTCCCT CTTCTCTTAT GGAGATC

(SEQ ID NO: 7).

Various alternative polyA sequences may be used in expression cassettes of the present disclosure, including without limitation, bovine growth hormone polyadenylation signal (bGHpA) (SEQ ID NO: 19), the SV40 early/late polyadenylation signal (SEQ ID NO: 20), and human growth hormone (HGH) polyadenylation signal (SEQ ID NO: 21):

1 TCGACTGTGC CTTCTAGTTG CCAGCCATCT GTTGTTTGCC CCTCCCCCGT GCCTTCCTTG

61 ACCCTGGAAG GTGCCACTCC CACTGTCCTT TCCTAATAAA ATGAGGAAAT TGCATCGCAT 121 TGTCTGAGTA GGTGTCATTC TATTCTGGGG GGTGGGGTGG GGCAGGACAG CAAGGGGGAG 181 GATTGGGAGG ACAATAGCAG GCATGCTGGG GATGCGGTGG GCTCTATGGC TTCTG

(SEQ ID NO: 19);

1 CAGACATGAT AAGATACATT GATGAGTTTG GACAAACCAC AACTAGAATG CAGTGAAAAA

61 AATGCTTTAT TTGTGAAATT TGTGATGCTA TTGCTTTATT TGTAACCATT ATAAGCTGCA

121 ATAAACAAGT TAACAACAAC AATTGCATTC ATTTTATGTT TCAGGTTCAG GGGGAGATGT

181 GGGAGGTTTT TTAAAGCAAG TAAAACCTCT ACAAATGTGG TA

(SEQ ID NO: 20);

1 CTGCCCGGGT GGCATCCCTG TGACCCCTCC CCAGTGCCTC TCCTGGCCCT GGAAGTTGCC

61 ACTCCAGTGC CCACCAGCCT TGTCCTAATA AAATTAAGTT GCATCATTTT GTCTGACTAG

121 GTGTCCTTCT ATAATATTAT GGGGTGGAGG GGGGTGGTAT GGAGCAAGGG GCCCAAGTTG

181 GGAAGAAACC TGTAGGGCCT GC

(SEQ ID NO: 21).

In some embodiments, the expression cassette comprises an active fragment of a polyA sequence. In particular embodiments, the active fragment of the polyA sequence comprises or consists of less than 20 base pair (bp), less than 50 bp, less than 100 bp, or less than 150 bp, e.g ., of any of the polyA sequences disclosed herein.

In some cases, expression of the transgene is increased by ensuring that the expression cassette does not contain competing ORFs. In an embodiment, the expression cassette comprises no start codon within 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 base pairs 5' of the start codon of the transgene. In some embodiment, the expression cassette comprises no start codon 5' of the start codon of the transgene. In some embodiments, the expression cassette comprises no start site 5' to the transgene capable of generating alternative mRNAs.

In an embodiment, the expression cassette comprises operatively linked, in the 5' to 3' direction, a first inverted terminal repeat, an enhancer/promoter region, introns, a consensus optimal Kozak sequence, the transgene, a 3' untranslated region including a full-length polyA sequence, and a second inverted terminal repeat, wherein the expression cassette comprises no start site 5' to the transgene capable of generating alternative mRNAs.

In some embodiments, the enhancer/promoter region comprises, in the 5' to 3' direction: a CMV IE enhancer and a chicken beta-actin promoter. In an embodiment, the enhancer/promoter region comprises a CAG promoter (SEQ ID NO: 22). As used herein “CAG promoter” refers to a polynucleotide sequence comprising a CMV early enhancer element, a chicken beta-actin promoter, the first exon and first intron of the chicken beta- actin gene, and a splice acceptor from the rabbit beta-globin gene.

1 CTAGTCGACA TTGATTATTG ACTAGTTATT AATAGTAATC AATTACGGGG TCATTAGTTC

61 ATAGCCCATA TATGGAGTTC CGCGTTACAT AACTTACGGT AAATGGCCCG CCTGGCTGAC

121 CGCCCAACGA CCCCCGCCCA TTGACGTCAA TAATGACGTA TGTTCCCATA GTAACGCCAA

181 TAGGGACTTT CCATTGACGT CAATGGGTGG AGTATTTACG GTAAACTGCC CACTTGGCAG

241 TACATCAAGT GTATCATATG CCAAGTACGC CCCCTATTGA CGTCAATGAC GGTAAATGGC

301 CCGCCTGGCA TTATGCCCAG TACATGACCT TATGGGACTT TCCTACTTGG CAGTACATCT

361 ACGTATTAGT CATCGCTATT ACCATGGTCG AGGTGAGCCC CACGTTCTGC TTCACTCTCC

421 CCATCTCCCC CCCCTCCCCA CCCCCAATTT TGTATTTATT TATTTTTTAA TTATTTTGTG

481 CAGCGATGGG GGCGGGGGGG GGGGGGGGGC GCGCGCCAGG CGGGGCGGGG CGGGGCGAGG

541 GGCGGGGCGG GGCGAGGCGG AGAGGTGCGG CGGCAGCCAA TCAGAGCGGC GCGCTCCGAA

601 AGTTTCCTTT TATGGCGAGG CGGCGGCGGC GGCGGCCCTA TAAAAAGCGA AGCGCGCGGC

661 GGGCGGGAGT CGCTGCGCGC TGCCTTCGCC CCGTGCCCCG CTCCGCCGCC GCCTCGCGCC

721 GCCCGCCCCG GCTCTGACTG ACCGCGTTAC TCCCACAGGT GAGCGGGCGG GACGGCCCTT

781 CTCCTCCGGG CTGTAATTAG CGCTTGGTTT AATGACGGCT TGTTTCTTTT CTGTGGCTGC

841 GTGAAAGCCT TGAGGGGCTC CGGGAGGGCC CTTTGTGCGG GGGGAGCGGC TCGGGGGGTG

901 CGTGCGTGTG TGTGTGCGTG GGGAGCGCCG CGTGCGGCTC CGCGCTGCCC GGCGGCTGTG

961 AGCGCTGCGG GCGCGGCGCG GGGCTTTGTG CGCTCCGCAG TGTGCGCGAG GGGAGCGCGG

1021 CCGGGGGCGG TGCCCCGCGG TGCGGGGGGG GCTGCGAGGG GAACAAAGGC TGCGTGCGGG

1081 GTGTGTGCGT GGGGGGGTGA GCAGGGGGTG TGGGCGCGTC GGTCGGGCTG CAACCCCCCC

1141 TGCACCCCCC TCCCCGAGTT GCTGAGCACG GCCCGGCTTC GGGTGCGGGG CTCCGTACGG

1201 GGCGTGGCGC GGGGCTCGCC GTGCCGGGCG GGGGGTGGCG GCAGGTGGGG GTGCCGGGCG

1261 GGGCGGGGCC GCCTCGGGCC GGGGAGGGCT CGGGGGAGGG GCGCGGCGGC CCCCGGAGCG

1321 CCGGCGGCTG TCGAGGCGCG GCGAGCCGCA GCCATTGCCT TTTATGGTAA TCGTGCGAGA

1381 GGGCGCAGGG ACTTCCTTTG TCCCAAATCT GTGCGGAGCC GAAATCTGGG AGGCGCCGCC

1441 GCACCCCCTC TAGCGGGCGC GGGGCGAAGC GGTGCGGCGC CGGCAGGAAG GAAATGGGCG

1501 GGGAGGGCCT TCGTGCGTCG CCGCGCCGCC GTCCCCTTCT CCCTCTCCAG CCTCGGGGCT

1561 GTCCGCGGGG GGACGGCTGC CTTCGGGGGG GACGGGGCAG GGCGGGGTTC GGCTTCTGGC

1621 GTGTGACCGG CGGCTCTAGA GCCTCTGCTA ACCATGTTCA TGCCTTCTTC TTTTTCCTAC

1681 AGCTCCTGGG CAACGTGCTG GTTATTGTGC TGTCTCATCA TTTTGGCAAA

(SEQ ID NO: 22).

In some embodiments, the enhancer/promoter region comprises a ubiquitous promoter. In some embodiments, the enhancer/promoter region comprises a CMV promoter (SEQ. ID NO: 23), an SV40 promoter (SEQ ID NO: 24), a PGK promoter (SEQ ID NO: 25), and/or a human beta-actin promoter (SEQ ID NO: 26). In some embodiments, the enhancer/promoter region comprises a polynucleotide that shares at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any one of SEQ ID NOs: 23-26:

1 GTGATGCGGT TTTGGCAGTA CATCAATGGG CGTGGATAGC GGTTTGACTC ACGGGGATTT 61 CCAAGTCTCC ACCCCATTGA CGTCAATGGG AGTTTGTTTT GGCACCAAAA TCAACGGGAC 121 TTTCCAAAAT GTCGTAACAA CTCCGCCCCA TTGACGCAAA TGGGCGGTAG GCGTGTACGG 181 TGGGAGGTCT ATATAAGCAG AGCT

(SEQ ID NO: 23); 1 GGTGTGGAAA GTCCCCAGGC TCCCCAGCAG GCAGAAGTAT GCAAAGCATG CATCTCAATT 61 AGTCAGCAAC CAGGTGTGGA AAGTCCCCAG GCTCCCCAGC AGGCAGAAGT ATGCAAAGCA 121 TGCATCTCAA TTAGTCAGCA ACCATAGTCC CGCCCCTAAC TCCGCCCATC CCGCCCCTAA 181 CTCCGCCCAG TTCCGCCCAT TCTCCGCCCC ATGGCTGACT AATTTTTTTT ATTTATGCAG 241 AGGCCGAGGC CGCCTCGGCC TCTGAGCTAT TCCAGAAGTA GTGAGGAGGC TTTTTTGGAG 301 GCCTAGGCTT TTGCAAA

(SEQ ID NO: 24);

1 GGGTAGGGGA GGCGCTTTTC CCAAGGCAGT CTGGAGCATG CGCTTTAGCA GCCCCGCTGG 61 GCACTTGGCG CTACACAAGT GGCCTCTGGC CTCGCACACA TTCCACATCC ACCGGTAGGC 121 GCCAACCGGC TCCGTTCTTT GGTGGCCCCT TCGCGCCACC TTCTACTCCT CCCCTAGTCA 181 GGAAGTTCCC CCCCGCCCCG CAGCTCGCGT CGTGCAGGAC GTGACAAATG GAAGTAGCAC 241 GTCTCACTAG TCTCGTGCAG ATGGACAGCA CCGCTGAGCA ATGGAAGCGG GTAGGCCTTT 301 GGGGCAGCGG CCAATAGCAG CTTTGCTCCT TCGCTTTCTG GGCTCAGAGG CTGGGAAGGG 361 GTGGGTCCGG GGGCGGGCTC AGGGGCGGGC TCAGGGGCGG GGCGGGCGCC CGAAGGTCCT 421 CCGGAGGCCC GGCATTCTGC ACGCTTCAAA AGCGCACGTC TGCCGCGCTG TTCTCCTCTT 481 CCTCATCTCC GGGCCTTTCG

(SEQ ID NO: 25);

1 CCTGCAGGGC CCACTAGTTC CATGTCCTTA TATGGACTCA TCTTTGCCTA TTGCGACACA 61 CACTCAATGA ACACCTACTA CGCGCTGCAA AGAGCCCCGC AGGCCTGAGG TGCCCCCACC 121 TCACCACTCT TCCTATTTTT GTGTAAAAAT CCAGCTTCTT GTCACCACCT CCAAGGAGGG 181 GGAGGAGGAG GAAGGCAGGT TCCTCTAGGC TGAGCCGAAT GCCCCTCTGT GGTCCCACGC 241 CACTGATCGC TGCATGCCCA CCACCTGGGT ACACACAGTC TGTGATTCCC GGAGCAGAAC 301 GGACCCTGCC CACCCGGTCT TGTGTGCTAC TCAGTGGACA GACCCAAGGC AAGAAAGGGT 361 GACAAGGACA GGGTCTTCCC AGGCTGGCTT TGAGTTCCTA GCACCGCCCC GCCCCCAATC 421 CTCTGTGGCA CATGGAGTCT TGGTCCCCAG AGTCCCCCAG CGGCCTCCAG ATGGTCTGGG 481 AGGGCAGTTC AGCTGTGGCT GCGCATAGCA GACATACAAC GGACGGTGGG CCCAGACCCA 541 GGCTGTGTAG ACCCAGCCCC CCCGCCCCGC AGTGCCTAGG TCACCCACTA ACGCCCCAGG 601 CCTGGTCTTG GCTGGGCGTG ACTGTTACCC TCAAAAGCAG GCAGCTCCAG GGTAAAAGGT 661 GCCCTGCCCT GTAGAGCCCA CCTTCCTTCC CAGGGCTGCG GCTGGGTAGG TTTGTAGCCT 721 TCATCACGGG CCACCTCCAG CCACTGGACC GCTGGCCCCT GCCCTGTCCT GGGGAGTGTG 781 GTCCTGCGAC TTCTAAGTGG CCGCAAGCCA CCTGACTCCC CCAACACCAC ACTCTACCTC 841 TCAAGCCCAG GTCTCTCCCT AGTGACCCAC CCAGCACATT TAGCTAGCTG AGCCCCACAG 901 CCAGAGGTCC TCAGGCCCTG CTTTCAGGGC AGTTGCTCTG AAGTCGGCAA GGGGGAGTGA 961 CTGCCTGGCC ACTCCATGCC CTCCAAGAGC TCCTTCTGCA GGAGCGTACA GAACCCAGGG 1021 CCCTGGCACC CGTGCAGACC CTGGCCCACC CCACCTGGGC GCTCAGTGCC CAAGAGATGT 1081 CCACACCTAG GATGTCCCGC GGTGGGTGGG GGGCCCGAGA GACGGGCAGG CCGGGGGCAG 1141 GCCTGGCCAT GCGGGGCCGA ACCGGGCACT GCCCAGCGTG GGGCGCGGGG GCCACGGCGC 1201 GCGCCCCCAG CCCCCGGGCC CAGCACCCCA AGGCGGCCAA CGCCAAAACT CTCCCTCCTC 1261 CTCTTCCTCA ATCTCGCTCT CGCTCTTTTT TTTTTTCGCA AAAGGAGGGG AGAGGGGGTA 1321 AAAAAATGCT GCACTGTGCG GCGAAGCCGG TGAGTGAGCG GCGCGGGGCC AATCAGCGTG 1381 CGCCGTTCCG AAAGTTGCCT TTTATGGCTC GAGCGGCCGC GGCGGCGCCC TATAAAACCC 1441 AGCGGCGCGA CGCGCCACCA CCGCCGAGAC CGCGTCCGCC CCGCGAGCAC AGAGCCTCGC 1501 CTTTGCCGAT CCGCCGCCCG TCCACACCCG CCGCCAGGTA AGCCCGGCCA GCCGACCGGG 1561 GCAGGCGGCT CACGGCCCGG CCGCAGGCGG CCGCGGCCCC TTCGCCCGTG CAGAGCCGCC 1621 GTCTGGGCCG CAGCGGGGGG CGCATGGGGG GGGAACCGGA CCGCCGTGGG GGGCGCGGGA 1681 GAAGCCCCTG GGCCTCCGGA GATGGGGGAC ACCCCACGCC AGTTCGGAGG CGCGAGGCCG 1741 CGCTCGGGAG GCGCGCTCCG GGGGTGCCGC TCTCGGGGCG GGGGCAACCG GCGGGGTCTT 1801 TGTCTGAGCC GGGCTCTTGC CAATGGGGAT CGCAGGGTGG GCGCGGCGGA GCCCCCGCCA 1861 GGCCCGGTGG GGGCTGGGGC GCCATTGCGC GTGCGCGCTG GTCCTTTGGG CGCTAACTGC 1921 GTGCGCGCTG GGAATTGGCG CTAATTGCGC GTGCGCGCTG GGACTCAAGG CGCTAACTGC 1981 GCGTGCGTTC TGGGGCCCGG GGTGCCGCGG CCTGGGCTGG GGCGAAGGCG GGCTCGGCCG 2041 GAAGGGGTGG GGTCGCCGCG GCTCCCGGGC GCTTGCGCGC ACTTCCTGCC CGAGCCGCTG 2101 GCCGCCCGAG GGTGTGGCCG CTGCGTGCGC GCGCGCCGAC CCGGCGCTGT TTGAACCGGG 2161 CGGAGGCGGG GCTGGCGCCC GGTTGGGAGG GGGTTGGGGC CTGGCTTCCT GCCGCGCGCC 2221 GCGGGGACGC CTCCGACCAG TGTTTGCCTT TTATGGTAAT AACGCGGCCG GCCCGGCTTC 2281 CTTTGTCCCC AATCTGGGCG CGCGCCGGCG CCCCCTGGCG GCCTAAGGAC TCGGCGCGCC 2341 GGAAGTGGCC AGGGCGGGGG CGACCTCGGC TCACAGCGCG CCCGGCTATT CTCGCAGCTC 2401 ACC

(SEQ ID NO: 26). Further exemplary promoters include, but are not limited to, human Elongation Factor

1 alpha promoter (EFS), SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, an endogenous cellular promoter that is heterologous to the gene of interest, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), a Rous sarcoma virus (RS V) promoter, synthetic promoters, hybrid promoters, and the like

In some embodiments, the 3' UTR comprises a polynucleotide (WPRE element) that shares at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 27:

1 ATTCGAGCAT CTTACCGCCA TTTATTCCCA TATTTGTTCT GTTTTTCTTG ATTTGGGTAT 61 ACATTTAAAT GTTAATAAAA CAAAATGGTG GGGCAATCAT TTACATTTTT AGGGATATGT 121 AATTACTAGT TCAGGTGTAT TGCCACAAGA CAAACATGTT AAGAAACTTT CCCGTTATTT 181 ACGCTCTGTT CCTGTTAATC AACCTCTGGA TTACAAAATT TGTGAAAGAT TGACTGATAT 241 TCTTAACTAT GTTGCTCCTT TTACGCTGTG TGGATATGCT GCTTTAATGC CTCTGTATCA 301 TGCTATTGCT TCCCGTACGG CTTTCGTTTT CTCCTCCTTG TATAAATCCT GGTTGCTGTC 361 TCTTTATGAG GAGTTGTGGC CCGTTGTCCG TCAACGTGGC GTGGTGTGCT CTGTGTTTGC 421 TGACGCAACC CCCACTGGCT GGGGCATTGC CACCACCTGT CAACTCCTTT CTGGGACTTT 481 CGCTTTCCCC CTCCCGATCG CCACGGCAGA ACTCATCGCC GCCTGCCTTG CCCGCTGCTG 541 GACAGGGGCT AGGTTGCTGG GCACTGATAA TTCCGTGGTG TTGTCGGGGA AGGGCC

(SEQ ID NO: 27).

In some embodiment, the expression cassette shares at least 95% identity to a sequence selected from SEQ ID NOs: 8-10. In an embodiment, the expression cassette shares complete identity to a sequence selected from SEQ ID NOs: 8-10, or shares 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: 8-10:

1 CTGCGCGCTC GCTCGCTCAC TGAGGCCGCC CGGGCAAAGC CCGGGCGTCG GGCGACCTTT 61 GGTCGCCCGG CCTCAGTGAG CGAGCGAGCG CGCAGAGAGG GAGTGGCCAA CTCCATCACT 121 AGGGGTTCCT TGTAGTTAAT GATTAACCCG CCATGCTACT TATCTACCAG GGTAATGGGG 181 ATCCTCTAGA ACTATAGCTA GTCGACATTG ATTATTGACT AGTTATTAAT AGTAATCAAT 241 TACGGGGTCA TTAGTTCATA GCCCATATAT GGAGTTCCGC GTTACATAAC TTACGGTAAA 301 TGGCCCGCCT GGCTGACCGC CCAACGACCC CCGCCCATTG ACGTCAATAA TGACGTATGT 361 TCCCATAGTA ACGCCAATAG GGACTTTCCA TTGACGTCAA TGGGTGGAGT ATTTACGGTA

421 AACTGCCCAC TTGGCAGTAC ATCAAGTGTA TCATATGCCA AGTACGCCCC CTATTGACGT

481 CAATGACGGT AAATGGCCCG CCTGGCATTA TGCCCAGTAC ATGACCTTAT GGGACTTTCC

541 TACTTGGCAG TACATCTACG TATTAGTCAT CGCTATTACC ATGGTCGAGG TGAGCCCCAC

601 GTTCTGCTTC ACTCTCCCCA TCTCCCCCCC CTCCCCACCC CCAATTTTGT AT T TAT T TAT

661 TTTTTAATTA TTTTGTGCAG CGATGGGGGC GGGGGGGGGG GGGGGGCGCG CGCCAGGCGG

721 GGCGGGGCGG GGCGAGGGGC GGGGCGGGGC GAGGCGGAGA GGTGCGGCGG CAGCCAATCA

781 GAGCGGCGCG CTCCGAAAGT TTCCTTTTAT GGCGAGGCGG CGGCGGCGGC GGCCCTATAA

841 AAAGCGAAGC GCGCGGCGGG CGGGAGTCGC TGCGCGCTGC CTTCGCCCCG TGCCCCGCTC

901 CGCCGCCGCC TCGCGCCGCC CGCCCCGGCT CTGACTGACC GCGTTACTCC CACAGGTGAG

961 CGGGCGGGAC GGCCCTTCTC CTCCGGGCTG TAATTAGCGC TTGGTTTAAT GACGGCTTGT

1021 TTCTTTTCTG TGGCTGCGTG AAAGCCTTGA GGGGCTCCGG GAGGGCCCTT TGTGCGGGGG

1081 GAGCGGCTCG GGGGGTGCGT GCGTGTGTGT GTGCGTGGGG AGCGCCGCGT GCGGCTCCGC

1141 GCTGCCCGGC GGCTGTGAGC GCTGCGGGCG CGGCGCGGGG CTTTGTGCGC TCCGCAGTGT

1201 GCGCGAGGGG AGCGCGGCCG GGGGCGGTGC CCCGCGGTGC GGGGGGGGCT GCGAGGGGAA

1261 CAAAGGCTGC GTGCGGGGTG TGTGCGTGGG GGGGTGAGCA GGGGGTGTGG GCGCGTCGGT

1321 CGGGCTGCAA CCCCCCCTGC ACCCCCCTCC CCGAGTTGCT GAGCACGGCC CGGCTTCGGG

1381 TGCGGGGCTC CGTACGGGGC GTGGCGCGGG GCTCGCCGTG CCGGGCGGGG GGTGGCGGCA

1441 GGTGGGGGTG CCGGGCGGGG CGGGGCCGCC TCGGGCCGGG GAGGGCTCGG GGGAGGGGCG

1501 CGGCGGCCCC CGGAGCGCCG GCGGCTGTCG AGGCGCGGCG AGCCGCAGCC ATTGCCTTTT

1561 ATGGTAATCG TGCGAGAGGG CGCAGGGACT TCCTTTGTCC CAAATCTGTG CGGAGCCGAA

1621 ATCTGGGAGG CGCCGCCGCA CCCCCTCTAG CGGGCGCGGG GCGAAGCGGT GCGGCGCCGG

1681 CAGGAAGGAA ATGGGCGGGG AGGGCCTTCG TGCGTCGCCG CGCCGCCGTC CCCTTCTCCC

1741 TCTCCAGCCT CGGGGCTGTC CGCGGGGGGA CGGCTGCCTT CGGGGGGGAC GGGGCAGGGC

1801 GGGGTTCGGC TTCTGGCGTG TGACCGGCGG CTCTAGAGCC TCTGCTAACC ATGTTCATGC

1861 CTTCTTCTTT TTCCTACAGC TCCTGGGCAA CGTGCTGGTT ATTGTGCTGT CTCATCATTT

1921 TGGCAAAGAA TTCGAGCGGC CGCCAGCCGC CACCATGGTC TGCTTCAGAC TGTTCCCTGT

1981 CCCTGGATCT GGTCTGGTGC TTGTGTGCTT GGTGCTGGGT GCTGTGAGAT CCTATGCCCT

2041 TGAGCTGAAC CTGACTGACT CAGAAAATGC CACTTGCCTG TATGCCAAGT GGCAGATGAA

2101 CTTCACTGTG AGATATGAGA CTACCAACAA GACCTACAAG ACTGTGACCA TCTCAGACCA

2161 TGGCACTGTC ACCTACAATG GATCAATCTG TGGTGATGAT CAGAATGGCC CAAAGATAGC

2221 AGTGCAGTTT GGGCCCGGTT TTTCCTGGAT TGCTAACTTC ACCAAGGCAG CCTCCACCTA

2281 CAGCATTGAC TCAGTCAGCT TCAGCTACAA CACTGGGGAT AACACCACCT TCCCTGACGC

2341 AGAGGACAAG GGAATCCTTA CTGTGGACGA ACTCCTGGCA ATCAGAATCC CCCTTAACGA

2401 CCTGTTCAGA TGCAACTCCC TTTCAACCCT T GAAAAGAAT GATGTGGTGC AACACTATTG

2461 GGACGTCCTG GTGCAAGCCT TTGTGCAGAA TGGGACAGTG AGTACCAACG AGTTCCTCTG

2521 TGACAAGGAC AAGACCAGCA CTGTGGCCCC C AC TAT C C AC ACCACTGTGC CCAGCCCTAC

2581 CACTACCCCC ACCCCTAAAG AGAAGCCAGA AGCTGGAACC TACTCAGTCA ACAATGGAAA

2641 TGACACATGC CTCCTTGCCA CCATGGGACT GCAGCTGAAC ATCACTCAGG ACAAGGTGGC

2701 CTCAGTGATT AACATCAACC CTAACACCAC TCATAGCACT GGGAGCTGCA GATCACATAC

2761 AGCTCTGCTG AGGCTCAACT CCTCCACCAT CAAGTACCTG GACTTTGTGT TTGCTGTGAA

2821 GAAT GAGAAC AGGTTCTACC TCAAGGAAGT GAACATTTCC ATGTACCTGG TCAATGGTTC

2881 AGTGTTCTCT ATTGCCAACA ACAATCTGAG CTACTGGGAT GCACCCCTGG GATCCTCCTA

2941 CATGTGCAAC AAGGAGCAGA CTGTGAGTGT GTCAGGTGCT TTTCAGATCA ACACTTTTGA

3001 CCTGAGGGTG CAGCCCTTCA ATGTGACTCA GGGAAAGTAC TCCACTGCAC AAGAGTGTTC

3061 CTTGGATGAT GACACTATCC TCATCCCCAT TATTGTGGGA GCTGGACTGT CAGGATTGAT

3121 TATAGTGATT GTGATTGCTT ATGTGATTGG AAG GAGAAAG AGCTATGCTG GCTACCAGAC

3181 CCTGTAAAAG GGCGAATTCC AGCACACGCG TCCTAGGAGC TCGAGTACTA CTGGCGGCCG

3241 TTACTAGTGG ATCCGCGGTA CAAGTAAGCA TGCAAGCTTC GAGGACGGGG TGAACTACGC

3301 CTGAATCAAG CTTATCGATA AATTCGAGCA TCTTACCGCC ATTTATTCCC ATATTTGTTC

3361 TGTTTTTCTT GATTTGGGTA TACATTTAAA TGTTAATAAA ACAAAATGGT GGGGCAATCA

3421 TTTACATTTT TAGGGATATG TAATTACTAG TTCAGGTGTA TTGCCACAAG ACAAACATGT

3481 TAAGAAACTT TCCCGTTATT TACGCTCTGT TCCTGTTAAT CAACCTCTGG AT T AC AAAAT

3541 TTGTGAAAGA TTGACTGATA TTCTTAACTA TGTTGCTCCT TTTACGCTGT GTGGATATGC

3601 TGCTTTAATG CCTCTGTATC ATGCTATTGC TTCCCGTACG GCTTTCGTTT TCTCCTCCTT

3661 GTATAAATCC TGGTTGCTGT CTCTTTATGA GGAGTTGTGG CCCGTTGTCC GTCAACGTGG

3721 CGTGGTGTGC TCTGTGTTTG CTGACGCAAC CCCCACTGGC TGGGGCATTG CCACCACCTG

3781 TCAACTCCTT TCTGGGACTT TCGCTTTCCC CCTCCCGATC GCCACGGCAG AACTCATCGC

3841 CGCCTGCCTT GCCCGCTGCT GGACAGGGGC TAGGTTGCTG GGCACTGATA ATTCCGTGGT 3901 GTTGTCGGGG AAGGGCCTCG ATACCGTCGA TATCGATCCT GGCTAATAAA GGAAATTTAT

3961 TTTCATTGCA ATAGTGTGTT GGAATTTTTT GTGTCTCTCA CTCGGAAGGA CATATGGGAG

4021 GGCAAATCAT TTAAAACATC AGAAT GAGT A TTTGGTTTAG AGTTTGGCAA CATATGCCCA

4081 TATGCTGGCT GCCATGAACA AAGGTTGGCT ATAAAGAGGT CATCAGTATA TGAAACAGCC

4141 CCCTGCTGTC CATTCCTTAT TCCATAGAAA AGCCTTGACT TGAGGTTAGA TTTTTTTTAT

4201 ATTTTGTTTT GTGTTATTTT TTTCTTTAAC ATCCCTAAAA TTTTCCTTAC ATGTTTTACT

4261 AGCCAGATTT TTCCTCCTCT CCTGACTACT CCCAGTCATA GCTGTCCCTC TTCTCTTATG

4321 GAGATCGAAG CAATTCGTTG ATCTGAATTT CGACCACCCA TAATAGATCT CCCATTACCC

4381 TGGTAGATAA GTAGCATGGC GGGTTAATCA TTAACTACAA GGAACCCCTA GTGATGGAGT

4441 TGGCCACTCC CTCTCTGCGC GCTCGCTCGC TCACTGAGGC CGGGCGACCA AAGGTCGCCC

4501 GACGCCCGGG CTTTGCCCGG GCGGCCTCAG TGAGCGAGCG AGCGCGCAG

(SEQ ID NO: 8);

1 CTGCGCGCTC GCTCGCTCAC TGAGGCCGCC CGGGCAAAGC CCGGGCGTCG GGCGACCTTT

61 GGTCGCCCGG CCTCAGTGAG CGAGCGAGCG CGCAGAGAGG GAGTGGCCAA CTCCATCACT

121 AGGGGTTCCT TGTAGTTAAT GATTAACCCG CCATGCTACT TAT CT ACC AG GGTAATGGGG

181 ATCCTCTAGA ACTATAGCTA GTCGACATTG ATTATTGACT AGTTATTAAT AGTAATCAAT

241 TACGGGGTCA TTAGTTCATA GCCCATATAT GGAGTTCCGC GTTACATAAC TTACGGTAAA

301 TGGCCCGCCT GGCTGACCGC CCAACGACCC CCGCCCATTG ACGTCAATAA TGACGTATGT

361 TCCCATAGTA ACGCCAATAG GGACTTTCCA TTGACGTCAA TGGGTGGAGT ATTTACGGTA

421 AACTGCCCAC TTGGCAGTAC ATCAAGTGTA TCATATGCCA AGTACGCCCC CTATTGACGT

481 CAATGACGGT AAATGGCCCG CCTGGCATTA TGCCCAGTAC ATGACCTTAT GGGACTTTCC

541 TACTTGGCAG TACATCTACG TATTAGTCAT CGCTATTACC ATGGTCGAGG TGAGCCCCAC

601 GTTCTGCTTC ACTCTCCCCA TCTCCCCCCC CTCCCCACCC CCAATTTTGT AT T TAT T TAT

661 TTTTTAATTA TTTTGTGCAG CGATGGGGGC GGGGGGGGGG GGGGGGCGCG CGCCAGGCGG

721 GGCGGGGCGG GGCGAGGGGC GGGGCGGGGC GAGGCGGAGA GGTGCGGCGG CAGCCAATCA

781 GAGCGGCGCG CTCCGAAAGT TTCCTTTTAT GGCGAGGCGG CGGCGGCGGC GGCCCTATAA

841 AAAGCGAAGC GCGCGGCGGG CGGGAGTCGC TGCGCGCTGC CTTCGCCCCG TGCCCCGCTC

901 CGCCGCCGCC TCGCGCCGCC CGCCCCGGCT CTGACTGACC GCGTTACTCC CACAGGTGAG

961 CGGGCGGGAC GGCCCTTCTC CTCCGGGCTG TAATTAGCGC TTGGTTTAAT GACGGCTTGT

1021 TTCTTTTCTG TGGCTGCGTG AAAGCCTTGA GGGGCTCCGG GAGGGCCCTT TGTGCGGGGG

1081 GAGCGGCTCG GGGGGTGCGT GCGTGTGTGT GTGCGTGGGG AGCGCCGCGT GCGGCTCCGC

1141 GCTGCCCGGC GGCTGTGAGC GCTGCGGGCG CGGCGCGGGG CTTTGTGCGC TCCGCAGTGT

1201 GCGCGAGGGG AGCGCGGCCG GGGGCGGTGC CCCGCGGTGC GGGGGGGGCT GCGAGGGGAA

1261 CAAAGGCTGC GTGCGGGGTG TGTGCGTGGG GGGGTGAGCA GGGGGTGTGG GCGCGTCGGT

1321 CGGGCTGCAA CCCCCCCTGC ACCCCCCTCC CCGAGTTGCT GAGCACGGCC CGGCTTCGGG

1381 TGCGGGGCTC CGTACGGGGC GTGGCGCGGG GCTCGCCGTG CCGGGCGGGG GGTGGCGGCA

1441 GGTGGGGGTG CCGGGCGGGG CGGGGCCGCC TCGGGCCGGG GAGGGCTCGG GGGAGGGGCG

1501 CGGCGGCCCC CGGAGCGCCG GCGGCTGTCG AGGCGCGGCG AGCCGCAGCC ATTGCCTTTT

1561 ATGGTAATCG TGCGAGAGGG CGCAGGGACT TCCTTTGTCC CAAATCTGTG CGGAGCCGAA

1621 ATCTGGGAGG CGCCGCCGCA CCCCCTCTAG CGGGCGCGGG GCGAAGCGGT GCGGCGCCGG

1681 CAGGAAGGAA ATGGGCGGGG AGGGCCTTCG TGCGTCGCCG CGCCGCCGTC CCCTTCTCCC

1741 TCTCCAGCCT CGGGGCTGTC CGCGGGGGGA CGGCTGCCTT CGGGGGGGAC GGGGCAGGGC

1801 GGGGTTCGGC TTCTGGCGTG TGACCGGCGG CTCTAGAGCC TCTGCTAACC ATGTTCATGC

1861 CTTCTTCTTT TTCCTACAGC TCCTGGGCAA CGTGCTGGTT ATTGTGCTGT CTCATCATTT

1921 TGGCAAAGAA TTCGAGCGGC CGCCAGCCGC CACCATGGTG TGCTTTAGAC TGTTTCCTGT

1981 GCCTGGTTCA GGGCTGGTCC TGGTCTGTCT GGTGCTGGGG GCTGTCAGAA GCTATGCCTT

2041 GGAGCTGAAC CTCACTGATA GTGAAAATGC CACTTGTCTG TATGCTAAGT GGCAGATGAA

2101 CTTCACTGTG AGATATGAAA CCACCAACAA GACTTACAAA ACAGTGACCA TCTCAGATCA

2161 TGGAACTGTG ACCTACAACG GCAGCATTTG TGGAGACGAC CAGAACGGAC CAAAAATCGC

2221 TGTCCAATTT GGGCCTGGAT TCTCCTGGAT TGCCAATTTC ACTAAAGCTG CCTCCACATA

2281 TTCAATTGAC TCAGTGTCCT TCTCCTACAA CACTGGGGAC AACACTACTT TCCCTGATGC

2341 TGAAGATAAG GGAATCTTGA CAGTGGATGA GCTGCTGGCT ATCAGGATCC CTTTGAATGA

2401 CCTGTTTAGG TGTAATTCAC TGAGCACTCT G GAGAAGAAC GACGTGGTGC AGCACTACTG

2461 GGACGTGCTG GTGCAGGCCT TTGTGCAGAA CGGCACTGTG TCCACCAACG AATTCCTGTG

2521 TGATAAGGAC AAAACTTCCA CTGTGGCACC TACAATTCAC ACTACTGTGC CTTCACCTAC

2581 C AC C AC T C C A ACTCCAAAGG AAAAGCCTGA AGCAGGAACC TACTCTGTGA ACAATGGCAA

2641 TGATACCTGT CTGTTGGCCA CCATGGGCCT CCAACTGAAC ATTACTCAGG ACAAGGTGGC

2701 CTCAGTGATT AACATTAACC CCAACACTAC CCACTCCACT GGCAGCTGTA GATCACACAC 2761 AGCCTTGCTC AGACTGAATA GCAGCACCAT CAAGTATTTG GATTTTGTGT TTGCAGTGAA 2821 GAATGAAAAC AGGTTCTACC TGAAGGAAGT CAACATCTCA ATGTACCTGG TGAACGGCTC 2881 AGTGTTCAGC ATTGCCAACA ACAACCTCTC CTATTGGGAC GCTCCACTGG GGAGCAGCTA 2941 CATGTGTAAC AAGGAACAGA CTGTGTCAGT GTCAGGAGCC TTCCAGATTA ACACCTTTGA 3001 TCTGAGGGTC CAACCCTTTA ATGTCACTCA AGGAAAGTAT AGCACTGCCC AGGAGTGCTC 3061 CCTGGATGAT GACACCATTC TGATTCCAAT CATTGTGGGT GCAGGACTTT CTGGGCTTAT 3121 TATTGTGATT GTGATTGCCT ATGTGATTGG CAGAAGGAAA TCCTATGCAG GGTACCAAAC 3181 TCTGTAAAAG GGCGAATTCC AGCACACGCG TCCTAGGAGC TCGAGTACTA CTGGCGGCCG 3241 TTACTAGTGG ATCCGCGGTA CAAGTAAGCA TGCAAGCTTC GAGGACGGGG TGAACTACGC 3301 CTGAATCAAG CTTATCGATA AATTCGAGCA TCTTACCGCC ATTTATTCCC ATATTTGTTC 3361 TGTTTTTCTT GATTTGGGTA TACATTTAAA TGTTAATAAA ACAAAATGGT GGGGCAATCA 3421 TTTACATTTT TAGGGATATG TAATTACTAG TTCAGGTGTA TTGCCACAAG ACAAACATGT 3481 TAAGAAACTT TCCCGTTATT TACGCTCTGT TCCTGTTAAT CAACCTCTGG ATTACAAAAT 3541 TTGTGAAAGA TTGACTGATA TTCTTAACTA TGTTGCTCCT TTTACGCTGT GTGGATATGC 3601 TGCTTTAATG CCTCTGTATC ATGCTATTGC TTCCCGTACG GCTTTCGTTT TCTCCTCCTT 3661 GTATAAATCC TGGTTGCTGT CTCTTTATGA GGAGTTGTGG CCCGTTGTCC GTCAACGTGG 3721 CGTGGTGTGC TCTGTGTTTG CTGACGCAAC CCCCACTGGC TGGGGCATTG CCACCACCTG 3781 TCAACTCCTT TCTGGGACTT TCGCTTTCCC CCTCCCGATC GCCACGGCAG AACTCATCGC 3841 CGCCTGCCTT GCCCGCTGCT GGACAGGGGC TAGGTTGCTG GGCACTGATA ATTCCGTGGT 3901 GTTGTCGGGG AAGGGCCTCG ATACCGTCGA TATCGATCCT GGCTAATAAA GGAAATTTAT 3961 TTTCATTGCA ATAGTGTGTT GGAATTTTTT GTGTCTCTCA CTCGGAAGGA CATATGGGAG 4021 GGCAAATCAT TTAAAACATC AGAATGAGTA TTTGGTTTAG AGTTTGGCAA CATATGCCCA 4081 TATGCTGGCT GCCATGAACA AAGGTTGGCT ATAAAGAGGT CATCAGTATA TGAAACAGCC 4141 CCCTGCTGTC CATTCCTTAT TCCATAGAAA AGCCTTGACT TGAGGTTAGA TTTTTTTTAT 4201 ATTTTGTTTT GTGTTATTTT TTTCTTTAAC ATCCCTAAAA TTTTCCTTAC ATGTTTTACT 4261 AGCCAGATTT TTCCTCCTCT CCTGACTACT CCCAGTCATA GCTGTCCCTC TTCTCTTATG 4321 GAGATCGAAG CAATTCGTTG ATCTGAATTT CGACCACCCA TAATAGATCT CCCATTACCC 4381 TGGTAGATAA GTAGCATGGC GGGTTAATCA TTAACTACAA GGAACCCCTA GTGATGGAGT 4441 TGGCCACTCC CTCTCTGCGC GCTCGCTCGC TCACTGAGGC CGGGCGACCA AAGGTCGCCC 4501 GACGCCCGGG CTTTGCCCGG GCGGCCTCAG TGAGCGAGCG AGCGCGCAG

(SEQ ID NO: 9); and

1 CTGCGCGCTC GCTCGCTCAC TGAGGCCGCC CGGGCAAAGC CCGGGCGTCG GGCGACCTTT 61 GGTCGCCCGG CCTCAGTGAG CGAGCGAGCG CGCAGAGAGG GAGTGGCCAA CTCCATCACT 121 AGGGGTTCCT TGTAGTTAAT GATTAACCCG CCATGCTACT TATCTACCAG GGTAATGGGG 181 ATCCTCTAGA ACTATAGCTA GTCGACATTG ATTATTGACT AGTTATTAAT AGTAATCAAT 241 TACGGGGTCA TTAGTTCATA GCCCATATAT GGAGTTCCGC GTTACATAAC TTACGGTAAA 301 TGGCCCGCCT GGCTGACCGC CCAACGACCC CCGCCCATTG ACGTCAATAA TGACGTATGT 361 TCCCATAGTA ACGCCAATAG GGACTTTCCA TTGACGTCAA TGGGTGGAGT ATTTACGGTA 421 AACTGCCCAC TTGGCAGTAC ATCAAGTGTA TCATATGCCA AGTACGCCCC CTATTGACGT 481 CAATGACGGT AAATGGCCCG CCTGGCATTA TGCCCAGTAC ATGACCTTAT GGGACTTTCC 541 TACTTGGCAG TACATCTACG TATTAGTCAT CGCTATTACC ATGGTCGAGG TGAGCCCCAC 601 GTTCTGCTTC ACTCTCCCCA TCTCCCCCCC CTCCCCACCC CCAATTTTGT ATTTATTTAT 661 TTTTTAATTA TTTTGTGCAG CGATGGGGGC GGGGGGGGGG GGGGGGCGCG CGCCAGGCGG 721 GGCGGGGCGG GGCGAGGGGC GGGGCGGGGC GAGGCGGAGA GGTGCGGCGG CAGCCAATCA 781 GAGCGGCGCG CTCCGAAAGT TTCCTTTTAT GGCGAGGCGG CGGCGGCGGC GGCCCTATAA 841 AAAGCGAAGC GCGCGGCGGG CGGGAGTCGC TGCGCGCTGC CTTCGCCCCG TGCCCCGCTC 901 CGCCGCCGCC TCGCGCCGCC CGCCCCGGCT CTGACTGACC GCGTTACTCC CACAGGTGAG 961 CGGGCGGGAC GGCCCTTCTC CTCCGGGCTG TAATTAGCGC TTGGTTTAAT GACGGCTTGT 1021 TTCTTTTCTG TGGCTGCGTG AAAGCCTTGA GGGGCTCCGG GAGGGCCCTT TGTGCGGGGG 1081 GAGCGGCTCG GGGGGTGCGT GCGTGTGTGT GTGCGTGGGG AGCGCCGCGT GCGGCTCCGC 1141 GCTGCCCGGC GGCTGTGAGC GCTGCGGGCG CGGCGCGGGG CTTTGTGCGC TCCGCAGTGT 1201 GCGCGAGGGG AGCGCGGCCG GGGGCGGTGC CCCGCGGTGC GGGGGGGGCT GCGAGGGGAA 1261 CAAAGGCTGC GTGCGGGGTG TGTGCGTGGG GGGGTGAGCA GGGGGTGTGG GCGCGTCGGT 1321 CGGGCTGCAA CCCCCCCTGC ACCCCCCTCC CCGAGTTGCT GAGCACGGCC CGGCTTCGGG 1381 TGCGGGGCTC CGTACGGGGC GTGGCGCGGG GCTCGCCGTG CCGGGCGGGG GGTGGCGGCA 1441 GGTGGGGGTG CCGGGCGGGG CGGGGCCGCC TCGGGCCGGG GAGGGCTCGG GGGAGGGGCG 1501 CGGCGGCCCC CGGAGCGCCG GCGGCTGTCG AGGCGCGGCG AGCCGCAGCC ATTGCCTTTT

1561 ATGGTAATCG TGCGAGAGGG CGCAGGGACT TCCTTTGTCC CAAATCTGTG CGGAGCCGAA

1621 ATCTGGGAGG CGCCGCCGCA CCCCCTCTAG CGGGCGCGGG GCGAAGCGGT GCGGCGCCGG

1681 CAGGAAGGAA ATGGGCGGGG AGGGCCTTCG TGCGTCGCCG CGCCGCCGTC CCCTTCTCCC

1741 TCTCCAGCCT CGGGGCTGTC CGCGGGGGGA CGGCTGCCTT CGGGGGGGAC GGGGCAGGGC

1801 GGGGTTCGGC TTCTGGCGTG TGACCGGCGG CTCTAGAGCC TCTGCTAACC ATGTTCATGC

1861 CTTCTTCTTT TTCCTACAGC TCCTGGGCAA CGTGCTGGTT ATTGTGCTGT CTCATCATTT

1921 TGGCAAAGAA TTCGAGCGGC CGCCAGCCGC CACCATGGTC TGTTTTAGGC TGTTCCCTGT

1981 CCCTGGTTCA GGACTGGTCT TAGTGTGTCT GGTGCTTGGA GCTGTCAGAA GCTATGCCCT

2041 GGAGCTGAAC CTGACTGACT CAGAAAATGC CACTTGCCTG TATGCCAAGT GGCAGATGAA

2101 CTTCACTGTC AGATATGAAA CCACCAACAA GACCTATAAG ACTGTGACCA TCTCAGACCA

2161 TGGCACTGTG ACTTACAATG GGTCAATTTG TGGAGATGAC CAGAATGGCC CTAAGATAGC

2221 TGTCCAGTTT GGTCCAGGAT TCAGCTGGAT TGCCAACTTC ACCAAGGCAG CCAGCACCTA

2281 CAGCATTGAC TCTGTGTCCT TCTCCTACAA CACAGGAGAC AACACCACTT TCCCTGATGC

2341 AGAG GAC AAA GGTATCCTGA CTGTGGATGA GTTGCTGGCA ATCAGGATCC CACTGAACGA

2401 TCTGTTCAGG TGCAACTCAC TGTCCACTCT G GAAAAGAAT GATGTGGTGC AGCACTATTG

2461 GGATGTGCTA GTCCAGGCCT TTGTCCAGAA TGGGACTGTG TCAACTAATG AGTTCCTGTG

2521 TGACAAGGAC AAGACAAGCA CTGTAGCCCC CACTATCCAT ACCACAGTAC CTAGCCCCAC

2581 CACTACTCCA ACCCCCAAGG AGAAGCCTGA GGCTGGCACC TACTCAGTGA ACAATGGGAA

2641 TGACACCTGT TTGCTGGCCA CTATGGGACT CCAACTGAAC ATCACCCAGG ACAAAGTGGC

2701 CTCTGTGATC AATATCAATC CCAACACCAC C C AC AG C AC T GGGTCCTGCA GAAGCCACAC

2761 TGCCCTCCTG AGGCTCAACT CATCAACTAT CAAGTACTTG GATTTTGTGT TTGCAGTGAA

2821 GAAT GAGAAC AGATTCTACC TCAAAGAGGT CAACATTTCA ATGTACCTGG T GAAT GG GAG

2881 TGTGTTCTCC ATTGCTAACA ACAACCTGAG CTACTGGGAT GCCCCTCTGG GCTCCTCATA

2941 CATGTGCAAC AAGGAACAGA CTGTGAGTGT GTCAGGGGCC TTCCAGATCA ACACTTTTGA

3001 CCTGAGAGTG CAGCCCTTTA ATGTGACACA GGGAAAGTAC AGCACTGCTC AGGAGTGCAG

3061 CCTGGATGAT GACACTATCC TGATCCCTAT CATTGTGGGG GCAGGCCTGT CTGGACTCAT

3121 TAT TGT GATT GTGATTGCCT ATGTGATAGG GAGAAGGAAG TCTTATGCTG GATACCAGAC

3181 CCTGTAAAAG GGCGAATTCC AGCACACGCG TCCTAGGAGC TCGAGTACTA CTGGCGGCCG

3241 TTACTAGTGG ATCCGCGGTA CAAGTAAGCA TGCAAGCTTC GAGGACGGGG TGAACTACGC

3301 CTGAATCAAG CTTATCGATA AATTCGAGCA TCTTACCGCC ATTTATTCCC ATATTTGTTC

3361 TGTTTTTCTT GATTTGGGTA TACATTTAAA TGTTAATAAA ACAAAATGGT GGGGCAATCA

3421 TTTACATTTT TAGGGATATG TAATTACTAG TTCAGGTGTA TTGCCACAAG ACAAACATGT

3481 TAAGAAACTT TCCCGTTATT TACGCTCTGT TCCTGTTAAT CAACCTCTGG AT T AC AAAAT

3541 TTGTGAAAGA TTGACTGATA TTCTTAACTA TGTTGCTCCT TTTACGCTGT GTGGATATGC

3601 TGCTTTAATG CCTCTGTATC ATGCTATTGC TTCCCGTACG GCTTTCGTTT TCTCCTCCTT

3661 GTATAAATCC TGGTTGCTGT CTCTTTATGA GGAGTTGTGG CCCGTTGTCC GTCAACGTGG

3721 CGTGGTGTGC TCTGTGTTTG CTGACGCAAC CCCCACTGGC TGGGGCATTG CCACCACCTG

3781 TCAACTCCTT TCTGGGACTT TCGCTTTCCC CCTCCCGATC GCCACGGCAG AACTCATCGC

3841 CGCCTGCCTT GCCCGCTGCT GGACAGGGGC TAGGTTGCTG GGCACTGATA ATTCCGTGGT

3901 GTTGTCGGGG AAGGGCCTCG ATACCGTCGA TATCGATCCT GGCTAATAAA GGAAATTTAT

3961 TTTCATTGCA ATAGTGTGTT GGAATTTTTT GTGTCTCTCA CTCGGAAGGA CATATGGGAG

4021 GGCAAATCAT TTAAAACATC AGAAT GAGT A TTTGGTTTAG AGTTTGGCAA CATATGCCCA

4081 TATGCTGGCT GCCATGAACA AAGGTTGGCT ATAAAGAGGT CATCAGTATA TGAAACAGCC

4141 CCCTGCTGTC CATTCCTTAT TCCATAGAAA AGCCTTGACT TGAGGTTAGA TTTTTTTTAT

4201 ATTTTGTTTT GTGTTATTTT TTTCTTTAAC ATCCCTAAAA TTTTCCTTAC ATGTTTTACT

4261 AGCCAGATTT TTCCTCCTCT CCTGACTACT CCCAGTCATA GCTGTCCCTC TTCTCTTATG

4321 GAGATCGAAG CAATTCGTTG ATCTGAATTT C GAC C AC C C A TAATAGATCT CCCATTACCC

4381 TGGTAGATAA GTAGCATGGC GGGTTAATCA TTAACTACAA GGAACCCCTA GTGATGGAGT

4441 TGGCCACTCC CTCTCTGCGC GCTCGCTCGC TCACTGAGGC CGGGCGACCA AAGGTCGCCC

4501 GACGCCCGGG CTTTGCCCGG GCGGCCTCAG TGAGCGAGCG AGCGCGCAG

(SEQ ID NO: 10).

In certain embodiments, the expression cassette comprises one or more modifications as compared to a sequence selected from SEQ ID NOs: 8-10, including but not limited to any of the modifications disclosed herein. In particular embodiments, the one or more modifications comprise one or more of: 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 of those 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 of those disclosed herein. An illustrative configuration of genetic elements within these exemplary expression cassettes is depicted in FIG. IB.

In an embodiment, the vector is an adeno-associated virus (AAV) vector. In an embodiment, the expression cassette comprises inverted terminal repeat (ITR)

sequences selected from SEQ ID NOs: 11 and 12:

1 CTGCGCGCTC GCTCGCTCAC TGAGGCCGCC CGGGCAAAGC CCGGGCGTCG GGCGACCTTT

61 GGTCGCCCGG CCTCAGTGAG CGAGCGAGCG CGCAGAGAGG GAGTGGCCAA CTCCATCACT

121 AGGGGTTCCT

(SEQ ID NO: 11);

1 AGGAACCCCT AGTGATGGAG TTGGCCACTC CCTCTCTGCG CGCTCGCTCG CTCACTGAGG

61 CCGGGCGACC AAAGGTCGCC CGACGCCCGG GCTTTGCCCG GGCGGCCTCA GTGAGCGAGC

121 GAGCGCGCAG

(SEQ ID NO: 12).

In related embodiments, the disclosure provides gene therapy vectors comprising an expression cassette disclosed herein. Generally, the gene therapy vectors described herein comprise an expression cassette comprising a polynucleotide encoding one or more isoforms of lysosome-associated membrane protein 2 (LAMP-2), and allows for the expression of LAMP-2 to partially or wholly rectify deficient LAMP -2 protein expression levels and/or autophagic flux in a subject in need thereof (e.g., a subject having Danon disease or another disorder characterized by deficient autophagic flux at least in part due to deficient LAMP -2 expression). In particular embodiments, the expression cassette comprises a polynucleotide sequence encoding LAMP -2 disclosed herein, e.g, SEQ ID NOs: 3-5, or a functional variant thereof. In some embodiments, the variant sequence has at least 90%, at least 95%, at least 98%, or at least 99% identity to any of SEQ ID NOs: 3-5. In some embodiments, the variant is a fragment of any of SEQ ID NOs: 3-5, e.g, a fragment having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the sequence of any of SEQ ID Nos: 3-5. The gene therapy vectors can be viral or non-viral vectors. Illustrative non-viral vectors include, e.g, naked DNA, cationic liposome complexes, cationic polymer complexes, cationic liposome-polymer complexes, and exosomes. Examples of viral vectors include, but are not limited to, adenoviral, retroviral, lentiviral, herpesvirus and adeno-associated virus (AAV) vectors.

In certain embodiments, the viral vector is an AAV vector. AAV is a 4.7 kb, single stranded DNA virus. Recombinant vectors based on AAV are associated with excellent clinical safety, since wild-type AAV is nonpathogenic and has no etiologic association with any known diseases. In addition, AAV offers the capability for highly efficient gene delivery and sustained transgene expression in numerous tissues. By an“AAV vector” is meant a vector derived from an adeno-associated virus serotype, including without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.lO,

AAVrh.74, etc. AAV vectors can have one or more of the AAV wild-type genes deleted in whole or part, e.g. , the rep and/or cap genes, but retain functional flanking inverted terminal repeat (ITR) sequences. Functional ITR sequences are necessary for the rescue, replication and packaging of the AAV virion. Thus, an AAV vector is defined herein to include at least those sequences required in cis for replication and packaging (e.g, functional ITRs) of the virus. The ITRs need not be the wild-type nucleotide sequences, and may be altered, e.g. by the insertion, deletion or substitution of nucleotides, as long as the sequences provide for functional rescue, replication and packaging. AAV vectors may comprise other

modifications, including but not limited to one or more modified capsid protein (e.g, VP1, VP2 and/or VP3). For example, a capsid protein may be modified to alter tropism and/or reduce immunogenicity.

Recombinant vectors based on AAV are associated with excellent clinical safety, since wild-type AAV is nonpathogenic and has no etiologic association with any known diseases. In addition, AAV offers the capability for highly efficient gene delivery and sustained transgene expression in numerous tissues. Various serotypes of AAV are known, including, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.lO, AAVrh.74, etc.. AAV vectors can have one or more of the AAV wild-type genes deleted in whole or part, e.g., the rep and/or cap genes, but retain functional flanking inverted terminal repeat (ITR) sequences. The serotype of a recombinant AAV vector is determined by its capsid. International Patent Publication No. W02003042397A2 discloses various capsid sequences including those of AAV1, AAV2, AAV3, AAV8, AAV9, and rhlO. International Patent Publication No. W02013078316A1 discloses the polypeptide sequence of the VPl from AAVrh74. Numerous diverse naturally occurring or genetically modified AAV capsid sequences are known in the art.

An exemplary, non-limiting capsid is an AAV9 capsid, having the sequence of SEQ ID NO: 28 (or the VP1, VP2, or VP3 fragments thereof). In some embodiments, the AAV vectors of the disclosure comprise capsid proteins that share at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity of the entire sequence of SEQ ID NO: 28, or over amino acids 138 to 736 of SEQ ID NO: 28, or over amino acids 203 to 736 of SEQ ID NO: 28.

1 MAADGYLPDW LEDNLSEGIR EWWALKPGAP QPKANQQHQD NARGLVLPGY KYLGPGNGLD 61 KGEPWAADA AALEHDKAYD QQLKAGDNPY LKYNHADAEF QERLKEDTSF GGNLGRAVFQ 121 AKKRLLEPLG LVEEAAKTAP GKKRPVEQSP QEPDSSAGIG KSGAQPAKKR LNFGQTGDTE 181 SVPDPQPIGE PPAAPSGVGS LTMASGGGAP VADNNEGADG VGSSSGNWHC DSQWLGDRVI 241 TTSTRTWALP TYNNHLYKQI SNSTSGGSSN DNAYFGYSTP WGYFDFNRFH CHFSPRDWQR 301 LINNNWGFRP KRLNFKLFNI QVKEVTDNNG VKTIANNLTS TVQVFTDSDY QLPYVLGSAH 361 EGCLPPFPAD VFMIPQYGYL TLNDGSQAVG RSSFYCLEYF PSQMLRTGNN FQFSYEFENV 421 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSKT INGSGQNQQT LKFSVAGPSN MAVQGRNYIP 481 GPSYRQQRVS TTVTQNNNSE FAWPGASSWA LNGRNSLMNP GPAMASHKEG EDRFFPLSGS 541 LIFGKQGTGR DNVDADKVMI TNEEEIKTTN PVATESYGQV ATNHQSAQAQ AQTGWVQNQG 601 ILPGMVWQDR DVYLQGPIWA KIPHTDGNFH PSPLMGGFGM KHPPPQILIK NTPVPADPPT 661 AFNKDKLNSF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYYKSNN VEFAWTEGV 721 YSEPRPIGTR YLTRNL

(SEQ ID NO: 28).

AAV expression vectors are constructed using known techniques to at least provide as operatively linked components in the direction of transcription, control elements including a transcriptional initiation region, the DNA of interest (i.e. the LAMP-2 gene) and a transcriptional termination region.

In some embodiments, the viral vector is an AAV9 vector. In some embodiments, the expression cassette of the viral vector is flanked by AAV2 inverted terminal repeats (ITRs). ITRs used in alternative embodiments of the disclosed vectors include, but are not limited to, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9. In some embodiments, the viral vector is an AAV2/9 vector. The notation AAV2/9 refers to an AAV vector have the ITRs of AAV2 and the capsid of AAV9. Other embodiments of the disclosure include without limitation AAV2/9, AAV5/9, AAVrh74, AAV2/rh74, AAV5/9, and AAV5/rh74 vectors. Other ITRs known in the art may be used. Exemplary ITRs (and other AAV components) useful in the vectors of the present disclosure include, without limitation, those described in US6936466B2, US9169494B2, US20050220766A1, US20190022249A1, and US7282199B2, which are each incorporated by reference herein in their entireties.

In some embodiments, the vector is a retroviral vector, or more specifically, a lentiviral vector. As used herein, the term“retrovirus” or“retroviral” refers an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and subsequently covalently integrates its genomic DNA into a host genome. Retrovirus vectors are a common tool for gene delivery (Miller, 2000, Nature. 357: 455-460). Once the virus is integrated into the host genome, it is referred to as a“provirus.” The provirus serves as a template for RNA polymerase II and directs the expression of RNA molecules encoded by the virus.

Illustrative retroviruses (family Retroviridae) include, but are not limited to: (1) genus gammaretrovirus, such as, Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), and feline leukemia virus (FLV), (2) genus spumavirus, such as, simian foamy virus, (3) genus lentivirus, such as, human immunodeficiency virus-l and simian

immunodeficiency virus.

As used herein, the term“lentiviral” or“lentivirus” refers to a group (or genus) of complex retroviruses. Illustrative lentiviruses include, but are not limited to: HIV (human immunodeficiency virus; including HIV type 1, and HIV type 2; visna-maedi virus (VMV) virus; the caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV). In one embodiment, HIV-based vector backbones (z.e., HIV cis-acting sequence elements) are preferred.

Retroviral vectors, and more particularly, lentiviral vectors, may be used in practicing the present invention. Accordingly, the term“retroviral vector,” as used herein is meant to include“lentiviral vector”; and the term“retrovirus” as used herein is meant to include “lentivirus.”

The term viral vector may refer either to a vector or viral particle capable of transferring a nucleic acid into a cell or to the transferred nucleic acid itself. Viral vectors contain structural and/or functional genetic elements that are primarily derived from a virus. The term“retroviral vector” refers to a viral vector containing structural and functional genetic elements, or portions thereof, that are primarily derived from a retrovirus. The term “lentiviral vector” refers to a viral vector containing structural and functional genetic elements, or portions thereof, including LTRs that are primarily derived from a lentivirus.

The term“hybrid” refers to a vector, LTR or other nucleic acid containing both retroviral, e.g ., lentiviral, sequences and non-lentiviral viral sequences. In one embodiment, a hybrid vector refers to a vector or transfer plasmid comprising retroviral, e.g. , lentiviral, sequences for reverse transcription, replication, integration and/or packaging.

In particular embodiments, the terms“lentiviral vector” and“lentiviral expression vector” may be used to refer to lentiviral transfer plasmids and/or infectious lentiviral particles. Where reference is made herein to elements such as cloning sites, promoters, regulatory elements, heterologous nucleic acids, etc., it is to be understood that the sequences of these elements are present in RNA form in the lentiviral particles of the invention and are present in DNA form in the DNA plasmids of the invention.

According to certain specific embodiments, most or all of the viral vector backbone sequences are derived from a lentivirus, e.g, HIV-l. However, it is to be understood that many different sources of lentiviral sequences can be used, and numerous substitutions and alterations in certain of the lentiviral sequences may be accommodated without impairing the ability of a transfer vector to perform the functions described herein. Moreover, a variety of lentiviral vectors are known in the art, see Naldini et al, (l996a, l996b, and 1998); Zufferey et ah, (1997); Dull et al, 1998, U.S. Pat. Nos. 6,013,516; and 5,994,136, many of which may be adapted to produce a viral vector or transfer plasmid of the present invention.

The LAMP-2B transgene sequences disclosed herein are, in various embodiments, used in any vector system known in the art or prospectively discovered. The invention is not limited to any particular viral vector described herein, as it is within the skill of those in the art to use a transgene sequence in other vector systems without undue experimentation and with a reasonable expectation of success.

Gene delivery viral vectors useful in the practice of the present invention can be constructed utilizing methodologies well known in the art of molecular biology. Typically, viral vectors carrying transgenes are assembled from polynucleotides encoding the transgene, suitable regulatory elements and elements necessary for production of viral proteins, which mediate cell transduction. Such recombinant viruses may be produced by techniques known in the art, e.g., by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of virus packaging cells include but are not limited to HeLa cells, SF9 cells (optionally with a baculovirus helper vector), 293 cells, etc. A

Herpesvirus-based system can be used to produce AAV vectors, as described in

US20170218395A1. Detailed protocols for producing such replication-defective recombinant viruses may be found for instance in W095/14785, W096/22378, U.S. Pat. No. 5,882,877, U.S. Pat. No. 6,013,516, U.S. Pat. No. 4,861,719, U.S. Pat. No. 5,278,056 and W094/19478, the complete contents of each of which is hereby incorporated by reference.

The present disclosure also provides pharmaceutical compositions comprising an expression cassette or vector (e.g, gene therapy vector) disclosed herein and one or more pharmaceutically acceptable carriers, diluents or excipients. In particular 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 of SEQ ID NOs: 3-5 and variants thereof. Provided are pharmaceutical compositions, e.g, for use in preventing or treating a disorder characterized by deficient autophagic flux (e.g, Danon disease) which comprises a therapeutically effective amount of an expression cassette or vector disclosed herein that comprises a nucleic acid sequence of a polynucleotide that encodes one or more isoforms of LAMP -2.

AAV vectors useful in the practice of the present invention can be packaged into AAV virions (viral particles) using various systems including adenovirus-based and helper- free systems. Standard methods in AAV biology include those described in Kwon and Schaffer. Pharm Res. (2008) 25(3):489-99; Wu et al. Mol. Ther. (2006) l4(3):316-27. Burger et al. Mol. Ther. (2004) 10(2):302-17; Grimm et al. Curr Gene Ther. (2003) 3(4):28l-304; Deyle DR, Russell DW. Curr OpinMol Ther. (2009) 11(4):442-447; McCarty et al. Gene Ther. (2001) 8(16): 1248-54; and Duan et al . Mol Ther. (2001) 4(4):383-9l. Helper-free systems included those described in US 6,004,797; US 7,588,772; and US 7,094,604;

The pharmaceutical compositions that contain the expression cassette or vector may be in any form that is suitable for the selected mode of administration, for example, for intraventricular, intramyocardial, intracoronary, intravenous, intra-arterial, intra-renal, intraurethral, epidural or intramuscular administration. The gene therapy vector comprising a polynucleotide encoding one or more LAMP-2 isoforms can be administered, as sole active agent, or in combination with other active agents, in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. In some embodiments, the pharmaceutical composition comprises cells transduced ex vivo with any of the gene therapy vectors of the disclosure.

In some embodiments, the viral vector ( e.g . AAV vector), or a pharmaceutical composition comprising that vector, is effective when administered systemically. For example, the viral vectors of the disclosure, in some cases, demonstrate efficacy when administered intravenously to subject (e.g., a primate, such as a non-human primate or a human). In some embodiments, the viral vectors of the disclosure are capable of inducing expression of LAMP-2B in various tissues when administered systemically (e.g, in heart, muscle, and/or lung). In particular embodiments, administration of an AAV9 vector comprising a transgene substantially identical to, or identical to, SEQ ID NO: 3 to a subject intravenously results in detectable expression of LAMP-2B in heart tissue. In some embodiments, expression of LAMP-2B is detectable in one or more, or all, of the left ventricle, the right ventricle, the left atrium, and the right atrium of the heart of the subject. In some embodiments, expression of LAMP-2B is detectable in sub-region 1 and/or sub-region 2 of the left ventricle of the heart of the subject.

“Detectable expression” typically refers to transgene expression at least 5%, 10%, 15%, 20% or more compared to a control subject or tissue not treated with the viral vector. In some embodiments, detectable expression means expression at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 5-fold, at least lO-fold, at least 20-fold, at least 50- fold, or at least lOO-fold greater than a no-vector control. Transgene expression can be determined as the increase over expression of the wild-type or endogenous gene in the cell (accounting for the potential that expression of the transgene may influence expression of the endogenous gene). Transgene expression can also be determined by RT-PCR detection of sequences that are present on the transgene mRNA transcript but not on the mRNA transcript of the endogenous gene. For example, the 3' UTR of the transcript may be used to determine the expression of the transgene independent of the expression of the endogenous gene (which may have a different 3' UTR). Expression of the polypeptide encoded by the transgene can be assessed by western blot or enzyme-linked immunosorbent assay (ELISA), as described in the examples that follow, or other methods known in the art. Antibodies cross-reactive to the wild-type and exogenous copies of the protein may be used. In some cases, an antibody specific to the exogenous protein can be identified and used to determine transgene expression. Those of skill in the art can design appropriate detection methodologies taking into account the target cell or tissue. In some cases, expression is measured quantitatively using a standard curve. Standard curves can be generated using purified LAMP -2 protein, by methods described in the examples or known in the art. Alternatively, expression of the transgene can be assessed by quantification of the corresponding mRNA.

In some embodiments, detectable expression of LAMP-2B in heart tissue occurs at doses, in vector genomes (vg) per kilogram weight of subject (kg), of 5xl0 ¹⁴ vg/kg or less, 3 ^c 10 ¹⁴ vg/kg or less, 2><l0 ¹⁴ vg/kg or less, lxlO ¹⁴ vg/kg or less, 9xl0 ¹³ vg/kg or less, 8xl0 ¹³ vg/kg or less, 7xl0 ¹³ vg/kg or less, 6xl0 ¹³ vg/kg or less, 5xl0 ¹³ vg/kg or less, 4xl0 ¹³ vg/kg or less, 3xl0 ¹³ vg/kg or less, 2xl0 ¹³ vg/kg or less, or lxlO ¹³ vg/kg or less.

In some embodiments, detectable expression of LAMP-2B in heart tissue occurs at doses, in vector genomes (vg) per kilogram weight of subject (kg), of lxlO ¹³ vg/kg to 2x 10 ¹³ vg/kg, 2xl0 ¹³ vg/kg to 3xl0 ¹³ vg/kg, 3xl0 ¹³ vg/kg to 4xl0 ¹³ vg/kg, 4xl0 ¹³ vg/kg to 5xl0 ¹³ vg/kg, 5xl0 ¹³ vg/kg to 6xl0 ¹³ vg/kg, 6xl0 ¹³ vg/kg to 7xl0 ¹³ vg/kg, 7xl0 ¹³ vg/kg to 8xl0 ¹³ vg/kg, 8x 10 ¹³ vg/kg to 9x 10 ¹³ vg/kg, 9x 10 ¹³ vg/kg to lxlO ¹⁴ vg/kg, lxlO ¹⁴ vg/kg to 2x 10 ¹⁴ vg/kg, 2xl0 ¹⁴ vg/kg to 3xl0 ¹⁴ vg/kg, or 3xl0 ¹⁴ vg/kg to 5xl0 ¹⁴ vg/kg.

In some embodiments, detectable expression of LAMP-2B in heart tissue occurs at doses, in vector genomes (vg) per kilogram weight of subject (kg), of lxlO ¹³ vg/kg to 3 x 10 ¹³ vg/kg, 3xl0 ¹³ vg/kg to 5xl0 ¹³ vg/kg, 5xl0 ¹³ vg/kg to 7xl0 ¹³ vg/kg, 7xl0 ¹³ vg/kg to 9xl0 ¹³ vg/kg, 9x 10 ¹³ vg/kg to 2x 10 ¹⁴ vg/kg, or 2x 10 ¹⁴ vg/kg to 5xl0 ¹⁴ vg/kg. In some embodiments, detectable expression of LAMP-2B in heart tissue occurs at doses, in vector genomes (vg) per kilogram weight of subject (kg), of 1 x 10 ¹³ vg/kg to 5x 10 ¹³ vg/kg, 5x 10 ¹³ vg/kg to 9x 10 ¹³ vg/kg, 9xl0 ¹³ vg/kg or to 5xl0 ¹⁴ vg/kg. In some embodiments, detectable expression of LAMP-2B in heart tissue occurs at doses, in vector genomes (vg) per kilogram weight of subject (kg), of lxlO ¹³ vg/kg to 9x 10 ¹³ vg/kg, or 9x 10 ¹³ vg/kg or to 5xl0 ¹⁴ vg/kg.

In some embodiments, detectable expression of LAMP-2B in heart tissue occurs at doses, in vector genomes (vg) per kilogram weight of subject (kg), of lxlO ¹³ vg/kg to 5x 10 ¹³ vg/kg, 5x 10 ¹³ vg/kg to 1 x 10 ¹⁴ vg/kg, or 1 x 10 ¹⁴ vg/kg to 5xl0 ¹⁴ vg/kg.

In some embodiments, detectable expression of LAMP-2B in heart tissue occurs at doses, in vector genomes (vg) per kilogram weight of subject (kg), of lxlO ¹³ vg/kg to 5xl0 ¹⁴ vg/kg. In some embodiments, detectable expression of LAMP-2B in heart tissue occurs at doses, in vector genomes (vg) per kilogram weight of subject (kg), of 1 ^c 10 ¹³ vg/kg to 1 ^c 10 ¹⁴ .

In various embodiments, the pharmaceutical compositions contain vehicles ( e.g ., carriers, diluents and excipients) that are pharmaceutically acceptable for a formulation capable of being injected. Exemplary excipients include a poloxamer. Formulation buffers for viral vectors (including AAV) general contains salts to prevent aggregation and other excipients (e.g. poloxamer) to reduce stickiness of the vector. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. Advantageously, the formulation is stable for storage and use when frozen (e.g. at less than 0°C, about -60°C, or about -72°C).

Exemplary methods of treating lysosomal disorders and/or Danon disease are provided in WO 2018/170239 Al, which is incorporated herein in its entirety. The transgenes, expression cassettes, and vectors of the disclosure are useful for both in vivo (e.g. systemic, particularly intravenous use) and also ex vivo use. LAMP-2B transgene and a functional promoter can be used to ex vivo gene-correct patients’ autologous hematopoietic stem and progenitor cells (HSPCs), which can then be re-transplanted in the patients to repopulate their bone marrow, which is a reservoir of“healthy” cells for the rest of the life of the patients. In some embodiments, lentiviral vectors are used for ex vivo gene corrected, but other non-viral or viral vectors may be used in place of a lentiviral vector. The disclosure are envisions allogeneic transplant using donor HSPCs. In some embodiments, the lentiviral vector is a self-inactivating (SIN) lentivirus vector. In some embodiments, the HSPCs are derived from peripheral blood mobilized using, e.g. , granulocyte-colony stimulating factor (G-CSF) and/or plerixafor.

In another aspect, the disclosure provides methods of preventing, mitigating, ameliorating, reducing, inhibiting, eliminating and/or reversing one or more symptoms of Danon disease or another autophagy disorder in a subject in need thereof, comprising administering to the subject a gene therapy vector of the disclosure. The term“Danon disease” refers to an X-linked dominant skeletal and cardiac muscle disorder with multisystem clinical manifestations. Danon disease mutations lead to an absence of lysosome-associated membrane protein 2 (LAMP -2) protein expression. Major clinical features include skeletal and cardiac myopathy, cardiac conduction abnormalities, cognitive difficulties, and retinal disease. Men are typically affected earlier and more severely than women.

In an embodiment, the vector is administered via a route selected from the group consisting of parenteral, intravenous, intra-arterial, intracardiac, intracoronary,

intramyocardial, intrarenal, intraurethral, epidural, and intramuscular. In an embodiment, the vector is administered multiple times. In an embodiment, the vector is administered by intramuscular injection of the vector. In an embodiment, the vector is administered by injection of the vector into skeletal muscle. In an embodiment, the expression cassette comprises a muscle-specific promoter, optionally a muscle creatine kinase (MCK) promoter or a MCK/SV40 hybrid promoter as described in Takeshita et al. Muscle creatine

kinase/SV40 hybrid promoter for muscle-targeted long-term transgene expression. IntJMol Med. 2007 Feb;l9(2):309-l5. In an embodiment, the vector is administered by intracardiac injection.

In an embodiment, the vector, e.g., AAV vector, is administered systemically, and more particularly, intravenously. Advantageously, the vector is administered at a dose (in vg per mL, vg/kg body mass, or vg/min/kg) less than the dose required to observe the same response when an original or wild-type LAMP-2B sequence is used. In particular

embodiments, the vector is an AAV2/9 vector comprising an expression cassette comprising a polynucleotide encoding LAMP-2B disclosed herein.

In some embodiments, the disclosure provides a method of expressing LAMP-2B in a subject, comprising systemically administering an adeno-associated viral (AAV) vector to the subject, wherein the AAV vector comprises an expression cassette comprising a transgene sharing at least 95% identity with SEQ ID NO: 3 or is identical to SEQ ID NO: 3, the transgene operatively linked to an enhancer/promoter region, wherein systemic

administration of the AAV vector to the subject results in increased expression of LAMP-2B compared to expression of LAMP-2B prior to administration of the AAV vector or expression of LAMP-2B in an untreated control subject. In some embodiments, the AAV vector is an AAV2/9 vector. In particular embodiments, the expression cassette comprises any of the elements disclosed herein. In some embodiment, systemic administration comprises intravenous administration. In some embodiments, the subject is exhibiting symptoms of Danon disease. In some embodiments, the subject suffers from, or is at risk for, Danon disease.

In some embodiments, the AAV vector is administered at a dose of between about 1 x 10 ¹² and 5x 10 ¹⁴ vector genomes (vg) of the AAV vector per kilogram (vg) of total body mass of the subject (vg/kg). In some embodiments, the AAV vector is administered at a dose of between about 1 ^c 10 ¹³ and 5xl0 ¹⁴ vg/kg. In some embodiments, the AAV vector is administered at a dose of between about 5xl0 ¹³ and 3xl0 ¹⁴ vg/kg. In some embodiments, the AAV vector is administered at a dose of between about 5x 10 ¹³ and 1 ^c 10 ¹⁴ vg/kg. In some embodiments, the AAV vector is administered at a dose of less than about 1 ^c 10 ¹² vg/kg, less than about 3 ^c 10 ¹² vg/kg, less than about 5xl0 ¹² vg/kg, less than about 7xl0 ¹² vg/kg, less than about lxlO ¹³ vg/kg, less than about 3xl0 ¹³ vg/kg, less than about 5xl0 ¹³ vg/kg, less than about 7 ^c 10 ¹³ vg/kg, less than about 1 ^c 10 ¹⁴ vg/kg, less than about 3 ^c 10 ¹⁴ vg/kg, less than about 5 ^c 10 ¹⁴ vg/kg, less than about 7x 10 ¹⁴ vg/kg, less than about 1 ^c 10 ¹⁵ vg/kg, less than about 3 ^c 10 ¹⁵ vg/kg, less than about 5xl0 ¹⁵ vg/kg, or less than about 7xl0 ¹⁵ vg/kg.

In some embodiments, the AAV vector is administered at a dose of about 1 ^c 10 ¹² vg/kg, about 3 ^c 10 ¹² vg/kg, about 5xl0 ¹² vg/kg, about 7xl0 ¹² vg/kg, about lxlO ¹³ vg/kg, about 3 ^c 10 ¹³ vg/kg, about 5xl0 ¹³ vg/kg, about 7xl0 ¹³ vg/kg, about lxlO ¹⁴ vg/kg, about 3 ^c 10 ¹⁴ vg/kg, about 5xl0 ¹⁴ vg/kg, about 7xl0 ¹⁴ vg/kg, about lxlO ¹⁵ vg/kg, about 3xl0 ¹⁵ vg/kg, about 5 ^c 10 ¹⁵ vg/kg, or about 7x 10 ¹⁵ vg/kg.

In some embodiments, the AAV vector is administered at a dose of lxlO ¹² vg/kg, 3 ^c 10 ¹² vg/kg, 5 ^c 10 ¹² vg/kg, 7xl0 ¹² vg/kg, lxlO ¹³ vg/kg, 3xl0 ¹³ vg/kg, 5xl0 ¹³ vg/kg, 7xl0 ¹³ vg/kg, lxlO ¹⁴ vg/kg, 3 ^c 10 ¹⁴ vg/kg, 5xl0 ¹⁴ vg/kg, 7xl0 ¹⁴ vg/kg, lxlO ¹⁵ vg/kg, 3xl0 ¹⁵ vg/kg, 5xl0 ¹⁵ vg/kg, or7xl0 ¹⁵ vg/kg.

In some embodiments, the lentiviral vector is administered at a dose of between about lxlO ¹² and 5x 10 ¹⁴ vector genomes (vg) of the lentiviral vector per kilogram (vg) of total body mass of the subject (vg/kg). In some embodiments, the lentiviral vector is administered at a dose of between about lxlO ¹³ and 5x 10 ¹⁴ vg/kg. In some embodimets, the lentiviral vector is administered at a dose of between about 5xl0 ¹³ and 3xl0 ¹⁴ vg/kg. In some embodiments, the lentiviral vector is administered at a dose of between about 5xl0 ¹³ and I ^c IO ¹⁴ vg/kg. In some embodiments, the lentiviral vector is administered at a dose of less than about 1 10 ¹² vg/kg, less than about 3 10 ¹² vg/kg, less than about 5 10 ¹² vg/kg, less than about 7 ^c 10 ¹² vg/kg, less than about 1 ^c 10 ¹³ vg/kg, less than about 3 ^c 10 ¹³ vg/kg, less than about 5 ^c 10 ¹³ vg/kg, less than about 7x 10 ¹³ vg/kg, less than about 1 ^c 10 ¹⁴ vg/kg, less than about 3 x 10 ¹⁴ vg/kg, less than about 5 ^c 10 ¹⁴ vg/kg, less than about 7x 10 ¹⁴ vg/kg, less than about 1 x 10 ¹⁵ vg/kg, less than about 3 ^c 10 ¹⁵ vg/kg, less than about 5 ^c 10 ¹⁵ vg/kg, or less than about 7 ^c 10 ¹⁵ vg/kg.

In some embodiments, the lentiviral vector is administered at a dose of about 1 ^c 10 ¹² vg/kg, about 3 ^c 10 ¹² vg/kg, about 5xl0 ¹² vg/kg, about 7xl0 ¹² vg/kg, about lxlO ¹³ vg/kg, about 3 ^c 10 ¹³ vg/kg, about 5xl0 ¹³ vg/kg, about 7xl0 ¹³ vg/kg, about lxlO ¹⁴ vg/kg, about 3 ^c 10 ¹⁴ vg/kg, about 5xl0 ¹⁴ vg/kg, about 7xl0 ¹⁴ vg/kg, about lxlO ¹⁵ vg/kg, about 3xl0 ¹⁵ vg/kg, about 5 ^c 10 ¹⁵ vg/kg, or about 7x 10 ¹⁵ vg/kg.

In some embodiments, the lentiviral vector is administered at a dose of lxlO ¹² vg/kg, 3 ^c 10 ¹² vg/kg, 5 ^c 10 ¹² vg/kg, 7xl0 ¹² vg/kg, lxlO ¹³ vg/kg, 3xl0 ¹³ vg/kg, 5xl0 ¹³ vg/kg, 7xl0 ¹³ vg/kg, lxlO ¹⁴ vg/kg, 3 ^c 10 ¹⁴ vg/kg, 5xl0 ¹⁴ vg/kg, 7xl0 ¹⁴ vg/kg, lxlO ¹⁵ vg/kg, 3xl0 ¹⁵ vg/kg, 5xl0 ¹⁵ vg/kg, or7xl0 ¹⁵ vg/kg.

In some embodiments, the viral vector is administered at a dose of between about lxlO ¹² and 5x 10 ¹⁴ vector genomes (vg) of the viral vector per kilogram (vg) of total body mass of the subject (vg/kg). In some embodiments, the viral vector is administered at a dose of between about lxlO ¹³ and 5xl0 ¹⁴ vg/kg. In some embodimets, the viral vector is administered at a dose of between about 5xl0 ¹³ and 3xl0 ¹⁴ vg/kg. In some embodiments, the viral vector is administered at a dose of between about 5x 10 ¹³ and lxlO ¹⁴ vg/kg. In some embodiments, the viral vector is administered at a dose of less than about lxlO ¹² vg/kg, less than about 3 ^c 10 ¹² vg/kg, less than about 5xl0 ¹² vg/kg, less than about 7xl0 ¹² vg/kg, less than about lxlO ¹³ vg/kg, less than about 3xl0 ¹³ vg/kg, less than about 5xl0 ¹³ vg/kg, less than about 7 ^c 10 ¹³ vg/kg, less than about lxlO ¹⁴ vg/kg, less than about 3 ^c 10 ¹⁴ vg/kg, less than about 5 ^c 10 ¹⁴ vg/kg, less than about 7x 10 ¹⁴ vg/kg, less than about lxlO ¹⁵ vg/kg, less than about 3 ^c 10 ¹⁵ vg/kg, less than about 5xl0 ¹⁵ vg/kg, or less than about 7xl0 ¹⁵ vg/kg.

In some embodiments, the viral vector is administered at a dose of about lxlO ¹² vg/kg, about 3 ^c 10 ¹² vg/kg, about 5xl0 ¹² vg/kg, about 7xl0 ¹² vg/kg, about lxlO ¹³ vg/kg, about 3 ^c 10 ¹³ vg/kg, about 5xl0 ¹³ vg/kg, about 7xl0 ¹³ vg/kg, about lxlO ¹⁴ vg/kg, about 3xl0 ¹⁴ vg/kg, about 5 10 ¹⁴ vg/kg, about Ί ^/ 10 ¹⁴ vg/kg, about 1 10 ¹⁵ vg/kg, about 3 10 ¹⁵ vg/kg, about 5 ^c 10 ¹⁵ vg/kg, or about 7x 10 ¹⁵ vg/kg.

In some embodiments, the viral vector is administered at a dose of 1 ^c 10 ¹² vg/kg, 3 ^c 10 ¹² vg/kg, 5 ^c 10 ¹² vg/kg, 7xl0 ¹² vg/kg, 1 ^c 10 ¹³ vg/kg, 3xl0 ¹³ vg/kg, 5xl0 ¹³ vg/kg, 7xl0 ¹³ vg/kg, lxlO ¹⁴ vg/kg, 3 ^c 10 ¹⁴ vg/kg, 5xl0 ¹⁴ vg/kg, 7xl0 ¹⁴ vg/kg, lxlO ¹⁵ vg/kg, 3xl0 ¹⁵ vg/kg, 5xl0 ¹⁵ vg/kg, or7xl0 ¹⁵ vg/kg.

In some embodiments, the AAV vector is administered systemically at a dose of between about lxlO ¹² and 5x 10 ¹⁴ vector genomes (vg) of the AAV vector per kilogram (vg) of total body mass of the subject (vg/kg). In some embodiments, the AAV vector is administered systemically at a dose of between about lxlO ¹³ and 5xl0 ¹⁴ vg/kg. In some embodiments, the AAV vector is administered systemically at a dose of between about 5xl0 ¹³ and 3xl0 ¹⁴ vg/kg. In some embodiments, the AAV vector is administered

systemically at a dose of between about 5xl0 ¹³ and lxlO ¹⁴ vg/kg. In some embodiments, the AAV vector is administered systemically at a dose of less than about lxlO ¹² vg/kg, less than about 3 x 10 ¹² vg/kg, less than about 5 ^c 10 ¹² vg/kg, less than about 7x 10 ¹² vg/kg, less than about lxlO ¹³ vg/kg, less than about 3xl0 ¹³ vg/kg, less than about 5xl0 ¹³ vg/kg, less than about 7 ^c 10 ¹³ vg/kg, less than about lxlO ¹⁴ vg/kg, less than about 3 ^c 10 ¹⁴ vg/kg, less than about 5 ^c 10 ¹⁴ vg/kg, less than about 7x 10 ¹⁴ vg/kg, less than about lxlO ¹⁵ vg/kg, less than about 3 ^c 10 ¹⁵ vg/kg, less than about 5xl0 ¹⁵ vg/kg, or less than about 7xl0 ¹⁵ vg/kg.

In some embodiments, the AAV vector is administered systemically at a dose of about lxlO ¹² vg/kg, about 3 ^c 10 ¹² vg/kg, about 5xl0 ¹² vg/kg, about 7xl0 ¹² vg/kg, about lxlO ¹³ vg/kg, about 3 ^c 10 ¹³ vg/kg, about 5xl0 ¹³ vg/kg, about 7xl0 ¹³ vg/kg, about lxlO ¹⁴ vg/kg, about 3 ^c 10 ¹⁴ vg/kg, about 5xl0 ¹⁴ vg/kg, about 7xl0 ¹⁴ vg/kg, about lxlO ¹⁵ vg/kg, about 3 x 10 ¹⁵ vg/kg, about 5 ^c 10 ¹⁵ vg/kg, or about 7x 10 ¹⁵ vg/kg.

In some embodiments, the AAV vector is administered systemically at a dose of lxlO ¹² vg/kg, 3 ^c 10 ¹² vg/kg, 5xl0 ¹² vg/kg, 7xl0 ¹² vg/kg, lxlO ¹³ vg/kg, 3xl0 ¹³ vg/kg, 5xl0 ¹³ vg/kg, 7 ^c 10 ¹³ vg/kg, lxlO ¹⁴ vg/kg, 3xl0 ¹⁴ vg/kg, 5xl0 ¹⁴ vg/kg, 7xl0 ¹⁴ vg/kg, lxlO ¹⁵ vg/kg, 3xl0 ¹⁵ vg/kg, 5xl0 ¹⁵ vg/kg, or7xl0 ¹⁵ vg/kg.

In some embodiments, the lentiviral vector is administered systemically at a dose of between about lxlO ¹² and 5x 10 ¹⁴ vector genomes (vg) of the lentiviral vector per kilogram (vg) of total body mass of the subject (vg/kg). In some embodiments, the lentiviral vector is administered systemically at a dose of between about lxlO ¹³ and 5/ 10 ¹⁴ vg/kg. In some embodimets, the lentiviral vector is administered systemically at a dose of between about 5 / 10 ¹ and 3/10 ¹⁴ vg/kg. In some embodiments, the lentiviral vector is administered systemically at a dose of between about 5><l0 ¹³ and lxlO ¹⁴ vg/kg. In some embodiments, the lentiviral vector is administered systemically at a dose of less than about lxlO ¹² vg/kg, less than about 3 ^c 10 ¹² vg/kg, less than about 5xl0 ¹² vg/kg, less than about 7xl0 ¹² vg/kg, less than about lxlO ¹³ vg/kg, less than about 3xl0 ¹³ vg/kg, less than about 5xl0 ¹³ vg/kg, less than about 7 ^c 10 ¹³ vg/kg, less than about lxlO ¹⁴ vg/kg, less than about 3 ^c 10 ¹⁴ vg/kg, less than about 5 ^c 10 ¹⁴ vg/kg, less than about 7x 10 ¹⁴ vg/kg, less than about lxlO ¹⁵ vg/kg, less than about 3 ^c 10 ¹⁵ vg/kg, less than about 5xl0 ¹⁵ vg/kg, or less than about 7xl0 ¹⁵ vg/kg.

In some embodiments, the lentiviral vector is administered systemically at a dose of about lxlO ¹² vg/kg, about 3xl0 ¹² vg/kg, about 5xl0 ¹² vg/kg, about 7xl0 ¹² vg/kg, about lxlO ¹³ vg/kg, about 3 ^c 10 ¹³ vg/kg, about 5xl0 ¹³ vg/kg, about 7xl0 ¹³ vg/kg, about lxlO ¹⁴ vg/kg, about 3 ^c 10 ¹⁴ vg/kg, about 5xl0 ¹⁴ vg/kg, about 7xl0 ¹⁴ vg/kg, about lxlO ¹⁵ vg/kg, about 3 ^c 10 ¹⁵ vg/kg, about 5xl0 ¹⁵ vg/kg, or about 7xl0 ¹⁵ vg/kg.

In some embodiments, the lentiviral vector is administered systemically at a dose of lxlO ¹² vg/kg, 3 ^c 10 ¹² vg/kg, 5xl0 ¹² vg/kg, 7xl0 ¹² vg/kg, lxlO ¹³ vg/kg, 3xl0 ¹³ vg/kg, 5xl0 ¹³ vg/kg, 7 ^c 10 ¹³ vg/kg, lxlO ¹⁴ vg/kg, 3xl0 ¹⁴ vg/kg, 5xl0 ¹⁴ vg/kg, 7xl0 ¹⁴ vg/kg, lxlO ¹⁵ vg/kg, 3xl0 ¹⁵ vg/kg, 5xl0 ¹⁵ vg/kg, or7xl0 ¹⁵ vg/kg.

In some embodiments, the viral vector is administered systemically at a dose of between about lxlO ¹² and 5x 10 ¹⁴ vector genomes (vg) of the viral vector per kilogram (vg) of total body mass of the subject (vg/kg). In some embodiments, the viral vector is administered systemically at a dose of between about lxlO ¹³ and 5xl0 ¹⁴ vg/kg. In some embodimets, the viral vector is administered systemically at a dose of between about 5xl0 ¹³ and 3 ^c 10 ¹⁴ vg/kg. In some embodiments, the viral vector is administered systemically at a dose of between about 5xl0 ¹³ and lxlO ¹⁴ vg/kg. In some embodiments, the viral vector is administered systemically at a dose of less than about lxlO ¹² vg/kg, less than about 3xl0 ¹² vg/kg, less than about 5x 10 ¹² vg/kg, less than about 7x 10 ¹² vg/kg, less than about lxlO ¹³ vg/kg, less than about 3xl0 ¹³ vg/kg, less than about 5xl0 ¹³ vg/kg, less than about 7xl0 ¹³ vg/kg, less than about lxlO ¹⁴ vg/kg, less than about 3xl0 ¹⁴ vg/kg, less than about 5xl0 ¹⁴ vg/kg, less than about 7x10 ¹⁴ vg/kg, less than about lxlO ¹⁵ vg/kg, less than about 3xl0 ¹⁵ vg/kg, less than about 5x 10 ¹⁵ vg/kg, or less than about 7x 10 ¹⁵ vg/kg.

In some embodiments, the viral vector is administered systemically at a dose of about lxlO ¹² vg/kg, about 3 ^c 10 ¹² vg/kg, about 5xl0 ¹² vg/kg, about 7xl0 ¹² vg/kg, about lxlO ¹³ vg/kg, about 3 ^c 10 ¹³ vg/kg, about 5xl0 ¹³ vg/kg, about 7xl0 ¹³ vg/kg, about lxlO ¹⁴ vg/kg, about 3 ^c 10 ¹⁴ vg/kg, about 5xl0 ¹⁴ vg/kg, about 7xl0 ¹⁴ vg/kg, about lxlO ¹⁵ vg/kg, about 3 x 10 ¹⁵ vg/kg, about 5 ^c 10 ¹⁵ vg/kg, or about 7x 10 ¹⁵ vg/kg.

In some embodiments, the viral vector is administered systemically at a dose of lxlO ¹² vg/kg, 3 ^c 10 ¹² vg/kg, 5xl0 ¹² vg/kg, 7xl0 ¹² vg/kg, lxlO ¹³ vg/kg, 3xl0 ¹³ vg/kg, 5xl0 ¹³ vg/kg, 7 ^c 10 ¹³ vg/kg, lxlO ¹⁴ vg/kg, 3xl0 ¹⁴ vg/kg, 5xl0 ¹⁴ vg/kg, 7xl0 ¹⁴ vg/kg, lxlO ¹⁵ vg/kg, 3xl0 ¹⁵ vg/kg, 5xl0 ¹⁵ vg/kg, or7xl0 ¹⁵ vg/kg.

In some embodiments, the AAV vector is administered intravenously at a dose of between about lxlO ¹² and 5x 10 ¹⁴ vector genomes (vg) of the AAV vector per kilogram (vg) of total body mass of the subject (vg/kg). In some embodiments, the AAV vector is administered intravenously at a dose of between about lxlO ¹³ and 5x 10 ¹⁴ vg/kg. In some embodiments, the AAV vector is administered intravenously at a dose of between about 5xl0 ¹³ and 3xl0 ¹⁴ vg/kg. In some embodiments, the AAV vector is administered

intravenously at a dose of between about 5x 10 ¹³ and lxlO ¹⁴ vg/kg. In some embodiments, the AAV vector is administered intravenously at a dose of less than about lxlO ¹² vg/kg, less than about 3 x 10 ¹² vg/kg, less than about 5 ^c 10 ¹² vg/kg, less than about 7x 10 ¹² vg/kg, less than about lxlO ¹³ vg/kg, less than about 3xl0 ¹³ vg/kg, less than about 5xl0 ¹³ vg/kg, less than about 7 ^c 10 ¹³ vg/kg, less than about lxlO ¹⁴ vg/kg, less than about 3 ^c 10 ¹⁴ vg/kg, less than about 5 ^c 10 ¹⁴ vg/kg, less than about 7x 10 ¹⁴ vg/kg, less than about lxlO ¹⁵ vg/kg, less than about 3 ^c 10 ¹⁵ vg/kg, less than about 5xl0 ¹⁵ vg/kg, or less than about 7xl0 ¹⁵ vg/kg.

In some embodiments, the AAV vector is administered intravenously at a dose of about lxlO ¹² vg/kg, about 3xl0 ¹² vg/kg, about 5xl0 ¹² vg/kg, about 7xl0 ¹² vg/kg, about lxlO ¹³ vg/kg, about 3 ^c 10 ¹³ vg/kg, about 5xl0 ¹³ vg/kg, about 7xl0 ¹³ vg/kg, about lxlO ¹⁴ vg/kg, about 3 ^c 10 ¹⁴ vg/kg, about 5xl0 ¹⁴ vg/kg, about 7xl0 ¹⁴ vg/kg, about lxlO ¹⁵ vg/kg, about 3 ^c 10 ¹⁵ vg/kg, about 5xl0 ¹⁵ vg/kg, or about 7xl0 ¹⁵ vg/kg.

In some embodiments, the AAV vector is administered intravenously at a dose of lxlO ¹² vg/kg, 3 ^c 10 ¹² vg/kg, 5xl0 ¹² vg/kg, 7xl0 ¹² vg/kg, lxlO ¹³ vg/kg, 3xl0 ¹³ vg/kg, 5xl0 ¹³ vg/kg, 7 ^c 10 ¹³ vg/kg, lxlO ¹⁴ vg/kg, 3xl0 ¹⁴ vg/kg, 5xl0 ¹⁴ vg/kg, 7xl0 ¹⁴ vg/kg, lxlO ¹⁵ vg/kg, 3xl0 ¹⁵ vg/kg, 5xl0 ¹⁵ vg/kg, or7xl0 ¹⁵ vg/kg.

In some embodiments, the lentiviral vector is administered intravenously at a dose of between about lxlO ¹² and 5x 10 ¹⁴ vector genomes (vg) of the lentiviral vector per kilogram (vg) of total body mass of the subject (vg/kg). In some embodiments, the lentiviral vector is administered intravenously at a dose of between about lxlO ¹³ and 5x 10 ¹⁴ vg/kg. In some embodimets, the lentiviral vector is administered intravenously at a dose of between about 5xl0 ¹³ and 3xl0 ¹⁴ vg/kg. In some embodiments, the lentiviral vector is administered intravenously at a dose of between about 5x 10 ¹³ and lxlO ¹⁴ vg/kg. In some embodiments, the lentiviral vector is administered intravenously at a dose of less than about lxlO ¹² vg/kg, less than about 3 ^c 10 ¹² vg/kg, less than about 5xl0 ¹² vg/kg, less than about 7xl0 ¹² vg/kg, less than about lxlO ¹³ vg/kg, less than about 3xl0 ¹³ vg/kg, less than about 5xl0 ¹³ vg/kg, less than about 7 ^c 10 ¹³ vg/kg, less than about lxlO ¹⁴ vg/kg, less than about 3 ^c 10 ¹⁴ vg/kg, less than about 5 ^c 10 ¹⁴ vg/kg, less than about 7x 10 ¹⁴ vg/kg, less than about lxlO ¹⁵ vg/kg, less than about 3 ^c 10 ¹⁵ vg/kg, less than about 5xl0 ¹⁵ vg/kg, or less than about 7xl0 ¹⁵ vg/kg.

In some embodiments, the lentiviral vector is administered intravenously at a dose of about lxlO ¹² vg/kg, about 3xl0 ¹² vg/kg, about 5xl0 ¹² vg/kg, about 7xl0 ¹² vg/kg, about lxlO ¹³ vg/kg, about 3 ^c 10 ¹³ vg/kg, about 5xl0 ¹³ vg/kg, about 7xl0 ¹³ vg/kg, about lxlO ¹⁴ vg/kg, about 3 ^c 10 ¹⁴ vg/kg, about 5xl0 ¹⁴ vg/kg, about 7xl0 ¹⁴ vg/kg, about lxlO ¹⁵ vg/kg, about 3 ^c 10 ¹⁵ vg/kg, about 5xl0 ¹⁵ vg/kg, or about 7xl0 ¹⁵ vg/kg.

In some embodiments, the lentiviral vector is administered intravenously at a dose of lxlO ¹² vg/kg, 3 ^c 10 ¹² vg/kg, 5xl0 ¹² vg/kg, 7xl0 ¹² vg/kg, lxlO ¹³ vg/kg, 3xl0 ¹³ vg/kg, 5xl0 ¹³ vg/kg, 7 ^c 10 ¹³ vg/kg, lxlO ¹⁴ vg/kg, 3xl0 ¹⁴ vg/kg, 5xl0 ¹⁴ vg/kg, 7xl0 ¹⁴ vg/kg, lxlO ¹⁵ vg/kg, 3xl0 ¹⁵ vg/kg, 5xl0 ¹⁵ vg/kg, or7xl0 ¹⁵ vg/kg.

In some embodiments, the viral vector is administered intravenously at a dose of between about lxlO ¹² and 5x 10 ¹⁴ vector genomes (vg) of the viral vector per kilogram (vg) of total body mass of the subject (vg/kg). In some embodiments, the viral vector is administered intravenously at a dose of between about lxlO ¹³ and 5x 10 ¹⁴ vg/kg. In some embodimets, the viral vector is administered intravenously at a dose of between about 5x 10 ¹³ and 3xl0 ¹⁴ vg/kg. In some embodiments, the viral vector is administered intravenously at a dose of between about 5xl0 ¹³ and lxlO ¹⁴ vg/kg. In some embodiments, the viral vector is administered intravenously at a dose of less than about lxlO ¹² vg/kg, less than about 3 10 ¹² vg/kg, less than about 5x 10 ¹² vg/kg, less than about 7x 10 ¹² vg/kg, less than about 1 ^c 10 ¹³ vg/kg, less than about 3xl0 ¹³ vg/kg, less than about 5xl0 ¹³ vg/kg, less than about 7xl0 ¹³ vg/kg, less than about lxlO ¹⁴ vg/kg, less than about 3xl0 ¹⁴ vg/kg, less than about 5xl0 ¹⁴ vg/kg, less than about 7xl0 ¹⁴ vg/kg, less than about lxlO ¹⁵ vg/kg, less than about 3xl0 ¹⁵ vg/kg, less than about 5x 10 ¹⁵ vg/kg, or less than about 7x 10 ¹⁵ vg/kg.

In some embodiments, the viral vector is administered intravenously at a dose of about lxlO ¹² vg/kg, about 3xl0 ¹² vg/kg, about 5xl0 ¹² vg/kg, about 7xl0 ¹² vg/kg, about lxlO ¹³ vg/kg, about 3 ^c 10 ¹³ vg/kg, about 5xl0 ¹³ vg/kg, about 7xl0 ¹³ vg/kg, about lxlO ¹⁴ vg/kg, about 3 ^c 10 ¹⁴ vg/kg, about 5xl0 ¹⁴ vg/kg, about 7xl0 ¹⁴ vg/kg, about lxlO ¹⁵ vg/kg, about 3 ^c 10 ¹⁵ vg/kg, about 5xl0 ¹⁵ vg/kg, or about 7xl0 ¹⁵ vg/kg.

In some embodiments, the viral vector is administered intravenously at a dose of lxlO ¹² vg/kg, 3 ^c 10 ¹² vg/kg, 5xl0 ¹² vg/kg, 7xl0 ¹² vg/kg, lxlO ¹³ vg/kg, 3xl0 ¹³ vg/kg, 5xl0 ¹³ vg/kg, 7 ^c 10 ¹³ vg/kg, lxlO ¹⁴ vg/kg, 3xl0 ¹⁴ vg/kg, 5xl0 ¹⁴ vg/kg, 7xl0 ¹⁴ vg/kg, lxlO ¹⁵ vg/kg, 3xl0 ¹⁵ vg/kg, 5xl0 ¹⁵ vg/kg, or7xl0 ¹⁵ vg/kg.

Systemic (or more particularly intravenous) administration in some embodiments results in expression of LAMP-2B polynucleotide as mRNA, in the form of an mRNA expressed from the transgene, in one or more tissues ( e.g . heart, muscle, and/or liver) of the subject. In some embodiments, expression of the LAMP-2B polynucleotide as mRNA is increased at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 1.6-fold, at least about 1.7-fold, at least about 1.8-fold, at least about 1.9-fold, at least about 2.0-fold, at least about 2.2-fold, at least about 2.3-fold, at least about 2.4-fold, at least about 2.5-fold, at least about 3-fold, or at least about 4-fold in the heart compared to expression in an untreated subject or a subject treated with a control vector. In some embodiments, expression of LAMP-2B polynucleotide as mRNA is increased at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2.0-fold, at least 2.2-fold, at least 2.3-fold, at least

2.4-fold, at least 2.5-fold, at least 3-fold, or at least 4-fold in the heart compared to expression in an untreated subject or a subject treated with a control vector. In some embodiments, expression ofLAMP-2B polynucleotide as mRNA is increased 1.2-fold, 1.3-fold, 1.4-fold,

1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 3 -fold, or 4-fold in the heart compared to expression in an untreated subject or a subject treated with a control vector.

In some embodiments, expression of LAMP-2B polynucleotide as mRNA is increased at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 1.6-fold, at least about 1.7-fold, at least about 1.8-fold, at least about 1.9-fold, at least about 2.0-fold, at least about 2.2-fold, at least about 2.3-fold, at least about 2.4-fold, at least about 2.5-fold, at least about 3-fold, or at least about 4-fold in the muscle compared to expression in an untreated subject or a subject treated with a control vector. In some embodiments, expression of LAMP-2B polynucleotide as mRNA is increased at least 1.2- fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2.0-fold, at least 2.2-fold, at least 2.3-fold, at least 2.4- fold, at least 2.5-fold, at least 3-fold, or at least 4-fold in the muscle compared to expression in an untreated subject or a subject treated with a control vector. In some embodiments, expression of LAMP-2B polynucleotide as mRNA is increased 1.2-fold, 1.3-fold, 1.4-fold,

1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold,

3 -fold, or 4-fold in the muscle compared to expression in an untreated subject or a subject treated with a control vector.

In some embodiments, the LAMP-2B transgene is expressed in the heart and not expressed in the liver of the subject. In some embodiments, expression of LAMP-2B polynucleotide as mRNA is observed to be at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 1.6-fold, at least about 1.7-fold, at least about 1.8-fold, at least about 1.9-fold, at least about 2.0-fold, at least about 2.2-fold, at least about 2.3-fold, at least about 2.4-fold, at least about 2.5-fold, at least about 3-fold, or at least about 4-fold in the heart compared to the liver. In some embodiments, expression of LAMP-2B polynucleotide as mRNA is observed to be at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9- fold, at least 2.0-fold, at least 2.2-fold, at least 2.3-fold, at least 2.4-fold, at least 2.5-fold, at least 3 -fold, or at least 4-fold in the heart compared to the liver. In some embodiments, expression of LAMP-2B polynucleotide as mRNA is observed to be 1.2-fold, 1.3-fold, 1.4- fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5- fold, 3 -fold, or 4-fold in the heart compared to the liver. In some embodiments, expression of wild-type or functional LAMP-2B protein is increased at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 1.6-fold, at least about 1.7-fold, at least about 1.8-fold, at least about 1.9-fold, at least about 2.0-fold, at least about 2.2-fold, at least about 2.3-fold, at least about 2.4-fold, at least about 2.5-fold, at least about 3-fold, or at least about 4-fold in the heart compared to expression in an untreated subject or a subject treated with a control vector. In some embodiments, expression of wild-type or functional LAMP-2B protein is increased at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7- fold, at least 1.8-fold, at least 1.9-fold, at least 2.0-fold, at least 2.2-fold, at least 2.3-fold, at least 2.4-fold, at least 2.5-fold, at least 3-fold, or at least 4-fold in the heart compared to expression in an untreated subject or a subject treated with a control vector. In some embodiments, expression of wild-type or functional LAMP-2B protein is increased 1.2-fold,

1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.2-fold, 2.3-fold,

2.4-fold, 2.5-fold, 3-fold, or 4-fold in the heart compared to expression in an untreated subject or a subject treated with a control vector.

In some embodiments, expression of wild-type or functional LAMP-2B protein is observed to be at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 1.6-fold, at least about 1.7-fold, at least about 1.8-fold, at least about 1.9-fold, at least about 2.0-fold, at least about 2.2-fold, at least about 2.3-fold, or at least 5-fold, in the heart compared to the liver. In some embodiments, expression of wild-type or functional LAMP-2B protein is observed to be at least 1.2-fold, at least 1.3-fold, at least

1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2.0-fold, at least 2.2-fold, at least 2.3-fold, at least 2.4-fold, at least 2.5-fold, at least

3 -fold, or at least 4-fold in the heart compared to the liver. In some embodiments, expression of wild-type or functional LAMP-2B protein is observed to be 1.2-fold, 1.3-fold, 1.4-fold,

1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold,

3 -fold, or 4-fold in the heart compared to the liver.

In some embodiments, administration of the gene therapy vector results in expression of wild-type or functional LAMP-2B protein in the liver of at most about 1. l-fold, at most about 1.2-fold, at most about 1.3-fold, at most about 1.4-fold, at most about 1.5-fold, at most about 1.6-fold, at most about 1.7-fold, at most about 1.8-fold, at most about 1.9-fold, or at most about 2-fold increased compared to expression in the liver of an untreated subject. In some embodiments, administration of the gene therapy vector results in expression of wild- type or functional LAMP-2B protein in the liver of at most 1.1 -fold, at most 1.2-fold, at most 1.3-fold, at most 1.4-fold, at most 1.5-fold, at most 1.6-fold, at most 1.7-fold, at most 1.8- fold, at most 1.9-fold, or at most 2-fold increased compared to expression in the liver of an untreated subject. In some embodiments, administration of the gene therapy vector results in expression of wild-type or functional LAMP-2B protein in the liver of l.l-fold, 1.2-fold, 1.3- fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, or 2-fold increased compared to expression in the liver of an untreated subject.

In an embodiment, the 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 an embodiment, the cells are stem cells, optionally pluripotent stem cells. In an embodiment, the stem cells are capable of differentiation into cardiac tissue. In an

embodiment, the stem cells are capable of differentiation into muscle tissue, e.g ., cardiac muscle tissue and/or skeletal muscle tissue. In an embodiment, the stem cells are autologous. In an embodiment, the stem cells are induced pluripotent stem cells (iPSCs).

In an embodiment, the disease or disorder is an autophagy disorder. In some embodiments, the autophagy disorder is selected from the group consisting of, but not limited to, end-stage heart failure, myocardial infarction, drug toxicities, diabetes, end-stage renal failure, and aging. In an embodiment, the subject is a mammal, e.g. , a human. In an embodiment, the subject is exhibiting symptoms of Danon disease or another autophagy disorder. In an embodiment, the subject has been identified as having reduced or non- detectable LAMP-2 expression. In an embodiment, the subject has been identified as having a mutated LAMP-2 gene.

Subjects/patients amenable to treatment using the methods described herein include, but are not limited to, individuals at risk of a disease or disorder characterized by insufficient autophagic flux (e.g, Danon disease as well as other known disorders of autophagy including, but not limited to, systolic and diastolic heart failure, myocardial infarction, drug toxicities (for example, anthracyclines chloroquine and its derivatives), diabetes, end-stage renal disease, and aging) but not showing symptoms, as well as subjects presently showing symptoms. Such subject may have been identified as having a mutated LAMP-2 gene or as having reduced or non-detectable levels of LAMP -2 expression.

In some embodiments, the patient is a human. In some embodiments, the patient is a pediatric, adolescent, or adult human. In some embodiments, the patient is 1, 2, 3, 4, 5, 6, 7,

8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 years old, or more than 20 years old. In some embodiments, the patient is 20 to 50 years old. In some embodiments, the patient is 50 to 65 years old. In some embodiments, the patient is 1 to 5, 2 to 6, 3 to 7, 4 to 8, 5 to 9, 6 to 10, 7 to

11, 8 to 12, 9 to 13, 10 to 14, 11 to 15, 12 to 16, 13 to 17, 14 to 18, 15 to 19, or 16 to 20 years old. In some embodiments, the patient is 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10, 10 to 11, 11 to

12, 12 to 13, 13 to 14, 14 to 15, 15 to 16, 16 to 17, 17 to 18, 18 to 19, 19 to 20, or 20 to 21 years old. In a particular embodiment, the patient is 15 to 16 years old.

In some embodiments, the patient is a human male. In some embodiments, the patient is a pediatric, adolescent, or adult human male. In some embodiments, the patient is a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 years old male, or a more than 20 years old male. In some embodiments, the patient is a 20 to 50 years old male. In some embodiments, the patient is a 50 to 65 years old male. In some embodiments, the patient is a 1 to 5, 2 to 6, 3 to 7, 4 to 8, 5 to 9, 6 to 10, 7 to 11, 8 to 12, 9 to 13, 10 to 14, 11 to 15, 12 to 16, 13 to 17, 14 to 18, 15 to 19, or 16 to 20 years old male. In some embodiments, the patient is a 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10, 10 to 11, 11 to 12, 12 to 13, 13 to 14, 14 to 15, 15 to 16, 16 to 17, 17 to 18, 18 to 19, 19 to 20, or 20 to 21 year old male. In a particular embodiment, the patient is 15 to 16 years old.

In some embodiments, the patient is a human female. In some embodiments, the patient is a pediatric, adolescent, or adult human female. In some embodiments, the patient is a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 years old female, or a more than 20 years old female. In some embodiments, the patient is a 20 to 50 years old female. In some embodiments, the patient is a 50 to 65 years old female.

In some embodiments, the subject is exhibiting symptoms of a disease or disorder characterized by insufficient autophagic flux ( e.g ., Danon 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). The symptoms may be actively manifesting, or may be suppressed or controlled (e.g., by medication) or in remission. The subject may or may not have been diagnosed with the disorder, e.g ., by a qualified physician.

Definitions

The terms“lysosome-associated membrane protein 2” and“LAMP -2”

interchangeably refer to nucleic acids and polypeptide polymorphic variants, alleles, mutants, and interspecies homologs that: (1) have an amino acid sequence that has greater than about 90% amino acid sequence identity, for example, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater 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 full-length, to an amino acid sequence encoded by a LAMP -2 nucleic acid (see, e.g. , GenBank Accession Nos.

NM_002294.2 (isoform A). NM_0l3995.2 (isoform B), NM_00l 122606.1 (isoform C)) or to an amino acid sequence of a LAMP-2 polypeptide ( see e.g. , GenBank Accession Nos.

NP_002285.l (isoform A), NP_05470l. l (isoform B), NP_00l 116078.1 (isoform C)); (2) bind to antibodies, e.g. , polyclonal antibodies, raised against an immunogen comprising an amino acid sequence of a LAMP -2 polypeptide (e.g, LAMP-2 polypeptides described herein); or an amino acid sequence encoded by a LAMP-2 nucleic acid (e.g, LAMP-2 polynucleotides described herein), and conservatively modified variants thereof; (3) specifically hybridize under stringent hybridization conditions to an anti-sense strand corresponding to a nucleic acid sequence encoding a 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 higher nucleotide sequence identity, preferably over a region of at least about 25, 50, 100, 200, 500, 1000, 2000 or more nucleotides, or over the full-length, to a LAMP-2 nucleic acid (e.g, LAMP-2 polynucleotides, as described herein, and LAMP -2 polynucleotides that encode LAMP-2 polypeptides, as described herein).

The terms“lysosome-associated membrane protein 2B” and“LAMP-2B”

interchangeably refer to nucleic acids and polypeptide polymorphic variants, alleles, mutants, and interspecies homologs that: (1) have an amino acid sequence that has greater than about 90% amino acid sequence identity, for example, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater 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 full-length, to an amino acid sequence encoded by a LAMP-2B nucleic acid (see e.g., NM 013995.2) or to an amino acid sequence of a LAMP-2B polypeptide (see e.g. , NP_05470l. l); (2) bind to antibodies, e.g. , polyclonal antibodies, raised against an immunogen comprising an amino acid sequence of a LAMP-2B polypeptide (e.g, LAMP-2B polypeptides described herein); or an amino acid sequence encoded by a LAMP-2B nucleic acid (e.g, LAMP-2B polynucleotides described herein), and conservatively modified variants thereof; (3) specifically hybridize under stringent hybridization conditions to an anti-sense strand corresponding to a nucleic acid sequence encoding a LAMP-2B 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 higher nucleotide sequence identity, preferably over a region of at least about 25, 50, 100, 200, 500, 1000, 2000 or more nucleotides, or over the full-length, to a LAMP-2B nucleic acid (e.g, LAMP-2B

polynucleotides, as described herein, and LAMP-2B polynucleotides that encode LAMP-2B polypeptides, as described herein).

The term“functional variant” in respect to a protein (e.g. a LAMP-2B) refers to a polypeptide sequence, or a fragment of a polypeptide sequence having at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, or at least about 80 amino acid resides, that retains one or more functional attributes of the protein. For example, a functional variant of LAMP-2B is a LAMP-2B (as defined herein) that retains one or more functions such as: (1) regulating human cardiomyocyte function (Chi et al. (2019) PNAS USA 116 (2) 556-565); (2) improving metabolic and physiological function in Danon disease (Adler et al. (2019) J. Am. College Cardiology S0735-l097(l9)31295-1); and/or (3) autophagy (Rowland et al. (2016) J. Cell Sci. (2016) 129, 2135-2143).

LAMP-2B has a lumenal domain (residues 29-375), a transmembrane domain

(residues 376-399), and a cytoplasmic domain (residues 400-410), see UniProt Accession No. P 13473. LAMP-2B functions in include chaperone-mediated autophagy, a process that mediates lysosomal degradation of proteins in response to various stresses and as part of the normal turnover of proteins with a long biological half-live (Cuervo et al. Science 273:501- 503(1996), Cuervo et al. J. Cell Sci. 113:4441-4450(2000), Bandyopadhyay et al. Mol. Cell. Biol. 28:5747-5763(2008), Li et al. Exp. Cell Res. 327:48-56(2014), Hubert et al. Biol. Open 5: 1516-1529(2016)). LAMP-2B may target GAPDH and MLLTl l for lysosomal

degradation. LAMP-2B may be required for the fusion of autophagosomes with lysosomes during autophagy. It has been suggested that cells that lack LAMP2 express normal levels of VAMP8, but fail to accumulate STX17 on autophagosomes, which is the most likely explanation for the lack of fusion between autophagosomes and lysosomes. LAMP-2B may be required for normal degradation of the contents of autophagosomes. LAMP-2B may be required for efficient MHCII-mediated presentation of exogenous antigens via its function in lysosomal protein degradation; antigenic peptides generated by proteases in the

endosomal/lysosomal compartment are captured by nascent MHCII subunits (Crotzer et al. Immunology 131 :318-330(2010)).

Functional variants of LAMP-2B therefore include fragments of LAMP-2B that are capable of mediating any of the foregoing functions. In some embodiments, the function fragment of LAMP-2B includes one or more of the lumenal, transmembrane, and cytoplasmic domains. In some embodiments, the functional variant of LAMP-2B comprises one or more C-terminal or N-terminal deletions with respect to native LAMP-2B. In some embodiments, the functional variant of LAMP-2B comprises one or more internal deletions with respect to native LAMP-2B.

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 specified percentage of amino acid residues or nucleotides that are the same (i.e. , share at least about 80% identity, for example, at least about 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity over a specified region to a reference sequence, e.g. , LAMP-2 polynucleotide or polypeptide sequence as described herein, when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Such sequences are then said to be“substantially identical.” This definition also refers to the compliment of a test sequence. Preferably, the identity exists over a region that is at least about 25 amino acids or nucleotides in length, for example, over a region that is 50, 100, 200, 300, 400 amino acids or nucleotides in length, or over the full-length of a reference sequence.

For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences 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 the default parameters are used.

A“comparison window,” as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g ., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. NatT. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wl), or by manual alignment and visual inspection (see, e.g. , Ausubel et ak, eds., 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 ak, J. Mol. Biol. 215:403-410 (1990) and Altschul et ak, 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 worldwide web at ncbi.nlm.nih.gov/).

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 the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, 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 sequence.

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

The terms“systemic administration” and“systemically administered” refer to a method of administering a compound or composition to a mammal so that the compound or composition is delivered to sites in the body, including the targeted site of pharmaceutical action, via the circulatory system. Systemic administration includes, but is not limited to, oral, intranasal, rectal and parenteral (e.g, other than through the alimentary tract, such as intramuscular, intravenous, intra-arterial, transdermal and subcutaneous) administration.

The term“co-administering” or“concurrent administration”, when used, for example with respect to the compounds (e.g, LAMP-2 polynucleotides) and/or analogs thereof and another active agent, refers to administration of the compound and/or analogs and the active agent such that both can simultaneously achieve a physiological effect. The two agents, however, need not be administered together. In certain embodiments, administration of one agent can precede administration of the other. Simultaneous physiological effect need not necessarily require presence of both agents in the circulation at the same time. However, in certain embodiments, co-administering typically results in both agents being simultaneously present in the body (e.g., in the plasma) 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.

The term“effective amount” or“pharmaceutically effective amount” refer to the amount and/or dosage, and/or dosage regime of one or more compounds (e.g, gene therapy vectors) necessary to bring about the desired result e.g, increased expression of one or more LAMP-2 isoforms in an amount sufficient to reduce the ultimate severity of a disease characterized by impaired or deficient autophagy (e.g, Danon disease).

The phrase“cause to be administered” refers to the actions taken by a medical professional (e.g, a physician), or a person controlling medical care of a subject, that control and/or permit the administration of the agent(s)/compound(s) at issue to the subject. Causing to be administered can involve diagnosis and/or determination of an appropriate therapeutic or prophylactic regimen, and/or prescribing particular agent(s)/compounds for a subject. Such prescribing can include, for example, drafting a prescription form, annotating a medical record, and the like.

As used herein, the terms“treating” and“treatment” refer to delaying the onset of, retarding or reversing the progress of, reducing the severity of, or alleviating or preventing either the disease or condition to which the term applies, or one or more symptoms of such disease or condition. The terms“treating” and“treatment” also include preventing, mitigating, ameliorating, reducing, inhibiting, eliminating and/or reversing one or more symptoms of the disease or condition.

The term“mitigating” refers to reduction or elimination of one or more symptoms of that pathology or disease, and/or a reduction in the rate or delay of onset or severity of one or more symptoms of that pathology or disease, and/or the prevention of that pathology or disease. In certain embodiments, the reduction or elimination of one or more symptoms of pathology or disease can include, e.g, measurable and sustained increase in the expression levels of one or more isoforms of LAMP-2.

As used herein, the phrase“consisting essentially of refers to the genera or species of active pharmaceutical agents recited in a method or composition, and further can include other agents that, on their own do not have substantial activity for the recited indication or purpose. The terms“subject,”“individual,” and“patient” interchangeably refer to a mammal, preferably a human or a non-human primate, but also domesticated mammals ( e.g ., canine or feline), laboratory mammals (e.g., mouse, rat, rabbit, hamster, guinea pig) and agricultural mammals (e.g, equine, bovine, porcine, ovine). In various embodiments, the subject can be a human (e.g, adult male, adult female, adolescent male, adolescent female, male child, female child).

The terms“gene transfer” or“gene delivery” refer to methods or systems for reliably inserting foreign DNA into host cells. 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 transferred genetic material into the genomic DNA of host cells.

The term“vector” is used herein to refer to a nucleic acid molecule capable transferring or transporting another nucleic acid molecule. The transferred nucleic acid is generally linked to, e.g, inserted into, the vector nucleic acid molecule. A vector may include sequences that direct autonomous replication or reverse transcription in a cell, or may include sequences sufficient to allow integration into host cell DNA.“vectors” include gene therapy vectors. As used herein, the term“gene therapy vector” refers to a vector capable of use in performing gene therapy, e.g, delivering a polynucleotide sequence encoding a therapeutic polypeptide to a subject. Gene therapy vectors may comprise a nucleic acid molecule (“transgene”) encoding a therapeutically active polypeptide, e.g, a LAMP-2B or other gene useful for replacement gene therapy when introduced into a subject. Useful vectors include, but are not limited to, viral vectors.

As used herein, the term“expression cassette” refers to a DNA segment that is capable in an appropriate setting of driving the expression of a polynucleotide (a“transgene”) encoding a therapeutically active polypeptide (e.g, LAMP-2B) that is incorporated in said expression cassette. When introduced into a host cell, an expression cassette inter alia is capable of directing the cell’s machinery to transcribe the transgene into RNA, which is then usually further processed and finally translated into the therapeutically active polypeptide.

The expression cassette can be comprised in a gene therapy vector. Generally, the term expression cassette excludes polynucleotide sequences 5' to the 5' ITR and 3' to the 3' ITR. All patents, patent publications, and other publications referenced and identified in the present specification are individually and expressly incorporated herein by reference in their entirety for all purposes.

EXAMPLES EXAMPLE 1: Enhanced Gene Expression using Lamp- 2 B Transgene Variants

A gene expression cassette depicted in FIG. 2 was constructed in a plasmid-based green fluorescence protein (GFP) reporter system for LAMP-2B transgene expression. The plasmid included a single open reading frame encoding the LAMP-2B transgene, a 2A peptide, and an enhanced green fluorescence protein (eGFP). Post-translational self-cleavage of the 2A peptide resulted in co-expression of LAMP-2B and eGFP in equimolar amounts. A wild-type LAMP-2B coding sequence (SEQ ID NO: 2) and three codon variants of the LAMP-2B coding sequence (codon variants 1, 2 and 3; SEQ ID NOs: 3-5, respectively) were tested as the transgenes. The three codon variants contained a reduced number of CpGs, removal of cryptic sites, and a reduced number of open reading frames as compared to the wild-type LAMP-2B coding sequence.

Forty wells of a CellBIND 96-well plate (NUNC #3300) were coated with 0.1% gelatin in water (Millipore ES-006-B) for 1 hour at room temperature. Approximately 88,000 induced pluripotent stem cell (iPSC)-derived cardiomyocytes (VWR MSPP-CMC 10001) were plated into each well in plating media (VWR #Ml00l) at 37°C and 5% carbon dioxide (CO2). After 4 hours, the media was changed to maintenance media (VWR #Ml003) that was pre-equilibrated to 37°C and 5% CO2. A transfection mixture was prepared by adding 6 pL of transfection reagent (ViaFect Promega #E4982) to 128 pL of 0.015 pg/pL plasmid (wildtype or codon variants 1, 2, or 3) in OptiMEM or OptiMEM + ViaFect only (negative control) and incubated for 10-20 min. 100 pL of this transfection mixture was added to 1 mL of maintenance media that was pre-equilibrated to 37°C and 5% CO2.

Approximately 28 hours after initial plating, 100 pL of this transfection mixture in maintenance media was added to each well. Approximately 48 hours after adding media with transfection mixture, the cells were imaged and analyzed on an automated confocal microscope (Perkin Elmer Operetta CLS, Harmony version 4.5 software) for GFP positive cells (FIG. 3) and their average fluorescent intensity (FIG. 4). Immunofluorescence images of the cells two or seven days after transfection are shown in FIG. 5 and FIG. 6, respectively.

FIG. 3 shows that the use of codon variants 1 (“CO 1”) or 2 (“CO 2”) resulted in expression of GFP in a significantly higher number of cells (~ 9-fold higher) as compared to the wild-type transgene. Similarly, FIG. 4 showed mean GFP intensity in cells transfected with codon variants 1 (“CO 1”) and 2 (“CO 2”) to be 1.5-fold higher than wild-type.

EXAMPLE 2: Optimized LAMP-2B Gene Therapy Vectors

Optimized AAV gene therapy vectors are produced by inserting the LAMP-2B optimized variant, COl sequence described in Example 1 into the expression cassette of a recombinant AAV vector. The AAV regulatory cassette is modified by removal of upstream cryptic ATG sequence, use of an optimized consensus Kozak sequence, and/or a full-length polyadenylation sequence. The vectors are tested in comparison to control recombinant AAV vectors containing one or more additional ATG sites upstream of the transgene, a non- optimal Kozak sequence, and/or a non-full-length polyadenylation sequence. Vectors are tested in vitro in Danon patient iPSC-derived cardiomyocytes and in a EAMR-2 knockout mouse model of Danon disease. The optimized AAV gene therapy cassettes and vectors are expected to result in a higher level of expression and/or expression in a higher percentage of cells as compared to the control recombinant AAV vectors.

EXAMPLE 3: In Vitro Evaluation of AAV9-LAMP-2B.vl.2 AAV gene therapy cassette and vector were produced by inserting the LAMP-2B variant sequence COl (SEQ ID NO: 3) into a recombinant AAV plasmid vector having no cryptic start sites upstream of the transgene, an optimized consensus Kozak sequence, and a full-length polyadenylation (poly A) sequence from rabbit globin (“LAMP-2B.V.1.2”;

expression cassette: SEQ ID NO: 8). LAMP-2B.vl.2 was compared to LAMP-2B vl.0, which is the regulatory cassette having a wild-type LAMP-2B transgene (transgene sequence: SEQ ID NO: 2) without an optimal Kozak sequence and a mini-poly A.

HEK293 cells were used to generate viral particles with three-plasmid, helper virus- free system was used to generate recombinant AAV particles containing serotype 9 capsid proteins and viral genomes that have AAV2 ITRs flanking the LAMP-2B expression cassette. The expression cassette contains the human codon-optimized LAMP-2B coding sequence (vl.2 or vl.0) driven by an upstream chimeric“CAG” promoter containing the CMV IE enhancer (CMV IE), the chicken b-actin (CBA) promoter, and a CBA intron splice donor (FIG. 1A). The expression cassette also includes a downstream WPRE element and is terminated by the rabbit beta-globin polyadenylation signal (RGpA). The HEK293 cells were transiently transfected with the LAMP-2B.vl.2 plasmid vector or the LAMP-2B.vl.O plasmid vector, a pAAV2/9 packaging plasmid, and pAd-Helper adenovirus helper plasmid.

CHO-Lec2 cells were seeded in a 24 well plate at 1.2 ^c 10 ⁵ cells/mL in MEM-a containing 10% FBS and 1% Normocin. The following day, CHO-Lec2 cells were transduced in serum-free MEMa medium with either AAV9-LAMP-2B.vl.O, AAV9-LAMP-2B.vl.2, or the same vector having GFP in place of the LAMP-2B transgene (at MOI of 3 ^c 10 ⁵ ). Seven days post-transduction, lysates were harvested using the Mammalian Cell Lysis kit (Sigma) and total protein was quantified using the MicroBCA kit per manufacturer’s instructions. Proteins were separated by SDS-PAGE, transferred onto nitrocellulose membranes and immunoblotted for LAMP-2B (1 :500) and GAPDH (1 : 10,000). CHO-Lec2 cells transduced with AAV9-optimized LAMP -2B .vl.2 showed increased expression compared to CHO-Lec2 cells transduced with the original AAV9-wild-type LAMP-2B.vl.O (FIG. 7A). LAMP-2B was not detected in cells transduced with AAV9-GFP vector alone (FIG. 7A).

LAMP-2B expression was also quantitated in cell lysates by ELISA. Briefly, a 96 well plate was coated with anti-LAMP-2B antibody (clone: H4B4), lysates were added to the wells, and detection was performed using anti-LAMP-2B polyclonal antibody (1 :500,

Thermo Fisher PA; 5-24575) followed by incubation with HRP-conjugated anti-rabbit antibody (1 :3000, Sigma). Transduction with AAV9-optimized LAMP-2B.vl.2 vector resulted in an approximately 7-fold increase in LAMP-2B expression compared to cells transduced with the AAV9-wild-type LAMP-2B.vl.O (FIG. 7B).

Cardiomyocytes were derived from iPSCs generated from individuals with Danon disease. Following rhythmic contraction and selection for purity, Danon disease

cardiomyocytes were transduced with various viral genome copies (vg) of AAV9-Luc (negative control), AAV9-wild-type LAMP-2B.vl.O or AAV9-optimized LAMP-2B.vl.2.

Ten days post-transduction, transduced cardiomyocytes were fixed with 4% paraformaldehyde, permeabilized, blocked for 30 min in 5% IgG-free BSA and incubated for 1 hour with either mouse anti-human LAMP-2B antibody (1 :25, clone: H4B4) or rabbit anti- a-actinin antibody (1 :200, #A78l 1, Sigma). Cells were washed with IX PBS to remove residual unbound primary antibody and then subjected to the appropriate anti -mouse

AlexaFluor tagged secondary antibody and 200 ng/mL DAPI for 60 minutes at room temperature. The wells were then washed with PBS prior to imaging. Human LAMP-2B expression was expressed at a higher level in cardiomyocytes transduced with low titer (l.56x l0 ⁸ vg/well) AAV9-optimized LAMP-2B.vl.2 vector compared to cardiomyocytes transduced with the highest titer (8.45 x lO ¹⁰ vg/well) of AAV9-wild-type LAMP-2B.vl.O (FIG. 8A and FIG. 8B)

Western blot analyses were performed on the transduced Danon disease

cardiomyocytes. AAV9-optimized LAMP-2B.vl.2 at 0.983 x lO ⁹ vg/well showed significant expression of LAMP-2B protein compared to no detection of LAMP-2B protein in cells transduced with either AAV9-wild-type LAM2B.vl.O (l.347x l0 ⁹ vg/well) or AAV9-Luc (1.167 ^c 10 ⁹ vg/well) vectors (FIG. 8C). Collectively, these results demonstrate that the optimized AAV9-LAMP-2B.vl.2 vector mediates human LAMP-2B expression in Danon disease cardiomyocytes at a significantly higher level than the original AAV9-wild-type LAMP-2B.vl.O vector.

EXAMPLE 4: In Vivo Evaluation of AA V9-LAMP-2B. vl.2 in Mouse Model of Danon Disease

LAMP-2-deficient mice were intravenously injected with 1 x 10 ¹³ vg/kg of AAV9 viral vectors containing original human LAMP-2B (AAV9-LAMP-2B.vl.O), optimized human LAMP-2B (AAV9-LAMP-2B.vl.2, codon variant 1 - SEQ ID NO: 3) or vehicle alone. Six weeks post-treatment, mice were sacrificed and heart tissue was collected for analysis of LAMP-2B expression.

Methods

For quantitative analyses of vector copy number, total DNA was isolated from frozen tissues using the DNeasy Blood and Tissue kit according to manufacturer’s guidelines. DNA concentration and integrity was assessed spectrophotometrically. qPCR was performed to calculate viral genome copies per pg of DNA using TaqPath ProAmp Master Mix (Applied Biosystems) with forward (5'-ATCATGCTATTGCTTCCCGTA-3'; SEQ ID NO: 36) and reverse (5'-GGGCCACAACTCCTCATAAA-3'; SEQ ID NO: 37) primers and a probe (5'- CCTCCTTGTATAAATCCTGGTTGCTGTCT-3'; SEQ ID NO: 38) for the WPRE gene. RNase P was used as an endogenous control (Thermofisher, #4403328). A standard curve was generated using a linearized plasmid that contained the vector genome (WPRE) used for virus production. Quantification of DNA per sample was calculated using TaqMan copy number reference assay and was represented as vector copy number per diploid nucleus (VCN/Diploid Nucleus).

RNA was extracted and purified from heart using RNeasy Fibrous Tissue Mini kit according to the manufacturer’s protocol. RNA concentration and integrity were assessed spectrophotometrically. RNA was reverse-transcribed using iScript cDNA Synthesis kit and cDNA was used as a template for quantitative real-time (qRT)-PCR. qRT-PCR was performed on cDNA using TaqPath ProAmp Master Mix with forward (5'- ATCATGCTATTGCTTCCCGTA-3 SEQ ID NO: 36) and reverse (5'- GGGCCACAACTCCTC AT AAA-3 SEQ ID NO: 37) primers and a probe (5'- CCTCCTTGTATAAATCCTGGTTGCTGTCT-3'; SEQ ID NO: 38) for the WPRE gene.

For protein extraction, tissues were flash-frozen and pulverized, and the subsequent tissue powder was digested in protein lysis buffer (100 mM Tris, 300 mM NaCl, 20 mM EDTA, 2% NP-40, 0.2% SDS) containing protease and phosphatase inhibitor cocktails.

Partial protein lysates were passed through a glass tissue grinder and sonicated with 3 bursts of 5 second on ice, with 10 seconds intervals in between at 30 amplitude microns power. Samples were centrifuged for 15 min at 12000 rpm and then the supernatant was collected. Concentration of protein in samples was determined by Lowry assay. Proteins (20 pg/sample) were separated using 10-20% SDS-PAGE and transferred to PVDF membranes by rapid dry transfer technique. Membranes were then blocked in 5% milk (non-fat dry milk solubilized in PBS containing 0.1% Tween-20) for lh, and incubated with anti-human LAMP-2B (1 : 100, H4B4), anti-mouse LAMP-2B (1 : 100) or anti-GAPDH (1 : 1000, #32233, Santa Cruz) antibodies overnight at 4°C. Membranes were washed and then incubated with the appropriate HRP-conjugated secondary antibodies (1 : 10,000) for 1 hour at room temperature. The blots were developed using WesternBright™ Sirius substrates followed by imaging on a BioRad gel imager. For immunofluorescence analyses, tissues were cryoprotected in 30% sucrose/PBS at 4°C, embedded in optimal cutting temperature (OCT) mounting media and then tissue was cut to 8-10 pm thickness on a standard cryotome. Cryosections were then fixed with 4% PFA for 5 min, permeabilized with 0.2% Triton-X for 5 min and blocked with 1% BSA, 3% serum, 1% cold water fish gelatin in PBS for 30 minutes. The sections were incubated with mouse anti-human LAMP-2B antibody (1 :50, H4B4) directly conjugated to Alexa Fluor 647 and rabbit anti-dystrophin antibody overnight at 4° C. The slides were then incubated with anti-rabbit Alexa Fluor 488 secondary antibody and DAPI (1 :2000, #D9542, Sigma) for 30 min at room temperature. Slides were then imaged using an Olympus FluoView F VI 000 confocal microscope. Scan speed, off set, voltage, and gain were kept constant during the acquisition of all images on a given day.

Results

Quantitative PCR was performed on cardiac tissue of AAV9-treated LAMP-2- deficient mice. Although similar viral copy numbers were observed in cardiac tissue of mice treated with wild-type and optimized LAMP-2B containing vector (FIG. 9A), transcription of AAV9-optimized LAMP-2B.vl.2 was increased nearly 7-fold compared to the AAV9-wild- type LAMP-2B.vl.O (FIG. 9B). Despite similar transduction of vl.O and vl.2 viral vectors in cardiac tissue, induction of human LAMP-2B mRNA expression was significantly enhanced using vl.2.

LAMP-2-deficient mice intravenously injected with AAV9-optimized LAMP-2B.vl .2 vector also showed significantly higher levels of human LAMP-2B protein in cardiac tissue compared to LAMP2-deficient mice treated with AAV9-wild-type LAMP-2B.vl.O or the vehicle control (FIG. 9C). Similar results were achieved with immunofluorescence staining: human LAMP-2B was highly induced in cardiac tissue of LAMP-2-deficient mice treated with AAV9-optimized LAMP-2B.vl .2 (FIG.9D). Collectively, these data show that viral transduction using the AAV9-optimized LAMP-2B.vl.2 vector leads to increased expression of human LAMP-2B protein in cardiac tissue in vivo compared to AAV9-wild-type LAMP- 2B.vl.O at the same dose. EXAMPLE 4: In Vivo Evaluation of AAV9-LAMP-2B.vl.2 in Non-Human Primates

Non-human primates were intravenously injected with 1 x 10 ¹³ vg/kg of either the AAV9 viral vector containing codon variant LAMP-2B (vl.2, codon variant 1 - SEQ ID NO: 3) described in Example 2, or vehicle control. Eight weeks post-treatment, the non human primates were humanely sacrificed, and heart, muscle, liver and brain tissue was collected for analysis of LAMP-2B expression.

Methods

For quantitative analyses of vector copy number, total DNA was isolated from frozen tissues using the Qiagen DNeasy kit according to manufacturer’s guidelines. DNA

concentration and integrity were assessed spectrophotometrically. Quantitative PCR on isolated DNA was performed using TaqMan ETniversal Master Mix II (Applied Biosystems) with forward (5'-ATCATGCTATTGCTTCCCGTA-3'; SEQ ID NO: 36) and reverse (5'- GGGCCACAACTCCTC AT AAA-3 SEQ ID NO: 37) primers and a probe (5'- CCTCCTTGTATAAATCCTGGTTGCTGTCT-3'; SEQ ID NO: 38) for the WPRE gene. RNaseP was used as an endogenous control (#4403328, ThermoFisher). A standard curve was generated using a linearized plasmid that contained the vector genome used for virus production. Quantification of DNA per sample was calculated using the TaqMan copy number reference assay and was represented as vector copy number per diploid nucleus (VCN/Diploid Nucleus).

RNA was extracted and purified from heart and skeletal muscle using the RNeasy Fibrous Tissue Mini kit (Qiagen) and from liver and brain using the RNeasy Lipid Tissue kit (Qiagen) according to manufacturer’s protocol. RNA concentration and integrity was assessed using the NanoDrop spectrophotometer. RNA was reverse-transcribed using

Superscript IV VILO master mix (ThermoFisher) and cDNA was used as a template for quantitative real-time (qRT)-PCR. qRT-PCR was performed on cDNA using TaqMan Universal Master Mix II with forward (5'-ATCATGCTATTGCTTCCCGTA-3'; SEQ ID NO: 36) and reverse (5 '-GGGCC AC AACTCCTC AT AAA-3 SEQ ID NO: 37) and a probe (5'- CCTCCTTGTATAAATCCTGGTTGCTGTCT-3'; SEQ ID NO: 38) of the WPRE gene. Human HPRT-l was used as an endogenous control. A standard curve was generated using a linearized plasmid that contained the vector genome used for virus production. For semi -quantitative analysis of mRNA using RNAScope technology, cardiac tissue was fixed in 10% neutral buffered formalin, embedded in paraffin and sectioned. Transgene mRNA was detected using WPRE-03 probe (#518628, ACD) with RNAscope 2.5 LS RED. Cells with greater than 1 dot were considered positive and the percentage of positive cells were binned into five categories: 0%, 1-25%, 26-50%, 51-75% or 100%.

For western blot analyses, 125 mg of cardiac tissue was homogenized in 500 pL of lysis buffer using the Next Advance Bullet System. Protein concentration was determined using the BCA kit (ThermoFisher) and proteins (50 pg/sample) were separated using SDS- PAGE and then transferred to nitrocellulose membranes. Membranes were then probed with mouse anti-human LAMP2 (1 : 100), washed and then incubated with HRP-conjugated anti mouse antibody. The blots were developed using ECL substrate and the BioRad ChemiDoc MP system.

For the LAMP-2B ELISA, protein extraction was performed as described above. A plate was coated with mouse anti-LAMP2 antibody (clone: H4B4, #NBP2-222l7, Novus Biologicals), 100 pg of tissue lysate was added to each well, and detection was performed using anti-LAMP2 polyclonal antibody (#AF6228, R&D Systems) followed by incubation with HRP-conjugated donkey anti -goat antibody (#APl80P, Millipore).

Results

Quantitative PCR was performed on various tissues of AAV9-treated primates. Viral copy numbers were increased in heart, muscle and liver tissue of primates injected with AAV9-LAMP-2B.vl.2 vector at l x lO ¹³ vg/kg compared to vehicle control (FIG. 10A). Vector genomes were detected in all cardiac chambers examined, including the left and right ventricles and left and right atriums (FIG. 10B). Vector mRNA was detected at significant levels in the heart, skeletal muscle and liver tissue of treated primates compared to the untreated vehicle control (FIG. 10C and FIG. 10D). In situ RNA analysis showed approximately 25-75% of heart and liver tissue expressing vector mRNA (FIG. 10E and FIG. 10F). These data demonstrate that systemic administration of 1 ^c 10 ¹³ vg/kg AAV9- optimized LAMP-2B.vl.2 to a primate results in efficient transduction and expression in heart tissue in vivo.

Western blot analyses showed that primates systemically treated with LAMP-2B.vl.2 at 1 x 10 ¹³ vg/kg showed increased human LAMP-2B protein in the left and right ventricles and left atrium of the heart compared to an untreated control (FIG. 10G and FIG. 10H). ELISA also showed that human LAMP-2B protein was increased in the left ventricle and atrium of the heart, as well as skeletal muscle tissue of primates treated with AAV9-LAMP- 2B.vl.2 vector compared to an untreated control (FIG. 101 and FIG. 10J). Vector transduction using AAV9.LAMP-2B.vl.2 leads to expression of human LAMP-2B protein in cardiac tissue of primates in vivo.