RU2822925C2 - Gene therapy vectors for treating danon's disease - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: described is a recombinant adeno-associated virus (rAAV) gene therapy vector. Virus contains polynucleotide containing 5'-IER, expressing cassette and 3'-IER, and capsid protein. Expression cassette contains a transgene coding membrane protein 2 associated with lysosomes (LAMP-2), or a functional version thereof. Expression cassette is flanked by 5'-IER and 3'-IER. Capsid protein includes capsid protein AAVrh.74 or its functional version. Capsid protein or its functional variant is characterized by at least 95% sequence identity with respect to the amino acid sequence SEQ ID NO: 2. Also described is a pharmaceutical composition containing said rAAV vector for gene therapy. Presented is a method of treating or preventing Danone's disease in a subject in need thereof, which involves administering an rAAV vector for gene therapy or a pharmaceutical composition to the subject. Also presented is a method of delivering a LAMP-2 polynucleotide coding a LAMP-2 protein to a subject and/or expressing the LAMP-2 protein in the subject, which involves administering an rAAV vector for gene therapy or a pharmaceutical composition to the subject.

EFFECT: invention extends the range of gene therapy products.

27 cl, 11 dwg, 3 tbl, 2 ex

Description

RELATED APPLICATIONS

This application claims benefit to U.S. Provisional Patent Application No. 62/934928, filed November 13, 2019, and U.S. Provisional Patent Application No. 62/804521, filed February 12, 2019, each of which is incorporated herein by reference in its entirety. .

LIST OF SEQUENCES

This application is submitted electronically via EFS-Web and includes the sequence submitted electronically in .txt format. The .txt file contains a sequence listing called ROPA 013 02WO ST25.txt of ~61 kilobytes, created on February 11, 2020. The sequence listing contained in this .txt file is part of the description and is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

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

BACKGROUND OF THE ART

Lysosome-associated membrane protein 2 (LAMP-2, also known as CD107b) is a gene that encodes a lysosome-associated membrane glycoprotein. Alternative splicing of the gene produces three isoforms: LAMP-2A, LAMP-2B and LAMP-2C. Loss-of-function mutations in LAMP-2 are associated with human diseases, including Danon disease, a familial cardiomyopathy associated with impaired autophagy. Danon disease is a rare but serious myopathy of cardiac and skeletal muscle, causing significant morbidity and early mortality due to arrhythmia and cardiomyopathy. The nature of X-linked inheritance explains the recorded differences in the phenotypic severity of this disease in men and women. Boucek et al. Genetics in Medicine 13:563–568 (2011). It is now understood that the cause of this disease is a primary deficiency of lysosome-associated membrane protein-2 (LAMP-2), which functions as a lysosomal membrane receptor for chaperone-mediated autophagy. Nishino et al. Nature 406:906–910 (2000).

SUMMARY OF THE INVENTION

The present invention provides such LAMP2-related gene therapy vectors, methods of using them, pharmaceutical compositions, and more. Although the clinical application of adeno-associated virus (AAV) vectors is known, selection of the preferred AAV serotype(s) for gene therapy remains a complex and unpredictable aspect.

The present invention provides improved vectors for gene therapy containing a polynucleotide sequence encoding a LAMP-2 polypeptide, methods of its use, pharmaceutical compositions and much more. In particular, the present invention provides recombinant vectors based on AAV serotype AAVrh74 expressing LAMP-2A LAMP-2B or LAMP-2C for use as a therapeutic agent for, for example, Danon's disease.

Other features and advantages of the present invention are apparent from and are covered by the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

In fig. 1 depicts an embodiment of an AAV-based vector characterized by serotype AAVrh74.

In fig. Figure 2 shows a histogram of the quantification of vector DNA in the organs most affected by Danon's disease using qPCR.

In fig. 3A shows a histogram of vector DNA quantification in cardiac regions by qPCR.

In fig. 3B shows a histogram of vector DNA quantification in muscle by qPCR.

In fig. Figure 4 shows a histogram of mRNA quantification in the organs most affected by Danon disease using RT-qPCR.

In fig. Figure 5A shows a histogram of mRNA quantification in cardiac regions by RT-qPCR.

In fig. Figure 5B shows a histogram of muscle mRNA quantification using RT-qPCR.

In fig. Figure 6A shows a photomicrograph of semiquantitative RNAscope analysis of mRNA in an untreated left ventricle.

In fig. . 6B shows micrographs of semiquantitative RNAscope analysis of mRNA in treated left ventricles.

In fig. Figure 7A shows a photomicrograph of semi-quantitative RNAscope analysis of mRNA in untreated quadriceps.

In fig. 7B shows micrographs of semi-quantitative RNAscope analysis of mRNA in treated quadriceps.

In fig. Figure 8 shows micrographs of semi-quantitative RNAscope analysis of mRNA in treated gastrocnemius muscle.

In fig. Figure 9 shows a histogram of the quantitative determination of protein in tissues most affected by Danon's disease using solid-phase ELISA

In fig. . 10A shows a histogram of protein quantification in cardiac regions by ELISA.

In fig. . 10B shows a histogram of muscle protein quantification using ELISA.

In fig. 11A-11D show line graphs of clinical pathology measurements in NHP serum during the study. Levels of clinical pathology were assessed as changes in alanine aminotransferase, ALT (Fig. 11A); aspartate aminotransferase, AST (Fig. 11B); leukocytes, WBC (Fig. 11C); and neutrophils (Fig. 11D) throughout the study.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides AAVrh74-based gene therapy vectors that utilize optimized expression cassettes to deliver a polynucleotide encoding one of the lysosome-associated membrane protein 2 (LAMP2) proteins, also known as CD107b. Typically, LAMP2 is human LAMP2, although expression of LAMP-2 from any mammal is contemplated. The native LAMP2 gene, due to alternative splicing, encodes three protein variants: LAMP-2A, LAMP-2B and LAMP-2C. LAMP-2B is associated with Danon disease. Although the present invention relates primarily to Danon disease, LAMP2 has been implicated in the development of various other diseases, including cancer. The described vectors can be used to treat any of these diseases.

The present invention further relates to AAVrh74 capsids or capsids having significant homology to the AAVrh74 capsid and retaining the function of the AAVrh74 capsid. The present invention provides the sequences listed in Table 1. Table 1 further provides polynucleotide sequences useful in various embodiments. These sequences are not intended to limit the present invention, since it is contemplated that these sequences may be replaced or modified by various promoters, enhancers, or other genetic elements.

The present invention provides recombinant adeno-associated virus (rAAV)-based gene therapy vectors. As used herein, the term “rAAV gene therapy vector” refers to the complete virus containing the nucleotide and protein components, including capsid proteins. In some embodiments, the capsid protein encodes a polynucleotide located on a plasmid in trans relative to the transfer plasmid. The polynucleotide sequence of the wild-type AAVrh74 capsid is as follows:

AAVrh74 capsid coding sequence (SEQ ID NO: 1)

The present invention further provides AAVrh74 VP3 protein sequences, including SEQ ID NOs: 2-4, and homologs or functional variants thereof.

AAVrh74 VP3 (SEQ ID NO: 2)

AAVrh74 VP3 (SEQ ID NO: 3)

AAVrh74 VP3 (SEQ ID NO: 4)

In some embodiments, the AAVrh74 capsid comprises the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the rAAV-based vector comprises a polypeptide that contains or substantially consists of, or additionally consists of, a sequence, such as at least 65%, of by at least 70%, by at least 75%, by at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or 89%, more typically case, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of VP3 AAVrh74 presented in SEQ ID NO: 2. B In some embodiments, the rAAV-based vector comprises a polypeptide that contains or substantially consists of, or further consists of, a sequence of, for example, at least 65%, at least 70%, at least 75%, at least 80 %, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or 89%, more typically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to SEQ ID NO: 3. In some embodiments, the rAAV-based vector contains a polypeptide that contains, or substantially consists of, or further consists of, a sequence, e.g. at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88 % or 89%, more typically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 4.

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

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

In one embodiment, the transgene encodes a polypepgide that has at least 95% identity to the amino acid sequence of SEQ ID NO: 6. In one embodiment, the transgene encodes a polypepgide that has at least 99% identity to the amino acid sequence of SEQ ID NO: 6. B In one embodiment, the polypeptide encoded by the transgene contains the amino acid sequence of SEQ ID NO: 6. In one embodiment, the polypeptide encoded by the transgene has at least 85%, 86%, 87%, 88%, 89%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or completely identical to SEQ ID NO:6.

Modifications to the LAMP-2B gene sequence including codon optimization, CpG depletion, removal of cryptic splice sites, and reduction of alternative open reading frames (ORFs) are described herein. Embodiments of the present invention provide a transgene encoding a lysosome-associated membrane protein 2 (LAMP-2) isoform or a functional variant thereof. In embodiments, the transgene is characterized by at least 85%, 86%, 87%, 88%, 89% , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or complete sequence identity selected from SEQ ID NO: 7-9. The present invention provides at least three sequence variants of the LAMP-2B gene (SEQ ID NO: 7-9):

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

93%, 94%, 95%, 96%, 97%, 98%, 99% or complete identity with respect to k SEQ ID NO: 9.

In some cases, the transgene contains a polynucleotide sequence that differs from the polynucleotide sequence of the reference sequence, for example, the "native" LAMP-2B sequence or the "wild-type" LAMP-2B sequence. In some embodiments, the transgene is characterized by no more than 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 to the reference sequence. In some embodiments, the reference sequence is SEQ ID NO: 6. For example, SEQ ID NO: 7 has 78.5% identity to k SEQ ID NO: 6.

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

In some embodiments, the transgene encodes a polypeptide similar or identical to a subsequence of any of SEQ ID NO: 6 or 16-18. In some embodiments, the transgene encodes a polypeptide containing a subsequence of any of SEQ ID NO: 6 or 16-18. In some embodiments, the subsequence may comprise any group of contiguous amino acids (AA) in a complete sequence 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 AK, at least about 100 AK, at least about 120 AK, at least about 130 AK, at least about 150 AK, at least about 170 AK, at least about 180 AK, at least about 200 AK, at least about 220 AK, at least about 230 AK, at least about 250 AK, at least about 270 AK, at least about 280 AK, at least about 300 AK, at least about 320 AK , at least about 330 AK, at least about 350 AK. at least about 370 AK, at least about 380 AK, or at least about 400 AK.

In some embodiments, the transgene encodes a LAMP-2 polypeptide that is N-terminally truncated by 1 to 10 amino acids (AA), 1 to 20 AA, 1 to 30 AA, 1 to 40 AA, or 1 to 50 AA, or shortened from the N-terminus to 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 AK; and/or shortened from the C-terminus by 1-10 amino acids (AA), 1-20 AA, 1-30 AA, 1-40 AA or 1-50 AA, or shortened from the C-terminus by 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 AK.

In some embodiments, the LAMP2 polypeptide subsequence comprises a functional variant of LAMP-2A, LAMP-2B, or LAMP-2C. As used herein, the term “functional variant” refers to a polypeptide sequence characterized by similarity to the reference sequence LAMP-2A, LAMP-2B or LAMP-2C and characterized by 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 an anti-LAMP2 antibody, and/or the ability to complement a defect in LAMP2 activity in a cell, tissue, and/or organism.

In some embodiments, the LAMP2 polypeptide subsequence comprises a functional LAMP-2A LAMP-2B or LAMP-2C fragment. As used herein, the term “functional fragment” refers to a polypeptide having sequence similarity to a subsequence of the reference LAMP-2A, LAMP-2B, or LAMP-2C and 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 an anti-LAMP2 antibody, and/or the ability to complement a defect in LAMP2 activity in a cell, tissue, and/or organism.

In one embodiment, the transgene is codon-optimized for expression in a human host cell. In one embodiment, the transgenic coding sequence is modified or "codon optimized" to enhance expression by replacing rarely occurring codons with more frequently occurring codons. A coding sequence is a fragment of an mRNA sequence that codes for amino acids for translation. During translation, each of the 61 trinucleotide codons is translated into one of 20 amino acids, resulting in degeneracy or redundancy of the genetic code. However, different cell types and different animal species use tRNAs (each carrying an anticodon) that encode the same amino acids at different frequencies. If a gene sequence contains codons that are sparsely represented by the corresponding tRNA, the ribosomal translation machinery may slow down, preventing efficient translation. Expression can be improved through species-specific “codon optimization,” where the coding sequence is changed to encode the same protein sequence but using codons that are well represented and/or used in highly expressed human proteins (Cid-Arregui et al, 2003; J Virol 77: 4928).

In some embodiments, the coding sequence of the transgene is modified by replacing codons rarely expressed in mammals or primates with codons commonly expressed in primates. For example, in some embodiments, the transgene encodes a polypeptide having at least 85% sequence identity with a reference polypeptide {eg, wild-type LAMP-2B; SEQ ID NO: 16) - for example, at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, at least 99% identity to a reference polypeptide, wherein at least one codon of the coding sequence characterized by a higher frequency of tRNA in humans compared to the corresponding codon in the sequence described above or herein.

In one embodiment, the transgene contains fewer alternative open reading frames than SEQ ID: 6. In one embodiment, the transgene is modified to enhance expression by stopping or removing open reading frames (ORFs) that do not encode the desired transgene. An open reading frame (ORF) is a nucleotide sequence that follows 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 could potentially be expressed in an expression cassette along with the gene of interest and could result in unwanted effects. In some embodiments, the transgene has been modified to remove open reading frames by further changing codon usage. This is done by eliminating one or more of the start codons (ATG, TTG, CTO) and/or introducing one or more of the stop codons (TAG, TAA or TGA) in the reverse orientation or outside the desired ORF, preserving the encoded amino acid sequence and, optionally, , maintaining heavily used codons in the gene of interest (i.e., avoiding codons with a frequency of <20%).

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

In one embodiment, the transgene contains fewer CpG sites than SEQ ID: 6. Without being bound by theory, the presence of CpG sites in a polynucleotide sequence is believed to be associated with adverse host immunological responses to a viral vector containing the polynucleotide sequence. In some embodiments, the transgene is designed to reduce the number of CpG sites. Representative methods are presented in US Patent Application Publication No. US 20020065236 A1.

In one embodiment, the transgene contains fewer cryptic splice sites than SEQ ID: 6. GeneArt® software can be used for optimization, for example, to increase GC content and/or remove cryptic splice sites to avoid transcriptional silencing and therefore increased expression transgenes. Alternatively, any optimization technique known in the art may be used. Removal of hidden splice sites is described, for example, in International Patent Application Publication No. WO 2004015106 A1.

Also described herein are expression cassettes and gene therapy vectors encoding LAMP-2B, for example, the codon-optimized LAMP-2B sequence described herein containing the consensus optimal Kozak sequence, a full-length polyadenylation (polyA) sequence (or substitution full-length polyA to truncated polyA) and not containing or containing a minimal number of start codons (i.e., ATG sites) above (i.e., in the 5' direction).

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

In one embodiment, the expression cassette contains 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)

In various embodiments, the expression cassette contains an alternative Kozak sequence operably linked to the transgene. In one embodiment, the Kozak sequence is an alternative Kozak sequence containing or consisting of any of the 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)

In SEQ ID NO: 21 lowercase letters indicate the most common base in a position in which the base may, however, vary; a capital letter indicates a highly conserved base; "R" indicates adenine or guanine. In SEQ ID NO: 21, the sequence in parentheses (gcc) is optional. In SEQ ID NO: 22-23, "N" refers to any base.

Instead of this consensus optimal Kozak sequence, a number of sequences can be used as the translation initiation site, and other sequences can be identified and tested by one skilled in the art. See Kozak M. An analysis of vertebrate mRNA sequences: intimations of translational control. J Cell Biol. 115(4):887903 (1991).

In one embodiment, the expression cassette contains a full-length polyA sequence operably linked to a trans gene. In one embodiment, the full-length polyA sequence comprises SEQ ID NO: 26.

Various alternative polyA sequences may be used in the expression cassettes of the present invention, including, but not limited to, bovine growth hormone polyadenylation (bGHpA) signal (SEQ ID NO: 27), SV40 early/latent polyadenylation signal (SEQ ID NO: 28 ) and human growth hormone (HGH) polyadenylation signal (SEQ ID NO: 29).

In some embodiments, the expression cassette contains an active fragment of a polyA sequence. In particular embodiments, the active fragment of the polyA sequence contains or consists of less than 20 base pairs (bp), less than 50 bp, less than 100 bp. or less than 150 bp, for example, any of the polyA sequences described herein.

In some cases, transgene expression is increased by ensuring that there are no competing ORFs in the expression cassette. 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 downstream of the start codon of the transgene. In one embodiment, the expression cassette does not contain a start codon upstream 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, for example, 300 base pairs or less in size.

In one embodiment, the expression cassette contains the following operably linked elements in the direction from the 5'-conn to the 3'-end: first inverted terminal repeat, enhancer-promoter region, introns, consensus optimal Kozak sequence, transgene, 3' untranslated region containing a full-length polyA sequence, and a second inverted terminal repeat, wherein the expression cassette does not contain a start codon in the direction 5'-end of the start codon of the transgene.

In one embodiment, the enhancer-promoter region comprises, in a 5' to 3' direction, the CMV IE enhancer and the chicken beta actin promoter. In one embodiment, the enhancer-promoter region comprises a CAG promoter. As used herein, the term “CAG promoter” refers to a polynucleotide sequence comprising a CMV early enhancer element, a chicken beta-actin promoter, the first exon and first intron of the chicken beta-actin gene, and a splice acceptor from the rabbit beta-globin gene.

In one embodiment, the expression cassette has at least 95% identity to a sequence selected from SEQ ID NO: 10-12. In one embodiment, the expression cassette is characterized by complete sequence identity selected from SEQ ID NO: 10-12, or is characterized by at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to the sequence selected from SEQ ID NO: 10-12. In certain embodiments, the expression cassette contains one or more modifications compared to the sequence selected from SEQ ID NO: 10-12. In particular embodiments, the one or more modifications comprise one or more of: deleting one or more (e.g., all) upstream ATG sequences, replacing the Kozak sequence with an optimized consensus Kozak sequence or another Kozak sequence, including, without limitation, any of the sequences, described herein, and/or replacing the polyadenylation sequence with a full-length polyadenylation sequence, including, but not limited to, any of such sequences described herein. A typical configuration of genetic elements in these typical expression cassettes is depicted in FIG. 1.

In one embodiment, the vector is an adeno-associated virus (AAV) vector. In one embodiment, the expression cassette contains ICP sequences selected from SEQ ID NOs: 13 and 14.

Related embodiments of the present invention provide gene therapy vectors containing an expression cassette described herein. Typically, the gene therapy vectors described herein contain an expression cassette containing a polynucleotide encoding one or more isoforms of lysosome-associated membrane protein 2 (LAMP-2), allowing expression of LAMP-2 for partial or complete replenishment levels of LAMP-2 protein expression and/or autophagic flux in a subject in need thereof (eg, a subject with Danon's disease or other disorder characterized by a deficiency of autophagic flux at least in part due to a deficiency in LAMP-2 expression).

LAMP-2A protein sequence

LAMP-2 B protein sequence

LAMP-2C protein sequence

In specific embodiments, the expression cassette contains a polynucleotide sequence encoding LAMP-2 described herein, for example, SEQ ID NO: 15-17, or a sequence characterized by at least 90%, at least 95%, at least 98 % or at least 99% identity to any of SEQ ID NO: 15-17. Vectors for gene therapy can be viral or non-viral vectors. Typical non-viral vectors include, for example, unbound DNA, cationic liposome complexes, cationic polymer complexes, cationic liposome polymer complexes, and exosomes. Examples of a viral vector include, but are not limited to, adenoviral, retroviral, lentiviral, herpesvirus, and adeno-associated virus (AAV) vectors.

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 containing native introns of the LAMP-2 gene allows expression of more than one isoform in the same cell using a single vector. In some embodiments, artificial introns, splice acceptors, and/or splice donors are used to optimize the length of the polynucleotide and/or optimize the ratio of isoforms expressed by a polynucleotide encoding two or more or all three of SEQ ID NOs: 15-17.

In some embodiments, the expression cassette, AAV capsid gene, and/or helper genes are delivered into cells using transduction, transfection, electroporation, lipofection, and any other methods known in the art. In some embodiments, the expression cassette, AAV capsid gene, and/or helper genes are delivered in a liposome or lipid nanoparticle (LNP). The expression cassette, AAV capsid gene, and/or helper genes may be present in DNA form, for example, on one or more plasmids, bacmids, or other DNA molecules. In some embodiments, the expression cassette, AAV capsid gene, and/or helper genes are delivered as RNA molecules. In some embodiments, the RNA molecules comprise one or more mRNA molecules, such as one or more in vitro transcribed mRNA molecules. In some embodiments, the mRNA molecules are modified mRNA molecules. Typical modifications include blocking nucleic acids, thiophosphate linkages, and modified nucleosides (eg, pseudouridine, 5-methylcytosine, or 5-methylcytidine). In some embodiments, the modified mRNA contains a cap, such as an ARCA cap. The expression cassette, AAV capsid gene and/or helper genes can be delivered in vitro or in vivo. In some embodiments, the AAV capsid gene comprises one or more of an AAV9 capsid gene and an AAVrh74 capsid gene.

Viral gene delivery vectors useful in the practice of the present invention can be constructed using techniques well known in the art of molecular biology. Typically, viral vectors carrying transgenes are assembled from polynucleotides encoding the transgene, suitable regulatory elements, and elements necessary to produce viral proteins that mediate cellular transduction. Such recombinant viruses can be produced using techniques known in the art, for example, by transfection of packaging cells or transient transfection with plasmids or helper viruses. Typical examples of virus packaging cells include, but are not limited to, HeLa cells, SF9 cells (optionally with a baculovirus helper vector), 293 cells, etc. The herpesvirus-based system can be used to produce AAV-based vectors, as described in US 20170218395 A1. Detailed protocols for the production of such recombinant replication-defective viruses can be found, for example, in US Patent W095/14785, W096/22378, US Patent No. 5882877, US Patent No. 60135116, US Patent No. 4861719, US Patent No. 5278056 and W094/19478 , the entire contents of each of which are incorporated herein by reference.

AAV is a 4.7 kb single-stranded DNA virus. Recombinant AAV-based vectors are associated with excellent clinical safety because wild-type AAV is non-pathogenic and has no etiological association with any known diseases. In addition, AAV enables highly efficient gene delivery and sustained transgene expression in many tissues. By "AAV vector" is meant a vector derived from an adeno-associated virus of a particular serotype, including, without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.10, AAVrh74, etc. .d. AAV vectors may be characterized by complete or partial deletion of one or more wild-type AAV genes, such as the rep and/or cap genes, but retaining functional flanking inverted terminal repeat (ITR) sequences. Functional ICP sequences are required for rescue, replication, and packaging of the AAV virion. Thus, the definition of an AAV vector as used herein includes at least those sequences that must be present in cis for replication and packaging (eg, functional ICPs) of the virus. ICPs do not need to be wild-type nucleotide sequences and can be modified, for example, by insertion, deletion or nucleotide substitution, as long as the sequences provide functional rescue, replication and packaging. AAV vectors may contain other modifications, including, without limitation, one or more modified capsid proteins (eg, VP1, VP2 and/or VP3). For example, the capsid protein can be modified to alter tropism and/or reduce immunogenicity. AAV expression vectors are constructed using known techniques that at least provide functionally related components in the direction of transcription, control elements including a transcription initiation region, DNA of interest (ie, the LAMP-2 gene), and a transcription termination region.

Adeno-associated virus (AAV) is a single-stranded DNA virus. The AAV genome is constructed from (+ or -) single-stranded deoxyribonucleic acid (ssDNA), which is approximately 4.7 kilobases in length. The genome contains inverted terminal repeats (ITRs) at both ends of the DNA strand and two open reading frames (ORFs): rep and cap. The first reading frame, rep, consists of four overlapping genes encoding Rep proteins essential for the AAV life cycle, and the second , cap, encodes three capsid proteins: VP1, VP2 and VP3. The cap gene is expressed as messenger RNA (mRNA) from the AAV p40 promoter. Alternatively, the mRNA is spliced to produce 2.3-kb transcripts. and 2.6 kb, with a transcript length of 2.3 kb. is more common. VP1 is expressed from only a 2.6-kb transcript, and the molecular weight of the VP1 protein is 87 kilodaltons (kDa). VP2 is expressed from an open reading frame that begins with an ACG codon rather than the canonical AUG codon due to the presence of an optimal Kozak sequence for translation initiation. The mass of VP2 is 72 kDa. VP3, which is only 62 kDa, is expressed from the ATC sequence present in the 2.3-kb transcript as well as the 2.6-kb transcript. The relative amounts of VP1:VP2:VP3 are 1:1:10. VP1, VP2 and VP3 interact with each other to form a capsid with icosahedral symmetry.

Recombinant AAV-based vectors are associated with excellent clinical safety because wild-type AAV is non-pathogenic and has no etiological association with any known diseases. In addition, AAV enables highly efficient gene delivery and sustained transgene expression in many tissues. Various serotypes of AAV are known, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.10, AAVrh74, etc. AAV vectors may be characterized by complete or partial deletion of one or more of the wild-type AAV genes, such as the rep and/or cap genes, but retain functional flanking sequences

inverted terminal repeats (ITRs). The serotype of a recombinant AAV vector is determined by its capsid. International Patent Publication No. WO2003042397A2 describes various capsid sequences, including the AAV1, AAV2, AAV3, AAV8, AAV9 and AAVrhlO capsid sequences. International patent publication No. WO 2013078316 A1 describes the polypeptide sequence VP1 AAVrh74. A large variety of natural or genetically modified AAV capsid sequences are known in the art.

The present invention also provides pharmaceutical compositions comprising an expression cassette or vector (eg, a gene therapy vector) described herein and one or more pharmaceutically acceptable carriers, diluents or excipients. In specific embodiments, the pharmaceutical composition comprises an AAV vector containing an expression cassette described herein, wherein, for example, said expression cassette contains a codon-optimized transgene encoding LAMP-2B, for example, any of SEQ ID NOs: 7-9. Pharmaceutical compositions are provided, for example, for use in the prevention or treatment of a disorder characterized by autophagic flux deficiency (eg, Danon disease), which contain a therapeutically effective amount of a vector containing a polynucleotide nucleotide sequence encoding one or more LAMP-2 isoforms.

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

In various embodiments, the pharmaceutical compositions contain carriers (eg, carriers, diluents, and excipients) that are pharmaceutically acceptable for injectable formulation. They can be, in particular, isotonic sterile solutions of salts (sodium dihydrogen phosphate or hydrogen phosphate, sodium chloride, potassium, calcium or magnesium, etc., or mixtures of such salts). Typical pharmaceutical forms suitable for injection use include, for example, sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the preparation of sterile injectable solutions or dispersions for immediate administration.

In yet another aspect, the present invention provides methods for preventing, mitigating, ameliorating, reducing, suppressing, eliminating, and/or reversing one or more of the symptoms of Danon's disease or other autophagy disorder in a subject in need thereof, comprising administering to the subject a gene therapy vector according to the present invention. The term Danon disease refers to an X-linked dominant disorder of skeletal muscle and cardiac muscle with multisystem clinical manifestations. Mutations that cause Danon disease result in a lack of expression of lysosome-associated membrane protein 2 (LAMP-2). Major clinical features include skeletal muscle and cardiac myopathy, cardiac conduction abnormalities, cognitive problems, and retinal disease. As a rule, this disease manifests itself earlier and more severely in men than in women.

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 tracts. In one embodiment, the vector is administered multiple times. In one embodiment, the vector is administered by intramuscular injection of the vector. In one embodiment, the vector is administered by injecting the vector into skeletal muscle. In one embodiment, the expression cassette comprises a muscle-specific promoter, optionally a muscle creatine kinase (MCK) promoter or a hybrid MCK/SV40 promoter as described by Takeshira et al. Muscle creatine kinase/SV40 hybrid promoter for muscle-targeted long-term transgene expression, hit J Mol Med. 2007 Feb;19(2):309-15. In one embodiment, the vector is administered by intracardiac injection.

In one embodiment, the present invention provides a method of treating a disease or disorder, not necessarily Danon disease, in a subject in need thereof, comprising contacting cells with a gene therapy vector of the present invention and administering said cells to the subject. In one embodiment, the cells are stem cells, optionally pluripotent stem cells. In one embodiment, the stem cells are capable of differentiating into cardiac tissue. In one embodiment, the stem cells are capable of differentiating into muscle tissue, such as cardiac muscle tissue and/or skeletal muscle tissue. In one embodiment, the stem cells are autologous. In one embodiment, the stem cells induce pluripotent stem cells (iPSCs).

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 disease, and aging. In one embodiment, the subject is a mammal, such as a human. In one embodiment, the subject exhibits symptoms of Danon disease or another autophagy disorder. In one embodiment, the subject is identified as having reduced or undetectable expression of LAMP-2. In one embodiment, the subject is identified by the presence of a mutant LAMP-2 gene.

Subjects/patients amenable to treatment using the methods described herein include individuals at risk for a disease or disorder characterized by deficient autophagic flux (e.g., Danon's disease, as well as other known autophagy disorders, including, but not limited to, systolic and diastolic heart failure, myocardial infarction, toxic effects of drugs (eg, anthracyclines, chloroquine and its derivatives), diabetes, end-stage renal disease and aging) who are asymptomatic, as well as subjects who are currently symptomatic. Such a subject can be identified by the presence of a mutant LAMP-2 gene or a reduced or undetectable level of LAMP-2 expression.

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

Definitions

The terms "lysosome-associated membrane protein 2" and "LAMP-2" interchangeably refer to nucleic acids and polymorphic variants, alleles, mutants, and interspecies homologs of polypeptides that: (1) contain an amino acid sequence characterized by greater than about 90% amino acid identity sequence, for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequence identity, preferably in a region that is at least about 25, 50 in length . , NM 001122606.1 (isoform C)) or to the amino acid sequence of the LAMP-2 polypeptide (see, for example, GenBank accession numbers NP_002285.1 (isoform A), NP_054701.1 (isoform B), NP 001116078.1 (isoform C)); (2) bind to antibodies, for example, polyclonal antibodies against an immunogen containing the amino acid sequence of a LAMP-2 polypeptide (for example, the LAMP-2 polypeptides described herein); or the amino acid sequence encoded by a LAMP-2 nucleic acid (eg, the LAMP-2 polynucleotides described herein) and conservatively modified variants thereof; (3) specifically hybridize under stringent hybridization conditions with an antisense strand corresponding to the nucleotide sequence encoding the LAMP-2 protein and its conservatively modified variants; (4) contain a nucleotide sequence having greater than about 90%, preferably greater than about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater nucleotide sequence identity, preferably in a region that is at least about 25, 50, 100, 200, 500, 1000, 2000 or more nucleotides in length, or in a full-length region, relative to the LAMP-2 LAMP-2 nucleic acid (e.g., LAMP-2 polynucleotide 2 described herein, and LAMP-2 polynucleotides encoding the LAMP-2 polypeptides described herein).

The terms "identity" or percentage "identity" in the context of two or more nucleotide or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of the same amino acid residues or nucleotides (i.e., having at least approximately 80% identity, for example, at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, 99% identity in a specified region with respect to a reference sequence, such as the LAMP-2 polynucleotide or polypeptide sequence described herein, when compared and aligned to provide maximum agreement within the comparison window or designated region measured using one from the following sequence comparison algorithms or by manual alignment and visual inspection, such sequences are said to be "substantially identical". This definition also refers to the sequence complementary to the test sequence. The identity preferably exists in a region of at least about 25 amino acids or nucleotides in length, for example, in a region of 50, 100, 200, 300, 400 amino acids or nucleotides in length, or throughout the full-length reference sequence.

When comparing sequences, typically one sequence acts as a reference sequence to which the test sequences are compared. When using a sequence comparison algorithm, the test and reference sequences are entered into the computer, and, if necessary, the subsequence coordinates and program parameters of the sequence processing algorithm are indicated. You can use the program's default settings or assign alternative settings. The sequence comparison algorithm then calculates the percentage identity of the test sequences relative to the reference sequence based on the program parameters. BLAST and BLAST 2.0 algorithms and default parameters were used to compare nucleic acid and protein sequences with LAMP-2 nucleic acids and proteins.

As used herein, the term "comparison window" refers to a segment of any of a number of contiguous positions selected from the group consisting of 20 to 600, typically from about 50 to about 200, more commonly from about 100 to about 150, at which the sequence can be compared to a reference sequence containing the same number of contiguous positions, after optimal alignment of the two sequences. Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed, for example, using the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), using the Needleman & Wunsch homologous alignment algorithm, J. Mol. Biol. 48:443 (1970), using the similarity search method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), through computer implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics software package, Genetics Computer Group, 575 Science Dr., Madison, WI, USA) or through manual alignment and visual inspection (see, for example, Ausubel et al, eds. Current Protocols in Molecular Biology (1995) supplement). Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms 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 analyzes is publicly available from the National Center for Biotechnology Information (at ncbi.nlm.nih.gov/).

An indication of the substantial identity of the two nucleotide sequences or polypeptides is that the polypeptide encoded by the first nucleic acid immunologically cross-reacts with antibodies against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypephyde, for example, if the two peptides differ only by conservative substitutions. Another indication of the virtual identity of two nucleotide sequences is that the two molecules or their complementary sequences hybridize to each other under stringent conditions. Another indication of the virtual identity of the two nucleotide sequences is that the same primers can be used to amplify these sequences.

As used herein, the term “administration” refers to local and systemic administration, such as, but not limited to, enteral, parenteral, pulmonary, and topical/transdermal administration. Routes of administration for compounds (e.g., a polynucleotide encoding one or more isoforms of LAMP-2) that find use in the methods described herein include: e.g., oral administration, nasal or inhalation administration, suppository administration, topical contact administration, transdermal delivery (eg, via transdermal patch), intrathecal (IT) administration, intravenous ("IV") administration, intraperitoneal ("IP") administration, intramuscular ("IM") administration, intralesional administration, or subcutaneous (“SC”) administration, or implantation of a slow release device, such as a mini-osmotic pump, depot compound, etc. subject. Administration can be accomplished by any route, including parenteral and transmucosal (eg, oral, nasal, vaginal, rectal, or transdermal routes). Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intrarenal, intraurethral, ingracardial, ingracoronary, ingramiocardial, intradermal, epidural, subcutaneous, intraperitoneal,

intraventricular, iontophoretic and intracranial. Other delivery methods include, but are not limited to, liposomal formulations, intravenous infusion, transdermal patches, etc. In some embodiments, the administered dose of the rAAV gene therapy vector is from about 1E+11 vector genomes (gv)/kg to about 1E+12 vg/kg, from about 1E+12 vg/kg to about 2E+12 v.g./kg, from approximately 2E+12 v.g./kg to approximately 3E+12 v.g./kg, from approximately 3E+12 v.g./kg to approximately 3E+13 v.g. .g./kg, from approximately 3E+13 to approximately 3E+14 v.g./kg. In some embodiments, the dose of the rAAV gene therapy vector administered is from about 3E+12 vg/kg to about 3E+14 vg/kg.

The terms “systemic administration” and “systemically administered” refer to a method of administering a compound or composition to mammals such that the compound or composition is delivered to specific areas of the body, including the target site of pharmaceutical activity, through the circulatory system. Systemic administration includes, but is not limited to, oral, intranasal, rectal, and parenteral (eg, non-gastrointestinal, eg, intramuscular, intravenous, intra-arterial, transdermal, and subcutaneous) administration.

The term "co-administration" or "simultaneous administration", for example, with respect to compounds (for example, LAMP-2 polynucleotides) and/or analogs thereof and another active agent, refers to the administration of said compound and/or analogs thereof and said active agent in a manner that the physiological effect of said compound and agent can be achieved simultaneously. However, the two agents do not need to be administered together. In certain embodiments, administration of one agent may precede administration of another. A simultaneous physiological effect does not necessarily require the simultaneous presence of both agents in the bloodstream. However, in certain embodiments, coadministration typically results in both agents being simultaneously present in the body (e.g., in blood plasma) in a significant proportion (e.g., 20% or more, e.g., 30% or 40% or more, e.g., 50 % or 60% or more, e.g., 70% or 80% or 90% or more) of their maximum serum concentration for any given dose.

The term "effective amount" or "pharmaceutically effective amount" refers to the amount and/or dosage and/or mode of administration of one or more compounds (for example, gene therapy vectors) necessary to achieve a desired result, for example, increased expression of one or more LAMP-2 isoforms in quantities sufficient to reduce the ultimate severity of a disease characterized by impaired or deficient autophagy (eg, Danon disease). In some embodiments, the effective amount is from about 1E+11 vg/kg to about 1E+12 vg/kg, from about 1E+12 vg/kg to about 2E+12 vg/kg. /kg, from approximately 2E+12 v.g./kg to approximately 3E+12 v.g./kg, from approximately 3E+12 v.g./kg to approximately 3E+13 v.g./kg or from approximately 3E+13 i.g./kg to approximately 3E+14 i.g./kg rAAV gene therapy vector. In some embodiments, the effective amount is from about 3E+12 vg/kg to about 3E+14 vg/kg rAAV gene therapy vector.

The phrase "cause for administration" refers to the actions taken by a health care professional (eg, physician) or person supervising the subject's medical care that controls and/or authorizes the administration of the agent(s)/compound(s) into the subject's tissue. Obtaining a reason for administration may include diagnosis and/or determination of an appropriate therapeutic or prophylactic regimen and/or administration of specific agent(s)/compounds to the subject. Such assignment may include, for example, preparing a prescription form, annotating a medical record, etc.

As used herein, the terms “treatment” and “treatment” refer to delaying the onset, slowing or reversing the progression, reducing the severity or alleviating or preventing the disease or condition to which the term applies, or one or more of the symptoms of such disease or condition. The terms "treatment" and "treatment" also include the prevention, mitigation, alleviation, reduction, inhibition, elimination and/or relief of one or more symptoms of a disease or condition.

The term "mitigation" refers to reducing the intensity or eliminating one or more symptoms of a specified pathology or disease and/or reducing the rate or severity or delaying the onset of one or more symptoms of a specified pathology or disease and/or preventing the specified pathology or disease. In certain embodiments, reduction in intensity or elimination of one or more symptoms of a pathology or disease may include, for example, a measurable and sustained increase in the expression levels of one or more LAMP-2 isoforms.

As used herein, the phrase "consisting primarily of" refers to the family or species of active pharmaceutical agents listed in the method or composition, and may further include other agents not of themselves essential activity for the indicated indication or purpose.

The terms “subject,” “individual,” and “patient” refer interchangeably to mammals, preferably human or non-human primates, but also to domestic mammals (eg, dogs or cats), laboratory mammals (eg, mice, rats) , rabbit, hamster, guinea pig) and agricultural mammals (e.g. horses, cattle, pigs, sheep). In various embodiments, the subject may be a person (eg, an adult male, an adult female, a male adolescent, a female adolescent, a male child, a female child).

The terms "gene transfer" or "gene delivery" refer to methods or systems for reliably introducing foreign DNA into host cells. Such methods may result in transient expression of unintegrated transferred DNA, extrachromosomal replication and expression of transferred replicons (eg, episomes), or integration of transferred genetic material into the genomic DNA of host cells.

The term "vector" is used herein to refer to a nucleic acid molecule capable of carrying or transporting another nucleic acid molecule. The transferred nucleic acid is typically associated, for example, with a vector nucleic acid molecule. The vector may contain sequences that direct autonomous replication or reverse transcription in the cell, or include sequences sufficient to ensure integration into the DNA of the host cell, "vectors" include vectors for gene therapy. As used herein, the term “gene therapy vector” refers to a vector that can be used in the implementation of gene therapy, for example, delivery to a subject of a polynucleotide sequence encoding a therapeutic polypeptide. Gene therapy vectors may contain a nucleic acid molecule ("transgene") encoding a therapeutically active polypeptide, for example, LAMP-2B or other gene useful for gene replacement therapy when administered to a subject. Suitable vectors include, but are not limited to, viral vectors.

As used herein, the term “expression cassette” refers to a segment of DNA that, in the appropriate situation, is capable of directing the expression of a polynucleotide (transgene) encoding a therapeutically active polypeptide (eg, LAMP-2B) included in said expression cassette. When introduced into a host cell, the expression cassette is, among other things, capable of using the cell's machinery to transcribe the transgene into RNA, which is then typically further processed and finally translated into a therapeutically active polypeptide. The expression cassette may be contained in a gene therapy vector. Generally, the term "expression cassette" excludes polynucleotide sequences located in the 5' direction of the 5' ICP and in the 3' direction of the 3' ICP.

All patents, patent publications, and other publications referenced or identified herein are hereby incorporated by reference in their entirety for all purposes.

EXAMPLES

EXAMPLE 1: Preclinical and Clinical Evaluation of AAVrh74-LAMP-2B

A plasmid vector containing a gene expression cassette was prepared as shown in FIG. 1. The transgene was modified to encode LAM2B-HA-FLAG such that the protein could be detected using anti-HA antibodies or anti-FLAG antibodies. The AAVrh74-LAMP2B viral vector was generated using a helper-free three-plasmid system, producing recombinant AAV particles containing serotype rh74 capsid proteins and viral genomes containing AAV2 ICP flanking the human LAMP-2B expression cassette. The viral vector was tested in non-human primates.

Pharmacological and toxicological studies were performed in LAMP-2B ^−/− and wild-type mice. Based on preclinical safety and efficacy data observed in studies in mice and non-human primates, clinical studies were performed in patients with Danon's disease.

EXAMPLE 2. DNA, RNA and protein expression in non-human primates following intravenous administration of 1×10 ¹³ i.g./kg AAV9.LAMP2B and LA Vrh 74. LAMP2B

Studies comparing the AAV9 and AAVrh74 vectors in non-human primates were performed on pairs of male and female African green monkeys (AGM). Subjects received AAV9.LAMP2B-HA-Flag or AAVrh74.LAMP2B-HA-Flag. "AAV9.LAMP2B-HA-Flag" was an adeno-associated virus serotype AAV9 vector encoding the C-terminus of LAMP2B fused to an HA moiety. "AAVrh74.LAMP2B-HA-Flag" was an adeno-associated virus serotype AAVrh74 vector encoding the C-terminus of LAMP2B fused to an HA-Flag marker. One subject was given vehicle control. Vectors were administered by intravenous injection of 2 ml of 1.85 × ¹⁰¹³ vector genomes (vg)/ml, as determined by quantitative polymerase chain reaction (qPCR) using a plasmid containing the WPRE sequence to generate a reference curve. This injection achieved the target vector dose, which was approximately 1.0 × 10 ¹³ i.g./kg. Due to lower body weight, female subjects received approximately 1.2 × 10 ¹³ i.g./kg of the respective vectors. A summary of this experiment is given in Table 2.

Two months after injection, subjects were humanely sacrificed and tissue was collected for DNA, RNA and protein analysis. The following tissues were examined: heart (left atrium, right atrium, left ventricle and right ventricle); skeletal muscles (quadriceps and gastrocnemius); liver (left, right, middle and quadrate lobes); brain (frontal lobe, parietal lobe, temporal lobe, occipital lobe, cortex, hippocampus, medulla oblongata and cerebellum); and gonads.

Vector DNA - quantitative PCR

DNA was isolated from frozen tissues using the Qiagen DNeasy® kit. DNA purity (A260/A280) and concentration were assessed using a NanoDrop OneTM spectrophotometer (Thermo). Quantitative PCR (qPCR) was performed using 20 ng DNA and TaqMan Universal Master Mix II (Thermo, 4440038) in a real-time PCR system (QuantStudio5, Thermo) using the following primers/probes:

WPRE (cassette):

Forward primer: 5`-ATCATGCTATTGCTTCCCGTA-3` (SEQ ID NO:30) Reverse primer: 5`-GGGCCACAACTCCTCATAAA-3` (SEQ ID NO:31) Probe: 5 ^, -CCTCCTTGTATAAAATCCTGGTTGCTGTCT-3 ^, (SEQ ID NO:32) RNaseP (Thermo housekeeping gene)

A standard curve was prepared using plasmid DNA containing the WPRE sequence. In fig. Figure 2 shows a histogram of the quantitative determination of vector DNA in organs most affected by Danon disease using qPCR.

LAMP2B mRNA - quantitative RT-PNR

RNA was isolated 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 using a NanoDrop One spectrophotometer. RNA was converted to cDNA using Superscript IV VILO master mix (Thermo). qPCR was performed using 10 ng RNA and TaqMan Universal Master Mix II (Thermo) in a real-time PCR system (QuantStudio5, Thermo) using the following primers/probes:

WPRE (cassette):

Forward primer: 5`-ATCATGCTATTGCTTCCCGTA- <sup>3`</sup> (SEQ ID NO:33) Reverse primer: 5`GGGCCAAASTSTCTAAA-3` (SEQ ID NO:34) Probe: 5 ^, -CCTCCTTGTATAAAATCCTGGTTGCTGTCT-3 ^, (SEQIDN0:35) HPRT-1 (housekeeping gene, Thermo)

A standard curve was prepared using plasmid DNA containing the WPRE sequence. In fig. 3A shows a histogram of vector DNA quantification in cardiac regions by qPCR. In fig. Figure 3 B shows a histogram of vector DNA quantification in muscle using qPCR. In fig. Figure 4 shows a histogram of mRNA quantification in the organs most affected by Danon disease using RT-qPCR. In fig. Figure 5A shows a histogram of mRNA quantification in cardiac regions by RT-qPCR. In fig. Figure 5 B shows a histogram of muscle mRNA quantification using RT-qPCR.

LAMP2B mRNA - RNAscope

5-mm tissue cubes were fixed in 10% neutral buffered formalin, embedded in paraffin, and cut into sections. Transgene mRNA was detected using the WPRE-O3 ZZ (ACD) probe using the RNAscope 2.5 LS RED assay. Semiquantitative visual scoring of one section of each tissue was performed, counting cells with ≥ 1 spot per cell as positive. The percentage of cells testing positive was categorized into five categories: 0%, 1–25%, 26–50%, 51–75%, or 100%.

In fig. Figure 6A shows a photomicrograph of semiquantitative RNAscope analysis of mRNA in an untreated left ventricle. In fig. 6B shows micrographs of semiquantitative RNAscope analysis of mRNA in treated left ventricles. In fig. Figure 7A shows a photomicrograph of semi-quantitative RNAscope analysis of mRNA in untreated quadriceps. In fig. Figure 7B shows micrographs of semi-quantitative RNAscope analysis of mRNA in treated quadriceps. In fig. Figure 8 shows micrographs of semi-quantitative RNAscope analysis of mRNA in treated gastrocnemius muscle. Summary results for vehicle control (Table 3A), AAV9 (Table 3A), and AAVrh74 (Table 3C) are shown.

LAMP2B protein - solid phase ELISA

Approximately 125 mg of tissue was homogenized in 500 ml lysis buffer using 0.9-2.00 mm stainless steel balls (Next Advance) and a Next Advance Bullet Blender 24. Lysis buffer contained 300 mM NaCl, 20 mM EDTD, 100 mM Tris, pH 8.0, 2% NP-40 and 0.2% SDS with Complete™ EDTA-free protease inhibitor and PhosSTOPTM phosphatase inhibitor. Total protein was assessed using BCA (Thermo). Each well was loaded with 100 mg of total protein. A standard curve was constructed using purified human LAMP2 protein (Origene). ELISA was performed using mouse monoclonal antibody (H4 B4, Novus Biologicals) as the capture antibody, goat polyclonal antibody (R&D Systems) as the detection antibody, and HRP-linked donkey anti-goat antibody (Millipore) as the secondary antibody. The plates were developed using TMB (Thermo) and quantified on a spectrophotometer (Spectramax M5 s).

In fig. Figure 9 shows a histogram of protein quantification in tissues most affected by Danon disease using solid-phase ELISA. In fig. 10A shows a histogram of protein quantification in cardiac regions by ELISA. In fig. 10B shows a histogram of muscle protein quantification using solid-phase ELISA.

Clinical pathology

The pathological effects of the vectors were assessed. In fig. 11A-11D show line graphs of clinical pathology measurements in NHP serum during the study. Levels of clinical pathology were assessed as changes in alanine aminotransferase, ALT (Fig. HA); aspartate aminotransferase, AST (Fig. 11B); white blood cell count, WBC (Fig. 11C); and neutrophil counts (Fig. 11D) throughout the study. B059 is a vehicle control medium. A991 and A602 are AAV9-treated animals. A710 and A981 are AAVrh74-treated animals.

conclusions

AAV-based gene therapy using the LAMP2B transgene is good

was tolerated in non-human primates at a vector dose of 1.0 × 10 ¹³ i.g./kg. This result is an important and unexpected result, since AAV-based gene therapy experiments with several other transgenes have demonstrated pathological effects at doses equal to 1.0 × 10 ¹³ vg/kg or lower. In addition, both AAV9 and AAVrh74 were well tolerated. Elevated levels of certain markers were observed in animals A602 and A710 on day 21, but these outliers may be due to experimental error or pathology that resolved on its own.

Both vectors were localized to and transduced into target tissues for the treatment of Danon disease (heart and muscle), but were present to a lesser extent in the brain or gonads. Expression in the gonads would be undesirable for safety reasons. As expected, significant amounts of vector accumulated in the liver, which is desirable since the liver is the tissue affected in Danon disease. The vector was present in every quadrant of the heart and in both the quadriceps and gastrocnemius muscles. This is a desirable outcome for the treatment of Danon disease.

The localization of any AAV vector serotype (eg, AAV9) is not predictive of the localization of other serotypes (eg, AAVrh74). This experiment demonstrated that AAVrh74 achieved the required localization for the treatment of Danon disease or other diseases with cardiac and muscle etiologies. LAMP2B transgene mRNA and protein were expressed in similar tissues in both the AAV9 and AAVrh74 groups. Expression of vectors of different serotypes was comparable. The number of animals in the study was too small to detect statistically significant trends in expression levels between AAV9 and AAVrh74. RNAscope analysis suggests that in the AAV9 and AAVrh74 groups, a similar proportion of cells in heart, muscle and liver tissue were infected.

These data demonstrate that AAVrh74 can be used as a vector to deliver LAMP2B to tissues relevant for the treatment of Danon disease. In these experiments, AAVrh74 was as effective as AAV9.

Claims (60)

1. A vector for gene therapy based on recombinant adeno-associated virus (rAAV), containing a polynucleotide containing a 5'-ICP, an expression cassette and a 3'-ICP; and capsid protein, wherein the expression cassette contains a transgene encoding lysosome-associated membrane protein 2 (LAMP-2), or a functional variant thereof, wherein the expression cassette is flanked by 5'-IKP and 3'-IKP, and wherein the capsid protein comprises an AAVrh.74 capsid protein or a functional variant thereof, wherein the capsid protein or a functional variant thereof has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 2. 2. rAAV vector for gene therapy according to claim 1, where LAMP-2 is selected from LAMP-2A, LAMP-2B and LAMP-2C. 3. rAAV gene therapy vector according to claim 1, wherein said capsid protein or functional variant thereof is characterized by 100% sequence identity to the amino acid sequence of SEQ ID NO: 2. 4. rAAV vector for gene therapy according to any one of paragraphs. 1-3, where each of the 5'-ICP and 3'-ICP is an AAV2 5'-ICP and an AAV2 3'-ICP, respectively, or variants thereof. 5. rAAV vector for gene therapy according to any one of paragraphs. 1-4, wherein LAMP-2 or a functional variant thereof is LAMP-2B or a functional variant thereof having at least 90% identity to SEQ ID NO: 16. 6. rAAV vector for gene therapy according to claim 5, where LAMP-2B or a functional variant thereof is characterized by 100% identity with respect to SEQ ID NO: 16. 7. rAAV vector for gene therapy according to any one of paragraphs. 1-6, wherein the transgene is 90% identical to SEQ ID NO: 6. 8. rAAV vector for gene therapy according to claim 7, where the transgene is characterized by 100% identity with respect to SEQ ID NO: 6. 9. rAAV vector for gene therapy according to claim 1, where: the capsid protein or a functional variant thereof has 100% sequence identity to the amino acid sequence of SEQ ID NO: 2; LAMP-2B or a functional variant thereof is 100% identical to SEQ ID NO: 16; And The transgene is 100% identical to SEQ ID NO: 6. 10. rAAV vector for gene therapy according to any one of paragraphs. 1-9, where the expression cassette contains: a consensus optimal Kozak sequence operably linked to the transgene, wherein said consensus optimal Kozak sequence comprises SEQ ID NO: 20; a full-length polyA sequence operably linked to the transgene, wherein said full-length polyA sequence comprises SEQ ID NO: 26; and/or does not contain a start codon in the 5' direction from the start codon of the transgene. 11. rAAV vector for gene therapy according to any one of paragraphs. 1-10, where the expression cassette contains the following functionally linked elements in the direction from the 5' end to the 3' end: first inverted terminal repeat, enhancer/promoter region, intron, consensus optimal Kozak sequence, transgene, 3' untranslated region, containing a full-length polyA sequence, and a second inverted terminal repeat, wherein the expression cassette does not contain a start codon in the 5' direction from the start codon of the transgene. 12. rAAV vector for gene therapy according to claim 11, where the enhancer/promoter region contains: in a 5' to 3' direction, a CMV IE enhancer and a chicken beta-actin promoter, wherein said enhancer/promoter region optionally further comprises a first exon and a first intron of a chicken beta-actin gene and a rabbit beta-globin gene splice acceptor; and/or a tissue-specific promoter capable of mediating increased expression in cardiac and/or skeletal muscle tissue compared to liver tissue. 13. rAAV vector for gene therapy according to claim 12, where: the capsid protein or a functional variant thereof has 100% sequence identity to the amino acid sequence of SEQ ID NO: 2; LAMP-2B or a functional variant thereof has 100% sequence identity to SEQ ID NO: 16; And The transgene is 100% identical to SEQ ID NO: 6. 14. Pharmaceutical composition containing rAAV vector for gene therapy according to any one of paragraphs. 1-13. 15. A method of treating or preventing Danon disease in a subject in need thereof, comprising administering to the subject an rAAV vector for gene therapy according to any one of claims. 1-13 or the pharmaceutical composition according to claim 14. 16. The method according to claim 15, characterized in that the rAAV gene therapy vector or pharmaceutical composition is administered by a parenteral route selected from intravenous, intraarterial, intracardiac, intracoronary, intramyocardial, intrarenal, intraurethral, epidural and intramuscular administration. 17. The method according to claim 15 or 16, characterized in that the subject is a human. 18. Method according to any one of paragraphs. 15-17, characterized in that the subject exhibits symptoms of Danon's disease; the subject is identified as having reduced or undetectable expression of endogenous LAMP-2B; and/or subject identified by the presence of a mutant LAMP-2B gene. 19. Method according to any one of paragraphs. 15-18, characterized in that the rAAV vector for gene therapy is administered at a dose of from approximately 3 × 10 ¹² vg/kg to approximately 3 × 10 ¹⁴ vg/kg. 20. Method according to any one of paragraphs. 15-19, characterized in that the introduction of an rAAV vector for gene therapy transduces one or more of the heart, muscle and liver; administration of the rAAV gene therapy vector causes expression of LAMP2B mRNA in one or more of the heart, muscle and liver; administration of the rAAV gene therapy vector causes expression of the LAMP2B protein in one or more of the heart, muscle and liver; administration of the rAAV gene therapy vector causes the rAAV gene therapy vector to infect 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; administration of the rAAV gene therapy vector causes transduction of the rAAV gene therapy vector in the gonads in an amount of less than 0.1 vector genome (vg) per diploid genome; administration of the rAAV vector for gene therapy causes the expression of LAMP2B mRNA in the gonads in an amount of less than 2 × 10 ⁴ copies of mRNA per μg of total RNA; administration of the rAAV vector for gene therapy does not induce LAMP2B protein expression in the brain and/or gonads; and/or administration of an rAAV gene therapy vector transduces and/or induces transgene expression at approximately the same level as administration of an AAV9 gene therapy vector containing the same expression cassette. 21. A method for delivering a LAMP-2 polynucleotide encoding the LAMP-2 protein to a subject and/or expressing the LAMP-2 protein in a subject, comprising administering to the subject an rAAV vector for gene therapy according to any one of claims. 1-13 or the pharmaceutical composition according to claim 14. 22. The method according to claim 21, characterized in that the rAAV gene therapy vector or pharmaceutical composition is administered by a parenteral route selected from intravenous, intraarterial, intracardiac, intracoronary, intramyocardial, intrarenal, intraurethral, epidural and intramuscular administration. 23. The method according to claim 21 or 22, characterized in that the subject suffers from or is at risk of an autophagy disorder selected from the group consisting of Danon disease, end-stage heart failure, heart attack myocardium, drug toxicity, diabetes, end-stage renal disease and aging. 24. Method according to any one of paragraphs. 21-23, characterized in that the subject is a human. 25. Method according to any one of paragraphs. 21-24, characterized in that the subject experiences symptoms of Danon's disease; the subject is identified as having reduced or undetectable expression of endogenous LAMP-2B; and/or subject identified by the presence of a mutant LAMP-2B gene. 26. Method according to any one of paragraphs. 21-24, characterized in that the rAAV gene therapy vector is administered at a dose of from about 3 × 10 ¹² vg/kg to about 3 × 10 ¹⁴ vg/kg, optionally at a dose of from about 3 × 10 ¹² vg/kg to approximately 1.2 × 10 ¹³ vg/kg or approximately 1.0 × 10 ¹³ vg/kg. 27. Method according to any one of paragraphs. 21-26, characterized in that the introduction of an rAAV vector for gene therapy transduces one or more of the heart, muscle and liver; administration of the rAAV gene therapy vector causes expression of LAMP2B mRNA in one or more of the heart, muscle and liver; administration of the rAAV gene therapy vector causes expression of the LAMP2B protein in one or more of the heart, muscle and liver; administration of the rAAV gene therapy vector causes the rAAV gene therapy vector to infect 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; administration of the rAAV gene therapy vector causes transduction of the rAAV gene therapy vector in the gonads in an amount of less than 0.1 vector genome (vg) per diploid genome; administration of the rAAV gene therapy vector causes expression of LAMP2B mRNA in the gonads at levels of less than 2 × 10 ⁴ mRNA copies per μg of total RNA; administration of the rAAV vector for gene therapy does not induce LAMP2B protein expression in the brain and/or gonads; and/or administration of an rAAV gene therapy vector transduces and/or induces transgene expression at approximately the same level as administration of an AAV9 gene therapy vector containing the same expression cassette.