Compositions and methods for treating tafazzin deficiency

Abstract

The invention provides compositions and methods for treating a disorder characterized by tafazzin deficiency. The compositions include a viral vector that contains sequences encoding tafazzin and a cell penetrating peptide, which can be administered systemically or locally to provide treatment for Barth Syndrome and tafazzin-related disorders.

Description

[0001] COMPOSITIONS AND METHODS FOR TREATING TAFAZZIN DEFICIENCY

[0002] Field of the Invention

[0003] The present application generally relates to treatment of disorders characterized by tafazzin deficiency. More specifically, the invention is directed to viral vectors that contain sequences encoding tafazzin and a cell penetrating peptide, which can be administered systemically or locally to provide treatment for Barth Syndrome and tafazzin-related disorders.

[0004] Sequence Listing

[0005] The present application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on February 6, 2025, is named “00398-569WO2_Sequence_Listing_2_6_25” and is 17,487 bytes in size.

[0006] Statement as to Federally Funded Research

[0007] This invention was made with government support under grant numbers 1 R61 HL154137-01 A1 and 4R33HL154137-03 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0008] Background of the Invention

[0009] Barth Syndrome is a disorder that is characterized by defects in the expression of tafazzin (TAZ), a mitochondrial acyltransferase that regulates the remodeling of cardiolipin (CL) and is essential for mitochondrial morphology and function. Clinical manifestations of Barth Syndrome include cardiomyopathy, muscular hypotonia, and neutropenia. There is currently no targeted treatment option available for Barth Syndrome and other disorders associated with TAZ deficiency.

[0010] Summary of the Invention

[0011] In one aspect, the invention provides viral vectors comprising sequences that encode tafazzin (TAZ) and a cell penetrating peptide (CPP).

[0012] In some embodiments, the TAZ is full-length TAZ.

[0013] In some embodiments, the TAZ comprises the amino acid sequence of SEQ ID NO: 1 .

[0014] In some embodiments, the TAZ lacks exon 5 (TAZ A5; isoform 2).

[0015] In some embodiments, the TAZ A5 comprises the amino acid sequence of SEQ ID NO: 3.

[0016] In some embodiments, the CPP is selected from the group consisting of cardiac targeting peptide (CTP), antennapedia permeability peptide (ANTP), human immunodeficiency virus (HIV) Tat permeability peptide, and Kaposi fibroblast growth factor 4 (FGF4)-permeability peptide, or a variant thereof.

[0017] In some embodiments, the CTP comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 5.

[0018] In some embodiments, the CTP comprises an amino acid sequence that is 100% identical to the sequence of SEQ ID NO: 5.

[0019] In some embodiments, the ANTP comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the ANTP comprises an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 6.

[0020] In some embodiments, the HIV Tat permeability peptide has an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 7.

[0021] In some embodiments, the HIV Tat permeability peptide comprises a sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 7.

[0022] In some embodiments, the Kaposi FGF4-permeability peptide has an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 8.

[0023] In some embodiments, the Kaposi FGF4-permeability peptide comprises an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 8.

[0024] In some embodiments, the TAZ and CPP are coupled by a peptide linker (TAZ-CPP).

[0025] In some embodiments, the peptide linker is a glycine-rich linker.

[0026] In some embodiments, the linker comprises the sequence of SEQ ID NO: 9 or SEQ ID NO: 10.

[0027] In some embodiments, the TAZ-CPP has an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 11 .

[0028] In some embodiments, the TAZ-CPP comprises an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 11 .

[0029] In some embodiments, the TAZ-CPP has an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 13.

[0030] In some embodiments, the TAZ-CPP comprises an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 13.

[0031] In some embodiments, the viral vector is a lentiviral vector.

[0032] In some embodiments, the viral vector is an adeno-associated viral vector.

[0033] In another aspect, the invention provides pharmaceutical compositions comprising (i) a viral vector described herein or a cell transduced by the viral vector, and (ii) a pharmaceutically acceptable carrier or diluent.

[0034] In another aspect, the invention provides methods of treating a subject having mitochondrial dysfunction or a TAZ deficiency, the method comprising administering to the subject a therapeutically effective amount of a viral vector described herein, a cell transduced by the viral vector, or a pharmaceutical composition described herein.

[0035] In some embodiments, the viral vector, cell, or pharmaceutical composition is administered to the subject by systemic administration or local administration.

[0036] In some embodiments, the local administration comprises intracoronary infusion.

[0037] In some embodiments, the cell is a bone marrow precursor cell or a descendant thereof.

[0038] In some embodiments, the subject has or is at risk of developing Barth syndrome, cardiomyopathy, aging-related condition, heart failure, diabetes, myocardial infarction, acquired mitochondrial disorder, or inherited mitochondrial disorder.

[0039] In some embodiments, the cardiomyopathy is chemotherapy-induced cardiomyopathy, diabetic cardiomyopathy, dilated cardiomyopathy, hypertensive cardiomyopathy, hypertrophic cardiomyopathy, ischemic cardiomyopathy, or noncompaction cardiomyopathy. In some embodiments, the inherited mitochondrial disorder is autosomal dominant optic atrophy (ADOA), Leber's hereditary optic neuropathy (LHON), LHON plus, Leigh syndrome, pyruvate dehydrogenase complex deficiency (PDCD), thymidine kinase 2 (TK2) deficiency, Alpers disease, Barth syndrome, a beta-oxidation defect, carnitine-acyl-carnitine deficiency, carnitine deficiency, a creatine deficiency syndrome, coenzyme Q10 deficiency, complex I deficiency, complex II deficiency, complex III deficiency, complex IV deficiency / COX deficiency, complex V deficiency, chronic progressive external ophthalmoplegia syndrome (CPEO), CPT I deficiency, CPT II deficiency, Kearns-Sayre syndrome (KSS), lactic acidosis, leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation (LBSL - leukodystrophy), long-chain acyl-CoA dehydrogenase deficiency (LCAD), long-chain L-3 hydroxyacyl-CoA dehydrogenase deficiency (LCHAD), Luft disease, multiple acyl-CoA dehydrogenase (MAD) deficiency, medium-chain acyl-CoA dehydrogenase deficiency (MCAD), mitochondrial encephalomyopathy lactic acidosis and stroke-like episodes (MELAS), myoclonic epilepsy and ragged- red fiber disease (MERRF), mitochondrial recessive ataxia syndrome (MIRAS), a mitochondrial cytopathy, mitochondrial DNA depletion, mitochondrial encephalopathy, mitochondrial neurogastrointestinal encephalopathy syndrome (MNGIE), neuropathy, ataxia, and retinitis pigmentosa (NARP), Pearson syndrome, pyruvate carboxylase deficiency, pyruvate dehydrogenase deficiency, a POLG mutation, short-chain acyl-CoA dehydrogenase deficiency (SCAD), 3-hydroxyacyl-CoA dehydrogenase (HADH) deficiency, very-long-chain acyl-CoA dehydrogenase deficiency (VLCAD), or Friedrich's ataxia.

[0040] Definitions

[0041] To facilitate the understanding of the invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by those of ordinary skill in the areas relevant to the invention.

[0042] As used herein, the terms “administration” or “administering” refer to providing or giving a subject a therapeutic agent (e.g., a viral vector described herein) that includes sequences that encode tafazzin (TAZ) (e.g., human TAZ FL or human TAZ A5) and a cell penetrating peptide (CPP), or a cell transduced by a viral vector expressing these sequences, by any effective route. As explained elsewhere herein, optionally the TAZ is linked to the CPP via a linker (e.g., a glycine-rich linker). Exemplary routes of administration are described herein and include systemic administration routes, such as intravenous injection and infusion, as well as local administration, e.g., administration to the heart by, e.g., intracoronary infusion, among others.

[0043] As used herein, the terms "cell penetrating peptide" and “CPP” refer to peptides that facilitate cellular or organelle uptake of the peptide itself and one or more cargo molecules (e.g., other peptides or polypeptides) that are linked to the cell penetrating peptides. In certain embodiments, the CPPs can comprise portions of cardiac targeting, Drosophila antennapedia, HIV Tat, or Kaposi FGF4 peptides, or variants thereof, which facilitate cellular or organelle uptake. As used herein, such peptide fragments may be referred to as cardiac targeting peptides, antennapedia permeability peptides, HIV Tat permeability peptides, and Kaposi FGF4 permeability peptides, respectively.

[0044] As used herein, the terms “conservative mutation,” “conservative substitution,” or “conservative amino acid substitution” refer to a substitution of one or more amino acids for one or more different amino acids that exhibit similar physicochemical properties, such as polarity, electrostatic charge, and / or steric volume. These properties are summarized for each of the twenty naturally occurring amino acids in table 1 below.

[0045] Table 1 . Representative physicochemical properties of naturally occurring amino acids

[0046] From this table it can be appreciated that conservative amino acid families include, e.g., (i) G, A, V, L, I, P, and M; (ii) D and E; (Hi) C, S and T ; (iv) H, K and R; (v) N and Q; and (vi) F, Y and W. A conservative mutation or substitution is therefore one that substitutes one amino acid for a member of the same amino acid family (e.g., a substitution of Ser for Thr or Lys for Arg). As used herein, the terms "effective amount," “therapeutically effective amount,” and a “sufficient amount” of a viral vector described herein refer to amounts that can result in a beneficial or desired physiological change in a subject. As such, an “effective amount” or synonym thereof depends upon the context in which it is being applied. For example, in the context of treating a TAZ deficiency (e.g., Barth Syndrome), it is an amount of a viral vector that achieves a treatment response as compared to a response obtained without such treatment. The amount will vary depending upon various factors, such as the pharmaceutical formulation, the route of administration, the type of disease or disorder, the identity of the subject (e.g., age, sex, weight) or host being treated, and the like, but can nevertheless be determined by those skilled in the art. Also, as used herein, a “therapeutically effective amount” of a viral vector of the present disclosure is an amount which results in a beneficial or desired result in a subject as compared to a control. As such, the amount needs not fully prevent or cure the disease or the condition but can also provide a partial benefit, such as a delay of onset or an alleviation or an improvement of at least one symptom of the disease or the condition. As defined herein, a “therapeutically effective amount” of viral vector of the present disclosure may be readily determined by those of ordinary skill in the art by methods that are known in the art. Dosage regimes may be adjusted to provide an optimum therapeutic response.

[0047] As used herein, the terms "fusion proteins" or "fusion peptides" refer to proteins or peptides created through the joining of two or more coding sequences or genes (e.g., a fusion gene), each of which codes for separate proteins or peptides. Translation of the fusion gene may result in one or more polypeptides comprising functional properties derived from each of the two or more genes. Optionally, the fused proteins or peptides are linked by a linker, such as a flexible peptide linker sequence (e.g., a glycine-rich sequence).

[0048] As used herein, the term “lentivirus” refers to a genus of the Retroviridae family. As used herein, the term “lentiviral vector” refers to a vector including one or more nucleic acid sequences derived from at least a portion of a lentivirus genome. In some embodiments, the lentiviral vector is a human immunodeficiency virus (HlV)-derived lentiviral vector. In some embodiments, the lentiviral vector is non- pathogenic. In some embodiments, the lentiviral vector is replication-defective. In some embodiments, the lentiviral vector is an HIV-1 -derived lentiviral vector. In some embodiments, the lentiviral vector is a third-generation lentiviral vector.

[0049] As used herein, the terms "peptide" and "polypeptide" are used in their broadest senses to refer to a sequence of subunit amino acids. The peptides or polypeptides of the invention may comprise L- amino acids, D-amino acids (which can be resistant to L-amino acid-specific proteases in vivo), or a combination of D- and L-amino acids. The terms peptide and polypeptide can be used interchangeably. The peptides and polypeptides described herein may be recombinantly expressed such as by the viral vectors described herein. The peptides and polypeptides may be linked to any other moiety as deemed useful for a given purpose. Such linkage can comprise covalent linkages or non-covalent linkages as is understood by those of skill in the art. As described elsewhere herein, peptides and / or polypeptides described herein can be linked to one another, optionally via a linker as described herein, in the form of a fusion protein or a fusion peptide.

[0050] “Percent (%) sequence identity” with respect to a reference polynucleotide or polypeptide (or peptide) sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to the nucleic acids or amino acids in the reference polynucleotide or polypeptide (or peptide) sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid or amino acid sequence identity can be achieved in various ways that are within the capabilities of those of skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, percent sequence identity values may be generated using the sequence comparison computer program BLAST. As an illustration, the percent sequence identity of a given nucleic acid or amino acid sequence, A, to, with, or against a given nucleic acid or amino acid sequence, B, (which can alternatively be phrased as a given nucleic acid or amino acid sequence, A that has a certain percent sequence identity to, with, or against a given nucleic acid or amino acid sequence, B) is calculated as follows:

[0051] 100 multiplied by (the fraction X / Y) where X is the number of nucleotides or amino acids scored as identical matches by a sequence alignment program (e.g., BLAST) in that program’s alignment of A and B, and where Y is the total number of nucleic acids in B. It will be appreciated that where the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, the percent sequence identity of A to B will not equal the percent sequence identity of B to A.

[0052] As used herein, the term “pharmaceutically acceptable” is used to describe compounds, materials, compositions, and / or dosage forms that are suitable for contact with the tissues of a subject, such as a mammal (e.g., a human) without excessive toxicity, irritation, allergic response, and other problems or complications commensurate with a reasonable benefit / risk ratio.

[0053] As used herein, the term “promoter” refers to a recognition site on DNA that is bound by an RNA polymerase. The polymerase drives transcription of a transgene that is operably associated with the promoter. The viral vectors described herein include promoter sequences that direct expression of transgene sequences encoding the TAZ-CPP fusions described herein.

[0054] As used herein, the terms “subject” and “patient” refer to an organism that receives treatment for a particular disease or condition as described herein (such as TAZ deficiency, e.g., Barth Syndrome). Examples of subjects and patients include mammals, such as humans, receiving treatment for a disease or condition described herein.

[0055] As used herein, the terms "tafazzin" and “TAZ” refer to a phospholipid-lysophospholipid transacylase that modifies cardiolipin (a membrane phospholipid) to its tetralinoleoyl form. In some embodiments, tafazzin can refer to full-length human tafazzin (TAZ FL) or human tafazzin lacking exon 5 (TAZ A5; isoform 2; TAZV2), both of which exhibit transacylase activity. TAZ FL has the amino acid sequence of SEQ ID NO: 1 and is encoded by the nucleic acid sequence of SEQ ID NO:2. TAZ A5 has the amino acid sequence of SEQ ID NO: 3 and is encoded by the nucleic acid sequence of SEQ ID NO:4. In some embodiments, the TAZ is a variant of a sequence defined herein.

[0056] As used herein, the terms “transduction” and “transduce” refer to a method of introducing a viral vector construct or a part thereof into a cell. When the vector construct is contained in a viral vector such as a lentiviral vector or an adenovirus vector, transduction refers to viral infection of the cell and subsequent transfer and integration of the vector construct or part thereof into the cell genome. As used herein, the term “transgene” refers to a recombinant nucleic acid (e.g., DNA or cDNA) encoding a gene product (e.g., TAZ, TAZ A5, or TAZV2 as described herein, which may be operably associated with a CPP as described herein, optionally via a linker). The gene product may be an RNA, peptide, or protein. In addition to the coding region for the gene product, the transgene may include or be operably linked to one or more elements to facilitate or enhance expression, such as a promoter, enhancer(s), and other functional elements.

[0057] As used herein, the terms “treat,” “treatment,” and “treating” refer to an approach for obtaining beneficial or desired results, e.g., clinical results. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms or conditions, diminishment of extent of disease or condition, stabilized (i.e. , not worsening) state of disease, disorder, or condition, preventing spread of disease or condition, delay or slowing the progression of disease or condition, amelioration or palliation of the disease or condition, and remission (whether partial or total), whether detectable or undetectable. “Ameliorating” or “palliating” a disease or condition means that the extent and / or undesirable clinical manifestations of the disease, disorder, or condition are lessened and / or time course of the progression is slowed or lengthened, as compared to the extent or time course in the absence of treatment. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder, as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

[0058] As used herein, the term "prophylactic treatment" refers to a treatment administered to a subject who does not display signs or symptoms of a disease or condition, or a subject who displays only early signs or symptoms of a disease or condition, such that treatment is administered for the purpose of diminishing, preventing, and / or decreasing the risk of further developing the disease or condition or of diminishing, preventing, and / or decreasing the risk of developing the disease or condition. Thus, a prophylactic treatment may function as a preventative treatment against a disease or condition.

[0059] As used herein, the term "therapeutic treatment" refers to a treatment administered to a subject who displays symptoms or signs of a disease or condition, and the therapeutic treatment is administered to the subject for the purpose of diminishing or eliminating the symptoms or the signs of the disease or the condition.

[0060] In some embodiments, a “variant” of a reference sequence defined herein comprises a sequence that has a specified level of sequence identity to the reference sequence (see, e.g., below). In other embodiments, a “variant” of a reference sequence defined herein has one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions, deletions, or insertions relative to the reference sequence. In some embodiments, such a variant has, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions relative to a reference sequence. In some embodiments, one or more of the substitutions is a conservative substitution. In some embodiments, each of the substitutions is a conservative substitution.

[0061] As used herein, the term “vector” refers to a nucleic acid, e.g., DNA or RNA, that may function as a vehicle for the delivery of a gene of interest into a cell (e.g., a mammalian cell, such as a human cell), such as for purposes of replication and / or expression. Vectors include viruses (e.g., lentiviruses, adeno- associated viruses, and adenoviruses) that are modified to carry and express transgenes in cells.

[0062] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. Hence “comprising A or B” means including A, or B, or A and B. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosed method, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0063] Brief Description of the Drawings

[0064] FIG. 1 is a graph showing the effects of lentiviral tafazzin treatment on MLCL / CL ratios in murine embryonic fibroblasts (MEFs).

[0065] FIG. 2 is a graph showing the effect of lentiviral tafazzin gene therapy on MLCL / CL ratio in treated mouse hearts. Analysis was performed with SPSS-29 software. Between the groups, one-way ANOVA was used followed by correction for multiple comparison testing using Bonferonni method. Within the groups multiple comparisons were carried out by repeated measure ANOVA with correction for multiple comparisons by the Bonferroni method. The results were considered significant when the corrected probability level was < 0.05 (*p < 0.05, **p < 0.01 , ***p < 0.001 ).

[0066] FIGs. 3-5 are graphs showing the effects of lentiviral tafazzin gene therapy on ejection fraction (FIG. 3), fractional shortening (FIG. 4), and global longitudinal strain (GLS; FIG. 5) in treated mouse hearts. Analysis was performed with SPSS-29 software. Between the groups, one-way ANOVA was used followed by correction for multiple comparison testing using Bonferonni method. Within the groups multiple comparisons were carried out by repeated measures ANOVA with correction for multiple comparisons by the Bonferroni method. The results were considered significant when the corrected probability level was < 0.05 (*p < 0.05, **p < 0.01 , ***p < 0.001 ). “* indicates the significant difference between wild type vs GFP and Empty virus, p < 0.001 ; ## indicates the significant difference between hTAZV2-Antp vs GFP and Empty Virus, p < 0.01 ; @@ indicates the significant difference between time point (TP)1 (week 4) vs TP3 (week 12) in GFP treated Lentivirus Mice within the group, p < 0.01 ; $$ indicates the significant difference between TP1 (week 4) vs TP4 (week 16) in GFP and Empty treated Lentivirus within the group, p < 0.01 .

[0067] FIG. 6 is a graph showing the effects of lentiviral tafazzin gene therapy on interventricular septum wall thickness in treated mouse hearts. Analysis was performed by SPSS-29 software. Between the groups, one-way ANOVA followed by multiple comparison using Bonferonni test was used. Within the groups multiple comparisons were carried out by repeated measures ANOVA and multiple comparisons were done by the Bonferroni test. The results were considered significant when the probability level was < 0.05 (7#p < 0.05, *7##p < 0.01 , **7###p < 0.001 ). “* Indicates the significant difference between the WT, and empty / GFP treated mice. ## indicates the significant difference between hTAZV2-Antp / hTAZV2- CTP and empty / GFP treated mice. FIG. 7 is a set of photomicrographs showing the GFP immune-positive cells in the heart section of GFP lentivirus-treated TAZ KO mouse at 20X (“G-A”) and 100x (“G-B”) total magnification and the negative control (no primary antibody) for GFP at 20X (“G-C”) and 100X (“G-D”) total magnification.

[0068] Detailed Description of the Invention

[0069] The invention provides viral vectors containing sequences that encode tafazzin (TAZ) and a cell penetrating peptide (CPP), compositions including the viral vectors, and methods of using the viral vectors and compositions for preventing or treating disorders or conditions such as those that are characterized by TAZ deficiency, such as Barth Syndrome (also see below). The invention is based, in part, on the inventor’s discovery that the presence of a CPP enhances the effects of TAZ that is produced after viral gene delivery. The vectors, compositions, and methods are described in more detail below.

[0070] Tafazzin

[0071] Tafazzin (TAZ) is a mitochondrial phospholipid transacylase that regulates the maturation of cardiolipin (CL), a phospholipid that is synthesized and functions in inner mitochondrial membranes. Failure to properly remodel CL results in defects of mitochondrial morphology and function, both of which have been implicated in cardiovascular disease.

[0072] TAZ-encoding constructs that can be used in the vectors, compositions, and methods described herein include polynucleotides that encode full length TAZ (TAZ FL), TAZ in which exon 5 sequences are deleted (TAZ A5; TAZV2), or variants thereof.

[0073] Human TAZ FL has the following amino acid sequence: MPLHVKWPFPAVPPLTWTLASSVVMGLVGTYSCFWTKYMNHLTVHNREVLYELIEKRGPATPLITVSNH QSCMDDPHLWGILKLRHIWNLKLMRWTPAAADICFTKELHSHFFSLGKCVPVCRGAEFFQAENEGKGVL DTGRHMPGAGKRREKGDGVYQKGMDFILEKLNHGDWVHIFPEGKVNMSSEFLRFKWGIGRLIAECHLN PIILPLWHVGMNDVLPNSPPYFPRFGQKITVLIGKPFSALPVLERLRAENKSAVEMRKALTDFIQEEFQHLK TQAEQLHNHLQPGR (SEQ ID NO: 1 ).

[0074] Human TAZ FL is encoded by the following nucleic acid sequence: ATGCCTCTGCACGTGAAGTGGCCGTTCCCCGCGGTGCCGCCGCTCACCTGGACCCTGGCCAGCAG CGTCGTCATGGGCTTGGTGGGCACCTACAGCTGCTTCTGGACCAAGTACATGAACCACCTGACCGT GCACAACAGGGAGGTGCTGTACGAGCTCATCGAGAAGCGAGGCCCGGCCACGCCCCTCATCACCG TGTCCAATCACCAGTCCTGCATGGACGACCCTCATCTCTGGGGGATCCTGAAACTCCGCCACATCTG GAACCTGAAGTTGATGCGTTGGACCCCTGCAGCTGCAGACATCTGCTTCACCAAGGAGCTACACTC CCACTTCTTCAGCTTGGGCAAGTGTGTGCCTGTGTGCCGAGGAGCAGAATTTTTCCAAGCAGAGAAT GAGGGGAAAGGTGTTCTAGACACAGGCAGGCACATGCCAGGTGCTGGAAAAAGAAGAGAGAAAGG AGATGGCGTCTACCAGAAGGGGATGGACTTCATTTTGGAGAAGCTCAACCATGGGGACTGGGTGCA TATCTTCCCAGAAGGGAAAGTGAACATGAGTTCCGAATTCCTGCGTTTCAAGTGGGGAATCGGGCG CCTGATTGCTGAGTGTCATCTCAACCCCATCATCCTGCCCCTGTGGCATGTCGGAATGAATGACGTC CTTCCTAACAGTCCGCCCTACTTCCCCCGCTTTGGACAGAAAATCACTGTGCTGATCGGGAAGCCCT TCAGTGCCCTGCCTGTACTCGAGCGGCTCCGGGCGGAGAACAAGTCGGCTGTGGAGATGCGGAAA GCCCTGACGGACTTCATTCAAGAGGAATTCCAGCATCTGAAGACTCAGGCAGAGCAGCTCCACAAC CACCTCCAGCCTGGGAGATAG (SEQ ID NO: 2). Human TAZ A5 (TAZV2) has the following amino acid sequence: MPLHVKWPFPAVPPLTWTLASSVVMGLVGTYSCFWTKYMNHLTVHNREVLYELIEKRGPATPLITVSNH QSCMDDPHLWGILKLRHIWNLKLMRWTPAAADICFTKELHSHFFSLGKCVPVCRGDGVYQKGMDFILEK LNHGDWVHIFPEGKVNMSSEFLRFKWGIGRLIAECHLNPIILPLWHVGMNDVLPNSPPYFPRFGQKITVLI GKPFSALPVLERLRAENKSAVEMRKALTDFIQEEFQHLKTQAEQLHNHLQPGR (SEQ ID NO: 3).

[0075] Human TAZ A5 (TAZV2) is encoded by the following nucleic acid sequence: ATGCCTCTGCACGTGAAGTGGCCGTTCCCCGCGGTGCCGCCGCTCACCTGGACCCTGGCCAGCAG CGTCGTCATGGGCTTGGTGGGCACCTACAGCTGCTTCTGGACCAAGTACATGAACCACCTGACCGT GCACAACAGGGAGGTGCTGTACGAGCTCATCGAGAAGCGAGGCCCGGCCACGCCCCTCATCACCG TGTCCAATCACCAGTCCTGCATGGACGACCCTCATCTCTGGGGGATCCTGAAACTCCGCCACATCTG GAACCTGAAGTTGATGCGTTGGACCCCTGCAGCTGCAGACATCTGCTTCACCAAGGAGCTACACTC CCACTTCTTCAGCTTGGGCAAGTGTGTGCCTGTGTGCCGAGGAGATGGCGTCTACCAGAAGGGGAT GGACTTCATTTTGGAGAAGCTCAACCATGGGGACTGGGTGCATATCTTCCCAGAAGGGAAAGTGAA CATGAGTTCCGAATTCCTGCGTTTCAAGTGGGGAATCGGGCGCCTGATTGCTGAGTGTCATCTCAAC CCCATCATCCTGCCCCTGTGGCATGTCGGAATGAATGACGTCCTTCCTAACAGTCCGCCCTACTTCC CCCGCTTTGGACAGAAAATCACTGTGCTGATCGGGAAGCCCTTCAGTGCCCTGCCTGTACTCGAGC GGCTCCGGGCGGAGAACAAGTCGGCTGTGGAGATGCGGAAAGCCCTGACGGACTTCATTCAAGAG GAATTCCAGCATCTGAAGACTCAGGCAGAGCAGCTCCACAACCACCTCCAGCCTGGGAGATAG (SEQ ID NO: 4).

[0076] Cell penetrating peptides (CPPs)

[0077] Cell penetrating peptides (CPPs) are short peptides, typically of 8 to 40 residues in length, that facilitate the delivery of molecular cargoes into cells or organelles. CPPs typically include multiple cationic amino acids, which may facilitate cell penetration. Without being bound by any particular theory, the CPPs used in conjunction with the viral vectors, compositions, and methods described herein may allow horizontal spread between targeted cells and adjacent cells, thereby amplifying the effect of the gene therapy.

[0078] CPPs that can be used in the invention include, in some embodiments, a cardiac targeting peptide (CTP) or a variant thereof, such as a peptide having, e.g., at least 70% (e.g., at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 5. In some embodiments, the CTP has 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 5. In some embodiments, the CTP has 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 5. In some embodiments, the CTP has 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 5. In some embodiments, the CTP has the amino acid sequence of: APWHLSSQYSRT (SEQ ID NO: 5).

[0079] In other embodiments, the CPP is an antennapedia permeability peptide (ANTP) or a variant thereof, such as a peptide having at least 70% (e.g., at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 6. In some embodiments, the ANTP has 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 6. In some embodiments, the ANTP has 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 6. In some embodiments, the ANTP has 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 6. In some embodiments, the ANTP has the amino acid sequence of: RQIKIWFQNRRMKWKK (SEQ ID NO: 6).

[0080] In other embodiments, the CPP is an human immunodeficiency virus (HIV) Tat permeability peptide or a variant thereof, such as a peptide having at least 70% (e.g., at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 7. In some embodiments, the HIV Tat permeability peptide has 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 7. In some embodiments, the HIV Tat permeability peptide has 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 7. In some embodiments, the HIV Tat permeability peptide has 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 7. In some embodiments, the HIV Tat permeability peptide has the amino acid sequence of: GRKKRRQRRRPPQ (SEQ ID NO: 7).

[0081] In other embodiments, the CPP is a Kaposi fibroblast growth factor 4 (FGF4)-permeability peptide or a variant thereof, such as a peptide having at least 70% (e.g., at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 8. In some embodiments, the Kaposi FGF4-permeability peptide has 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 8. In some embodiments, the Kaposi FGF4-permeability peptide has 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 8. In some embodiments, the Kaposi FGF4-permeability peptide has 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 8. In some embodiments, the Kaposi FGF4-permeability peptide has the amino acid sequence of: AAVALLPAVLLALLAP (SEQ ID NO: 8).

[0082] In some embodiments, CPPs used in the invention have one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions relative to a reference sequence (e.g., any one of SEQ ID NOs: 5-8), provided that the function of the CPP is substantially maintained. In some embodiments, the CPP includes 1 , 2, or 3 such substitutions.

[0083] Linkers and Fusion Proteins / Peptides

[0084] In some embodiments, TAZ-encoding constructs that can be used with the compositions and methods described herein include polynucleotides that encode TAZ (e.g., as described above) coupled to a CPP (e.g., a CTP, an ANTP, an HIV Tat permeability peptide, or a Kaposi FGF4-permeability peptide; e.g., see above, or a variant thereof) by a peptide linker (TAZ-CPP), such as a linker including multiple (e.g., 3, 4, 5, or 6) glycine residues. In certain embodiments, the peptide linker used to couple TAZ to a CPP is VESGGGGSPG (SEQ ID NO: 9). In certain embodiments, the peptide linker used to couple TAZ to a CPP is VEESGGGGS (SEQ ID NO: 10). In certain embodiments, the peptide linker is a variant of SEQ ID NO: 9 or SEQ ID NO: 10 (e.g., a variant having 1 , 2, 3, 4, or 5 substitutions, e.g., conservative substitutions).

[0085] Examples of fusion proteins including TAZ A5 and a CTP CPP or TAZ A5 and an ANTP CPP are provided below. These descriptions can be adapted to describe fusions of TAZ FL or TAZ A5 in combination with other CPPs (e.g., CPPs described herein) and / or linkers (e.g., linkers described herein).

[0086] In certain embodiments, the TAZ-CPP is TAZ A5-CTP (TAZV2-CTP) or a variant thereof having at least 70% (e.g., at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 11 . In some embodiments, the TAZ-CPP has an amino acid sequence of SEQ ID NO: 11 or is a variant thereof having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 11. In some embodiments, the TAZ-CPP has an amino acid sequence of SEQ ID NO: 11 or is a variant thereof having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 11 . In some embodiments, the TAZ-CPP has an amino acid sequence of SEQ ID NO: 11 or is a variant thereof having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 11. In some embodiments, the TAZ-CPP has the amino acid sequence of:

[0087] MPLHVKWPFPAVPPLTWTLASSVVMGLVGTYSCFWTKYMNHLTVHNREVLYELIEKRGPATPLITVSNH QSCMDDPHLWGILKLRHIWNLKLMRWTPAAADICFTKELHSHFFSLGKCVPVCRGDGVYQKGMDFILEK LNHGDWVHIFPEGKVNMSSEFLRFKWGIGRLIAECHLNPIILPLWHVGMNDVLPNSPPYFPRFGQKITVLI GKPFSALPVLERLRAENKSAVEMRKALTDFIQEEFQHLKTQAEQLHNHLQPGRVESGGGGSPGAPWHL SSQYSRT (SEQ ID NO: 11 ).

[0088] In certain embodiments, the TAZ A5-CTP is encoded by the nucleic acid sequence of: ATGCCTCTGCACGTGAAGTGGCCGTTCCCCGCGGTGCCGCCGCTCACCTGGACCCTGGCCAGCAG CGTCGTCATGGGCTTGGTGGGCACCTACAGCTGCTTCTGGACCAAGTACATGAACCACCTGACCGT GCACAACAGGGAGGTGCTGTACGAGCTCATCGAGAAGCGAGGCCCGGCCACGCCCCTCATCACCG TGTCCAATCACCAGTCCTGCATGGACGACCCTCATCTCTGGGGGATCCTGAAACTCCGCCACATCTG GAACCTGAAGTTGATGCGTTGGACCCCTGCAGCTGCAGACATCTGCTTCACCAAGGAGCTACACTC CCACTTCTTCAGCTTGGGCAAGTGTGTGCCTGTGTGCCGAGGAGATGGCGTCTACCAGAAGGGGAT GGACTTCATTTTGGAGAAGCTCAACCATGGGGACTGGGTGCATATCTTCCCAGAAGGGAAAGTGAA CATGAGTTCCGAATTCCTGCGTTTCAAGTGGGGAATCGGGCGCCTGATTGCTGAGTGTCATCTCAAC CCCATCATCCTGCCCCTGTGGCATGTCGGAATGAATGACGTCCTTCCTAACAGTCCGCCCTACTTCC CCCGCTTTGGACAGAAAATCACTGTGCTGATCGGGAAGCCCTTCAGTGCCCTGCCTGTACTCGAGC GGCTCCGGGCGGAGAACAAGTCGGCTGTGGAGATGCGGAAAGCCCTGACGGACTTCATTCAAGAG GAATTCCAGCATCTGAAGACTCAGGCAGAGCAGCTCCACAACCACCTCCAGCCTGGGAGAGTCGAG TCCGGCGGAGGCGGCTCTCCTGGCGCCCCATGGCATCTGAGCAGCCAGTATAGCCGTACCTAA (SEQ ID NO: 12).

[0089] In certain embodiments, the TAZ-CPP is TAZ A5-ANTP (TAZV2-Antp) or a variant thereof having at least 70% (e.g., at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 13. In some embodiments, the TAZ-CPP has an amino acid sequence of SEQ ID NO: 13 or is a variant thereof having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 13. In some embodiments, the TAZ-CPP has an amino acid sequence of SEQ ID NO: 13 or is a variant thereof having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 13. In some embodiments, the TAZ-CPP has an amino acid sequence of SEQ ID NO: 13 or is a variant thereof having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 13. In some embodiments, the TAZ-CPP has the amino acid sequence of:

[0090] MPLHVKWPFPAVPPLTWTLASSVVMGLVGTYSCFWTKYMNHLTVHNREVLYELIEKRGPATPLITVSNH QSCMDDPHLWGILKLRHIWNLKLMRWTPAAADICFTKELHSHFFSLGKCVPVCRGDGVYQKGMDFILEK LNHGDWVHIFPEGKVNMSSEFLRFKWGIGRLIAECHLNPIILPLWHVGMNDVLPNSPPYFPRFGQKITVLI GKPFSALPVLERLRAENKSAVEMRKALTDFIQEEFQHLKTQAEQLHNHLQPGRVEESGGGGSRQIKIWF QNRRMKWKK (SEQ ID NO: 13).

[0091] In certain embodiments, the TAZ A5-ANTP is encoded by the nucleic acid sequence of: ATGCCTCTGCACGTGAAGTGGCCGTTCCCCGCGGTGCCGCCGCTCACCTGGACCCTGGCCAGCAG CGTCGTCATGGGCTTGGTGGGCACCTACAGCTGCTTCTGGACCAAGTACATGAACCACCTGACCGT GCACAACAGGGAGGTGCTGTACGAGCTCATCGAGAAGCGAGGCCCGGCCACGCCCCTCATCACCG TGTCCAATCACCAGTCCTGCATGGACGACCCTCATCTCTGGGGGATCCTGAAACTCCGCCACATCTG GAACCTGAAGTTGATGCGTTGGACCCCTGCAGCTGCAGACATCTGCTTCACCAAGGAGCTACACTC CCACTTCTTCAGCTTGGGCAAGTGTGTGCCTGTGTGCCGAGGAGATGGCGTCTACCAGAAGGGGAT GGACTTCATTTTGGAGAAGCTCAACCATGGGGACTGGGTGCATATCTTCCCAGAAGGGAAAGTGAA CATGAGTTCCGAATTCCTGCGTTTCAAGTGGGGAATCGGGCGCCTGATTGCTGAGTGTCATCTCAAC CCCATCATCCTGCCCCTGTGGCATGTCGGAATGAATGACGTCCTTCCTAACAGTCCGCCCTACTTCC CCCGCTTTGGACAGAAAATCACTGTGCTGATCGGGAAGCCCTTCAGTGCCCTGCCTGTACTCGAGC GGCTCCGGGCGGAGAACAAGTCGGCTGTGGAGATGCGGAAAGCCCTGACGGACTTCATTCAAGAG GAATTCCAGCATCTGAAGACTCAGGCAGAGCAGCTCCACAACCACCTCCAGCCTGGGAGAGTCGAG GAGTCCGGCGGAGGCGGATCTCGCCAGATAAAGATTTGGTTCCAGAATCGGCGCATGAAGTGGAAG AAGTAA (SEQ ID NO: 14).

[0092] Expression and delivery of TAZ-CPP into mammalian cells

[0093] Viral vectors known in the art can be used to introduce TAZ-CPP transgenes into cells and to direct expression of TAZ-CPP in cells for the therapeutic purposes described herein. Examples of viral vectors that can be used include lentiviral vectors, adeno-associated viral vectors, and adenovirus vectors (see, e.g., Bulcha et al., Nature 6:53, 2021 ).

[0094] Lentiviral vectors

[0095] The delivery vector used in the compositions and methods described herein can be a lentiviral vector, which is a retroviral vector. Lentiviral vectors transduce a wide range of dividing and non-dividing cell types with high efficiency, conferring stable, long-term expression of the transgene encoding the polypeptide or RNA.

[0096] In some embodiments, the lentiviral vectors are HIV (e.g., HIV-1 )-derived vectors. In some embodiments, the lentiviral vectors are replication defective. In some embodiments, the lentiviral vectors are non-pathogenic. In some embodiments, the lentiviral vectors used in the compositions and methods described herein can be third generation lentiviral vectors, which are generated using a vector or transfer plasmid containing the transgene of interest (e.g., transgene encoding TAZ-CPP), two packaging plasmids - one encoding Rev and another encoding Gag and Pol - and an envelope plasmid expressing vesicular stomatitis virus glycoprotein (VSV-G). Details of such vectors are well known in the art and can readily be adapted for use in the invention (see, e.g., Bulcha et al., supra, and AddGene.org).

[0097] In some examples, the vector plasmid may contain one or more of 5’-Long terminal repeat (LTR), psi sequence, Rev Responsive Element (RRE), central polypurine tract (cPPT), woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and 3’-self inactivating LTR (SIN-LTR). The lentiviral vectors described herein can include a promoter sequence. The promoter may be a ubiquitous promoter such as cytomegalovirus (CMV) promoter, Rous sarcoma virus (RSV) promoter, elongation factor 1 alpha (EF1 a) promoter, and phosphoglycerate kinase 1 (PGK) promoter. A person skilled in the art will be familiar with a number of promoters that will be suitable in the vector constructs described herein. Enhancer elements can be used to increase expression of modified DNA molecules or increase the lentiviral integration efficiency. The lentiviral vectors can include a cPPT sequence, which enhances vector integration. The lentiviral vectors can also include a WPRE, which promotes nuclear export of transcripts and / or increases the efficacy of polyadenylation of the nascent transcript.

[0098] Other lentiviral vectors known in the art can be adapted for use in the invention by those of skill in the art.

[0099] Adeno-associated viral vectors

[0100] Transgenes of the compositions and methods described herein can be incorporated into recombinant adeno-associated viral (rAAV) vectors and / or virions in order to facilitate their introduction into a cell. rAAV vectors useful in the compositions and methods herein are recombinant nucleic acid constructs that include a heterologous sequence to be expressed (e.g., transgene encoding TAZ or TAZ- CPP) and viral sequences that facilitate integration and expression of the heterologous genes. The viral sequences may include those sequences of AAV that are required in cis for replication and packaging of the DNA into a virion. Such rAAV vectors may also contain marker or reporter genes. Useful rAAV vectors have one or more of the AAV wildtype genes deleted in whole or in part but retain functional flanking inverted terminal repeat (ITR) sequences. The AAV ITRs may be of any serotype suitable for a particular application. Methods for using rAAV vectors are described for example, in Tai et al., J. Biomed. Sci. 7:279 (2000), and Monahan and Samulski, Gene Delivery 7:24 (2000), the disclosures of each of which are incorporated herein by reference as they pertain to AAV vectors for gene delivery.

[0101] The capsid proteins of AAV compose the exterior, non-nucleic acid portion of the virion and are encoded by the AAV cap gene. The cap gene encodes three viral coat proteins, virion protein 1 (VP1 ), VP2, and VP3, which are required for virion assembly. The construction of rAAV virions has been described, for example, in US 5,173,414; US 5,139,941 ; US 5,863,541 ; US 5,869,305; US 6,057,152; and US 6,376,237; as well as in Rabinowitz et al., J. Virol. 76:791 (2002) and Bowles et al., J. Virol. 77:423 (2003), the disclosures of each of which are incorporated herein by reference as they pertain to AAV vectors for gene delivery. rAAV virions useful in conjunction with the compositions and methods described herein include those derived from a variety of AAV serotypes including AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , AAV12, and AAV13. Construction and use of AAV vectors and AAV proteins of different serotypes are described, for example, in Chao et al., Mol. Ther. 2:619 (2000); Davidson et al., Proc. Natl. Acad. Sci. USA 97:3428 (2000); Xiao et al., J. Virol. 72:2224 (1998); Halbert et al., J. Virol. 74:1524 (2000); Halbert et al., J. Virol. 75:6615 (2001 ); and Auricchio et al., Hum. Molec. Genet. 10:3075 (2001 ), the disclosures of each of which are incorporated herein by reference as they pertain to AAV vectors for gene delivery.

[0102] Pseudotyped rAAV vectors may also be used. Pseudotyped vectors include AAV vectors of a given serotype pseudotyped with a capsid gene derived from a serotype other than the given serotype (e.g., AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , AAV12, AAV13, among others). Techniques involving the construction and use of pseudotyped rAAV virions are known in the art and are described, for example, in Duan et al., J. Virol. 75:7662 (2001 ); Halbert et al., J. Virol. 74:1524 (2000); Zolotukhin et al., Methods, 28:158 (2002); and Auricchio et al., Hum. Molec. Genet. 10:3075 (2001 ).

[0103] AAV virions that have mutations within the virion capsid may be used to infect particular cell types more effectively than non-mutated capsid virions. For example, suitable AAV mutants may have ligand insertion mutations for the facilitation of targeting AAV to specific cell types. The construction and characterization of AAV capsid mutants including insertion mutants, alanine screening mutants, and epitope tag mutants is described in Wu et al., J. Virol. 74:8635 (2000). Other rAAV virions that can be used in methods described herein include those capsid hybrids that are generated by molecular breeding of viruses as well as by exon shuffling. See, e.g., Soong et al., Nat. Genet. 25:436 (2000) and Kolman and Stemmer, Nat. Biotechnol. 19:423 (2001 ).

[0104] Adenoviral vectors

[0105] Adenoviral vectors can also be used to deliver transgenes encoding TAZ-CPP into a cell.

[0106] Adenovirus genome contains early (E1 -4) and late (L1 -5) transcripts flanked by ITRs. Deletion in or of the E1 region renders a recombinant adenoviral vector replication-defective. The replication-defective adenoviral vector may also include deletion in or of the E3 region. Alternatively, a recombinant adenoviral vector may be replication-competent and contain one or more of the E1 , E3, or E4 regions. The recombinant adenoviral vectors useful in conjunction with the compositions and methods described herein can include an expression cassette that replaces or disrupts all or a portion of the E1 region of the adenovirus. The expression cassette may include a promoter (e.g., a CMV promoter) that stimulates expression of a transgene (e.g., transgene encoding TAZ-CPP). While at least 57 different adenovirus serotypes have been identified, serotype 2 (Ad2) and serotype 5 (Ad5) are commonly used for gene therapy. Ad5-based vectors bind the Coxsackie-Adenovirus Receptor (CAR) receptor to enter a cell. A person skilled in the art will be familiar with other serotypes and receptors that will be suitable for the adenoviral vector described herein.

[0107] Pharmaceutical compositions

[0108] The viral vectors described herein can be formulated, for example, into pharmaceutical compositions for administration to a subject, such as a human subject with a disorder characterized by TKZ. deficiency (e.g., Barth Syndrome; also see above), in a biologically compatible form suitable for administration in vivo.

[0109] In some embodiments, the pharmaceutical composition containing the viral vector encoding TAZ and a CPP include a pharmaceutically acceptable carrier, which does not produce significantly adverse, allergic, or other untoward reactions that may outweigh the benefit of administration, whether for research, prophylactic, and / or therapeutic treatments. Exemplary pharmaceutically acceptable carriers and formulations are disclosed in Remington's Pharmaceutical Sciences, 18th Ed., Mack Printing Company, 1990, which is incorporated by reference herein for its teachings regarding the same. Moreover, formulations can be prepared to meet sterility, pyrogenicity, general safety, and purity standards as required by the United States FDA's Division of Biological Standards and Quality Control and / or other relevant U.S. and foreign regulatory agencies.

[0110] Exemplary, generally used, pharmaceutically acceptable carriers may optionally include bulking agents or fillers, solvents or co-solvents, dispersion media, coatings, surfactants, antioxidants (e.g., ascorbic acid, methionine, and vitamin E), preservatives, isotonic agents, absorption delaying agents, salts, stabilizers, buffering agents, chelating agents (e.g., EDTA), gels, binders, disintegration agents, and / or lubricants.

[0111] Exemplary buffering agents include citrate buffers, succinate buffers, tartrate buffers, fumarate buffers, gluconate buffers, oxalate buffers, lactate buffers, acetate buffers, phosphate buffers, histidine buffers, and / or trimethylamine salts.

[0112] Exemplary preservatives include phenol, benzyl alcohol, meta-cresol, methylparaben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides, hexamethonium chloride, alkyl parabens (such as methyl or propyl paraben), catechol, resorcinol, cyclohexanol, and / or 3-pentanol.

[0113] Exemplary isotonic agents include polyhydric sugar alcohols comprising, but not limited to, trihydric or higher sugar alcohols, (e.g., glycerin, erythritol, arabitol, xylitol, sorbitol, and / or mannitol).

[0114] Exemplary stabilizers include organic sugars, polyhydric sugar alcohols, polyethylene glycol, sulfur-containing reducing agents, amino acids, low molecular weight polypeptides, proteins, immunoglobulins, hydrophilic polymers, and / or polysaccharides.

[0115] Methods of treatment

[0116] Patient selection

[0117] Subjects that can be treated using the vectors, compositions, or methods described herein are subjects having or at risk of developing a disorder characterized by TAZ deficiency (e.g., Barth syndrome). The vectors, compositions, and methods described herein can be used to treat subjects with cardiomyopathy, an aging-related condition, heart failure, diabetes, myocardial infarction, an acquired mitochondrial disorder, and / or an inherited mitochondrial disorder.

[0118] In more detail, Barth syndrome is an X-linked recessive disorder that results from defects in the TAZ gene. Barth syndrome is characterized by impaired lipid metabolism, leading to mitochondrial dysfunction, and has clinical manifestations in heart and skeletal muscle. Patients with Barth syndrome may experience, for example, neutropenia, muscular hypotonia, cardiomyopathy, cardiac failure, endomyocardial fibroelastosis, dilated cardiomyopathy, and / or hypertrabeculation. The vectors, compositions, and methods of the invention can be used in the prevention, inhibition, amelioration, or treatment of any one or more of these or other features of Barth syndrome.

[0119] Other indications associated with TAZ deficiency include cardiomyopathy, aging-related condition, heart failure, diabetes, myocardial infarction, acquired mitochondrial disorder, and inherited mitochondrial disorder. The vectors, compositions, and methods of the invention can be used in the prevention, inhibition, amelioration, or treatment of any one or more of these conditions, or one or more symptoms thereof.

[0120] Examples of cardiomyopathy include, e.g., dilated cardiomyopathy, hypertrophic cardiomyopathy, noncompaction cardiomyopathy, ischemic cardiomyopathy, hypertensive cardiomyopathy, diabetic cardiomyopathy, and chemotherapy induced cardiomyopathy. In certain embodiments, the cardiomyopathy may be associated with a TAZ gene mutation. In certain embodiments, the cardiomyopathy may not be associated with a TAZ gene mutation.

[0121] Examples of inherited mitochondrial disorders include autosomal dominant optic atrophy (ADOA), Leber's hereditary optic neuropathy (LHON), LHON plus, Leigh syndrome, pyruvate dehydrogenase complex deficiency (PDCD), thymidine kinase 2 (TK2) deficiency, Alpers disease, Barth syndrome, a beta-oxidation defect, carnitine-acyl-carnitine deficiency, carnitine deficiency, a creatine deficiency syndrome, coenzyme Q10 deficiency, complex I deficiency, complex II deficiency, complex III deficiency, complex IV deficiency / COX deficiency, complex V deficiency, chronic progressive external ophthalmoplegia syndrome (CPEO), CPT I deficiency, CPT II deficiency, Kearns-Sayre syndrome (KSS), lactic acidosis, leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation (LBSL - leukodystrophy), long-chain acyl-CoA dehydrogenase deficiency (LCAD), long-chain L-3 hydroxyacyl-CoA dehydrogenase deficiency (LCHAD), Luft disease, multiple acyl-CoA dehydrogenase (MAD) deficiency, medium-chain acyl-CoA dehydrogenase deficiency (MCAD), mitochondrial encephalomyopathy lactic acidosis and stroke-like episodes (MELAS), myoclonic epilepsy and ragged- red fiber disease (MERRF), mitochondrial recessive ataxia syndrome (MIRAS), a mitochondrial cytopathy, mitochondrial DNA depletion, mitochondrial encephalopathy, mitochondrial neurogastrointestinal encephalopathy syndrome (MNGIE), neuropathy, ataxia, and retinitis pigmentosa (NARP), Pearson syndrome, pyruvate carboxylase deficiency, pyruvate dehydrogenase deficiency, a POLG mutation, short-chain acyl-CoA dehydrogenase deficiency (SCAD), 3-hydroxyacyl-CoA dehydrogenase (HADH) deficiency, very-long-chain acyl-CoA dehydrogenase deficiency (VLCAD), Friedrich's ataxia, and / or any other suitable inherited mitochondrial disorder.

[0122] Routes of administration

[0123] The viral vectors and compositions described herein can be administered to subjects with a disorder characterized by TAZ deficiency or as described elsewhere herein by any of a variety of routes selected to be appropriate by those of skill in the art. Thus, for example, the vectors or compositions can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intravesicularly, intrapericardially, intraumbilically, intraocularly, mucosally, orally, subcutaneously, or subconjunctivally. In some embodiments, viral vectors or compositions described herein are administered to a subject systemically (e.g., intravenously). In some embodiments, viral vectors or compositions described herein are administered ex vivo by infecting bone marrow precursor cells, which are in turn administered to subjects. In some embodiments, viral vectors or compositions described herein are administered locally by intracoronary infusion.

[0124] The most suitable route for administration in any given case will depend on the particular viral vector administered, the subject, pharmaceutical formulation methods, administration methods (e.g., administration time and administration route), the subject’s age, body weight, sex, severity of the disorder being treated, the subject’s diet, and the subject’s excretion rate. Multiple routes of administration may be used to treat a single subject, e.g., intravenous injection and intraosseous injection, intravenous injection and intracoronary infusion, or intraosseous injection and intracoronary infusion. Multiple routes of administration may be used to treat a single subject at one time, or the subject may receive treatment via one route of administration first and receive treatment via another route of administration during a second appointment, e.g., 1 week later, 2 weeks later, 1 month later, 6 months later, or 1 year later.

[0125] Dosing

[0126] For administration, effective amounts and therapeutically effective amounts (also referred to herein as doses) can be initially estimated based on results from in vitro assays or animal model studies. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC50 as determined in cell culture. Such information can be used to determine useful doses in subjects of interest more accurately. Clinical studies can further be carried out to determine dosages.

[0127] The actual dose amount administered to a particular subject can be determined by a physician, a veterinarian, or a researcher, taking into account parameters such as physical and physiological factors including body weight, severity of condition, type of disease, previous or concurrent therapeutic interventions, idiopathy of the subject, and / or route of administration.

[0128] Doses can range, for example, from 0.1 mg / kg / day to 5 mg / kg / day, from 0.5 mg / kg / day to 1 mg / kg / day, from 0.1 mg / kg / day to 5 g / kg / day, or from 0.5 mg / kg / day to 1 pg / kg / day. In other non-limiting examples, a dose can comprise 1 g / kg / day, 5 pg / kg / day, 10 pg / kg / day, 50 pg / kg / day, 100 pg / kg / day, 200 pg / kg / day, 350 g / kg / day, 500 g / kg / day, 1 mg / kg / day, 5 mg / kg / day, 10 mg / kg / day, 50 mg / kg / day, 100 mg / kg / day, 200 mg / kg / day, 350 mg / kg / day, 500 mg / kg / day, or 1000 mg / kg / day. Therapeutically effective amounts can be achieved by administering single or multiple doses during the course of a treatment regimen (i.e., days, weeks, months, etc.).

[0129] Combination therapy

[0130] The viral vectors described herein can be used in combination therapy with different treatments that are administered to a subject as part of a defined treatment regimen for a particular disease or condition. For example, subjects that are being treated for myocardial infarction, heart failure, arrhythmias, or neutropenia using standard treatments may benefit from simultaneous administration of the viral vector described herein. Subjects that are placed on extracorporeal membrane oxygenation (ECMO) after myocardial infarction may also be treated by the viral vector described herein. The treatment regimen defining the dose and frequency of administration of each treatment may allow the effects of the separate therapies on the subject to overlap. The therapeutic effects of administration of two or more treatments in combination may be greater than what would be observed with one treatment delivered alone or in the absence of the other. The effect of the two or more treatments can be partially additive, wholly additive, or greater than additive (e.g., synergistic). Two or more treatments may be administered by the same route or by different routes. Sequential or substantially simultaneous administration of each therapeutic composition may be affected by the suitable route of administration for each.

[0131] Examples

[0132] The following examples are put forth so as to provide those of ordinary skill in the art with a description of how the compositions and methods described herein may be used and evaluated and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention.

[0133] Example 1. CPPs enhance the efficacy of TAZ gene therapy

[0134] Objective

[0135] The objective of this study was to explore whether a cell penetrating peptide (CPP) may be useful to enhance tafazzin (TAZ) gene therapy.

[0136] Materials and Methods

[0137] Lentiviral constructs

[0138] We developed third generation lentiviral constructs using the pLJM1 -eGFP plasmid as a backbone (Addgene #19319; Sancak Y, Peterson TR, Shaul YD, Lindquist RA, Thoreen CC, Bar-Peled L, Sabatini DM. Science. 2008 Jun 13. 320(5882): 1496-501) and replaced the eGFP expression sequences with sequences enclosing human TAZ lacking exon 5 in native form (hTAZV2) or modified at the carboxyl terminus to contain a CPP, either CTP (APWHLSSQYSRT; M. Zahid, B. E. Phillips, S. M. Albers, N. Giannoukakis, S. C. Watkins and P. D. Robbins. PLoS One 2010 Vol. 5 Issue 8 Pages e12252. Accession Number: 20808875 PMCID: 2923200 DOI: 10.1371 / journal. pone.0012252) (hTAZV2-CTP) or ANTP (RQIKIWFQNRRMKWKK; S. H. Min, D. M. Kim, M. N. Kim, J. Ge, D. C. Lee, I. Y. Park, et al. Biomaterials 2010 Vol. 31 Issue 7 Pages 1858-64 Accession Number: 19954838 DOL S0142- 9612(09)01247-2 [pii] 10.1016 / j.biomaterials.2009.11 .019) (hTAZV2-Antp).

[0139] Efficacy of lentiviral hTAZV2, hTAZV2-CTP, and hTAZV2-ANTP in MEFs

[0140] We tested the efficacy of these lentiviral constructs in murine embryonic fibroblasts (MEFs) from wildtype (WT) and TAZ knockout (KO) mice. GFP and empty constructs were used as negative controls and the ratio of monolysocardiolipin (MLCL) to cardiolipin (CL) was measured.

[0141] Efficacy of lentiviral hTAZV2, hTAZV2-CTP, and hTAZV2-ANTP in mice

[0142] We tested the efficacy of the lentiviral constructs in vivo using 4-week-old WT and TAZ KO mice. After measuring the baseline heart function via echocardiogram and taking blood samples, the 4-week- old mice were treated with systemic administration of the lentiviral constructs (empty, GFP, hTAZV2, hTAZV2-CTP, and hTAZV2-ANTP). Echocardiograms were performed and blood samples were taken every 4 weeks and up to 16 weeks of age. At the end of the treatment, the heart tissues were collected for histology and cardiolipin profiling.

[0143] Immunohistochemistry of Lentivirally Infected Mouse Hearts

[0144] Hearts were carefully dissected out and fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4. The fixative was changed within 24 h, followed by changes with fresh fixative every day for 5 days, and then every third day for another week. Paraffin blocks were made, and coronal sections of 5 pm thickness were cut and stained for the expression of GFP by standard immunohistochemical techniques.

[0145] Briefly, sections were treated with 10 mM sodium citrate buffer, pH 6.0, for antigen retrieval to increase the tissue antigenicity. After three washes with TBS-T (Tris Buffered Saline, pH 7.5, 0.05% Tween-20), the sections were incubated in 10% normal goat serum (PK-6101 , VECTASTAIN, ABC Kit, Vector laboratories, Newark, CA, USA) for 2 h at room temperature to reduce the non-specific staining. Subsequently, sections were incubated in anti-GFP antibody (1 :500, Ab290, Abeam, Waltham, MA, USA) for 24 h at 4 °C temperature, followed by repeated washing. Thereafter, the sections were treated for 4 h with biotinylated secondary antibody (PK-6101 , VECTASTAIN, ABC Kit, Vector Laboratories, Newark, CA, USA). The primary and secondary antibodies were diluted in the blocking buffer (goat serum in TBS- T) and after washing with TBS-T, the sections were incubated with the avidin-biotin-peroxidase complex (PK-6101 , VECTASTAIN, ABC Kit, Vector Laboratories, Newark, CA, USA) for 2 h at room temperature. The binding sites of the antigen-antibody interaction were visualized by using the chromogen diaminobenzidine tetrahydrochloride (DAB). The sections were then air-dried, dehydrated in ethanol, mounted with DPX (Distyrene Plasticizer Xylene), and observed under a microscope.

[0146] Results

[0147] Efficacy of lentiviral hTAZV2, hTAZV2-CTP, and hTAZV2-ANTP on cardiolipin remodeling in MEFs

[0148] We found that treatment of TAZ KO MEFs with lentivirally encoded hTAZV2 or hTAZV2-CPPs reduces the MLCL / CL ratio in the cells significantly (FIG. 1), thereby rescuing the defect in cardiolipin remodeling.

[0149] Efficacy of lentiviral hTAZV2, hTAZV2-CTP, and hTAZV2-ANTP on cardiolipin remodeling in TAZ KO mouse hearts

[0150] We have found that treatment of TAZ KO mice with lentivirally encoded hTAZV2 or hTAZV2- CPPs reduces the MLCL / CL ratio in the hearts significantly (FIG. 2), thereby rescuing the defect in cardiolipin remodeling.

[0151] Efficacy of lentiviral hTAZV2, hTAZV2-CTP, and hTAZV2-ANTP on left ventricular ejection fraction in TAZ KO mice

[0152] After the treatment, the ejection fraction, fractional shortening, and global longitudinal strain continued to decrease in the empty virus or GFP treated mice, whereas they remained stable in those treated with lentivirally encoded hTAZV2-ANTP (FIGs. 3-5). The other proteins, hTAZV2 and hTAZV2- CTP, despite rescuing the defect in cardiolipin remodeling, did not completely rescue the defects in ejection fraction (FIG. 3), fractional shortening (FIG. 4), or global longitudinal strain (FIG. 5). In addition, the interventricular septum (IVS) becomes significantly thicker over time in the TAZ KO mice treated with empty vector and GFP virus, while treatment with hTAZV2-Antp or hTAZV2-CTP prevents pathological wall thickening and the wall thickness in not significantly different from WT mice (FIG. 6).

[0153] The immunohistochemical staining of heart sections for GFP revealed detectable expression in the GFP-treated mice but not in the controls (FIG. 7). These findings confirm the viral infection and protein expression implied by the change in MLCL / CL ratio.

[0154] Conclusion

[0155] These findings show that CPPs can be used to enhance TAZ gene therapies for the treatment for Barth Syndrome and other conditions that may be treated based on TAZ gene therapy.

[0156] Other Embodiments

[0157] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the invention that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims. Some embodiments are within the scope of the following numbered paragraphs.

[0158] 1 . A viral vector comprising sequences that encode tafazzin (TAZ) and a cell penetrating peptide (CPP).

[0159] 2. The viral vector of paragraph 1 , wherein the TAZ is full-length TAZ.

[0160] 3. The viral vector of paragraph 2, wherein the TAZ comprises the amino acid sequence of SEQ

[0161] ID NO: 1.

[0162] 4. The viral vector of paragraph 1 , wherein the TAZ lacks exon 5 (TAZ A5; isoform 2).

[0163] 5. The viral vector of paragraph 4, wherein the TAZ A5 comprises the amino acid sequence of

[0164] SEQ ID NO: 3.

[0165] 6. The viral vector of any one of paragraphs 1 to 5, wherein the CPP is selected from the group consisting of cardiac targeting peptide (CTP), antennapedia permeability peptide (ANTP), human immunodeficiency virus (HIV) Tat permeability peptide, and Kaposi fibroblast growth factor 4 (FGF4)- permeability peptide, or a variant thereof.

[0166] 7. The viral vector of paragraph 6, wherein the CTP comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 5.

[0167] 8. The viral vector of paragraph 7, wherein the CTP comprises an amino acid sequence that is 100% identical to the sequence of SEQ ID NO: 5.

[0168] 9. The viral vector of paragraph 6, wherein the ANTP comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 6.

[0169] 10. The viral vector of paragraph 9, wherein the ANTP comprises an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 6. 11 . The viral vector of paragraph 6, wherein the HIV Tat permeability peptide has an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 7.

[0170] 12. The viral vector of paragraph 11 , wherein the HIV Tat permeability peptide comprises a sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 7.

[0171] 13. The viral vector of paragraph 6, wherein the Kaposi FGF4-permeability peptide has an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 8.

[0172] 14. The viral vector of paragraph 13, wherein the Kaposi FGF4-permeability peptide comprises an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 8.

[0173] 15. The viral vector of any one of paragraphs 1 to 14, wherein the TAZ and CPP are coupled by a peptide linker (TAZ-CPP).

[0174] 16. The viral vector of paragraph 15, wherein the peptide linker is a glycine-rich linker.

[0175] 17. The viral vector of paragraph 15 or 16, wherein the linker comprises the sequence of SEQ ID NO: 9 or SEQ ID NO: 10.

[0176] 18. The viral vector of any one of paragraphs 1 to 17, where in the TAZ-CPP has an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 11 .

[0177] 19. The viral vector of paragraph 18, wherein the TAZ-CPP comprises an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 11 .

[0178] 20. The viral vector of any one of paragraphs 1 to 17, where in the TAZ-CPP has an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 13.

[0179] 21 . The viral vector of paragraph 20, wherein the TAZ-CPP comprises an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 13.

[0180] 22. The viral vector of any one of paragraphs 1 to 21 , wherein the viral vector is a lentiviral vector.

[0181] 23. The viral vector of any one of paragraphs 1 to 21 , wherein the viral vector is an adeno- associated viral vector.

[0182] 24. A pharmaceutical composition comprising (i) a viral vector of any one of paragraphs 1 to 23 or a cell transduced by the viral vector, and (ii) a pharmaceutically acceptable carrier or diluent.

[0183] 25. A method of treating a subject having mitochondrial dysfunction or a TAZ deficiency, the method comprising administering to the subject a therapeutically effective amount of a viral vector of any one of paragraphs 1 to 23, a cell transduced by the viral vector, or a pharmaceutical composition of paragraph 24.

[0184] 26. The method of paragraph 25, wherein the viral vector, cell, or pharmaceutical composition is administered to the subject by systemic administration or local administration.

[0185] 27. The method of paragraph 26, wherein the local administration comprises intracoronary infusion.

[0186] 28. The method of any one of paragraphs 25 to 27, wherein the cell is a bone marrow precursor cell or a descendant thereof.

[0187] 29. The method of any one of paragraphs 25 to 28, wherein the subject has or is at risk of developing Barth syndrome, cardiomyopathy, aging-related condition, heart failure, diabetes, myocardial infarction, acquired mitochondrial disorder, or inherited mitochondrial disorder. 30. The method of paragraph 29, wherein the cardiomyopathy is chemotherapy-induced cardiomyopathy, diabetic cardiomyopathy, dilated cardiomyopathy, hypertensive cardiomyopathy, hypertrophic cardiomyopathy, ischemic cardiomyopathy, or noncompaction cardiomyopathy.

[0188] 31 . The method of paragraph 29, wherein the inherited mitochondrial disorder is autosomal dominant optic atrophy (ADOA), Leber's hereditary optic neuropathy (LHON), LHON plus, Leigh syndrome, pyruvate dehydrogenase complex deficiency (PDCD), thymidine kinase 2 (TK2) deficiency, Alpers disease, Barth syndrome, a beta-oxidation defect, carnitine-acyl-carnitine deficiency, carnitine deficiency, a creatine deficiency syndrome, coenzyme Q10 deficiency, complex I deficiency, complex II deficiency, complex III deficiency, complex IV deficiency / COX deficiency, complex V deficiency, chronic progressive external ophthalmoplegia syndrome (CPEO), CPT I deficiency, CPT II deficiency, Kearns- Sayre syndrome (KSS), lactic acidosis, leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation (LBSL - leukodystrophy), long-chain acyl-CoA dehydrogenase deficiency (LCAD), long-chain L-3 hydroxyacyl-CoA dehydrogenase deficiency (LCHAD), Luft disease, multiple acyl-CoA dehydrogenase (MAD) deficiency, medium-chain acyl-CoA dehydrogenase deficiency (MCAD), mitochondrial encephalomyopathy lactic acidosis and stroke-like episodes (MELAS), myoclonic epilepsy and ragged-red fiber disease (MERRF), mitochondrial recessive ataxia syndrome (MIRAS), a mitochondrial cytopathy, mitochondrial DNA depletion, mitochondrial encephalopathy, mitochondrial neurogastrointestinal encephalopathy syndrome (MNGIE), neuropathy, ataxia, and retinitis pigmentosa (NARP), Pearson syndrome, pyruvate carboxylase deficiency, pyruvate dehydrogenase deficiency, a POLG mutation, short-chain acyl-CoA dehydrogenase deficiency (SCAD), 3-hydroxyacyl-CoA dehydrogenase (HADH) deficiency, very-long-chain acyl-CoA dehydrogenase deficiency (VLCAD), or Friedrich's ataxia.

[0189] Other embodiments are within the scope of the claims.

Claims

CLAIMS1 . A viral vector comprising sequences that encode tafazzin (TAZ) and a cell penetrating peptide (CPP).

2. The viral vector of claim 1 , wherein the TAZ is full-length TAZ.

3. The viral vector of claim 2, wherein the TAZ comprises the amino acid sequence of SEQ ID NO: 1 .

4. The viral vector of claim 1 , wherein the TAZ lacks exon 5 (TAZ A5; isoform 2).

5. The viral vector of claim 4, wherein the TAZ A5 comprises the amino acid sequence of SEQ ID NO: 3.

6. The viral vector of any one of claims 1 to 5, wherein the CPP is selected from the group consisting of cardiac targeting peptide (CTP), antennapedia permeability peptide (ANTP), human immunodeficiency virus (HIV) Tat permeability peptide, and Kaposi fibroblast growth factor 4 (FGF4)-permeability peptide, or a variant thereof.

7. The viral vector of claim 6, wherein the CTP comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 5.

8. The viral vector of claim 7, wherein the CTP comprises an amino acid sequence that is 100% identical to the sequence of SEQ ID NO: 5.

9. The viral vector of claim 6, wherein the ANTP comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 6.

10. The viral vector of claim 9, wherein the ANTP comprises an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 6.11 . The viral vector of claim 6, wherein the HIV Tat permeability peptide has an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 7.

12. The viral vector of claim 11 , wherein the HIV Tat permeability peptide comprises a sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 7.

13. The viral vector of claim 6, wherein the Kaposi FGF4-permeability peptide has an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 8.

14. The viral vector of claim 13, wherein the Kaposi FGF4-permeability peptide comprises an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 8.

15. The viral vector of claim 1 , wherein the TAZ and CPP are coupled by a peptide linker (TAZ-CPP).

16. The viral vector of claim 15, wherein the peptide linker is a glycine-rich linker.

17. The viral vector of claim 15, wherein the linker comprises the sequence of SEQ ID NO: 9 or SEQ ID NO: 10.

18. The viral vector of claim 1 , where in the TAZ-CPP has an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 11 .

19. The viral vector of claim 18, wherein the TAZ-CPP comprises an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 11 .

20. The viral vector of claim 1 , where in the TAZ-CPP has an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 13.21 . The viral vector of claim 20, wherein the TAZ-CPP comprises an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 13.

22. The viral vector of claim 1 , wherein the viral vector is a lentiviral vector.

23. The viral vector of claim 1 , wherein the viral vector is an adeno-associated viral vector.

24. A pharmaceutical composition comprising (i) a viral vector of claim 1 or a cell transduced by the viral vector, and (ii) a pharmaceutically acceptable carrier or diluent.

25. A method of treating a subject having mitochondrial dysfunction or a TAZ deficiency, the method comprising administering to the subject a therapeutically effective amount of a viral vector of claim 1 , a cell transduced by the viral vector, or a pharmaceutical composition comprising the vector or a cell comprising the vector.

26. The method of claim 25, wherein the viral vector, cell, or pharmaceutical composition is administered to the subject by systemic administration or local administration.

27. The method of claim 26, wherein the local administration comprises intracoronary infusion.

28. The method of claim 25, wherein the cell is a bone marrow precursor cell or a descendant thereof.

29. The method of claim 25, wherein the subject has or is at risk of developing Barth syndrome, cardiomyopathy, aging-related condition, heart failure, diabetes, myocardial infarction, acquired mitochondrial disorder, or inherited mitochondrial disorder.

30. The method of claim 29, wherein the cardiomyopathy is chemotherapy-induced cardiomyopathy, diabetic cardiomyopathy, dilated cardiomyopathy, hypertensive cardiomyopathy, hypertrophic cardiomyopathy, ischemic cardiomyopathy, or noncompaction cardiomyopathy.31 . The method of claim 29, wherein the inherited mitochondrial disorder is autosomal dominant optic atrophy (ADOA), Leber's hereditary optic neuropathy (LHON), LHON plus, Leigh syndrome, pyruvate dehydrogenase complex deficiency (PDCD), thymidine kinase 2 (TK2) deficiency, Alpers disease, Barth syndrome, a beta-oxidation defect, carnitine-acyl-carnitine deficiency, carnitine deficiency, a creatine deficiency syndrome, coenzyme Q10 deficiency, complex I deficiency, complex II deficiency, complex III deficiency, complex IV deficiency / COX deficiency, complex V deficiency, chronic progressive external ophthalmoplegia syndrome (CPEO), OPT I deficiency, OPT II deficiency, Kearns-Sayre syndrome (KSS), lactic acidosis, leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation (LBSL - leukodystrophy), long-chain acyl-CoA dehydrogenase deficiency (LOAD), long-chain L-3 hydroxyacyl-CoA dehydrogenase deficiency (LCHAD), Luft disease, multiple acyl-CoA dehydrogenase (MAD) deficiency, medium-chain acyl-CoA dehydrogenase deficiency (MCAD), mitochondrial encephalomyopathy lactic acidosis and stroke-like episodes (MELAS), myoclonic epilepsy and ragged-red fiber disease (MERRF), mitochondrial recessive ataxia syndrome (MIRAS), a mitochondrial cytopathy, mitochondrial DNA depletion, mitochondrial encephalopathy, mitochondrial neurogastrointestinal encephalopathy syndrome (MNGIE), neuropathy, ataxia, and retinitis pigmentosa (NARP), Pearson syndrome, pyruvate carboxylase deficiency, pyruvate dehydrogenase deficiency, a POLG mutation, short-chain acyl-CoA dehydrogenase deficiency (SCAD), 3-hydroxyacyl-CoA dehydrogenase (HADH) deficiency, very-long-chain acyl-CoA dehydrogenase deficiency (VLCAD), or Friedrich's ataxia.

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