RU2808459C2 - Gene therapy vectors for treatment of danon's disease - Google Patents

Abstract

FIELD: molecular biology.

SUBSTANCE: gene therapy vector containing an expression cassette that contains a transgene encoding lysosome-associated membrane protein 2B (LAMP-2B) or a functional variant thereof is described, wherein said transgene is codon-optimized for expression in a human host cell, and wherein the sequence of said transgene has at least 95% identity to the sequence selected from SEQ ID NO: 3-5. A pharmaceutical composition for treating or preventing Danon disease, containing a gene therapy vector, is also described. A method of treating or preventing Danon disease or other autophagic disorder in a subject in need thereof, comprising administering to said subject a gene therapy vector or pharmaceutical composition, is presented.

EFFECT: claimed invention expands the arsenal of remedies for Danon's disease.

30 cl, 10 dwg, 4 ex

Description

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application No. 62/697302, filed July 12, 2018, which is incorporated herein by reference in its entirety.

LIST OF SEQUENCES

This application was filed electronically via the EFS-Web system and includes an electronically filed sequence listing in .txt format. The specified .txt file includes a sequence list called “ROPA_011_01WO_SeqList_ST25.txt”, created on July 11, 2019 and having a size of ~62 kilobytes. The sequence listing contained in said .txt file is included as part of this specification and is incorporated herein by reference in its entirety.

TECHNICAL FIELD

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

BACKGROUND OF THE ART

LAMP-2 (lysosome-associated membrane protein 2, also known as CD107b) is a gene that encodes a lysosome-associated membrane glycoprotein. Alternative splicing of this 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, an inherited cardiomyopathy associated with impaired autophagy.

International Patent Application Publication No. WO2017127565A1 discloses that LAMP-2 is overexpressed in human induced pluripotent stem cells (hiPSCs) derived from patients with LAMP-2 mutations, as described in Hashem, et al., Stem Cells. July 2015; 33(7):2343-50, leads to reduced levels of oxidative stress and apoptotic cell death, confirming the importance of LAMP-2B in the pathophysiology of the disease.

There remains a need in the art for gene therapy vectors for LAMP-2. According to the present invention, there are proposed, among other things, such gene therapy vectors, methods of their use, and pharmaceutical compositions.

BRIEF DESCRIPTION OF THE INVENTION

According to the present invention, there are proposed, among other things, improved gene therapy vectors containing a polynucleotide sequence encoding the LAMP-2 polypeptide, methods of their use, and pharmaceutical compositions.

According to one aspect of the present invention, there is provided a gene therapy vector comprising an expression cassette containing a transgene encoding a lysosome-associated membrane protein 2 (LAMP-2) isoform or a functional variant thereof, wherein said transgene is codon-optimized for expression in a human host cell.

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

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

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

In an embodiment, the sequence of said transgene has at least 95% identity to a sequence selected from SEQ ID NO: 3-5.

In an embodiment, the sequence of said transgene is at least 99% identical to the sequence selected from SEQ ID NO: 3-5.

In an embodiment, said transgene comprises a sequence selected from SEQ ID NO: 3-5.

In an embodiment, said transgene has at least 95% sequence identity to SEQ ID NO: 3.

In an embodiment, the sequence of said transgene is at least 99% identical to SEQ ID NO: 3.

In an embodiment, said transgene contains a sequence identical to SEQ ID NO: 3.

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

In an embodiment, said expression cassette comprises a full-length (poly)A sequence operably linked to the transgene, wherein said full-length (poly)A sequence optionally comprises SEQ ID NO: 7.

In an embodiment, said expression cassette does not contain a start site in the 5′ direction from the transgene capable of producing alternative mRNAs.

According to an embodiment, said expression cassette contains, in the direction from the 5′ to the 3′ end, functionally linked: first inverted terminal repeat, enhancer/promoter region, consensus optimal Kozak sequence, transgene, untranslated 3′ region, including full-length (poly)A -sequence, and a second inverted terminal repeat.

In an embodiment, said enhancer/promoter region comprises, in a 5′ to 3′ direction, a cytomegalovirus immediate-early (CMV IE) enhancer and a chicken beta-actin promoter.

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

In an embodiment, the sequence of said expression cassette is identical to the sequence selected from SEQ ID NO: 8-10.

According to a second aspect of the present invention, there is provided a method of preventing, mitigating, ameliorating, reducing, inhibiting, eliminating and/or reversing one or more symptoms of Danon's disease or other autophagic disorder in a subject in need thereof, comprising administering to said subject any gene therapy vector as described herein.

In an embodiment, the vector is administered by a route selected from the group consisting of intravenous, intraarterial, intracardiac, intracoronary, intramyocardial, intrarenal, intraurethral, epidural, and intramuscular administration.

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

In an embodiment, said subject is a human.

In an embodiment, the subject exhibits symptoms of Danon disease or other autophagic disorder.

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

In an embodiment, the subject has been identified as having a mutated LAMP-2 gene.

According to a third aspect of the present invention, there is provided a pharmaceutical composition for use in preventing, mitigating, ameliorating, reducing, inhibiting, eliminating and/or reversing one or more symptoms of Danon's disease or other autophagic disorder, comprising any gene therapy vector as described herein.

Other features and advantages of the present invention will be apparent from, and are incorporated into, the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

In fig. 1A is a diagram of an exemplary embodiment of a viral vector as described herein.

In fig. 1B is a diagram of an exemplary embodiment of an adeno-associated virus (AAV) gene therapy vector expression cassette.

In fig. 2 shows a plasmid-based green fluorescent protein (GFP) reporter system expression cassette used to test and compare constructs with wild-type LAMP-2B or codon-optimized LAMP-2B.

In fig. 3 is a graph showing the transfection efficiency of LAMP-2B constructs tested using a plasmid-based GFP reporter transfection system and measured as the number of GFP ⁺ cells per well. The wild-type (WT) LAMP-2B construct is compared to three codon-optimized (“CO”) constructs, CO 1 , CO 2 , and CO 3 , with a no-vector control (designated “ViaFect only”).

In fig. Figure 4 is a graph showing the level of gene expression in cells transfected with plasmid-based GFP reporter systems used to test LAMP-2B constructs, measured as the average fluorescence intensity of GFP ⁺ cells in absorbance units (a.u.). Expression of GFP by cells transfected with the wild-type (WT) LAMP-2B construct is compared with GFP expression by three codon-optimized (“CO”) constructs, CO 1 , CO 2 , and CO 3 , or a no-vector control (designated “ViaFect only”) ").
In fig. Figure 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 DNA-free constructs or LAMP-2B constructs expressing wild-type LAMP-2B, CO 1 variant, CO 2 variant, or CO 3 variant.

In fig. Figure 6 shows immunofluorescence images of iPSC-derived cardiomyocytes seven days after transfection with a plasmid-based GFP reporter system. Cells transfected with DNA-free constructs or LAMP-2B constructs expressing wild-type LAMP-2B, CO 1 variant, CO 2 variant, or CO 3 variant.

In fig. 7A shows an immunoblot of human LAMP-2B protein in CHO-Lec2 cells transduced with viral AAV9 vectors containing the wild-type LAMP-2B v1.0 transgene (AAV9 1.0), the optimized LAMP-2B v1.2 transgene (AAV9 1.2), or the transgene GFP (AAV9 GFP). Molecular weight (MW) markers and a control sample of recombinant LAMP-2B protein (LAMP2B (positive control)) were also included.

In fig. 7B shows quantification of LAMP-2B protein using ELISA in CHO-Lec2 cells transduced with AAV9 with wild-type LAMP-2B (v1.0), AAV9 with optimized LAMP-2B (v1.2), or AAV9–GFP (GFP) vectors ).

In fig. Figure 8A shows immunofluorescence images of iPSC-derived cardiomyocytes from a Danon disease patient transduced with the indicated amounts of AAV9–Luc (Luc), AAV9 with wild-type LAMP-2B (LAMP2B v1.0), or AAV9 with optimized LAMP-2B (LAMP2B v1.2) vectors. .

In fig. 8B shows quantification of human LAMP-2B immunofluorescence in Danon disease patient iPSC-derived cardiomyocytes transduced with AAV9–Luc, AAV9 with wild-type LAMP-2B (v1.0), or AAV9 with optimized LAMP-2B (v1.2) vectors. .

In fig. Figure 8C shows an immunoblot of human LAMP-2B protein in cardiomyocytes derived from iPSCs from a Danon disease patient transduced with AAV9–Luc, AAV9 with wild-type LAMP-2B (v1.0), or AAV9 with optimized LAMP-2B (v1.2) vectors.

In fig. 9A shows PCR quantification of viral vector DNA in cardiac tissue recovered from LAMP-2-deficient mice treated with AAV9 with wild-type LAMP-2B (v1.0), AAV9 with optimized LAMP-2B (v1.2), or control AAV9 - the basis. Vector copy number was quantified as VCN/diploid nucleus in cardiac tissue. Control wild-type mice that were not injected with vector were included as controls (WT).

In fig. 9B shows quantitative RT-PCR analyzes on transgene mRNA, which were performed using RT-PCR using specific probes for the WPRE element in cardiac tissue isolated from LAMP-2-deficient mice treated with AAV9 with wild-type LAMP-2B (v1 .0), AAV9 with optimized LAMP-2B (v1.2) or control AAV9 - basis (Base). mRNA expression was quantified as the number of vector genomes (veg) per μg of total cellular RNA, using a standard curve to convert copy number to number of vector genomes.

In fig. 9C shows an immunoblot of LAMP-2B protein in cardiac tissue recovered from LAMP-2-deficient mice treated with wild-type AA9-LAMP-2B (v1.0), AAV9 optimized LAMP-2B (v1.2), or control AAV9 − basis (Baseline), or untreated wild-type mice (No Treatment).

In fig. 9D shows immunofluorescence images of human LAMP-2B protein in cardiac tissue extracted from LAMP-2-deficient mice treated with AAV9 with wild-type LAMP-2B (v1.0), AAV9 with optimized LAMP-2B (v1.2), or control AAV9 is the basis.

In fig. 10A shows PCR quantitation of viral vector DNA in heart, muscle, liver and brain tissue recovered from primates treated with AAV9 vector with optimized human LAMP-2B (Treatment) or a backbone control without vector (No Treatment). Individuals are indicated by black or white squares.

In fig. 10B shows PCR quantitation of viral vector DNA in cardiac chambers extracted from primates treated with AAV9 vector with optimized human LAMP-2B (Treatment) or a backbone control without vector (No Treatment). Individuals are designated B059 (male, M), A991 (female, F), and A602 (male, M).

In fig. 10C shows quantitative RT-PCR assays for transgenic mRNA performed using RT-PCR using WPRE element-specific probes in heart, muscle, liver, and brain tissue isolated from primates treated with the LAMP-optimized AAV9 vector. -2B human (Treatment) or vector-free control (no treatment).

In fig. 10D shows quantitative RT-PCR assays for transgenic mRNA in heart chambers extracted from primates injected with AAV9 vector with optimized human LAMP-2B (Treatment) or a backbone control without vector (No Treatment).

In fig. 10E shows the percentage of cells expressing transgenic mRNA in situ in heart, muscle, and liver tissue isolated from primates injected with an AAV9 vector with optimized human LAMP-2B (Treatment) or a backbone control without vector (No Treatment). Individuals are designated B059 (male, M), A991 (female, F), and A602 (male, M).

In fig. 10F shows in situ staining of transgenic mRNA in heart tissue extracted from primates injected with AAV9 vector with optimized human LAMP-2B or a vector-free control (No Treatment). Individuals are designated B059 (male, M), A991 (female, F), and A602 (male, M).

In fig. 10G shows the fold change in LAMP-2B protein levels as determined by Western blotting in heart, muscle, and liver tissue isolated from primates treated with the AAV9 vector with optimized human LAMP-2B or not treated with the vector (No Treatment).

In fig. 10H shows the fold change in LAMP-2B protein levels by Western blotting in cardiac chambers removed from primates treated with the AAV9 vector with optimized human LAMP-2B or not treated with the vector (No Treatment).

In fig. 10I shows the quantification of LAMP-2B protein using ELISA in heart, muscle and liver tissue isolated from primates treated with an AAV9 vector with optimized human LAMP-2B or not treated with the vector (No Treatment).

In fig. 10J shows quantification of LAMP-2B protein using ELISA in heart chambers removed from primates treated with an AAV9 vector with optimized human LAMP-2B or no vector (No Treatment).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides improved polynucleotide sequences, expression cassettes and vectors encoding an isoform of LAMP-2 (eg, LAMP-2B), as well as related pharmaceutical compositions, and their use for the treatment of diseases and disorders associated with LAMP-2 deficiency or mutation. 2. The present inventors have discovered that modifications to the LAMP-2B gene sequence result in increased expression of the transgene. In addition, the presence of specific sequence elements in the expression cassettes of gene therapy vectors encoding LAMP-2B leads to improved transgene expression. Accordingly, LAMP-2 polynucleotide sequences, expression cassettes and vectors as described herein provide advantages for gene therapy over previously known gene therapy vectors, including the ability to achieve higher levels of LAMP-2 expression in therapeutically relevant tissues.

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

1 MVCFRLFPVP GSGLVLVCLV LGAVRSYALE LNLTDSENAT CLYAKWQMNF TVRYETTNKT

61 YKTVTISDHG TVTYNGSICG DDQNGPKIAV QFGPGFSWIA NFTKAASTYS IDSVSFSYNT

121 GDNTTFPDAE DKGILTVDEL LAIRIPLNDL FRCNSLSTLE KNDVVQHYWD VLVQAFVQNG

181 TVSTNEFLCD KDKTSTVAPT IHTTVPSPTT TPTPKEKPEA GTYSVNNGND TCLLATMGLQ

241 LNITQDKVAS VININPNTTH STGSCRSHTA LLRLNSSTIK YLDFVFAVKN ENRFYLKEVN

301 ISMYLVNGSV 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 GTTCTTTGTAC 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 TTGTCTTTTGC 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).

The present invention provides modified polynucleotide sequences encoding an isoform of lysosome-associated membrane protein 2 (LAMP-2) or a functional variant thereof. In some embodiments, the modified polynucleotide sequences comprise one or more of the following modifications relative to the wild-type polynucleotide encoding the LAMP-2 isoform: 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. Embodiments of the present invention provide a polynucleotide sequence or transgene encoding LAMP-2B or a functional variant thereof and containing one or more nucleotide substitutions relative to SEQ ID NO:2. In embodiments, the sequence of said transgene is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, 99% identical or completely identical to the sequence selected from SEQ ID NO: 3-5. The present invention provides at least three exemplary transgene sequence variants encoding LAMP-2B (SEQ ID NO: 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 GCCAGAGCT

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 CTACACACA

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

In some embodiments, the transgene is similar or identical to a subsequence from any of SEQ ID NOs: 3-5. In some embodiments, the transgene comprises a subsequence from any of SEQ ID NOs: 3-5. In various embodiments, the subsequence may comprise any set of consecutive nucleotides (NT) in a complete sequence of at least about 50 NT, at least about 100 NT, at least about 150 NT, at least about 250 NT, about 200 HT, at least about 350 HT, at least about 450 HT, at least about 400 HT, at least about 450 HT, at least about 550 HT, at least about 600 HT, 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 HT, at least about 950 HT or at least about 1000 HT.

In some embodiments, the sequence of said transgene has at least 95% identity to a subsequence that contains nucleotides 1-500, 250-750, 500-1000, or 750-1240 from any of SEQ ID NO: 3-5. In an embodiment, the sequence of said transgene is at least 99% identical to a subsequence that contains nucleotides 1-500, 250-750, 500-1000, or 750-1240 from any of SEQ ID NOs: 3-5. In an embodiment, said transgene comprises a sequence that comprises nucleotides 1-500, 250-750, 500-1000, or 750-1240 of any of SEQ ID NO: 3-5. In an embodiment, the sequence of said transgene is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, 99% identical or completely identical to the subsequence that contains nucleotides 1-500, 250-750, 500-1000 or 750-1240 from any of SEQ ID NO: 3-5. In embodiments, the sequence of said transgene is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, 99% identical or completely identical to the subsequence that contains nucleotides 1-500, 250-750, 500-1000, or 750-1240 of SEQ ID NO: 3. In an embodiment, the sequence of the specified transgene is at least 85%, 86% , 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical or completely identical to the subsequence that contains nucleotides 1– 500, 250-750, 500-1000 or 750-1240 from SEQ ID NO: 3.

In some 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. Accordingly, in specific embodiments, said expression cassette is an optimized polynucleotide sequence that encodes any of LAMP-2A, LAMP-2B, or LAMP-2C, or a functional fragment or variant thereof, and contains one or more modifications relative to the corresponding polynucleotide sequence wild type, including one or more of the following modifications: codon optimization of the transgene sequence encoding LAMP-2A, LAMP-2B or LAMP-2C; said expression cassette or transgene sequence contains fewer CpG sites than the corresponding wild-type sequence; said expression cassette or transgene sequence contains fewer CpG sites than the corresponding wild-type sequence; said expression cassette or transgene sequence contains fewer cryptic splice sites than the corresponding wild-type sequence; and/or said expression cassette or transgene sequence contains fewer open reading frames than the corresponding wild-type sequence. In specific 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 shown in SEQ ID NO: 29 and 30, respectively. The wild-type sequences of human LAMP-2A and LAMP-2C proteins are given in SEQ ID NO: 34 and 35, respectively. Sequences of wild-type LAMP-2 isoforms and coding sequences are also publicly available. Although specific embodiments are described herein in relation to LAMP-2B, it should be understood that any embodiment may alternatively use LAMP-2A or LAMP-2C.

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) at least throughout nucleotides 1–1080. Accordingly, those skilled in the art will appreciate that transgenes, expression cassettes, and vectors as described herein can be adapted to express the indicated LAMP-2 isoforms by substituting the 3′ end (nucleotide 1081 to the last nucleotide) of LAMP-2. 2A (SEQ ID NO: 29) or from wild-type LAMP-2C (SEQ ID NO: 30) nucleotides 1081–1233 of LAMP-2B (e.g., optimized LAMP-2B presented in any of SEQ ID NO: 3–5). For example, embodiments of the present invention use nucleotides 1-1080 of the optimized LAMP-2B gene sequences, SEQ ID NO: 3-5, which are respectively represented by SEQ ID NO: 31-33.

In an embodiment, the sequence of said transgene has at least 95% identity to a sequence selected from SEQ ID NOs: 31-33. In an embodiment, the sequence of said transgene is at least 99% identical to the sequence selected from SEQ ID NOs: 31-33. In an embodiment, said transgene comprises a sequence selected from SEQ ID NO: 31-33. In an embodiment, the sequence of said transgene is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, 99% identical or completely identical to SEQ ID NO: 31. In an embodiment, the sequence of said transgene is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98%, 99% identical or completely identical to SEQ ID NO: 32. In an embodiment, the sequence of said transgene is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical or completely identical to SEQ ID NO: 33. In some cases, the specified transgene is characterized by a polynucleotide sequence , different from the polynucleotide sequence of the reference sequence, for example, the “natural” or “wild type” LAMP-2B sequence. In some embodiments, the sequence of said transgene is 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% identical to the reference sequence. In some embodiments, the reference sequence is SEQ ID NO: 2. For example, SEQ ID NO: 3 is 78.5% identical to SEQ ID NO: 2.

In some cases, the transgene is characterized by a polynucleotide sequence different from the polynucleotide sequence of the reference sequence, for example, the "natural" or "wild type" LAMP-2A sequence. In some embodiments, the sequence of said transgene is 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% identical to the reference sequence. In some embodiments, the reference sequence is the wild-type human LAMP-2A sequence from SEQ ID NO: 29.

In some cases, the transgene is characterized by a polynucleotide sequence different from the reference polynucleotide sequence, for example, the "natural" or "wild type" LAMP-2C sequence. In some embodiments, the sequence of said transgene is 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% identical to the reference sequence. In some embodiments, the reference sequence is the wild-type human LAMP-2A sequence from SEQ ID NO: 30.

In an embodiment, the transgene is codon-optimized for expression in a human host cell. In an embodiment, the coding sequence of the transgene is modified or "codon-optimized" to enhance expression by replacing infrequently presented codons with more frequently presented codons. The coding sequence is the part of the mRNA sequence that codes for amino acids for translation. During translation, each of the 61 trinucleotide codons is translated into one of 20 amino acids, which leads to degeneracy, or redundancy, of the genetic code. However, different cell types and different animal species use tRNAs (each carrying an anticodon) encoding the same amino acids at different frequencies. If a gene sequence contains codons that are rarely present in the corresponding tRNA, the ribosomal translation machinery may operate more slowly, making efficient translation difficult. Expression can be improved by species-specific “codon optimization,” where the coding sequence is changed to encode the same protein sequence, but using codons that are frequently found and/or used in 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 rarely expressed in a mammal or primate with codons commonly expressed in primates. For example, in some embodiments, the transgene encodes a polypeptide that is at least 85% sequence identical to a reference polypeptide ( e.g. , wild type LAMP-2B; SEQ ID NO: 3)—e.g., at least 90% identical, possesses at least 95% identical to, at least 98% identical to, or at least 99% identical to the sequence of a reference polypeptide - and at least one codon of the specified coding sequence occurs in human tRNA with a higher frequency than the corresponding codon in sequence described above or in the text of this document.

In an embodiment, the transgene contains fewer alternative open reading frames than SEQ ID: 2. In an embodiment, the transgene is modified to increase expression by terminating or removing open reading frames (ORFs) that do not encode the desired transgene. An open reading frame (ORF) is a nucleic acid sequence located after a start codon and does not contain a stop codon. ORFs may be in forward or reverse orientation, and may be in-frame or out-of-frame with respect to the gene of interest. Such open reading frames could potentially be expressed in an expression cassette along with the gene of interest and could cause unwanted side effects. In some embodiments, the transgene is modified to remove open reading frames by further changing codon usage. This may be accomplished by eliminating one or more start codons (ATG) and/or introducing one or more stop codons (TAG, TAA or TGA) in the reverse orientation or out of frame with respect to the desired ORF, while maintaining the encoded amino acid sequence and, optionally, , maintaining frequently used codons in the gene of interest ( i.e. avoiding codons occurring at <20% frequency).

In some embodiments, said expression cassette contains a maximum of one, a maximum of two, a maximum of three, a maximum of four, or a maximum of five start codons in the 5′ direction relative to the start codon of the transgene. In some embodiments, said expression cassette does not contain a start codon in the 5′ direction relative to the start codon of said transgene. In some embodiments, one or more ATG codons in the 5′UTR, promoter, enhancer, promoter/enhancer element, or other sequences located 5′ to the start codon of the transgene are retained after removal of one or more cryptic start sites. In some embodiments, the expression cassette does not contain hidden start sites 5' of the transgene that can produce erroneous mRNAs.

In embodiments of the present invention, the coding sequence of a transgene can be optimized either by codon optimization, or by removal of non-transgenic ORFs, or by both techniques. In some cases, non-transgenic ORFs are removed or minimized after codon optimization to remove ORFs introduced during codon optimization.

In an embodiment, the transgene contains fewer CpG sites than SEQ ID: 2. Without being bound by any theory, the presence of CpG sites in a polynucleotide sequence is believed to be associated with undesirable host immunological responses to a viral vector containing the polynucleotide sequence. In some embodiments, the transgene is designed such that the number of CpG sites is reduced. Examples of methods are provided in published US patent application US20020065236A1.

In an embodiment, the transgene contains fewer cryptic splice sites than SEQ ID: 2. GeneArt® software may be used for optimization, for example, to increase GC content and/or remove cryptic splice sites to avoid transcriptional silencing and thus increase transgene expression. Alternatively, any optimization method known in the art may be used. Removal of hidden splice sites is described, for example, in published international patent application WO2004015106A1.

The present invention also provides expression cassettes and gene therapy vectors encoding LAMP-2B. In some embodiments, the expression cassettes and gene therapy vectors comprise a codon-optimized LAMP-2B polynucleotide sequence, or a variant of a LAMP-2B polynucleotide sequence, or a transgene sequence as described herein.

In specific embodiments, the expression cassette or gene therapy vector encoding LAMP-2B comprises: a consensus optimal Kozak sequence, a full-length polyadenylation ((poly)A) sequence (or replacement of a full-length (poly)A with a truncated (poly)A), and minimal or no upstream ( i.e. 5′) or hidden start codons ( i.e. ATG sites). In some embodiments, the expression cassette does not contain a start site 5′ of the transgene capable of producing alternative mRNAs. In some embodiments, the expression cassette or gene therapy vector comprises a LAMP-2B coding sequence, such as a codon optimized sequence or polynucleotide variant of LAMP-2B or a transgene sequence as described herein.

In some cases, said expression cassette comprises two or more first inverted terminal repeats, an enhancer/promoter region, a consensus optimal Kozak sequence, a transgene ( eg, a transgene encoding LAMP-2B as described herein), a 3′ untranslated region including a full-length (poly)A sequence, and a second inverted terminal repeat. In some embodiments, one or both inverted terminal repeats (ITRs) is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, or AAV9 ITR, or any ITR known in the art. In some embodiments, the expression cassette contains exactly two ITRs. In some embodiments, both ITRs are ITRs AAV2, AAV5, or AAV9. In some embodiments, both ITRs are AAV2 ITRs.

In an embodiment, said expression cassette contains a Kozak sequence operably linked to the transgene. In an embodiment, said 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 operably linked to the transgene. In an embodiment, said Kozak sequence is an alternative Kozak sequence containing or consisting of any of the 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 does not contain a Kozak sequence.

In SEQ ID NO: 14 lowercase letters indicate the most common bases in positions where other base variants are, however, possible; capital letters indicate highly conserved bases; designated adenine or guanine. In SEQ ID NO: 14, the sequence in parentheses (gcc) is optional. In SEQ ID NO: 15-17, "N" denotes any base.

Instead of the specified consensus optimal Kozak sequence, various sequences may be used as the translation initiation site, and other sequences will be able to be identified and tested by those skilled in the art. See Kozak M. An analysis of vertebrate mRNA sequences: intimations of translational control. J Cell Biol. 115(4):887–903 (1991).

In an embodiment, said expression cassette contains a full-length (poly)A sequence operably linked to the transgene. In an embodiment, said full-length (poly)A 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 (poly)A sequences may be used in the expression cassettes herein, including, but not limited to, bovine growth hormone polyadenylation (bGHpA) signal (SEQ ID NO: 19), 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 CTGCCCCGGGT GGCATCCCTG TGACCCCTCC CCAGTGCCTC TCCTGGCCCT GGAAGTTGCC

61 ACTCCAGTGC CCACCAGCCT TGTCCTAATA AAATTAAGTT GCATCATTTTT 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 (poly)A sequence. In certain embodiments, the active fragment of said (poly)A sequence contains or consists of less than 20 base pairs (bp), less than 50 bp, less than 100 bp. or less than 150 bp, for example, from any of the (poly)A sequences as described herein.

In some cases, expression of the transgene is increased to ensure that the expression cassette is free of competing ORFs. In an embodiment, said expression cassette does not contain a start codon within 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 base pairs in the 5′ direction from the start codon of the transgene. In a certain embodiment, said expression cassette does not contain a start codon 5′ from the start codon of the transgene. In some embodiments, the expression cassette does not contain a start site 5′ from the transgene capable of producing alternative mRNAs.

According to an embodiment, said expression cassette contains, in the direction from the 5′ to the 3′ end, functionally linked: first inverted terminal repeat, enhancer/promoter region, introns, consensus optimal Kozak sequence, transgene, untranslated 3′ region, including a full-length (poly )A sequence, and a second inverted terminal repeat, wherein said expression cassette does not contain, in the 5′ direction of the transgene, a start site capable of producing alternative mRNAs.

In some embodiments, the enhancer/promoter region comprises, in a 5′ to 3′ direction: a cytomegalovirus mid-early gene enhancer (CMV IE) and a chicken beta-actin promoter. In an embodiment, said 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 a chicken beta-actin gene, and a splice acceptor from a 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 GGTAAAATGGC

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 AGAGGTGCGGG CGGCAGCCAA TCAGAGCGGC GCGCTCCCGAA

601 AGTTTCCTTT TATGGCGAGG CGGCGGCGGC GGCGGCCCTA TAAAAAGCGA AGCCGCGGC

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 common promoter. In some embodiments, said 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, said enhancer/promoter region comprises a polynucleotide that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identical to any of SEQ ID NO: 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 AATTTTTTT ATTTATGCAG

241 AGGCCGAGGC CGCCTCGGCC TCTGAGCTAT TCCAGAAGTA GTGAGGAGGC TTTTTTGGAG

301 GCCTAGGCTT TTGCAAA

(SEQ ID NO: 24);

1 GGGTAGGGGA GGCGCTTTTC CCAAGGCAGT CTGGAGCATG CGCTTTAGCA GCCCCCGCTGG

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 GGGTTGGGGGC 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).

Additional examples of promoters include, but are not limited to, human elongation factor 1-alpha (EFS) promoter, SV40 early promoter, murine mammary cancer virus long terminal repeat (LTR) promoter; adenoviral major late promoter (Ad MLP); herpes simplex virus (HSV) promoter, endogenous cellular promoter heterologous to the gene of interest, cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), Rous sarcoma virus (RSV) promoter, synthetic promoters, hybrid promoters, and etc.

In some embodiments, said 3′-UTR comprises a polynucleotide (WPRE element) that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100 in sequence % identical to 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 AACCCTTGGA 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 a certain embodiment, the sequence of said expression cassette has at least 95% identity to a sequence selected from SEQ ID NOs: 8-10. In an embodiment, the sequence of said expression cassette is completely identical to the sequence selected from SEQ ID NO: 8-10, or at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the sequence selected from SEQ ID NO: 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 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 CGGGAGCCGAA

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 TGAAAAGAAT GATGTGGTGC AACACTATTG

2461 GGACGTCCTG GTGCAAGCCT TTGTGCAGAA TGGGACAGTG AGTACCAACG AGTTCCTCTG

2521 TGACAAGGAC AAGACCAGCA CTGTGGCCCC CACTATCCAC 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 GAATGAGAAC AGGTTCTACC TCAAGGAAGT GAACATTTCC ATGTACCTGG TCAATGGTTC

2881 AGTGTTCTCT ATTGCCAACA ACAATCTGAG CTACTGGGAT GCACCCCTGG GATCCTCCTA

2941 CATGTGCAAC AAGGAGCAGA CTGTGAGTGT GTCAGGTGCT TTTCAGATCA ACACTTTTTGA

3001 CCTGAGGGTG CAGCCCTTCA ATGTGACTCA GGGAAAGTAC TCCACTGCAC AAGAGTGTTC

3061 CTTGGATGAT GACACTATCC TCATCCCCAT TATTGTGGGA GCTGGACTGT CAGGATTGAT

3121 TATAGTGATT GTGATTGCTT ATGTGATTGG AAGGAGAAAG AGCTATGCTG GCTACCAGAC

3181 CCTGTAAAAG GGCGAATTCC AGCACACGCG TCCTAGGAGC TCGAGTACTA CTGGCGGCCG

3241 TTACTAGTGG ATCCGCGGTA CAAGTAAGCA TGCAAGCTTC GAGGACGGGG TGAACTACGC

3301 CTGAATCAAG CTTATCGATA AATTCGAGCA TCTTACCGCC ATTTATTCCC ATATTTTGTTC

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 GACGCCGGG 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 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 CGGGAGCCGAA

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 GGAGAAGAAC GACGTGGTGC AGCACTACTG

2461 GGACGTGCTG GTGCAGGCCT TTGTGCAGAA CGGCACTGTG TCCACCAACG AATTCCTGTG

2521 TGATAAGGAC AAAACTTCCA CTGTGGCACC TACAATTCAC ACTACTGTGC CTTCACCTAC

2581 CACCACTCCA 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 ATATTTTGTTC

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 GACGCCGGG 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 CGGGAGCCGAA

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 AGAGGACAAA GGTATCCTGA CTGTGGATGA GTTGCTGGCA ATCAGGATCC CACTGAACGA

2401 TCTGTTCAGG TGCAACTCAC TGTCCACTCT GGAAAAGAAT 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 CCACAGCACT GGGTCCTGCA GAAGCCACAC

2761 TGCCCTCCTG AGGCTCAACT CATCAACTAT CAAGTACTTG GATTTTGTGT TTGCAGTGAA

2821 GAATGAGAAC AGATTCTACC TCAAAGAGGT CAACATTTCA ATGTACCTGG TGAATGGGAG

2881 TGTGTTCTCC ATTGCTAACA ACAACCTGAG CTACTGGGAT GCCCCTCTGG GCTCCTCATA

2941 CATGTGCAAC AAGGAACAGA CTGTGAGTGT GTCAGGGGCC TTCCAGATCA ACACTTTTTGA

3001 CCTGAGAGTG CAGCCCTTTA ATGTGACACA GGGAAAGTAC AGCACTGCTC AGGAGTGCAG

3061 CCTGGATGAT GACACTATCC TGATCCCTAT CATTGTGGGG GCAGGCCTGT CTGGACTCAT

3121 TATTGTGATT 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 ATATTTTGTTC

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 GACGCCGGG CTTTGCCCGG GCGGCCTCAG TGAGCGAGCG AGCGCGCAG

(SEQ ID NO: 10).

In some embodiments, said expression cassette contains one or more modifications relative to the sequence selected from SEQ ID NOs: 8-10, including, but not limited to, any of the modifications as described herein. In specific embodiments, the one or more modifications include any one or more of: deleting one or more ( e.g., all) 5' ATG sequences, replacing the Kozak sequence with an optimized consensus Kozak sequence, or another Kozak sequence, including , but not limited to, any of those described herein, and/or replacing a polyadenylation sequence with a full-length polyadenylation sequence or another polyadenylation sequence, including, but not limited to, any of those described herein. An exemplary configuration of genetic elements according to these example expression cassettes is shown in FIG. 1B .

In an embodiment, the vector is an adeno-associated virus (AAV) vector. In an embodiment, said 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).

Related embodiments of the present invention provide gene therapy vectors containing an expression cassette as described herein. In general, the gene therapy vectors described herein contain an expression cassette containing a polynucleotide encoding one or more isoforms of lysosome-associated membrane protein 2 (LAMP-2) and allow expression of LAMP-2 to partially or completely correct deficient expression levels LAMP-2 protein and/or autophagic flux in a subject in need thereof ( eg, a subject with Danon disease or another disorder characterized by a deficiency of autophagic flux due at least in part to deficient expression of LAMP-2). In specific embodiments, said expression cassette comprises a polynucleotide sequence encoding LAMP-2 as described herein, for example, SEQ ID NO: 3-5 or a functional variant thereof. In some embodiments, the sequence variant is at least 90%, at least 95%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 3-5. In some embodiments, the embodiment is a fragment of any of SEQ ID NOs: 3-5, for example, a fragment of at least 50%, at least 60%, at least 70%, at least 80% , at least 90% or at least 95% identical to the sequence of any of SEQ ID Nos: 3-5. Said gene therapy vectors may be viral or non-viral vectors. Exemplary non-viral vectors include, for example, deproteinized DNA, cationic liposomal complexes, cationic polymer complexes, cationic liposomal-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 some embodiments, the viral vector is an AAV vector. AAV is a 4.7 kb single-stranded DNA virus. Recombinant AAV-based vectors have been associated with a very good level of clinical safety, since wild-type AAV is not pathogenic and has not been etiologically linked to any known diseases. In addition, AAV allows for highly efficient gene delivery and sustained transgene expression in multiple tissues. By "AAV vector" is meant a vector derived from an adeno-associated virus serotype, including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.10, AAVrh.74, and etc. In AAV vectors, one or more of the wild-type AAV genes, such as the rep and/or cap genes, may be partially or completely deleted while retaining functional flanking inverted terminal repeat (ITR) sequences. Functional ITR sequences are required for AAV virion rescue, replication, and packaging. Accordingly, an AAV vector is defined herein to include at least the required sequences in cis for replication and packaging ( eg, a functional ITR) of the virus. These ITRs need not be wild-type nucleotide sequences and may be altered, for example, by insertion, deletion or nucleotide substitution, as long as the sequences provide functional rescue, replication and packaging. AAV vectors may contain other modifications, including, but not limited to, one or more modified capsid proteins ( eg, VP1, VP2 and/or VP3). For example, the capsid protein may be modified to alter tropism and/or reduce immunogenicity.

Recombinant AAV-based vectors are associated with a very good level of clinical safety, since wild-type AAV is not pathogenic and has not been etiologically linked to any known diseases. In addition, AAV allows for highly efficient gene delivery and sustained transgene expression in multiple tissues. Various serotypes of AAV are known, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.10, AAVrh.74, etc. In AAV vectors, one or more of the wild-type AAV genes, such as the rep and/or cap genes, may be partially or completely deleted while retaining functional flanking inverted terminal repeat (ITR) sequences. The serotype of the recombinant AAV vector is determined by the capsid. International patent publication WO2003042397A2 describes various capsid sequences, including AAV1, AAV2, AAV3, AAV8, AAV9 and rh10 capsids. International patent publication WO2013078316A1 describes the sequence of the VP1 polypeptide from AAVrh74. Many different naturally occurring or genetically modified AAV capsid sequences are known in the art.

A non-limiting example of a capsid is the AAV9 capsid of SEQ ID NO: 28 (or VP1, VP2 or VP3 fragments thereof). In some embodiments, the AAV vectors described herein contain at least 93%, at least 94%, at least 95%, at least 96%, at least 97% capsid proteins, at least 98%, at least 99%, or at least 100% identical to SEQ ID NO: 28 throughout the entire sequence or amino acids 138-736 or 203-736.

1 MAADGYLPDW LEDNLSEGIR EWWALKPGAP QPKANQQHQD NARGLVLPGY KYLGPGNGLD

61 KGEPVNAADA 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 VEFAVNTEGV

721 YSEPRPIGTR YLTRNL

(SEQ ID NO: 28).

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

In some embodiments, the viral vector is an AAV9 vector. In some embodiments, the viral vector expression cassette is flanked by AAV2 inverted terminal repeats (ITRs). ITRs used in alternative embodiments of the proposed 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 term AAV2/9 refers to an AAV vector with ITR repeats from AAV2 and a capsid from AAV9. Other embodiments of the present invention include, but are not limited to, AAV2/9, AAV5/9, AAVrh74, AAV2/rh74, AAV5/9 and AAV5/rh74 vectors. Other ITRs known in the art may be used. Examples of ITRs (and other AAV components) suitable for use in vectors according to the present disclosure include, without limitation, those described in references: US6936466B2, US9169494B2, US20050220766A1, US20190022249A1 and US7282199B2, each of which is incorporated by reference herein in its entirety.

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

Exemplary retroviruses (family Retroviridae) include, but are not limited to, the following: (1) the genus Gammaretrovirus, e.g., Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), murine mammary tumor virus (MuMTV), virus gibbon leukemia virus (GaLV) and feline leukemia virus (FLV), (2) the genus Spumavirus, such as simian foamy virus, (3) the genus Lentivirus, such as human immunodeficiency virus-1 and simian immunodeficiency virus.

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

Retroviral vectors and, more specifically, lentiviral vectors can be used in the practice of the present invention. Accordingly, the term “retroviral vector” as used herein includes “lentiviral vector”; and the term “retrovirus” as used herein includes “lentivirus”.

The term "viral vector" can refer either to a vector or viral particle capable of carrying a nucleic acid into a cell, or to the nucleic acid itself being transferred. Viral vectors contain structural and/or functional genetic elements that are primarily derived from the virus. The term "retroviral vector" refers to a viral vector containing structural and functional genetic elements or parts thereof that are primarily derived from a retrovirus. The term "lentiviral vector" refers to a viral vector containing structural and functional genetic elements or parts thereof, including LTRs, which are primarily derived from a lentivirus. The term “hybrid” refers to a vector, LTR, or other nucleic acid containing both retroviral, eg, lentiviral, and non-lentiviral viral sequences. In one embodiment, a hybrid vector refers to a transfer vector or plasmid containing retroviral, eg, lentiviral sequences for reverse transcription, replication, integration, and/or packaging.

In specific embodiments, the terms “lentiviral vector” and “lentiviral expression vector” may refer to lentiviral transfer plasmids and/or infectious lentiviral particles. When elements such as cloning sites, promoters, regulatory elements, heterologous nucleic acids, and the like are mentioned herein, it is understood that the sequences of such elements are present in the form of RNA in the lentiviral particles of the present invention and in the form of DNA in DNA plasmids according to the present invention.

In certain specific embodiments, most or all of the viral vector backbone sequences are derived from a lentivirus, such as HIV-1. However, it should be understood that many different sources of lentiviral sequences can be used, and numerous substitutions and changes can be included in some lentiviral sequences without impairing the ability of the transfer vector to perform the functions described herein. In addition, various lentiviral vectors are known in the art, see references: Naldini et al. , (1996a, 1996b, 1998); Zufferey et al., (1997); Dull et al. , 1998, US patents No. 6013516; and No. 5994136, many of which can be adapted to produce a viral vector or plasmid for transfer according to the present invention.

The LAMP-2B transgene sequences as described herein are used, according to various embodiments, in any vector system that is known in the art or will be discovered in the future. The present invention is not limited to any particular viral vector described herein, as those skilled in the art are aware of methods for using the transgene sequence in other vector systems without undue experimentation and with a reasonable expected level of success.

Viral gene delivery vectors suitable for use in the practice of the present invention can be constructed using techniques well known in the field of molecular biology. Typically, viral vectors carrying transgenes are assembled from polynucleotides encoding the transgene, suitable regulatory elements, and elements necessary for the production of viral proteins that mediate cell transduction. Such recombinant viruses can be produced using techniques known in the art, for example, by transfection of packaging cells or transient transfection with helper plasmids or viruses. Typical examples of packaging cells for viruses include, but are not limited to, HeLa cells, SF9 cells (optionally with a baculovirus helper vector), 293 cells, and the like. The herpesvirus-based system can be used to produce AAV vectors as described in US20170218395A1. Detailed protocols for the production of such replication-defective recombinant viruses can be found, for example, in the references: W095/14785, W096/22378, US Patent No. 5882877, US Patent No. 6013516, US Patent No. 4861719, US Patent No. 5278056 and W094/19478. the contents of each of which are incorporated herein by reference in their entirety.

The present invention also provides pharmaceutical compositions comprising an expression cassette or vector ( eg, a gene therapy vector) as described herein and one or more pharmaceutically acceptable carriers, diluents or excipients. In specific embodiments, the pharmaceutical composition comprises an AAV vector containing an expression cassette as described herein, for example, containing a codon-optimized transgene encoding LAMP-2B, for example, any of SEQ ID NO: 3-5 and variants thereof. Pharmaceutical compositions are provided, for example, for use in the prevention or treatment of a disorder characterized by insufficient autophagic flux ( e.g., Danon disease), which contain a therapeutically effective amount of an expression cassette or vector as described herein, which contains a polynucleotide nucleic acid sequence, encoding one or more of the LAMP-2 isoforms.

AAV vectors suitable for use in the present invention can be packaged into AAV virions (viral particles) using a variety of systems, including adenovirus-based systems and helper virus-free systems. Standard methods in the field of AAV biology include those described in: Kwon, Schaffer. Pharm Res. (2008) 25(3):489-99; Wu et al. Mol. Ther. (2006) 14(3):316-27. Burger et al. Mol. Ther. (2004) 10(2):302-17; Grimm et al. Curr Gene Ther. (2003) 3(4):281-304; Deyle DR, Russell DW. Curr Opin Mol 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):383-91. Systems without helper viruses included those described in US 6,004,797; US 7588772; and US 7094604;

Pharmaceutical compositions that contain said expression cassette or said vector may be presented in any form that is suitable for the chosen route of administration, for example, intraventricular, intramyocardial, intracoronary, intravenous, intraarterial, intrarenal, intraurethral, epidural or intramuscular administration. A gene therapy vector containing a polynucleotide encoding one or more isoforms of LAMP-2 can be administered to animals and humans as the sole active agent or in combination with other active agents, in a single dosage form, as a mixture with standard pharmaceutical carriers. In some embodiments, the pharmaceutical composition comprises cells transduced ex vivo with any of the gene therapy vectors described herein.

In some embodiments, said viral vector ( eg, an AAV vector) or a pharmaceutical composition containing said vector is effective when administered systemically. For example, viral vectors according to the present description in some cases demonstrate effectiveness when administered intravenously to a subject ( eg, a primate, such as a non-human primate or a human). In some embodiments, the viral vectors described herein are capable of inducing LAMP-2B expression in various tissues when administered systemically ( eg, heart, muscle, and/or lung). In certain embodiments, intravenous administration of an AAV9 vector containing a transgene substantially identical to or identical to SEQ ID NO: 3 to a subject results in detectable expression of LAMP-2B in cardiac tissue. In some embodiments, LAMP-2B expression is detected in one or more, or all, of the left ventricle, right ventricle, left atrium, and right atrium of the heart of the subject. In some embodiments, LAMP-2B expression is detected in subregion 1 and/or subregion 2 of the left ventricle of the heart of the subject.

“Detectable expression” generally refers to transgene expression that is at least 5%, 10%, 15%, 20% or more of that in a control subject not treated with the specified viral vector or in control tissue not treated the specified viral vector. In some embodiments, detectable expression means expression of at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 5-fold, at least at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold higher than control expression without vector. Transgene expression can be defined as an increase in expression relative to the expression of a wild-type gene or an endogenous gene in a cell (taking into account the possibility of the expression of a transgene influencing the expression of an endogenous gene). Transgene expression can also be determined using RT-PCR detection of sequences present on the transgene mRNA transcript, but not on the endogenous gene mRNA transcript. For example, the 3′-UTR of a transcript can be used to determine the expression of a 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 using Western blotting or enzyme-linked immunosorbent assay (ELISA) as described in the examples below or other methods known in the art. Antibodies capable of cross-reacting with the wild-type protein and exogenous copies of the protein may be used. In some cases, an antibody specific for the exogenous protein can be identified and used to determine transgene expression. Those skilled in the art can develop suitable detection methods depending on the target cell or tissue. In some cases, expression is quantified using a standard curve. Standard curves can be generated using purified LAMP-2 protein in the methods described in the examples or known in the art. Alternatively, transgene expression can be assessed by quantifying the corresponding mRNA.

In some embodiments, the detectable expression of LAMP-2B in cardiac tissue at doses expressed as number of vector genomes (vg) per kilogram of subject body weight (kg) is 5×10 ¹⁴ vg/kg or less, 3×10 ¹⁴ vg/kg or less, 2×10 ¹⁴ vg/kg or less, 1×10 ¹⁴ vg/kg or less, 9×10 ¹³ vg/kg or less, 8×10 ¹³ vg/kg or less, 7×10 ¹³ vg/kg or less, 6×10 ¹³ vg/kg or less, 5×10 ¹³ vg/kg or less, 4×10 ¹³ vg/kg or less, 3×10 ¹³ vg/kg or less, 2×10 ¹³ vg/kg or less, or 1×10 ¹³ vg/kg or less.

In some embodiments, the detectable expression of LAMP-2B in cardiac tissue at doses expressed as number of vector genomes (vg) per kilogram of subject body weight (kg) is from 1 x 10 ¹³ vg/kg to 2 x 10 ¹³ vg/kg. from 2×10 ¹³ vg/kg to 3×10 ¹³ vg/kg, from 3×10 ¹³ vg/kg to 4×10 ^{13 vg/kg, from 4×10 13 vg} /kg to 5×10 ¹³ vg/kg , from 5×10 ¹³ vg/kg to 6×10 ¹³ vg/kg, from 6×10 ¹³ vg/kg to 7×10 ¹³ vg/kg, from 7×10 ¹³ vg/kg to 8×10 ¹³ vg/ kg, from 8×10 ¹³ vg/kg to 9×10 ¹³ vg/kg, from 9×10 ¹³ vg/kg to 1×10 ¹⁴ vg/kg, from 1×10 ¹⁴ vg/kg to 2×10 ¹⁴ vg /kg, from 2×10 ¹⁴ vg/kg to 3×10 ¹⁴ vg/kg or from 3×10 ¹⁴ vg/kg to 5×10 ¹⁴ vg/kg.

In some embodiments, the detectable expression of LAMP-2B in cardiac tissue at doses expressed as vector genomes (vg) per kilogram of subject body weight (kg) is from 1 x 10 ¹³ vg/kg to 3 x 10 ¹³ vg/kg. from 3×10 ¹³ vg/kg to 5×10 ¹³ vg/kg, from 5×10 ¹³ vg/kg to 7×10 ^{13 vg/kg, from 7×10 13 vg} /kg to 9×10 ¹³ vg/kg , from 9×10 ¹³ vg/kg to 2×10 ¹⁴ vg/kg or from 2×10 ¹⁴ vg/kg to 5×10 ¹⁴ vg/kg. In some embodiments, the detectable expression of LAMP-2B in cardiac tissue at doses expressed as vector genomes (vg) per kilogram of subject body weight (kg) is from 1 x 10 ¹³ vg/kg to 5 x 10 ¹³ vg/kg. from 5×10 ¹³ vg/kg to 9×10 ¹³ vg/kg, 9×10 ¹³ vg/kg or up to 5×10 ¹⁴ vg/kg. In some embodiments, the detectable expression of LAMP-2B in cardiac tissue at doses expressed as vector genomes (vg) per kilogram of subject body weight (kg) is from 1 x 10 ¹³ vg/kg to 9 x 10 ¹³ vg/kg. or 9×10 ¹³ vg/kg, or up to 5×10 ¹⁴ vg/kg.

In some embodiments, the detectable expression of LAMP-2B in cardiac tissue at doses expressed as vector genomes (vg) per kilogram of subject body weight (kg) is from 1 x 10 ¹³ vg/kg to 5 x 10 ¹³ vg/kg. from 5×10 ¹³ vg/kg to 1×10 ¹⁴ vg/kg, or from 1×10 ¹⁴ vg/kg to 5×10 ¹⁴ vg/kg.

In some embodiments, the detectable expression of LAMP-2B in cardiac tissue at doses expressed as vector genomes (vg) per kilogram of subject body weight (kg) ranges from 1×10 ¹³ vg/kg to 5×10 ¹⁴ vg/kg. In some embodiments, the detectable expression of LAMP-2B in cardiac tissue at doses expressed as vector genomes (vg) per kilogram of subject body weight (kg) is from 1×10 ¹³ vg/kg to 1×10 ¹⁴ .

In various embodiments, said pharmaceutical compositions contain bases ( eg, carriers, diluents, and excipients) that are pharmaceutically acceptable for injectable formulation. Examples of excipients include poloxamer. Formulation buffers for viral vectors (including AAV) generally include salts to prevent aggregation and other excipients ( eg , poloxamer) to reduce vector clumping. Said substances may be, in particular, isotonic sterile saline solutions (monosodium or disodium phosphate, sodium chloride, potassium, calcium or magnesium chloride, etc., or mixtures of such salts) or dry, in particular freeze-dried compositions, which allow reconstitute injectable solutions by adding, depending on the situation, sterile water or physiological saline solution. Preferably, the composition is stable during storage and use after freezing ( eg , at less than 0°C, about -60°C, or about -72°C).

Examples of methods for the treatment of lysosomal disorders and/or Danon disease are given in the source: WO 2018/170239 A1, which is included in this document in its entirety. Transgenes, expression cassettes and vectors according to the present description are suitable for use both in vivo ( for example , systemic, in particular, intravenous use) and ex vivo . The LAMP-2B transgene and functional promoter can be used to gene correct ex vivo autologous hematopoietic stem and progenitor cells (HSPCs) from patients, which can then be transplanted back into patients to repopulate their bone marrow, thereby creating a reservoir of “healthy” cells in patients for the rest of their lives. In some embodiments, lentiviral vectors are used for ex vivo gene editing, however, other non-viral or viral vectors may be used in place of the lentiviral vector. The present invention provides for allogeneic transplantation using donor HSPCs. In some embodiments, the lentiviral vector is a self-inactivating (SIN) lentiviral vector. In some embodiments, the HSPCs are derived from peripheral blood cells mobilized using, for example, granulocyte colony-stimulating factor (G-CSF) and/or plerixafor.

According to another aspect of the present invention, methods are provided for preventing, mitigating, ameliorating, reducing, inhibiting, eliminating and/or reversing one or more symptoms of Danon disease or other autophagic disorder in a subject in need thereof, comprising administering to said subject a gene therapy vector as described herein. The term “Danon disease” refers to an X-linked dominant disorder of the cardiac and skeletal muscles with multisystem clinical manifestations. Mutations in Danon disease result in a lack of expression of lysosome-associated membrane protein 2 (LAMP-2). Major clinical features include cardiac and skeletal muscle myopathy, cardiac conduction abnormalities, cognitive difficulties, and retinal disease. As a rule, in men the lesion develops earlier and is more severe than in women.

In an embodiment, the vector is administered by a route selected from the group consisting of parenteral, intravenous, intraarterial, intracardiac, intracoronary, intramyocardial, intrarenal, intraurethral, epidural, and intramuscular administration. According to an embodiment, said vector is introduced repeatedly. According to an embodiment, said vector is administered by intramuscular injection of the vector. In an embodiment, said vector is administered by injecting the vector into skeletal muscle. In an embodiment, said expression cassette comprises a muscle-specific promoter, optionally a muscle creatine kinase (MCK) promoter or a hybrid MCK/SV40 promoter as described in Takeshita et al. Muscle creatine kinase/SV40 hybrid promoter for muscle-targeted long-term transgene expression. Int J Mol Med. 2007 Feb;19(2):309-15. In an embodiment, said vector is administered by intracardiac injection.

In an embodiment, said vector, for example an AAV vector, is administered systemically, and more particularly intravenously. Preferably, said vector is administered at a lower dose (in g per ml, vg/kg body weight or vg/min/kg) than required to achieve the same observed response using the original or wild-type LAMP-2B sequence. In specific embodiments, the vector is an AAV2/9 vector containing an expression cassette containing a polynucleotide encoding LAMP-2B as described herein.

According to some embodiments of the present invention, there is provided a method of expressing LAMP-2B in a subject, comprising systemically administering an adeno-associated virus (AAV) vector to said subject, wherein said AAV vector contains an expression cassette containing a transgene having a sequence of at least 95% identical to SEQ ID NO: 3 or identical to SEQ ID NO: 3, operably linked to an enhancer/promoter region, wherein systemic administration of the AAV vector to the subject results in increased LAMP-2B expression compared to LAMP-2B expression prior to AAV administration -vector or LAMP-2B expression in an untreated control subject. In some embodiments, said AAV vector is an AAV2/9 vector. In specific embodiments, said expression cassette comprises any of the elements described herein. In a certain embodiment, systemic administration includes intravenous administration. In some embodiments, the subject exhibits symptoms of Danon disease. In some embodiments, the subject has Danon disease or is at risk of developing Danon disease.

In some embodiments, the AAV vector is administered at a dose of about 1 x 10 ¹² to 5 x 10 ¹⁴ vector genomes (vg) of the AAV vector per kilogram (kg) of the subject's total body weight (vg/kg). In some embodiments, the AAV vector is administered at a dose of about 1 x 10 ¹³ to 5 x 10 ¹⁴ vg/kg. In some embodiments, the AAV vector is administered at a dose of about 5 x 10 ¹³ to 3 x 10 ¹⁴ vg/kg. In some embodiments, the AAV vector is administered at a dose of about 5×10 ¹³ to 1×10 ¹⁴ vg/kg. In some embodiments, the AAV vector is administered at a dose of less than about 1 x 10 ¹² vg/kg, less than about 3 x 10 ¹² vg/kg, less than about 5 x 10 ¹² vg/kg, less than about 7 ×10 ¹² vg/kg, less than about 1×10 ¹³ vg/kg, less than about 3×10 ¹³ vg/kg, less than about 5×10 ¹³ vg/kg, less than about 7×10 ¹³ vg/kg , less than about 1×10 ¹⁴ vg/kg, less than about 3×10 ¹⁴ vg/kg, less than about 5×10 ¹⁴ vg/kg, less than about 7×10 ¹⁴ vg/kg, less than about 1× 10 ¹⁵ vg/kg, less than about 3 x 10 ¹⁵ vg/kg, less than about 5 x 10 ¹⁵ vg/kg, or less than about 7 x 10 ¹⁵ vg/kg.

In some embodiments, the AAV vector is administered at a dose of about 1x1012 ^vg /kg, about ^3x1012 vg/kg, about ^5x1012 vg/kg, about ^7x1012 vg/kg, about 1×10 ¹³ vg/kg, approximately 3×10 ¹³ vg/kg, approximately 5×10 ¹³ vg/kg, approximately 7×10 ¹³ vg/kg, approximately 1×10 ¹⁴ vg/kg, approximately 3×10 ¹⁴ vg /kg, approximately 5×10 ¹⁴ vg/kg, approximately 7×10 ¹⁴ vg/kg, approximately 1×10 ^{15 vg/kg, approximately 3×10 15 vg} /kg, approximately 5×10 ¹⁵ vg/kg or approximately 7 ×10 ¹⁵ vg/kg.

In some embodiments, said AAV vector is administered at a dose of ^1x1012vg /kg, ^3x1012vg /kg, ^5x1012vg /kg, ^7x1012vg /kg, ^1x1013vg /kg , 3×10 ¹³ vg/kg, 5×10 ¹³ vg/kg, 7×10 ¹³ vg/kg, 1×10 ¹⁴ vg/kg, 3×10 ¹⁴ vg/kg, 5×10 ¹⁴ vg/kg, 7 ×10 ¹⁴ vg/kg, 1x10 ¹⁵ vg/kg, 3x10 ¹⁵ vg/kg, 5x10 ¹⁵ vg/kg or 7x10 ¹⁵ vg/kg.

In some embodiments, the lentiviral vector is administered at a dose of about 1 x 10 ¹² to 5 x 10 ¹⁴ vector genomes (vg) of the lentiviral vector per kilogram (kg) of the subject's total body weight (vg/kg). In some embodiments, the lentiviral vector is administered at a dose of about 1 x 10 ¹³ to 5 x 10 ¹⁴ vg/kg. In some embodiments, the lentiviral vector is administered at a dose of about 5 x 10 ¹³ to 3 x 10 ¹⁴ vg/kg. In some embodiments, the lentiviral vector is administered at a dose of about 5 x 10 ¹³ to 1 x 10 ¹⁴ vg/kg. In some embodiments, said lentiviral vector is administered at a dose of less than about 1 x 10 ¹² vg/kg, less than about 3 x 10 ¹² vg/kg, less than about 5 x 10 ¹² vg/kg, less than about 7 x 10 ¹² vg/kg, less than about 1×10 ¹³ vg/kg, less than about 3×10 ¹³ vg/kg, less than about 5×10 ¹³ vg/kg, less than about 7×10 ¹³ vg/kg, less than about 1×10 ¹⁴ vg/kg, less than about 3×10 ¹⁴ vg/kg, less than about 5×10 ¹⁴ vg/kg, less than about 7×10 ¹⁴ vg/kg, less than about 1×10 ¹⁵ vg /kg, less than about 3×10 ¹⁵ vg/kg, less than about 5×10 ¹⁵ vg/kg, or less than about 7×10 ¹⁵ vg/kg.

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

In some embodiments, said lentiviral vector is administered at a dose of ^1x1012vg /kg, ^3x1012vg /kg, ^5x1012vg /kg, ^7x1012vg /kg, ^1x1013vg /kg, 3×10 ¹³ vg/kg, 5×10 ¹³ vg/kg, 7×10 ¹³ vg/kg, 1×10 ¹⁴ vg/kg, 3×10 ¹⁴ vg/kg, 5×10 ¹⁴ vg/kg, 7× 10 ¹⁴ vg/kg, 1x10 ¹⁵ vg/kg, 3x10 ¹⁵ vg/kg, 5x10 ¹⁵ vg/kg or 7x10 ¹⁵ vg/kg.

In some embodiments, the viral vector is administered at a dose of about 1 x 10 ¹² to 5 x 10 ¹⁴ vector genomes (vg) of the viral vector per kilogram (kg) of the subject's total body weight (vg/kg). In some embodiments, the viral vector is administered at a dose of about 1 x 10 ¹³ to 5 x 10 ¹⁴ vg/kg. In some embodiments, the viral vector is administered at a dose of about 5 x 10 ¹³ to 3 x 10 ¹⁴ vg/kg. In some embodiments, the viral vector is administered at a dose of about 5×10 ¹³ to 1×10 ¹⁴ vg/kg. In some embodiments, the viral vector is administered at a dose of less than about 1 x 10 ¹² vg/kg, less than about 3 x 10 ¹² vg/kg, less than about 5 x 10 ¹² vg/kg, less than about 7 x 10 ¹² vg/kg, less than about 1×10 ¹³ vg/kg, less than about 3×10 ¹³ vg/kg, less than about 5×10 ¹³ vg/kg, less than about 7×10 ¹³ vg/kg, less than about 1×10 ¹⁴ vg/kg, less than about 3×10 ¹⁴ vg/kg, less than about 5×10 ¹⁴ vg/kg, less than about 7×10 ¹⁴ vg/kg, less than about 1×10 ¹⁵ vg /kg, less than about 3×10 ¹⁵ vg/kg, less than about 5×10 ¹⁵ vg/kg, or less than about 7×10 ¹⁵ vg/kg.

In some embodiments, the viral vector is administered at a dose of about 1 x 10 ¹² vg/kg, about 3 x 10 ¹² vg/kg, about 5 x 10 ¹² vg/kg, about 7 x 10 ¹² vg/kg, about 1 ×10 ¹³ vg/kg, approximately 3×10 ¹³ vg/kg, approximately 5×10 ¹³ vg/kg, approximately 7×10 ¹³ vg/kg, approximately 1×10 ¹⁴ vg/kg, approximately 3×10 ¹⁴ vg/ kg, approximately 5×10 ¹⁴ vg/kg, approximately 7×10 ¹⁴ vg/kg, approximately 1×10 ^{15 vg/kg, approximately 3×10 15 vg} /kg, approximately 5×10 ¹⁵ vg/kg or approximately 7× 10 ¹⁵ vg/kg.

In some embodiments, said viral vector is administered at a dose of ^1x1012vg /kg, ^3x1012vg /kg, ^5x1012vg /kg, ^7x1012vg /kg, ^1x1013vg /kg, 3×10 ¹³ vg/kg, 5×10 ¹³ vg/kg, 7×10 ¹³ vg/kg, 1×10 ¹⁴ vg/kg, 3×10 ¹⁴ vg/kg, 5×10 ¹⁴ vg/kg, 7× 10 ¹⁴ vg/kg, 1x10 ¹⁵ vg/kg, 3x10 ¹⁵ vg/kg, 5x10 ¹⁵ vg/kg or 7x10 ¹⁵ vg/kg.

In some embodiments, the AAV vector is administered systemically at a dose of about 1 x 10 ¹² to 5 x 10 ¹⁴ vector genomes (vg) of the AAV vector per kilogram (kg) of the subject's total body weight (vg/kg). In some embodiments, the AAV vector is administered systemically at a dose of from about 1 x 10 ¹³ to 5 x 10 ¹⁴ vg/kg. In some embodiments, the AAV vector is administered systemically at a dose of about 5×10 ¹³ to 3×10 ¹⁴ vg/kg. In some embodiments, the AAV vector is administered systemically at a dose of about 5×10 ¹³ to 1×10 ¹⁴ vg/kg. In some embodiments, the AAV vector is administered systemically 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× 10 ¹² vg/kg, less than about 1×10 ¹³ vg/kg, less than about 3×10 ¹³ vg/kg, less than about 5×10 ¹³ vg/kg, less than about 7×10 ¹³ vg/kg, less than about 1×10 ¹⁴ vg/kg, less than about 3×10 ¹⁴ vg/kg, less than about 5×10 ¹⁴ vg/kg, less than about 7×10 ¹⁴ vg/kg, less than about 1×10 ¹⁵ vg/kg, less than about 3 x 10 ¹⁵ vg/kg, less than about 5 x 10 ¹⁵ vg/kg, or less than about 7 x 10 ¹⁵ vg/kg.

In some embodiments, said AAV vector is administered systemically at a dose of about 1 x 10 ¹² vg/kg, about 3 x 10 ¹² vg/kg, about 5 x 10 ¹² vg/kg, about 7 x 10 ¹² vg/kg, approximately 1×10 ¹³ vg/kg, approximately 3×10 ¹³ vg/kg, approximately 5×10 ¹³ vg/kg, approximately 7×10 ¹³ vg/kg, approximately 1×10 ¹⁴ vg/kg, approximately 3×10 ¹⁴ vg/kg, approximately 5×10 ¹⁴ vg/kg, approximately 7×10 ¹⁴ vg/kg, approximately 1×10 15 vg/kg, approximately 3×10 ¹⁵ vg/kg, approximately 5× ^{10 15 vg} /kg or approximately 7×10 ¹⁵ vg/kg.

In some embodiments, said AAV vector is administered systemically at a dose of ^1x1012vg /kg, ^3x1012vg /kg, ^5x1012vg /kg, ^7x1012vg /kg, ^1x1013vg / kg, 3×10 ¹³ vg/kg, 5×10 ¹³ vg/kg, 7×10 ¹³ vg/kg, 1×10 ¹⁴ vg/kg, 3×10 ¹⁴ vg/kg, 5×10 ¹⁴ vg/kg, 7x10 ¹⁴ vg/kg, 1x10 ¹⁵ vg/kg, 3x10 ¹⁵ vg/kg, 5x10 ¹⁵ vg/kg or 7x10 ¹⁵ vg/kg.

In some embodiments, the lentiviral vector is administered systemically at a dose of about 1 x 10 ¹² to 5 x 10 ¹⁴ vector genomes (vg) of the lentiviral vector per kilogram (kg) of the subject's total body weight (vg/kg). In some embodiments, the lentiviral vector is administered systemically at a dose of from about 1×10 ¹³ to 5×10 ¹⁴ vg/kg. In some embodiments, the lentiviral vector is administered systemically at a dose of about 5×10 ¹³ to 3×10 ¹⁴ vg/kg. In some embodiments, the lentiviral vector is administered systemically at a dose of from about 5×10 ¹³ to 1×10 ¹⁴ vg/kg. In some embodiments, said lentiviral vector is administered systemically at a dose of less than about 1 x 10 ¹² vg/kg, less than about 3 x 10 ¹² vg/kg, less than about 5 x 10 ¹² vg/kg, less than about 7 x 10 ¹² vg/kg, less than about 1×10 ¹³ vg/kg, less than about 3×10 ¹³ vg/kg, less than about 5×10 ¹³ vg/kg, less than about 7×10 ¹³ vg/kg, less less than about 1×10 ¹⁴ vg/kg, less than about 3×10 ¹⁴ vg/kg, less than about 5×10 ¹⁴ vg/kg, less than about 7×10 ¹⁴ vg/kg, less than about 1×10 ¹⁵ vg/kg, less than about 3×10 ¹⁵ vg/kg, less than about 5×10 ¹⁵ vg/kg, or less than about 7×10 ¹⁵ vg/kg.

In some embodiments, the lentiviral vector is administered systemically at a dose of about ^1x1012 vg/kg, about ^3x1012 vg/kg, about ^5x1012 vg/kg, about ^7x1012 vg/kg, about 1×10 ¹³ vg/kg, approximately 3×10 ¹³ vg/kg, approximately 5×10 ¹³ vg/kg, approximately 7×10 ¹³ vg/kg, approximately 1×10 ¹⁴ vg/kg, approximately 3×10 ¹⁴ vg /kg, approximately 5×10 ¹⁴ vg/kg, approximately 7×10 ¹⁴ vg/kg, approximately 1×10 ^{15 vg/kg, approximately 3×10 15 vg} /kg, approximately 5×10 ¹⁵ vg/kg or approximately 7 ×10 ¹⁵ vg/kg.

In some embodiments, said lentiviral vector is administered systemically at a dose of ^1x1012vg /kg, ^3x1012vg /kg, ^5x1012vg /kg, ^7x1012vg /kg, ^1x1013vg /kg , 3×10 ¹³ vg/kg, 5×10 ¹³ vg/kg, 7×10 ¹³ vg/kg, 1×10 ¹⁴ vg/kg, 3×10 ¹⁴ vg/kg, 5×10 ¹⁴ vg/kg, 7 ×10 ¹⁴ vg/kg, 1x10 ¹⁵ vg/kg, 3x10 ¹⁵ vg/kg, 5x10 ¹⁵ vg/kg or 7x10 ¹⁵ vg/kg.

In some embodiments, the viral vector is administered systemically at a dose of about 1 x 10 ¹² to 5 x 10 ¹⁴ vector genomes (vg) of the viral vector per kilogram (kg) of the subject's total body weight (vg/kg). In some embodiments, the viral vector is administered systemically at a dose of about 1 x 10 ¹³ to 5 x 10 ¹⁴ vg/kg. In some embodiments, the viral vector is administered systemically at a dose of about 5 x 10 ¹³ to 3 x 10 ¹⁴ vg/kg. In some embodiments, the viral vector is administered systemically at a dose of about 5 x 10 ¹³ to 1 x 10 ¹⁴ vg/kg. In some embodiments, the viral vector is administered systemically at a dose of less than about 1 x 10 ¹² vg/kg, less than about 3 x 10 ¹² vg/kg, less than about 5 x 10 ¹² vg/kg, less than about 7 x 10 ¹² vg/kg, less than about 1×10 ¹³ vg/kg, less than about 3×10 ¹³ vg/kg, less than about 5×10 ¹³ vg/kg, less than about 7×10 ¹³ vg/kg, less less than about 1×10 ¹⁴ vg/kg, less than about 3×10 ¹⁴ vg/kg, less than about 5×10 ¹⁴ vg/kg, less than about 7×10 ¹⁴ vg/kg, less than about 1×10 ¹⁵ vg/kg, less than about 3×10 ¹⁵ vg/kg, less than about 5×10 ¹⁵ vg/kg, or less than about 7×10 ¹⁵ vg/kg.

In some embodiments, the viral vector is administered systemically at a dose of about 1x1012 ^vg /kg, about ^3x1012 vg/kg, about ^5x1012 vg/kg, about ^7x1012 vg/kg, about 1×10 ¹³ vg/kg, approximately 3×10 ¹³ vg/kg, approximately 5×10 ¹³ vg/kg, approximately 7×10 ¹³ vg/kg, approximately 1×10 ¹⁴ vg/kg, approximately 3×10 ¹⁴ vg /kg, approximately 5×10 ¹⁴ vg/kg, approximately 7×10 ¹⁴ vg/kg, approximately 1×10 ^{15 vg/kg, approximately 3×10 15 vg} /kg, approximately 5×10 ¹⁵ vg/kg or approximately 7 ×10 ¹⁵ vg/kg.

In some embodiments, said viral vector is administered systemically at a dose of ^1x1012vg /kg, ^3x1012vg /kg, ^5x1012vg /kg, ^7x1012vg /kg, ^1x1013vg /kg , 3×10 ¹³ vg/kg, 5×10 ¹³ vg/kg, 7×10 ¹³ vg/kg, 1×10 ¹⁴ vg/kg, 3×10 ¹⁴ vg/kg, 5×10 ¹⁴ vg/kg, 7 ×10 ¹⁴ vg/kg, 1x10 ¹⁵ vg/kg, 3x10 ¹⁵ vg/kg, 5x10 ¹⁵ vg/kg or 7x10 ¹⁵ vg/kg.

In some embodiments, the AAV vector is administered intravenously at a dose of about 1 x 10 ¹² to 5 x 10 ¹⁴ vector genomes (vg) of the AAV vector per kilogram (kg) of the subject's total body weight (vg/kg). In some embodiments, the AAV vector is administered intravenously at a dose of about 1 x 10 ¹³ to 5 x 10 ¹⁴ vg/kg. In some embodiments, the AAV vector is administered intravenously at a dose of about 5 x 10 ¹³ to 3 x 10 ¹⁴ vg/kg. In some embodiments, the AAV vector is administered intravenously at a dose of about 5×10 ¹³ to 1×10 ¹⁴ vg/kg. In some embodiments, the AAV vector is administered intravenously 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× 10 ¹² vg/kg, less than about 1×10 ¹³ vg/kg, less than about 3×10 ¹³ vg/kg, less than about 5×10 ¹³ vg/kg, less than about 7×10 ¹³ vg/kg, less than about 1×10 ¹⁴ vg/kg, less than about 3×10 ¹⁴ vg/kg, less than about 5×10 ¹⁴ vg/kg, less than about 7×10 ¹⁴ vg/kg, less than about 1×10 ¹⁵ vg/kg, less than about 3 x 10 ¹⁵ vg/kg, less than about 5 x 10 ¹⁵ vg/kg, or less than about 7 x 10 ¹⁵ vg/kg.

In some embodiments, said AAV vector is administered intravenously at a dose of about 1x1012 ^vg /kg, about ^3x1012 vg/kg, about ^5x1012 vg/kg, about ^7x1012 vg/kg, approximately 1×10 ¹³ vg/kg, approximately 3×10 ¹³ vg/kg, approximately 5×10 ¹³ vg/kg, approximately 7×10 ¹³ vg/kg, approximately 1×10 ¹⁴ vg/kg, approximately 3×10 ¹⁴ vg/kg, approximately 5×10 ¹⁴ vg/kg, approximately 7×10 ¹⁴ vg/kg, approximately 1×10 15 vg/kg, approximately 3×10 ¹⁵ vg/kg, approximately 5× ^{10 15 vg} /kg or approximately 7×10 ¹⁵ vg/kg.

In some embodiments, said AAV vector is administered intravenously at a dose of ^1x1012vg /kg, ^3x1012vg /kg, ^5x1012vg /kg, ^7x1012vg /kg, ^1x1013vg / kg, 3×10 ¹³ vg/kg, 5×10 ¹³ vg/kg, 7×10 ¹³ vg/kg, 1×10 ¹⁴ vg/kg, 3×10 ¹⁴ vg/kg, 5×10 ¹⁴ vg/kg, 7x10 ¹⁴ vg/kg, 1x10 ¹⁵ vg/kg, 3x10 ¹⁵ vg/kg, 5x10 ¹⁵ vg/kg or 7x10 ¹⁵ vg/kg.

In some embodiments, the lentiviral vector is administered intravenously at a dose of about 1 x 10 ¹² to 5 x 10 ¹⁴ vector genomes (vg) of the lentiviral vector per kilogram (kg) of the subject's total body weight (vg/kg). In some embodiments, the lentiviral vector is administered intravenously at a dose of about 1 x 10 ¹³ to 5 x 10 ¹⁴ vg/kg. In some embodiments, the lentiviral vector is administered intravenously at a dose of about 5 x 10 ¹³ to 3 x 10 ¹⁴ vg/kg. In some embodiments, the lentiviral vector is administered intravenously at a dose of about 5×10 ¹³ to 1×10 ¹⁴ vg/kg. In some embodiments, the lentiviral vector is administered intravenously at a dose of less than about 1 x 10 ¹² vg/kg, less than about 3 x 10 ¹² vg/kg, less than about 5 x 10 ¹² vg/kg, less than about 7 x 10 ¹² vg/kg, less than about 1×10 ¹³ vg/kg, less than about 3×10 ¹³ vg/kg, less than about 5×10 ¹³ vg/kg, less than about 7×10 ¹³ vg/kg, less less than about 1×10 ¹⁴ vg/kg, less than about 3×10 ¹⁴ vg/kg, less than about 5×10 ¹⁴ vg/kg, less than about 7×10 ¹⁴ vg/kg, less than about 1×10 ¹⁵ vg/kg, less than about 3×10 ¹⁵ vg/kg, less than about 5×10 ¹⁵ vg/kg, or less than about 7×10 ¹⁵ vg/kg.

In some embodiments, the lentiviral vector is administered intravenously at a dose of about 1x1012 ^vg /kg, about 3x1012 ^vg /kg, about ^5x1012 vg/kg, about ^7x1012 vg/kg, about 1×10 ¹³ vg/kg, approximately 3×10 ¹³ vg/kg, approximately 5×10 ¹³ vg/kg, approximately 7×10 ¹³ vg/kg, approximately 1×10 ¹⁴ vg/kg, approximately 3×10 ¹⁴ vg /kg, approximately 5×10 ¹⁴ vg/kg, approximately 7×10 ¹⁴ vg/kg, approximately 1×10 ^{15 vg/kg, approximately 3×10 15 vg} /kg, approximately 5×10 ¹⁵ vg/kg or approximately 7 ×10 ¹⁵ vg/kg.

In some embodiments, said lentiviral vector is administered intravenously at a dose of ^1x1012vg /kg, ^3x1012vg /kg, ^5x1012vg /kg, ^7x1012vg /kg, ^1x1013vg /kg , 3×10 ¹³ vg/kg, 5×10 ¹³ vg/kg, 7×10 ¹³ vg/kg, 1×10 ¹⁴ vg/kg, 3×10 ¹⁴ vg/kg, 5×10 ¹⁴ vg/kg, 7 ×10 ¹⁴ vg/kg, 1x10 ¹⁵ vg/kg, 3x10 ¹⁵ vg/kg, 5x10 ¹⁵ vg/kg or 7x10 ¹⁵ vg/kg.

In some embodiments, the viral vector is administered intravenously at a dose of about 1 x 10 ¹² to 5 x 10 ¹⁴ vector genomes (vg) of the viral vector per kilogram (kg) of the subject's total body weight (vg/kg). In some embodiments, the viral vector is administered intravenously at a dose of about 1 x 10 ¹³ to 5 x 10 ¹⁴ vg/kg. In some embodiments, the viral vector is administered intravenously at a dose of about 5 x 10 ¹³ to 3 x 10 ¹⁴ vg/kg. In some embodiments, the viral vector is administered intravenously at a dose of about 5 x 10 ¹³ to 1 x 10 ¹⁴ vg/kg. In some embodiments, the viral vector is administered intravenously at a dose of less than about 1 x 10 ¹² vg/kg, less than about 3 x 10 ¹² vg/kg, less than about 5 x 10 ¹² vg/kg, less than about 7 x 10 ¹² vg/kg, less than about 1×10 ¹³ vg/kg, less than about 3×10 ¹³ vg/kg, less than about 5×10 ¹³ vg/kg, less than about 7×10 ¹³ vg/kg, less less than about 1×10 ¹⁴ vg/kg, less than about 3×10 ¹⁴ vg/kg, less than about 5×10 ¹⁴ vg/kg, less than about 7×10 ¹⁴ vg/kg, less than about 1×10 ¹⁵ vg/kg, less than about 3×10 ¹⁵ vg/kg, less than about 5×10 ¹⁵ vg/kg, or less than about 7×10 ¹⁵ vg/kg.

In some embodiments, the viral vector is administered intravenously at a dose of about 1x1012 ^vg /kg, about ^3x1012 vg/kg, about ^5x1012 vg/kg, about ^7x1012 vg/kg, about 1×10 ¹³ vg/kg, approximately 3×10 ¹³ vg/kg, approximately 5×10 ¹³ vg/kg, approximately 7×10 ¹³ vg/kg, approximately 1×10 ¹⁴ vg/kg, approximately 3×10 ¹⁴ vg /kg, approximately 5×10 ¹⁴ vg/kg, approximately 7×10 ¹⁴ vg/kg, approximately 1×10 ^{15 vg/kg, approximately 3×10 15 vg} /kg, approximately 5×10 ¹⁵ vg/kg or approximately 7 ×10 ¹⁵ vg/kg.

In some embodiments, said viral vector is administered intravenously at a dose of ^1x1012vg /kg, ^3x1012vg /kg, ^5x1012vg /kg, ^7x1012vg /kg, ^1x1013vg /kg , 3×10 ¹³ vg/kg, 5×10 ¹³ vg/kg, 7×10 ¹³ vg/kg, 1×10 ¹⁴ vg/kg, 3×10 ¹⁴ vg/kg, 5×10 ¹⁴ vg/kg, 7 ×10 ¹⁴ vg/kg, 1x10 ¹⁵ vg/kg, 3x10 ¹⁵ vg/kg, 5x10 ¹⁵ vg/kg or 7x10 ¹⁵ vg/kg.

Systemic (or more specifically, intravenous) administration, according to some embodiments, results in the expression of the LAMP-2B polynucleotide as mRNA, in the form of mRNA expressed from a transgene, in one or more tissues ( eg, heart, muscle, and/or liver) of the subject. . In some embodiments, expression of the LAMP-2B polynucleotide as mRNA in the heart is increased by at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1-fold .5 times, at least about 1.6 times, at least about 1.7 times, at least about 1.8 times, at least about 1.9 times, at least about 2 ,0 times, at least about 2.2 times, at least about 2.3 times, at least about 2.4 times, at least about 2.5 times, at least about 3 -fold or at least approximately 4-fold compared to expression in an untreated subject or in a subject treated with a control vector. In some embodiments, expression of the LAMP-2B polynucleotide as mRNA in the heart is increased by at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold , at least 1.6 times, at least 1.7 times, at least 1.8 times, at least 1.9 times, at least 2.0 times, 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 compared to expression in in an untreated subject or in a subject treated with a control vector. In some embodiments, expression of the LAMP-2B polynucleotide as mRNA in the heart is increased by 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold , 1.8x, 1.9x, 2.0x, 2.2x, 2.3x, 2.4x, 2.5x, 3x or 4x compared to expression in an untreated subject or a subject treated with a control vector.

In some embodiments, expression of the LAMP-2B polynucleotide as mRNA in muscle is increased by at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1-fold .5 times, at least about 1.6 times, at least about 1.7 times, at least about 1.8 times, at least about 1.9 times, at least about 2 ,0 times, at least about 2.2 times, at least about 2.3 times, at least about 2.4 times, at least about 2.5 times, at least about 3 -fold or at least approximately 4-fold compared to expression in an untreated subject or in a subject treated with a control vector. In some embodiments, expression of the LAMP-2B polynucleotide as mRNA in muscle is increased by at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold , at least 1.6 times, at least 1.7 times, at least 1.8 times, at least 1.9 times, at least 2.0 times, 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 compared to expression in in an untreated subject or in a subject treated with a control vector. In some embodiments, expression of the LAMP-2B polynucleotide as mRNA in muscle is increased by 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold , 1.8x, 1.9x, 2.0x, 2.2x, 2.3x, 2.4x, 2.5x, 3x or 4x 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 is not expressed in the liver of the subject. In some embodiments, the heart exhibits 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 times, at least about 1.7 times, at least about 1.8 times, at least about 1.9 times, at least about 2.0 times, at least about 2.2 times, at least about 2.3 times, at least about 2.4 times, at least about 2.5 times, at least about 3 times, or at least about 4-fold increased expression of the LAMP-2B polynucleotide as mRNA compared to expression in the liver. In some embodiments, 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 are observed in the heart. multiple, at least 1.7 times, at least 1.8 times, at least 1.9 times, at least 2.0 times, at least 2.2 times, at least 2.3-fold, at least 2.4-fold, at least 2.5-fold, at least 3-fold, or at least 4-fold increased expression of the LAMP-2B polynucleotide as mRNA compared to the expression in the liver. In some embodiments, the heart exhibits 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 increased expression of the LAMP-2B polynucleotide as mRNA versus liver expression.

In some embodiments, expression of wild-type LAMP-2B protein or functional LAMP-2B protein in the heart is increased by at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5 times, at least about 1.6 times, at least about 1.7 times, at least about 1.8 times, at least about 1.9 times, at least about 2.0 times, at least about 2.2 times, at least about 2.3 times, at least about 2.4 times, at least about 2.5 times, at least about 3-fold or at least about 4-fold compared to expression in an untreated subject or in a subject treated with a control vector. In some embodiments, expression of wild-type LAMP-2B protein or functional LAMP-2B protein in the heart is increased by at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5 times, at least 1.6 times, at least 1.7 times, at least 1.8 times, at least 1.9 times, at least 2.0 times , at least 2.2 times, at least 2.3 times, at least 2.4 times, at least 2.5 times, at least 3 times or at least 4 times compared to expression in an untreated subject or a subject treated with a control vector. In some embodiments, expression of wild-type LAMP-2B protein or functional LAMP-2B protein in the heart is increased by 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7x, 1.8x, 1.9x, 2.0x, 2.2x, 2.3x, 2.4x, 2.5x, 3- fold or 4-fold compared to expression in an untreated subject or a subject treated with a control vector.

In some embodiments, the heart exhibits 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 times, at least about 1.7 times, at least about 1.8 times, at least about 1.9 times, at least about 2.0 times, at least about 2.2-fold, at least about 2.3-fold, or at least 5-fold increased expression of wild-type LAMP-2B protein or functional LAMP-2B protein compared to expression in the liver. In some embodiments, 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 are observed in the heart. multiple, at least 1.7 times, at least 1.8 times, at least 1.9 times, at least 2.0 times, at least 2.2 times, at least 2.3-fold, at least 2.4-fold, at least 2.5-fold, at least 3-fold, or at least 4-fold increased expression of wild-type LAMP-2B protein or functional LAMP- protein 2B compared to liver expression. In some embodiments, the heart exhibits 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 increased wild-type LAMP-2B protein expression or functional LAMP-2B protein compared with liver expression.

In some embodiments, administration of said gene therapy vector results in the expression of wild-type LAMP-2B protein or functional LAMP-2B protein in the liver by a maximum of about 1.1-fold, a maximum of about 1.2-fold, a maximum of about 1.3-fold, a maximum of approximately 1.4 times, a maximum of approximately 1.5 times, a maximum of approximately 1.6 times, a maximum of approximately 1.7 times, a maximum of approximately 1.8 times, a maximum of approximately 1.9 times, or a maximum of approximately 2-fold increased compared to expression in the liver of an untreated subject. In some embodiments, administration of said gene therapy vector results in the expression of wild-type LAMP-2B protein or functional LAMP-2B protein in the liver by a maximum of 1.1-fold, a maximum of 1.2-fold, a maximum of 1.3-fold, a maximum of 1. 4-fold, maximum 1.5-fold, maximum 1.6-fold, maximum 1.7-fold, maximum 1.8-fold, maximum 1.9-fold, or maximum 2-fold increased compared to liver expression untreated subject. In some embodiments, administration of said gene therapy vector results in the expression of wild-type LAMP-2B protein or functional LAMP-2B protein in the liver by 1.1-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.

According to an embodiment of the present invention, there is provided 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 in accordance with the present invention and administering said cells to said subject. In an embodiment, said cells are stem cells, optionally pluripotent stem cells. In an embodiment, said stem cells are capable of differentiating into cardiac tissue. In an embodiment, said stem cells are capable of differentiating into muscle tissue, such as cardiac muscle tissue and/or skeletal muscle tissue. According to an embodiment, said stem cells are autologous. In an embodiment, said stem cells are induced pluripotent stem cells (iPSCs).

In an embodiment, the disease or disorder is an autophagic disorder. In some embodiments, the autophagic disorder is selected from the group consisting of, but not limited to, end-stage heart failure, myocardial infarction, drug toxicity, diabetes, end-stage renal disease, and aging. In an embodiment, said subject is a mammal, such as a human. In an embodiment, the subject exhibits symptoms of Danon disease or other autophagic disorder. In an embodiment, the subject has been identified as having reduced or undetectable LAMP-2 expression. In an embodiment, the subject has been identified as having a mutated LAMP-2 gene.

Subjects/patients suitable for treatment using the methods described herein include, but are not limited to, individuals who are at risk for developing a disease or disorder characterized by deficient autophagic flux ( e.g., Danon disease, as well as other known autophagy disorders, including, but not limited to, systolic and diastolic heart failure, myocardial infarction, drug toxicities (eg, anthracyclines, chloroquine and its derivatives), diabetes, end-stage renal failure, and aging) without symptoms, and subjects in whom symptoms are observed at the time in question. Such a subject could be identified as having a mutated LAMP-2 gene, or having reduced or undetectable levels of LAMP-2 expression.

In some embodiments, said patient is a human. In some embodiments, the patient is a child, adolescent, or adult. In some embodiments, the patient's age is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 years, or more than 20 years. In some embodiments, said patient is between 20 and 50 years old. In some embodiments, said patient is between 50 and 65 years of age. In some embodiments, the age of said patient is 1-5, 2-6, 3-7, 4-8, 5-9, 6-10, 7-11, 8-12, 9-13, 10-14, 11- 15, 12–16, 13–17, 14–18, 15–19 or 16–20 years. In some embodiments, the patient's age is 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, 14-15, 15- 16, 16–17, 17–18, 18–19, 19–20 or 20–21 years old. In a specific embodiment, the age of said patient is 15-16 years.

In some embodiments, the patient is a male human. In some embodiments, the patient is a child, adolescent, or adult male. In some embodiments, the patient is a male human aged 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 years, or more than 20 years. In some embodiments, the patient is a male human between 20 and 50 years of age. In some embodiments, the patient is a male human between 50 and 65 years of age. In some embodiments, the patient is a male human aged 1-5, 2-6, 3-7, 4-8, 5-9, 6-10, 7-11, 8-12, 9-13, 10- 14, 11–15, 12–16, 13–17, 14–18, 15–19 or 16–20 years. In some embodiments, the patient is a male human aged 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, 14- 15, 15–16, 16–17, 17–18, 18–19, 19–20 or 20–21 years. In a specific embodiment, the age of said patient is 15-16 years.

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

In some embodiments, the subject has symptoms of a disease or disorder characterized by deficient 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 failure and aging). These symptoms may be active, or may be suppressed or controlled ( eg with medication) or in remission. Said subject may or may not have been diagnosed with said disorder, for example, by a qualified medical professional.

Definitions

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

The terms "lysosome-associated membrane protein 2B" and "LAMP-2B" are used interchangeably and refer to nucleic acids and polymorphic variants, alleles, mutants and interspecies homologs of polypeptides that: (1) have an amino acid sequence greater than about 90%, for example, by 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more, preferably over an area of at least about 25, 50, 100, 200, 300 , 400 or more amino acids, or throughout the entire sequence identical to the amino acid sequence encoded by the LAMP-2B nucleic acid (see, for example , NM_013995.2) or the amino acid sequence of the LAMP-2B polypeptide (see, for example , NP_054701.1); (2) bind to antibodies, for example, raised polyclonal antibodies against an immunogen containing the amino acid sequence of a LAMP-2B polypeptide ( eg, the LAMP-2B polypeptides described herein); or the amino acid sequence encoded by a LAMP-2B nucleic acid ( eg, the LAMP-2B polynucleotides described herein) and conservatively modified variants thereof; (3) specifically hybridize under stringent hybridization conditions with an antisense strand corresponding to the nucleic acid sequence encoding the LAMP-2B protein and conservatively modified variants thereof; (4) have a nucleic acid sequence that is greater than about 90%, preferably greater than about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical, preferably over a region of at least about 25, 50, 100, 200, 500, 1000, 2000 or more nucleotides, or throughout the entire sequence, of the 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" with respect to a protein ( eg , LAMP-2B) refers to a polypeptide sequence or fragment of a polypeptide sequence containing 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 residues that retain one or more functional features of the protein. For example, a functional variant of LAMP-2B is LAMP-2B (as defined herein) that retains one or more of the following functions: (1) regulation of human cardiomyocyte function (Chi et al. (2019) PNAS USA 116 ( 2) 556-565); (2) improved metabolic and physiological function in Danon disease (Adler et al. (2019) J. Am. College Cardiology S0735-1097(19)31295-1); and/or (3) autophagy (Rowland et al. (2016) J. Cell Sci. (2016) 129, 2135-2143).

LAMP-2B contains a luminal domain (residues 29–375), a transmembrane domain (residues 376–399), and a cytoplasmic domain (residues 400–410), see UniProt accession number P13473. LAMP-2B functions include chaperone-mediated autophagy, a process that mediates lysosomal protein degradation in response to various types of stress and as part of normal protein turnover with a long biological half-life (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 can target GAPDH and MLLT11 for lysosomal degradation. LAMP-2B may be required for the fusion of autophagosomes with lysosomes in autophagy. It has been proposed that cells lacking LAMP2 express normal levels of VAMP8 but fail to accumulate STX17 on autophagosomes, which is the most likely explanation for the lack of autophagosome fusion with lysosomes. LAMP-2B may be required for normal degradation of autophagosome contents. LAMP-2B may be required for efficient MHCII-mediated presentation of exogenous antigens through its function in lysosomal protein degradation; antigenic peptides generated by proteases in the endosomal/lysosomal compartment are captured by the resulting MHCII subunits (Crotzer et al. Immunology 131:318-330(2010)).

Functional variants of LAMP-2B accordingly include fragments of LAMP-2B that are capable of mediating any of the above-described functions. In some embodiments, said functional fragment of LAMP-2B includes any one or more of a luminal, transmembrane, and cytoplasmic domain. In some embodiments, said functional variant of LAMP-2B comprises one or more C-terminal or N-terminal deletions relative to native LAMP-2B. In some embodiments, said functional variant of LAMP-2B contains one or more internal deletions relative to native LAMP-2B.

The terms "identical" or percentage of "identity" in the context of two or more nucleic acid or polypeptide sequences refer to two or more sequences or subsequences that are the same or contain a certain percentage of the same amino acid or nucleotide residues ( i.e., at least about 80% identical) identical, e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the reference sequence over a defined region, e.g. , LAMP-2 polynucleotide or polypeptide sequences as described herein, when compared and aligned to achieve the best match within a comparison window or specified region, as assessed using one of the sequence comparison algorithms described below or by manual alignment and visual inspection. In this case, such sequences are said to be “substantially identical.” This definition also applies to the complement of the test sequence. Preferably, the identity is observed over a region of at least about 25 amino acids or nucleotides, for example, over a region of 50, 100, 200, 300, 400 amino acids or nucleotides, or throughout the entire reference sequence.

When comparing sequences, typically one sequence acts as a reference sequence to which the test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into the computer, subsequence coordinates are assigned, if necessary, and software parameters for the sequence comparison algorithm are assigned. The program's default parameters may be used, or alternative parameters may be assigned. The sequence comparison algorithm then calculates the percentage identity between the test sequences and the reference sequence based on the program parameters. When comparing nucleic acid and protein sequences to LAMP-2 nucleic acids and proteins, the BLAST and BLAST 2.0 algorithms and default parameters are used.

A “comparison window” as used herein refers to a segment containing any number of contiguous positions selected from the group consisting of 20 to 600, typically from about 50 to about 200, more commonly from about 100 to about 150, within which sequence comparison can be made with a reference sequence containing the same number of contiguous positions, after the two sequences have been subjected to optimal alignment. Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be carried out, for example, using a local homology algorithm according to the source: Smith & Waterman, Adv. Appl. Math. 2:482 (1981), using an algorithm for regions of homology according to the source: Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), using the similarity search method according to the source: Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), using computerized versions of the specified algorithms (GAP, BESTFIT, FASTA and TFASTA from the Wisconsin Genetics software package, Genetics Computer Group, 575 Science Drive, Madison, Wis.) or by manual alignment and visual inspection (see ., for example , Ausubel et al., eds., Current Protocols in Molecular Biology (1995 supplement). Examples of algorithms suitable for determining percent sequence identity and sequence similarity are provided by the BLAST and BLAST 2.0 algorithms, which are described in the source: Altschul et al., J. Mol. Biol. 215:403-410 (1990) and Altschul et al., Nucleic Acids Res. 25:3389-3402 (1977), respectively. Open access software for BLAST analysis is provided by the National Center for Biotechnology Information (on the Internet at ncbi.nlm.nih.gov/).

An indication that two nucleic acid or polypeptide sequences are substantially identical is immunological cross-reactivity of the polypeptide encoded by the first nucleic acid to antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Accordingly, the polypeptide is typically substantially identical to a second polypeptide, for example, if 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. 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, “administration” refers to local and systemic administration, including, for example , enteral, parenteral, pulmonary, and local/transdermal administration. Routes of administration of compounds ( e.g., a polynucleotide encoding one or more isoforms of LAMP-2) suitable for use in the methods described herein include, for example, oral (oral (PO)) administration, nasal or inhalation administration, administration suppository, local contact, transdermal delivery ( eg, transdermal patch), intrathecal (i/t) administration, intravenous (“i.v.”) administration, intraperitoneal (“i.p.”) administration, intramuscular (“i.v.”) m") administration, intralesional or subcutaneous (“SC”) administration, or implantation of a slow release device, such as an osmotic mini-pump, depot formulation, or the like, into a subject. Administration may be via any route, including parenteral and transmucosal ( eg, oral, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intrarenal, intraurethral, intracardiac, intracoronary, intramyocardial, intradermal, epidural, subcutaneous, intraperitoneal, intraventricular, iontophoretic and intracranial. Other delivery methods include, but are not limited to, liposomal formulations, intravenous infusion, transdermal patches, and the like.

The terms “systemic administration” and “systemically administered” refer to a method of administering a compound or composition to a mammal, providing delivery of the compound or composition to sites in the body, including the target site of pharmaceutical action, through the circulatory system. Systemic administration includes, but is not limited to, oral, intranasal, rectal, and parenteral ( eg, non-gastrointestinal, eg, intramuscular, intravenous, intra-arterial, transdermal, and subcutaneous) administration.

The term "co-administration" or "simultaneous administration", for example, as applied to compounds ( eg, LAMP-2 polynucleotides) and/or analogs thereof and another active agent, refers to the administration of said compound and/or analogs and said active agent in a manner so that they can both simultaneously provide a physiological effect. The two agents, however, are not necessarily administered together. In some embodiments, administration of one agent may precede administration of another agent. A simultaneous physiological effect does not necessarily require the presence of both agents in the bloodstream at the same time. However, in some embodiments, coadministration typically results in a significant portion of both agents being present in the body ( e.g., plasma) at the same time ( e.g., 20% or more, e.g., 30% or 40% or more, e.g. 50% or 60% or more, e.g., 70% or 80% or 90% or more) relative to the maximum serum concentration at any given dose.

The term “effective amount” or “pharmaceutically effective amount” refers to the amount and/or dosage and/or dosage regimen of one or more compounds ( e.g., gene therapy vectors) necessary to achieve a desired result, e.g., increased expression of one or more LAMP isoforms -2 in an amount sufficient to reduce the maximum severity of a disease characterized by impaired or insufficient autophagy ( for example, Danon disease).

The expression "reason for administration" refers to the actions taken by a medical professional ( eg, an attending physician) or person supervising the subject's medical care, controlling and/or allowing the administration of the subject agent(s)/compound(s) to the subject. Determining the reason for administration may include establishing a diagnosis, and/or determining an appropriate therapeutic or prophylactic regimen, and/or prescribing specific agent(s)/compounds to the subject. Such assignment may include, for example, writing a prescription, annotating a medical record, etc.

As used herein, the terms “treat” and “treating” refer to delaying the onset, slowing or reversing the progression, reducing the severity, or mitigating or preventing either the disease or condition to which the term is used, or one or more symptoms of such disease or condition. The terms “treat” and “treating” also include preventing, mitigating, alleviating, reducing, inhibiting, eliminating and/or reversing one or more symptoms of a specified disease or condition.

The term "mitigation" refers to the reduction or elimination of one or more symptoms of a specified pathology or specified disease, and/or the reduction in the level or delay of the occurrence, or reduction in the severity of one or more symptoms of a specified pathology or specified disease, and/or the prevention of a specified pathology or specified condition . In some embodiments, said reduction or elimination of one or more symptoms of a pathology or disease may include, for example, a measurable and sustained increase in the expression levels of one or more LAMP-2 isoforms.

As used herein, the expression “substantially” refers to the family or kind of active pharmaceutical agents referred to in connection with the method or composition, and may additionally include other agents that do not themselves have substantially any activity relevant to the indicated indication or purpose.

The terms "subject", "individual" and "patient" are used interchangeably and refer to a mammal, preferably a human or non-human primate, as well as domesticated mammals ( eg, canine or feline), laboratory mammals ( eg, mouse, rat, rabbit , hamster, guinea pig) and agricultural mammals ( eg horses, cattle, pigs, sheep). In various embodiments, the subject may be a human ( eg, adult male, adult female, male adolescent, female adolescent, male child, female child).

The terms "gene transfer" or "gene delivery" refer to methods or systems for reliable insertion of foreign DNA into host cells. Such methods may allow for transient expression of unintegrated transferred DNA, extrachromosomal replication and expression of transferred replicons ( eg , 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 of carrying or transporting another nucleic acid molecule. The transferred nucleic acid is typically linked to, for example inserted into, a vector nucleic acid molecule. The vector may include sequences that direct autonomous replication or reverse transcription in the cell, or may include sequences sufficient to ensure integration into the DNA of the host cell. “Vectors” include gene therapy vectors. As used herein, the term “gene therapy vector” refers to a vector suitable for use in gene therapy, for example, for delivering a polynucleotide sequence encoding a therapeutic polypeptide to a subject. Gene therapy vectors may contain a nucleic acid molecule ("transgene") encoding a therapeutically active polypeptide, for example, LAMP-2B or other gene suitable for gene replacement therapy when administered to a subject. Suitable vectors include, but are not limited to, viral vectors.

As used herein, the term "expression cassette" refers to a segment of DNA that is capable, under suitable conditions, of directing the expression of a polynucleotide ("transgene") encoding a therapeutically active polypeptide ( eg, LAMP-2B) that is included in said expression cassette. When introduced into a host cell, an expression cassette, including , can direct the cell's machinery to transcribe the transgene into RNA, which is then typically processed and finally translated into a therapeutically active polypeptide. Said expression cassette may be part of a gene therapy vector. In general, the term “expression cassette” excludes polynucleotide sequences located in the 5′ direction of the 5′-ITR and in the 3′ direction of the 3′-ITR.

All patents, patent publications and other publications mentioned and referred to herein are individually and expressly incorporated herein by reference in their entirety and for all purposes.

EXAMPLES

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

The gene expression cassette shown in FIG. 2 was inserted into a plasmid-based green fluorescent protein (GFP) reporter system to express the LAMP-2B transgene. This plasmid included a single open reading frame encoding the LAMP-2B transgene, peptide 2A and enhanced green fluorescent protein (eGFP). Post-translational self-cleavage of peptide 2A resulted in coexpression of LAMP-2B and eGFP in equimolar amounts. The 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 NO: 3–5, respectively) were tested as transgenes. These three codon variants were characterized by a reduced number of CpGs, deletion of cryptic sites, and a reduced number of open reading frames relative 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-CMC10001) were seeded into each well in seeding medium (VWR #M1001) at 37ºC with 5% carbon dioxide (CO ₂ ). After 4 hours, the medium was replaced with maintenance medium (VWR #M1003), which had been pre-equilibrated to 37ºC and 5% CO ₂ . Prepare a transfection mixture by adding 6 μl of transfection reagent (ViaFect Promega #E4982) to 128 μl of 0.015 μg/μl plasmid (wild type or variant codons 1, 2, or 3) in OptiMEM or OptiMEM + ViaFect alone (negative control), and incubated for 10–20 min. 100 μl of the indicated transfection mixture was added to 1 ml of maintenance medium, which had previously been equilibrated to 37ºC and 5% CO ₂ .

Approximately 28 hours after initial seeding, 100 μl of the indicated transfection mixture in maintenance medium was added to each well. Approximately 48 hours after the addition of transfection mixture medium, cells were imaged and analyzed for GFP-positive cells using an automated confocal microscope (Perkin Elmer Operetta CLS, Harmony software version 4.5) (Fig. 3 ) and mean intensity measured. fluorescence ( Fig . 4 ). Immunofluorescence images of cells two days or seven days after transfection are shown in Fig. 5 and fig. 6 , respectively.

In fig. Figure 3 shows that the use of codon 1 (“CO 1”) or 2 (“CO 2”) variants resulted in GFP expression in a significantly larger (~9-fold) number of cells compared to expression of the wild-type transgene. Likewise, in FIG. Figure 4 shows that the average GFP intensity in cells transfected with variant codons 1 (“CO 1”) and 2 (“CO 2”) was 1.5-fold higher than expression of the wild-type transgene.

EXAMPLE 2: Optimized Gene Therapy Vectors with LAMP-2B

Optimized AAV gene therapy vectors are prepared by inserting an optimized variant of LAMP-2B, the CO1 sequence described in Example 1, into the expression cassette of a recombinant AAV vector. The AAV regulatory cassette is modified by removing the 5' cryptic ATG sequence, using an optimized Kozak consensus sequence, and/or a full-length polyadenylation sequence. Vectors are tested against control recombinant AAV vectors containing one or more additional ATG sites 5' of the transgene, a suboptimal Kozak sequence, and/or a partial polyadenylation sequence. The vectors are tested in vitro in iPSC-derived cardiomyocytes from a Danon disease patient and in a LAMP-2 ^−/− knockout mouse model of Danon disease. Optimized AAV gene therapy cassettes and vectors are expected to provide higher levels of expression and/or expression in a greater percentage of cells compared to control recombinant AAV vectors.

EXAMPLE 3: In Vitro Evaluation of AAV9-LAMP-2B.v1.2

The gene therapy AAV cassette and vector were prepared by inserting the LAMP-2B CO1 sequence variant (SEQ ID NO: 3) into a recombinant plasmid AAV vector without hidden start sites 5' from the transgene, with an optimized Kozak consensus sequence and a full-length polyadenylation sequence (( rabbit poly)A) globin (“LAMP-2B.v.1.2”; expression cassette: SEQ ID NO: 8). LAMP-2B.v1.2 was compared with LAMP-2B v1.0, which is a regulatory cassette containing the wild-type LAMP-2B transgene (transgene sequence: SEQ ID NO: 2) without the optimal Kozak sequence and mini-(poly)A .

HEK293 cells were used to produce virus particles with a three-plasmid system without a helper virus, producing recombinant AAV particles containing serotype 9 capsid proteins and viral genomes containing the AAV2 ITR flanking a LAMP-2B expression cassette. Said expression cassette contains a codon-optimized human LAMP-2B coding sequence (v1.2 or v1.0) driven by a 5' chimeric "CAG" promoter containing the cytomegalovirus mid-early gene enhancer (CMV IE) (CMV IE) , the chicken β-actin (CBA) promoter and splice donor of the CBA intron ( Figure 1A ). This expression cassette also includes a 3' WPRE element and ends with a rabbit beta-globin polyadenylation (RGpA) signal. HEK293 cells were transiently transfected with plasmid vector LAMP-2B.v1.2 or plasmid vector LAMP-2B.v1.0, packaging plasmid pAAV2/9, and adenoviral helper plasmid pAd-Helper.

CHO-Lec2 cells were seeded in a 24-well plate at a density of 1.2 x 10 ⁵ cells/ml in MEM-α containing 10% FBS and 1% normocin. The next day, CHO-Lec2 cells were transduced in serum-free MEMα medium with either AAV9-LAMP-2B.v1.0 or AAV9-LAMP-2B.v1.2, or the same vector containing GFP instead of the LAMP-2B transgene (with MOI = 3×10 ⁵ ). Seven days after transduction, lysates were collected using a mammalian cell lysis kit (Sigma) and total protein was quantified using a MicroBCA kit according to the manufacturer's instructions. Proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted for LAMP-2B (1:500) and GAPDH (1:10,000). CHO-Lec2 cells transduced with AAV9 with optimized LAMP-2B.v1.2 showed increased expression compared to CHO-Lec2 cells transduced with original AAV9 with wild-type LAMP-2B.v1.0 ( Figure 7A ). LAMP-2B was not detected in cells transduced with the AAV9–GFP vector alone (Fig. 7A ).

LAMP-2B expression was also quantitatively analyzed in cell lysates using 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 polyclonal anti-LAMP-2B antibody (1:500, Thermo Fisher PA; 5-24575) followed by incubation with HRP-conjugated anti-rabbit antibody (1:3000, Sigma). Transduction with the LAMP-2B.v1.2-optimized AAV9 vector resulted in an approximately 7-fold increase in LAMP-2B expression compared with expression in cells transduced with wild-type AAV9-LAMP-2B.v1.0 ( Fig . 7B ).

Cardiomyocytes were derived from iPSCs from individuals with Danon disease. After rhythmic contraction and purity selection, cardiomyocytes from Danon disease patients were transduced with different viral genome copy numbers (vg) using AAV9–Luc (negative control), AAV9 with wild-type LAMP-2B.v1.0, or AAV9 with optimized LAMP-2B .v1.2. Ten days after transduction, transduced cardiomyocytes were fixed with 4% paraformaldehyde, permeabilized, blocked for 30 min in 5% BSA without IgG, and incubated for 1 h with either mouse anti-LAMP-2B antibody (1:25, clone: H4B4) or with rabbit anti-α-actinin antibody (1:200, #A7811, Sigma). Cells were washed with 1X PBS to remove residual unbound primary antibody and then treated with appropriate AlexaFluor-labeled anti-mouse AB secondary antibody and 200 ng/ml DAPI for 60 min at room temperature. Wells were then washed with PBS before imaging. A higher level of human LAMP-2B expression was observed in cardiomyocytes transduced with a low titer (1.56 x 10 ⁸ /well) of AAV9 vector with optimized LAMP-2B.v1.2, compared with cardiomyocytes transduced with a maximum titer (8, 45×10 ¹⁰ vg/well) AAV9 with wild-type LAMP-2B.v1.0 ( Fig . 8A and Fig. 8B ).

Western blot analysis was performed on transduced cardiomyocytes from patients with Danon disease. AAV9 with optimized LAMP-2B.v1.2 at a dose of 0.983×10 ⁹ vg/well demonstrated significant expression of LAMP-2B protein, while LAMP-2B protein was not detected in cells transduced with the AAV9 vector with wild-type LAM2B.v1.0 (1.347× ¹⁰⁹ vg/well) or the AAV9–Luc vector (1.167× ¹⁰⁹ vg/well) ( Fig . 8C ). Taken together, these results demonstrate that the optimized AAV9-LAMP-2B.v1.2 vector mediates human LAMP-2B expression in cardiomyocytes from patients with Danon disease at a significantly higher level than the original AAV9 vector with wild-type LAMP-2B.v1.0 .

EXAMPLE 4: Evaluation of AAV9-LAMP-2B.v1.2 in an in vivo mouse model of Danon disease

LAMP-2-deficient mice were intravenously injected with 1 × 10 ¹³ vg/kg viral AAV9 vectors containing original human LAMP-2B (AAV9–LAMP-2B.v1.0), optimized human LAMP-2B (AAV9–LAMP-2B. v1.2, codon option 1 – SEQ ID NO: 3) or a separate base. Six weeks after treatment, mice were sacrificed and heart tissues were collected for analysis of LAMP-2B expression.

Methods

For quantitative vector copy number assays, total DNA was isolated from frozen tissues using the DNeasy Blood and Tissue Isolation Kit according to the manufacturer's instructions. DNA concentration and integrity were assessed spectrophotometrically. qPCR was performed to calculate the number of viral genome copies per μg of DNA using TaqPath ProAmp Master Mix (Applied Biosystems) with forward (5′-ATCATGCTATTGCTTCCCGTA-3′; SEQ ID NO: 36) and reverse (5′-GGGCCACAACTCCCTCATAAA-3 '; SEQ ID NO: 37) primers and probe (5'-CCTCCTTGTATAAAATCCTGGTTGCTGTCT-3'; SEQ ID NO: 38) for the WPRE gene. RNase P (Thermofisher, #4403328) was used as an endogenous control. A standard curve was generated using a linearized plasmid that contained the vector genome (WPRE) used to produce the virus. The amount of DNA per sample was calculated using the TaqMan copy number reference kit and expressed as vector copy number per diploid core (VCN/diploid core).

RNA was extracted and purified from the heart using the RNeasy Fibrous Tissue Mini Kit according to the manufacturer's protocol. RNA concentration and integrity were assessed spectrophotometrically. The RNA was reverse transcribed using the iScript cDNA synthesis kit, and the cDNA was used as a template for quantitative real-time PCR (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′-GGGCCACAACTCCTCATAAA-3′; SEQ ID NO: 37) primers and a probe (5′-CCTCCTTGTATAAAATCCTGGTTGCTGTCT-3′; SEQ ID NO: 38) for the WPRE gene.

For protein extraction, tissues were flash frozen and finely ground, and the resulting 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 inhibitor cocktails and phosphatases. Partial protein lysates were passed through a glass tissue homogenizer and treated on ice with 3 pulses of ultrasound lasting 5 seconds with 10-second breaks at a power with an amplitude of 30 μm. Samples were centrifuged for 15 minutes at 12,000 rpm, after which the supernatant was collected. The protein concentration in the samples was determined using the Lowry method. Proteins (20 μg/sample) were separated on 10–20% SDS-PAGE and transferred to PVDF membranes by rapid dry transfer. The membranes were then blocked in 5% milk (nonfat dry milk solubilized in PBS with 0.1% Tween-20) for 1 hour and incubated with anti-human LAMP-2B (1:100, H4B4), anti-LAMP-2B mouse (1:100) or anti-GAPDG antibodies (1:1000, #32233, Santa Cruz) overnight at 4°C. Membranes were washed and then incubated with appropriate HRP-conjugated secondary antibodies (1:10,000) for 1 hour at room temperature. Blots were developed on WesternBright™ Sirius substrates followed by visualization in a BioRad gel scanner.

For immunofluorescence analyses, tissue was cryoprotected in 30% sucrose/PBS at 4°C, embedded in OCT mounting medium (“Optimal Cutting Temperature Compound”), and then tissue sections 8–10 μm thick were prepared on a conventional microtome. Cryosections were then fixed with 4% PFA for 5 minutes, permeabilized with 0.2% Triton-X for 5 minutes, and blocked with 1% BSA, 3% serum, 1% cold-water fish gelatin in PBS for 30 minutes. Sections were incubated with a mouse anti-human LAMP-2B antibody (1:50, H4B4) directly conjugated to Alexa Fluor 647 and a rabbit anti-dystrophin antibody overnight at 4°C. The slides were then incubated with a secondary antibody with Alexa Fluor 488 against AT rabbit and DAPI (1:2000, #D9542, Sigma) for 30 minutes at room temperature. The slides were then imaged using an Olympus FluoView FV1000 confocal microscope. Scan speed, offset, voltage, and gain were kept constant while all images were captured on a given day.

results

Quantitative PCR was performed on cardiac tissue from AAV9-treated LAMP-2-deficient mice. Although cardiac tissue from mice treated with vector containing wild-type LAMP-2B and optimized LAMP-2B ( Fig . 9A ) exhibited similar virus copy numbers, transcription of AAV9 with optimized LAMP-2B.v1.2 was increased nearly 7-fold compared to wild-type AAV9–LAMP-2B.v1.0 ( Figure 9B ). Despite similar transduction by viral vectors v1.0 and v1.2 in cardiac tissue, induction of human LAMP-2B mRNA expression was significantly enhanced by v1.2.

LAMP-2-deficient mice that were intravenously injected with an AAV9 vector with optimized LAMP-2B.v1.2 also had significantly higher levels of human LAMP-2B protein in cardiac tissue compared with levels in LAMP-2-deficient mice. treated with wild-type AAV9-LAMP-2B.v1.0 or control–backbone ( Fig . 9C ). Similar results were achieved by immunofluorescence staining: significant induction of human LAMP-2B occurred in cardiac tissue of LAMP-2-deficient mice treated with AAV9 with optimized LAMP-2B.v1.2 ( Fig . 9D ). Taken together, these data demonstrate that viral transduction using an AAV9 vector with optimized LAMP-2B.v1.2 results in increased expression of human LAMP-2B protein in cardiac tissue in vivo compared with AAV9 with wild-type LAMP-2B.v1.0 kind of at the same dose.

EXAMPLE 4: In Vivo Evaluation of AAV9-LAMP-2B.v1.2 in Non-Human Primates

Non-human primates were intravenously injected with 1 x 10 ¹³ vg/kg with either the AAV9 viral vector containing the LAMP-2B variant codons (v1.2, variant codons 1 - SEQ ID NO: 3) described in Example 2, or a backbone control . Eight weeks after treatment, nonhuman primates were humanely sacrificed and heart, muscle, liver, and brain tissues were collected for analysis of LAMP-2B expression.

Methods

For quantitative vector copy number analysis, total DNA was isolated from frozen tissues using the Qiagen DNeasy kit according to the manufacturer's instructions. DNA concentration and integrity were assessed spectrophotometrically. Quantitative PCR on isolated DNA was performed using TaqMan Universal Master Mix II (Applied Biosystems) with forward (5′-ATCATGCTATTGCTTCCCGTA-3′; SEQ ID NO: 36) and reverse (5′-GGGCCACAACTCCTCATAAA-3′; SEQ ID NO: 37) primers and probe (5′-CCTCCTTGTATAAAATCCTGGTTGCTGTCT-3′; SEQ ID NO: 38) for the WPRE gene. RNase P (#4403328, ThermoFisher) was used as an endogenous control. A standard curve was constructed using a linearized plasmid that contained the vector genome used to produce the virus. The amount of DNA per sample was calculated using the TaqMan copy number reference kit and expressed 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 the manufacturer's protocol. RNA concentration and integrity were assessed using a NanoDrop spectrophotometer. RNA was reverse transcribed using SuperScript IV VILO master mix (ThermoFisher) and cDNA was used as template for quantitative real-time PCR (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 primers (5′-GGGCCACAACTCCTCATAAA-3′; SEQ ID NO: 37 ) and a probe (5′-CCTCCTTGTATAAAATCCTGGTTGCTGTCT-3′; SEQ ID NO: 38) for the WPRE gene. Human HPRT-1 was used as an endogenous control. A standard curve was constructed using a linearized plasmid that contained the vector genome used to produce the virus.

For semiquantitative mRNA analysis using RNAScope technology, cardiac tissue was fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned. The mRNA transgene was detected using the WPRE-O3 probe (#518628, ACD) in the RNAscope 2.5 LS RED assay. Cells with more than 1 spot were considered positive, and the percentage of positive cells was divided into five categories: 0%, 1–25%, 26–50%, 51–75%, or 100%.

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

For the LAMP-2B ELISA, protein extraction was performed as described above. The plate was coated with a mouse anti-LAMP2 antibody (clone: H4B4, #NBP2-22217, Novus Biologicals), 100 μg of tissue lysate was added to each well, and detection was performed using a polyclonal anti-LAMP2 antibody (#AF6228, R&D Systems) followed by incubation with HRP -conjugated donkey anti-goat Ig antibody (#AP180P, Millipore).

results

Quantitative PCR was performed on different tissues from AAV9-treated primates. Virus copy numbers increased in the heart, muscle and liver tissue of primates injected with the AAV9-LAMP-2B.v1.2 vector at a dose of 1 x 10 ¹³ vg/kg, compared with those in vehicle-injected controls ( Fig . 10A ). Vector genomes were detected in all heart chambers examined, including the left and right ventricles, as well as the left and right atria (Fig. 10B ). Vector mRNA was detected at significant levels in heart, skeletal muscle, and liver tissue of treated primates compared to levels for untreated controls ( FIG . 10C and FIG. 10D ). In situ RNA analysis revealed that approximately 25–75% of heart and liver tissue expressed vector mRNA ( Fig . 10E and Fig. 10F ). These data demonstrate that systemic administration of 1 x 10 ¹³ vg/kg AAV9 optimized LAMP-2B.v1.2 to primates results in efficient transduction and expression in cardiac tissue in vivo .

Western blot analysis showed that primates treated systemically with LAMP-2B.v1.2 at a dose of 1 x 10 ¹³ vg/kg had increased levels of human LAMP-2B protein in the left and right ventricles and left atrium of the heart compared with control without treatment ( Fig . 10G and Fig. 10H ). The ELISA also showed that human LAMP-2B protein levels were increased in the left ventricle and atrium of the heart, as well as skeletal muscle tissue of primates treated with the AAV9–LAMP-2B.v1.2 vector, compared to untreated controls ( Fig . 10I and Fig. 10J ). Vector transduction using AAV9.LAMP-2B.v1.2 results in expression of human LAMP-2B protein in primate cardiac tissue in vivo .

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LIST OF SEQUENCES

<110> ROKIT PHARMACEUTICALS, LTD.

<120> GENOTHERAPY VECTORS FOR THE TREATMENT OF DANON'S DISEASE

<130> ROPA-011/01WO 329592-2071

<150> US 62/697302

<151> 2018-07-12

<160> 38

<170> PatentIn, version 3.5

<210> 1

<211> 409

<212> PROTEIN

<213> Homo sapiens

<400> 1

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

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ctgggagctg tgcggtctta tgcattggaa cttaatttga cagattcaga aaatgccact

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tgcctttatg caaaatggca gatgaatttc acagttcgct atgaaactac aaataaaact

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tataaaactg taaccatttc agaccatggc actngtgacat ataatggaag catttgtggg

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gatgatcaga atggtcccaa aatagcagtg cagttcggac ctggcttttc ctggattgcg

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aattttacca aggcagcatc tacttattca attgacagcg tctcattttc ctacaacact

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ggtgataaca caacatttcc tgatgctgaa gataaaggaa ttcttactgt tgatgaactt

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ttggccatca gaattccatt gaatgacctt tttagatgca atagtttatc aactttggaa

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aagaatgatg ttgtccaaca ctactgggat gttcttgtac aagcttttgt ccaaaatggc

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acagtgagca caaatgagtt cctgtgtgat aaagacaaaa cttcaacagt ggcacccacc

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atacacacca ctgtgccatc tcctactaca acacctactc caaaggaaaa accagaagct

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ggaacctatt cagttaataa tggcaatgat acttgtctgc tggctaccat ggggctgcag

720

ctgaacatca ctcaggataa ggttgcttca gttattaaca tcaaccccaa tacaactcac

780

tccacaggca gctgccgttc tcacactgct ctacttagac tcaatagcag caccattaag

840

tatctagact ttgtctttgc tgtgaaaaat gaaaaccgat tttatctgaa ggaagtgaac

900

atcagcatgt atttggttaa tggctccgtt ttcagcattg caaataacaa tctcagctac

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tgggatgccc ccctgggaag ttcttatatg tgcaacaaag agcagactgt ttcagtgtct

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ggagcatttc agataaatac ctttgatcta agggttcagc ctttcaatgt gacacaagga

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aagtattcta cagcccaaga gtgttcgctg gatgatgaca ccattctaat cccaattata

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gttggtgctg gtctttcagg cttgattatc gttatagtga ttgcttacgt aattggcaga

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agaaaaagtt atgctggata tcagactctg taa

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ctgggtgctg tgagatccta tgcccttgag ctgaacctga ctgactcaga aaatgccact

120

tgcctgtatg ccaagtggca gatgaacttc actngtgagat atgagactac caacaagacc

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tacaagactg tgaccatctc agaccatggc actgtcacct acaatggatc aatctgtggt

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gatgatcaga atggcccaaa gatagcagtg cagtttgggc ccggtttttc ctggattgct

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aacttcacca aggcagcctc cacctacagc attgactcag tcagcttcag ctacaacact

360

ggggataaca ccaccttccc tgacgcagag gacaagggaa tccttactgt ggacgaactc

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ctggcaatca gaatccccct taacgacctg ttcagatgca actccctttc aacccttgaa

480

aagaatgatg tggtgcaaca ctattgggac gtcctggtgc aagcctttgt gcagaatggg

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acagtgagta ccaacgagtt cctctgtgac aaggacaaga ccagcactgt ggcccccact

600

atccacacca ctgtgcccag ccctaccact acccccaccc ctaaagagaa gccagaagct

660

ggaacctact cagtcaacaa tggaaatgac acatgcctcc ttgccaccat gggactgcag

720

ctgaacatca ctcaggacaa ggtggcctca gtgattaaca tcaaccctaa caccactcat

780

agcactggga gctgcagatc acatacagct ctgctgaggc tcaactcctc caccatcaag

840

tacctggact ttgtgtttgc tgtgaagaat gagaacaggt tctacctcaa ggaagtgaac

900

atttccatgt acctggtcaa tggttcagtg ttctctattg ccaacaacaa tctgagctac

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tgggatgcac ccctgggatc ctcctacatg tgcaacaagg agcagactgt gagtgtgtca

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ggtgcttttc agatcaacac ttttgacctg agggtgcagc ccttcaatgt gactcaggga

1080

aagtactcca ctgcacaaga gtgttccttg gatgatgaca ctatcctcat ccccattatt

1140

gtgggagctg gactgtcagg attgattata gtgattgtga ttgcttatgt gattggaagg

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agaaagagct atgctggcta ccagaccctg taa

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atggtgtgct ttagactgtt tcctgtgcct ggttcagggc tggtcctggt ctgtctggtg

60

ctggggggctg tcagaagcta tgccttggag ctgaacctca ctgatagtga aaatgccact

120

tgtctgtatg ctaagtggca gatgaacttc actngtgagat atgaaaccac caacaagact

180

tacaaaacag tgaccatctc agatcatgga actgtgacct acaacggcag catttgtgga

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gacgaccaga acggaccaaa aatcgctgtc caatttgggc ctggattctc ctggattgcc

300

aatttcacta aagctgcctc cacatattca attgactcag tgtccttctc ctacaacact

360

ggggacaaca ctactttccc tgatgctgaa gataagggaa tcttgacagt ggatgagctg

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ctggctatca ggatcccttt gaatgacctg tttaggtgta attcactgag cactctggag

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aagaacgacg tggtgcagca ctactgggac gtgctggtgc aggcctttgt gcagaacggc

540

actgtgtcca ccaacgaatt cctgtgtgat aaggacaaaa cttccactgt ggcacctaca

600

attcacacta ctgtgccttc acctaccacc actccaactc caaaggaaaa gcctgaagca

660

ggaacctact ctgtgaacaa tggcaatgat acctgtctgt tggccaccat gggcctccaa

720

ctgaacatta ctcaggacaa ggtggcctca gtgattaaca ttaaccccaa cactacccac

780

tccactggca gctgtagatc acacacagcc ttgctcagac tgaatagcag caccatcaag

840

tatttggatt ttgtgtttgc agtgaagaat gaaaacaggt tctacctgaa ggaagtcaac

900

atctcaatgt acctggtgaa cggctcagtg ttcagcattg ccaacaacaa cctctcctat

960

tgggacgctc cactggggag cagctacatg tgtaacaagg aacagactgt gtcagtgtca

1020

ggagccttcc agattaacac ctttgatctg agggtccaac cctttaatgt cactcaagga

1080

aagtatagca ctgcccagga gtgctccctg gatgatgaca ccattctgat tccaatcatt

1140

gtgggtgcag gactttctgg gcttattatt gtgattgtga ttgcctatgt gattggcaga

1200

aggaaatcct atgcagggta ccaaactctg taa

1233

<210> 5

<211> 1233

<212> DNA

<213> Artificial sequence

<220>

<223> Option LAMP-2

<400> 5

atggtctgtt ttaggctgtt ccctgtccct ggttcaggac tggtcttagt gtgtctggtg

60

cttggagctg tcagaagcta tgccctggag ctgaacctga ctgactcaga aaatgccact

120

tgcctgtatg ccaagtggca gatgaacttc actgtcagat atgaaaccac caacaagacc

180

tataagactg tgaccatctc agaccatggc actgtgactt acaatgggtc aatttgtgga

240

gatgaccaga atggccctaa gatagctgtc cagtttggtc caggattcag ctggattgcc

300

aacttcacca aggcagccag cacctacagc attgactctg tgtccttctc ctacaacaca

360

ggagacaaca ccactttccc tgatgcagag gacaaaggta tcctgactgt ggatgagttg

420

ctggcaatca ggatcccact gaacgatctg ttcaggtgca actcactgtc cactctggaa

480

aagaatgatg tggtgcagca ctattgggat gtgctagtcc aggcctttgt ccagaatggg

540

actgtgtcaa ctaatgagtt cctgtgtgac aaggacaaga caagcactgt agcccccact

600

atccatacca cagtacctag ccccaccact actccaaccc ccaaggagaa gcctgaggct

660

ggcacctact cagtgaacaa tgggaatgac acctgtttgc tggccactat gggactccaa

720

ctgaacatca cccaggacaa agtggcctct gtgatcaata tcaatcccaa caccacccac

780

agcactgggt cctgcagaag ccacactgcc ctcctgaggc tcaactcatc aactatcaag

840

tacttggatt ttgtgtttgc agtgaagaat gagaacagat tctacctcaa agaggtcaac

900

atttcaatgt acctggtgaa tgggagtgtg ttctccatg ctaacaacaa cctgagctac

960

tgggatgccc ctctgggctc ctcatacatg tgcaacaagg aacagactgt gagtgtgtca

1020

ggggccttcc agatcaacac ttttgacctg agagtgcagc cctttaatgt gacacaggga

1080

aagtacagca ctgctcagga gtgcagcctg gatgatgaca ctatcctgat ccctatcatt

1140

gtggggggcag gcctgtctgg actcattatt gtgattgtga ttgcctatgt gatagggaga

1200

aggaagtctt atgctggata ccagaccctg taa

1233

<210> 6

<211> 13

<212> DNA

<213> Unknown

<220>

<223> Kozak sequence

<400> 6

gccgccacca tgg

13

<210> 7

<211> 387

<212> DNA

<213> Unknown

<220>

<223> Full-length (poly)A sequence

<400> 7

tggctaataa aggaaattta ttttcattgc aatagtgtgt tggaattttt tgtgtctctc

60

actcggaagg acatatggga gggcaaatca tttaaaacat cagaatgagt atttggttta

120

gagtttggca acatatgccc atatgctggc tgccatgaac aaaggttggc tataaagagg

180

tcatcagtat atgaaacagc cccctgctgt ccattcctta ttccatagaa aagccttgac

240

ttgaggttag atttttttta tattttgttt tgtgttattt ttttctttaa catccctaaa

300

attttcctta catgttttac tagccagatt tttcctcctc tcctgactac tcccagtcat

360

agctgtccct cttctcttat ggagatc

387

<210> 8

<211> 4549

<212> DNA

<213> Artificial sequence

<220>

<223> Expression Cassette

<400> 8

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt

60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact

120

aggggttcct tgtagttaat gattaacccg ccatgctact tatctaccag ggtaatgggg

180

atcctctaga actatagcta gtcgacattg attattgact agttattaat agtaatcaat

240

tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa

300

tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt

360

tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta

420

aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt

480

caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc

540

tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac

600

gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat

660

tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggggcgcg cgccaggcgg

720

ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca

780

gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa

840

aaagcgaagc gcgcggcggg cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc

900

cgccgccgcc tcgcgccgcc cgccccggct ctgactgacc gcgttactcc cacaggtgag

960

cgggcgggac ggcccttctc ctccggggctg taattagcgc ttggtttaat gacggcttgt

1020

ttcttttctg tggctgcgtg aaagccttga ggggctccgg gagggccctt tgtgcggggg

1080

gagcggctcg gggggtgcgt gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggctccgc

1140

gctgcccggc ggctgtgagc gctgcgggcg cggcgcgggg ctttgtgcgc tccgcagtgt

1200

gcgcgagggg agcgcggccg ggggcggtgc cccgcggtgc ggggggggct gcgaggggaa

1260

caaaggctgc gtgcggggtg tgtgcgtggg ggggtgagca gggggtgtgg gcgcgtcggt

1320

cgggctgcaa ccccccctgc acccccctcc ccgagttgct gagcacggcc cggcttcggg

1380

tgcggggctc cgtacggggc gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca

1440

ggtggggggtg ccgggcgggg cggggccgcc tcgggccggg gagggctcgg gggaggggcg

1500

cggcggcccc cggagcgccg gcggctgtcg aggcgcggcg agccgcagcc attgcctttt

1560

atggtaatcg tgcgagaggg cgcagggact tcctttgtcc caaatctgtg cggagccgaa

1620

atctgggagg cgccgccgca ccccctctag cgggcgcggg gcgaagcggt gcggcgccgg

1680

caggaaggaa atgggcgggg agggccttcg tgcgtcgccg cgccgccgtc cccttctccc

1740

tctccagcct cggggctgtc cgcgggggga cggctgcctt cgggggggac ggggcagggc

1800

ggggttcggc ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc atgttcatgc

1860

cttcttcttt ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt

1920

tggcaaagaa ttcgagcggc cgccagccgc caccatggtc tgcttcagac tgttccctgt

1980

ccctggatct ggtctggtgc ttgtgtgctt ggtgctgggt gctgtgagat cctatgccct

2040

tgagctgaac ctgactgact cagaaaatgc cacttgcctg tatgccaagt ggcagatgaa

2100

cttcactgtg agatatgaga ctaccaacaa gacctacaag actgtgacca tctcagacca

2160

tggcactgtc acctacaatg gatcaatctg tggtgatgat cagaatggcc caaagatagc

2220

agtgcagttt gggcccggtt tttcctggat tgctaacttc accaaggcag cctccaccta

2280

cagcattgac tcagtcagct tcagctacaa cactggggat aacaccacct tccctgacgc

2340

agaggacaag ggaatcctta ctgtggacga actcctggca atcagaatcc cccttaacga

2400

cctgttcaga tgcaactccc tttcaaccct tgaaaagaat gatgtggtgc aacactattg

2460

ggacgtcctg gtgcaagcct ttgtgcagaa tgggacagtg agtaccaacg agttcctctg

2520

tgacaaggac aagaccagca ctgtggcccc cactatccac accactgtgc ccagccctac

2580

cactaccccc acccctaaag agaagccaga agctggaacc tactcagtca acaatggaaa

2640

tgacacatgc ctccttgcca ccatgggact gcagctgaac atcactcagg acaaggtggc

2700

ctcagtgatt aacatcaacc ctaacaccac tcatagcact gggagctgca gatcacatac

2760

agctctgctg aggctcaact cctccaccat caagtacctg gactttgtgt ttgctgtgaa

2820

gaatgagaac aggttctacc tcaaggaagt gaacatttcc atgtacctgg tcaatggttc

2880

agtgttctct attgccaaca acaatctgag ctactgggat gcacccctgg gatcctccta

2940

catgtgcaac aaggagcaga ctgtgagtgt gtcaggtgct tttcagatca acacttttga

3000

cctgaggtg cagcccttca atgtgactca gggaaagtac tccactgcac aagagtgttc

3060

cttggatgat gacactatcc tcatccccat tattgtggga gctggactgt caggattgat

3120

tatagtgatt gtgattgctt atgtgattgg aaggagaaag agctatgctg gctaccagac

3180

cctgtaaaag ggcgaattcc agcacacgcg tcctaggagc tcgagtacta ctggcggccg

3240

ttactagtgg atccgcggta caagtaagca tgcaagcttc gaggacgggg tgaactacgc

3300

ctgaatcaag cttatcgata aattcgagca tcttaccgcc atttattccc atatttgttc

3360

tgtttttctt gatttgggta tacatttaaa tgttaataaa acaaaatggt ggggcaatca

3420

tttacatttt tagggatatg taattactag ttcaggtgta ttgccacaag acaaacatgt

3480

taagaaactt tcccgttatt tacgctctgt tcctgttaat caacctctgg attacaaaat

3540

ttgtgaaaga ttgactgata ttcttaacta tgttgctcct tttacgctgt gtggatatgc

3600

tgctttaatg cctctgtatc atgctattgc ttcccgtacg gctttcgttt tctcctcctt

3660

gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtcc gtcaacgtgg

3720

cgtggtgtgc tctgtgtttg ctgacgcaac ccccactggc tggggcattg ccaccacctg

3780

tcaactcctt tctgggactt tcgctttccc cctcccgatc gccacggcag aactcatcgc

3840

cgcctgcctt gcccgctgct ggacaggggc taggttgctg ggcactgata attccgtggt

3900

gttgtcgggg aagggcctcg ataccgtcga tatcgatcct ggctaataaa ggaaatttat

3960

tttcattgca atagtgtgtt ggaatttttt gtgtctctca ctcggaagga catatgggag

4020

ggcaaatcat ttaaaacatc agaatgagta tttggtttag agtttggcaa catatgccca

4080

tatgctggct gccatgaaca aaggttggct ataaagaggt catcagtata tgaaacagcc

4140

ccctgctgtc cattccttat tccatagaaa agccttgact tgaggttaga tttttttttat

4200

attttgtttt gtgttatttt tttctttaac atccctaaaa ttttccttac atgtttttact

4260

agccagattt ttcctcctct cctgactact cccagtcata gctgtccctc ttctcttatg

4320

gagatcgaag caattcgttg atctgaattt cgaccaccca taatagatct cccattaccc

4380

tggtagataa gtagcatggc gggttaatca ttaactacaa ggaaccccta gtgatggagt

4440

tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc

4500

gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcag

4549

<210> 9

<211> 4549

<212> DNA

<213> Artificial sequence

<220>

<223> Expression Cassette

<400> 9

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt

60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact

120

aggggttcct tgtagttaat gattaacccg ccatgctact tatctaccag ggtaatgggg

180

atcctctaga actatagcta gtcgacattg attattgact agttattaat agtaatcaat

240

tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa

300

tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt

360

tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta

420

aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt

480

caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc

540

tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac

600

gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat

660

tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggggcgcg cgccaggcgg

720

ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca

780

gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa

840

aaagcgaagc gcgcggcggg cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc

900

cgccgccgcc tcgcgccgcc cgccccggct ctgactgacc gcgttactcc cacaggtgag

960

cgggcgggac ggcccttctc ctccggggctg taattagcgc ttggtttaat gacggcttgt

1020

ttcttttctg tggctgcgtg aaagccttga ggggctccgg gagggccctt tgtgcggggg

1080

gagcggctcg gggggtgcgt gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggctccgc

1140

gctgcccggc ggctgtgagc gctgcgggcg cggcgcgggg ctttgtgcgc tccgcagtgt

1200

gcgcgagggg agcgcggccg ggggcggtgc cccgcggtgc ggggggggct gcgaggggaa

1260

caaaggctgc gtgcggggtg tgtgcgtggg ggggtgagca gggggtgtgg gcgcgtcggt

1320

cgggctgcaa ccccccctgc acccccctcc ccgagttgct gagcacggcc cggcttcggg

1380

tgcggggctc cgtacggggc gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca

1440

ggtggggggtg ccgggcgggg cggggccgcc tcgggccggg gagggctcgg gggaggggcg

1500

cggcggcccc cggagcgccg gcggctgtcg aggcgcggcg agccgcagcc attgcctttt

1560

atggtaatcg tgcgagaggg cgcagggact tcctttgtcc caaatctgtg cggagccgaa

1620

atctgggagg cgccgccgca ccccctctag cgggcgcggg gcgaagcggt gcggcgccgg

1680

caggaaggaa atgggcgggg agggccttcg tgcgtcgccg cgccgccgtc cccttctccc

1740

tctccagcct cggggctgtc cgcgggggga cggctgcctt cgggggggac ggggcagggc

1800

ggggttcggc ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc atgttcatgc

1860

cttcttcttt ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt

1920

tggcaaagaa ttcgagcggc cgccagccgc caccatggtg tgctttagac tgtttcctgt

1980

gcctggttca gggctggtcc tggtctgtct ggtgctgggg gctgtcagaa gctatgcctt

2040

ggagctgaac ctcactgata gtgaaaatgc cacttgtctg tatgctaagt ggcagatgaa

2100

cttcactgtg agatatgaaa ccaccaacaa gacttacaaa acagtgacca tctcagatca

2160

tggaactgtg acctacaacg gcagcatttg tggagacgac cagaacggac caaaaatcgc

2220

tgtccaattt gggcctggat tctcctggat tgccaatttc actaaagctg cctccacata

2280

ttcaattgac tcagtgtcct tctcctacaa cactggggac aacactactt tccctgatgc

2340

tgaagataag ggaatcttga cagtggatga gctgctggct atcaggatcc ctttgaatga

2400

cctgtttagg tgtaattcac tgagcactct ggagaagaac gacgtggtgc agcactactg

2460

ggacgtgctg gtgcaggcct ttgtgcagaa cggcactgtg tccaccaacg aattcctgtg

2520

tgataaggac aaaacttcca ctgtggcacc tacaattcac actactgtgc cttcacctac

2580

caccactcca actccaaagg aaaagcctga agcaggaacc tactctgtga acaatggcaa

2640

tgatacctgt ctgttggcca ccatgggcct ccaactgaac attactcagg acaaggtggc

2700

ctcagtgatt aacattaacc ccaacactac ccactccact ggcagctgta gatcacacac

2760

agccttgctc agactgaata gcagcaccat caagtatttg gattttgtgt ttgcagtgaa

2820

gaatgaaaac aggttctacc tgaaggaagt caacatctca atgtacctgg tgaacggctc

2880

agtgttcagc attgccaaca acaacctctc ctattgggac gctccactgg ggagcagcta

2940

catgtgtaac aaggaacaga ctgtgtcagt gtcaggagcc ttccagatta acacctttga

3000

tctgaggtc caacccttta atgtcactca aggaaagtat agcactgccc aggagtgctc

3060

cctggatgat gacaccattc tgattccaat cattgtgggt gcaggacttt ctgggcttat

3120

tattgtgatt gtgattgcct atgtgattgg cagaaggaaa tcctatgcag ggtaccaaac

3180

tctgtaaaag ggcgaattcc agcacacgcg tcctaggagc tcgagtacta ctggcggccg

3240

ttactagtgg atccgcggta caagtaagca tgcaagcttc gaggacgggg tgaactacgc

3300

ctgaatcaag cttatcgata aattcgagca tcttaccgcc atttattccc atatttgttc

3360

tgtttttctt gatttgggta tacatttaaa tgttaataaa acaaaatggt ggggcaatca

3420

tttacatttt tagggatatg taattactag ttcaggtgta ttgccacaag acaaacatgt

3480

taagaaactt tcccgttatt tacgctctgt tcctgttaat caacctctgg attacaaaat

3540

ttgtgaaaga ttgactgata ttcttaacta tgttgctcct tttacgctgt gtggatatgc

3600

tgctttaatg cctctgtatc atgctattgc ttcccgtacg gctttcgttt tctcctcctt

3660

gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtcc gtcaacgtgg

3720

cgtggtgtgc tctgtgtttg ctgacgcaac ccccactggc tggggcattg ccaccacctg

3780

tcaactcctt tctgggactt tcgctttccc cctcccgatc gccacggcag aactcatcgc

3840

cgcctgcctt gcccgctgct ggacaggggc taggttgctg ggcactgata attccgtggt

3900

gttgtcgggg aagggcctcg ataccgtcga tatcgatcct ggctaataaa ggaaatttat

3960

tttcattgca atagtgtgtt ggaatttttt gtgtctctca ctcggaagga catatgggag

4020

ggcaaatcat ttaaaacatc agaatgagta tttggtttag agtttggcaa catatgccca

4080

tatgctggct gccatgaaca aaggttggct ataaagaggt catcagtata tgaaacagcc

4140

ccctgctgtc cattccttat tccatagaaa agccttgact tgaggttaga tttttttttat

4200

attttgtttt gtgttatttt tttctttaac atccctaaaa ttttccttac atgtttttact

4260

agccagattt ttcctcctct cctgactact cccagtcata gctgtccctc ttctcttatg

4320

gagatcgaag caattcgttg atctgaattt cgaccaccca taatagatct cccattaccc

4380

tggtagataa gtagcatggc gggttaatca ttaactacaa ggaaccccta gtgatggagt

4440

tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc

4500

gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcag

4549

<210> 10

<211> 4549

<212> DNA

<213> Artificial sequence

<220>

<223> Expression Cassette

<400> 10

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt

60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact

120

aggggttcct tgtagttaat gattaacccg ccatgctact tatctaccag ggtaatgggg

180

atcctctaga actatagcta gtcgacattg attattgact agttattaat agtaatcaat

240

tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa

300

tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt

360

tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta

420

aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt

480

caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc

540

tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac

600

gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat

660

tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggggcgcg cgccaggcgg

720

ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca

780

gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa

840

aaagcgaagc gcgcggcggg cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc

900

cgccgccgcc tcgcgccgcc cgccccggct ctgactgacc gcgttactcc cacaggtgag

960

cgggcgggac ggcccttctc ctccggggctg taattagcgc ttggtttaat gacggcttgt

1020

ttcttttctg tggctgcgtg aaagccttga ggggctccgg gagggccctt tgtgcggggg

1080

gagcggctcg gggggtgcgt gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggctccgc

1140

gctgcccggc ggctgtgagc gctgcgggcg cggcgcgggg ctttgtgcgc tccgcagtgt

1200

gcgcgagggg agcgcggccg ggggcggtgc cccgcggtgc ggggggggct gcgaggggaa

1260

caaaggctgc gtgcggggtg tgtgcgtggg ggggtgagca gggggtgtgg gcgcgtcggt

1320

cgggctgcaa ccccccctgc acccccctcc ccgagttgct gagcacggcc cggcttcggg

1380

tgcggggctc cgtacggggc gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca

1440

ggtggggggtg ccgggcgggg cggggccgcc tcgggccggg gagggctcgg gggaggggcg

1500

cggcggcccc cggagcgccg gcggctgtcg aggcgcggcg agccgcagcc attgcctttt

1560

atggtaatcg tgcgagaggg cgcagggact tcctttgtcc caaatctgtg cggagccgaa

1620

atctgggagg cgccgccgca ccccctctag cgggcgcggg gcgaagcggt gcggcgccgg

1680

caggaaggaa atgggcgggg agggccttcg tgcgtcgccg cgccgccgtc cccttctccc

1740

tctccagcct cggggctgtc cgcgggggga cggctgcctt cgggggggac ggggcagggc

1800

ggggttcggc ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc atgttcatgc

1860

cttcttcttt ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt

1920

tggcaaagaa ttcgagcggc cgccagccgc caccatggtc tgttttaggc tgttccctgt

1980

ccctggttca ggactggtct tagtgtgtct ggtgcttgga gctgtcagaa gctatgccct

2040

ggagctgaac ctgactgact cagaaaatgc cacttgcctg tatgccaagt ggcagatgaa

2100

cttcactgtc agatatgaaa ccaccaacaa gacctataag actgtgacca tctcagacca

2160

tggcactgtg acttacaatg ggtcaatttg tggagatgac cagaatggcc ctaagatagc

2220

tgtccagttt ggtccaggat tcagctggat tgccaacttc accaaggcag ccagcaccta

2280

cagcattgac tctgtgtcct tctcctacaa cacaggagac aacaccactt tccctgatgc

2340

agaggacaaa ggtatcctga ctgtggatga gttgctggca atcaggatcc cactgaacga

2400

tctgttcagg tgcaactcac tgtccactct ggaaaagaat gatgtggtgc agcactattg

2460

ggatgtgcta gtccaggcct ttgtccagaa tgggactgtg tcaactaatg agttcctgtg

2520

tgacaaggac aagacaagca ctgtagcccc cactatccat accaccagtac ctagccccac

2580

cactactcca acccccaagg agaagcctga ggctggcacc tactcagtga acaatgggaa

2640

tgacacctgt ttgctggcca ctatgggact ccaactgaac atcacccagg acaaagtggc

2700

ctctgtgatc aatatcaatc ccaacaccac ccacagcact gggtcctgca gaagccacac

2760

tgccctcctg aggctcaact catcaactat caagtacttg gattttgtgt ttgcagtgaa

2820

gaatgagaac agattctacc tcaaagaggt caacatttca atgtacctgg tgaatgggag

2880

tgtgttctcc attgctaaca acaacctgag ctactgggat gcccctctgg gctcctcata

2940

catgtgcaac aaggaacaga ctgtgagtgt gtcaggggcc ttccagatca acacttttga

3000

cctgagagtg cagcccttta atgtgacaca gggaaagtac agcactgctc aggagtgcag

3060

cctggatgat gacactatcc tgatccctat cattgtgggg gcaggcctgt ctggactcat

3120

tattgtgatt gtgattgcct atgtgatagg gagaaggaag tcttatgctg gataccagac

3180

cctgtaaaag ggcgaattcc agcacacgcg tcctaggagc tcgagtacta ctggcggccg

3240

ttactagtgg atccgcggta caagtaagca tgcaagcttc gaggacgggg tgaactacgc

3300

ctgaatcaag cttatcgata aattcgagca tcttaccgcc atttattccc atatttgttc

3360

tgtttttctt gatttgggta tacatttaaa tgttaataaa acaaaatggt ggggcaatca

3420

tttacatttt tagggatatg taattactag ttcaggtgta ttgccacaag acaaacatgt

3480

taagaaactt tcccgttatt tacgctctgt tcctgttaat caacctctgg attacaaaat

3540

ttgtgaaaga ttgactgata ttcttaacta tgttgctcct tttacgctgt gtggatatgc

3600

tgctttaatg cctctgtatc atgctattgc ttcccgtacg gctttcgttt tctcctcctt

3660

gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtcc gtcaacgtgg

3720

cgtggtgtgc tctgtgtttg ctgacgcaac ccccactggc tggggcattg ccaccacctg

3780

tcaactcctt tctgggactt tcgctttccc cctcccgatc gccacggcag aactcatcgc

3840

cgcctgcctt gcccgctgct ggacaggggc taggttgctg ggcactgata attccgtggt

3900

gttgtcgggg aagggcctcg ataccgtcga tatcgatcct ggctaataaa ggaaatttat

3960

tttcattgca atagtgtgtt ggaatttttt gtgtctctca ctcggaagga catatgggag

4020

ggcaaatcat ttaaaacatc agaatgagta tttggtttag agtttggcaa catatgccca

4080

tatgctggct gccatgaaca aaggttggct ataaagaggt catcagtata tgaaacagcc

4140

ccctgctgtc cattccttat tccatagaaa agccttgact tgaggttaga tttttttttat

4200

attttgtttt gtgttatttt tttctttaac atccctaaaa ttttccttac atgtttttact

4260

agccagattt ttcctcctct cctgactact cccagtcata gctgtccctc ttctcttatg

4320

gagatcgaag caattcgttg atctgaattt cgaccaccca taatagatct cccattaccc

4380

tggtagataa gtagcatggc gggttaatca ttaactacaa ggaaccccta gtgatggagt

4440

tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc

4500

gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcag

4549

<210> 11

<211> 130

<212> DNA

<213> Unknown

<220>

<223> Inverted terminal repeat (ITR) sequence

<400> 11

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt

60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact

120

aggggttcct

130

<210> 12

<211> 130

<212> DNA

<213> Unknown

<220>

<223> Inverted terminal repeat (ITR) sequence

<400> 12

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg

60

ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc

120

gagcgcgcag

130

<210> 13

<400> 13

000

<210> 14

<211> 13

<212> RNA

<213> Unknown

<220>

<223> Alternate Kozak sequence

<220>

<221> Other signs

<222> (1)..(3)

<223> May be missing

<220>

<221> Other signs

<222> (1)..(1)

<223> g represents the most common base,

but other options are possible

<220>

<221> Other signs

<222> (2)..(3)

<223> c represents the most common base,

but other options are possible

<220>

<221> other signs

<222> (4)..(4)

<223> g represents the most common base,

but other options are possible

<220>

<221> Other signs

<222> (5)..(6)

<223> c represents the most common base,

but other options are possible

<220>

<221> Other signs

<222> (8)..(9)

<223> c represents the most common base,

but other options are possible

<400> 14

nnnnnnrnna ugg

13

<210> 15

<211> 8

<212> RNA

<213> Unknown

<220>

<223> Alternate Kozak sequence

<220>

<221> Other signs

<222> (3)..(4)

<223> n represents a, c, g or u

<220>

<221> Other signs

<222> (8)..(8)

<223> n represents a, c, g or u

<400> 15

agnnaugn

8

<210> 16

<211> 7

<212> RNA

<213> Unknown

<220>

<223> Alternate Kozak sequence

<220>

<221> Other signs

<222> (2)..(3)

<223> n represents a, c, g or u

<400> 16

annaugg

7

<210> 17

<211> 7

<212> RNA

<213> Unknown

<220>

<223> Alternate Kozak sequence

<400> 17

accaugg

7

<210> 18

<211> 10

<212> RNA

<213> Unknown

<220>

<223> Alternate Kozak sequence

<400> 18

gacaccaugg

10

<210> 19

<211> 235

<212> DNA

<213> Bos taurus

<400> 19

tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg

60

accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat

120

tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag

180

gattgggagg acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctg

235

<210> 20

<211> 222

<212> DNA

<213> Monkey virus 40

<400> 20

cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa

60

aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca

120

ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag ggggagatgt

180

gggaggtttt ttaaagcaag taaaacctct acaaatgtgg ta

222

<210> 21

<211> 202

<212> DNA

<213> Homo sapiens

<400> 21

ctgcccgggt ggcatccctg tgacccctcc ccagtgcctc tcctggccct ggaagttgcc

60

actccagtgc ccaccagcct tgtcctaata aaattaagtt gcatcatttt gtctgactag

120

gtgtccttct ataatattat ggggtggagg ggggtggtat ggagcaaggg gcccaagttg

180

ggaagaaacc tgtagggcct gc

202

<210> 22

<211> 1730

<212> DNA

<213> Unknown

<220>

<223> CAG promoter

<400> 22

ctagtcgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc

60

atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac

120

cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa

180

tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag

240

tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc

300

ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct

360

acgtattagt catcgctatt accatggtcg aggtgagccc cacgttctgc ttcactctcc

420

ccatctcccc cccctcccca cccccaattt tgtatttatt tattttttaa ttattttgtg

480

cagcgatggg ggcggggggg gggggggggc gcgcgccagg cggggcgggg cggggcgagg

540

ggcggggcgg ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa

600

agtttccttt tatggcgagg cggcggcggc ggcggcccta taaaaagcga agcgcgcggc

660

gggcgggagt cgctgcgcgc tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc

720

gcccgccccg gctctgactg accgcgttac tcccacaggt gagcgggcgg gacggccctt

780

ctcctccggg ctgtaattag cgcttggttt aatgacggct tgtttctttt ctgtggctgc

840

gtgaaagcct tgaggggctc cgggaggggcc ctttgtgcgg ggggagcggc tcggggggtg

900

cgtgcgtgtg tgtgtgcgtg gggagcgccg cgtgcggctc cgcgctgccc ggcggctgtg

960

agcgctgcgg gcgcggcgcg gggctttgtg cgctccgcag tgtgcgcgag gggagcgcgg

1020

ccggggggcgg tgccccgcgg tgcggggggg gctgcgaggg gaacaaaggc tgcgtgcggg

1080

gtgtgtgcgt gggggggtga gcaggggggtg tgggcgcgtc ggtcgggctg caaccccccc

1140

tgcacccccc tccccgagtt gctgagcacg gcccggcttc gggtgcgggg ctccgtacgg

1200

ggcgtggcgc ggggctcgcc gtgccgggcg gggggtggcg gcaggtgggg gtgccgggcg

1260

gggcggggcc gcctcgggcc ggggagggct cgggggaggg gcgcggcggc ccccggagcg

1320

ccggcggctg tcgaggcgcg gcgagccgca gccattgcct tttatggtaa tcgtgcgaga

1380

gggcgcaggg acttcctttg tcccaaatct gtgcggagcc gaaatctggg aggcgccgcc

1440

gcaccccctc tagcgggcgc ggggcgaagc ggtgcggcgc cggcaggaag gaaatgggcg

1500

ggggagggcct tcgtgcgtcg ccgcgccgcc gtccccttct ccctctccag cctcggggct

1560

gtccgcgggg ggacggctgc cttcgggggg gacggggcag ggcggggttc ggcttctggc

1620

gtgtgaccgg cggctctaga gcctctgcta accatgttca tgccttcttc tttttcctac

1680

agctcctggg caacgtgctg gttattgtgc tgtctcatca ttttggcaaa

1730

<210> 23

<211> 204

<212> DNA

<213> Human cytomegalovirus

<400> 23

gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt

60

ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac

120

tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg

180

tgggaggtct atataagcag agct

204

<210> 24

<211> 317

<212> DNA

<213> Monkey virus 40

<400> 24

ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt

60

agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca

120

tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa

180

ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag

240

aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag

300

gcctaggctt ttgcaaa

317

<210> 25

<211> 500

<212> DNA

<213> Unknown

<220>

<223> PGK promoter

<400> 25

gggtagggga ggcgcttttc ccaaggcagt ctggagcatg cgctttagca gccccgctgg

60

gcacttggcg ctacacaagt ggcctctggc ctcgcacaca ttccacatcc accggtaggc

120

gccaaccggc tccgttcttt ggtggcccct tcgcgccacc ttctactcct cccctagtca

180

ggaagttccc ccccgccccg cagctcgcgt cgtgcaggac gtgacaaatg gaagtagcac

240

gtctcactag tctcgtgcag atggacagca ccgctgagca atggaagcgg gtaggccttt

300

ggggcagcgg ccaatagcag ctttgctcct tcgctttctg ggctcagagg ctgggaaggg

360

gtgggtccgg gggcgggctc aggggcgggc tcaggggcgg ggcgggcgcc cgaaggtcct

420

ccggaggccc ggcattctgc acgcttcaaa agcgcacgtc tgccgcgctg ttctcctctt

480

cctcatctcc gggcctttcg

500

<210> 26

<211> 2403

<212> DNA

<213> Homo sapiens

<400> 26

cctgcagggc ccactagttc catgtcctta tatggactca tctttgccta ttgcgacaca

60

cactcaatga acacctacta cgcgctgcaa agagccccgc aggcctgagg tgcccccacc

120

tcaccactct tcctattttt gtgtaaaaat ccagcttctt gtcaccacct ccaaggaggg

180

ggaggaggag gaaggcaggt tcctctaggc tgagccgaat gcccctctgt ggtcccacgc

240

cactgatcgc tgcatgccca ccacctgggt acacacagtc tgtgattccc ggagcagaac

300

ggaccctgcc cacccggtct tgtgtgctac tcagtggaca gacccaaggc aagaaagggt

360

gacaaggaca gggtcttccc aggctggctt tgagttccta gcaccgcccc gcccccaatc

420

ctctgtggca catggagtct tggtccccag agtcccccag cggcctccag atggtctggg

480

agggcagttc agctgtggct gcgcatagca gacatacaac ggacggtggg cccagaccca

540

ggctgtgtag acccagcccc cccgccccgc agtgcctagg tcacccacta acgccccagg

600

cctggtcttg gctgggcgtg actgttaccc tcaaaagcag gcagctccag ggtaaaaggt

660

gccctgccct gtagagccca ccttccttcc cagggctgcg gctgggtagg tttgtagcct

720

tcatcacggg ccacctccag ccactggacc gctggcccct gccctgtcct ggggagtgtg

780

gtcctgcgac ttctaagtgg ccgcaagcca cctgactccc ccaacaccac actctacctc

840

tcaagcccag gtctctccct agtgacccac ccagcacatt tagctagctg agccccacag

900

ccagaggtcc tcaggccctg ctttcagggc agttgctctg aagtcggcaa gggggagtga

960

ctgcctggcc actccatgcc ctccaagagc tccttctgca ggagcgtaca gaacccaggg

1020

ccctggcacc cgtgcagacc ctggcccacc ccacctgggc gctcagtgcc caagagatgt

1080

cccacacctag gatgtcccgc ggtgggtggg gggcccgaga gacgggcagg ccggggggcag

1140

gcctggccat gcggggccga accgggcact gcccagcgtg gggcgcgggg gccacggcgc

1200

gcgcccccag cccccggggcc cagcacccca aggcggccaa cgccaaaact ctccctcctc

1260

ctcttcctca atctcgctct cgctcttttt ttttttcgca aaaggagggg agagggggta

1320

aaaaaatgct gcactgtgcg gcgaagccgg tgagtgagcg gcgcggggcc aatcagcgtg

1380

cgccgttccg aaagttgcct tttatggctc gagcggccgc ggcggcgccc tataaaaccc

1440

agcggcgcga cgcgccacca ccgccgagac cgcgtccgcc ccgcgagcac agagcctcgc

1500

ctttgccgat ccgccgcccg tccacacccg ccgccaggta agcccggcca gccgaccggg

1560

gcaggcggct cacggcccgg ccgcaggcgg ccgcggcccc ttcgcccgtg cagagccgcc

1620

gtctgggccg cagcgggggg cgcatggggg gggaaccgga ccgccgtggg gggcgcggga

1680

gaagcccctg ggcctccgga gatgggggac accccacgcc agttcggagg cgcgaggccg

1740

cgctcgggag gcgcgctccg ggggtgccgc tctcggggcg ggggcaaccg gcggggtctt

1800

tgtctgagcc gggctcttgc caatggggat cgcagggtgg gcgcggcgga gcccccgcca

1860

ggcccggtgg gggctggggc gccattgcgc gtgcgcgctg gtcctttggg cgctaactgc

1920

gtgcgcgctg ggaattggcg ctaattgcgc gtgcgcgctg ggactcaagg cgctaactgc

1980

gcgtgcgttc tggggcccgg ggtgccgcgg cctgggctgg ggcgaaggcg ggctcggccg

2040

gaaggggtgg ggtcgccgcg gctcccgggc gcttgcgcgc acttcctgcc cgagccgctg

2100

gccgcccgag ggtgtggccg ctgcgtgcgc gcgcgccgac ccggcgctgt ttgaaccggg

2160

cggaggcggg gctggcgccc ggttgggagg gggttggggc ctggcttcct gccgcgcgcc

2220

gcggggacgc ctccgaccag tgtttgcctt ttatggtaat aacgcggccg gcccggcttc

2280

ctttgtcccc aatctgggcg cgcgccggcg ccccctggcg gcctaaggac tcggcgcgcc

2340

ggaagtggcc agggcggggg cgacctcggc tcacagcgcg cccggctatt ctcgcagctc

2400

acc

2403

<210> 27

<211> 596

<212> DNA

<213> Woodchuck hepatitis virus

<400> 27

attcgagcat cttaccgcca tttattccca tatttgttct gtttttcttg atttgggtat

60

acatttaaat gttaataaaa caaaatggtg gggcaatcat ttacattttt agggatatgt

120

aattactagt tcaggtgtat tgccacaaga caaacatgtt aagaaacttt cccgttattt

180

acgctctgtt cctgttaatc aacctctgga ttacaaaatt tgtgaaagat tgactgatat

240

tcttaactat gttgctcctt ttacgctgtg tggatatgct gctttaatgc ctctgtatca

300

tgctattgct tcccgtacgg ctttcgtttt ctcctccttg tataaatcct ggttgctgtc

360

tctttatgag gagttgtggc ccgttgtccg tcaacgtggc gtggtgtgct ctgtgtttgc

420

tgacgcaacc cccactggct ggggcattgc caccacctgt caactccttt ctgggacttt

480

cgctttcccc ctcccgatcg ccacggcaga actcatcgcc gcctgccttg cccgctgctg

540

gacaggggct aggttgctgg gcactgataa ttccgtggtg ttgtcgggga agggcc

596

<210> 28

<211> 736

<212> PROTEIN

<213> Adeno-associated virus 9

<400> 28

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Gln Pro

20 25 30

Lys Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ala Gly Ile Gly

145 150 155 160

Lys Ser Gly Ala Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro Pro

180 185 190

Ala Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Val Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Asn Ser Thr Ser Gly Gly Ser Ser Asn Asp Asn

260 265 270

Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg

275 280 285

Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn

290 295 300

Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile

305 310 315 320

Gln Val Lys Glu Val Thr Asp Asn Asn Gly Val Lys Thr Ile Ala Asn

325 330 335

Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Asp Tyr Gln Leu

340 345 350

Pro Tyr Val Leu Gly Ser Ala His Glu Gly Cys Leu Pro Pro Phe Pro

355 360 365

Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asp

370 375 380

Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe

385 390 395 400

Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Glu

405 410 415

Phe Glu Asn Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu

420 425 430

Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser

435 440 445

Lys Thr Ile Asn Gly Ser Gly Gln Asn Gln Gln Thr Leu Lys Phe Ser

450 455 460

Val Ala Gly Pro Ser Asn Met Ala Val Gln Gly Arg Asn Tyr Ile Pro

465 470 475 480

Gly Pro Ser Tyr Arg Gln Gln Arg Val Ser Thr Thr Val Thr Gln Asn

485 490 495

Asn Asn Ser Glu Phe Ala Trp Pro Gly Ala Ser Ser Trp Ala Leu Asn

500 505 510

Gly Arg Asn Ser Leu Met Asn Pro Gly Pro Ala Met Ala Ser His Lys

515 520 525

Glu Gly Glu Asp Arg Phe Phe Pro Leu Ser Gly Ser Leu Ile Phe Gly

530 535 540

Lys Gln Gly Thr Gly Arg Asp Asn Val Asp Ala Asp Lys Val Met Ile

545 550 555 560

Thr Asn Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu Ser

565 570 575

Tyr Gly Gln Val Ala Thr Asn His Gln Ser Ala Gln Ala Gln Ala Gln

580 585 590

Thr Gly Trp Val Gln Asn Gln Gly Ile Leu Pro Gly Met Val Trp Gln

595 600 605

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620

Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Met

625 630 635 640

Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala

645 650 655

Asp Pro Pro Thr Ala Phe Asn Lys Asp Lys Leu Asn Ser Phe Ile Thr

660 665 670

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680 685

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn

690 695 700

Tyr Tyr Lys Ser Asn Asn Val Glu Phe Ala Val Asn Thr Glu Gly Val

705 710 715 720

Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu

725 730 735

<210> 29

<211> 1233

<212> DNA

<213> Homo sapiens

<400> 29

atggtgtgct tccgcctctt cccggttccg ggctcagggc tcgttctggt ctgcctagtc

60

ctgggagctg tgcggtctta tgcattggaa cttaatttga cagattcaga aaatgccact

120

tgcctttatg caaaatggca gatgaatttc acagtacgct atgaaactac aaataaaact

180

tataaaactg taaccatttc agaccatggc actngtgacat ataatggaag catttgtggg

240

gatgatcaga atggtcccaa aatagcagtg cagttcggac ctggcttttc ctggattgcg

300

aattttacca aggcagcatc tacttattca attgacagcg tctcattttc ctacaacact

360

ggtgataaca caacatttcc tgatgctgaa gataaaggaa ttcttactgt tgatgaactt

420

ttggccatca gaattccatt gaatgacctt tttagatgca atagtttatc aactttggaa

480

aagaatgatg ttgtccaaca ctactgggat gttcttgtac aagcttttgt ccaaaatggc

540

acagtgagca caaatgagtt cctgtgtgat aaagacaaaa cttcaacagt ggcacccacc

600

atacacacca ctgtgccatc tcctactaca acacctactc caaaggaaaa accagaagct

660

ggaacctatt cagttaataa tggcaatgat acttgtctgc tggctaccat ggggctgcag

720

ctgaacatca ctcaggataa ggttgcttca gttattaaca tcaaccccaa tacaactcac

780

tccacaggca gctgccgttc tcacactgct ctacttagac tcaatagcag caccattaag

840

tatctagact ttgtctttgc tgtgaaaaat gaaaaccgat tttatctgaa ggaagtgaac

900

atcagcatgt atttggttaa tggctccgtt ttcagcattg caaataacaa tctcagctac

960

tgggatgccc ccctgggaag ttcttatatg tgcaacaaag agcagactgt ttcagtgtct

1020

ggagcatttc agataaatac ctttgatcta agggttcagc ctttcaatgt gacacaagga

1080

aagtattcta cagctcaaga ctgcagtgca gatgacgaca acttccttgt gcccatagcg

1140

gtgggagctg ccttggcagg agtacttatt ctagtgttgc tggcttattt tattggtctc

1200

aagcaccatc atgctggata tgagcaattt tag

1233

<210> 30

<211> 1236

<212> DNA

<213> Homo sapiens

<400> 30

atggtgtgct tccgcctctt cccggttccg ggctcagggc tcgttctggt ctgcctagtc

60

ctgggagctg tgcggtctta tgcattggaa cttaatttga cagattcaga aaatgccact

120

tgcctttatg caaaatggca gatgaatttc acagtacgct atgaaactac aaataaaact

180

tataaaactg taaccatttc agaccatggc actngtgacat ataatggaag catttgtggg

240

gatgatcaga atggtcccaa aatagcagtg cagttcggac ctggcttttc ctggattgcg

300

aattttacca aggcagcatc tacttattca attgacagcg tctcattttc ctacaacact

360

ggtgataaca caacatttcc tgatgctgaa gataaaggaa ttcttactgt tgatgaactt

420

ttggccatca gaattccatt gaatgacctt tttagatgca atagtttatc aactttggaa

480

aagaatgatg ttgtccaaca ctactgggat gttcttgtac aagcttttgt ccaaaatggc

540

acagtgagca caaatgagtt cctgtgtgat aaagacaaaa cttcaacagt ggcacccacc

600

atacacacca ctgtgccatc tcctactaca acacctactc caaaggaaaa accagaagct

660

ggaacctatt cagttaataa tggcaatgat acttgtctgc tggctaccat ggggctgcag

720

ctgaacatca ctcaggataa ggttgcttca gttattaaca tcaaccccaa tacaactcac

780

tccacaggca gctgccgttc tcacactgct ctacttagac tcaatagcag caccattaag

840

tatctagact ttgtctttgc tgtgaaaaat gaaaaccgat tttatctgaa ggaagtgaac

900

atcagcatgt atttggttaa tggctccgtt ttcagcattg caaataacaa tctcagctac

960

tgggatgccc ccctgggaag ttcttatatg tgcaacaaag agcagactgt ttcagtgtct

1020

ggagcatttc agataaatac ctttgatcta agggttcagc ctttcaatgt gacacaagga

1080

aagtattcta cagctgaaga atgttctgct gactctgacc tcaactttct tattcctgtt

1140

gcagtgggtg tggccttggg cttccttata attgttgtct ttatctctta tatgattgga

1200

agaaggaaaa gtcgtactgg ttatcagtct gtgtaa

1236

<210> 31

<211> 1000

<212> DNA

<213> Artificial sequence

<220>

<223> Option LAMP-2

<400> 31

atggtctgct tcagactgtt ccctgtccct ggatctggtc tggtgcttgt gtgcttggtg

60

ctgggtgctg tgagatccta tgcccttgag ctgaacctga ctgactcaga aaatgccact

120

tgcctgtatg ccaagtggca gatgaacttc actngtgagat atgagactac caacaagacc

180

tacaagactg tgaccatctc agaccatggc actgtcacct acaatggatc aatctgtggt

240

gatgatcaga atggcccaaa gatagcagtg cagtttgggc ccggtttttc ctggattgct

300

aacttcacca aggcagcctc cacctacagc attgactcag tcagcttcag ctacaacact

360

ggggataaca ccaccttccc tgacgcagag gacaagggaa tccttactgt ggacgaactc

420

ctggcaatca gaatccccct taacgacctg ttcagatgca actccctttc aacccttgaa

480

aagaatgatg tggtgcaaca ctattgggac gtcctggtgc aagcctttgt gcagaatggg

540

acagtgagta ccaacgagtt cctctgtgac aaggacaaga ccagcactgt ggcccccact

600

atccacacca ctgtgcccag ccctaccact acccccaccc ctaaagagaa gccagaagct

660

ggaacctact cagtcaacaa tggaaatgac acatgcctcc ttgccaccat gggactgcag

720

ctgaacatca ctcaggacaa ggtggcctca gtgattaaca tcaaccctaa caccactcat

780

agcactggga gctgcagatc acatacagct ctgctgaggc tcaactcctc caccatcaag

840

tacctggact ttgtgtttgc tgtgaagaat gagaacaggt tctacctcaa ggaagtgaac

900

atttccatgt acctggtcaa tggttcagtg ttctctattg ccaacaacaa tctgagctac

960

tgggatgcac ccctgggatc ctcctacatg tgcaacaagg

1000

<210> 32

<211> 1000

<212> DNA

<213> Artificial sequence

<220>

<223> Option LAMP-2

<400> 32

atggtgtgct ttagactgtt tcctgtgcct ggttcagggc tggtcctggt ctgtctggtg

60

ctggggggctg tcagaagcta tgccttggag ctgaacctca ctgatagtga aaatgccact

120

tgtctgtatg ctaagtggca gatgaacttc actngtgagat atgaaaccac caacaagact

180

tacaaaacag tgaccatctc agatcatgga actngtgacct acaacggcag catttgtgga

240

gacgaccaga acggaccaaa aatcgctgtc caatttgggc ctggattctc ctggattgcc

300

aatttcacta aagctgcctc cacatattca attgactcag tgtccttctc ctacaacact

360

ggggacaaca ctactttccc tgatgctgaa gataagggaa tcttgacagt ggatgagctg

420

ctggctatca ggatcccttt gaatgacctg tttaggtgta attcactgag cactctggag

480

aagaacgacg tggtgcagca ctactgggac gtgctggtgc aggcctttgt gcagaacggc

540

actgtgtcca ccaacgaatt cctgtgtgat aaggacaaaa cttccactgt ggcacctaca

600

attcacacta ctgtgccttc acctaccacc actccaactc caaaggaaaa gcctgaagca

660

ggaacctact ctgtgaacaa tggcaatgat acctgtctgt tggccaccat gggcctccaa

720

ctgaacatta ctcaggacaa ggtggcctca gtgattaaca ttaaccccaa cactacccac

780

tccactggca gctgtagatc acacacagcc ttgctcagac tgaatagcag caccatcaag

840

tatttggatt ttgtgtttgc agtgaagaat gaaaacaggt tctacctgaa ggaagtcaac

900

atctcaatgt acctggtgaa cggctcagtg ttcagcattg ccaacaacaa cctctcctat

960

tgggacgctc cactggggag cagctacatg tgtaacaagg

1000

<210> 33

<211> 1000

<212> DNA

<213> Artificial sequence

<220>

<223> Option LAMP-2

<400> 33

atggtctgtt ttaggctgtt ccctgtccct ggttcaggac tggtcttagt gtgtctggtg

60

cttggagctg tcagaagcta tgccctggag ctgaacctga ctgactcaga aaatgccact

120

tgcctgtatg ccaagtggca gatgaacttc actgtcagat atgaaaccac caacaagacc

180

tataagactg tgaccatctc agaccatggc actgtgactt acaatgggtc aatttgtgga

240

gatgaccaga atggccctaa gatagctgtc cagtttggtc caggattcag ctggattgcc

300

aacttcacca aggcagccag cacctacagc attgactctg tgtccttctc ctacaacaca

360

ggagacaaca ccactttccc tgatgcagag gacaaaggta tcctgactgt ggatgagttg

420

ctggcaatca ggatcccact gaacgatctg ttcaggtgca actcactgtc cactctggaa

480

aagaatgatg tggtgcagca ctattgggat gtgctagtcc aggcctttgt ccagaatggg

540

actgtgtcaa ctaatgagtt cctgtgtgac aaggacaaga caagcactgt agcccccact

600

atccatacca cagtacctag ccccaccact actccaaccc ccaaggagaa gcctgaggct

660

ggcacctact cagtgaacaa tgggaatgac acctgtttgc tggccactat gggactccaa

720

ctgaacatca cccaggacaa agtggcctct gtgatcaata tcaatcccaa caccacccac

780

agcactgggt cctgcagaag ccacactgcc ctcctgaggc tcaactcatc aactatcaag

840

tacttggatt ttgtgtttgc agtgaagaat gagaacagat tctacctcaa agaggtcaac

900

atttcaatgt acctggtgaa tgggagtgtg ttctccatg ctaacaacaa cctgagctac

960

tgggatgccc ctctgggctc ctcatacatg tgcaacaagg

1000

<210> 34

<211> 410

<212> PROTEIN

<213> Homo sapiens

<400> 34

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

Lys His His His Ala Gly Tyr Glu Gln Phe

405 410

<210> 35

<211> 411

<212> PROTEIN

<213> Homo sapiens

<400> 35

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

Arg Arg Lys Ser Arg Thr Gly Tyr Gln Ser Val

405 410

<210> 36

<211> 21

<212> DNA

<213> Woodchuck hepatitis virus

<400> 36

atcatgctat tgcttcccgt a

21

<210> 37

<211> 20

<212> DNA

<213> Woodchuck hepatitis virus

<400> 37

gggccacaac tcctcataaa

20

<210> 38

<211> 29

<212> DNA

<213> Woodchuck hepatitis virus

<400> 38

cctccttgta taaatcctgg ttgctgtct

29

<---

Claims (30)

1. A gene therapy vector comprising an expression cassette that contains a transgene encoding lysosome-associated membrane protein 2B (LAMP-2B) or a functional variant thereof, wherein said transgene is codon-optimized for expression in a human host cell, and wherein the sequence of said transgene has at least 95% identity to a sequence selected from SEQ ID NO: 3-5. 2. Gene therapy vector according to claim 1, characterized in that LAMP-2B contains SEQ ID NO: 1. 3. Gene therapy vector according to any one of paragraphs. 1 and 2, characterized in that said expression cassette contains fewer CpG sites than SEQ ID: 2. 4. Gene therapy vector according to any one of paragraphs. 1-3, characterized in that said expression cassette contains fewer cryptic splice sites than SEQ ID: 2. 5. Gene therapy vector according to any one of paragraphs. 1-4, characterized in that said expression cassette encodes fewer alternative open reading frames than SEQ ID: 2. 6. Gene therapy vector according to any one of paragraphs. 1-5, characterized in that the sequence of said transgene is at least 99% identical to the sequence selected from SEQ ID NO: 3-5. 7. Gene therapy vector according to any one of paragraphs. 1-6, characterized in that said transgene contains a sequence selected from SEQ ID NO: 3-5. 8. Gene therapy vector according to any one of paragraphs. 1–5, characterized in that the sequence of said transgene has at least 95% identity with SEQ ID NO: 3. 9. Gene therapy vector according to claim 8, characterized in that said transgene contains SEQ ID NO: 3. 10. Gene therapy vector according to any one of paragraphs. 1-9, characterized in that said expression cassette contains a consensus optimal Kozak sequence, which optionally contains SEQ ID NO: 6. 11. Gene therapy vector according to any one of paragraphs. 1-10, characterized in that said expression cassette contains a full-length (poly)A sequence, which optionally contains SEQ ID NO: 7. 12. Gene therapy vector according to any one of paragraphs. 1–11, characterized in that the specified expression cassette does not contain a start site in the direction 5′ from the transgene capable of producing alternative mRNAs. 13. Gene therapy vector according to any one of paragraphs. 1–12, characterized in that said expression cassette contains, in the direction from the 5′ to the 3′ end: a first inverted terminal repeat, an enhancer/promoter region, introns, a consensus optimal Kozak sequence, a transgene, an untranslated 3′ region, including a full-length (poly)A sequence, and a second inverted terminal repeat. 14. The gene therapy vector according to claim 13, characterized in that said enhancer/promoter region contains, in the direction from the 5′- to 3′-end, the enhancer of the middle and early genes of cytomegalovirus (CMV IE) and the chicken beta-actin promoter, and at the same time Optionally, the enhancer/promoter region further comprises the first exon and first intron of the chicken beta-actin gene and a splice acceptor of the rabbit beta-globin gene. 15. The gene therapy vector according to claim 1, characterized in that the sequence of said expression cassette has at least 95% identity with the sequence selected from SEQ ID NO: 8-10. 16. Gene therapy vector according to claim 15, characterized in that the sequence of the specified expression cassette is completely identical to the sequence selected from SEQ ID NO: 8-10. 17. The gene therapy vector according to claim 16, characterized in that the sequence of said expression cassette is identical to SEQ ID NO: 8. 18. Gene therapy vector according to any one of paragraphs. 1-17, characterized in that said vector is an adeno-associated virus (AAV)-based vector. 19. The gene therapy vector according to claim 18, characterized in that the AAV vector is an AAV9 vector or an AAVrh.74 vector. 20. Pharmaceutical composition for the treatment or prevention of Danon disease, containing a gene therapy vector according to any one of paragraphs. 1–19. 21. A method of treating or preventing Danon disease or other autophagic disorder in a subject in need thereof, comprising administering to said subject a gene therapy vector according to any one of claims. 1–19 or the pharmaceutical composition according to claim 20. 22. The method according to claim 21, characterized in that the specified vector or pharmaceutical composition is administered by a route selected from the group consisting of intravenous, intraarterial, intracardiac, intracoronary, intramyocardial, intrarenal, intraurethral, epidural and intramuscular administration. 23. The method of claim 21 or 22, characterized in that said autophagic disorder is selected from the group consisting of end-stage heart failure, myocardial infarction, drug toxicity, diabetes, end-stage renal failure and aging. 24. Method according to any one of paragraphs. 21–23, characterized in that said subject is a human. 25. Method according to any one of paragraphs. 21-24, characterized in that said subject exhibits symptoms of Danon disease or other autophagic disorder. 26. Method according to any one of paragraphs. 21-25, characterized in that said subject has been identified as having reduced or undetectable expression of LAMP-2. 27. Method according to any one of paragraphs. 21-26, characterized in that said subject has been identified as having a mutated LAMP-2 gene. 28. Method according to any one of paragraphs. 21-27, characterized in that administration of said gene therapy vector results in increased expression of LAMP-2B polynucleotide and/or LAMP-2B protein in the heart compared to its expression in an untreated subject. 29. Method according to any one of paragraphs. 21–28, characterized in that the introduction of said gene therapy vector results in at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1. 5 times, at least about 1.6 times, at least about 1.7 times, at least about 1.8 times, at least about 1.9 times, 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 a fold or at least approximately 4-fold increase in the expression of LAMP-2B polynucleotide and/or LAMP-2B protein in the heart compared to its expression in an untreated subject. 30. Method according to any one of paragraphs. 21-29, characterized in that administration of said gene therapy vector results in a maximum of about 1.1-fold, a maximum of about 1.2-fold, a maximum of about 1.3-fold, a maximum of about 1.4-fold, or a maximum of about 1, A 5-fold increase in the expression of LAMP-2B polynucleotide and/or LAMP-2B protein in the liver compared to expression in the liver of an untreated subject.