Gene therapy vectors for treatment of danon disease

Abstract

To provide a gene therapy vector comprising a polynucleotide sequence encoding a LAMP-2 polypeptide for use as a therapeutic in the treatment of Danon Disease, as well as methods of use thereof, pharmaceutical compositions, and more.SOLUTION: The disclosure provides a recombinant AAV vector having AAVrh74 serotype expressing LAMP-2A, LAMP-2B, or LAMP-2C for use as a therapeutic in, for example, Danon Disease.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 934,928, filed November 13, 2019, and U.S. Provisional Patent Application No. 62 / 804,521, filed February 12, 2019, each of which is incorporated by reference in its entirety herein.

[0002] Sequence Listing This application has been filed electronically via EFS-Web and contains an electronically submitted sequence listing in .txt format. The .txt file contains a sequence listing entitled "ROPA_013_02WO_ST25.txt" which was created on February 11, 2020 and has a size of approximately 61 kilobytes. The sequence listing contained in this .txt file is a part of the present specification and is incorporated by reference in its entirety.

[0003] FIELD OF THEINVENTION The present invention relates generally to gene therapy for diseases associated with mutations in lysosome-associated membrane protein 2 (LAMP-2, also known as CD107b). [Background technology]

[0004] background Lysosome-associated membrane protein 2 (LAMP-2, also known as CD107b) is a gene encoding a lysosome-associated membrane glycoprotein. Alternative splicing of the gene generates three isoforms: LAMP-2A, LAMP-2B, and LAMP-2C. Loss-of-function mutations in LAMP-2 are associated with human diseases, including Danon disease, a familial cardiomyopathy associated with impaired autophagy. Danon disease is a rare but severe cardiac and skeletal myopathy that causes significant morbidity and premature death due to arrhythmias and cardiomyopathy. X-linked inheritance is responsible for the reported differences in phenotypic severity between males and females. Boucek et al. Genetics in Medicine 13:563-568 (2011). It is understood that the disease is caused by a primary deficiency of lysosome-associated membrane protein-2 (LAMP-2), which functions as a lysosomal membrane receptor during chaperone-mediated autophagy. Nishino et al. Nature 406:906-910 (2000) (Non-Patent Document 2). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Boucek et al. Genetics in Medicine 13:563-568(2011) [Non-Patent Document 2] Nishino et al. Nature 406:906-910(2000) Summary of the Invention

[0006] The present disclosure provides such gene therapy vectors related to LAMP2, methods of use thereof, pharmaceutical compositions, and others. Although the clinical use of adeno-associated virus (AAV) vectors is known, the selection of a preferred serotype of AAV for gene therapy remains difficult and unpredictable.

[0007] The present disclosure provides improved gene therapy vectors, including polynucleotide sequences encoding LAMP-2 polypeptides, methods of use thereof, pharmaceutical compositions, and the like. In particular, the present disclosure provides recombinant AAV vectors having the AAVrh74 serotype expressing LAMP-2A, LAMP-2B, or LAMP-2C for use as therapeutic agents in, for example, Danon disease.

[0008] [The present invention 1001] A recombinant adeno-associated virus (rAAV) gene therapy vector comprising a polynucleotide comprising a 5'ITR, an expression cassette, and a 3'ITR; and a capsid protein, the expression cassette comprises a transgene encoding lysosomal-associated membrane protein 2 (LAMP-2) or a functional variant thereof; the expression cassette is flanked by the 5' ITR and the 3' ITR; The capsid protein comprises an AAVrh.74 capsid protein or a functional variant thereof. rAAV gene therapy vectors. [The present invention 1002] 1001. The rAAV gene therapy vector of the present invention, wherein said LAMP-2 is selected from LAMP-2A, LAMP-2B, and LAMP-2C. [The present invention 1003] 1001. The rAAV gene therapy vector of the present invention, wherein the capsid protein has at least 95% sequence identity with an amino acid sequence selected from SEQ ID NO:2-4. [The present invention 1004] The rAAV gene therapy vector of the present invention, wherein said capsid protein shares at least 95% sequence identity with SEQ ID NO:2. [The present invention 1005] The rAAV gene therapy vector of the present invention 1004, wherein said capsid protein shares at least 97% sequence identity with SEQ ID NO:2. [The present invention 1006] The rAAV gene therapy vector of the present invention, wherein the capsid protein shares at least 99% sequence identity with SEQ ID NO:2. [The present invention 1007] The rAAV gene therapy vector of any one of claims 1001 to 1006, wherein the capsid protein is an AAVrh.74 capsid protein. [The present invention 1008] The rAAV gene therapy vector of any of claims 1001 to 1007, wherein the 5'ITR and the 3'ITR are, respectively, the 5'ITR of AAV2 and the 3'ITR of AAV2, or variants thereof. [The present invention 1009] 1008. The rAAV gene therapy vector of the present invention, wherein the 5'ITR shares at least 98% identity with SEQ ID NO:13 and the 3'ITR shares at least 98% identity with SEQ ID NO:14. [The present invention 1010] The rAAV gene therapy vector of any of claims 1001 to 1009, wherein the transgene is codon-optimized for expression in a human host cell. [The present invention 1011] The rAAV gene therapy vector of any of claims 1001 to 1010, wherein the expression cassette contains fewer CpG sites than SEQ ID NO:6. [The present invention 1012] The rAAV gene therapy vector of any of claims 1001 to 1011, wherein the expression cassette contains fewer cryptic splice sites than SEQ ID NO:6. [The present invention 1013] The rAAV gene therapy vector of any of claims 1001 to 1012, wherein the expression cassette encodes an alternative open reading frame less than SEQ ID NO:6. [The present invention 1014] The rAAV gene therapy vector of any of claims 1001 to 1013, wherein the transgene shares at least 95% identity with a sequence selected from SEQ ID NOs:7 to 9. [The present invention 1015] The rAAV gene therapy vector of the present invention, wherein the transgene shares at least 99% identity with a sequence selected from SEQ ID NOs:7-9. [The present invention 1016] The rAAV gene therapy vector of the present invention, wherein the transgene comprises a sequence selected from SEQ ID NO:7-9. [The present invention 1017] Any of the rAAV gene therapy vectors of claims 1001 to 1016, wherein the expression cassette comprises a consensus optimal Kozak sequence operably linked to the introduced gene, and the consensus optimal Kozak sequence comprises SEQ ID NO:20. [The present invention 1018] The rAAV gene therapy vector of any of claims 1001 to 1017, wherein the expression cassette comprises a full-length polyA sequence operably linked to the introduced gene, and the full-length polyA sequence comprises SEQ ID NO:26. [The present invention 1019] The rAAV gene therapy vector of any of claims 1001 to 1018, wherein the expression cassette does not contain a start codon 5' to the start codon of the transgene. [The present invention 1020] The rAAV gene therapy vector of any of claims 1001 to 1019, wherein the expression cassette comprises, in the 5' to 3' direction, a first inverted terminal sequence operably linked, an enhancer / promoter region, an intron, a consensus optimal Kozak sequence, the transgene, a 3' untranslated region comprising a full-length polyA sequence, and a second inverted terminal sequence, and the expression cassette does not comprise a start codon 5' to the start codon of the transgene. [The present invention 1021] The rAAV gene therapy vector of the present invention 1020, wherein the enhancer / promoter region comprises, in the 5' to 3' direction, a CMV IE enhancer and a chicken beta-actin promoter, and optionally, the enhancer / promoter region further comprises the first exon and first intron of a chicken beta-actin gene and a splice acceptor of a rabbit beta-globin gene. [The present invention 1022] The rAAV gene therapy vector of the present invention 1020, wherein the enhancer / promoter region comprises a tissue-specific promoter capable of mediating increased expression in cardiac tissue and / or skeletal muscle tissue compared to liver tissue. [The present invention 1023] The rAAV gene therapy vector of any of claims 1001 to 1021, wherein the expression cassette shares at least 95% identity with a sequence selected from SEQ ID NOs: 10 to 12. [The present invention 1024] The rAAV gene therapy vector of the present invention, wherein the expression cassette comprises a sequence selected from SEQ ID NO:10-12. [The present invention 1025] A pharmaceutical composition comprising any one of the rAAV gene therapy vectors of the present invention 1001 to 1024. [The present invention 1026] A method for treating or preventing Danon disease or another autophagy disorder in a subject in need thereof, comprising administering to the subject an rAAV gene therapy vector of any of claims 1001 to 1024 or a pharmaceutical composition of claim 1025. [The present invention 1027] The method of claim 1026, wherein the rAAV gene therapy vector or pharmaceutical composition is administered via a route selected from the group consisting of intravenous, intraarterial, intracardiac, intracoronary, intramyocardial, intrarenal, intraurethral, ​​epidural, and intramuscular. [The present invention 1028] The method of claim 1026 or 1027, wherein said autophagy disorder is selected from the group consisting of end-stage heart failure, myocardial infarction, drug toxicity, diabetes, end-stage renal failure, and aging. [The present invention 1029] The method of any one of claims 1026 to 1028, wherein the subject is a human. [The present invention 1030] The method of any of claims 1026 to 1029, wherein the subject exhibits symptoms of Danon disease or another autophagy disorder. [The present invention 1031] Any of the methods of claims 1026 to 1030, wherein the subject is identified as having reduced or undetectable expression of endogenous LAMP-2. [The present invention 1032] The method of any one of claims 1026 to 1031, wherein the subject is identified as having a mutated LAMP-2 gene. [The present invention 1033] The rAAV gene therapy vector is about 3×10 12 vg / kg ~ approx. 3×10 14 The method of any of claims 1026 to 1032, wherein the compound is administered at a dose of 100 mg / kg. [The present invention 1034] The rAAV gene therapy vector is about 3×10 12 vg / kg ~ approx. 1.2×10 13 The method of any of claims 1026 to 1033, wherein the compound is administered at a dose of 100 mg / kg. [The present invention 1035] The rAAV gene therapy vector is about 1.0×10 13 The method of any of claims 1026 to 1033, wherein the compound is administered at a dose of 100 mg / kg. [The present invention 1036] The dose of the rAAV gene therapy vector administered is optionally about 1.0×10 13 6. The method of any of claims 1026 to 1035, which does not cause clinical pathology when administered at a dose of 1000 mg / kg. [The present invention 1037] The method of any of claims 1026 to 1036, wherein administration of the rAAV gene therapy vector transduces one or more of the heart, muscle, and liver. [The present invention 1038] Any of the methods of claims 1026 to 1037, wherein administration of the rAAV gene therapy vector induces LAMP2B mRNA expression in one or more of the heart, muscle, and liver. [The present invention 1039] Any of the methods of claims 1026 to 1038, wherein administration of the rAAV gene therapy vector causes LAMP2B protein expression in one or more of the heart, muscle, and liver. [The present invention 1040] Any of the methods of claims 1026 to 1039, wherein administration of the rAAV gene therapy vector causes infection of at least about 10%, at least about 20%, or at least about 30% of cells in one or more of the heart, muscle, and liver with the rAAV gene therapy vector. [The present invention 1041] Any of the methods of claims 1026 to 1040, wherein administration of the rAAV gene therapy vector results in transduction of the rAAV gene therapy vector in the gonads at less than 0.1 vector genomes (vg) per diploid genome. [The present invention 1042] The administration of the rAAV gene therapy vector is 2×10 4 Any of the methods of claims 1026 to 1041, which induces LAMP2B mRNA expression in the gonads with less than 100 mRNA copies. [The present invention 1043] Any of the methods of claims 1026 to 1042, wherein administration of the rAAV gene therapy vector does not cause LAMP2B protein expression in the brain and / or gonads. [The present invention 1044] Any of the methods of claims 1026 to 1043, wherein administration of the rAAV gene therapy vector transduces and / or induces transgene expression at approximately the same level as an AAV9 gene therapy vector having the same expression cassette. [The present invention 1045] A method for delivering a LAMP-2 polynucleotide encoding a LAMP-2 protein to a cell, comprising contacting the cell with an rAAV gene therapy vector of any one of claims 1001 to 1024 or the pharmaceutical composition of claim 1025, wherein the cell is optionally selected from a cardiac cell, a lung cell, and / or a muscle cell. [The present invention 1046] A method for transducing a cell, comprising contacting the cell with an rAAV gene therapy vector of any one of claims 1001 to 1024 or the pharmaceutical composition of claim 1025, wherein the cell is optionally selected from a cardiac cell, a lung cell, and / or a muscle cell. [The present invention 1047] A method for delivering a LAMP-2 polynucleotide encoding a LAMP-2 protein to a tissue and / or expressing a LAMP-2 protein in a tissue, comprising contacting the tissue with an rAAV gene therapy vector of any one of claims 1001 to 1024 or the pharmaceutical composition of claim 1025, wherein the tissue is optionally selected from cardiac tissue, lung tissue, and / or muscle tissue. [The present invention 1048] A method for delivering a LAMP-2 polynucleotide encoding a LAMP-2 protein to a subject and / or expressing a LAMP-2 protein in a subject, the method comprising administering to the subject an rAAV gene therapy vector of any of claims 1001 to 1024 or a pharmaceutical composition of claim 1025. [The present invention 1049] The method of claim 1048, wherein the rAAV gene therapy vector or pharmaceutical composition is administered via a route selected from the group consisting of intravenous, intraarterial, intracardiac, intracoronary, intramyocardial, intrarenal, intraurethral, ​​epidural, and intramuscular. [The present invention 1050] The method of claim 1048 or claim 1049, wherein the subject is suffering from or at risk of an autophagy disorder selected from the group consisting of Danon disease, end-stage heart failure, myocardial infarction, drug toxicity, diabetes, end-stage renal failure, and aging. [The present invention 1051] The method of any one of claims 1048 to 1050, wherein the subject is a human. [The present invention 1052] The method according to any one of claims 1048 to 1051, wherein the subject exhibits a symptom of the autophagy disorder. [The present invention 1053] The method of any of claims 1048 to 1052, wherein the subject is identified as having reduced or undetectable expression of endogenous LAMP-2. [The present invention 1054] The method according to any one of claims 1048 to 1053, wherein the subject is identified as having a mutated LAMP-2 gene. [The present invention 1055] The rAAV gene therapy vector is about 3×10 12 vg / kg ~ approx. 3×10 14 The method of any of claims 1048 to 1054, wherein the compound is administered at a dose of 100 mg / kg. [The present invention 1056] The rAAV gene therapy vector is about 3×10 12 vg / kg ~ approx. 1.2×10 13 The method of any of claims 1048 to 1055, wherein the compound is administered at a dose of 100 mg / kg. [The present invention 1057] The rAAV gene therapy vector is about 1.0×10 13 The method of any of claims 1048 to 1056, wherein the compound is administered at a dose of 100 mg / kg. [The present invention 1058] The dose of the rAAV gene therapy vector administered is optionally about 1.0×10 13 8. The method of any of claims 1048 to 1057, which does not cause clinical pathology when administered at a dose of 1000 to 1500 vg / kg. [The present invention 1059] The method of any of claims 1048 to 1058, wherein administration of the rAAV gene therapy vector transduces one or more of the heart, muscle, and liver. [The present invention 1060] Any of the methods of claims 1048 to 1059, wherein administration of the rAAV gene therapy vector causes LAMP2B mRNA expression in one or more of the heart, muscle, and liver. [The present invention 1061] Any of the methods of claims 1048 to 1060, wherein administration of the rAAV gene therapy vector causes LAMP2B protein expression in one or more of the heart, muscle, and liver. [The present invention 1062] Any of the methods of claims 1048 to 1061, wherein administration of the rAAV gene therapy vector causes infection with the rAAV gene therapy vector in at least about 10%, at least about 20%, or at least about 30% of cells in one or more of the heart, muscle, and liver. [The present invention 1063] Any of the methods of claims 1048 to 1062, wherein administration of the rAAV gene therapy vector results in transduction of the rAAV gene therapy vector in the gonads at less than 0.1 vector genomes (vg) per diploid genome. [The present invention 1064] The administration of the rAAV gene therapy vector is 2×10 4 Any of the methods of claims 1048 to 1063, which induces LAMP2B mRNA expression in the gonads with less than 100 mRNA copies. [The present invention 1065] Any of the methods of claims 1048 to 1064, wherein administration of the rAAV gene therapy vector does not cause LAMP2B protein expression in the brain and / or gonads. [The present invention 1066] Any of the methods of claims 1048 to 1065, wherein administration of the rAAV gene therapy vector transduces and / or induces transgene expression at approximately the same level as an AAV9 gene therapy vector having the same expression cassette. Other features and advantages of the invention will be apparent from and are encompassed by the following detailed description and claims. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 describes an embodiment of an AAV vector having the AAVrh74 serotype. [Diagram 2] FIG. 2 shows a bar graph of vector DNA quantification by qPCR in organs most affected by Danon disease. [Diagram 3] Figure 3A shows a bar graph of vector DNA quantification in heart regions by qPCR, and Figure 3B shows a bar graph of vector DNA quantification in muscle by qPCR. [Figure 4] FIG. 4 shows a bar graph of mRNA quantification in the organs most affected by Danon disease by RT-qPCR. [Diagram 5] Figure 5A shows a bar graph of mRNA quantification in heart regions by RT-qPCR, and Figure 5B shows a bar graph of mRNA quantification in muscle by RT-qPCR. [Figure 6A] FIG. 6A shows photomicrographs of semi-quantitative mRNA analysis by RNAscope in untreated left ventricles. [Figure 6B] FIG. 6B shows photomicrographs of semi-quantitative mRNA analysis by RNAscope in untreated left ventricles. [Figure 7A] FIG. 7A shows photomicrographs of semi-quantitative mRNA analysis by RNAscope in untreated quadriceps muscle. [Figure 7B] FIG. 7B shows photomicrographs of semi-quantitative mRNA analysis by RNAscope in untreated quadriceps muscle. [Figure 8]FIG. 8 shows photomicrographs of semi-quantitative mRNA analysis by RNAscope in untreated gastrocnemius muscle. [Figure 9] FIG. 9 shows a bar graph of protein quantification in the most affected tissues in Danon disease by ELISA. [Figure 10A] FIG. 10A shows a bar graph of protein quantification in heart regions by ELISA. [Figure 10B] FIG. 10B shows a bar graph of protein quantification in muscle by ELISA. [Figure 11] 11A-11D show line graphs of clinical pathology measurements in NHP serum over the course of the study. Clinical pathology levels were assessed as changes during the study period: (FIG. 11A) alanine aminotransferase, ALT; (FIG. 11B) aspartate aminotransferase, AST; (FIG. 11C) white blood cells, WBC; and (FIG. 11D) neutrophils. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Detailed Description of the Invention The present disclosure provides an AAVrh74-based gene therapy vector that uses an optimized expression cassette to deliver a polynucleotide encoding one of the lysosomal associated membrane protein 2 (LAMP2) proteins, also known as CD107b. Generally, LAMP2 is human LAMP2, but expression of any mammalian LAMP-2 is contemplated. The native LAMP2 gene encodes three variants, LAMP-2A, LAMP-2B, and LAMP-2C, by alternative splicing. LAMP-2B is associated with Danon disease. Although the present disclosure is primarily concerned with Danon disease, LAMP2 has been implicated in a variety of other diseases, including cancer. The disclosed vectors can be used to treat any of these diseases.

[0011] The present disclosure further relates to AAVrh74 capsid or capsids that have substantial homology with AAVrh74 capsid and retain the function of AAVrh74 capsid. The present disclosure provides the sequences listed in Table 1. Table 1 further provides polynucleotide sequences used in various embodiments. The sequences are not intended to limit the present invention, as substitution or modification of these sequences with different promoters, enhancers, or other genetic elements is contemplated.

[0012] (Table 1) Sequence TIFF2025063228000002.tif230166

[0013] The present disclosure provides a recombinant adeno-associated virus (rAAV) gene therapy vector. As used herein, "rAAV gene therapy vector" refers to a complete virus that includes nucleic acid and protein components, including capsid protein. In some embodiments, the capsid protein is encoded by a polynucleotide that is provided on a plasmid in trans to a transfer plasmid. The polynucleotide sequence of wild type AAVrh74 cap is as follows: AAVrh74 capsid coding sequence (SEQ ID NO:1) TIFF2025063228000003.tif193159TIFF2025063228000004.tif98159

[0014] The disclosure further provides the protein sequence of AAVrh74 VP3, including SEQ ID NOs:2-4, and homologs or functional variants thereof. AAVrh74 VP3 (SEQ ID NO: 2) TIFF2025063228000005.tif69158 AAVrh74 VP3 (SEQ ID NO:3) TIFF2025063228000006.tif55160 AAVrh74 VP3 (SEQ ID NO: 4) TIFF2025063228000007.tif41159

[0015] In certain instances, the AAVrh74 capsid comprises the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the rAAV vector comprises a polypeptide comprising, consisting essentially of, or even consisting of a sequence that is, e.g., at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, and more usually 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of AAVrh74 VP3 set forth in SEQ ID NO:2. In some embodiments, the rAAV vector comprises a polypeptide comprising, or consisting essentially of, or even consisting of a sequence that is, e.g., at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, and more usually 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:3. In some embodiments, the rAAV vector comprises a polypeptide comprising, or consisting essentially of, or even consisting of a sequence that is, e.g., at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, and more usually 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:4.

[0016] The wild-type polypeptide sequence of LAMP-2B (SEQ ID NO:5) and the wild-type polynucleotide sequence of LAMP-2B (SEQ ID NO:6) are, respectively: The file is TIFF2025063228000008.tif226159.

[0017] In one embodiment, the transgene shares at least 95% identity with the polynucleotide sequence of SEQ ID NO:5. In one embodiment, the transgene shares at least 99% identity with the polynucleotide sequence of SEQ ID NO:5. In one embodiment, the transgene comprises the polynucleotide sequence of SEQ ID NO:5. In embodiments, the transgene shares at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or complete identity with SEQ ID NO:5.

[0018] In one embodiment, the transgene shares at least 95% identity with the amino acid sequence of SEQ ID NO:6. In one embodiment, the transgene shares at least 99% identity with the amino acid sequence of SEQ ID NO:6. In one embodiment, the polypeptide encoded by the transgene comprises the amino acid sequence of SEQ ID NO:6. In an embodiment, the polypeptide encoded by the transgene shares at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or complete identity with SEQ ID NO:6.

[0019] Disclosed herein are modifications to the gene sequence of LAMP-2B, including codon optimization, CpG depletion, removal of cryptic splice sites, and reduction of alternative open reading frames (ORFs). In embodiments, the disclosure provides a transgene encoding an isoform of lysosomal associated membrane protein 2 (LAMP-2) or a functional variant thereof. In embodiments, the transgene shares at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or complete identity with a sequence selected from SEQ ID NOs: 7-9. The disclosure provides at least three variant gene sequences of LAMP-2B (SEQ ID NOs: 7-9): TIFF2025063228000009.tif84159TIFF2025063228000010.tif232160TIFF2025063228000011.tif182160.

[0020] In one embodiment, the transgene shares at least 95% identity with a sequence selected from SEQ ID NOs:7-9. In one embodiment, the transgene shares at least 99% identity with a sequence selected from SEQ ID NOs:7-9. In one embodiment, the transgene comprises a sequence selected from SEQ ID NOs:7-9. In an embodiment, the transgene shares at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or complete identity with SEQ ID NO:7. In an embodiment, the transgene shares at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or complete identity with SEQ ID NO:8. In embodiments, the transgene shares at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or complete identity with SEQ ID NO:9.

[0021] In some cases, the transgene has a polynucleotide sequence that differs from the polynucleotide sequence of a reference sequence, e.g., a "natural" or "wild-type" LAMP-2B sequence. In some embodiments, the transgene shares up to 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95% identity with the reference sequence. In some embodiments, the reference sequence is SEQ ID NO:6. For example, SEQ ID NO:7 shares 78.5% identity with SEQ ID NO:6.

[0022] In some embodiments, the transgene is similar or identical to a subsequence of any one of SEQ ID NOs:5, or 7-9. In some embodiments, the transgene comprises a subsequence of any one of SEQ ID NOs:5, or 7-9. In various embodiments, a subsequence can comprise any set of contiguous nucleotides (nt) having a length of at least about 50 nt, at least about 100 nt, at least about 150 nt, at least about 250 nt, at least about 200 nt, at least about 350 nt, at least about 450 nt, at least about 400 nt, at least about 450 nt, at least about 550 nt, at least about 600 nt, at least about 650 nt, at least about 600 nt, at least about 650 nt, at least about 700 nt, at least about 750 nt, at least about 800 nt, at least about 850 nt, at least about 900 nt, at least about 950 nt, at least about 1000 nt, at least about 1050 nt, at least about 1100 nt, at least about 1150 nt, or at least about 1200 nt in the complete sequence.

[0023] In some embodiments, the transgene encodes a polypeptide similar or identical to a subsequence of any one of SEQ ID NOs:6, or 16-18. In some embodiments, the transgene encodes a polypeptide comprising a subsequence of any one of SEQ ID NOs:6, or 16-18. In some embodiments, a subsequence can include any set of consecutive amino acids (aa) in the complete sequence having a length of at least about 20 aa, at least about 30 aa, at least about 50 aa, at least about 70 aa, at least about 80 aa, at least about 100 aa, at least about 120 aa, at least about 130 aa, at least about 150 aa, at least about 170 aa, at least about 180 aa, at least about 200 aa, at least about 220 aa, at least about 230 aa, at least about 250 aa, at least about 270 aa, at least about 280 aa, at least about 300 aa, at least about 320 aa, at least about 330 aa, at least about 350 aa, at least about 370 aa, at least about 380 aa, or at least about 400 aa.

[0024] In some embodiments, the transgene comprises an N-terminal truncation of 1-10 amino acids (aa), 1-20 aa, 1-30 aa, 1-40 aa, or 1-50 aa, or an N-terminal truncation of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 48, 50 or more aa, and / or or a LAMP-2 polypeptide comprising a C-terminal truncation of 1-10 amino acids (aa), 1-20 aa, 1-30 aa, 1-40 aa, or 1-50 aa, or a C-terminal truncation of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 48, 50 aa or more.

[0025] In some embodiments, a subsequence of a LAMP2 polypeptide comprises a functional variant of LAMP-2A, LAMP-2B, or LAMP-2C. As used herein, a "functional variant" refers to a polypeptide that shares sequence similarity with a reference LAMP-2A, LAMP-2B, or LAMP-2C and has at least one biological property of LAMP-2A, LAMP-2B, or LAMP-2C. The biological property may include the ability to specifically interact with one or more binding partners, the ability to bind to an anti-LAMP2 antibody, and / or the ability to complement a deficiency in LAMP2 activity in a cell, tissue, and / or organism.

[0026] In some embodiments, a subsequence of a LAMP2 polypeptide comprises a functional fragment of LAMP-2A, LAMP-2B, or LAMP-2C. As used herein, a "functional fragment" refers to a polypeptide that shares sequence similarity with a subsequence of a reference LAMP-2A, LAMP-2B, or LAMP-2C and has at least one biological property of LAMP-2A, LAMP-2B, or LAMP-2C. The biological property may include the ability to specifically interact with one or more binding partners, the ability to bind to an anti-LAMP2 antibody, and / or the ability to complement a deficiency in LAMP2 activity in a cell, tissue, and / or organism.

[0027] In one embodiment, the transgene is codon-optimized for expression in a human host cell. In one embodiment, the transgene coding sequence is modified or "codon-optimized" to enhance expression by replacing rarely represented codons with more frequently represented codons. The coding sequence is the portion of the mRNA sequence that codes for amino acids for translation. During translation, each of the 61 trinucleotide codons is translated into one of the 20 amino acids, resulting in degeneracy or redundancy in the genetic code. However, different cell types and different animal species utilize tRNAs (each with an anticodon) that code for the same amino acid at different frequencies. If a gene sequence contains a codon that is rarely represented by the corresponding tRNA, the ribosomal translation machinery may slow down, preventing efficient translation. Expression can be improved for a particular species through "codon optimization," in which the coding sequence is modified to encode the same protein sequence but utilize codons utilized by highly represented and / or highly expressed human proteins (Cid-Arregui et al., 2003; J. Virol. 77:4928).

[0028] In some embodiments, the coding sequence of the transgene is modified to replace a codon that is rarely expressed in a mammal or primate with a codon that is frequently expressed in a primate. For example, in some embodiments, the transgene encodes a polypeptide having at least 85% sequence identity, e.g., at least 90% sequence identity, at least 95% sequence identity, at least 98% identity, or at least 99% identity, with a reference polypeptide (e.g., wild-type LAMP-2B, SEQ ID NO:16), and at least one codon of the coding sequence has a higher tRNA frequency in humans than the corresponding codon in the sequences described above or disclosed herein.

[0029] In one embodiment, the transgene contains fewer alternative open reading frames than SEQ ID NO:6. In one embodiment, the transgene is modified to enhance expression by terminating or removing open reading frames (ORFs) that do not code for the desired transgene. An open reading frame (ORF) is a nucleic acid sequence following 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" relative to the gene of interest. Such open reading frames may be expressed in the expression cassette along with the gene of interest, potentially resulting in unwanted adverse effects. In some embodiments, the transgene is modified to remove open reading frames by further altering the codon usage. This is accomplished by removing one or more start codons (ATG, TTG, CTG) and / or introducing one or more stop codons (TAG, TAA, or TGA) in a reverse or out-of-frame orientation relative to the desired ORF, while retaining the amino acid sequence encoded in the gene of interest and, optionally, maintaining highly utilized codons (i.e., avoiding codons with a frequency <20%).

[0030] In variations of the present disclosure, the transgene coding sequence may be optimized by either codon optimization and removal of non-transgene ORFs, or using both techniques. In some cases, non-transgene ORFs are removed or minimized after codon optimization to remove ORFs introduced during codon optimization.

[0031] In one embodiment, the transgene contains fewer CpG sites than SEQ ID NO:6. Without being bound by theory, it is believed that the presence of CpG sites in the polynucleotide sequence is associated with an undesirable immune response of the host to the viral vector containing the polynucleotide sequence. In some embodiments, the transgene is designed to reduce the number of CpG sites. Exemplary methods are provided in U.S. Patent Application Publication No. US20020065236A1.

[0032] In one embodiment, the transgene contains fewer cryptic splice sites than SEQ ID NO:6. For optimization, GeneArt® software can be used to remove cryptic splice sites, for example, to increase GC content and / or avoid transcriptional silencing and thus increase expression of the transgene. Alternatively, any optimization method known in the art can be used. Removal of cryptic splice sites is described, for example, in International Patent Application Publication WO2004015106A1.

[0033] Also disclosed herein are expression cassettes and gene therapy vectors encoding LAMP-2B, e.g., the codon-optimized LAMP-2B sequence disclosed herein, which includes a consensus optimal Kozak sequence, a full-length polyadenylation (polyA) sequence (or a full-length polyA replacement for the truncated polyA), and minimal or no upstream (i.e., 5') start codon (i.e., an ATG site).

[0034] In some cases, the expression cassette contains two or more of a first inverted terminal sequence, an enhancer / promoter region, a consensus optimal Kozak sequence, a transgene (e.g., a transgene encoding LAMP-2B as disclosed herein), a 3' untranslated region comprising a full-length polyA sequence, and a second inverted terminal sequence.

[0035] In one embodiment, the expression cassette comprises a Kozak sequence operably linked to the transgene. In one embodiment, the Kozak sequence is a consensus optimal Kozak sequence comprising or consisting of SEQ ID NO:20. GCCGCCACCATGG (SEQ ID NO:20)

[0036] In various embodiments, the expression cassette comprises an alternative Kozak sequence operably linked to the transgene, hi one embodiment, the Kozak sequence is an alternative Kozak sequence comprising or consisting of any one of SEQ ID NOs:21-25. (gcc)gccRccAUGG (SEQ ID NO:21) AGNNAUGN(SEQ ID NO:22) ANNAUGG(SEQ ID NO:23) ACCAUGG (SEQ ID NO: 24) GACACCAUGG (SEQ ID NO:25)

[0037] In SEQ ID NO:21, lower case letters indicate the most common base at positions where the base can still be varied, and upper case letters indicate highly conserved bases. "R" indicates adenine or guanine. In SEQ ID NO:21, the sequence in brackets (gcc) is optional. In SEQ ID NO:22-23, "N" indicates any base.

[0038] A variety of sequences can be used as translation initiation sites in place of this consensus optimal Kozak sequence, and it is within the skill of the art to identify and test other sequences. See Kozak M. An analysis of vertebrate mRNA sequences: intimations of translational control. J. Cell Biol. 115(4):887-903 (1991).

[0039] In one embodiment, the expression cassette comprises a full length polyA sequence operably linked to the transgene. In one embodiment, the full length polyA sequence comprises SEQ ID NO:26. TIFF2025063228000012.tif55159

[0040] A variety of alternative polyA sequences may be used in the expression cassettes of the present disclosure, including, but not limited to, the bovine growth hormone polyadenylation signal (bGHpA) (SEQ ID NO:27), the SV40 early / late polyadenylation signal (SEQ ID NO:28), and the human growth hormone (HGH) polyadenylation signal (SEQ ID NO:29). TIFF2025063228000013.tif103160

[0041] In some embodiments, the expression cassette comprises an active fragment of a polyA sequence. In certain embodiments, the active fragment of a polyA sequence comprises or consists of, for example, less than 20 base pairs (bp), less than 50 bp, less than 100 bp, or less than 150 bp of any of the polyA sequences disclosed herein.

[0042] In some cases, expression of the transgene is increased by ensuring that the expression cassette does not contain competing ORFs. In one embodiment, the expression cassette does not contain a start codon within 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, or 300 base pairs 5' of the start codon of the transgene. In one embodiment, the expression cassette does not contain a start codon 5' of the start codon of the transgene. In some embodiments, the expression cassette does not contain alternative transcripts. In some embodiments, the expression cassette does not contain alternative transcripts, except for small transcripts, e.g., 300 base pairs or less.

[0043] In one embodiment, the expression cassette comprises, in a 5' to 3' direction, a first inverted terminal sequence operably linked to an enhancer / promoter region, an intron, a consensus optimal Kozak sequence, a transgene, a 3' untranslated region comprising a full-length polyA sequence, and a second inverted terminal sequence, wherein the expression cassette does not comprise a start codon 5' to the start codon of the transgene.

[0044] In one embodiment, the enhancer / promoter region comprises, from 5' to 3', the CMV IE enhancer and the chicken beta-actin promoter.In one embodiment, the enhancer / promoter region comprises the CAG promoter.As used herein, "CAG promoter" refers to a polynucleotide sequence that comprises the CMV early enhancer element, the chicken beta-actin promoter, the first exon and the first intron of the chicken beta-actin gene, and the splice acceptor of the rabbit beta-globin gene.

[0045] In one embodiment, the expression cassette shares at least 95% identity with a sequence selected from SEQ ID NOs: 10-12. In one embodiment, the expression cassette shares complete identity with a sequence selected from SEQ ID NOs: 10-12, or shares at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with a sequence selected from SEQ ID NOs: 10-12. In certain embodiments, the expression cassette comprises one or more modifications compared to a sequence selected from SEQ ID NOs: 10-12. In certain embodiments, the one or more modifications comprise one or more of the following: removal of one or more (e.g., all) upstream ATG sequences, replacement of the Kozak sequence with an optimized consensus Kozak sequence or another Kozak sequence, including but not limited to any of those disclosed herein, and / or replacement of the polyadenylation sequence with a full length polyadenylation sequence or another polyadenylation sequence, including but not limited to any of those disclosed herein. Exemplary arrangements of the genetic elements within these exemplary expression cassettes are set forth in FIG. TIFF2025063228000014.tif172159TIFF2025063228000015.tif245160TIFF2025063228000016.tif239159TIFF2025063228000017.tif245159TIFF2025063228000018.tif245160TIFF2025063228000019.tif239159TIFF2025063228000020.tif245160TIFF2025063228000021.tif187160

[0046] In one embodiment, the vector is an adeno-associated virus (AAV) vector. In one embodiment, the expression cassette comprises an ITR sequence selected from SEQ ID NOs: 13 and 14. TIFF2025063228000022.tif41158

[0047] In a related embodiment, the present disclosure provides a gene therapy vector comprising the expression cassette disclosed herein. Generally, the gene therapy vector described herein comprises an expression cassette comprising a polynucleotide encoding one or more isoforms of lysosomal associated membrane protein 2 (LAMP-2), and expresses LAMP-2 to allow partial or complete regulation of deficient LAMP-2 protein expression levels and / or autophagy flux in a subject in need thereof (e.g., a subject with Danon's disease or another disorder characterized by defective autophagy flux at least partially due to deficient LAMP-2 expression).

[0048] LAMP-2A protein sequence TIFF2025063228000023.tif55159 LAMP-2B protein sequence TIFF2025063228000024.tif55159 LAMP-2C protein sequence TIFF2025063228000025.tif55159

[0049] In certain embodiments, the expression cassette comprises a polynucleotide sequence encoding LAMP-2 disclosed herein, such as SEQ ID NO:15-17, or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% identity to any of SEQ ID NO:15-17. The gene therapy vector may be a viral vector or a non-viral vector. Exemplary non-viral vectors include, for example, naked DNA, cationic liposome complexes, cationic polymer complexes, cationic liposome-polymer complexes, and exosomes. Exemplary viral vectors include, but are not limited to, adenovirus, retrovirus, lentivirus, herpes virus, and adeno-associated virus (AAV) vectors.

[0050] In some embodiments, the expression cassette comprises a polynucleotide sequence encoding one or more, two or more, or all three of SEQ ID NOs: 15-17. In some embodiments, a polynucleotide sequence comprising a natural intron of the LAMP-2 gene allows expression of multiple isoforms in the same cell using one vector. In some embodiments, artificial introns, splice acceptors, and / or splice donors are used to optimize the length of the polynucleotide and / or to optimize the ratio of isoforms expressed by the polynucleotide encoding two or more, or all three of SEQ ID NOs: 15-17.

[0051] In some embodiments, the expression cassette, AAV capsid gene, and / or helper gene are delivered to cells using transduction, transfection, electroporation, lipofection, and any other method known in the art. In some embodiments, the expression cassette, AAV capsid gene, and / or helper gene are delivered to liposomes or lipid nanoparticles (LNPs). The expression cassette, AAV capsid gene, and / or helper gene may be provided as DNA, for example, on one or more plasmids, bacmids, or other DNA molecules. In some embodiments, the expression cassette, AAV capsid gene, and / or helper gene are delivered as RNA molecules. In some embodiments, the RNA molecule comprises one or more mRNA molecules, for example, one or more in vitro transcribed mRNA molecules. In some embodiments, the mRNA molecule is a modified mRNA molecule. Exemplary modifications include locked nucleic acids, phosphothiolate linkages, and modified nucleosides (e.g., pseudouridine, 5-methylcytosine, or 5-methylcytidine). In some embodiments, the modified mRNA comprises a cap, e.g., an ARCA cap. The expression cassette, AAV capsid gene, and / or helper gene may be delivered in vitro or in vivo. In some embodiments, the AAV capsid gene comprises one or more of the AAV9 capsid gene and the AAVrh74 capsid gene.

[0052] Gene delivery viral vectors useful for carrying out the present invention can be constructed using methodologies well known in the art of molecular biology. Typically, viral vectors carrying transgenes are constructed from polynucleotides that code for the transgene, appropriate regulatory factors, and elements required for the production of viral proteins, which mediate cell transduction. Such recombinant viruses can be produced by techniques well known in the art, for example, by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of viral packaging cells include, but are not limited to, HeLa cells, SF9 cells (optionally with baculovirus helper vectors), 293 cells, etc. AAV vectors can be produced using herpes virus-based systems, as described in US20170218395A1. Detailed protocols for producing such replication-defective recombinant viruses can be found, for example, in WO95 / 14785, WO96 / 22378, U.S. Pat. No. 5,882,877, U.S. Pat. No. 6,013,516, U.S. Pat. No. 4,861,719, U.S. Pat. No. 5,278,056 and WO94 / 19478, the complete contents of each of which are incorporated herein by reference.

[0053] AAV is a 4.7 kb single-stranded DNA virus. Because wild-type AAV is non-pathogenic and has no known etiological association with disease, recombinant vectors based on AAV are associated with excellent clinical safety. Furthermore, AAV offers the capacity for highly efficient gene delivery and sustained transgene expression in multiple tissues. By "AAV vector" is meant vectors derived from adeno-associated virus serotypes, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.10, AAVrh74, and the like. AAV vectors can have one or more AAV wild-type genes deleted in whole or in part, e.g., the rep gene and / or the cap gene, but retain functionally adjacent inverted terminal repeat (ITR) sequences. Functional ITR sequences are necessary for rescue, replication, and packaging of AAV virions. Thus, an AAV vector is defined herein to include at least those sequences required in cis for viral replication and packaging (e.g., functional ITRs). The ITRs need not be wild-type nucleotide sequences and may be modified, for example, by insertion, deletion, or substitution of nucleotides, so long as the sequences provide functional rescue, replication, and packaging. AAV vectors may include other modifications, including, but not limited to, one or more modified capsid proteins (e.g., VP1, VP2, and / or VP3). For example, capsid proteins may be modified to change tropism and / or reduce immunogenicity. AAV expression vectors are constructed using well-known techniques to provide at least the following control elements as operably linked components in the direction of transcription: a transcription initiation region, a DNA of interest (i.e., the LAMP-2 gene), and a transcription termination region.

[0054] Adeno-associated virus (AAV) is a single-stranded DNA virus. The AAV genome is constructed of single-stranded deoxyribonucleic acid (ssDNA), either positive or negative sense, and is approximately 4.7 kilobases in length. The genome contains inverted terminal repeats (ITRs) at both ends of the DNA strand, as well as two open reading frames (ORFs): rep and cap. The first, rep, is composed of four overlapping genes that code for the Rep protein required for the AAV life cycle, while the second, cap, codes for three capsid proteins: VP1, VP2, and VP3. The cap gene is expressed as messenger RNA (mRNA) from the AAV p40 promoter. The mRNA is alternatively spliced ​​into 2.3 kb and 2.6 kb transcripts, with the 2.3 kb transcript being the more abundant. VP1 is expressed only from the 2.6 kb transcript, and the VP1 protein has a molecular weight of 87 kilodaltons (kDa). VP2 is expressed from an open reading frame beginning with an ACG codon rather than the canonical AUG codon due to the presence of an optimal Kozak sequence for translation initiation. VP2 is 72 kDa. VP3, of only 62 kDa, is expressed from a 2.3 kb transcript as well as an ATG sequence present in a 2.6 kb transcript. The relative abundance of VP1:VP2:VP3 is 1:1:10. VP1, VP2, and VP3 interact to form a capsid with icosahedral symmetry.

[0055] Since wild-type AAV is non-pathogenic and has no known etiological association with disease, recombinant vectors based on AAV are associated with excellent clinical safety. Furthermore, AAV offers the capacity 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, AAVrh74, etc. AAV vectors can have one or more AAV wild-type genes, e.g., rep and / or cap genes, deleted in whole or in part, but retain functionally adjacent inverted terminal repeat (ITR) sequences. The serotype of a recombinant AAV vector is determined by its capsid. International Patent Publication WO2003042397A2 discloses various capsid sequences, including AAV1, AAV2, AAV3, AAV8, AAV9, and AAVrh10. International Patent Publication WO2013078316A1 discloses the polypeptide sequence of VP1 from AAVrh74. Many different naturally occurring or genetically modified AAV capsid sequences are known in the art.

[0056] The present disclosure also provides pharmaceutical compositions comprising an expression cassette or vector (e.g., a gene therapy vector) disclosed herein and one or more pharma- ceutically acceptable carriers, diluents, or excipients. In certain embodiments, the pharmaceutical compositions include an AAV vector comprising an expression cassette disclosed herein, e.g., the expression cassette comprises a codon-optimized transgene encoding LAMP-2B, e.g., any of SEQ ID NOs:7-9. Pharmaceutical compositions are provided comprising a therapeutically effective amount of a vector comprising a nucleic acid sequence of a polynucleotide encoding one or more isoforms of LAMP-2 for use in the prevention or treatment of disorders characterized by defects in autophagic flux (e.g., Danon disease).

[0057] The pharmaceutical composition containing the expression cassette or vector may be in any form suitable for the selected mode of administration, for example, intracerebroventricular, intramyocardial, intracoronary, intravenous, intraarterial, intrarenal, intraurethral, ​​epidural, or intramuscular administration. Gene therapy vectors containing polynucleotides encoding one or more LAMP-2 isoforms can be administered to animals and humans as the sole active agent or in combination with other active agents, in unit dosage forms, in admixture with conventional pharmaceutical supports. In some embodiments, the pharmaceutical composition comprises cells transduced ex vivo with any of the gene therapy vectors disclosed herein.

[0058] In various embodiments, the pharmaceutical composition contains pharma- ceutically acceptable vehicles (e.g., carriers, diluents, and excipients) for injection preparations.These may be, in particular, isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium, or magnesium chloride, etc., or mixtures of such salts).Exemplary pharmaceutical forms suitable for injection include, for example, sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for extemporaneous preparation of sterile injections or dispersions.

[0059] In another aspect, the present disclosure provides a method for preventing, alleviating, ameliorating, reducing, suppressing, eliminating, and / or reversing one or more symptoms of Danon disease or another autophagy disorder in a subject in need thereof, comprising administering to the subject a gene therapy vector of the present disclosure. The term "Danon disease" refers to an X-linked dominant skeletal and myocardial disorder with multisystem clinical manifestations. Danon disease mutations result in the absence of lysosomal associated membrane protein 2 (LAMP-2) protein expression. Major clinical features include skeletal and cardiac myopathy, cardiac conduction abnormalities, cognitive impairment, and retinal disease. Males are usually affected earlier and more severely than females.

[0060] In one embodiment, the vector is administered via a route selected from the group consisting of parenteral, intravenous, intraarterial, intracardiac, intracoronary, intramyocardial, intrarenal, intraurethral, ​​epidural, and intramuscular. In one embodiment, the vector is administered multiple times. In one embodiment, the vector is administered by intramuscular injection of the vector. In one embodiment, the vector is administered by injection of the vector into a skeletal muscle. In one embodiment, the expression cassette comprises a muscle-specific promoter, optionally a muscle creatine kinase (MCK) promoter, or the MCK / SV40 hybrid promoter 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 one embodiment, the vector is administered by intracardiac injection.

[0061] In one embodiment, the present disclosure provides a method of treating a disease or disorder, optionally Danon disease, in a subject in need thereof, comprising contacting a cell with a gene therapy vector according to the present disclosure and administering the cell to the subject. In one embodiment, the cell is a stem cell, optionally a pluripotent stem cell. In one embodiment, the stem cell can differentiate into cardiac tissue. In one embodiment, the stem cell can differentiate into muscle tissue, for example, cardiac muscle tissue and / or skeletal muscle tissue. In one embodiment, the stem cell is autologous. In one embodiment, the stem cell is an induced pluripotent stem cell (iPSC).

[0062] In one embodiment, the autophagy disorder is selected from the group consisting of end-stage heart failure, myocardial infarction, drug toxicity, diabetes, end-stage renal failure, and aging. In one embodiment, the subject is a mammal, such as a human. In one embodiment, the subject is symptomatic of Danon disease or another autophagy disorder. In one embodiment, the subject is identified as having reduced or undetectable LAMP-2 expression. In one embodiment, the subject is identified as having a mutant LAMP-2 gene.

[0063] Subjects / patients who can be treated using the methods described herein include individuals who are at risk but do not exhibit symptoms of diseases or disorders characterized by insufficient autophagy flux, such as Danon disease, including but not limited to systolic and diastolic heart failure, myocardial infarction, drug toxicity (e.g., anthracyclines, chloroquine, and derivatives thereof), diabetes, end-stage renal disease, and aging, as well as individuals who currently exhibit symptoms. Such subjects can be identified as having a mutant LAMP-2 gene or having reduced or undetectable levels of LAMP-2 expression.

[0064] In some embodiments, the subject exhibits symptoms of a disease or disorder characterized by insufficient autophagic flux (e.g., Danon disease and other known autophagy disorders, including, but not limited to, systolic and diastolic heart failure, myocardial infarction, drug toxicity, diabetes, end-stage renal disease, and aging). The symptoms may be actively manifest, or may be suppressed or controlled (e.g., by a drug), or may be in remission. The subject may or may not have been diagnosed with the disorder, e.g., by a licensed physician.

[0065] definition The terms "lysosome-associated membrane protein 2" and "LAMP-2" refer interchangeably to nucleic acid and polypeptide polymorphic variants, alleles, mutants, and interspecies homologs, and include (1) the amino acid sequence encoded by a LAMP-2 nucleic acid (see, e.g., GenBank Accession Nos. NM_002294.2 (isoform A), NM_013995.2 (isoform B), NM_001122606.1 (isoform C)) or the amino acid sequence of a LAMP-2 polypeptide (see, e.g., GenBank Accession Nos. NP_001122606.1 (isoform D)). NP_054701.1 (isoform B), NP_001116078.1 (isoform C)), preferably over a region of at least about 25, 50, 100, 200, 300, 400 or more amino acids, or over the entire length, having an amino acid sequence identity of greater than about 90%, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more, to a LAMP-2 polypeptide (e.g., see NP_054701.1 (isoform A), NP_054701.1 (isoform B), NP_001116078.1 (isoform C)); (3) an antibody, e.g., a polyclonal antibody, that is raised against an immunogen that includes an amino acid sequence of a LAMP-2 polypeptide described herein, or an amino acid sequence encoded by a LAMP-2 nucleic acid (e.g., a LAMP-2 polynucleotide described herein), and conservatively modified variants thereof; (4) an antibody that specifically hybridizes under stringent hybridization conditions to an antisense strand corresponding to a nucleic acid sequence encoding a LAMP-2 protein, and conservatively modified variants thereof; The nucleic acid has a nucleic acid sequence that has greater than about 90%, preferably greater than about 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, or more nucleotide sequence identity over a region of at least about 25, 50, 100, 200, 500, 1000, 2000 or more nucleotides, or over the entire length, with a nucleic acid (e.g., a LAMP-2 polynucleotide described herein, and a LAMP-2 polynucleotide encoding a LAMP-2 polypeptide described herein).

[0066] The term "identical" or "percent identity" refers to two or more sequences or subsequences that are the same or have a certain percentage of the same amino acid residues or nucleotides (i.e., share at least about 80% identity, e.g., at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity over a specified region with a reference sequence, e.g., a LAMP-2 polynucleotide or polypeptide sequence described herein), when compared and sequenced for maximum correspondence in a comparison window or designated region, as measured using the following sequence comparison algorithm, or by manual sequence comparison and visual inspection. Such sequences are said to be "substantially identical". This definition also refers to the complement of the test sequence. Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length, for example, over a region that is 50, 100, 200, 300, 400 amino acids or nucleotides in length, or over the entire length of the reference sequence.

[0067] For sequence comparison, typically, one sequence acts as a reference sequence, and test sequence is compared to it.When using sequence comparison algorithm, test sequence and reference sequence are input into computer, subsequence coordinates are designated as necessary, and sequence algorithm program parameters are designated.Default program parameters can be used, or alternative parameters can be designated.The sequence comparison algorithm then calculates the sequence identity percentage of test sequence to reference sequence based on program parameters.For sequence comparison of nucleic acid and protein to LAMP-2 nucleic acid and protein, BLAST and BLAST 2.0 algorithms and default parameters are used.

[0068] As used herein, a "comparison window" includes reference to any one segment of a number of contiguous positions selected from the group consisting of 20 to 600, usually about 50 to about 200, more usually about 100 to about 150, and a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods for alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wl), or by manual sequence comparison 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 the BLAST and BLAST 2.0 algorithms, described in Altschul et al., J. Mol. Biol. 215:403-410 (1990) and Altschul et al., Nucleic Acids Res. 25:3389-3402 (1977), respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (World Wide Web, ncbi.nlm.nih.gov / ).

[0069] An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with the antibody produced against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, when the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequences.

[0070] As used herein, "administration" refers to local and systemic administration, including, for example, enteral administration, parenteral administration, pulmonary administration, and topical / transdermal administration. Routes of administration of compounds (e.g., polynucleotides encoding one or more LAMP-2 isoforms) that find use in the methods described herein include, for example, oral (per os (PO)), nasal or inhalation administration, administration as a suppository, topical contact, transdermal delivery (e.g., via a transdermal patch), intrathecal (IT), intravenous ("iv"), intraperitoneal ("ip"), intramuscular ("im"), intralesional, or subcutaneous ("sc") administration, or implantation of a sustained release device, e.g., a mini-osmotic pump, administration of a depot formulation, and the like, to a subject. Administration may be by any route, including parenteral and transmucosal (e.g., oral, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intrarenal, intraurethral, ​​intracardiac, intracoronary, intramyocardial, intradermal, epidural, subcutaneous, intraperitoneal, intraventricular, ion exchange, and intracranial. Other delivery modes include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, and the like. In some embodiments, the dose of rAAV gene therapy vector administered is about 1E+11 vector genomes (vg) / kg to about 1E+12vg / kg, about 1E+12vg / kg to about 2E+12vg / kg, about 2E+12vg / kg to about 3E+12vg / kg, about 3E+12vg / kg to about 3E+13vg / kg, or about 3E+13vg / kg to about 3E+14vg / kg. In some embodiments, the dose of rAAV gene therapy vector administered is about 3E+12 vg / kg to about 3E+14 vg / kg.

[0071] The terms "systemic administration" and "administered systemically" refer to a method of administering a compound or composition to a mammal such that the compound or composition is delivered via the circulatory system to a site within the body, including the target site of pharmaceutical action. Systemic administration includes, but is not limited to, oral, intranasal, rectal and parenteral (e.g., outside the digestive tract, such as intramuscular, intravenous, intraarterial, transdermal, and subcutaneous) administration.

[0072] The term "co-administration" or "simultaneous administration", for example, when used with respect to a compound (e.g., a LAMP-2 polynucleotide) and / or an analog thereof and another active agent, refers to administration of the compound and / or an analog and an active agent such that both can achieve a physiological effect simultaneously. However, the two agents do not have to be administered together. In certain embodiments, administration of one agent can precede administration of the other. A simultaneous physiological effect does not necessarily require the presence of both agents in the circulation at the same time. However, in certain embodiments, co-administration typically results in both agents being simultaneously present in the body (e.g., in plasma) at a significant fraction (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 more, e.g., 90% or more) of their maximum serum concentration for any given dose.

[0073] The term "effective amount" or "pharmaceutical effective amount" refers to the amount and / or dosage and / or dosing regimen of one or more compounds (e.g., gene therapy vectors) necessary to produce a desired result, e.g., an increase in expression of one or more LAMP-2 isoforms in an amount sufficient to reduce the ultimate severity of a disorder or disease characterized by defects in autophagy (e.g., Danon disease). In some embodiments, an effective amount is about 1E+11vg / kg to about 1E+12vg / kg, about 1E+12vg / kg to about 2E+12vg / kg, about 2E+12vg / kg to about 3E+12vg / kg, about 3E+12vg / kg to about 3E+13vg / kg, or about 3E+13vg / kg to about 3E+14vg / kg of a rAAV gene therapy vector. In some embodiments, an effective amount is about 3E+12vg / kg to about 3E+14vg / kg of a rAAV gene therapy vector.

[0074] The phrase "administering" refers to the act of a medical professional (e.g., a physician) or someone who controls the medical care of a subject, controlling and / or authorizing the administration of the agent(s) / compound(s) in question to a subject. Administering may involve diagnosing and / or determining the appropriate therapeutic or prophylactic regimen, and / or prescribing a particular agent(s) / compound to the subject. Such prescribing may include, for example, drafting a prescription, annotating medical records, etc.

[0075] As used herein, the terms "treat" and "treatment" refer to slowing the onset, slowing or reversing the progression, reducing the severity, or alleviating or preventing either the disease or condition to which the term applies, or one or more symptoms of the disease or condition. The terms "treat" and "treatment" also include preventing, alleviating, ameliorating, reducing, inhibiting, eliminating, and / or reversing one or more symptoms of a disease or condition.

[0076] The term "amelioration" refers to the reduction or elimination of one or more symptoms of the pathology or disease, and / or the reduction in incidence or delay in onset, or reduction in severity, of one or more symptoms of the pathology or disease, and / or the prevention of the pathology or disease. In certain embodiments, the reduction or elimination of one or more symptoms of the pathology or disease may include, for example, a measurable and sustained increase in the expression level of one or more isoforms of LAMP-2.

[0077] As used herein, the phrase "consisting essentially of" refers to the genus or species of active pharmaceutical agents recited in a method or composition, and may further include other agents that do not themselves have substantial activity for the recited indication or purpose.

[0078] The terms "subject," "individual," and "patient" refer interchangeably to a mammal, preferably a human or non-human primate, but also to domesticated mammals (e.g., dogs or cats), laboratory mammals (e.g., mice, rats, rabbits, hamsters, guinea pigs), and agricultural mammals (e.g., horses, cows, pigs, cattle). In various embodiments, the subject may be a human (e.g., adult male, adult female, adolescent male, adolescent female, male child, female child).

[0079] The term "gene transfer" or "gene delivery" refers to a method or system for reliably inserting foreign DNA into a host cell. Such methods may result in transient expression of the non-integrated introduced DNA, extrachromosomal replication and expression of the introduced replicon (e.g., episome), or integration of the introduced genetic material into the genomic DNA of the host cell.

[0080] The term "vector" is used herein to refer to a nucleic acid molecule that can transmit or transport another nucleic acid molecule. The introduced nucleic acid is generally linked, e.g., inserted, into the vector nucleic acid molecule. The vector may contain sequences that direct autonomous replication or reverse transcription in a cell, or may contain sequences sufficient to allow integration into the host cell DNA. "Vector" includes gene therapy vectors. As used herein, the term "gene therapy vector" refers to a vector that can be used in performing gene therapy, e.g., delivering a polynucleotide sequence that encodes a therapeutic polypeptide to a subject. A gene therapy vector may contain a nucleic acid molecule ("transgene") that encodes a therapeutically active polypeptide, e.g., LAMP-2B, or other gene that is useful for replacement gene therapy when introduced into a subject. Useful vectors include, but are not limited to, viral vectors.

[0081] As used herein, the term "expression cassette" refers to a DNA segment capable in an appropriate setting of driving the expression of a polynucleotide (transgene) encoding a therapeutically active polypeptide (e.g., LAMP-2B) incorporated into the expression cassette. When introduced into a host cell, the expression cassette can specifically direct the cellular machinery to transcribe the transgene into RNA, which is then typically further treated and ultimately translated into a therapeutically active polypeptide. The expression cassette may be included in a gene therapy vector. Generally, the term expression cassette excludes the polynucleotide sequences 5' to 5' ITR and 3' to 3' ITR.

[0082] All patents, patent publications, and other publications referenced and identified in this specification are individually and expressly incorporated by reference herein in their entirety for all purposes. EXAMPLES

[0083] Example 1: Preclinical and clinical evaluation of AAVrh74-LAMP-2B A plasmid vector containing the gene expression cassette depicted in Figure 1 is generated. The transgene is modified to encode LAM2B-HA-FLAG, so that the protein can be detected using either anti-HA or anti-FLAG antibodies. AAVrh74-LAMP2B viral vectors are generated using a three-plasmid helper virus-free system to generate recombinant AAV particles containing serotype rh74 capsid protein and viral genome with AAV2 ITR flanking the human LAMP-2B expression cassette. The viral vectors are tested in non-human primates.

[0084] Pharmacology and toxicity studies were performed using LAMP-2B - / - The study will be conducted in mice and wild-type mice. Based on the preclinical safety and efficacy data observed in mouse and non-human primate studies, clinical trials will be conducted in patients with Danon disease.

[0085] Example 2: 1 x 1013 DNA, RNA, and protein expression in non-human primates following intravenous administration of vg / kg AAV9.LAMP2B and AAVrh74.LAMP2B A non-human primate study of AAV9 versus AAVrh74 vectors was conducted in paired male and female African green monkeys (AGM). Subjects received either AAV9.LAMP2B-HA-Flag or AAVrh74.LAMP2B-HA-Flag. "AAV9.LAMP2B-HA-Flag" is an AAV9 serotype adeno-associated viral vector encoding LAMP2B C-terminally fused to an HA-Flag tag. "AAVrh74.LAMP2B-HA-Flag" is an AAVrh74 serotype adeno-associated viral vector encoding LAMP2B C-terminally fused to an HA-Flag tag. One subject received a vehicle control. 1.85 × 10 13 The vector was administered by intravenous injection of 2 mL of vector genome (vg) / mL to generate a reference curve. This injection was administered at the target dose of vector, i.e., approximately 1.0 × 10 13 vg / kg. Due to the lower body weight of female subjects, each vector achieved approximately 1.2 × 10 13 The study is summarized in Table 2.

[0086] (Table 2) TIFF2025063228000026.tif62155

[0087] Subjects were humanely sacrificed 2 months after injection and tissues were collected for DNA, RNA, and protein analysis. The following tissues were examined: heart (left atrium, right atrium, left ventricle, and right ventricle); skeletal muscle (quadriceps and gastrocnemius); liver (left lobe, right lobe, intermediate lobe, and quadriceps lobe); brain (frontal lobe, parietal lobe, temporal lobe, occipital lobe, cortex, hippocampus, medulla, and cerebellum); and gonads.

[0088] Vector DNA - quantitative PCR DNA was extracted from frozen tissues using the Qiagen DNeasy® kit. DNA purity (A260 / A280) and concentration were assessed on a NanoDrop One™ spectrophotometer (Thermo). Quantitative PCR (qPCR) was performed on 20 ng of DNA using the TaqMan Universal Master MixII (Thermo, 4440038) on a real-time PCR system (QuantStudio5, Thermo) with the following primers / probes: WPRE (cassette): TIFF2025063228000027.tif23134

[0089] RNaseP (housekeeping gene, Thermo) Plasmid DNA containing the WPRE sequence was used to generate a standard curve. Figure 2 shows a bar graph of vector DNA quantification in organs most affected by Danon disease by qPCR.

[0090] LAMP2B mRNA-quantitative RT-PCR RNA was extracted from heart and muscle tissues using the RNeasy Fibrous Tissue kit (Qiagen), and from liver and brain using the RNeasy Lipid Tissue kit (Qiagen). Purity (A260 / A280) and concentration were determined on a NanoDrop One spectrophotometer. RNA was converted to cDNA using Superscript IV VILO Master Mix (Thermo). qPCR was performed on 10 ng of RNA using the following primers / probes in TaqMan Universal Master MixII (Thermo) on a real-time PCR system (QuantStudio5, Thermo). WPRE (cassette): TIFF2025063228000028.tif23134

[0091] HPRT-1 (housekeeping gene, Thermo) Plasmid DNA containing the WPRE sequence was used to generate a standard curve. Figure 3A shows a bar graph of vector DNA quantification in the region of the heart by qPCR. Figure 3B shows a bar graph of vector DNA quantification in muscle by qPCR. Figure 4 shows a bar graph of mRNA quantification in the organs most affected by Danon disease by RT-qPCR. Figure 5A shows a bar graph of mRNA quantification in the region of the heart by RT-qPCR. Figure 5B shows a bar graph of mRNA quantification in muscle by RT-qPCR.

[0092] LAMP2B mRNA-RNAscope Five-mm tissue cubes were fixed in 10% neutral buffered formalin, paraffin embedded, and sectioned. Transgene mRNA was detected using a WPRE-O3 ZZ probe (ACD) in RNAscope 2.5 LS RED. Semiquantitative visual assessment of one section from each tissue was performed, with cells with ≥1 dot per cell considered positive. The percentage of positive cells was divided into five categories: 0%, 1–25%, 26–50%, 51–75%, or 100%.

[0093] Figure 6A shows a photomicrograph of semi-quantitative mRNA analysis by RNAscope in untreated left ventricle. Figure 6B shows a photomicrograph of semi-quantitative mRNA analysis by RNAscope in untreated left ventricle. Figure 7A shows a photomicrograph of semi-quantitative mRNA analysis by RNAscope in untreated quadriceps. Figure 7B shows a photomicrograph of semi-quantitative mRNA analysis by RNAscope in untreated quadriceps. Figure 8 shows a photomicrograph of semi-quantitative mRNA analysis by RNAscope in untreated gastrocnemius. Results are summarized for vehicle control (Table 3A), AAV9 (Table 3A), and AAVrh74 (Table 3C).

[0094] (Table 3A) TIFF2025063228000029.tif40156

[0095] (Table 3B) TIFF2025063228000030.tif134157

[0096] (Table 3C) TIFF2025063228000031.tif138155

[0097] LAMP2B protein-ELISA Approximately 125 mg of tissue was homogenized in 500 μL of lysis buffer using 0.9-2.00 mm stainless steel beads (Next Advance) and a Next Advance Bullet Blender 24. The lysis buffer contains 300 mM NaCl, 20 mM EDTA, 100 mM Tris pH 8.0, 2% NP-40, and 0.2% SDS with Complete™ EDTA-free protease inhibitors and PhosSTOP™ phosphatase inhibitors. Total protein was assessed by BCA (Thermo). 100 mg of total protein was loaded per well. A standard curve was constructed using purified human LAMP2 protein (Origene). ELISA was performed using mouse monoclonal antibody H4B4 Biologicals (AstraZeneca Biologicals) as capture antibody, goat polyclonal antibody (R&D Systems) as detection antibody, and HRP-conjugated antibody: donkey anti-goat (Millipore) as secondary antibody. Plates were developed using TMB (Thermo) and quantified on a spectrophotometer (Spectramax M5c).

[0098] Figure 9 shows bar graphs of protein quantification in the tissues most affected by Danon's disease by ELISA. Figure 10A shows bar graphs of protein quantification in the area of ​​the heart by ELISA. Figure 10B shows bar graphs of protein quantification in muscle by ELISA.

[0099] clinical pathology The pathological effects of the vectors were evaluated. Figures 11A-11D show line graphs of clinical pathology measurements in NHP serum over the course of the study. Clinical pathology levels were assessed as changes during the study period: (Figure 11A) alanine aminotransferase, ALT; (Figure 11B) aspartate aminotransferase, AST; (Figure 11C) white blood cells, WBC; and (Figure 11D) neutrophils. B059 is the vehicle control. A991 and A602 are AAV9-treated animals. A710 and A981 are AAVrh74-treated animals.

[0100] conclusion AAV-based gene therapy using the LAMP2B transgene was performed with a vector dose of 1.0 × 10 13 vg / kg. Experiments using AAV gene therapy with several other transgenes have been performed at 1.0 × 10 13 This is an important and unexpected result, as AAV9 and AAVrh74 showed pathological effects at doses below 1000 vg / kg. Furthermore, both AAV9 and AAVrh74 were well tolerated. Although elevated levels of certain markers were observed in A602 and A710 animals at day 21, these outliers may be due to experimental error or self-resolving pathology.

[0101] Both vectors localize and transduce target tissues for the treatment of Danon disease (heart and muscle), but to a lesser extent in the brain or gonads. Expression in the gonads is undesirable for safety reasons. As expected, substantial amounts of vector accumulate in the liver, which is desirable since the liver is the tissue affected by Danon disease. The vector is present within each quadrant of the heart and in both the quadriceps and gastrocnemius muscles, a desired outcome for the treatment of Danon disease.

[0102] The localization of an AAV vector serotype (e.g., AAV9) is not predictive of the localization of other vectors (e.g., AAVrh74). This experiment demonstrates that AAVrh74 achieves the desired localization for the treatment of Danon disease or other diseases with pathogenesis involving heart and muscle tissues. Both LAMP2B transgene mRNA and protein are expressed in the same set of tissues in both the AAV9 and AAVrh74 groups. Expression is comparable between vector serotypes. The number of animals in the study is too small to discern statistically significant trends in expression levels between AAV9 and AAVrh74. RNAscope suggests that similar amounts of cells are infected in heart, muscle, and liver tissues in the AAV9 and AAVrh74 groups.

[0103] These data demonstrate that AAVrh74 can be used as a vector to deliver LAMP2B to tissues relevant for the treatment of Danon disease. AAVrh74 was non-inferior to AAV9 in these experiments.

[0104] Sequence information SEQUENCE LISTING <110> SPACECRAFT SEVEN, LLC <120> GENE THERAPY VECTORS FOR TREATMENT OF DANON DISEASE <150> US 62 / 804,521 <151> 2019-02-12 <150> US 62 / 934,928 <151> 2019-11-13 <160> 35 <170> PatentIn version 3.5 <210> 1 <211> 2217 <212> DNA <213> Non-human primate Adeno-Associated Virus <400> 1 atggctgccg atggttatct tccagattgg ctcgaggaca acctctctga gggcattcgc 60 gagtggtggg acctgaaacc tggagccccg aaacccaaag ccaaccagca aaagcaggac 120 aacggccggg gtctggtgct tcctggctac aagtacctcg gacccttcaa cggactcgac 180 aagggggagc ccgtcaacgc ggcggacgca gcggccctcg agcacgacaa ggcctacgac 240 cagcagctcc aagcgggtga caatccgtac ctgcggtata atcacgccga cgccgagttt 300 caggagcgtc tgcaagaaga tacgtctttt gggggcaacc tcgggcgcgc agtcttccag 360 gccaaaaagc gggttctcga acctctgggc ctggttgaat cgccggttaa gacggctcct 420 ggaaagaaga gaccggtaga gccatcaccc cagcgctctc cagactcctc tacgggcatc 480 ggcaagaaag gccagcagcc cgcaaaaaag agactcaatt ttgggcagac tggcgactca 540 gagtcagtcc ccgaccctca accaatcgga gaaccaccag caggcccctc tggtctggga 600 tctggtacaa tggctgcagg cggtggcgct ccaatggcag acaataacga aggcgccgac 660 ggagtgggta gttcctcagg aaattggcat tgcgattcca catggctggg cgacagagtc 720 atcaccacca gcacccgcac ctgggccctg cccacctaca acaaccacct ctacaagcaa atctccaacg ggacctcggg aggaagcacc aacgacaaca cctacttcgg ctacagcacc ccctgggggt attttgactt caacagattc cactgccact tttcaccacg tgactggcag cgactcatca acaacaactg gggattccgg cccaagaggc tcaacttcaa gctcttcaac atccaagtca aggaggtcac gcagaatgaa ggcaccaaga ccatcgccaa taaccttacc agcacgattc aggtctttac ggactcggaa taccagctcc cgtacgtgct cggctcggcg caccagggct gcctgcctcc gttcccggcg gacgtcttca tgattcctca gtacgggtac 1140 ctgactctga acaatggcag tcaggctgtg ggccggtcgt ccttctctg cctggagtc tttccttctc aaatgctgag aacgggcaac aactttgaat tcagctacaa cttcgaggac gtgcccttcc acagcagcta cgcgcacagc cagagcctgg accggctgat gaaccctctc 1320 atcgaccagt acttgtacta cctgtcccgg actcaaagca cgggcggtac tgcaggaact cagcagttgc tattttctca ggccggggcct aacaacatgt cggctcaggc caagaactgg ctacccggtc cctgctaccg gcagcaacgc gtctccacga cactgtcgca gaacaac 1500 agcaactttg cctggacggg tgccaccaag tatcatctga atggcagaga ctctctggtg 1560 aatcctggcg ttgccatggc tacccacaag gacgacaag agcgattttt tccatccagc 1620 ggagtcttaa tgtttgggaa acagggagct ggaaaagaca acgtggacta tagcagcgtg 1680 atgctaacca gcgaggaaga aataagacc accaacccag tggccacaga acagtacgg 1740 gtggtggccg ataacctgca acagcaaaac gccgctccta ttgtaggggc cgtcaatagt 1800 caaggagcct tacctggcat ggtgtggcag aaccgggacg tgtacctgca gggtcccatc 1860 tgggccaaga ttcctcatac ggacggcaac tttcatccct cgccgctgat gggaggcttt 1920 ggactgaagc atccgcctcc tcagatcctg attaaaaaaca cacctgttcc cgcggatcct 1980 ccgaccacct tcaatcaggc caagctggct tctttcatca cgcagtacag taccggccag 2040 gtcagcgtgg agatcgagtg ggagctgcag aaggaaca gcaaacgctg gaacccagag 2100 attcagtaca cttccaacta ctacaaatct acaaatgtgg actttgctgt caatactgag 2160 ggtacttatt ccgagcctcg ccccattggc acccgttacc tcacccgtaa tctgtaa 2217 <210> 2 <211> 535 <212> PRT <213> Non-human primate adeno-associated Virus <400> 2 Met Ala Ala Gly Gly Gly Ala Pro Met Ala Asp Asn Asn Glu Gly Ala 1 5 10 15 Asp Gly Val Gly Ser Ser Ser Gly Asn Trp His Cys Asp Ser Thr Trp 20 25 30 Leu Gly Asp Arg Val Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro 35 40 45 Thr Tyr Asn Asn His Leu Tyr Lys Gln Ile Ser Asn Gly Thr Ser Gly 50 55 60 Gly Ser Thr Asn Asp Asn Thr Tyr Phe Gly Tyr Ser Thr Pro Trp Gly 65 70 75 80 Tyr Phe Asp Phe Asn Arg Phe His Cys His Phe Ser Pro Arg Asp Trp 85 90 95 Gln Arg Leu Ile Asn Asn Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn 100 105 110 Phe Lys Leu Phe Asn Ile Gln Val Lys Glu Val Thr Gln Asn Glu Gly 115 120 125 Thr Lys Thr Ile Ala Asn Asn Leu Thr Ser Thr Ile Gln Val Phe Thr 130 135 140 Asp Ser Glu Tyr Gln Leu Pro Tyr Val Leu Gly Ser Ala His Gln Gly 145 150 155 160 Cys Leu Pro Pro Phe Pro Ala Asp Val Phe Met Ile Pro Gln Tyr Gly 165 170 175 Tyr Leu Thr Leu Asn Asn Gly Ser Gln Ala Val Gly Arg Ser Ser Phe 180 185 190 Tyr Cys Leu Glu Tyr Phe Pro Ser Gln Met Leu Arg Thr Gly Asn Asn 195 200 205 Phe Glu Phe Ser Tyr Asn Phe Glu Asp Val Pro Phe His Ser Ser Tyr 210 215 220 Ala His Ser Gln Ser Leu Asp Arg Leu Met Asn Pro Leu Ile Asp Gln 225 230 235 240 Tyr Leu Tyr Tyr Leu Ser Arg Thr Gln Ser Thr Gly Gly Thr Ala Gly 245 250 255 Thr Gln Gln Leu Leu Phe Ser Gln Ala Gly Pro Asn Asn Met Ser Ala 260 265 270 Gln Ala Lys Asn Trp Leu Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val 275 280 285 Ser Thr Thr Leu Ser Gln Asn Asn Asn Ser Asn Phe Ala Trp Thr Gly 290 295 300 Ala Thr Lys Tyr His Leu Asn Gly Arg Asp Ser Leu Val Asn Pro Gly 305 310 315 320 Val Ala Met Ala Thr His Lys Asp Asp Glu Glu Arg Phe Phe Pro Ser 325 330 335 Ser Gly Val Leu Met Phe Gly Lys Gln Gly Ala Gly Lys Asp Asn Val 340 345 350 Asp Tyr Ser Ser Val Met Leu Thr Ser Glu Glu Glu Ile Lys Thr Thr 355 360 365 Asn Pro Val Ala Thr Glu Gln Tyr Gly Val Val Ala Asp Asn Leu Gln 370 375 380 Gln Gln Asn Ala Ala Pro Ile Val Gly Ala Val Asn Ser Gln Gly Ala 385 390 395 400 Leu Pro Gly Met Val Trp Gln Asn Arg Asp Val Tyr Leu Gln Gly Pro 405 410 415 Ile Trp Ala Lys Ile Pro His Thr Asp Gly Asn Phe His Pro Ser Pro 420 425 430 Leu Met Gly Gly Phe Gly Leu Lys His Pro Pro Pro Gln Ile Leu Ile 435 440 445 Lys Asn Thr Pro Val Pro Ala Asp Pro Pro Thr Thr Phe Asn Gln Ala 450 455 460 Lys Leu Ala Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val Ser Val 465 470 475 480 Glu Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro 485 490 495 Glu Ile Gln Tyr Thr Ser Asn Tyr Tyr Lys Ser Thr Asn Val Asp Phe 500 505 510 Ala Val Asn Thr Glu Gly Thr Tyr Ser Glu Pro Arg Pro Ile Gly Thr 515 520 525 Arg Tyr Leu Thr Arg Asn Leu 530 535 <210> 3 <211> 398 <212> PRT <213> Non-human primate adeno-associated virus <400> 3 Ser Thr Ile Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr Val 1 5 10 15 Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp Val 20 25 30 Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser Gln 35 40 45 Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser Gln 50 55 60 Met Leu Arg Thr Gly Asn Asn Phe Glu Phe Ser Tyr Asn Phe Glu Asp 65 70 75 80 Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg Leu 85 90 95 Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr Gln 100 105 110 Ser Thr Gly Gly Thr Ala Gly Thr Gln Gln Leu Leu Phe Ser Gln Ala 115 120 125 Gly Pro Asn Asn Met Ser Ala Gln Ala Lys Asn Trp Leu Pro Gly Pro 130 135 140 Cys Tyr Arg Gln Gln Arg Val Ser Thr Thr Leu Ser Gln Asn Asn Asn 145 150 155 160 Ser Asn Phe Ala Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly Arg 165 170 175 Asp Ser Leu Val Asn Pro Gly Val Ala Met Ala Thr His Lys Asp Asp 180 185 190 Glu Glu Arg Phe Phe Pro Ser Ser Gly Val Leu Met Phe Gly Lys Gln 195 200 205 Gly Ala Gly Lys Asp Asn Val Asp Tyr Ser Ser Val Met Leu Thr Ser 210 215 220 Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr Gly 225 230 235 240 Val Val Ala Asp Asn Leu Gln Gln Gln Asn Ala Ala Pro Ile Val Gly 245 250 255 Ala Val Asn Ser Gln Gly Ala Leu Pro Gly Met Val Trp Gln Asn Arg 260 265 270 Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His Thr Asp 275 280 285 Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu Lys His 290 295 300 Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala Asp Pro 305 310 315 320 Pro Thr Thr Phe Asn Gln Ala Lys Leu Ala Ser Phe Ile Thr Gln Tyr 325 330 335 Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln Lys Glu 340 345 350 Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr Tyr 355 360 365 Lys Ser Thr Asn Val Asp Phe Ala Val Asn Thr Glu Gly Thr Tyr Ser 370 375 380 Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu 385 390 395 <210> 4 <211> 332 <212> PRT <213> Non-human primate adeno-associated virus <400> 4 Arg Thr Gly Asn Asn Phe Glu Phe Ser Tyr Asn Phe Glu Asp Val Pro 1 5 10 15 Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg Leu Met Asn 20 25 30 Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr Gln Ser Thr 35 40 45 Gly Gly Thr Ala Gly Thr Gln Gln Leu Leu Phe Ser Gln Ala Gly Pro 50 55 60 Asn Asn Met Ser Ala Gln Ala Lys Asn Trp Leu Pro Gly Pro Cys Tyr 65 70 75 80 Arg Gln Gln Arg Val Ser Thr Thr Leu Ser Gln Asn Asn Asn Ser Asn 85 90 95 Phe Ala Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly Arg Asp Ser 100 105 110 Leu Val Asn Pro Gly Val Ala Met Ala Thr His Lys Asp Asp Glu Glu 115 120 125 Arg Phe Phe Pro Ser Ser Gly Val Leu Met Phe Gly Lys Gln Gly Ala 130 135 140 Gly Lys Asp Asn Val Asp Tyr Ser Ser Val Met Leu Thr Ser Glu Glu 145 150 155 160 Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr Gly Val Val 165 170 175 Ala Asp Asn Leu Gln Gln Gln Asn Ala Ala Pro Ile Val Gly Ala Val 180 185 190 Asn Ser Gln Gly Ala Leu Pro Gly Met Val Trp Gln Asn Arg Asp Val 195 200 205 Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His Thr Asp Gly Asn 210 215 220 Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu Lys His Pro Pro 225 230 235 240 Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala Asp Pro Pro Thr 245 250 255 Thr Phe Asn Gln Ala Lys Leu Ala Ser Phe Ile Thr Gln Tyr Ser Thr 260 265 270 Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser 275 280 285 Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr Tyr Lys Ser 290 295 300 Thr Asn Val Asp Phe Ala Val Asn Thr Glu Gly Thr Tyr Ser Glu Pro 305 310 315 320 Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu 325 330 <210> 5 <211> 409 <212> PRT <213> Homo sapiens <400> 5 Met Val Cys Phe Arg Leu Phe Pro Val Pro Gly Ser Gly Leu Val Leu 1 5 10 15 Val Cys Leu Val Leu Gly Ala Val Arg Ser Tyr Ala Leu Glu Leu Asn 20 25 30 Leu Thr Asp Ser Glu Asn Ala Thr Cys Leu Tyr Ala Lys Trp Gln Met 35 40 45 Asn Phe Thr Val Arg Tyr Glu Thr Thr Asn Lys Thr Tyr Lys Thr Val 50 55 60 Thr Ile Ser Asp His Gly Thr Val Thr Tyr Asn Gly Ser Ile Cys Gly 65 70 75 80 Asp Asp Gln Asn Gly Pro Lys Ile Ala Val Gln Phe Gly Pro Gly Phe 85 90 95 Ser Trp Ile Ala Asn Phe Thr Lys Ala Ala Ser Thr Tyr Ser Ile Asp 100 105 110 Ser Val Ser Phe Ser Tyr Asn Thr Gly Asp Asn Thr Thr Phe Pro Asp 115 120 125 Ala Glu Asp Lys Gly Ile Leu Thr Val Asp Glu Leu Leu Ala Ile Arg 130 135 140 Ile Pro Leu Asn Asp Leu Phe Arg Cys Asn Ser Leu Ser Thr Leu Glu 145 150 155 160 Lys Asn Asp Val Val Gln His Tyr Trp Asp Val Leu Val Gln Ala Phe 165 170 175 Val Gln Asn Gly Thr Val Ser Thr Asn Glu Phe Leu Cys Asp Lys Asp 180 185 190 Lys Thr Ser Thr Val Ala Pro Thr Ile His Thr Thr Val Pro Ser Pro 195 200 205 Thr Thr Thr Pro Thr Pro Lys Glu Lys Pro Glu Ala Gly Thr Tyr Ser 210 215 220 Val Asn Asn Gly Asn Asp Thr Cys Leu Leu Ala Thr Met Gly Leu Gln 225 230 235 240 Leu Asn Ile Thr Gln Asp Lys Val Ala Ser Val Ile Asn Ile Asn Pro 245 250 255 Asn Thr Thr His Ser Thr Gly Ser Cys Arg Ser His Thr Ala Leu Leu 260 265 270 Arg Leu Asn Ser Ser Thr Ile Lys Tyr Leu Asp Phe Val Phe Ala Val 275 280 285 Lys Asn Glu Asn Arg Phe Tyr Leu Lys Glu Val Asn Ile Ser Met Tyr 290 295 300 Leu Val Asn Gly Ser Val Phe Ser Ile Ala Asn Asn Asn Leu Ser Tyr 305 310 315 320 Trp Asp Ala Pro Leu Gly Ser Ser Tyr Met Cys Asn Lys Glu Gln Thr 325 330 335 Val Ser Val Ser Gly Ala Phe Gln Ile Asn Thr Phe Asp Leu Arg Val 340 345 350 Gln Pro Phe Asn Val Thr Gln Gly Lys Tyr Ser Thr Ala Gln Glu Cys 355 360 365 Ser Leu Asp Asp Asp Thr Ile Leu Ile Pro Ile Ile Val Gly Ala Gly 370 375 380 Leu Ser Gly Leu Ile Ile Val Ile Val Ile Ala Tyr Val Ile Gly Arg 385 390 395 400 Arg Light Ser Tyr Ala Gly Tyr Gln Thr 405 <210> 6 <211> 1233 <212> DNA <213> Homo sapiens <400> 6 atggtgtgct tccgcctctt cccggttccg ggctcagggc tcgttctggt ctgcctagtc ctgggagctg tgcggtctta tgcattgga cttaatttga cagattcaga aaatgccact tgcctttatg caaaatggca gatgaatttc acagttcgct atgaaactac aaataaaact 240. tataaaactg taaccatttc agaccatggc actgtgacat father catttgtggg 300. gatgatcaga atggtcccaa aatagcagtg cagttcggac ctggcttttc ctggattgcg aattttacca aggcagcatc tacttattca attgacagcg tctcattttc ctacaacact ggtgataaca caacatttcc tgatgctgaa gataagga ttcttactgt tgatgaactt ttggccatca gattccatt gatgacctt tttagatgca atagtttatc aactttggaa 480 aagaatgatg ttgtccaaca ctactgggat gttcttgtac aagctttgt ccaaaatggc 540 acagtgagca caatgagtt cctgtgtgat aaagacaaaa cttcacagt ggcacccacc 600 atacacacca ctgtgccatc tcctactaca acacctactc aaggaaaa accagaagct 660 ggaacctatt cagttaataa tggcaatgat acttgtctgc tggctaccat ggggctgcag 720 ctgaacatca ctcaggataa ggttgctca gttattaca tcaacccaa tacaactcac 780 tccacaggca gctgccgttc tcacactgct ctacttagac tcatagcag caccattaag 840 tatctagact ttgtctttgc tgtgaaaaat gaaaaccgat tttatctgaa ggaagtgaac 900 atcagcatgt atttggttaa tggctccgtt ttcagcattg caaatacaa tctcagctac 960 tgggatgccc ccctgggaag ttcttatg tgcacaag agcagactgt ttcagtgtct 1020 ggagcatttc agataatac ctttgatcta agggttcagc ctttcaatgt jagaagga 1080 aagtattcta cagcccaaga gtgttcgctg gatgatgaca ccattctaat cccaattata 1140 gttggtgctg gtctttcagg cttgattatc gttatagtga ttgcttacgt aattggcaga agaaaaagtt atgctggata tcagactctg taa <210> 7 <211> 1233 <212> DNA <213> Artificial Sequence <220> <223> Made in Lab - Engineered Lamp-2B Coding Sequence <400> 7 atggtctgct tcagactgtt ccctgtccct ggatctggtc tggtgcttgt gtgcttggtg ctgggtgctg tgagatccta tgcccttgag ctgaacctga ctgactcaga aaatgccact tgcctgtatg ccaagtggca gatgaacttc actgtgagat atgagactac caacaagacc 240. to take away tgaccatctc to take away actgtcacct acaatggatc aatctgtggt gatgatcaga atggcccaaa gatagcagtg cagtttgggc ccggtttttc ctggattgct aacttcacca aggcagcctc cacctacagc attgactcag tcagcttcag ctacaacact ggggataaca ccaccttccc tgacgcagag gacaaggga tccttactgt ggacgaactc ctggcaatca gaatccccct taacgacctg ttcagatgca actccctttc aacccttgaa 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 agcagactgt gagtgtgtca 1020 ggtgcttttc agatcaacac ttttgacctg agggtgcagc ccttcaatgt gactcaggga 1080 aagtactcca ctgcacaaga gtgttccttg gatgatgaca ctatcctcat ccccattatt 1140 gtgggagctg gactgtcagg attgattata gtgattgtga ttgcttatgt gattggaagg 1200 agaaagagct atgctggcta ccagaccctg taa 1233 <210> 8 <211> 1233 <212> DNA <213> Artificial Sequence <220> <223> Made in Lab - Engineered Lamp-2B coding sequence <400> 8 atggtgtgct ttagactgtt tcctgtgcct ggttcagggc tggtcctggt ctgtctggtg 60 ctgggggctg tcagaagcta tgccttggag ctgaacctca ctgatagtga aaatgccact 120 tgtctgtatg ctaagtggca gatgaacttc actgtgagat atgaaaccac caacaagact 180 tacaaaacag tgaccatctc agatcatgga actgtgacct 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 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> 9 <211> 1233 <212> DNA <213> Artificial Sequence <220> <223> Made in Lab - Engineered Lamp-2B coding sequence <400> 9 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 acctgttgc tggccactat gggactccaa 720 ctgaacatca cccaggacaa agtggcctct gtgatcaata tcaatccca caccacccac 780 agcactgggt cctgcagaag ccacactgcc ctcctgaggc tcactcatc accatcaag 840 tacttggatt tgtgtttgc agtgaagaat gagaacagat tctactca agaggtcac 900 atttcaatgt acctggtgaa tgggagtgtg ttctccattg ctaacaaa cctgagctac 960 tgggatgccc ctctgggctc ctcatacatg tgcacaagg aacagactgt gagtgtgtca 1020 ggggccttcc agatcacac tttgacctg agagtgcagc cctttaatgt gacacaggga 1080 aagtacagca ctgctcagga gtgcagcctg gatgatgaca ctatcctgat ccctatcatt 1140 gtgggggcag gcctgtctgg actcattatt gtgattgtga tgctgt gatgggaga 1200 Aggaagtctt atgctggata ccagaccctg taa 1233 <210> 10 <211> 4549 <212> DNA <213> Artificial Sequence <220> <223> Made in Lab - Engineered expression cassette <400> 10 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc cgggcgtcg ggcgacctt 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 attacggta 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 ctccgggctg 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 ggtgggggtg 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 tcctgacgc 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 <h2 style=";text-align:left;direction:ltr">agctctgctg aggctcaact cctccaccat caagtacctg gactttgtgt ttgctgtgaa 2820<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gaatgagaac aggttctacc tcaaggaagt gaacatttcc atgtacctgg tcaatggttc 2880<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> agtgttctct attgccaaca acaatctgag ctactgggat gcacccctgg gatcctccta 2940<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> catgtgcaac aagggagcaga ctgtgagtgt gtcaggtgct tttcagatca acacttttga 3000<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> cctgagggtg cagcccttca atgtgactca gggaaagtac tccactgcac aagagtgttc 3060<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> cttggatgat gacactatcc tcatccccat tattgtggga gctggactgt caggattgat 3120<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tatagtgatt gtgattgctt atgtgattgg aaggagaaag agctatgctg gctaccagac 3180<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> cctgtaaaag ggcgaattcc agcacacgcg tcctaggagc tcgagtacta ctggcggccg 3240<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ttactagtgg atccgcggta caagtaagca tgcaagcttc gaggacgggg tgaactacgc 3300<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ctgaatcaag ctttatcgata aattcgagca tcttaccgcc atttattccc atatttgttc 3360<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tgtttttctt gatttgggta tacatttaaa tgttaataaa acaaaatggt ggggcaatca 3420<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tttacatttt tagggatatg taattactag ttcaggtgta ttgccacaag acaaacatgt 3480<h2 style=";text-align:left;direction:ltr"> taagaaactt tcccgttatt tacgctctgt tcctgttaat caacctctgg attacaaaat 3540 ttgtgaaaga ttgactgata ttcttaacta tgttgctcct tttacgctgt gtggatatgc 3600 tgctttaatg cctctgtatc atgctattgc ttcccgtacg gctttcgttt tctctcctt 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 ggaatttat 3960 tttcattgca atagtgtgtt ggaatttttt gtgtctctca ctcggagagga catatgggag 4020 ggcaaatcat ttaaaacatc agaatgagta tttggtttag agtttggcaa catatgccca 4080 tatgctggct gccatgaaca aaggttggct ataaagaggt catcagtata tgaaacagcc 4140 ccctgctgtc cattccttat tccatagaaa agccttgact tgaggttaga ttttttttat 4200 attttgtttt gtgttatttt tttctttaac atccctaaaa ttttccttac atgttttact 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> 4549 <212> DNA <213> Artificial Sequence <220> <223> Made in Lab - Engineered expression cassette <400> 11 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 attacggta 420 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt 480 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc 540 tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac 600 gttctgcttc actctcccca tctcccccc ctccccaccc ccaatttgtgt atttatttat 660 ttttaatta ttttgtgcag cgatgggggc gggggggggg ggggggcgcg cgccaggcgg 720 ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca 780 gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa 840 aaagcgaagc gcgcggcggg cggggcgc tgcgcgctgc cttcgcccg tgcccgctc 900 cgccgccgcc tcgcgccgcc cgccccggct ctgactgacc gcgttactcc cacaggtgag 960 cgggcgggac ggcccttctc ctccgggctg 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 ggtgggggtg 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 2400 cctgtttagg tgtaattcac tgagcactct ggaagaac gacgtggtgc agcactactg 2460 ggacgtgctg gtgcaggcct ttgtgcagaa cggcactgtg tccaccaacg aattcctgtg 2520 tgataaggac aaaacttcca ctgtggcacc tacaattcac actactgtgc cttcacctac 2580 caccactcca actccaaagg aaaagcctga aggaggaacc tactctgtga aaatggcaa 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 aaagaacaga ctgtgtcagt gtcaggagcc ttccagatta acacctttga 3000 tctgagggtc caacccttta atgtcactca aggaaagtat agcactgccc aggagtgctc 3060 <h2 style=";text-align:left;direction:ltr">cctggatgat gacaccattc tgattccaat cattgtgggt gcaggacttt ctgggcttat 3120<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tattgtgatt gtgattgcct atgtgattgg cagaaggaaa tcctatgcag ggtaccaaac 3180<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tctgtaaaag ggcgaattcc agcacacgcg tcctaggagc tcgagtacta ctggcggccg 3240<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ttactagtgg atccgcggta caagtaagca tgcaagcttc gaggacgggg tgaactacgc 3300<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ctgaatcaag ctttatcgata aattcgagca tcttaccgcc atttattccc atatttgttc 3360<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tgtttttctt gatttgggta tacatttaaa tgttaataaa acaaaatggt ggggcaatca 3420<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tttacatttt tagggatatg taattactag ttcaggtgta ttgccacaag acaaacatgt 3480<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> taagaaactt tcccgttatt tacgctctgt tcctgttaat caacctctgg attacaaaat 3540<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ttgtgaaaga ttgactgata ttcttaacta tgttgctcct tttacgctgt gtggatatgc 3600<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tgctttaatg cctctgtatc atgctattgc ttcccgtacg gctttcgttt tctcctcctt 3660<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtcc gtcaacgtgg 3720<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> cgtggtgtgc tctgtgtttg ctgacgcaac ccccactggc tggggcattg ccaccacctg 3780<h2 style=";text-align:left;direction:ltr"> 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 ttttttttat 4200 attttgtttt gtgttatttt tttctttaac atccctaaaa ttttccttac atgttttact 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> 12 <211> 4549 <212> DNA <213> Artificial Sequence <220> <223> Made in Lab - Engineered expression cassette <400> 12 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 tctcccccc ctccccaccc ccaatttgtgt atttatttat 660 ttttaatta ttttgtgcag cgatgggggc gggggggggg ggggggcgcg cgccaggcgg 720 ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca 780 gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa 840 aaagcgaagc gcgcggcggg cggggcgc tgcgcgctgc cttcgcccg tgcccgctc 900 cgccgccgcc tcgcgccgcc cgccccggct ctgactgacc gcgttactcc cacaggtgag 960 cgggcgggac ggcccttc ctccgggctg taattagcgc ttggtttaat gacggcttgt 1020 ttctttctg tggctgcgtg aaagccttga ggggctccgg gagggccctt tgtgcggggg 1080 gagcggctcg gggggtgcgt gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggctccgc 1140 gctgcccggc ggctgtgagc gctgcgggcg cggcgcggg cttttgcgc 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 ggtgggggtg 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 accacagtac ctagccccac 2580 cactactcca acccccaagg agaagcctga ggctggcacc tactcagtga acaatgggaa 2640 tgacacctgt ttgctggcca ctatgggact ccaactgac atcacccagg acaaagtggc ctctgtgatc aatatcaatc ccaacaccac ccacagcact gggtcctgca gaagccacac tgccctcctg aggctcaact catcaactt caagtacttg gattttgtgt ttgcagtgaa gaatgagaac agattctacc tcaaagaggt caacatttca atgtacctgg tgaatgggag 2940. tgtgttctcc attgctaaca acaacctgag ctactgggat gcccctctgg gctcctcata catgtgcaac aaggaacaga ctgtgagtgt gtcaggggcc ttccagatca acacttttga cctgagagtg cagcccttta atgtgacaca gggaaagtac agcactgctc agcagtgcag 3060 cctggatgat gacactatcc tgatccctat cattgtgggg gcaggcctgt ctggactcat 3180. gtgattgcct gtgattgcct gagaaggaag tcttatgctg gataccagac cctgtaaaag ggcgaattcc agcacacgcg tcctaggagc tcgagtacta ctggcggccg 3240 ttactagtgg atccgcggta caagtaagca tgcaagcttc gaggacgggg tgaactacgc ctgaatcaag cttatcgata aattcgagca tcttaccgcc atttattccc atatttgttc tgtttttctt gatttgggta tacatttaaa tgttaataaa acaaaatggt ggggcaatca 3420 tttacattt 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 cctttatga ggagttgtgg cccgttgtcc gtcaacgtgg 3720 cgtggtgtgc tctgtgtttg ctgacgcaac ccccactggc tggggcattg ccaccacctg 3780 tcaactcctt tctgggactt tcgctttccc cctcccgatc gccacggcag aactcatcgc 3840 cgcctgcctt gccgctgct ggacaggggc taggttgctg ggcactgata attccgtggt 3900 gttgtcgggg aagggcctcg ataccgtcga tatcgatcct ggctaataaa ggaaatttat 3960 tttcattgca atagtgtgtt ggaattttt gtgtctctca ctcggaagga catatgggag 4020 ggcaaatcat ttaaaacatc agaatgagta tttggtttag agtttggcaa catatgccca 4080 tatgctggct gccatgaaca aaggttggct ataaagaggt catcagtata tgaaacagcc 4140 ccctgctgtc cattccttat tccatagaaa agccttgact tgaggttaga ttttttttat 4200 attttgtttt gtgttatttt tttctttaac atccctaaaa ttttccttac atgttttact 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> 13 <211> 130 <212> DNA <213> Adeno-associated viurs <400> 13 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120 aggggttcct 130 <210> 14 <211> 130 <212> DNA <213> Adeno-associated virus <400> 14 aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60 ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc 120 gagcgcgcag 130 <210> 15 <211> 410 <212> PRT <213> Homo sapiens <400> 15 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> 16 <211> 410 <212> PRT <213> Homo sapiens <400> 16 Met Val Cys Phe Arg Leu Phe Pro Val Pro Gly Ser Gly Leu Val Leu 1 5 10 15 Val Cys Leu Val Leu Gly Ala Val Arg Ser Tyr Ala Leu Glu Leu Asn 20 25 30 Leu Thr Asp Ser Glu Asn Ala Thr Cys Leu Tyr Ala Lys Trp Gln Met 35 40 45 Asn Phe Thr Val Arg Tyr Glu Thr Thr Asn Lys Thr Tyr Lys Thr Val 50 55 60 Thr Ile Ser Asp His Gly Thr Val Thr Tyr Asn Gly Ser Ile Cys Gly 65 70 75 80 Asp Asp Gln Asn Gly Pro Lys Ile Ala Val Gln Phe Gly Pro Gly Phe 85 90 95 Ser Trp Ile Ala Asn Phe Thr Lys Ala Ala Ser Thr Tyr Ser Ile Asp 100 105 110 Ser Val Ser Phe Ser Tyr Asn Thr Gly Asp Asn Thr Thr Phe Pro Asp 115 120 125 Ala Glu Asp Lys Gly Ile Leu Thr Val Asp Glu Leu Leu Ala Ile Arg 130 135 140 Ile Pro Leu Asn Asp Leu Phe Arg Cys Asn Ser Leu Ser Thr Leu Glu 145 150 155 160 Lys Asn Asp Val Val Gln His Tyr Trp Asp Val Leu Val Gln Ala Phe 165 170 175 Val Gln Asn Gly Thr Val Ser Thr Asn Glu Phe Leu Cys Asp Lys Asp 180 185 190 Lys Thr Ser Thr Val Ala Pro Thr Ile His Thr Thr Val Pro Ser Pro 195 200 205 Thr Thr Thr Pro Thr Pro Lys Glu Lys Pro Glu Ala Gly Thr Tyr Ser 210 215 220 Val Asn Asn Gly Asn Asp Thr Cys Leu Leu Ala Thr Met Gly Leu Gln 225 230 235 240 Leu Asn Ile Thr Gln Asp Lys Val Ala Ser Val Ile Asn Ile Asn Pro 245 250 255 Asn Thr Thr His Ser Thr Gly Ser Cys Arg Ser His Thr Ala Leu Leu 260 265 270 Arg Leu Asn Ser Ser Thr Ile Lys Tyr Leu Asp Phe Val Phe Ala Val 275 280 285 Lys Asn Glu Asn Arg Phe Tyr Leu Lys Glu Val Asn Ile Ser Met Tyr 290 295 300 Leu Val Asn Gly Ser Val Phe Ser Ile Ala Asn Asn Asn Leu Ser Tyr 305 310 315 320 Trp Asp Ala Pro Leu Gly Ser Ser Tyr Met Cys Asn Lys Glu Gln Thr 325 330 335 Val Ser Val Ser Gly Ala Phe Gln Ile Asn Thr Phe Asp Leu Arg Val 340 345 350 Gln Pro Phe Asn Val Thr Gln Gly Lys Tyr Ser Thr Ala Gln Glu Cys 355 360 365 Ser Leu Asp Asp Asp Thr Ile Leu Ile Pro Ile Ile Val Gly Ala Gly 370 375 380 Leu Ser Gly Leu Ile Ile Val Ile Val Ile Ala Tyr Val Ile Gly Arg 385 390 395 400 Arg Lys Ser Tyr Ala Gly Tyr Gln Thr Leu 405 410 <210> 17 <211> 411 <212> PRT <213> Homo sapiens <400> 17 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 Only Leu Gly Leu Ile Ile Val Val Where You Will Be Tyr With Gly 385 390 395 400 Arg Arg Lys Ser Arg Thr Gly Tyr Gln Ser Val 405 410 <210> 18 <211> 1730 <212> DNA <213> Artificial Sequence <220> <223> Made in Lab - promoter sequence <400> 18 ctagtcgaca ttgattattg actagttatt atagtaatc aattacgggg tcattagttc 60 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac 120 cgcccaacga cccccgccca tgacgtcaa taatgacgta tgttcccata gtaacgccaa 180 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag 240 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc 300 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg fotcatct 360 acgtattagt catcgctatt accatggtcg aggtgagccc cacgttctgc ttcactctcc 420 ccatctcccc cccctcccca cccccaattt tgtatttatt tattttttaa ttattttgtg 480 cagcgatggg ggcgggggg ggggggggc gcgcgccagg cggggcgggg cggggcgagg 540 ggcggggcgg ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa 600 agtttccttt tatggcgagg cggcggcgg ggcggcccta taaaaagcga agcgcgcgg 660 gggcgggagt cgctgcgcgc tgccttgcc ccgtgccccg ctccgccgcc gcctcgcgcc 720 gcccgccccg gctctgactg accgcgttac tcccacaggt gagcgggcgg gacggccctt 780 ctcctccggg ctgtaattag cgcttggttt aatgacggct tgtttctttt ctgtggctgc 840 gtgaaagcct tgaggggctc cgggagggcc ctttgtgcgg ggggagcggc tcgggggtg 900 cgtgcgtgtg tgtgtgcgtg gggagcgccg cgtgcggctc cgcgctgccc ggcggctgtg 960 agcgctgcgg gcgcggcgcg gggctttgtg cgctccgcag tgtgcgcgag gggagcgcgg 1020 ccgggggcgg tgccccgcgg tgcggggggg gctgcgaggg gaacaaaggc tgcgtgcggg 1080 gtgtgtgcgt gggggggtga gcagggggtg 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 gggagggcct 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> 19 <211> 596 <212> DNA <213> Woodchuck hepatitis virus <400> 19 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> 20 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> Kozak consensus sequence <400> 20 gccgccacca tgg 13 <210> 21 <211> 13 <212> RNA <213> Artificial Sequence <220> <223> Kozak consensus sequence <400> 21 gccgccrcca ugg 13 <210> 22 <211> 8 <212> RNA <213> Artificial Sequence <220> <223> Kozak consensus sequence <220> <221> misc_feature <222> (3)..(4) <223> n is a, c, g, or u <220> <221> misc_feature <222> (8)..(8) <223> n is a, c, g, or u <400> 22 agnnaugn 8 <210> 23 <211> 7 <212> RNA <213> Artificial Sequence <220> <223> Kozak consensus sequence <220> <221> misc_feature <222> (2)..(3) <223> n is a, c, g, or u <400> 23 annaugg 7 <210> 24 <211> 7 <212> RNA <213> Artificial Sequence <220> <223> Kozak consensus sequence <400> 24 accaugg 7 <210> 25 <211> 10 <212> RNA <213> Artificial Sequence <220> <223> Kozak consensus sequence <400> 25 gacaccaugg 10 <210> 26 <211> 387 <212> DNA <213> Mus musculus <400> 26 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 attttttta tattttgttt tgtgttattt ttttctttaa catccctaaa 300 atttcctta catgttttac tagccagatt tttcctcctc tcctgactac tcccagtcat 360 agctgtccct cttctcttat ggagatc 387 <210> 27 <211> 235 <212> DNA <213> Taurus boss <400> 27 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> 28 <211> 222 <212> DNA <213> Simian virus 40 <400> 28 cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa 60 aatgcttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca 120 ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag ggggagatgt 180 gggaggtttt ttaaagcaag taaaacctct acaaatgtgg ta 222 <210> 29 <211> 202 <212> DNA <213> Homo sapiens <400> 29 ctgcccgggt ggcatccctg tgacccctcc ccagtgcctc tcctggccct ggaagttgcc 60 actccagtgc ccaccagcct tgtcctaata aaattaagtt gcatcattttt gtctgactag 120 gtgtccttct ataatattat ggggtggagg ggggtggtat ggagcaaggg gcccaagttg 180 ggaagaaacc tgtagggcct gc 202 <210> 30 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 30 atcatgctat tgcttcccgt a 21 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 31 gggccacaac tcctcataaa 20 <210> 32 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 32 cctccttgta taaatcctgg ttgctgtct 29 <210> 33 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 33 atcatgctat tgcttcccgt a 21 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 34 gggccacaac tcctcataaa 20 <210> 35 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 35 cctccttgta taaatcctgg ttgctgtct 29

Claims

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

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

3. 2. The rAAV gene therapy vector of claim 1, wherein the capsid protein has at least 95% sequence identity to an amino acid sequence selected from SEQ ID NOs: 2-4.

4. 4. The rAAV gene therapy vector of claim 3, wherein the capsid protein shares at least 95% sequence identity with SEQ ID NO:

2.

5. The rAAV gene therapy vector of claim 4, wherein the capsid protein shares at least 97% sequence identity with SEQ ID NO:

2.

6. 6. The rAAV gene therapy vector of claim 5, wherein the capsid protein shares at least 99% sequence identity with SEQ ID NO:

2.

7. The rAAV gene therapy vector of any one of claims 1 to 6, wherein said capsid protein is AAVrh.74 capsid protein.

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

9. 9. The rAAV gene therapy vector of claim 8, wherein the 5' ITR shares at least 98% identity with SEQ ID NO:13 and the 3' ITR shares at least 98% identity with SEQ ID NO:

14.

10. The rAAV gene therapy vector of any one of claims 1 to 9, wherein the transgene is codon-optimized for expression in a human host cell.

11. The rAAV gene therapy vector of any one of claims 1 to 10, wherein the expression cassette contains fewer CpG sites than SEQ ID NO:

6.

12. The rAAV gene therapy vector of any one of claims 1 to 11, wherein the expression cassette contains fewer cryptic splice sites than SEQ ID NO:

6.

13. The rAAV gene therapy vector of any one of claims 1 to 12, wherein the expression cassette encodes an alternative open reading frame less than SEQ ID NO:

6.

14. The rAAV gene therapy vector of any one of claims 1 to 13, wherein the transgene shares at least 95% identity with a sequence selected from SEQ ID NOs: 7-9.

15. The rAAV gene therapy vector of claim 14, wherein the transgene shares at least 99% identity with a sequence selected from SEQ ID NOs: 7-9.

16. The rAAV gene therapy vector of claim 15, wherein the transgene comprises a sequence selected from SEQ ID NOs: 7-9.

17. 17. The rAAV gene therapy vector of any one of claims 1 to 16, wherein said expression cassette comprises a consensus optimal Kozak sequence operably linked to said transgene, said consensus optimal Kozak sequence comprising SEQ ID NO:

20.

18. 18. The rAAV gene therapy vector of any one of claims 1 to 17, wherein said expression cassette comprises a full length polyA sequence operably linked to said transgene, said full length polyA sequence comprising SEQ ID NO:

26.

19. The rAAV gene therapy vector of any one of claims 1 to 18, wherein the expression cassette does not contain a start codon 5' to the start codon of the transgene.

20. 20. The rAAV gene therapy vector of any one of claims 1 to 19, wherein the expression cassette comprises, in the 5' to 3' direction, a first inverted terminal sequence operably linked, an enhancer / promoter region, an intron, a consensus optimal Kozak sequence, the transgene, a 3' untranslated region comprising a full-length polyA sequence, and a second inverted terminal sequence, wherein the expression cassette does not comprise a start codon 5' to the start codon of the transgene.

21. The rAAV gene therapy vector of claim 20, wherein the enhancer / promoter region comprises, in the 5' to 3' direction, a CMV IE enhancer and a chicken beta-actin promoter, and optionally, the enhancer / promoter region further comprises the first exon and first intron of a chicken beta-actin gene and a splice acceptor of a rabbit beta-globin gene.

22. The rAAV gene therapy vector of claim 20, wherein the enhancer / promoter region comprises a tissue-specific promoter capable of mediating increased expression in cardiac tissue and / or skeletal muscle tissue compared to liver tissue.

23. 22. The rAAV gene therapy vector of any one of claims 1 to 21, wherein the expression cassette shares at least 95% identity with a sequence selected from SEQ ID NOs: 10-12.

24. 24. The rAAV gene therapy vector of claim 23, wherein the expression cassette comprises a sequence selected from SEQ ID NOs: 10-12.

25. A pharmaceutical composition comprising an rAAV gene therapy vector according to any one of claims 1 to 24.

26. 27. A method of treating or preventing Danon disease or another autophagy disorder in a subject in need thereof, comprising administering to the subject a rAAV gene therapy vector of any one of claims 1-24 or a pharmaceutical composition of claim 25.

27. 27. The method of claim 26, wherein the rAAV gene therapy vector or pharmaceutical composition is administered via a route selected from the group consisting of intravenous, intraarterial, intracardiac, intracoronary, intramyocardial, intrarenal, intraurethral, ​​epidural, and intramuscular.

28. 28. The method of claim 26 or claim 27, wherein the autophagy disorder is selected from the group consisting of end-stage heart failure, myocardial infarction, drug toxicity, diabetes, end-stage renal failure, and aging.

29. The method of any one of claims 26 to 28, wherein the subject is a human.

30. The method of any one of claims 26 to 29, wherein the subject exhibits symptoms of Danon disease or another autophagy disorder.

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

32. The method of any one of claims 26 to 31, wherein the subject is identified as having a mutated LAMP-2 gene.

33. The rAAV gene therapy vector is about 3×10 １２ vg / kg ~ approx. 3×10 １４ 33. The method of any one of claims 26 to 32, wherein the administration is in a dose of 100 mg / kg.

34. The rAAV gene therapy vector is about 3×10 １２ vg / kg ~ approx. 1.2×10 １３ 34. The method of any one of claims 26 to 33, wherein the administration is in a dose of 100 mg / kg.

35. The rAAV gene therapy vector is about 1.0×10 １３ 34. The method of any one of claims 26 to 33, wherein the administration is in a dose of 100 mg / kg.

36. When the rAAV gene therapy vector is administered, the dose is optionally about 1.0×10 １３ 36. The method of any one of claims 26 to 35, which does not cause clinical pathology when administered at a dose of 1000 mg / kg.

37. The method of any one of claims 26-36, wherein administration of the rAAV gene therapy vector transduces one or more of the heart, muscle, and liver.

38. The method of any one of claims 26-37, wherein administration of the rAAV gene therapy vector induces LAMP2B mRNA expression in one or more of the heart, muscle, and liver.

39. The method of any one of claims 26-38, wherein administration of the rAAV gene therapy vector induces LAMP2B protein expression in one or more of the heart, muscle, and liver.

40. 40. The method of any one of claims 26-39, wherein administration of the rAAV gene therapy vector results in infection of at least about 10%, at least about 20%, or at least about 30% of cells in one or more of the heart, muscle, and liver with the rAAV gene therapy vector.

41. 41. The method of any one of claims 26-40, wherein administration of the rAAV gene therapy vector results in transduction of the rAAV gene therapy vector in the gonads at less than 0.1 vector genomes (vg) per diploid genome.

42. The administration of the rAAV gene therapy vector is 2×10 ４ The method according to any one of claims 26 to 41, wherein less than one mRNA copy causes LAMP2B mRNA expression in the gonads.

43. The method of any one of claims 26 to 42, wherein administration of the rAAV gene therapy vector does not result in LAMP2B protein expression in the brain and / or gonads.

44. The method of any one of claims 26-43, wherein administration of the rAAV gene therapy vector transduces and / or induces transgene expression at approximately the same level as an AAV9 gene therapy vector carrying the same expression cassette.

45. 26. A method of delivering a LAMP-2 polynucleotide encoding a LAMP-2 protein to a cell, comprising contacting said cell with an rAAV gene therapy vector according to any one of claims 1 to 24 or a pharmaceutical composition according to claim 25, wherein said cell is optionally selected from a cardiac cell, a lung cell, and / or a muscle cell.

46. 26. A method of transducing a cell, comprising contacting said cell with an rAAV gene therapy vector of any one of claims 1 to 24 or the pharmaceutical composition of claim 25, wherein said cell is optionally selected from a cardiac cell, a lung cell, and / or a muscle cell.

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

48. A method of delivering a LAMP-2 polynucleotide encoding a LAMP-2 protein to a subject and / or expressing a LAMP-2 protein in a subject, comprising administering to said subject an rAAV gene therapy vector described in any one of claims 1 to 24 or a pharmaceutical composition described in claim 25.

49. The method of claim 48, wherein the rAAV gene therapy vector or pharmaceutical composition is administered via a route selected from the group consisting of intravenous, intraarterial, intracardiac, intracoronary, intramyocardial, intrarenal, intraurethral, ​​epidural, and intramuscular.

50. 50. The method of claim 48 or claim 49, wherein the subject is suffering from or at risk for an autophagy disorder selected from the group consisting of Danon disease, end stage heart failure, myocardial infarction, drug toxicity, diabetes, end stage renal failure, and aging.

51. The method of any one of claims 48 to 50, wherein the subject is a human.

52. The method of any one of claims 48 to 51, wherein the subject exhibits symptoms of the autophagy disorder.

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

54. The method of any one of claims 48 to 53, wherein the subject is identified as having a mutated LAMP-2 gene.

55. The rAAV gene therapy vector is about 3×10 １２ vg / kg ~ approx. 3×10 １４ 55. The method of any one of claims 48 to 54, wherein the administration is in a dose of 100 mg / kg.

56. The rAAV gene therapy vector is about 3×10 １２ vg / kg ~ approx. 1.2×10 １３ 56. The method of any one of claims 48 to 55, wherein the method is administered in a dose of 100 mg / kg.

57. The rAAV gene therapy vector is about 1.0×10 １３ 57. The method of any one of claims 48 to 56, wherein the method is administered in a dose of 100 mg / kg.

58. When the rAAV gene therapy vector is administered, the dose is optionally about 1.0×10 １３ 58. The method of any one of claims 48 to 57, which does not cause clinical pathology when administered at a dose of 1000 mg / kg.

59. 59. The method of any one of claims 48-58, wherein administration of the rAAV gene therapy vector transduces one or more of the heart, muscle, and liver.

60. 60. The method of any one of claims 48-59, wherein administration of the rAAV gene therapy vector induces LAMP2B mRNA expression in one or more of the heart, muscle, and liver.

61. 61. The method of any one of claims 48-60, wherein administration of the rAAV gene therapy vector induces LAMP2B protein expression in one or more of the heart, muscle, and liver.

62. The method of any one of claims 48-61, wherein administration of the rAAV gene therapy vector results in infection of at least about 10%, at least about 20%, or at least about 30% of cells in one or more of the heart, muscle, and liver with the rAAV gene therapy vector.

63. 63. The method of any one of claims 48-62, wherein administration of the rAAV gene therapy vector results in transduction of the rAAV gene therapy vector in the gonads at less than 0.1 vector genomes (vg) per diploid genome.

64. The administration of the rAAV gene therapy vector is 2×10 ４ 64. The method of any one of claims 48 to 63, wherein less than one mRNA copy causes LAMP2B mRNA expression in the gonads.

65. The method of any one of claims 48-64, wherein administration of the rAAV gene therapy vector does not result in LAMP2B protein expression in the brain and / or gonads.

66. The method of any one of claims 48-65, wherein administration of the rAAV gene therapy vector transduces and / or induces transgene expression at approximately the same level as an AAV9 gene therapy vector carrying the same expression cassette.

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