Gene therapy for barth syndrome

Abstract

Provided herein is a recombinant AAV (rAAV) comprising an AAV capsid and a vector genome packaged therein, wherein the vector genome comprises an AAV 5ʹ inverted terminal repeat (ITR), an expression cassette comprising engineered nucleic acid sequence encoding human Tafazzin and operably linked thereto regulatory sequences, and an AAV 3ʹ ITR. Also provided is a pharmaceutical composition comprising a rAAV as described herein in a formulation buffer, and a method of treatment of Barth Syndrome or a disease associated with a mutation in a Tafazzin (TAZ) gene.

Description

[0001] GENE THERAPY FOR BARTH SYNDROME

[0002] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0003] The electronic sequence listing filed herewith named "UPN-25- 1 101 1 PCT.xml" with size of 103,237 bytes, created on date of December 5, 2025, and the contents of the electronic sequence listing (e.g.. the sequences and text therein) are incorporated herein by reference in entirety.

[0004] BACKGROUND OF THE INVENTION

[0005] Barth syndrome (BTHS) is a pediatric-onset X-linked mitochondrial cardiomyopathy exclusively affecting male patients, caused by pathogenic variants in Tafazzin (TAZ gene), and characterized by cardiomyopathy, skeletal myopathy, growth retardation, neutropenia, characteristic dysmorphism (Chin, M.T., and Conway, S.J.. Role of Tafazzin in Mitochondrial Function, Development and Disease, J. Dev. Biol. 2020, 8, 10). Approximately 70% of affected patients present cardiac symptoms during their first year, and 100% by age 5. Other disease related manifestations include recurrent infections, as well as a mild proximal myopathy. The cause of death is usually sudden cardiac arrest due to ventricular arrythmia or bacterial sepsis (neutropenia related). There is initial good response to heart failure treatment, with deterioration after months-years leading to cardiac transplant. There is no disease modifying treatment addressing the cause of the disease and the mitochondrial dysfunction of cardiomyocytes, and therefore a largely unaddressed unmet need in patients. Barth syndrome is a multisystem disorder management of which requires multidisciplinary approach to the organ-specific manifestations, e.g., cardiological, hematological, nutritional, muscular, metabolic.

[0006] Tafazzin (TAZ) gene encodes a phospholipid-lysophospholipid transacylase that metabolizes monolysocardiolipin in mature cardiolipin, which is a key inner membrane mitochondrial component. A disease-causing mutation in TAZ gene normally results in a loss of function, and has low prevalence, with estimate incidence 1 / 300-400,000.

[0007] There is not standard treatment or cure for Barth Syndrome and TAZ-related disorders. A continuing need in the art exists for compositions and methods for effective treatment for Barth Syndrome and TAZ-related disorders.

[0008] SUMMARY OF THE INVENTION

[0009] In another aspect provided herein is a composition comprising a recombinant adeno- associated virus (rAAV) comprising an adeno-associated virus (AAV) capsid and a nucleic acid molecule comprising an expression cassette in tire capsid, wherein the capsid is a clade F capsid comprising an AVIRGDV (SEQ ID NO: 25) peptide inserted between amino acid position 588 and 589 with reference to AAV9, and further wherein the expression cassette is a nucleic sequence comprising a cDNA sequence comprising SEQ ID NO: 1 encoding a human Tafazzin (hTafazzin) operably linked to at least one regulatory control sequence. In certain embodiments, the composition comprises the rAAV which is for use in the treatment of Barth Syndrome or a disease associated with a mutation in a Tafazzin (TAZ) gene. In certain embodiments, the composition comprises the rAAV which is formulated for use at a dose at 2 x 1012GC / kg. In certain embodiments, tire composition comprises the rAAV which is formulated for use at a dose at 6 x 1012GC / kg. In certain embodiments, tire composition comprises the rAAV which is formulated for intravenous (IV) injection. In certain embodiments, the at least one regulatory sequence comprises a CB7 hybrid promoter comprising a cytomegalovirus immediate-early (CMV IE) enhancer and the chicken P-actin promoter, optionally with spacer sequence, optionally with a chimeric intron comprising chicken beta actin intron and further comprising a chicken beta-actin splicing donor including the exon sequence, chicken beta actin intron, and rabbit beta-globin splicing acceptor. In certain embodiments, the at least one regulatory sequence comprises rabbit beta-globin polyadenylation (polyA) signal sequence.

[0010] In another aspect provided herein is a composition comprising a recombinant adeno- associated virus (rAAV) comprising an adeno-associated virus (AAV) capsid and a nucleic acid molecule comprising an expression cassette in the capsid, wherein the capsid is a clade F capsid comprising an RGDYREV (SEQ ID NO: 42) peptide inserted between amino acid position 588 and 589 with reference to AAV9 (SEQ ID NO: 23), and further wherein the expression cassette is a nucleic sequence comprising a cDNA sequence comprising SEQ ID NO: 1 encoding a human Tafazzin (hTafazzin) operably linked to at least one regulator ’ control sequence.

[0011] In certain embodiments, the composition comprises the rAAV which is for use in the treatment of Barth Syndrome or a disease associated with a mutation in a Tafazzin (TAZ) gene. In certain embodiments, the composition comprises the rAAV which is formulated for use at a dose at 2 x 1012GC / kg. In certain embodiments, the composition comprises the rAAV which is formulated for use at a dose at 6 x 1012GC / kg. In certain embodiments, the composition comprises the rAAV which is formulated for intravenous (IV) injection.

[0012] In certain embodiments, provided herein is a method of treating Barth Syndrome or a disease associated with a mutation in Tafazzin (TAZ) gene in a subject in a need thereof, said method comprising administering to tire subject the composition as describe herein. In certain embodiments, provided herein is a composition as described herein for use in preparing a medicament for treatment of Barth Syndrome or a disease associated with a mutation in Tafazzin (TAZ) gene. In another aspect provided herein is an improved method of delivering human Tafazzin to a target tissue to afford expression levels at least 100 time to 5000 times higher than a reference unmodified clade F capsid, the method comprising delivering a composition comprising an effective amount of a recombinant adeno-associated virus (rAAV) comprising an adeno- associated virus (AAV) capsid and a nucleic acid molecule comprising an expression cassette in the capsid, wherein the capsid is a clade F capsid comprising an AVIRGDV (SEQ ID NO: 25) peptide inserted between amino acid position 588 and 589 with reference to AAV9, and further wherein the expression cassette is a nucleic sequence comprising a cDNA sequence comprising SEQ ID NO: 1 encoding a human Tafazzin (hTafazzin) operably linked to at least one regulatory control sequence. In certain embodiments, the AAV capsid is produced by a nucleic acid sequence encoding amino acid sequence of SEQ ID NO: 38.

[0013] In another aspect provided herein is an improved method of delivering human Tafazzin to a target tissue to afford expression levels at least 100 time to 5000 times higher than a reference unmodified clade F capsid, the method comprising delivering a composition comprising an effective amount of a recombinant adeno-associated virus (rAAV) comprising an adeno- associated virus (AAV) capsid and a nucleic acid molecule comprising an expression cassette in the capsid, wherein the capsid is a clade F capsid comprising an RGDYREV (SEQ ID NO: 44) peptide inserted amino acid between position 588 and 589 with reference to AAV9 (SEQ ID NO: 23), and further wherein the expression cassette is a nucleic sequence comprising a cDNA sequence comprising SEQ ID NO: 1 encoding a human Tafazzin (hTafazzin) operably linked to at least one regulatory control sequence.

[0014] In another aspect provided herein is a use of a rAAV vector for treating a condition associated with dysfunctional Tafazzin gene comprising delivering a rAAV in a recombinant adeno-associated virus (rAAV) comprising an adeno-associated virus (AAV) capsid and a nucleic acid molecule comprising an expression cassette in the capsid, wherein the capsid is a clade F capsid comprising an AVIRGDV (SEQ ID NO: 25) peptide inserted between amino acid position 588 and 589 with reference to AAV9 (SEQ ID NO: 23), and further wherein the expression cassette is a nucleic sequence comprising a cDNA sequence comprising SEQ ID NO: 1 encoding a human Tafazzin (hTafazzin) operably linked to at least one regulatory control sequence, wherein the dose is from about 2 x 1012GC / kg to 5 x 1013GC / kg.

[0015] In another aspect provided herein is a use of a recombinant adeno-associated virus (rAAV) in tire manufacture of a medicament for treatment of Barth Syndrome or a disease associated with a mutation in Tafazzin (TAZ) gene, wherein the rAAV comprises an adeno- associated virus (AAV) capsid and a nucleic acid molecule comprising an expression cassette in the capsid, wherein the capsid is a clade F capsid comprising an AVIRGDV (SEQ ID NO: 25) peptide inserted between amino acid position 588 and 589 with reference to AAV9 (SEQ ID NO: 23), and further wherein the expression cassette is a nucleic sequence comprising a cDNA sequence comprising SEQ ID NO: 1 encoding a human Tafazzin (hTafazzin) operably linked to at least one regulatory control sequence, wherein a dose of the rAAV is 2 x 1012GC / kg to 5 x 1013GC / kg.

[0016] In another aspect provided herein is a use of a composition as described herein in the manufacture of a medicament for treatment of Barth Syndrome or a disease associated with a mutation in Tafazzin (TAZ) gene.

[0017] In another aspect provided herein is a use of a composition as described herein in the manufacture of a medicament for improved delivery of human Tafazzin to a target tissue to afford expression levels at least 100 times to 5000 times higher than a reference unmodified clade F capsid.

[0018] These and other aspects of the invention are apparent from tire following detailed description of the invention.

[0019] BRIEF DESCRIPTION OF THE FIGURES

[0020] FIG. 1A shows representative images of In Situ Hybridization (ISH) analysis in heart and liver tissue collected from mice following rAAV administration.

[0021] FIG. IB shows results of ISH analysis plotted as percent positive cardiomyocytes following rAAV administration in mice.

[0022] FIG. 2A shows analysis of cardiac function plotted as percent of fractional shortening in hearts in mice following administration with rAAV. hTafazzin or PBS (control) in TAZKD (TAZ knockdown) and wild-type (WT) mice. FIG. 2B shows analysis of cardiac function plotted as relative wall thickness (mm) of hearts in mice following administration with rAAV.hTafazzin or PBS (control) in TAZKD and wild-type (WT) mice. FIG. 2C shows results of the LC / MS (mass spectrometry) analysis of measured levels of monolysocardiolipin (MLCL) in ratio to cardiolipin (CL) (MLCL:CL) plotted as percent ratio standardized against MCLC:CL levels as measured in WT-PBS mice. FIG. 2C shows measured levels of GDF-15 plotted as percent standardized against GDF-15 levels as measured in WT-PBS mice.

[0023] FIG. 3 shows a proposed study schematic.

[0024] FIG. 4A shows TAZ DNA levels in left ventricle, plotted as GC / diploid cell, following A A Vhu68. hTafazzin. AAV9-IIRGDPA.hTafazzin, AAV9-AVIRGDV.hTafazzin administration. FIG. 4B shows TAZ RNA levels in left ventricle, plotted as vector GC / 100 ng total RNA (as normalized to U6), following AAVhu68. hTafazzin, AAV9-IIRGDPA.hTafazzin, AAV9- AVIRGDV.hTafazzin administration. FIG. 4C shows TAZ DNA levels in septum, plotted as GC / diploid cell, following AAVhu68.hTafazzin, AAV9-IIRGDPA.hTafazzin, AAV9- AVIRGDV hTafazzin administration. FIG. 4D shows TAZ RNA levels in septum, plotted as vector GC / 100 ng total RNA (as normalized to U6). following AAVhu68.hTafazzin, AAV9- IIRGDPA.hTafazzin, AAV9- AVIRGDV. hTafazzin administration. FIG. 4E shows TAZ DNA levels in left atrium, plotted as GC / diploid cell, following AAVhu68.hTafazzin, AAV9- IIRGDPA. hTafazzin, AAV9- AVIRGDV. hTafazzin administration. FIG. 4F shows TAZ RNA levels in left atrium, plotted as vector GC / 100 ng total RNA (as normalized to U6), following AAVhu68. hTafazzin, AAV9-IIRGDP A. hTafazzin, AAV9- AVIRGDV. hTafazzin administration. FIG. 4G shows TAZ DNA levels in liver, plotted as GC / diploid cell, following AAVhu68.hTafazzin, AAV9-IIRGDPA.hTafazzin, AAV9- AVIRGDV.hTafazzin administration. FIG. 4H shows TAZ RNA levels in liver, plotted as vector GC / 100 ng total RNA (as normalized to U6), following A AVhu68. hTafazzin, AAV9-IIRGDPA. hTafazzin, AAV9- AVIRGDV.hTafazzin administration. FIG. 41 shows TAZ DNA levels in diaphragm, plotted as GC / diploid cell, following AAVhu68. hTafazzin, AAV9-IIRGDPA. hTafazzin, AAV9- AVIRGDV.hTafazzin administration. FIG. 4J shows TAZ RNA levels in diaphragm, plotted as vector GC / 100 ng total RNA (as normalized to U6), following AAVhu68. hTafazzin, AAV9- IIRGDPA.hTafazzin, AAV9- AVIRGDV.hTafazzin administration. FIG. 4K shows TAZ DNA levels in quadriceps, plotted as GC / diploid cell, following AAVhu68.hTafazzin. AAV9- IlRGDPA.hTafazzin, AAV9- AVIRGDV.hTafazzin administration. FIG. 4L shows TAZ RNA levels in quadriceps, plotted as vector GC / 100 ng total RNA (as normalized to U6), following AAVhu68. hTafazzin, A AV9-IIRGDP A. hTafazzin, AAV9- AVIRGDV.hTafazzin administration.

[0025] FIG. 5 A shows results of ISH analysis, plotted as percent ISH-positive cells in tissue of left ventricle of the heart. FIG. 5B shows results of ISH analysis, plotted as percent ISH-positive cells in tissue of intraventricular septum of the heart. FIG. 5C shows results of ISH analysis, plotted as percent ISH-positive cells in tissue of right ventricle of the heart. FIG. 5D shows results of ISH analysis, plotted as percent ISH-positive cells in diaphragm tissue. FIG. 5E shows results of ISH analysis, plotted as percent ISH-positive cells in quadricep tissue.

[0026] FIG. 6 shows representative microscopy images of hepatocytes at day 90 following AAVhu68. hTafazzin, A AV9-IIRGDP A. hTafazzin, AAV9- AVIRGDV.hTafazzin administration. These results show a more transcriptionally active hepatocytes at day 90 following rAAV9- AVIRGDV.hT afazzin administration.

[0027] FIG. 7A shows measured levels of aspartate aminotransferase (AST) in blood samples on DO to D90 following AAVhu68.hTafazzin, AAV9-IIRGDPA.11T afazzin, AAV9- AVIRGDV. hTafazzin administration. FIG. 7B shows measured levels of alanine aminotransferase (ALT) in blood samples on DO to D90 following A AVhu68. hTafazzin, AAV9- IIRGDPA.hTafazzin, AAV9- AVIRGDV.hTafazzin administration. FIG. 7C shows measured levels of platelet count in blood samples on DO to D90 following AAVhu68.hTafazzin, AAV9- IIRGDPA.hTafazzin, AAV9- AVIRGDV.hTafazzin administration. FIG. 7D shows measured levels of d dimer in blood samples on DO to D90 following AAVhu68.hTafazzin, AAV9- IIRGDPA.hTafazzin, AAV9- AVIRGDV.hTafazzin administration. FIG. 7E shows measured levels of troponin I in blood samples on DO to D90 following AAVhu68.hTafazzin, AAV9- IIRGDPA.hTafazzin, AAV9- AVIRGDV.hTafazzin administration.

[0028] FIG. 8 shows representative microscopy images of H&E pathology' analysis of liver, heart and nervous tissues.

[0029] FIG. 9A shows heart / body weight ratio, plotted as percent wild-type, in wild-type and TAZKD mice. FIG. 9B shows tafazzin / viculin levels ratio, plotted as percent wild-type, as measured by western blot in heart tissue in wild-type and TAZKD mice. FIG. 9C shows levels of cardiolipin, plotted as percent wild-type, as measured in heart in wild-type and TAZKD mice. FIG. 9D shows levels of serum biomarker GDF-15, plotted as concentration (pg / mL), in wildtype and TAZKD mice.

[0030] FIG. 10A shows baseline serum levels of GDF-15 (before treatment). FIG. 10B shows serum levels of GDF-15 in mice on day 110 following administration of AAV in TAZKD mice.

[0031] FIG. 11A shows percent ISH (in situ hybridization)-positive cells (hTafazzin) as measured in heart tissue on day 110 following AAV administration. FIG. 1 IB shows expression levels of tafazzin (tafazzin / vinculin), as measured by western blot, and plotted as percent PBS.

[0032] FIG. 12A shows interventricular septum thickness (IVSd) of tire left ventricular wall, plotted as mm. FIG. 12B shows end-diastolic left ventricular posterior wall thickness (LVPWd) of the left ventricular wall, plotted as mm. FIG. 12C shows left ventricular internal end diastolic diameter (LVIDd; i.e., left ventricular lumen size), plotted as mm. FIG. 12D shows left ventricular internal diameter end systole (LVIDs; i.e., left ventricular lumen size), plotted as mm. FIG. 12E shows left ventricular mass (LVM) as estimated by Echo, plotted as mg. FIG. 12F shows left ventricular mass index (LVMI) as estimated by Echo, plotted as AU. FIG. 12G shows relative wall thickness (RWT; i.e., systolic function), plotted as AU. FIG. 12H shows fractional shortening (FS; i.e., systolic function), plotted as percent.

[0033] FIG. 13A shows representative microscopic images of ISH analysis of cardiac tissue (left ventricle). FIG. 13B shows quantified results of ISH analy sis, plotted as percent ISH-positive cells, in mice following rAAV administration.

[0034] FIG. 14A shows DNA biodistribution in various tissues on day 90 following AAV administration, plotted as vector genome copies (GC) / diploid cell. FIG. 14B shows DNA distribution on day 90 following rAAV administration, plotted as fold over hu68. FIG. 14C shows RNA biodistribution in various tissues on day 90 following rAAV administration, plotted as vector RNA copies / 100 ng total RNA (normalized to U6 delta CT). FIG. 14D shows RNA distribution on day 90 following rAAV administration, plotted as fold over hu68.

[0035] FIG. 15 shows a study schematic.

[0036] FIG. 16 shows RNA levels, plotted as transcript copies per / 100 ng RNA, as examined in brain (frontal cortex), heart, liver, diaphragm, skeletal muscle tissues.

[0037] FIG. 17 shows RNA levels, plotted as transcript copies per / 100 ng RNA, as examined in diaphragm, biceps brachii, biceps femoris, deltoid, gastrocnemius, gluteus maximus, soleus, and vastus lateralis tissues.

[0038] FIG. 18 shows RNA levels, plotted as transcript copies per / 100 ng RNA, as examined in gastrocnemius muscle tissues following biopsies collected on days 0, 30, 60, and 90.

[0039] FIG. 19A shows representative images of the In Situ Hybridization (ISH) analysis of gastrocnemius tissue as examined on day 30 and day 60. FIG. 19B shows results of in situ hybridization (ISH) analysis in gastrocnemius tissue, plotted as percent hTafazzin-positive fibers as normalized to an AAVhu68 control.

[0040] FIG. 20A shows representative images of the In Situ Hybridization (ISH) analysis of gastrocnemius, vastus lateralis, pectoralis. heart, soleus, and diaphragm tissues. FIG. 20B shows results of ISH analysis, plotted as percent ISH-positive myofibers in tissue from biceps brachii, deltoid, diaphragm, gastrocnemius, pectoralis, soleus, and vastus lateralis.

[0041] FIG. 21 shows results of ISH analysis, plotted as percent ISH-positive cells in tissue collected from necropsy on day 90 following rAAV administration (left ventricle, septum, biceps brachii, deltoid, diaphragm, gastrocnemius, soleus, vastus, pectoralis, and liver).

[0042] FIG. 22 shows results of RT-PCR, plotted as copies / 100 ng input RNA in tissue collected at 3 months following AAV administration (IV) 3xl013GC / kg (3E13 / Kg).

[0043] FIG. 23 A shows dose-dependent normalization of the cardiolipin content in heart 100 days post administration of rAAV in mice, plotted as percent wild type (WT)-PBS. FIG. 23B shows a dose-dependent reduction of GDF-15 in serum, a mitochondrial function biomarker following administration of rAAV in mice, plotted as pg / mL of GDF-15.

[0044] FIGs. 24A to 24E show representative microscopy images showing a dose-dependent increase in human tafazzin expression, in heart according to in situ hybridization (ISH).

[0045] FIG. 25A shows measured AST (aspartate transaminase) levels, plotted as IU / L, in NHP on Day -5 to Day 90 at various doses. FIG. 25B shows measured AST levels, plotted as IU / L, in NHP on Day -5 to Day 90 post rAAV administration at 6xl012GC / kg. FIG. 26A and FIG. 26B show representative microscopy images showing huma tafazzin mRNA levels in heart tissue (gray against black background, according to in situ hybridization (ISH), 4x magnification), post administration of rAAV at 6xl012GC / kg and 12xl013GC / kg. FIG. 26C shows tafazzin RNA in various tissues (left ventricle, right ventricle, septum, quadricep, bicep, liver), plotted as copies / lOOng RNA, as measured by RTqPCR.

[0046] FIG. 27A shows vector DNA biodistribution in various tissues on day 90 post rAAV administration. FIG. 27B shows RNA biodistribution in various tissues on day 90 post rAAV administration.

[0047] FIG. 28A shows RNA biodistribution in heart tissue (left ventricle) on day 90 and day 180 post administrator with rAAV at various doses (vehicle. 2xl012GC / kg, 6xl012GC / kg. 2xl013GC / kg). FIG. 28B shows RNA biodistribution in liver on day 90 and day 180 post administrator with rAAV at various doses (vehicle, 2xlO12GC / kg, 6xl012GC / kg, 2xl013GC / kg).

[0048] FIG. 29 shows results of the ISH analysis (tafazzin In situ hybridization VisioPharm quantification), plotted as percent ISH+ (ISH positive) cells in various tissues (left ventricle, septum, quadricep, bicep, lover and pectoralis) post at day 90 and day 180 post rAAV administration at various doses (from left to right for each tissue: vehicle, 2xlO12GC / kg, 6xl012GC / kg, 2xl013GC / kg).

[0049] FIG. 30A shows measured ALP (alkaline phosphatase) levels, plotted as IU / L. in NHP on Day -6 to Day 90 at various doses of administered rAAV. FIG. 30B shows measured BUN (blood urea nitrogen) levels, plotted as mg / dL, in NHP on Day -6 to Day 90 at various doses of administered rAAV. FIG. 30C shows measured GGT (gamma-glutamyl transferase) levels, plotted as IU / L, in NHP on Day -6 to Day 90 at various doses of administered rAAV. FIG. 30D shows measured platelets levels, plotted as 1 7pL. in NHP on Day -6 to Day 90 at various doses of administered rAAV. FIG. 30E shows measured WBC (white blood cell) levels, plotted as 103 / pL. in NHP on Day -6 to Day 90 at various doses of administered rAAV. FIG. 30F shows measured DDimer levels, plotted as ng / mL. in NHP on Day -6 to Day 90 at various doses of administered rAAV. FIG. 30G shows measured PT (prothrombin time) levels, plotted as seconds (sec), in NHP on Day -6 to Day 90 at various doses of administered rAAV. FIG. 30H shows measured aPTT (activated partial thromboplastin time) levels, plotted as seconds (sec), in NHP on Day -6 to Day 90 at various doses of administered rAAV. FIG. 301 shows measured ALT (alanine aminotransferase) levels, plotted as IU / L, in NHP on Day -6 to Day 90 at various doses of administered rAAV.

[0050] FIG. 31A shows measured serum GDF-15 concentration, plotted as pg / mmol, on days 25, 55, and 85. FIG. 31B shows measured serum GDF-15 concentration, plotted as pg / mmol, on day 85. TAZKD animals displayed significantly elevated GDF-15 levels in all groups at baseline timepoint.

[0051] FIG. 32A shows a plot of measured body weights of mice administered with rAAV at various doses. FIG. 32B shows as plot of measured body weights of mice on day 91 post rAAV administration.

[0052] FIG. 33A shows body weight at scheduled necropsy date, plotted as grams (g). FIG. 33B shows a plot of heart weight to brain weight ratio, plotted as mg / mg. FIG. 33C shows a plot of heart weight to necropsy body weight, plotted as mg / g.

[0053] FIG. 34A shows measured ALP (alkaline phosphatase) levels, plotted as IU / L, in mice at various doses of administered rAAV. FIG. 34B shows measured ALT (alanine aminotransferase) levels, plotted as IU / L, in mice at various doses of administered rAAV. FIG. 34C shows measured ALB (albumin) levels, plotted as g / dL, in mice at various doses of administered rAAV. FIG. 34D shows measured GLOB (globulin) levels, plotted as g / dL, in mice at various doses of administered rAAV. FIG. 34E shows measured MG levels, plotted as mg / dL, in mice at various doses of administered rAAV. FIG. 34F shows measured WBC (white blood cells) levels, plotted as 103 / pL, in mice at various doses of administered rAAV. FIG. 34G shows measured HB (hemoglobin) levels, plotted as g / dL, in mice at various doses of administered rAAV. FIG. 34H shows measured PLT (platelet count) levels, plotted as 103 / pL, in mice at various doses of administered rAAV. FIG. 341 shows measured MON (monocytes) levels, plotted as 103 / pL, in mice at various doses of administered rAAV. FIG. 34J shows measured EOS (eosinophil) levels, plotted as 103 / pL, in mice at various doses of administered rAAV.

[0054] DETAILED DESCRIPTION OF THE INVENTION

[0055] Provided herein are recombinant, replication-defective adeno-associated virus (rAAV) vectors, nucleic acid sequences, expression cassettes, and vector expressing human functional Tafazzin (hTafazzin) protein and compositions containing the same. Also provided herein are methods useful for the treatment of Barth Syndrome or a disease associated with a mutation in Tafazzin (TAZ) gene and / or alleviating symptoms thereof. In certain embodiments, the human Tafazzin (hTafazzin) protein is delivered via the rAAV as provided herein. Also provided herein are pharmaceutical compositions, formulations containing same, and in particularly, an aqueous liquid suspension. Uses of these compositions are also provided.

[0056] The nucleic acid sequences provided herein are useful for packaging functional hTafazzin coding sequence into suitable vector (e.g., rAAV) or a genetic element useful for manufacture (e.g., plasmid).

[0057] In one aspect, provided herein is a recombinant adeno-associated virus (rAAV) comprising an adeno-associated virus (AAV) capsid and a nucleic acid molecule comprising an expression cassette, wherein the expression cassette is a nucleic sequence comprising: (i) a cDNA sequence comprising SEQ ID NO: 1 encoding a human Tafazzin (hTAfazzin), and (ii) regulatory control sequences operably linked to the sequences of (i), wherein the regulatory control sequences comprise an optional enhancer, a promoter, an optional intron, and a poly adenylation (poly A) signal sequence. In certain embodiments, the rAAV comprises nucleic acid molecule comprises a vector genome comprising AAV 5' inverted terminal repeat (ITR), the expression cassette, and an AAV 3' ITR, optionally wherein the AAV ITRs are from AAV2. In certain embodiments, the rAAV comprises expression cassette comprising regulatory control sequences comprising cytomegalovirus immediate early (CMV IE) enhancer, a chicken beta actin promoter, an intron, optionally a chicken beta actin intron, and a rabbit beta globin polyA signal sequence. In certain embodiments, tire rAAV comprises expression cassette comprising regulatory control sequences comprising a CB7 hybrid promoter comprising a cytomegalovirus immediate-early (CMV IE) enhancer and the chicken P-actin promoter, optionally with spacer sequence, optionally with a chimeric intron comprising chicken beta actin intron and further comprising a chicken beta-actin splicing donor (including tire exon sequence, chicken beta actin intron) and rabbit beta-globin splicing acceptor). In certain embodiments, the rAAV comprises expression cassette comprising regulatory control sequences comprising (a) a nucleic acid sequence of SEQ ID NO: 11 comprising a CB7 hybrid promoter, and (b) a nucleic acid sequence of SEQ ID NO: 12 comprising a rabbit beta-globin polyA signal sequence. In certain embodiments, the rAAV comprises expression cassette comprising regulatory control sequences comprising a cardiac promoter, optionally a cardiac troponin (cTnT) promoter, further optionally a chicken cTnT promoter. In certain embodiments, the rAAV comprises expression cassette comprising regulator}' control sequences comprising a WRPE element, optionally a mutant WPRE element.

[0058] In certain embodiments, the expression cassette comprises nucleic acid sequence of SEQ ID NO: 6, or a nucleic acid sequence at least 99% identical to SEQ ID NO: 6. In certain embodiments, the vector genome comprises nucleic acid sequence of SEQ ID NO: 5 or a sequence at least 99% identical to SEQ ID NO: 5. In certain embodiments, tire capsid is a wildtype or mutant Clade F AAV capsid, optionally an AAVhu68 capsid, an AAV9 capsid, a mutant AAV9 capsid, an AAVhu95 capsid, or AAVhu96 capsid. In certain embodiments, the capsid AAV capsid is a mutant AAV9 capsid comprising AVIRGDV (SEQ ID NO: 25).

[0059] In a further aspect, provided herein is a composition and pharmaceutical composition comprising a rAAV or a vector as described herein and an aqueous suspension media. In certain embodiments, the rAAV or the composition thereof is for use in the treatment of Barth Syndrome (BTHS) or a disease associated with a mutation in a Tafazzin (TAZ) gene. In certain embodiments, the disease associated with a mutation in a TAZ gene is dilated cardiomyopathy (DCM), hypertrophic DCM, endocardial fibroelastosis, left ventricular noncompaction (LVNC), familial dilated cardiomyopathy, and isolated noncompaction of left ventricular myocardium. In certain embodiments, the composition and pharmaceutical composition is formulated for intravenous injection, optionally wherein the rAAV suspension is formulated to have a pH of 6.5 to 7.5, optionally 6.8 to 7.2.

[0060] In another aspect, provided herein is a method for treating or ameliorating or improving one or more symptoms of Barth Syndrome in a subject in need thereof. In a further aspect, provided herein is a method of treating or ameliorating or improving one or more symptoms of a disease associated with a mutation in a Tafazzin (TAZ) gene in a subject.

[0061] In another aspect, provide herein is a recombinant nucleic acid molecule an AAV vector genome which is: (a) an AAV - 5' inverted terminal repeat (ITR); (b) an expression cassette which is a nucleic acid sequence comprising: (i) a cDNA sequence comprising SEQ ID NO: 1 encoding a human Tafazzin (hTAfazzin), and (ii) regulatory control sequences operably linked to the sequences of (i), wherein the regulatory control sequences comprise an optional enhancer, a promoter, an optional intron, and a poly adenylation (polyA) signal sequence; and (c) an AAV - 3' ITR. In certain embodiments, the recombinant nucleic acid molecule comprises AAV vector genome comprises a nucleic acid sequence of SEQ ID NO: 6 or SEQ ID NO: 5. In certain embodiments, tire recombinant nucleic acid molecule is a plasmid.

[0062] In another aspect, provided herein is a packaging host cell for rAAV production outside the human body (e.g., in culture) which comprises a nucleic acid molecule as described herein. In certain embodiments, the packaging host cell further comprises AAV rep coding sequences operably linked to sequences which express rep protein in the packaging host cell, an AAV capsid coding sequences operably linked to sequences which express AAV capsid proteins in the packaging host cell, and helper virus functions necessary to permit packaging of the expression cassette and AAV ITRs into tire AAV capsid. In certain embodiments, a packaging cell is provided which comprises tire expression cassette, vector genome or plasmid. In certain embodiments, an rAAV production is provided which comprises a cell culture which may be adherent or in suspension comprising the packaging host cell. In another aspect, provided herein is a production system useful for producing rAAV as described herein, wherein the production system comprises a cell culture comprising: (a) a nucleic acid sequence encoding a AAV capsid protein; (b) the vector genome; and (c) sufficient AAV rep functions and helper functions to permit packaging of the vector genome into the AAV capsid.

[0063] In another aspect, provided herein is a recombinant AAV (rAAV) for use in preparing a medicament for treatment of Barth Syndrome or a disease associated with a mutation in Tafazzin (TAZ) gene.

[0064] In one embodiment, the functional hTafazzin has an amino acid sequence of SEQ ID NO: 2 or an amino acid sequence at least about 95 % (e.g., at least 95%, 96%, 97%, 98%, 99%. or 99.9%) identical thereto.

[0065] In certain embodiments, a functional hT fazzin protein ameliorates symptoms or delays progression of Barth Syndrome or a disease associated with a mutation in a Tafazzin (TAZ) gene in an animal model. One exemplified animal model is a TAZ-knock down (TAZ-kd or TAZKD) mouse. Another exemplified animal model is a TAZ -knock-out (TAZ-ko) mouse. Other suitable models may be used. See also, jax_org / strain / 014648#; Soustek, M.S., et al., Characterization of a Transgenic Short Hairpin RNA-Induced Murine Model of Tafazzin Deficiency, Hum Gene Ther. 2011 Jul; 22(7): 865-871; Redelsperger, I. M., et al., (2016), Stability of Doxycycline in Feed and Water and Minimal Effective Doses in Tetracycline-Inducible Systems, Journal of the American Association for Laboratory Animal Science, 55(4), 467-474; and Phoon CK, et al., Tafazzin knockdown in mice leads to a developmental cardiomyopathy with early diastolic dysfunction preceding myocardial noncompaction, J Am Heart Assoc, 2012 Apr;l(2):jah3- c000455, Epub 2012 Apr 24. which arc incorporated herein by reference in their entireties. The Barth Syndrome symptoms or progression may be evaluated using various assays / methods, including but not limited to. a survival plot (e.g., Kaplan-Meier survival plot), monitoring body weights, echocardiogram (echo) and electrocardiogram (EKG or ECG), and oxidative stress levels (i.e., GDF-15). In certain embodiment, administration or expression of a functional hTAfazzin protein in an animal model leads to amelioration of Barth Syndrome symptoms or delay in Barth Syndrome progression shown by an assay result which is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, or more than 100% of that obtained in a corresponding wildtype animal. In certain embodiment, administration or expression of a functional hTafazzin protein in a Barth Syndrome animal model leads to amelioration of Barth Syndrome symptoms or delay in Barth Syndrome progression shown by an improved assay result which is at least about 10%. 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, or more than 100% of that obtained from a corresponding non-treated Barth Syndrome animal.

[0066] Provided herein are coding sequence(s), expression cassette(s), and a vector genome(s) encoding the functional hTafazzin, wherein the sequence(s), expression cassette(s), or a vector genome(s) comprises nucleic acid sequence comprising cDNA sequence comprising SEQ ID NO: 1 encoding hTafazzin. In certain embodiments, provided herein is hTafazzin engineered coding sequence comprising a cDNA sequence comprising SEQ ID NO: 1.

[0067] With regard to the description of these various embodiments, it is intended that each of the compositions herein described, is useful, in another embodiment, in tire methods of the invention. In addition, it is also intended that each of the compositions herein described as useful in tire methods, is, in another embodiment, itself an embodiment of the invention.

[0068] Unless defined otherw ise in this specification, 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 and by reference to published texts, which provide one skilled in the art with a general guide to many of the terms used in the present application.

[0069] Nucleic Acid Molecules

[0070] In one aspect, provided herein is a hTafazzin coding sequence which is an engineered hTafazzin coding sequence. In one embodiment, the engineered sequence is useful to improve production, transcription, expression or safety in a subject. In another embodiment, the engineered sequence is useful to increase efficacy of the resulting therapeutic compositions or treatment. In a further embodiment, the engineered sequence is useful to increase the efficacy of the functional hTafazzin protein being expressed, but may also pennit a lower dose of a therapeutic reagent that delivers the functional protein to increase safety.

[0071] In one aspect, provided herein is a recombinant nucleic acid molecule comprising an engineered hTafazzin coding sequence which encodes a functional hTafazzin. In certain embodiments, the engineered hTafazzin coding sequence comprises a nucleic acid sequence of SEQ ID NO: 1. In certain embodiments, the engineered hTafazzin coding sequence comprises nucleic acid sequence of about 90%, at least 95% identical, at least 97% identical, at least 98% identical, or 99% to 100% identical to SEQ ID NO: 1 and which expresses the functional hTafazzin protein.

[0072] In certain embodiments, the engineered hTafazzin coding sequence is SEQ ID NO: 1 or a nucleic acid sequence at least 99.9% identical to SEQ ID NO: I which encodes an amino acid sequence of SEQ ID NO: 2. A “nucleic acid’’, as described herein, can be RNA, DNA, or a modification thereof, and can be single or double stranded, and can be selected, for example, from a group including: nucleic acid encoding a protein of interest, oligonucleotides, nucleic acid analogues, for example peptide-nucleic acid (PNA), pseudocomplementary PNA (pc-PNA), locked nucleic acid (LNA) etc. Such nucleic acid sequences include, for example, but are not limited to, nucleic acid sequence encoding proteins, for example that act as transcriptional repressors, antisense molecules, ribozymes, small inhibitory nucleic acid sequences, for example but are not limited to RNAi, shRNAi, siRNA, micro RNAi (rnRNAi), antisense oligonucleotides etc.

[0073] In certain embodiments, the recombinant nucleic acid molecule comprises expression cassette comprising at least one miRNA target sequences operably linked to a transgene, optionally in its 3' UTR and / or its 5' UTR. In certain embodiments, the recombinant nucleic acid molecule comprises a vector genome (comprising an expression cassette) which comprises at least one miRNA seed, binding site or full sequence. MicroRNAs (or miRNA or miR) are 19-25 nucleotide noncoding RNAs that bind to the sites of nucleic acid targets and down-regulate gene expression either by reducing nucleic acid molecule stability or by inhibiting translation. In some embodiments, a microRNA sequence comprises a seed region, e.g., a sequence in the region of positions 2-8 of the mature microRNA, which has Watson-Crick sequence fully or partially complementarity to the miRNA target sequence of tire nucleic acid. Such at least one miRNA may be used in combinations, including in an expression cassette or a vector genome also comprising a coding sequence for therapeutic protein, enzyme, or other moiety, and which is operably linked to the coding sequence. In certain embodiments, the vector genome may contain one miRNA to eight miRNA sequences, which are the same or different. In certain embodiments, the miRNA is miR-122 miRNA which modulates, e.g., reduces, tire expression of a gene product in the liver. Optionally, the vector genome does not contain therapeutic transgenes other than miRNA sequences.

[0074] Nucleic acid sequences described herein can be cloned using routine molecular biology techniques, or generated de novo by DNA synthesis, which can be performed using routine procedures by service companies having business in the field of DNA synthesis and / or molecular cloning (e.g., GeneArt, GenScript, Life Technologies, Eurofins). The nucleic acid sequences encoding the RNA or DNA (e.g., cDNA) described herein are assembled and placed into any suitable genetic element, e.g., naked DNA, phage, transposon, cosmid, episome, etc., which transfers the sequences carried thereon to a host cell, e.g., for generating non-viral delivery systems (e.g.. RNA-based systems, naked DNA, or the like), or for generating viral vectors in a packaging host cell, and / or for delivery to a host cells in a subject. In one embodiment, the genetic element is a vector. In one embodiment, the genetic element is a plasmid. The methods used to make such engineered constructs are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).

[0075] It should be understood that the hTafazzin coding sequences described herein are intended to be applied to other compositions, regiments, aspects, embodiments and methods described across the Specification.

[0076] Expression Cassette and Vector Genome

[0077] Provided herein is a nucleic acid molecule comprising the engineered hTaffazin coding sequence under control of regulatory sequences which direct the functional hTafazzin expression in a target cell, also termed as an expression cassette. In certain embodiments, the expression cassette comprises an open reading frame (ORF) for a functional hTafazzin coding sequence which encode functional hTafazzin, wherein the ORF is operably linked to regulatory control sequences which direct expression of the functional hTaffazin protein in a cell, and w herein the regulatory’ control sequences comprise a promoter, or a hybrid promoter, optionally an enhancer, optionally an intron, and a poly adenylation (polyA) sequence. In certain embodiments, tire expression cassette is a nucleic sequence comprising: (i) a cDNA sequence comprising SEQ ID NO: 1 encoding a human Tafazzin (hTAfazzin), and (ii) regulatory control sequences operably linked to the sequences of (i), w herein the regulatory control sequences comprise an optional enhancer, a promoter, an optional intron, and a polyadenylation (polyA) signal sequence.

[0078] As used herein, an “expression cassette" refers to a nucleic acid molecule which comprises a biologically useful nucleic acid sequence (e.g., a gene cDNA encoding a protein, enzyme or other useful gene product, mRNA, etc.) and include regulatory sequences operably linked thereto which control, direct, enable and / or modulate transcription, translation, and / or expression of the nucleic acid sequence and its gene product. As used herein, “operably linked7’ sequences include both regulatory sequences that are contiguous or non-contiguous with the nucleic acid sequence and regulatory sequences that act in trans or cis nucleic acid sequence. Such regulatory sequences typically include, e.g., one or more of a promoter, an enhancer, an intron, a Kozak sequence, a polyadenylation sequence, and a TATA signal. The expression cassette may contain regulatory sequences upstream (5’ to or also 5') of the gene sequence, e.g., one or more of a promoter, an enhancer, an intron, etc., and one or more of an enhancer, or regulatory sequences downstream (3’ to or 3') a gene sequence, e.g.. 3’ untranslated region (3’ UTR) comprising a polyadenylation site, among other elements. In certain embodiments, the regulatory sequences are operably linked to the nucleic acid sequence of a gene product, w herein the regulatory sequences are separated from nucleic acid sequence of a gene product by an intervening nucleic acid sequences, i.e., 5 ’-untranslated regions (5’UTR). In certain embodiments, the expression cassette comprises nucleic acid sequence of one or more of gene products. In some embodiments, the expression cassette can be a monocistronic or a bicistronic expression cassette. In other embodiments, the term '‘transgene” refers to one or more DNA sequences from an exogenous source which are inserted into a target cell.

[0079] As used herein, the term “regulatory sequence / s”, or “regulatory control sequence / s “or “expression control sequence / s” refers to nucleic acid sequences, including, but not limiting to, e.g., initiator sequences, enhancer sequences, promoter sequences, intron sequences, and polyA signal sequences which direct, enable, induce, repress, or otherw ise control the transcription, translation and / or expression of nucleic acid sequences encoding a product to which they are operably linked.

[0080] Typically, such an expression cassette can be used for generating a viral vector and contains the coding sequence for the gene product described herein flanked by packaging signals of the viral genome and other expression control sequences such as those described herein. In certain embodiments, a vector genome may contain tw o or more expression cassettes.

[0081] The term “exogenous” as used to describe a nucleic acid sequence or protein means that the nucleic acid or protein does not naturally occur in the position in w'hich it exists in a chromosome, or host cell. An exogenous nucleic acid sequence also refers to a sequence derived from and inserted into the same host cell or subject, but which is present in a non-natural state, e.g., a different copy number, or under the control of different regulatory elements.

[0082] The term “heterologous” as used to describe a nucleic acid sequence or protein means that the nucleic acid or protein w as derived from a different organism or a different species of the same organism than the host cell or subject in which it is expressed. The term “heterologous” when used with reference to a protein or a nucleic acid in a plasmid, expression cassette, or vector, indicates that the protein or the nucleic acid is present with another sequence or subsequence which w ith which the protein or nucleic acid in question is not found in the same relationship to each other in nature.

[0083] The expression cassette may contain regulatory' sequences upstream (5' to) of the gene sequence, e g., one or more of a promoter, a hybrid promoter, an enhancer, an intron, etc., and one or more of an enhancer, or regulatory sequences downstream (3' to) a gene sequence, e.g., 3' untranslated region (3' UTR) comprising a polyadenylation (polyA) site, among other elements.

[0084] In certain embodiments, the regulatory sequences comprise one or more of a promoter, an enhancer, an intron, a transcription factor, a transcription terminator, an efficient RNA processing signals such as splicing and polyadenylation site signals (polyA), a sequences that stabilize cytoplasmic mRNA, for example Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE). and sequences that enhance translation efficiency (i.e., Kozak consensus sequence). In certain embodiments the selected promoter is a constitutive promoter. In certain embodiments, the promoter is a ubiquitous promoter. For example such promoters may include chicken beta-actin (CB) promoter, human cytomegalovirus (CMV) promoter, ubiquitin C promoter (UbC), the early and late promoters of simian virus 40 (SV40), U6 promoter, metallothionein promoters, EFla promoter, ubiquitin promoter, hypoxanthine phosphoribosyl transferase (HPRT) promoter, dihydrofolate reductase (DHFR) promoter (Scharfmann et al., Proc. Natl. Acad. Sci. USA 88:4626-4630 (1991), adenosine deaminase promoter, phosphoglycerol kinase (PGK) promoter, pyruvate kinase promoter phosphoglycerol mutase promoter, the p-actin promoter (Lai et al.. Proc. Natl. Acad. Sci. USA 86: 10006-10010 (1989)), the long terminal repeats (LTR) of Moloney Leukemia Virus and other retroviruses, the thymidine kinase promoter of Herpes Simplex Virus and other constitutive promoters known to those of skill in the art. In certain embodiments, tire promoter is a CB7 promoter, also referred to as hybrid CB7 promoter, comprising a cytomegalovirus immediate-early (CMV IE) enhancer and the chicken P-actin promoter, optionally with spacer sequence, optionally with a chimeric intron comprising chicken beta actin intron and further comprising a chicken beta-actin splicing donor (including the exon sequence, chicken beta actin intron) and rabbit beta-globin splicing acceptor), or a CBh promoter [SJ Gray et al, Hu Gene Ther, 201 1 Sep: 22(9): 143-1153],

[0085] In certain embodiments, the promoter is a tissue- or cell specific-promoter. In certain embodiments, the promoter is cardiac specific promoter, e.g., cardiac troponin T (cTNT), desmin (DES), alpha-myosin heavy chain (a-MHC), myosin light chain 2 (MLC-2) promoters. Sec also, Pacak, C.A., et al., Tissue specific promoters improve specificity of AAV9 mediated transgene expression following intra-vascular gene delivery in neonatal mice, Genetic V accines and Therapy 2008, 6: 13. In certain embodiments, the expression cassette comprises a promoter which is a chicken cardiac Troponin T promoter (also referred to as chicken TnT or chTnT). In certain embodiments, the promoter is a hybrid promoter. In certain embodiments, the promoter is a hybrid cardiac promoter. As used herein, the term “hybrid promoter” refers to a regulatory control sequence comprising a hybrid between an enhancer, a spacer sequence, and a promoter sequence. In certain embodiments, the hybrid cardiac promoter comprises a CMV IE enhancer sequence, a spacer sequence, a chicken cardiac troponin T promoter. See also, International Patent Application No. PCT / US2022 / 082384. filed December 24, 2022, now published WO 2023 / 122804 AL which are incorporated herein by reference in its entirety. In one embodiment, expression of the gene product is controlled by a regulatable promoter that provides tight control over tire transcription of the sequence encoding the gene product, e.g., a pharmacological agent, or transcription factors activated by a pharmacological agent or in alternative embodiments, physiological cues. Promoter systems that are non-leaky and that can be tightly controlled are preferred. Examples of regulatable promoters which are liganddependent transcription factor complexes that include, without limitation, members of the nuclear receptor superfamily activated by their respective ligands (e.g., glucocorticoid, estrogen, progestin, retinoid, ecdysone, and analogs and mimetics thereof) and rTTA activated by tetracycline. In one aspect, the gene switch is an EcR-based gene switch. Examples of such systems include, without limitation, the systems described in US Patent Nos. 6,258,603, 7,045,315, U.S. Published Patent Application Nos. 2006 / 0014711. 2007 / 0161086. and International Published Application No. WO 01 / 70816. Examples of chimeric ecdysone receptor systems are described in U.S. Pat. No. 7,091,038, U.S. Published Patent Application Nos. 2002 / 0110861, 2004 / 0033600, 2004 / 0096942, 2005 / 0266457, and 2006 / 0100416, and International Published Application Nos. WO 01 / 70816, WO 02 / 066612, WO 02 / 066613, WO 02 / 066614, WO 02 / 066615, WO 02 / 29075, and WO 2005 / 108617, each of which is incorporated by reference in its entirety. An example of a non-steroidal ecdysone agonist-regulated system is the RheoSwitch® Mammalian Inducible Expression System (New England Biolabs. Ipswich, MA).

[0086] Still other promoter systems may include response elements including but not limited to a tetracycline (tet) response element (such as described by Gossen & Bujard (1992, Proc. Natl. Acad. Sci. USA 89:5547-551); or a hormone response element such as described by Lee et al. (1981, Nature 294:228-232); Hynes ct al. (1981, Proc. Natl. Acad. Sci. USA 78:2038-2042); Klock et al. (1987, Nature 329:734-736); and Israel & Kaufman (1989, Nucl. Acids Res. 17:2589-2604) and other inducible promoters known in the art. These response elements may include, a hypoxia response element (HRE) that binds HIF-Ia and p. a metal-ion response element such as described by Mayo et al. (1982, Cell 29:99-108); Brinster et al. (1982, Nature 296:39-42) and Searle et al. (1985, Mol. Cell. Biol. 5: 1480-1489); or a heat shock response element such as described by Nouer et al. (in: Heat Shock Response, ed. Nouer, L., CRC, Boca Raton, Fla., ppI67-220, 1991).

[0087] Using such promoters, expression of the transgene can be controlled, for example, by the Tet-on / off system (Gossen et al., 1995, Science 268: 1766-9: Gossen et al.. 1992, Proc. Natl. Acad. Sci. USA., 89(12):5547-51); the TetR-KRAB system (Urrutia R., 2003. Genome Biol., 4(10):231 ; Deuschle U et al., 1995, Mol Cell Biol. (4): 1907-14); the mifepristone (RU486) regulatable system (Geneswitch: Wang Y et al., 1994, Proc. Natl. Acad. Sci. USA., 91(17):8180- 4; Schillinger et al., 2005, Proc. Natl. Acad. Sci. U S A. 102(39): 13789-94); and the humanized tamoxifen-dep regulatable system (Roscilli et al., 2002, Mol. Ther. 6(5):653-63).

[0088] In another aspect, the gene switch is based on heterodimerization of FK506 binding protein (FKBP) with FKBP rapamycin associated protein (FRAP) and is regulated through rapamycin or its non-immunosuppressive analogs. Examples of such systems, include, without limitation, the ARGENT™ Transcriptional Technology (ARIAD Pharmaceuticals, Cambridge, Mass.) and the systems described in U.S. Pat. Nos. 6,015,709, 6,117,680, 6,479,653, 6,187,757, and 6,649,595, U.S. Publication No. 2002 / 0173474, U.S. Publication No. 200910100535, U.S. Patent No. 5,834,266, U.S. Patent No. 7,109,317, U.S. Patent No. 7,485.441, U.S.PatentNo. 5,830,462, U.S. Patent No. 5,869,337. U.S. Patent No. 5,871,753, U.S. Patent No. 6,011,018, U.S. Patent No. 6,043,082, U.S. Patent No. 6,046.047, U.S. Patent No. 6,063,625, U.S. Patent No. 6,140,120, U.S. Patent No. 6,165,787, U.S. Patent No. 6,972,193, U.S. Patent No. 6,326,166, U.S. Patent No. 7,008,780, U.S. Patent No. 6,133,456, U.S. Patent No. 6,150,527, U.S. Patent No. 6,506,379, U.S. Patent No. 6,258,823, U.S. Patent No. 6,693,189, U.S. Patent No. 6,127,521, U.S. Patent No. 6,150,137, U.S. Patent No. 6,464,974, U.S. Patent No. 6,509,152, U.S. Patent No. 6,015,709, U.S. Patent No. 6,117,680, U.S. Patent No. 6,479,653, U.S. Patent No. 6,187,757, U.S. Patent No. 6,649,595, U.S. Patent No. 6,984,635. U.S. Patent No. 7,067,526, U.S. Patent No. 7,196,192, U.S. Patent No. 6.476.200, U.S. Patent No. 6,492,106, WO 94 / 18347, WO 96 / 20951, WO 96 / 06097. WO 97 / 31898, WO 96 / 41865, WO 98 / 02441. WO 95 / 33052, WO 99110508, WO 99110510, WO 99 / 36553, WO 99 / 41258, WO 01114387, ARGENT™ Regulated Transcription Plasmid Kit, Takara Bio iDimerize regulated transcription kit, or comparable kits from QuantiTect, Sensiscript, or the like, each of which is incorporated herein by reference in its entirety. These systems arc designed to be induced by rapamycin or one of its analogs, referred to as "rapalogs". Examples of suitable rapamycins are provided in the documents listed above in connection with the description of the ARGENT™ system. In one embodiment, the molecule is rapamycin [e.g., marketed as Rapamune™ by Pfizer], In another embodiment, a rapalog known as AP21967 [ARIAD] is used. Examples of these dimerizer molecules include, but are not limited to rapamycin, FK506, FK1012 (a homodimer of FK506), rapamycin analogs (“rapalogs”) which are readily prepared by chemical modifications of the natural product to add a "bump" that reduces or eliminates affinity for endogenous FKBP and / or FRAP. Examples of rapalogs include, but are not limited to such as AP26113 (Ariad), AP1510 (Amara, J.F., et al., 1997, Proc Natl Acad Sci USA, 94(20): 10618-23) AP22660, AP22594, AP21370. AP22594, AP23054, AP1855, AP1856, AP1701, AP1861. AP1692 and AP1889. with designed 'bumps' that minimize interactions with endogenous FKBP. Still other rapalogs may be selected, e.g.. AP23573 [Merck], In certain embodiments, rapamycin or a suitable analog may be delivered locally or systemically to the AAV-transfected cells.

[0089] In certain embodiments, the expression cassette comprises one or more expression enhancers. In one embodiment, the expression cassette contains two or more expression enhancers. These enhancers may be the same or may differ from one another. In a further embodiment, the enhancer(s) is selected from one or more of an APB enhancer, an ABPS enhancer, an alpha mic / bik enhancer, a TTR enhancer, an en34 enhancer, an ApoE enhancer, a cytomegalovirus immediate early (CMV IE) enhancer, or an RSV enhancer. In yet another embodiment, the regulatory elements comprise an intron. In a further embodiment, the intron is selected from chicken beta actin intron (CBA), human beta globin, IVS2, SV40 (Promega), chimeric intron available from Promega®, bGH, alpha-globulin, beta-globulin, collagen, ovalbumin, or p53. See, e.g., WO 2011 / 126808. In one embodiment, the regulatory elements comprise a polyA. In a further embodiment, the polyA is a synthetic polyA or from bovine growth hormone (bGH), human growth hormone (hGH), SV40, rabbit -globin (RBG or rBG), or modified RBG (mRBG). Optionally, one or more sequences may be selected to stabilize mRNA. An example of such a sequence is a modified WPRE sequence, which may be engineered upstream of the polyA sequence and downstream of the coding sequence [see, e.g., MA Zanta- Boussif, et al. Gene Therapy (2009) 16: 605-619.

[0090] In certain embodiments, the expression cassettes may include one or more expression enhancers such as post-transcriptional regulatory element from hepatitis viruses of woodchuck (WPRE), human (HPRE), ground squirrel (GPRE) or arctic ground squirrel (AGSPRE); or a synthetic post-transcriptional regulatory element. These expression-enhancing elements are particularly advantageous when placed in a 3' UTR and can significantly increase mRNA stability and / or protein yield. In certain embodiments, the expressions cassettes provided include a regulator sequence that is a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) or a variant thereof. Suitable WPRE sequences are provided in the vector genomes described herein and are known in the art (e.g., such as those are described in US Patent Nos. 6,136,597, 6,287,814, and 7,419,829, which are incorporated by reference). In certain embodiments, the WPRE is a variant that has been mutated to eliminate expression of the woodchuck hepatitis B virus X (WHX) protein, including, for example, mutations in tire start codon of the WHX gene. See also, Kingsman S.M., Mitrophanous K., & Olsen J.C. (2005), Potential Oncogene Activity of the Woodchuck Hepatitis Post-Transcriptional Regulatory Element (Wpre)." Gene Ther. 12( 1): 3-4; and Zanta-Boussif M.A.. Charrier S.. Brice-Ouzet A., Martin S., Opolon P., Thrasher A.J., Hope T.J., & Galy A. (2009), Validation of a Mutated PreSequence Allowing High and Sustained Transgene Expression While Abrogating Whv-X Protein Synthesis: Application to the Gene Therapy of Was, Gene Ther. 16(5): 605- 19, both of which are incorporated herein by reference in its entirety. In other embodiments, enhancers are selected from a non-viral source. In certain embodiments, no WPRE sequence is present.

[0091] In certain embodiments, the expression cassette comprising hTafazzin coding sequence and may include other regulatory sequences. The regulatory sequences necessary are operably linked to the hTafazzin coding sequence in a manner which permits its transcription, translation and / or expression in target cell. In certain embodiment, the target cell may be a cardiac muscle cell and / or a skeletal muscle cell. In certain embodiments, the target cell is cardiac tissue cell. In certain embodiments, the target cell is a skeletal muscle tissue cell. In certain embodiments, the target cell is heart cell. In certain embodiments, the target cell is any other cell which expresses a hTafazzin in a subject without Barth Syndrome or a disease associated with a mutation in a TAZ gene.

[0092] In certain embodiments, the regulatory sequences comprise an enhancer, wherein the enhancer is a cytomegalovirus immediate early enhancer (CMV IE enhancer). In certain embodiments, the regulatory' sequences comprise an enhancer, wherein the enhancer is a CMV IE enhancer comprising nucleic acid of SEQ ID NO: 7. In certain embodiments, the regulatory sequences comprise a promoter, wherein the promoter is a chicken beta actin promoter. In certain embodiments, the regulatory sequences comprise a promoter, wherein the promoter is a chicken beta actin promoter comprising SEQ ID NO: 8. In certain embodiments, the regulatory sequences comprise an intron, wherein the intron is a chicken beta actin intron. In certain embodiments, the regulatory sequences comprise an intron, wherein the intron is a chicken beta actin intron comprising SEQ ID NO: 9. In certain embodiments, the regulatory sequences comprise an intron, wherein the intron is a chimeric intron comprising a chicken beta actin intron and further comprising a chicken beta-actin splicing donor (including the exon sequence, chicken beta actin intron) and rabbit beta-globin splicing acceptor). In certain embodiments, the regulatory sequences comprise an intron, wherein the intron is a chimeric intron comprising SEQ ID NO: 10. In certain embodiments, the regulatory sequences comprise a promoter which is a hybrid CB7 promoter comprising a cytomegalovirus immediate-early (CMV IE) enhancer and the chicken f>- actin promoter, optionally with spacer sequence, optionally with a chimeric intron comprising chicken beta actin intron and further comprising a chicken beta-actin splicing donor (including the exon sequence, chicken beta actin intron) and rabbit beta-globin splicing acceptor). In certain embodiments, the regulatory sequences comprise a promoter which is a hybrid CB7 promoter comprising SEQ ID NO: 11. In certain embodiments, the regulatory sequences comprise a polyadenylation (poly A) signal sequence, wherein the polyadenylation signal sequence is a rabbit beta globin (rBG) polyA signal sequence. In certain embodiments, the regulatory sequences comprise a polyA signal sequence, wherein the polyadenylation signal sequence is a rabbit beta globin polyA signal sequence comprising SEQ ID NO: 12.

[0093] In certain embodiments, the expression cassette comprises an optional enhancer, a promoter, an optional intron, at least one ORF which comprises a nucleic acid sequence encoding hTafazzin, and a polyadenylation (polyA) sequence. In certain embodiments, the expression cassette comprises nucleic sequence comprising (i) a cDNA sequence comprising SEQ ID NO: 1 encoding a human Tafazzin (hTAfazzin), and (ii) regulatory control sequences operably linked to tire sequences of (i), wherein the regulatory’ control sequences comprise an optional enhancer, a promoter, an optional intron, and a polyadenylation (polyA) signal sequence. In certain embodiments, the expression cassette comprises an enhancer, a promoter, an intron, a cDNA sequence comprising SEQ ID NO: 1 encoding hTafazzin, and a polyA signal sequence. In certain embodiments, the expression cassette comprises an optional enhancer, a cardiac promoter, an optional intron, a cDNA sequence comprising SEQ ID NO: 1 encoding hTafazzin, and a polyA signal sequence. In certain embodiments, the expression cassette comprises an enhancer, a chicken beta-actin promoter, an intron, a cDNA sequence comprising SEQ ID NO: 1 encoding hTafazzin, and a polyA signal sequence. In certain embodiments, the expression cassette comprises a cytomegalovirus immediate early (CMV IE) enhancer, a chicken beta-actin promoter, a chicken beta actin intron, a cDNA sequence comprising SEQ ID NO: 1 encoding hTafazzin, and a rabbit beta globin polyA signal sequence. In certain embodiments, the expression cassette comprises a CB7 hybrid promoter comprising a cytomegalovirus immediate- early (CMV IE) enhancer and the chicken -actin promoter, optionally with spacer sequence, optionally with a chimeric intron comprising chicken beta actin intron and further comprising a chicken beta-actin splicing donor (including the exon sequence, chicken beta actin intron) and rabbit beta-globin splicing acceptor), a cDNA sequence comprising SEQ ID NO: 1 encoding hTafazzin, and a rabbit beta globin polyA signal sequence.

[0094] In certain embodiments, the expression cassette comprises a cytomegalovirus immediate early (CMV IE) enhancer (SEQ ID NO: 7), a chicken beta-actin promoter (SEQ ID NO: 8), a chicken beta actin intron (SEQ ID NO: 9), a cDNA sequence comprising SEQ ID NO: 1 encoding hTafazzin, and a rabbit beta globin polyA signal sequence (SEQ ID NO: 12).

[0095] In certain embodiments, the expression cassette comprises a CB7 hybrid promoter (SEQ ID NO: 11) comprising a CMV IE enhancer, a chicken beta-actin promoter, and a chimeric intron comprising chicken beta actin splicing donor including chicken beta actin intron and rabbit beta globin splicing acceptor, a cDNA sequence comprising SEQ ID NO: 1 encoding hTafazzin, and a rabbit beta globin polyA signal sequence (SEQ ID NO: 12). In certain embodiments, the expression cassette comprises nucleic acid sequence of SEQ ID NO: 6 or a sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99 to at least 100% identical thereto. In certain embodiments, the expression cassette comprises nucleic acid sequence of SEQ ID NO: 6 or a sequence at least 99% identical thereto. In certain embodiments, the expression cassette comprises nucleic acid sequence of SEQ ID NO: 6.

[0096] In a further aspect, provided herein is a vector genome comprising at least one of an AAV 5' inverted terminal repeat (ITR) or an AAV 3' ITR on the vector genome at an extreme 5' end and / or an extreme 3' end of the vector genome. In certain embodiments the vector genome comprises an AAV 5' inverted terminal repeat (ITR), an expression cassette, and an AAV3' ITR. In certain embodiments, tire vector genome comprises an AAV 5' inverted terminal repeat (ITR), an expression cassette, and an AAV3' ITR, wherein the expression cassette comprises a nucleic acid sequence encoding a functional hTaffazin gene operably linked regulatory sequences which direct expression thereof in a cell. In certain embodiments, the vector genome comprises an expression cassette having a nucleic acid sequence of SEQ ID NO: 6 or a sequence at least about 90% identical to SEQ ID NO: 6. In certain embodiments, the vector genome comprises a nucleic acid molecule comprising, 5' to 3', AAV5' ITR - CB7 hybrid promoter - engineered hTaffazin coding sequence - rabbit beta globin poly A - AAV3' ITR. In certain embodiments, the vector genome comprises nucleic acid sequence of SEQ ID NO: 5. In certain embodiments, the vector genome comprises nucleic acid sequence of at least 95%, at least 96%, at least 97%, at least 98%, at least 99 to at least 100% identical to SEQ ID NO: 5.

[0097] As used herein, a “vector genome” refers to the nucleic acid sequence packaged inside a parvovirus (e.g., rAAV) capsid which forms a viral particle. Such a nucleic acid sequence contains AAV inverted terminal repeat sequences (ITRs). In the examples herein, a vector genome contains, at a minimum, from 5’ to 3‘, an AAV 5' ITR (also referred to as 5’ ITR), coding sequence(s) (i.e., transgene(s)), and an AAV 3’ ITR (also referred to as 3’ ITR). ITRs from AAV2, a different source AAV than the capsid, or other than full-length ITRs may be selected. In certain embodiments, the ITRs are from the same AAV source as the AAV which provides the rep function during production or a transcomplementing AAV. Further, other ITRs, e.g., self-complementary (scAAV) ITRs, may be used. Both single-stranded AAV and self- complementary (sc) AAV are encompassed with the rAAV. The transgene is a nucleic acid coding sequence, heterologous to the vector sequences, which encodes a polypeptide, protein, functional RNA molecule (e.g., miRNA, miRNA inhibitor) or other gene product, of interest. The nucleic acid coding sequence is operatively linked to regulatory components in a manner which permits transgene transcription, translation, and / or expression in a cell of a target tissue. Suitable components of a vector genome are discussed in more detail herein. In one example, a “vector genome” contains, at a minimum, from 5' to 3’, a vector-specific sequence, a nucleic acid sequence encoding hTafazzin operably linked to regulatory control sequences (which direct their expression in a target cell), where the vector-specific sequence may be a terminal repeat sequence which specifically packages the vector genome into a viral vector capsid or envelope protein. For example, AAV inverted terminal repeats are utilized for packaging into AAV and certain other parvovirus capsids. In certain embodiments, tire vector genome is an expression cassette having inverted terminal repeat (ITR) sequences necessary for packaging the vector genome into the AAV capsid at the extreme 5’ and 3’ end and containing therebetween a hTaffazin gene as described herein operably linked to sequences which direct expression thereof. In certain embodiments, a vector genome may comprise at a minimum from 5’ to 3', an AAV 5' ITR, coding sequence(s). and an AAV 3’ ITR. In certain embodiments, the ITRs are from AAV2. a different source AAV than the capsid, or other than full-length ITRs may be selected. In certain embodiments, the ITRs are from the same AAV source as the AAV which provides the rep function during production or a transcomplementing AAV. Further, other ITRs may be used.

[0098] The AAV sequences of the vector typically comprise the cis-acting 5' and 3' inverted terminal repeat sequences (See, e.g., B. J. Carter, in “Handbook of Parvoviruses”, ed., P. Tijsser, CRC Press, pp. 155 168 (1990)). The ITR sequences are about 145 bp in length. Preferably, substantially the entire sequences encoding the ITRs are used in the molecule, although some degree of minor modification of these sequences is permissible. The ability to modify these ITR sequences is within the skill of the art. (See, e.g., texts such as Sambrook et al, “Molecular Cloning. A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory. New York (1989); and K. Fisher et al., J. Virol., 70:520 532 (1996)). An example of such a molecule employed is a “cis- acting” plasmid containing the transgcnc, in which the selected transgcnc sequence and associated regulatory elements are flanked by the 5' and 3' AAV ITR sequences. In one embodiment, the ITRs are from an AAV different than that supplying a capsid. In one embodiment, the ITR sequences from AAV2. However. ITRs from other AAV sources may be selected. A shortened version of the 5’ ITR, termed AITR, has been described in which the D- sequence and terminal resolution site (trs) are deleted. In certain embodiments, the vector genome includes a shortened AAV2 ITR of 130 base pairs, wherein the external A elements is deleted. Without wishing to be bound by theory, it is believed that the shortened ITR reverts back to the wild-type length of 145 base pairs during vector DNA amplification using the internal (A’) element as a template. In other embodiments, full-length AAV 5’ and 3‘ ITRs are used. Where the source of the ITRs is from AAV2 and the AAV capsid is from another AAV source, the resulting vector may be termed pseudotyped. However, other configurations of these elements may be suitable. In certain embodiments, the provided herein is rAAV comprising a nucleic acid molecule comprising a vector genome comprising at least one AAV ITR at the extreme 5' and / or extreme 3' end of tire nucleic acid molecule which is the vector genome and an expression cassette. In certain embodiments, the vector genome is a nucleic acid molecule which comprises a 5' - AAV ITR, the expression cassette and a 3' - AAV ITR. In certain embodiments, wherein the vector genome comprises a nucleic acid sequence encoding a gene product operably linked to regulatory sequences which enables expression of transgene in a target cell, (e.g., by directing transcription, translation and / or expression).

[0099] In certain embodiments, provided herein is an rAAV comprising an AAV capsid and a nucleic acid molecule comprising expression cassette. In certain embodiments, tire rAAV comprises vector genome comprising a nucleic acid molecule comprising. 5' to 3'. AAV- 5' ITR - an optional enhancer - a promoter - an optional intron - coding sequence (e.g., test transgene) - polyadenylation (polyA) signal sequence - AAV3' - ITR. In other embodiments, the orientation of the ITRs may change from the orientation presented in the vector genome of the nucleic acid used in production (e.g., a plasmid). Thus, in certain embodiments, the rAAV may comprise a vector genome flanked by 3' and 5' AAV ITRs, respectively. In certain embodiments, the rAAV may comprise a vector genome flanked by two 5' AAV ITRs. In certain embodiments, the rAAV may comprise a vector genome flanked by two 3' AAV ITRs. In other embodiments, an rAAV as provided herein may be partially truncated such that the 5' AAV ITR and / or the 3' AAV ITR is not detectable in the vector genome packaged in a final rAAV product.

[0100] It should be understood that the compositions in the expression cassette and vector genomes described herein are intended to be applied to other compositions, regiments, aspects, embodiments and methods described across the Specification.

[0101] Vector

[0102] In one aspect, provided herein is a vector comprising an engineered open reading frame (ORF) for human Tafazzin (hTafazzin), wherein the ORF has a hTafazzin coding sequence, which is a nucleic acid sequence encoding a functional hTafazzin, wherein ORF is operably linked to regulatory control sequences which direct the expression of the hTafazzin in a cell, wherein the hTafazzin coding sequence comprises nucleic acid sequence of SEQ ID NO: 1, which encode amino acid sequence of SEQ ID NO: 2, and wherein the regulatory control sequences include an optional enhancer, a promoter an optional intron.

[0103] In certain embodiments, the vector comprises hTafazzin coding sequence comprising a nucleic acid sequence comprising cDNA sequence comprising SEQ ID NO: 1 or a sequence at least 99% to 100% identical to SEQ ID NO: 1 and which expresses the functional hTafazzin protein. In certain embodiments, the vector comprises hTafazzin coding sequence comprising a nucleic acid sequence comprising cDNA sequence comprising SEQ ID NO: 1 which encodes an amino acid sequence of SEQ ID NO: 2.

[0104] A " vector’ as used herein is a biological or chemical moiety comprising a nucleic acid sequence which can be introduced into an appropriate target cell for replication or expression of said nucleic acid sequence. Examples of a vector includes but not limited to a recombinant virus, a plasmid, Lipoplexes, a Polymersome, Polyplexes, a dendrimer, a cell penetrating peptide (CPP) conjugate, a magnetic particle, or a nanoparticle. In one embodiment, a vector is a nucleic acid molecule into which an exogenous or heterologous or engineered nucleic acid encoding a functional hTafazzin may be inserted, which can then be introduced into an appropriate target cell. Such vectors preferably have one or more origin of replication, and one or more site into which the recombinant DNA can be inserted. Vectors often have means by which cells with vectors can be selected from those without, e.g., they encode drug resistance genes. Common vectors include plasmids, viral genomes, and "artificial chromosomes". Conventional methods of generation, production, characterization or quantification of the vectors are available to one of skill in the art.

[0105] In one embodiment, the vector is a non-viral plasmid that comprises an expression cassette described thereof, e.g., "‘naked DNA”, ""naked plasmid DNA”; coupled with various compositions and nano particles, including, e.g., micelles, liposomes, cationic lipid - nucleic acid compositions, poly-glycan compositions and other polymers, lipid and / or cholesterol-based - nucleic acid conjugates, and other constructs such as are described herein. See, e.g., X. Su, et al, Mol. Pharmaceutics, 2011, 8 (3), pp 774-787; web publication: March 21, 2011; WO2013 / 182683, WO 2010 / 053572 and WO 2012 / 170930, all of which arc incorporated herein by reference.

[0106] In certain embodiments, the vector described herein is a “replication-defective virus" or a “viral vector” which refers to a synthetic or artificial viral particle in which an expression cassette containing a nucleic acid sequence encoding hTafazzin is packaged in a viral capsid or envelope, where any viral genomic sequences also packaged within the viral capsid or envelope are replication-deficient; i.e., they cannot generate progeny virions but retain the ability to infect target cells. In one embodiment, the genome of tire viral vector does not include genes encoding the enzymes required to replicate (the genome can be engineered to be "gutless" - containing only the nucleic acid sequence encoding hTafazzin flanked by the signals required for amplification and packaging of the artificial genome), but these genes may be supplied during production. Therefore, it is deemed safe for use in gene therapy since replication and infection by progeny virions cannot occur except in the presence of the viral enzyme required for replication. As used herein, a recombinant virus vector is an adeno-associated virus (AAV), an adenovirus, a bocavirus, a hybrid AAV / bocavirus, a herpes simplex virus or a lentivirus.

[0107] As used herein, the term ‘“host cell” may refer to the packaging cell line in which a vector (e.g., a recombinant AAV) is produced. A host cell may be a prokaryotic or eukaryotic cell (e.g., human, insect, or yeast) that contains exogenous or heterologous DNA that has been introduced into the cell by any means, e.g., electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, transfection, liposome delivery, membrane fusion techniques, high velocity' DNA-coated pellets, viral infection and protoplast fusion. Examples of host cells may include, but are not limited to an isolated cell, a cell culture, an Escherichia coli cell, a yeast cell, a human cell, a non-human cell, a mammalian cell, a non-mammalian cell, an insect cell, an HEK-293 cell, a liver cell, a kidney cell, a cell of the central nervous system, a heart cell, or a stem cell.

[0108] It should be understood that the compositions in the vector described herein are intended to be applied to other compositions, regiments, aspects, embodiments and methods described across the Specification.

[0109] Recombinant Adeno-associated Virus (rAAV)

[0110] Provided herein is a recombinant adeno-associated virus (rAAV) useful for treating Barth Syndrome or a disease associated with a mutation in a Tafazzin gene (TAZ), e.g., such as caused by a complete or partial loss-of-function mutation. The rAAV comprises (a) an AAV capsid; and (b) a vector genome packaged in the AAV capsid of (a). Suitably, the AAV capsid selected targets the cells to be treated. In certain embodiments, tire capsid is from Clade F. However, in certain embodiments, another AAV capsid source may be selected, i.e., Clade A. In certain embodiments, tire AAV capsid is AAVhu68 capsid. In certain embodiments, the AAV capsid is AAVhu95 capsid. In certain embodiments, the AAV capsid is AAVhu96 capsid. In certain embodiments, the AAV capsid is an AAV9 capsid. In certain embodiments, the AAV capsid is a mutant AAV9 capsid. The vector genome comprises an AAV 5’ (also referenced to as AAV 5') inverted terminal repeat (ITR), an engineered nucleic acid sequence encoding a hTafazzin as described herein, a regulatory' sequence which direct expression of hTafazzin in a target cell, and an AAV 3’ (also referenced to as AAV 3') ITR.

[0111] In one aspect, the rAAV.hTafazzin is for use in the treatment of Barth Syndrome. In certain embodiments, the rAAV.hTafazzin is for tire use in treatment of a disease associated with a defect (e.g.. a gene mutation) in Tafazzin (TAZ). In certain embodiments, the rAAV.hTafazzin is for the use in treatment of dilated cardiomyopathy (DCM). In certain embodiments, the rAAV.hTafazzin is for the use in treatment of hypertrophic DCM. In certain embodiments, the rAAV.hTafazzin is for the use in treatment of endocardial fibroelastosis. In certain embodiments, the rAAV.hTafazzin is for tire use in treatment of left ventricular noncompaction (LVNC). In certain embodiments, the rAAV.hTafazzin is for tire use in treatment familial dilated cardiomyopathy associated with a mutation in TAZ gene. In certain embodiments, the rAAV.hTafazzin is for the use in treatment of isolated noncompaction of left ventricular myocardium.

[0112] In certain embodiments, the rAAV comprises an adeno-associated virus (AAV) capsid and a nucleic acid molecule comprising an expression cassette, wherein the expression cassette is a nucleic sequence comprising: (i) a cDNA sequence comprising SEQ ID NO: 1 encoding a human Tafazzin (hTAfazzin), and (ii) regulatory control sequences operably linked to the sequences of (i), wherein the regulatory control sequences comprise an optional enhancer, a promoter, an optional intron, and a polyadenylation (polyA) signal sequence (rAAV.hTafazzin).

[0113] In certain embodiments, tire rAAV comprises a nucleic acid molecule comprising a vector genome comprising at least one AAV ITR at the extreme 5' and / or extreme 3' end of the nucleic acid molecule which is the vector genome and an expression cassette. In certain embodiments, the vector genome is a nucleic acid molecule which comprises a 5' - AAV ITR, the expression cassette and a 3' - AAV ITR. In certain embodiments, the rAAV comprises an expression cassette comprising an engineered nucleic acid sequence comprising open reading frame (ORF) for a functional hTafazzin coding sequence which encode functional hTafazzin, wherein the ORF is operably linked to regulatory control sequences which direct expression of the functional hTafazzin protein in a cell, and wherein the regulatory control sequences comprise an optional enhancer, a promoter, an optional intron, and a polyadenylation (polyA) sequences. In certain embodiments, the rAAV comprises the vector genome comprising an expression cassette having a nucleic acid sequence of SEQ ID NO: 6 or a sequence at least about 90% identical to SEQ ID NO: 6.

[0114] In certain embodiments, the rAAV comprises vector genome comprising a nucleic acid molecule comprising, 5’ to 3’, AAV- 5’ ITR (also referred to as 5' AAV ITRs or 5’ AAV ITRs) - an optional enhancer - a promoter - an optional intron - engineered hTafazzin coding sequence - polyadenylation (polyA) signal sequence - AAV3’ - ITR (also referred to as 3' AAV ITRs or 3’ AAV ITRs). In other embodiments, the orientation of the ITRs may change from the orientation presented in the vector genome of the nucleic acid used in production (e g., a plasmid). Thus, in certain embodiments, the rAAV may comprise a vector genome flanked by 3’ and 5' AAV ITRs, respectively. In certain embodiments, the rAAV may comprise a vector genome flanked by two 5' AAV ITRs. In certain embodiments, the rAAV may comprise a vector genome flanked by two 3' AAV ITRs. In other embodiments, an rAAV as provided herein may be partially truncated such that the 5' AAV ITR and / or the 3' AAV ITR is not detectable in the final rAAV product.

[0115] In certain embodiments, the rAAV comprises vector genome comprising a nucleic acid molecule comprising an enhancer - a promoter - an optional intron - engineered hTafazzin coding sequence -polyA signal sequence. In certain embodiments, tire rAAV comprises vector genome comprising a nucleic acid molecule comprising an enhancer - a cardiac promoter - an optional intron - engineered hTafazzin coding sequence -polyA signal sequence. In certain embodiments, the rAAV comprises vector genome comprising a nucleic acid molecule comprising an enhancer - a promoter - an intron - engineered hTafazzin coding sequence - polyA signal sequence. In certain embodiments, the rAAV comprises vector genome comprising a nucleic acid molecule comprising a cytomegalovirus immediate early (CMV IE) enhancer - a chicken beta actin promoter - a chicken beta actin intron - engineered hTafazzin coding sequence - rabbit beta globin polyA signal sequence. In certain embodiments, the rAAV comprises vector genome comprising a nucleic acid molecule comprising a cytomegalovirus immediate early (CMV IE) enhancer - a chicken beta actin promoter - a chimeric intron comprising chicken beta actin splicing donor including chicken beta actin intron and rabbit beta globin splicing acceptor - engineered hTafazzin coding sequence - rabbit beta globin polyA signal sequence. In certain embodiments, tire rAAV comprises vector genome comprising a nucleic acid molecule comprising a CB7 hybrid promoter - engineered hTafazzin coding sequence - rabbit beta globin polyA signal sequence. In certain embodiments, the rAAV comprises a vector genome comprising nucleic acid molecule comprising nucleic acid sequence of SEQ ID NO: 6 or a sequence of at least 95%, at least 96%, at least 97%, at least 98%, at least 99 to at least 100% identical to SEQ ID NO: 6 (rAAV. CB7.CI.TAZ.RBG). In certain embodiments, the rAAV comprises a vector genome comprising nucleic acid sequence of SEQ ID NO: 5 or a sequence of at least 95%, at least 96%, at least 97%, at least 98%, at least 99 to at least 100% identical to SEQ ID NO: 5 (rAAV. CB7.CI.TAZ.RBG).

[0116] In certain embodiments, the rAAV comprises vector genome comprising a nucleic acid molecule comprising, 5’ to 3’, an AAV ITR - an enhancer - a promoter - an optional intron - engineered hTafazzin coding sequence -polyA signal sequence - AAV ITR. In certain embodiments, the rAAV comprises vector genome comprising a nucleic acid molecule comprising, 5’ to 3', AAV ITR - an enhancer - a cardiac promoter - an optional intron - engineered hTafazzin coding sequence -polyA signal sequence - AAV ITR. In certain embodiments, tire rAAV comprises vector genome comprising a nucleic acid molecule comprising, 5’ to 3’, AAV ITR - an enhancer - a promoter - an intron - engineered hTafazzin coding sequence -polyA signal sequence - AAV ITR. In certain embodiments, the rAAV comprises vector genome comprising a nucleic acid molecule comprising, 5’ to 3’, AAV ITR - a cytomegalovirus immediate early (CMV IE) enhancer - a chicken beta actin promoter - a chicken beta actin intron - engineered hTafazzin coding sequence - rabbit beta globin polyA signal sequence - AAV ITR. In certain embodiments, the rAAV comprises vector genome comprising a nucleic acid molecule comprising, 5’ to 3’, AAV ITR - a cytomegalovirus immediate early (CMV IE) enhancer - a chicken beta actin promoter - a chimeric intron comprising chicken beta actin splicing donor including chicken beta actin intron and rabbit beta globin splicing acceptor - engineered hTafazzin coding sequence - rabbit beta globin polyA signal sequence - AAV ITR. In certain embodiments, the rAAV comprises vector genome comprising a nucleic acid molecule comprising, 5’ to 3', AAV ITR - a CB7 hybrid promoter - engineered hTafazzin coding sequence - rabbit beta globin polyA signal sequence - AAV ITR. In certain embodiments, the rAAV comprises a vector genome comprising nucleic acid sequence of SEQ ID NO: 5 or a sequence of at least 95%, at least 96%, at least 97%, at least 98%, at least 99 to at least 100% identical to SEQ ID NO: 5 (rAAV. CB7.CI.TAZ.RBG).

[0117] In certain embodiments, tire AAV capsid for the compositions and methods described herein is chosen based on the target cell. In certain embodiments, the AAV capsid transduces a muscle cell. In certain embodiment, the AAV capsid transduces a heart cell. In certain embodiments, the AAV capsid transduces a skeletal muscle cell. In certain embodiments, other AAV capsid may be chosen.

[0118] In certain embodiments, the Clade F AAV capsid is an AAVhu68 capsid [See, e.g., US2020 / 0056159; PCT / US21 / 55436; SEQ ID NO: 13 and 14 for nucleic acid sequence; SEQ ID NO: 15 for amino acid sequence], an AAVhu95 capsid [See, e.g., US Provisional Application No. 63 / 251,599, filed October 2, 2201, International Patent Application No. PCT / US2022 / 077315, filed September 30, 2022; SEQ ID NOs: 16 and 17 (hu95 nucleic acid sequence)] and SEQ ID NO: 18 (hu95 amino acid sequence), or an AAVhu96 capsid [See. e g., US Provisional Application No. 63 / 251,599, filed October 2, 2201, and International Patent Application No. PCT / US2022 / 077315, filed September 30, 2022; SEQ ID NOs: 19 and 20 (hu96 nucleic acid sequence) and SEQ ID NO: 21 (hu96 amino acid sequence)], AAV9 capsid (SEQ ID NO: 22 for nucleic acid sequence; SEQ ID NO: 23 for amino acid sequence) [See, e.g., US 7,906,111] or engineered mutants and variants thereof [see, e.g., W02020 / 200499; W02003 / 054197], See also, International Patent Application No. PCT / US2022 / 077315, filed September 30, 2022, which is incorporated herein by reference in its entirety. See also, US Provisional Patent Application No. 63 / 387.941, filed December 17. 2022. and US Provisional Patent Application No. 63 / 514,404 filed July 19, 2023, which are incorporated herein by reference in its entirety. In certain embodiments, the AAV capsid is a mutant Clade F capsid. In one embodiment, the mutant capsid is a mutant AAV9 capsid. In another embodiment, the mutant capsid is a mutant AAVhu68 capsid. In certain embodiments, the AAV capsid is a mutant Clade F capsid comprising embedded ”RGD“ motif (i.e., XXXRGDX). Examples of suitable mutant capsids are provided, e.g., in International Patent Application No. PCT / US2023 / 084192, filed December 15, 2023, now published WO 2024 / 130067A2, International Patent Application No. PCT / US2024 / 0559I0 filed November 14, 2024, now published WO 2025 / 106661 Al, which are all incorporated by reference herein in their entireties. US Provisional Patent Application No. 63 / 785,501, filed April 11, 2025, and US Provisional Patent Application No. 63 / 803,163, filed May 9, 2025 are incorporated herein by reference. Examples of other suitable mutant capsids are provided, e.g., WO 2024 / 173802 A2, filed February 16. 2024. WO 2023 / 230409, filed May 11, 2023, WO 2023 / 196967 Al, filed April 7, 2023, US 2023 / 039476, filed March 29, 2022, WO 2023 / 039476, filed September 8, 2022, WO 2024040193 A2, filed Aug 16, 2023, which are all incorporated herein by reference.

[0119] In certain embodiments, the exogenous targeting peptide, wherein tire exogenous targeting peptide is “Xn - n-mer - Xm”, wherein the Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, wherein the n-mer is AVIRGDV (SEQ ID NO: 25), IIRGDPA (SEQ ID NO: 24), RGDYREV (SEQ ID NO: 42), RGDYHQV (SEQ ID NO: 44), RGDLHGY (SEQ ID NO: 27), PYQRGDH (SEQ ID NO: 29), or a n-mer sequence of at least 6. at least 7, or full length consecutive amino acids of any one of the n-mers, and wherein Xm is 0, 1 , 2, or 3 amino acid residues independently selected from any amino acid. In certain embodiments, a mutant AAV9 capsid comprises exogenous peptide with linkers of various length (s) which is inserted (and / or engineered) in a suitable location of HVRVIII region of amino acid 588 and 589 (Q-A), based on the residue position in the VP1 of an AAV9 capsid (e.g., SEQ ID NO: 23). In certain embodiments, a mutant AAV9 capsid comprises the exogenous targeting peptide which comprises “n - AVIRGDV (SEQ ID NO: 25) - Xm”. wherein tire Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, wherein the Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein the exogenous targeting peptide provide significant transduction advantages in the muscle cell, including the cardiac muscle cell and / or skeletal muscle cell, as compared to a AAV9 capsid. In certain embodiments, a mutant AAV9 capsid comprises the exogenous targeting peptide which comprises “n - IIRGDPA (SEQ ID NO: 24) - Xm”. wherein tire Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, wherein the Xm is 0, 1, 2. or 3 amino acid residues independently selected from any amino acid, wherein the exogenous targeting peptide provide significant transduction advantages in the muscle cell, including the cardiac muscle cell and / or skeletal muscle cell, as compared to a AAV9 capsid. In certain embodiments, a mutant AAV9 capsid comprises the exogenous targeting peptide which comprises “n - RGDYREV (SEQ ID NO: 42) - Xm”, wherein tire Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, wherein the Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein the exogenous targeting peptide provide significant transduction advantages in the muscle cell, including the cardiac muscle cell and / or skeletal muscle cell, as compared to a AAV9 capsid. In certain embodiments, a mutant AAV9 capsid comprises the exogenous targeting peptide which comprises “n - RGDYHQV (SEQ ID NO: 44) - Xm”, wherein the Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, wherein the Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein the exogenous targeting peptide provide significant transduction advantages in the muscle cell, including the cardiac muscle cell and / or skeletal muscle cell, as compared to a AAV9 capsid. In certain embodiments, a mutant AAV9 capsid comprises the exogenous targeting peptide which comprises “n - RGDLHGY (SEQ ID NO: 27) - Xm”, wherein the Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, wherein the Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein the exogenous targeting peptide provide significant transduction advantages in the muscle cell, including the cardiac muscle cell and / or skeletal muscle cell, as compared to a AAV9 capsid. In certain embodiments, a mutant AAV 9 capsid comprises tire exogenous targeting peptide which comprises “n - PYQRGDH (SEQ ID NO: 29) - Xm”, wherein the Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, wherein the Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein the exogenous targeting peptide provide significant transduction advantages in the muscle cell, including tire cardiac muscle cell and / or skeletal muscle cell, as compared to a AAV9 capsid.

[0120] In certain embodiments, the mutant capsids described herein are characterized by having a deamidation pattern similar to their parental AAV, e.g., such as described in US 2020 / 0056159, published Feb 20, 2020 (AAVhu68; highly deamidated in N57, N329, N452 and N512), with minor optional amounts of deamidation); US 2020 / 0407750, published Dec 31, 2020 (AAV9, highly deamidated in N57, N329, N452 and N512), each of which is incorporated herein by reference.

[0121] In certain embodiments, the mutant AAV capsid comprises an exogenous targeting peptide which is immediately preceded by flanking amino acids which are mutated, as compared to parental AAV capsid (e.g., AAV9 capsid). In certain embodiments, the mutated flanking amino acids, together with 1, 2, 3, 4, 5, or 6 inserted amino acids comprise the exogenous targeting peptide. In certain embodiments, the entirety of the exogenous targeting peptide is inserted into the parental AAV capsid. In still other embodiments, the sequence inserted into a capsid may comprise all or a fragment of tire exogenous targeting peptide at the carboxy (COO-) or amino terminus (N-) (i.e., via insertion of the 5' or 3' coding sequences therefor) and further comprises 0-3 flanking amino acid residues as provided in the above formulae. In certain embodiments, engineered rAAV capsids comprising the targeting peptides, as provided herein, demonstrate reduced transduction (i.e., de-targeted / ing) to liver as compared to its parental capsid (e.g., AAV9 or another clade F capsid (e.g., AAVhu68)).

[0122] In certain embodiments, tire rAAV comprises a mutant AAV9 capsid wherein the mutant AAV9 capsid protein comprises exogenous peptide which is immediately preceded by “AQ” (e.g., “AQ-IIRGDPA (SEQ ID NO: 24)”, “AQ-IIRGDPA (SEQ ID NO: 24)-AQ”, or “IIRGDPA (SEQ ID NO: 24)-AQ”. etc.). In certain embodiments, the rAAV comprises a mutant AAV9 capsid wherein the mutant AAV9 capsid protein comprises exogenous peptide which is immediately preceded by the native residues of the AAV9 which may be unmodified at the amino (N-) terminus, and / or at the carboxy (COO-) terminus. In certain embodiments, the rAAV comprises a mutant AAV9 capsid wherein the mutant AAV9 capsid protein comprises exogenous peptide which is immediately preceded by the native residues of tire AAV9 which may be mutated at the amino (N-) terminus, and / or at the carboxy (COO-) terminus. In certain embodiments, the rAAV comprises a mutant AAV9 capsid wherein the mutant AAV9 capsid protein comprises exogenous peptide wherein the exogenous peptide is flanked by: (i) “SAQ” at amino (N-) terminus of the exogenous peptide, (ii) “AQA” at carboxy (COO-) terminus. In certain embodiments, the rAAV comprises a mutant AAV9 capsid wherein the mutant AAV9 capsid protein comprises exogenous peptide wherein the exogenous peptide is flanked by mutated trimcr “ENT” at amino (N-) terminus of tire exogenous peptide. In certain embodiments, tire rAAV comprises a mutant AAV9 capsid wherein the AAV9 capsid protein comprises exogenous peptide wherein the exogenous peptide is flanked by mutated trimer “SHQ”. SWQ”. SAI”, “GAQ”, “FAQ”, “QAQ”. “AAQ”, “SGQ”, “SGM” at amino (N-) terminus of the exogenous peptide. In certain embodiments, the rAAV comprises a mutant AAV9 capsid wherein the mutant AAV9 capsid protein comprises exogenous peptide wherein the exogenous peptide is flanked by mutated trimer “QQA”, “NQA”, “AMA”, “AQC”, “GQA”, “ARA”, “GRA” at carboxy (COO-) tenninus.

[0123] In certain embodiments, the AAV capsid is a non-clade F capsid, for example a Clade A, B, C, D, or E capsid. In certain embodiment, the non-Clade F capsid is an AAV 1 or a variation thereof. In certain embodiment, the AAV capsid transduces a target cell other than the heart cells. In certain embodiments, the AAV capsid is a Clade A capsid (e.g.. AAV 1, AAV6, AAVrh91), a Clade B capsid (e.g., AAV 2), a Clade C capsid (e.g., hu53), a Clade D capsid (e.g., AAV7), or a Clade E capsid (e.g., rhlO).

[0124] As used herein, the term ‘“clade” as it relates to groups of AAV refers to a group of AAV which are phylogenetically related to one another as determined using a Neighbor-Joining algorithm by a bootstrap value of at least 75% (of at least 1000 replicates) and a Poisson correction distance measurement of no more than 0.05, based on alignment of the AAV vpl amino acid sequence. The Neighbor-Joining algorithm has been described in the literature. See, e.g., M. Nei and S. Kumar, Molecular Evolution and Phylogenetics (Oxford University Press, New York (2000). Computer programs are available that can be used to implement this algorithm. For example, the MEGA v2. 1 program implements the modified Nei-Gojobori method. Using these techniques and computer programs, and the sequence of an AAV vpl capsid protein, one of skill in the art can readily determine whether a selected AAV is contained in one of the clades identified herein, in another clade, or is outside these clades. See, e.g., G Gao, et al, J Virol, 2004 Jun; 78(10): 6381-6388, which identifies Clades A, B, C, D, E and F, and provides nucleic acid sequences of novel AAV, GenBank Accession Numbers AY530553 to AY530629. See, also, WO 2005 / 033321.

[0125] A rAAV is composed of an AAV capsid and a vector genome. An AAV capsid is an assembly of a heterogeneous population of vpl. a heterogeneous population of vp2, and a heterogeneous population of vp3 proteins. As used herein when used to refer to vp capsid proteins, the term “heterogeneous” or any grammatical variation thereof, refers to a population consisting of elements that are not the same, for example, having vpl, vp2 or vp3 monomers (proteins) with different modified amino acid sequences.

[0126] As used herein when used to refer to vp capsid proteins, tire term “heterogeneous” or any grammatical variation thereof, refers to a population consisting of elements tlrat are not tire same, for example, having vpl, vp2 or vp3 (also referenced as VP1, VP2, VP3, or Vpl, Vp2, Vp3) monomers (proteins) with different modified amino acid sequences. The term “heterogeneous population” as used in connection with vpl, vp2 and vp3 proteins (alternatively termed isoforms), refers to differences in the amino acid sequence of the vpl, vp2 and vp3 proteins within a capsid. The AAV capsid contains subpopulations within the vpl proteins, within the vp2 proteins and within the vp3 proteins which have modifications from the predicted amino acid residues. These subpopulations include, at a minimum, certain deamidated asparagine (N or Asn) residues. For example, certain subpopulations comprise at least one, two, three or four highly deamidated asparagines (N) positions in asparagine - glycine pairs and optionally further comprising other deamidated amino acids, wherein the deamidation results in an amino acid change and other optional modifications. In certain embodiments, AAV capsids are provided which have a heterogeneous population of AAV capsid isoforms (i.e., VP1, VP2, VP3) which contain multiple highly deamidated “NG” positions. In certain embodiments, the highly deamidated positions are in the locations identified below, with reference to the predicted full-length VP1 amino acid sequence. In other embodiments, the capsid gene is modified such that the referenced “NG” is ablated and a mutant “NG” is engineered into another position. In certain embodiments, certain subpopulations comprise three or four highly deamidated asparagines in positions: N57, N329, N452 and N512, and optional deamidation in other positions within the capsid sequence. In certain embodiments, the VP1, VP2 VP3 proteins are a heterogenous population having deamidation in about 50% to about 100% of positions N57 (VP1 only), N329, N452, and / or N512, based on the AAV9 capsid residue positions. In certain embodiments, the capsid comprises VP proteins which are highly deamidated in all of these positions. In certain embodiments, the percentage of deamidation in one or more of these highly deamidated positions is over 50%, over 55%, over 60%, over 65%, over 70%, over 75%, over 80%, over 85%, over 90%, over 95%, or about 70% to about 100%, or values therebetween.

[0127] As used herein, the terms “target cell” and “target tissue” can refer to any cell or tissue which is intended to be transduced by the subject AAV vector or in which expression of hTAfazzin is desired. The term may refer to any one or more of muscle, liver, lung, airway epithelium, central nervous system, neurons, eye (ocular cells), or heart. In certain embodiments, the term “target cell” is intended to reference the cells of the subject being treated for Barth Syndrome or a disease associated with a mutation in a TAZ gene. In certain embodiments, the vector is delivered to a target cell ex vivo. In certain embodiments, the vector is delivered to the target cell in vivo. In certain embodiments, the target cell is a muscle cell. In certain embodiments, the target cell is a skeletal muscle cell. In certain embodiments, the target cells is heart cell.

[0128] Additionally, provided herein, is an rAAV production system useful for producing a rAAV as described herein. The production system comprises a cell culture comprising (a) a nucleic acid sequence encoding an AAV capsid protein; (b) the vector genome; and (c) sufficient AAV rep functions and helper functions to permit packaging of the vector genome into the AAV capsid. In certain embodiments, the vector genome is SEQ ID NO: 5. In certain embodiments, the cell culture is a human embryonic kidney 293 cell culture. In certain embodiments, the AAV rep is from a different AAV. In certain embodiments, wherein the AAV rep is from AAV2. In certain embodiments, the AAV rep coding sequence and cap genes are on the same nucleic acid molecule, wherein there is optionally a spacer between the rep sequence and cap gene. For use in producing an AAV viral vector (e.g., a recombinant (r) AAV), the vector genomes can be carried on any suitable vector, e.g., a plasmid, which is delivered to a packaging host cell in culture or in suspension. The plasmids useful in this invention may be engineered such that they are suitable for replication and packaging in vitro in prokaryotic cells, insect cells, mammalian cells, among others. Suitable transfection techniques and packaging host cells are known and / or can be readily designed by one of skill in the art.

[0129] In certain embodiments, a plasmid useful in producing an rAAV particle is provided which comprises a vector genome comprising a AAV 5' ITR, an expression cassette, and a AAV3' ITR, wherein expression cassette is a nucleic sequence comprising :(i) a cDNA sequence comprising SEQ ID NO: 1 encoding a human Tafazzin (hTAfazzin), and (ii) regulatory control sequences operably linked to the sequences of (i), wherein the regulatory control sequences comprise an optional enhancer, a promoter, an optional intron, and a polyadenylation (poly A) signal sequence. In certain embodiments, a nucleic acid molecule (e g., a plasmid) useful in rAAV production comprises a vector genome comprising a AAV5’ ITR, a CB7 hybrid promoter, a hTafazzin coding sequence, a rabbit beta globin polyA sequence, and a AAV3’ ITR. In certain embodiments, a nucleic acid (e.g., a plasmid) useful in rAAV production comprises a vector genome comprising nucleic acid sequence of SEQ ID NO: 5.

[0130] As used herein, a " vector genome” refers to the nucleic acid sequence packaged inside the rAAV capsid which forms a viral particle. Such a nucleic acid sequence contains AAV inverted terminal repeat sequences (ITRs). In the examples herein, a vector genome contains, at a minimum, from 5’ to 3’, an AAV 5’ ITR, expression cassette comprising coding sequence(s) (i.e., transgene(s)), and an AAV 3’ ITR. In certain embodiments, the ITRs are from AAV2, a different source AAV than the capsid, or other than full-length ITRs may be selected. In certain embodiments, the ITRs are from the same AAV source as the AAV which provides the rep function during production or a trans-complementing AAV. Further, other ITRs. e.g., self- complementary (scAAV) ITRs. may be used. Both single-stranded AAV and self-complementary (sc) AAV are encompassed with the rAAV. The transgene is a nucleic acid coding sequence, heterologous to the vector sequences, which encodes a polypeptide, protein, functional RNA molecule (e.g., miRNA, rniRNA inhibitor) or other gene product, of interest. The nucleic acid coding sequence is operatively linked to regulatory components in a manner which permits transgene transcription, translation, and / or expression in a cell of a target tissue. Suitable components of a vector genome are discussed in more detail herein. In one example, a “vector genome"’ contains, at a minimum, from 5’ to 3’. a vector-specific sequence, a nucleic acid sequence encoding protein of interest operably linked to regulatory control sequences (which direct their expression in a target cell), where the vector-specific sequence may be a terminal repeat sequence which specifically packages the vector genome into a viral vector capsid or envelope protein. For example, AAV inverted terminal repeats are utilized for packaging into AAV and certain other parvovirus capsids.

[0131] In certain embodiments, non-viral genetic elements used in manufacture of a rAAV, will be referred to as vectors (e.g., production vectors). In certain embodiments, these vectors are plasmids, but the use of other suitable genetic elements is contemplated. Such production plasmids may encode sequences expressed during rAAV production, e.g., AAV capsid or rep proteins required for production of a rAAV, which are not packaged into the rAAV. Alternatively, such a production plasmid may carry the vector genome which is packaged into the rAAV.

[0132] Methods for generating and isolating AAVs suitable for use as vectors are known in the art. See generally, e.g., Grieger & Samulski, 2005, Adeno-associated virus as a gene therapy vector: Vector development, production and clinical applications, Adv. Biochem. Engin / Biotechnol. 99: 119-145; Buning et al., 2008, Recent developments in adeno-associated virus vector technology, J. Gene Med. 10:717-733; and the references cited below7, each of w hich is incorporated herein by reference in its entirety. As used herein, a gene therapy vector refers to a rAAV as described herein, which is suitable for use in treating a patient. For packaging a gene into virions, the ITRs are the only AAV components required in cis in the same construct as the nucleic acid molecule containing the gene. The cap and rep genes can be supplied in trans.

[0133] In one embodiment, the expression cassettes described herein are engineered into a genetic element (e.g., a shuttle plasmid) which transfers the immunoglobulin construct sequences carried thereon into a packaging host cell for production of a viral vector. In one embodiment, the selected genetic element may be delivered to an AAV packaging cell by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection and protoplast fusion. Stable AAV packaging cells can also be made. The methods used to make such constructs are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Molecular Cloning: A Laboratory Manual, ed. Green and Sambrook, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).

[0134] The term ‘‘AAV intermediate” or “AAV vector intermediate” refers to an assembled rAAV capsid which lacks the desired genomic sequences packaged therein. These may also be termed an “empty” capsid. Such a capsid may contain no detectable genomic sequences of an expression cassette, or only partially packaged genomic sequences which are insufficient to achieve expression of the gene product. These empty capsids are non-functional to transfer the gene of interest to a host cell. The recombinant adeno-associated virus (AAV) described herein may be generated using techniques which are known. See, e.g., WO 2003 / 042397; WO 2005 / 033321, WO 2006 / 110689; US 7588772 B2. Such a method involves culturing a host cell which contains a nucleic acid sequence encoding an AAV capsid protein; a functional rep gene; an expression cassette composed of, at a minimum, AAV inverted terminal repeats (ITRs) and a transgene; and sufficient helper functions to permit packaging of the expression cassette into the AAV capsid protein. Methods of generating the capsid, coding sequences therefor, and methods for production of rAAV viral vectors have been described. See, e.g., Gao, et al, Proc. Natl. Acad. Sci. U.S.A. 100 (10), 6081-6086 (2003) and US 2013 / 0045186A1.

[0135] In certain embodiment, the rAAV are generated (manufactured) using triple transfection techniques. In certain embodiments the rAAV are generated using a stable mammalian cell line. In certain embodiments, the stable cell line comprises one or more of: (a) a first plurality of polynucleotide molecules which comprise a coding sequence for at least one adeno-associated virus (AAV) replicase (Rep) protein necessary for production of a replication-defective rAAV vector (Rep52 and Rep78), wherein said rep proteins coding sequences are operably linked to a doxycycline-inducible promoter which directs expression of the rep proteins in the cell line; (b) at least a second plurality of polynucleotide molecules each encoding adenovirus (Ad) helper proteins necessary for production of a replication-defective rAAV vector comprising at least an Ad E2A DNA Binding Protein (DBP) coding sequence, and Ad E4ORF6 coding sequence, wherein the Ad E2A DBP coding sequences and the Ad E4ORF6 coding sequences are operably linked to a doxycycline-inducible promoter which direct expression of the Ad helper proteins in the cell line; (c) a nucleic acid molecule comprising an Ad El coding sequence operably linked to a constitutive promoter which directs expression of the Ad El in the cell line; (d) at least a third plurality of nucleic acid molecules each of which comprises an AAV VP1 coding sequence which encodes AAV VP 1 proteins, AAV VP2 proteins and AAV VP3 proteins which self-assemble to form an AAV capsid following expression in the cell, said AAV VP 1 coding sequence being operably linked to a promoter which directs expression of the VP 1 coding sequences in the cell line.

[0136] In one embodiment, a production cell culture useful for producing a recombinant AAV having a capsid selected from an AAVhu68, an AAV9, a mutant AAV9, an AAVhu95 or an AAVhu96 is provided. Such a cell culture contains a nucleic acid which expresses the AAVhu68 capsid protein (or alternatively AAV9 capsid, mutant AAV9 capsid, AAVhu95 capsid, or AAVhu96 capsid) in the host cell (e.g., SEQ ID NO: 13 or SEQ ID NO: 14 for AAVhu68: SEQ ID NO: 16 or SEQ ID NO: 17 for AAVhu95; SEQ ID NO: 19 or SEQ ID NO: 20 for AAVhu96; SEQ ID NO: 31 encoding amino acid sequence of SEQ ID NO: 32 for AAV- RGDLHGY; SEQ ID NO: 33 encoding amino acid sequence of SEQ ID NO: 34 for AAV- PYQRGDH; SEQ ID NO: 35 encoding amino acid sequence of SEQ ID NO: 36 for AAV-IIRGDPA; SEQ ID NO: 37 encoding for amino acid sequence of SEQ ID NO: 38 for AAV-AVIRGDV: SEQ ID NO: 45 encoding for an amino acid sequence of SEQ ID NO: 46 for AAV- RGDYREV: SEQ ID NO: 47 encoding for an amino acid sequence of SEQ ID NO: 48 for AAV-RGDYHQV); a nucleic acid molecule suitable for packaging into the AAVhu68 capsid, e.g., a vector genome which contains AAV ITRs and a non- AAV nucleic acid sequence encoding a gene operably linked to regulatory sequences which direct expression of the gene in a host cell; and sufficient AAV rep functions and adenovirus helper functions to permit packaging of tire vector genome into the recombinant AAVhu68 (or AAV9. mutant AAV9, AAVhu95, AAVhu96 capsid. In one embodiment, tire cell culture is composed of mammalian cells (e.g.. human embryonic kidney 293 cells, among others) or insect cells (e.g., Spodoptera frugiperda (Sf9) cells). In certain embodiments, baculovirus provides the helper functions necessary for packaging the vector genome into the recombinant AAVhu68 capsid, AAV9 capsid, mutant AAV9 capsid, AAVhu95 capsid or AAVhu96 capsid.

[0137] Optionally the rep functions are provided by an AAV other than AAV2, selected to complement the source of tire ITRs.

[0138] In one embodiment, cells are manufactured in a suitable cell culture (e.g., HEK 293 or Sf9) or suspension. Methods for manufacturing the gene therapy vectors described herein include methods well known in the art such as generation of plasmid DNA used for production of the gene therapy vectors, generation of the vectors, and purification of the vectors. In some embodiments, the gene therapy vector is an AAV vector and the plasmids generated are an AAV cis-plasmid encoding the AAV vector genome and the gene of interest, an AAV trans-plasmid containing AAV rep and cap genes, and an adenovirus helper plasmid. The vector generation process can include method steps such as initiation of cell culture, passage of cells, seeding of cells, transfection of cells with the plasmid DNA, post-transfection medium exchange to serum free medium, and the harvest of vector-containing cells and culture media. The harvested vectorcontaining cells and culture media are referred to herein as crude cell harvest. In yet another system, the gene therapy vectors are introduced into insect cells by infection with baculovirus- based vectors. For reviews on these production systems, see generally, e.g., Zhang et al., 2009, Adenovirus-adeno-associated virus hybrid for large-scale recombinant adeno-associated virus production, Human Gene Therapy 20:922-929, the contents of each of which is incorporated herein by reference in its entirety. Methods of making and using these and other AAV production systems are also described in the following US patents, the contents of each of which is incorporated herein by reference in its entirety: US Patent Nos. 5,139,941; 5,741,683; 6,057,152; 6,204,059; 6,268,213; 6,491,907; 6,660,514; 6,951,753; 7,094,604; 7,172,893; 7,201,898; 7,229,823; and 7,439.065.

[0139] The crude cell harvest may thereafter be subject method steps such as concentration of the vector harvest, diafiltration of the vector harvest, microfluidization of the vector harvest, nuclease digestion of the vector harvest, filtration of microfluidized intermediate, crude purification by chromatography, crude purification by ultracentrifugation, buffer exchange by tangential flow filtration, and / or formulation and filtration to prepare bulk vector. An affinity chromatography purification followed anion exchange resin chromatography are used to purity' the vector drug product and to remove empty capsids. These methods are described in more detail in International Patent Application No. PCT / US2016 / 065970, filed December 9. 2016, and US 11,098.286 B2. entitled “Scalable Purification Method for AAV9”, which are incorporated by reference. Purification methods for AAV8, International Patent Application No.

[0140] PCT / US2016 / 065976, filed December 9, 2016, and US 11,015,174 B2, entitled “Scalable Purification Method for AAV8”, which are incorporated herein by reference. Purification methods for rhlO, International Patent Application No. PCT / US16 / 066013, filed December 9, 2016, and US 11,028,372 B2, entitled “Scalable Purification Method for AAVrhlO”, which are incorporated herein by reference. Purification methods for AAV1, International Patent Application No. PCT / US2016 / 065974, filed December 9, 2016, and US 11,015,173 B2, entitled “Scalable Purification Method for AAV 1”, which are incorporated herein by reference. Other suitable methods may be selected. See also, International Patent Application No.

[0141] PCT / US2023 / 076186, filed October 6, 2023, now published WO 2024 / 081551, which is incorporated herein by reference.

[0142] To calculate empty’ and full particle content, VP3 band volumes for a selected sample (e.g., in examples herein an iodixanol gradient-purified preparation where # of genome copies (GC) = # of particles) are plotted against GC particles loaded. The resulting linear equation (y = mx+c) is used to calculate the number of particles in the band volumes of the test article peaks. The number of particles (pt) per 20 pL loaded is then multiplied by 50 to give particles (pt) / mL. Pt / mL divided by GC / mL gives the ratio of particles to genome copies (pt / GC). Pt / mL-GC / mL gives empty' pt / mL. Empty pt / mL divided by pt / rnL and x 100 gives the percentage of empty particles.

[0143] Generally, methods for assaying for empty- capsids and AAV vector particles with packaged genomes have been known in the art. See, e g., Grimm et al., Gene Therapy (1999) 6: 1322-1330: Sommer et al., Molec. Ther. (2003) 7: 122-128. To test for denatured capsid, the methods include subjecting the treated AAV stock to SDS-polyacrylamide gel electrophoresis, consisting of any gel capable of separating the three capsid proteins, for example, a gradient gel containing 3-8% Tris-acetate in the buffer, then running tire gel until sample material is separated, and blotting the gel onto nylon or nitrocellulose membranes, preferably nylon. Anti-AAV capsid antibodies are then used as the primary antibodies that bind to denatured capsid proteins, preferably an anti -AAV capsid monoclonal antibody, most preferably the Bl anti-AAV-2 monoclonal antibody (Wobus et al., J. Virol. (2000) 74:9281-9293). A secondary antibody is then used, one that binds to the primary antibody and contains a means for detecting binding with the primary antibody, more preferably an anti-IgG antibody containing a detection molecule covalently bound to it, most preferably a sheep anti-mouse IgG antibody covalently linked to horseradish peroxidase. A method for detecting binding is used to semi-quantitatively determine binding between the primary and secondary antibodies, preferably a detection method capable of detecting radioactive isotope emissions, electromagnetic radiation, or colorimetric changes, most preferably a chemiluminescence detection kit. For example, for SDS-PAGE, samples from column fractions can be taken and heated in SDS-PAGE loading buffer containing reducing agent (e.g., DTT), and capsid proteins were resolved on pre-cast gradient polyacrylamide gels (e.g., Novex). Silver staining may be performed using SilverXpress (Invitrogen, CA) according to the manufacturer's instructions or other suitable staining method, i.e., SYPRO ruby or coomassie stains. In one embodiment, the concentration of AAV vector genomes (vg) in column fractions can be measured by quantitative real time PCR (Q-PCR). Samples are diluted and digested with DNase I (or another suitable nuclease) to remove exogenous DNA. After inactivation of the nuclease, the samples are further diluted and amplified using primers and a TaqMan™ Anorogenic probe specific for the DNA sequence between the primers. The number of cycles required to reach a defined level of Auorescence (threshold cycle, Ct) is measured for each sample on an Applied Biosystems Prism 7700 Sequence Detection System. Plasmid DNA containing identical sequences to that contained in the AAV vector is employed to generate a standard curve in the Q-PCR reaction. The cycle threshold (Ct) values obtained from tire samples are used to determine vector genome titer by normalizing it to the Ct value of the plasmid standard curve. End-point assays based on the digital PCR can also be used.

[0144] In one aspect, an optimized q-PCR method is used which utilizes a broad spectrum serine protease, e.g., proteinase K (such as is commercially available from Qiagen). More particularly, tire optimized qPCR genome titer assay is similar to a standard assay, except that after tire DNase I digestion, samples are diluted with proteinase K buffer and treated with proteinase K followed by heat inactivation. Suitably samples are diluted with proteinase K buffer in an amount equal to the sample size. The proteinase K buffer may be concentrated to 2-fold or higher. Typically, proteinase K treatment is about 0.2 mg / mL, but may be varied from 0. 1 mg / mL to about 1 mg / mL. The treatment step is generally conducted at about 55 °C for about 15 minutes, but may be performed at a lower temperature (e.g., about 37 °C to about 50 °C) over a longer time period (e.g., about 20 minutes to about 30 minutes), or a higher temperature (e.g.. up to about 60 °C) for a shorter time period (e.g., about 5 to 10 minutes). Similarly, heat inactivation is generally at about 95 °C for about 15 minutes, but the temperature may be lowered (e.g., about 70 to about 90 °C) and the time extended (e.g., about 20 minutes to about 30 minutes). Samples are then diluted (e.g., 1000-fold) and subjected to TaqMan analysis as described in the standard assay.

[0145] Additionally, or alternatively, droplet digital PCR (ddPCR) may be used. For example, methods for determining single-stranded and self-complementary AAV vector genome titers by ddPCR have been described. See, e.g., M. Lock et al, Hu Gene Therapy Methods, Hum Gene Ther Methods. 2014 Apr;25(2): 115-25. doi: 10. 1089 / hgtb.2013.131. Epub 2014 Feb 14.

[0146] In brief, the method for separating the rAAV capsid (mutant Clade F, AAVhu68 AAV9, AAVhu95 or AAVhu96) particles having packaged genomic sequences from genome-deficient AAV intermediates involves subjecting a suspension comprising recombinant AAV viral particles and AAV capsid intermediates to fast performance liquid chromatography, wherein the AAV viral particles and AAV intermediates are bound to a strong anion exchange resin equilibrated (e.g., at a high pH of about 10.2), and subjected to a salt gradient while monitoring eluate for ultraviolet absorbance at about 260 nanometers (nm) and about 280 nm. In other embodiments, isocratic methods may be used. Although less optimal for the mutant Clade F rAAV, or for hu68 or AAV9, the pH may be in the range of about 10 to 10.4. In this method, the AAV full capsids are collected from a fraction which is eluted when the ratio of A260 / A280 reaches an inflection point. In one example, for the Affinity Chromatography step, the diafiltered product may be applied to an affinity resin (Life Technologies) that efficiently captures the AAV seroty pe. Under these ionic conditions, a significant percentage of residual cellular DNA and proteins flow through the column, while AAV particles are efficiently captured.

[0147] The rAAV.hTafazzin A (e.g., rAAV. CB7.CI.TAZ.RBG) is suspended in a suitable physiologically compatible composition (e.g.. a buffered saline). This composition may be frozen for storage, later thawed and optionally diluted with a suitable diluent. Alternatively, the vector may be prepared as a composition which is suitable for delivery to a patient without proceeding through the freezing and thawing steps.

[0148] Pharmaceutical Composition

[0149] In one aspect, provided herein is a pharmaceutical composition comprising a vector as described herein in a formulation buffer. In one embodiment, provided is a pharmaceutical composition comprising a rAAV as described herein in a formulation buffer. In one embodiment, the rAAV is formulated at about 1 x 109genome copies (GC) / mL to about 1 x 1014GC / mL. In a further embodiment, the rAAV is formulated at about 3 x 109GC / mL to about 3 x 1013GC / mL. In yet a further embodiment, tire rAAV is formulated at about 1 x 109GC / mL to about 1 x 1013GC / mL. In one embodiment, the rAAV is formulated at least about 1 x 1011GC / mL.

[0150] Provided herein also is a composition comprising an rAAV or a vector as described herein and an aqueous suspension media. In certain embodiments, the suspension is formulated for intravenous administration or intramuscular administration. In one aspect, the compositions contain at least one rAAV stock and an optional carrier, excipient and / or preservative.

[0151] As used herein, “carrier” includes solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in tire art. Supplementary active ingredients can also be incorporated into the compositions. The phrase “pharmaceutically-acceptable” refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a host. Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, may be used for the introduction of the compositions of the present invention into suitable host cells. In particular, the rAAV vector delivered vector genomes may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.

[0152] In one embodiment, a composition includes a final formulation suitable for delivery to a subject, e.g., is an aqueous liquid suspension buffered to a physiologically compatible pH and salt concentration. Optionally, one or more surfactants are present in the formulation. In another embodiment, the composition may be transported as a concentrate which is diluted for administration to a subject. In other embodiments, the composition may be ly ophilized and reconstituted at the time of administration.

[0153] A suitable surfactant, or combination of surfactants, may be selected from among nonionic surfactants that are nontoxic. In one embodiment, a difunctional block copolymer surfactant terminating in primary hydroxyl groups is selected, e.g., such as Pluronic® F68 [BASF], also known as Poloxamer 188, which has a neutral pH, has an average molecular weight of 8400. Other surfactants and other Poloxamers may be selected, i.e., nonionic triblock copolymers composed of a central hydrophobic chain of polyoxypropylene (polypropylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly (ethylene oxide)), SOLUTOL HS 15 (Macrogol-15 Hydroxystcaratc). LABRASOL (Polyoxy capryllic glyceride), polyoxy 10 oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid esters), ethanol and polyethylene glycol. In one embodiment, the formulation contains a poloxamer. These copolymers are commonly named with the letter "P" (for poloxamer) followed by three digits: the first two digits x 100 give the approximate molecular mass of the polyoxypropylene core, and tire last digit x 10 gives the percentage polyoxyethylene content. In one embodiment Poloxamer 188 is selected. In one embodiment, the surfactant may be present in an amount up to about 0.0005 % to about 0.001% (based on weight ratio, w / w %) of tire suspension. In another embodiment, the surfactant may be present in an amount up to about 0.0005 % to about 0.001% (based on volume ratio, v / v %) of the suspension. In yet another embodiment, the surfactant may be present in an amount up to about 0.0005 % to about 0.001% of tire suspension, wherein n % indicates n gram per 100 rnL of the suspension.

[0154] In another embodiment, the composition includes a carrier, diluent, excipient and / or adjuvant. Suitable carriers may be readily selected by one of skill in tire art in view of the indication for which tire transfer virus is directed. For example, one suitable carrier includes saline, which may be formulated with a variety of buffering solutions (e.g., phosphate buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The buffer / carrier should include a component that prevents the rAAV, from sticking to the infusion tubing but does not interfere with the rAAV binding activity in vivo. A suitable surfactant, or combination of surfactants, maybe selected from among non-ionic surfactants that are nontoxic. In one embodiment, a difunctional block copolymer surfactant terminating in primary hydroxyl groups is selected, e.g., such as Poloxamer 188 (also known under the commercial names Pluronic® F68 (BASF], Lutrol® F68, Synperonic® F68, Kolliphor® Pl 88) which has a neutral pH, has an average molecular weight of 8400. Other surfactants and other Poloxamers may? be selected, i.e., nonionic triblock copolymers composed of a central hydrophobic chain of polyoxy propylene (poly (propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(cthylcnc oxide)), SOLUTOL HS 15 (Macrogol-15 Hydroxystearate), LABRASOL (Polyoxy capryllic glyceride), polyoxy -oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid esters), ethanol and polyethylene glycol. In one embodiment, the formulation contains a poloxamer. These copolymers are commonly named with the letter "P" (for poloxamer) followed by three digits: the first two digits x 100 give the approximate molecular mass of the polyoxypropylene core, and the last digit x 10 gives the percentage poly oxy ethylene content. In one embodiment Poloxamer 188 is selected. The surfactant may be present in an amount up to about 0.0005 % to about 0.001% of the suspension.

[0155] In certain embodiments, the composition containing the rAAV.hTafazzin is delivered at a pH in the range of 6.8 to 8, or 7.2 to 7.8, or 7.5 to 8. In certain embodiments, the composition containing the rAAV.hTafazzin is delivered intravenously at a pH of about 6.5 to about 7.5. In certain embodiments, the composition containing the rAAV.hTafazzin A is delivered intravenously at a pH of about 6.8 to about 7.2. However, other pHs within the broadest ranges and these subranges may be selected for other route of delivery.

[0156] In certain embodiments, the formulation may contain a buffered saline aqueous solution not comprising sodium bicarbonate. Such a formulation may contain a buffered saline aqueous solution comprising one or more of sodium phosphate, sodium chloride, potassium chloride, calcium chloride, magnesium chloride and mixtures thereof, in water, such as a Harvard’s buffer. In one embodiment, the buffer is PBS.

[0157] Optionally, the compositions may contain, in addition to tire rAAV and carrier(s), other conventional pharmaceutical ingredients, such as preservatives, or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0158] The compositions may comprise a pharmaceutically acceptable carrier, such as defined above. Suitably, the compositions described herein comprise an effective amount of one or more AAV suspended in a pharmaceutically suitable carrier and / or admixed with suitable excipients designed for delivery to the subject via injection, or for delivery by another route and / or device.

[0159] In one embodiment, a therapeutically effective amount of said vector is included in the pharmaceutical composition. The selection of the carrier is not a limitation of the present invention. As used herein, a “therapeutically effective amount” refers to the amount of the composition comprising the nucleic acid sequence encoding hTafazzin (or an rAAV or a vector thereof) which delivers and expresses in tire target cells an amount of protein sufficient to achieve efficacy. In one embodiment, the dosage of the vector is about 1 x 109GC / kg mass to about 1 x 1014GC / kg, including all integers or fractional amounts within the range and the endpoints. In certain embodiments, the dosage is about 2 x 1012GC / kg mass. In certain embodiments, the dosage is about 6 x 1012GC / kg mass. In certain embodiments, the dosage is about 2 x 1013GC / kg mass. In certain embodiments, the dosage is about 3 x 1013GC / kg mass. In certain embodiments, the dosage is about 5 x 1013GC / kg mass. In certain embodiments, an effective amount may be determined based on an animal model, rather than a human patient. In certain embodiments, the dosage is determined based on an animal model, e.g., following reverse transcription polymerase chain reaction (RT-PCR) analysis measuring the amount of specific RNA. In certain embodiments, the dosage is selected based on increase in fold efficiency (i.e., amount RNA), e.g., about 20-fold to about 1000-fold, or about 50-fold, about 200-fold, about 5000-fold, or fold values therebetween.

[0160] In one embodiment, provided herein is a composition comprising rAAV, as described herein, wherein the rAAV is at a dose about 1 x 109GC / kg mass to about 1 x 1014GC / kg, including all integers or fractional amounts within tire range and tire endpoints. In one embodiment, provided herein is a composition comprising rAAV, as described herein, wherein the rAAV is at a dose about 2 xlO12GC / kg mass to about 2 x 1013GC / kg, including all integers or fractional amounts within the range and the endpoints. In one embodiment, provided herein is a composition comprising rAAV, as described herein, wherein the rAAV is at a dose about 2 x 1012GC / kg mass. In one embodiment, provided herein is a composition comprising rAAV, as described herein, wherein the rAAV is at a dose about 6 x 1012GC / kg mass. In one embodiment, provided herein is a composition comprising rAAV, as described herein, wherein the rAAV is at a dose about 2 x 1013GC / kg mass. In one embodiment, provided herein is a composition comprising rAAV, as described herein, wherein the rAAV is at a dose about 3 x 1013GC / kg mass. In one embodiment, provided herein is a composition comprising rAAV, as described herein, wherein the rAAV is at a dose 5 x 1013GC / kg mass.

[0161] The dosage is adjusted to balance the therapeutic benefit against any side effects and such dosages may vary depending upon the therapeutic application for which the recombinant vector is employed. The levels of expression of the transgene product can be monitored to determine the frequency of dosage resulting in viral vectors, preferably AAV vectors containing the minigene. Optionally, dosage regimens similar to those described for therapeutic purposes may be utilized for immunization using the compositions of the invention.

[0162] As used herein, the term “dosage” or “amount” can refer to the total dosage or amount delivered to the subject in the course of treatment, or the dosage or amount delivered in a single unit (or multiple unit or split dosage) administration.

[0163] Also, the replication-defective virus compositions can be formulated in dosage units to contain an amount of replication-defective virus that is in the range of about 1.0 x 109GC to about 1.0 x 1016GC (to treat an average subject of 70 kg in body weight) including all integers or fractional amounts within the range, and preferably 1.0 x 1012GC to 1.0 x 1014GC for a human patient. In one embodiment, the compositions are formulated to contain at least IxlO9, 2xl09, 3xl09, 4xl09, 5xl09, 6xl09, 7xl09, 8xl09, or 9xl09GC per dose including all integers or fractional amounts within the range. In another embodiment, the compositions are formulated to contain at least IxlO10, 2xlO10, 3xl010, 4xlO10, 5xl010, 6xlO10, 7xlO10, 8xl010, or 9xlO10GC per dose including all integers or fractional amounts within the range. In another embodiment, the compositions are formulated to contain at least IxlO11, 2xlOn, 3xl0n, 4x10", 5xl0n, 6xlOn, 7x10”, 8x 10". or 9x 10" GC per dose including all integers or fractional amounts within tire range. In another embodiment, the compositions are formulated to contain at least IxlO12. 2xl012, 3xl012, 4xl012, 5xl012, 6xl012, 7xl012, 8xl012, or 9xl012GC per dose including all integers or fractional amounts within the range. In another embodiment, the compositions are formulated to contain at least IxlO13, 2xl013, 3xl013, 4xl013, 5xl013, 6xl013, 7xl013, 8xl013, or 9xl013GC per dose including all integers or fractional amounts within the range. In another embodiment, the compositions are fonnulated to contain at least IxlO14, 2xl014, 3xl014, 4x1014. 5xl014, 6xl014, 7xl014, 8xl014, or 9xl014GC per dose including all integers or fractional amounts within the range. In another embodiment, the compositions are formulated to contain at least IxlO15, 2xl015, 3xl015, 4xl015, 5xl015, 6xl015, 7xl015, 8xl015, or 9xl015GC per dose including all integers or fractional amounts within the range. In one embodiment, for human application tire dose can range from IxlO10to about IxlO12GC per dose including all integers or fractional amounts within the range.

[0164] It should be understood that the compositions in the pharmaceutical composition described herein are intended to be applied to other compositions, regiments, aspects, embodiments and methods described across the Specification.

[0165] Methods and Uses

[0166] In one aspect, a method is provided herein is a method of treating a human subject diagnosed with Barth Syndrome or a disease associated with a mutation in the TAZ gene. Further provided herein are use of an rAAV in the manufacture (preparing) of a medicament for the treatment a human subject diagnosed with Barth Syndrome or a disease associated with a mutation in the TAZ gene.

[0167] In certain embodiments, provided herein is also a use of a rAAV vector for treating a condition associated with dysfunctional Tafazzin gene comprising delivering a rAAV in a recombinant adeno-associated virus (rAAV) comprising an adeno-associated virus (AAV) capsid and a nucleic acid molecule comprising an expression cassette in the capsid, wherein the capsid is a clade F capsid comprising an AVIRGDV (SEQ ID NO: 25) peptide inserted between amino acid position 588 and 589 with reference to AAV9 (SEQ ID NO: 23), and further wherein the expression cassette is a nucleic sequence comprising a cDNA sequence comprising SEQ ID NO: 1 encoding a human Tafazzin (hTafazzin) operably linked to at least one regulatory control sequence , wherein the dose is from about 2 x 1012GC / kg to 5 x 1013GC / kg. In certain embodiments, the rAAV dose is from about 2 x 1012GC / kg to 2 x 1013GC / kg. In certain embodiments, the rAAV dose is from about 2 x 1012GC / kg to 6 x 1012GC / kg. In certain embodiments, the rAAV dose is from about 6 x 1012GC / kg to 2 x 1013GC / kg. In certain embodiments, the rAAV dose is from about 6 x 1012GC / kg to 5 x 1013GC / kg. In certain embodiments, the rAAV dose is from about 3 x 1012GC / kg to 1 x 1013GC / kg.

[0168] The method comprises administering to a subject a suspension of a vector or an rAAV as described herein. In certain embodiments, the rAAV comprises AAV capsid and a nucleic acid molecule comprising an expression cassete in tire capsid, wherein tire capsid is a clade F capsid comprising an AVIRGDV(SEQ ID NO: 25) peptide inserted between amino acid positions 588 and 589 with reference to AAV9, and further wherein the expression cassette is a nucleic acid sequence comprising a cDNA sequence comprising SEQ ID NO: 1 encoding a human Tafazzin operably linked to at least one regulatory control sequence, optionally wherein the at least one regulatory sequence comprises CB7 promoter. In certain embodiments, the rAAV comprises AAV capsid and a nucleic acid molecule comprising an expression cassette in the capsid, wherein the capsid is a clade F capsid comprising an AVIRGDV(SEQ ID NO: 25) peptide inserted between amino acid positions 588 and 589 with reference to AAV9, and further wherein the expression cassete is a nucleic acid sequence comprising a cDNA sequence comprising SEQ ID NO: 1 encoding a human Tafazzin operably linked to at least one regulatory control sequence, wherein the at least one regulatory sequence comprises CB7 promoter. In certain embodiments, the rAAV comprises AAV capsid and a nucleic acid molecule comprising an expression cassete in the capsid, wherein tire capsid is a clade F capsid comprising an AVIRGDV(SEQ ID NO: 25) peptide inserted between amino acid positions 588 and 589 with reference to AAV9, and further wherein the expression cassete is a nucleic acid sequence comprising a cDNA sequence comprising SEQ ID NO: 1 encoding a human Tafazzin operably linked to at least one regulatory control sequence, wherein the at least one regulatory sequence comprises CB7 promoter comprising a cytomegalovirus immediate-early (CMV IE) enhancer and the chicken P-actin promoter, optionally with spacer sequence, optionally with a chimeric intron comprising chicken beta actin intron and further comprising a chicken beta-actin splicing donor (including the exon sequence, chicken beta actin intron) and rabbit beta-globin splicing acceptor). In certain embodiments, the rAAV comprises AAV capsid and a nucleic acid molecule comprising an expression cassete in the capsid, wherein the capsid is a clade F capsid comprising an AVIRGDV(SEQ ID NO: 25) peptide inserted between amino acid positions 588 and 589 with reference to AAV9. and further wherein the expression cassete is a nucleic acid sequence comprising a cDNA sequence comprising SEQ ID NO: 1 encoding a human Tafazzin operably linked to at least one regulatory control sequence, wherein tire at least one regulatory sequence comprises CB7 promoter, and rabbit beta globin polyA signal sequence. In a further embodiment, tire method comprises administering to a subject a suspension of a rAAV as described herein in a formulation buffer at a minimum dose (e.g., a minimum effective dose (MED)) of rAAV is about 2xl012GC / kg. In a further embodiment, tire method comprises administering to a subject a suspension of a rAAV as described herein in a formulation buffer at a minimum dose (e.g., a minimum effective dose (MED), see also Example 5, incorporated herein by reference) of rAAV is 2xl012GC / kg. In a further embodiment, the rAAV is formulated at about 2 x 1012GC / kg or about 6 x 1012GC / kg. In a further embodiment, tire rAAV is formulated at 2 x 1012GC / kg or 6 x 1012GC / kg. In a further embodiment, the rAAV is fonnulated at a dose of greater than or equal to about 2 x 1012GC / kg. In a further embodiment, the rAAV is fonnulated at a dose of greater than or equal to 2 x 1012GC / kg. In a further embodiment, the rAAV is formulated at a dose of less than or equal to about 6 x 1012GC / kg. In a further embodiment, the rAAV is formulated at a dose of less than or equal to 6 x 1012GC / kg. In a further embodiment, the rAAV is formulated at a dose of greater than or equal to about 6 x 1012GC / kg. In a further embodiment, the rAAV is fonnulated at a dose of greater than or equal to 6 x 1012GC / kg. In a further embodiment, the rAAV is formulated at a dose greater than or equal to 2 x 1012GC / kg and less than or equal to 6 x 1012GC / kg. In a further embodiment, the rAAV is fonnulated at a dose greater than or equal to 2 x 1012GC / kg and less than or equal to 2 x 1013GC / kg. In a further embodiment, the rAAV is formulated at a dose greater than or equal to 2 x 1012GC / kg and less than or equal to 5 x 1013GC / kg. In a further embodiment, the rAAV is formulated at a dose range of 2 x 1012GC / kg to 6 x 1012GC / kg, including endpoints of the range. In a further embodiment, the rAAV is formulated at a dose range of 2 x 1012GC / kg to 2 x 1013GC / kg, including all integers or fractional amounts within the range and the endpoints of the range. In a further embodiment, tire rAAV is formulated at a dose range of 2 x 1012GC / kg to 5 x 101GC / kg, including endpoints of the range. In a further embodiment, the rAAV is formulated at a dose range of 3 x 1012GC / kg to 2 x 1013GC / kg, including endpoints of the range. In a further embodiment, the rAAV is formulated at a dose range of 4 x 1012GC / kg to 2 x 1013GC / kg, including endpoints of the range. In a further embodiment, the rAAV is formulated at a dose range of 5 x 1012GC / kg to 2 x 1013GC / kg, including endpoints of the range. In a further embodiment, the rAAV is formulated at a dose range of 6 x 1012GC / kg to 2 x 1013GC / kg, including endpoints of the range.

[0169] In other embodiments, about 1 x 109genome copies (GC) / kg to about 1 x 1014GC / kg. In a further embodiment, tire rAAV is formulated at 2 x 1013GC / kg. In a further embodiment, the rAAV is formulated at 3 x 1013GC / kg.

[0170] In certain embodiments, the method of treatment of Barth Syndrome or a disease associated with a mutation in TAZ gene further comprises monitoring hTafazzin expression and percent cardiomyocyte transduction using endomyocardial biopsy.

[0171] The methods and compositions described herein may be used for treatment of any of tire stages of Barth Syndrome or a disease associated with a mutation in a TAZ gene. In certain embodiments, the patient is an infant, a toddler, or the patient is from 3 years to 6 years of age, from 3 years to 12 years of age. from 3 years to 18 years of age. from 3 years to 20 years of age. In certain embodiments, patients are older than 18 years of age. In certain embodiments, the methods and compositions may be used for treatment of Barth Syndrome or a disease associated with mutation in TAZ gene. In certain embodiments, tire methods and compositions may be used for treatment of TAZ gene associates X-linked disease presenting with cardiomyopathy, skeletal muscle weakness, neutropenia, and abnormal growth in patients. In certain embodiments, the methods and compositions may be used for treatment of disease associated with mutation in TAZ gene, e.g., associated with R57L, H69Q, R94S, Cl 18R, G167R, I179N, L180R, L182P, H184R, G210, or another mutation in TAZ gene. See also, uniprot.org / uniprotkb / Q16635 / entry #disease_variants, which is incorporated herein by reference in its entirety. In certain embodiments, diseases associated with a mutation in a TAZ gene include dilated cardiomyopathy, left ventricular noncompaction (LVNC), familial dilated cardiomyopathy, isolated noncompaction of left ventricular myocardium (INVM). muscular dystrophy, neuropathy, lipodystrophy, segmental progeroid. See also, medlineplus^gov / genetics / gene / tafazzin / #conditions, which is incorporated herein by reference.

[0172] Symptoms of Barth Syndrome or a disease associated with a mutation in a Tafazzin (TAZ) gene include dilated cardiomyopathy with endocardial fibroelastosis, a predominantly proximal skeletal myopathy, growth retardation, neutropenia, organic aciduria, particularly excess of 3-methylglutaconic acid, hypertrophic cardiomyopathy, isolated left ventricular noncompaction, ventricular arrhythmia, motor delay, poor appetite, fatigue and exercise intolerance, hypoglycemia, lactic acidosis, hyperammonemia, and dramatic late catch-up growth after growth delay throughout childhood. See also, Schlame, M., and Xu, Y., The Function of Tafazzin, a Mitochondrial Phospholipid-Lysophospholipid Acyltransferase, J. Mol. Biol., 2020, Aug 21; 432(18):5043-5051, and Chin, M.T., and Conway, S.J., Role of Tafazzin in Mitochondrial Function, Development and Disease, J. Dev. Biol. 2020, 8, 10 which arc incorporated herein by reference in their entireties.

[0173] In certain embodiments, the methods and compositions described herein are used to ameliorate or improve one or more symptoms of Barth Syndrome or a disease associated with a mutation in a Tafazzin (TAZ) gene including cardiac function and skeletal muscle performance. In certain embodiments, the methods and compositions described herein may be used to ameliorate one or more symptoms of Barth Syndrome or a disease associated with a mutation in a Tafazzin (TAZ) gene including increased average life span, and / or reduction in progression towards heart failure.

[0174] The compositions described herein may be used in a regimen involving co-administration of other active agents. Any suitable method or route can be used to administer such other agents. Routes of administration include, for example, systemic, oral, intravenous, intraperitoneal. subcutaneous, or intramuscular administration. Optionally, the AAV compositions described herein may also be administered by one of these routes.

[0175] In certain embodiments, co-therapies or co-treatments may be utilized, which comprise co-administration with another active agent. In certain embodiments, the co-therapy may further comprise physical therapy, or antibiotics. In certain embodiments, the co-treatment may further comprise implantable cardioverter defibrillators (ICD), pacemakers (PM) and / or cardiac resynchronization therapy (CRT).

[0176] Optionally, an immunosuppressive co-therapy may be used in a subject in need. Immunosuppressants for such co-therapy include, but are not limited to, a glucocorticoid, steroids, antimetabolites, T-cell inhibitors, a macrolide (e.g., a rapamycin or rapalog), and cytostatic agents including an alkylating agent, an anti-metabolite, a cytotoxic antibiotic, an antibody, or an agent active on immunophilin. The immune suppressant may include a nitrogen mustard, nitrosourea, platinum compound, methotrexate, azathioprine, mercaptopurine, fluorouracil, dactinomycin, an anthracycline, mitomycin C, bleomycin, mithramycin, IL-2 receptor- (CD25-) or CD3-directed antibodies, anti-IL-2 antibodies, ciclosporin, tacrolimus, sirolimus, IFN- , IFN-y, an opioid, or TNF-a (tumor necrosis factor-alpha) binding agent. In certain embodiments, the immunosuppressive therapy may be started 0, 1, 2, 3, 4, 5, 6, 7, or more days prior to or after the gene therapy administration. Such immunosuppressive therapy may involve administration of one, two or more drugs (e.g., glucocorticoids, prednelisone, micophenolate mofetil (MMF) and / or sirolimus (i.e., rapamycin)). Such immunosuppressive drugs may be administrated to a subject in need once, twice or for more times at the same dose or an adjusted dose. Such therapy may involve co-administration of tw o or more drugs, tire (e.g., prednelisone, micophenolate mofetil (MMF) and / or sirolimus (i.e., rapamycin)) on the same day. One or more of these drugs may be continued after gene therapy administration, at the same dose or an adjusted dose. Such therapy may be for about 1 week (7 days), about 60 days, or longer, as needed. In certain embodiments, a tacrolimus-free regimen is selected.

[0177] In one embodiment, the rAAV as described herein is administrated once to the subject in need. In another embodiment, the rAAV is administrated more than once to the subject in need.

[0178] “Patient” or “subject”, as used herein interchangeably, means a mammalian animal, including a human, a veterinary or farm animal, a domestic animal or pet, and animals normally used for clinical research. In one embodiment, the subject of these methods and compositions is a human patient. In one embodiment, the subject of these methods and compositions is a male or female human patient. In certain embodiment, the subject of these methods and compositions is diagnosed with Barth Syndrome or a disease associated with a mutation in a Tafazzin (TAZ) gene and / or with symptoms of Barth Syndrome or a disease associated with a mutation in a Tafazzin (TAZ) gene.

[0179] In certain embodiments, provided herein is an improved method of delivering human Tafazzin to a target tissue to afford expression levels at least 100 time to 5000 times higher than a reference unmodified clade F capsid, the vector genome, the method comprising delivering a composition comprising 2 x 1012GC / kg to 2 x 1013GC / kg of a recombinant adeno-associated virus (rAAV) comprising an adeno-associated virus (AAV) capsid and a nucleic acid molecule comprising an expression cassette in the capsid, wherein the capsid is a clade F capsid comprising an AVIRGDV (SEQ ID NO: 25) peptide inserted amino acid between position 588 and 589 with reference to AAV9 (SEQ ID NO: 23). and further wherein the expression cassette is a nucleic sequence comprising a cDNA sequence comprising SEQ ID NO: 1 encoding a human Tafazzin (hTafazzin) operably linked to at least one regulatory control sequence.

[0180] It should be understood that the compositions in the method described herein are intended to be applied to other compositions, regiments, aspects, embodiments and methods described across the Specification.

[0181] Kit

[0182] In certain embodiments, a kit is provided which includes a concentrated vector suspended in a formulation (optionally frozen), optional dilution buffer, and devices and components required for intravenous administration. In another embodiment, the kit may additional or alternatively include components for intravenous delivery. In one embodiment, the kit provides sufficient buffer to allow for injection. Such buffer may allow for about a 1 : 1 to a 1 :5 dilution of the concentrated vector, or more. In other embodiments, higher or lower amounts of buffer or sterile water are included to allow for dose titration and other adjustments by the treating clinician. In still other embodiments, one or more components of the device are included in the kit. Suitable dilution buffer is available, such as, a saline, a phosphate buffered saline (PBS) or a glycerol / PBS.

[0183] It should be understood that the compositions in kit described herein are intended to be applied to other compositions, regiments, aspects, embodiments and methods described across tire Specification.

[0184] As used herein, a "‘neutralizing antibody" or “N Ab" binds specifically to a viral capsid or envelope and interferes with the infectivity of the virus or a recombinant viral vector having the viral capsid or envelope, thus preventing the recombinant viral vector from delivering effective amounts of a gene product encoded by an expression cassette in its vector genome. Various methods for assessing neutralizing antibodies in a patient’s sera may be utilized. The term method and assay may be used interchangeably. As used herein, tire term "neutralization assay" and "serum virus neutralization assay" refers to a serological test to detect the presence of systemic antibodies that may prevent infectivity of a virus. Such assays may also qualitatively or quantitatively discern the binding capacity (e.g., magnitude) or efficiency of the antibodies to neutralize a target. Immunological assays may include enzyme immunoassay (EIA), radioimmunoassay (RIA), which uses radioactive isotopes, fluoroimmunoassay (FIA) which uses fluorescent materials, chemiluminescent immunoassay (CLIA) which uses chemiluminescent materials and counting immunoassay (CIA) which employs particle-counting techniques, other modified assays such as western blot, immunohistochemistry (IHC) and agglutination. One of the most common enzyme immunoassays is enzyme-linked immunosorbent assay (ELISA).

[0185] ■‘Neutralizing antibody titer” (NAb titer) a measurement of how much neutralizing antibody (e g., anti-AAV NAb) is produced which neutralizes the physiologic effect of its targeted epitope (e.g., an AAV). Anti-AAV NAb titers may be measured as described in, e.g., Calcedo, R., et al., Worldwide Epidemiology of Neutralizing Antibodies to Adeno-Associated Viruses. Journal of Infectious Diseases, 2009, 199 (3): p. 381-390, w hich is incorporated by reference herein.

[0186] As used herein, a “subpopulation” of vp proteins refers to a group of vp proteins which has at least one defined characteristic in common and which consists of at least one group member to less than all members of the reference group, unless otherwise specified. For example, a “subpopulation” of vpl proteins is at least one (1) vpl protein and less than all vpl proteins in an assembled AAV capsid, unless otherw ise specified. A “subpopulation” of vp3 proteins may be one (1) vp3 protein to less than all vp3 proteins in an assembled AAV capsid, unless otherwise specified. For example, vpl proteins may be a subpopulation of vp proteins; vp2 proteins may be a separate subpopulation of vp proteins, and vp3 are yet a further subpopulation of vp proteins in an assembled AAV capsid. In another example, vpl, vp2 and vp3 proteins may contain subpopulations having different modifications, e.g., at least one, two, three or four highly deamidated asparagines, e g., at asparagine - glycine pairs. Unless otherwise specified, highly deamidated refers to at least 45% deamidated, at least 50% deamidated, at least 60% deamidated, at least 65% deamidated, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 97%, 99%, up to about 100% deamidated, 50% to 100% deamidated, 70% to 100% deamidated, 75% to 100% deamidated, or 70% to 90% deamidated at a referenced amino acid position, as compared to the predicted amino acid sequence at the reference amino acid position. Such percentages may be determined using 2D-geL mass spectrometry techniques, or other suitable techniques. As used herein, a ‘‘stock” of rAAV refers to a population of rAAV. Despite heterogeneity in their capsid proteins due to deamidation, rAAV in a stock are expected to share an identical vector genome. A stock can include rAAV having capsids with, for example, heterogeneous deamidation patterns characteristic of the selected AAV capsid proteins and a selected production system. The stock may be produced from a single production system or pooled from multiple rims of the production system. A variety of production systems, including but not limited to those described herein, may be selected. See, e.g., WO 2019 / 168961, published September 6, 2019, including Table G providing the deamidation pattern for AAV9 and WO 2020 / 160582, filed September 7, 2018. See, also, e.g., WO 2020 / 223231, published November 5, 2020 (rh91, including table with deamidation pattern). US Provisional Patent Application No. 63 / 065,616, filed August 14, 2020, and US Provisional Patent Application No. 63 / 109.734, filed November 4, 2020, and International Patent Application No. PCT / US21 / 45945, filed August 13, 2021, which are all incorporated herein by reference in its entirety.

[0187] The abbreviation “sc” refers to self-complementary. “Self-complementary AAV” refers a construct in which a coding region carried by a recombinant AAV nucleic acid sequence has been designed to form an intra-molecular double-stranded DNA template. Upon infection, rather than waiting for cell mediated synthesis of the second strand, the two complementary halves of scAAV will associate to fonn one double stranded DNA (dsDNA) unit that is ready for immediate replication and transcription. See, e.g., D M McCarty et al, “Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis”. Gene Therapy, (August 2001), Vol 8, Number 16, Pages 1248- 1254. Self-complementary AAVs are described in, e.g., U.S. Patent Nos. 6,596,535; 7,125,717; and 7,456,683, each of which is incorporated herein by reference in its entirety.

[0188] The term “heterologous” when used with reference to a protein or a nucleic acid indicates that the protein or the nucleic acid comprises two or more sequences or subsequences which are not found in the same relationship to each other in nature. For instance, the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid. For example, in one embodiment, the nucleic acid has a promoter from one gene arranged to direct the expression of a coding sequence from a different gene. Thus, with reference to the coding sequence, the promoter is heterologous.

[0189] A “replication-defective virus” or “viral vector” refers to a synthetic or artificial viral particle in which an expression cassette containing a gene of interest is packaged in a viral capsid or envelope, where any viral genomic sequences also packaged within the viral capsid or envelope are replication-deficient; i.e., they cannot generate progeny virions but retain the ability to infect target cells. In one embodiment, the genome of the viral vector does not include genes encoding the enzymes required to replicate (tire genome can be engineered to be “gutless” - containing only the transgene of interest flanked by the signals required for amplification and packaging of the artificial genome), but these genes may be supplied during production. Therefore, it is deemed safe for use in gene therapy since replication and infection by progeny virions cannot occur except in tire presence of the viral enzyme required for replication.

[0190] As used herein, the terms “rAAV” and “artificial AAV” used interchangeably, mean, without limitation, a AAV comprising a capsid protein and a vector genome packaged therein, wherein the vector genome comprising a nucleic acid heterologous to the AAV. In one embodiment, the capsid protein is a non-naturally occurring capsid. Such an artificial capsid may be generated by any suitable technique, using a selected AAV sequence (e.g., a fragment of a vpl capsid protein) in combination with heterologous sequences which may be obtained from a different selected AAV, non-contiguous portions of the same AAV, from a non-AAV viral source, or from a non-viral source. An artificial AAV may be, without limitation, a pseudotyped AAV, a chimeric AAV capsid, a recombinant AAV capsid, or a “humanized” AAV capsid. Pseudotyped vectors, wherein tire capsid of one AAV is replaced with a heterologous capsid protein, are useful in the invention. In one embodiment, AAV2 / 5 and AAV2 / 8 are exemplary pseudotyped vectors. The selected genetic element may be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection and protoplast fusion. The methods used to make such constructs are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).

[0191] The term “nuclease-resistant” indicates that the AAV capsid has assembled around the expression cassette which is designed to deliver a transgene to a host cell and protects these packaged genomic sequences from degradation (digestion) during nuclease incubation steps designed to remove contaminating nucleic acids which may be present from the production process.

[0192] In many instances, rAAV particles are referred to as DNase resistant. However, in addition to this endonuclease (DNase), other endo- and exo- nucleases may also be used in tire purification steps described herein, to remove contaminating nucleic acids. Such nucleases may be selected to degrade single stranded DNA and / or double-stranded DNA, and RNA. Such steps may contain a single nuclease, or mixtures of nucleases directed to different targets, and may be endonucleases or exonucleases. As used herein, tire term “host cell” may refer to tire packaging cell line in which the rAAV is produced from the plasmid. In the alternative, tire term “host cell” may refer to the target cell in which expression of the transgene is desired.

[0193] As used herein, a “variant capsid” or a “variant AAV” or “variant AAV capsid” refers to a modified capsid, engineered capsid or a mutated capsid, wherein the capsid protein comprises an insertion of a tissue-specific targeting peptide, wherein modified insert is not a naturally occurring mutant.

[0194] The term “expression” is used herein in its broadest meaning and comprises the production of RNA or of RNA and protein. Expression may be transient or may be stable. The term “substantial homology” or “substantial similarity,” when referring to a nucleic acid, or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 95 to 99% of the aligned sequences. Preferably, the homology is over full-length sequence, or an open reading frame thereof, or another suitable fragment which is at least 15 nucleotides in length. Examples of suitable fragments are described herein.

[0195] The term “heterologous” as used to describe a nucleic acid sequence or protein means that the nucleic acid or protein was derived from a different organism or a different species of the same organism than the host cell or subject in which it is expressed. The term "heterologous" when used with reference to a protein or a nucleic acid in a plasmid, expression cassette, or vector, indicates that the protein or the nucleic acid is present with another sequence or subsequence which with which the protein or nucleic acid in question is not found in the same relationship to each other in nature.

[0196] As described above, the terms “increase” “decrease” “reduce” “ameliorate” “improve” “delay” or any grammatical variation thereof, or any similar terms indication a change, means a variation of about 5000 fold, about 4000 fold, about 2000 fold, about 1000 fold, about 500 fold, about 200 fold, about 100 fold, 5 fold, about 2 fold, about 1 fold, or values therebetween, or about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 5 % compared to the corresponding reference (e.g., untreated control or a subject in normal condition without Barth Syndrome or a disease associated with a mutation in a Tafazzin (TAZ) gene), unless otherwise specified.

[0197] The rAAV and compositions provided herein are useful in a systemic gene therapy approach (e.g., intravenous / IV administration) with a reduced dose and improved safety compared to existing clade F-based vectors (AAV9 family). The resulting vector is formulated for delivery to a human subject for treatment of one or more symptoms associated with Barth syndrome (an X-linked, multisystem disorder), including, e.g., cardiomyopathy, skeletal myopathy, neutropenia, and / or developmental delay. Improvement may be observed, e.g., by detecting expression levels of functional Tafazzin (TAZ), and / or improvement in cardiac symptoms such as decreased or slowing of progression in dilated or hypertrophic cardiomyopathy, endomyocardial fibroelastosis, and / or decrease of a developmental cardiomyopathy known as left ventricular myocardial hypertrabeculation / noncompaction (“spongy myocardium”).

[0198] As used herein, the term “administration” or any grammatical variations thereof refers to delivery' of composition described herein to a subject.

[0199] The term “percent (%) identity”, “sequence identity”, “percent sequence identity”, or “percent identical” in the context of nucleic acid sequences refers to the residues in tire two sequences which are the same when aligned for correspondence. The length of sequence identity comparison may be over the full-length of the genome, the full-length of a gene coding sequence, or a fragment of at least about 500 to 5000 nucleotides, is desired. However, identity among smaller fragments, e.g., of at least about nine nucleotides, usually at least about 20 to 24 nucleotides, at least about 28 to 32 nucleotides, at least about 36 or more nucleotides, may also be desired.

[0200] Percent identity may be readily determined for amino acid sequences over the full-length of a protein, polypeptide, about 32 amino acids, about 330 amino acids, or a peptide fragment thereof or the corresponding nucleic acid sequence coding sequences. A suitable amino acid fragment may be at least about 7 amino acids in length, and may be up to about 700 amino acids.

[0201] Examples of suitable fragments are described herein. By tire term “highly conserved” is meant at least 80% identity', preferably at least 90% identity', and more preferably, over 97% identity. Identity’ is readily determined by one of skill in tire art by resort to algorithms and computer programs known by those of skill in the art.

[0202] Generally, when referring to “identity”, “homology”, or “similarity” between two different sequences, “identity”, “homology” or “similarity” is determined in reference to “aligned” sequences. “Aligned” sequences or “alignments” refer to multiple nucleic acid sequences or protein (amino acids) sequences, often containing corrections for missing or additional bases or amino acids as compared to a reference sequence.

[0203] Identity may be determined by preparing an aligmnent of the sequences and through the use of a variety of algorithms and / or computer programs known in tire art or commercially available (e.g., BLAST. ExPASy; Clustal Omega; FASTA; using, e.g., Needleman-Wunsch algorithm, Smith- Waterman algorithm). Alignments are performed using any of a variety of publicly or commercially available Multiple Sequence Alignment Programs. Multiple sequence alignment programs are available for nucleic acid sequences. Examples of such programs include, “Clustal Omega”, “Clustal W”, “MUSCLE”, “CAP Sequence Assembly”, “BLAST”, “MAP”, and “MEME”, which are accessible through Web Servers on tire internet. Other sources for such programs are known to those of skill in the art. Alternatively, Vector NTI utilities are also used. There are also a number of algorithms known in the art that can be used to measure nucleotide sequence identity, including those contained in the programs described above. As another example, polynucleotide sequences can be compared using Fasta™, a program in GCG Version 10. 1. Fasta™ provides alignments and percent sequence identity of the regions of the best overlap between the query' and search sequences. For instance, percent sequence identity between nucleic acid sequences can be determined using Fasta™ with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) as provided in GCG Version 10. 1, herein incorporated by reference. Sequence alignment programs are also available for amino acid sequences, e.g., the “Clustal Omega”, “Clustal X”, “MUSCLE”, “MAP”, “PIMA”, “MSA”, “BLOCKMAKER”, “MEME”, and “Match-Box” programs. Generally, any of these programs are used at default settings, although one of skill in the art can alter these settings as needed. Alternatively, one of skill in the art can utilize another algorithm or computer program which provides at least tire level of identity’ or alignment as that provided by tire referenced algorithms and programs. See, e.g., J. D. Thomson et al, Nucl. Acids. Res., “A comprehensive comparison of multiple sequence alignments”, 27(13):2682-2690 (1999).

[0204] As used herein, the term “administration” or any grammatical variations thereof refers to delivery of composition described herein to a subject.

[0205] As used throughout this specification and the claims, the terms “comprise” and “contain” and its variants including, “comprises”, “comprising”, “contains” and “containing”, among other variants, is inclusive of other components, elements, integers, steps and the like. The term “consists of’ or “consisting of’ are exclusive of other components, elements, integers, steps and the like.

[0206] It is to be noted that the term “a” or “an”, refers to one or more, for example, “an enhancer”, is understood to represent one or more enhancer(s). As such, the terms “a” (or “an”), “one or more,” and “at least one” is used interchangeably herein.

[0207] As used herein, the term "about" or refers to a variant of ±10% from the reference integer and values therebetw een, unless otherwise specified. For example, “about” 500 pM includes ±50 (i.e.. 450 - 550, which includes the integers therebetween). For other values, particularly when reference is to a percentage (e.g., 90% of taste), the term “about” is inclusive of all values within the range including both the integer and fractions. As described above, the term “about” when used to modify a numerical value means a variation of ±10%, (±10%, e.g., ±1, ±2, ±3, ±4, ±5, ±6, ±7, ±8, ±9, ±10, or values therebetween) from the reference given, unless otherwise specified.

[0208] In certain instances, the term “E±#” or the term “e±#” is used to reference an exponent. For example, “5E10” or “5el0” is 5 x 10lu. These terms may be used interchangeably.

[0209] As used throughout this specification and the claims, the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). Herein, “up to” a number (for example, up to 50) includes the number (for example, 50). The term “in the range” or “within a range” (and similar statements) includes the endpoints of the stated range.

[0210] With regard to the description of various embodiments herein, it is intended that each of the compositions herein described, is useful, in another embodiment, in the methods of the invention. In addition, it is also intended that each of the compositions herein described as useful in the methods, is, in another embodiment, itself an embodiment of the invention.

[0211] Unless defined otherwise in this specification, 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 and by reference to published texts, which provide one skilled in the art with a general guide to many of the terms used in the present application.

[0212] Additional embodiments of the present invention are listed in the enumerated paragraphs below, without limitation:

[0213] El. A recombinant adeno-associated virus (rAAV) comprising an adeno-associated virus (AAV) capsid and a nucleic acid molecule comprising an expression cassette in the capsid, wherein the expression cassette is a nucleic sequence comprising a cDNA sequence comprising S EQ ID NO: 1 encoding a human Tafazzin (hTAfazzin) operably linked to regulatory control sequences.

[0214] E2. The rAAV of El, wherein the rAAV comprises a nucleic acid molecule comprising a vector genome, wherein the vector genome comprises the expression cassette and at least one of an AAV 5' inverted terminal repeat (ITR) or an AAV 3' ITR on the vector genome at an extreme 5' end and / or an extreme 3' end of the vector genome.

[0215] E3. The rAAV of El or E2, wherein the regulatory control sequences comprise a cytomegalovirus immediate early (CMV IE) enhancer, a chicken beta actin promoter, and an intron, optionally wherein an intron is a chicken beta actin intron. E4. The rAAV of El to E3, wherein the regulatory control sequences comprise an intron, wherein the intron is a chimeric intron comprising chicken beta actin splicing donor including chicken beta actin intron and rabbit beta globin splicing acceptor.

[0216] E5. The rAAV of El to E4, wherein regulatory control sequences further comprise a rabbit beta globin polyadenylation (poly A) signal sequence.

[0217] E6. The rAAV of El to E5, wherein the regulatory control sequences comprise an enhancer, wherein the enhancer is the CMV IE enhancer having a nucleic acid sequence of SEQ ID NO: 7.

[0218] E7. The rAAV of El to E5, wherein the regulatory control sequences comprise a promoter, wherein the promoter is the chicken beta actin promoter having a nucleic acid sequence of SEQ ID NO: 8.

[0219] E8. The rAAV of El to E5, wherein the regulatory7control sequences comprise an intron, wherein the intron is the chicken beta actin intron having a nucleic acid sequence of SEQ ID NO: 9.

[0220] E9. The rAAV of El to E5, wherein the regulatory control sequences comprise an intron, wherein the intron is a chimeric intron having nucleic acid sequence of SEQ ID NO: 10.

[0221] E10. The rAAV of El to E5, wherein the regulatory sequences comprise: (a) a nucleic acid sequence of SEQ ID NO: 11 comprising the CMV IE enhancer, the chicken beta-actin promoter, and tire chimeric intron comprising chicken beta actin splicing donor including chicken beta actin intron and rabbit beta globin splicing acceptor, and (b) a nucleic acid sequence of SEQ ID NO: 12 comprising a rabbit beta-globin polyA signal sequence.

[0222] El l. The rAAV of E2, wherein the regulatory7sequences comprise the promoter which is a cardiac promoter.

[0223] El 2. The rAAV of El 1, wherein the cardiac promoter is a cardiac troponin T promoter.

[0224] El 3. The rAAV of E12, wherein the cardiac troponin T promoter is a chicken cardiac troponin T promoter.

[0225] E14. The rAAV of El l to E13, wherein the regulatory control sequences comprise a hybrid cardiac promoter comprising a cytomegalovirus immediate early (CMV IE) enhancer, an optional spacer sequence, and a chicken cardiac troponin T promoter.

[0226] E15. The rAAV of El to E2, or El l to E14, wherein the regulatory7control sequences further comprise a WPRE element.

[0227] E 16. The rAAV of E 15 , wherein the regulatory control sequences comprising a WPRE element which is a mutant WPRE element.

[0228] E17. The rAAV of El to E16, wherein the at least one AAV ITR is from AAV2. El 8. The rAAV of El to E10, wherein the expression cassette comprises nucleic acid sequence of SEQ ID NO: 6, or a nucleic acid sequence at least 99% identical to SEQ ID NO: 6.

[0229] El 9. The rAAV of E2 to E10, wherein the vector genome comprises nucleic acid sequence of SEQ ID NO: 5 or a sequence at least 99% identical to SEQ ID NO: 5.

[0230] E20. The rAAV of El to El 9, wherein the AAV capsid is a mutant Clade F AAV capsid comprising an AVIRGDV (SEQ ID NO: 25) peptide insert.

[0231] E21. The rAAV of El to E19, wherein the AAV capsid is AAVhu68.

[0232] E22. The rAAV of, wherein the AAV capsid is a mutant AAV9 capsid.

[0233] E23. The rAAV of El to E22, wherein tire expression cassette comprises a nucleic acid sequence of SEQ ID NO: 6 and the rAAV has a mutant AAV9 capsid comprising an AVIRGDV (SEQ ID NO: 25) peptide insert.

[0234] E24. The rAAV of E 1 to E23, which is for use in the treatment of Barth Syndrome or a disease associated with a mutation in a Tafazzin (TAZ) gene.

[0235] E25. The rAAV of E24, wherein the disease is associated with a mutation in a Tafazzin (TAZ) gene is selected from dilated cardiomyopathy (DCM), hypertrophic DCM, endocardial fibroelastosis, left ventricular noncompaction (LVNC), familial dilated cardiomyopathy, and isolated noncompaction of left ventricular myocardium.

[0236] E26. A composition comprising a stock of rAAV of E 1 to E25 and an aqueous suspension media.

[0237] E27. The composition of E26, wherein the suspension is formulated for intravenous (IV) injection.

[0238] E28. A pharmaceutical composition comprising a rAAV of El to E25 in a formulation buffer.

[0239] E29. The pharmaceutical composition of E28, which is formulated for delivery via intravenous (IV) injection.

[0240] E30. The pharmaceutical composition of E28 or claimE 29. which is formulated to have pH of about 6.5 to about 7.5.

[0241] E31. The pharmaceutical composition of E28 to E30, which is formulated to have pH of about 6.8 to about 7.2.

[0242] E32. A recombinant nucleic acid molecule comprising an AAV vector genome which is:

[0243] (a) an AAV - 5' inverted terminal repeat (ITR);

[0244] (b) an expression cassette which is a nucleic acid sequence comprising:

[0245] (i) a cDNA sequence comprising SEQ ID NO: 1 encoding a human Tafazzin (hTAfazzin), and (ii) regulatory control sequences operably linked to the sequences of (i), wherein the regulatory control sequences comprise an optional enhancer, a promoter, an optional intron, and a polyadenylation (poly A) signal sequence; and

[0246] (c) an AAV - 3' ITR.

[0247] E33. The recombinant nucleic acid molecule of E32, wherein the regulatory sequences comprise a CB7 hybrid promoter comprising a CMV IE enhancer, a chicken beta-actin promoter, and a chimeric intron comprising chicken beta actin splicing donor including chicken beta actin intron and rabbit beta globin splicing acceptor.

[0248] E34. The recombinant nucleic acid molecule of E32, wherein the regulatory sequences comprise a promoter which is a cardiac troponin T promoter, optionally a chicken cardiac troponin T promoter.

[0249] E35. The recombinant nucleic acid molecule of E32 or E34, wherein the regulatory control sequences comprise a hybrid cardiac promoter comprising a cytomegalovirus immediate early (CMV IE) enhancer, an optional spacer sequence, and a chicken cardiac troponin T promoter.

[0250] E36. The recombinant nucleic acid molecule of E32 or E33, wherein the AAV vector genome comprises a nucleic acid sequence of SEQ ID NO: 6 or SEQ ID NO: 5.

[0251] E37. The recombinant nucleic acid molecule of E32 or E36. wherein the recombinant nucleic acid molecule is a plasmid.

[0252] E38. A packaging host cell in culture comprising a nucleic acid molecule of E32 to E37.

[0253] E39. The packaging host cell of E38, which further comprises AAV rep coding sequences operably linked to sequences which express rep protein in the packaging host cell, an AAV capsid coding sequences operably linked to sequences which express AAV capsid proteins in tire packaging host cell, and helper virus functions necessary to permit packaging of the expression cassette and AAV ITRs into the AAV capsid.

[0254] E40. The packaging host cell of E38 or E39, wherein the AAV capsid isa mutant AAV9 capsid comprising AVIRGDV (SEQ ID NO: 25).

[0255] E41. An rAAV production system useful for producing the rAAV of El to E25, wherein the production system comprises a cell culture comprising the packaging host cell of E38 to E40.

[0256] E42. An rAAV production system useful for producing the rAAV of El to E25, wherein tire production system comprises a cell culture comprising:

[0257] (a) a nucleic acid sequence encoding a AAV capsid protein;

[0258] (b) the vector genome; and (c) sufficient AAV rep functions and helper functions to permit packaging of the vector genome into the AAV capsid.

[0259] E43. The rAAV production system of E42, wherein the AAV capsid is selected from AAVhu68, AAV9, or a mutant AAV9 comprising AVIRGDV (SEQ ID NO: 25).

[0260] E44. The rAAV production system o E42 orE43 wherein the vector genome comprises nucleic acid sequence of SEQ ID NO: 5.

[0261] E45. A method of treating Barth Syndrome in a subject in a need thereof, said method comprising administering to the subject a suspension of a rAAV of El to E25 in a formulation buffer.

[0262] E46. A method of treating a disease associated with a mutation in Tafazzin (TAZ) gene in a subject, said method comprising administering to the subject a suspension of a rAAV of El to E25 in a formulation buffer.

[0263] E47. The method of E45 or E46, wherein the method comprises amelioration or improvement of one or more symptoms of Barth Syndrome or a disease associated with a mutation in Tafazzin (TAZ) gene in a subject.

[0264] E48. A recombinant AAV (rAAV) of El to E25 or a composition of E26 or E27 for use in preparing a medicament for treatment of Barth Syndrome or a disease associated with a mutation in Tafazzin (TAZ) gene.

[0265] E49. A composition comprising a recombinant adeno-associated virus (rAAV) comprising an adeno-associated virus (AAV) capsid and a nucleic acid molecule comprising an expression cassette in the capsid, wherein the capsid is a clade F capsid comprising an AVIRGDV (SEQ ID NO: 25) peptide inserted between amino acid position 588 and 589 with reference to AAV9 (SEQ ID NO: 23), and further wherein the expression cassette is a nucleic sequence comprising a cDNA sequence comprising SEQ ID NO: 1 encoding a human Tafazzin (hTafazzin) operably linked to at least one regulatory control sequence, wherein the composition comprises rAAV which is for use at a dose of 2 x 1012GC / kg to 2 x 1013GC / kg.

[0266] E50. An improved method of delivering human Tafazzin to a target tissue to afford expression levels at least 100 times to 5000 times higher than a reference unmodified clade F capsid, the method comprising delivering a composition comprising 2 x 1012GC / kg to 2 x 1013GC / kg of a recombinant adeno-associated virus (rAAV) comprising an adeno-associated virus (AAV) capsid and a nucleic acid molecule comprising an expression cassette in the capsid, wherein the capsid is a clade F capsid comprising an AVIRGDV (SEQ ID NO: 25) peptide inserted amino acid between position 588 and 589 with reference to AAV9 (SEQ ID NO: 23), and further wherein the expression cassette is a nucleic sequence comprising a cDNA sequence comprising SEQ ID NO: 1 encoding a human Tafazzin (hTafazzin) operably linked to at least one regulatory control sequence.

[0267] E51. The composition according to E49 or the method according to E50, wherein the at least one regulatory sequence comprises a CB7 hybrid promoter comprising a cytomegalovirus immediate-early (CMV IE) enhancer and the chicken P-actin promoter, optionally with spacer sequence, optionally with a chimeric intron comprising chicken beta actin intron and further comprising a chicken beta-actin splicing donor including the exon sequence, chicken beta actin intron, and rabbit beta-globin splicing acceptor.

[0268] E52. The composition according to E49 or E51, or tire method according or claim 2, wherein the at least one regulatory sequence comprises rabbit beta-globin polyadenylation (poly A) signal sequence.

[0269] E53. The composition according to any one of E49, E51, E52 or the method according to any one of E50-52, wherein the capsid is produced by a sequence encoding SEQ ID NO: 38.

[0270] E54. The composition according to any one of E49, E51-E53, wherein the rAAV is for use in the treatment of Barth Syndrome or a disease associated with a mutation in a Tafazzin (TAZ) gene

[0271] E55. The composition according to any one of E49, E51-E54, wherein the rAAV is formulated for use at a dose at 6 x 1012GC / kg.

[0272] E56. The composition according to any one of E49, E51-E55, wherein the rAAV is formulated for intravenous (IV) injection.

[0273] E57. A method of treating Barth Syndrome or a disease associated with a mutation in Tafazzin (TAZ) gene in a subject in a need thereof, said method comprising administering to the subject the composition according to any one of claims E49, E51, E54 to E56.

[0274] E58. A composition according to any one of E49, E51-E57 for use in preparing a medicament for treatment of Barth Syndrome or a disease associated with a mutation in Tafazzin (TAZ) gene.

[0275] E59. Use of a rAAV vector for treating a condition associated with dysfunctional Tafazzin gene comprising delivering a recombinant adeno-associated virus (rAAV) comprising an adeno-associated virus (AAV) capsid and a nucleic acid molecule comprising an expression cassette in the capsid, wherein the capsid is a clade F capsid comprising an AVIRGDV (SEQ ID NO: 25) peptide inserted between amino acid position 588 and 589 with reference to AAV9 (SEQ ID NO: 23), and further wherein tire expression cassette is a nucleic sequence comprising a cDNA sequence comprising SEQ ID NO: 1 encoding a human Tafazzin (hTafazzin) operably linked to at least one regulatory control sequence, wherein a dose of rAAV is 2 x 1012GC / kg to 5 x 1013GC / kg. E60. Use of a recombinant adeno-associated virus (rAAV) in the manufacture of a medicament for treatment of Barth Syndrome or a disease associated with a mutation in Tafazzin (TAZ) gene, wherein the rAAV comprises an adeno-associated virus (AAV) capsid and a nucleic acid molecule comprising an expression cassette in the capsid, wherein the capsid is a clade F capsid comprising an AVIRGDV (SEQ ID NO: 25) peptide inserted between amino acid position 588 and 589 with reference to AAV9 (SEQ ID NO: 23), and further wherein the expression cassette is a nucleic sequence comprising a cDNA sequence comprising SEQ ID NO: 1 encoding a human Tafazzin (hTafazzin) operably linked to at least one regulatory’ control sequence, wherein a dose of the rAAV is 2 x 1012GC / kg to 5 x 1013GC / kg.

[0276] E61. Use of a composition according to any one of E49, E51-E57 in the manufacture of a medicament for treatment of Barth Syndrome or a disease associated with a mutation in Tafazzin (TAZ) gene.

[0277] E62. Use of a composition according to any one of E49, E51-E57 in tire manufacture of a medicament for improved delivery of human Tafazzin to a target tissue to afford expression levels at least 100 times to 5000 times higher than a reference unmodified clade F capsid.

[0278] EXAMPLES

[0279] The following examples are provided to illustrate certain aspects of the claimed invention. The invention is not limited to these examples. We compared a clade F benchmark vector (AAVhu68) with engineered cardiotropic capsids. All the vectors expressed an engineered human TAZ transgene (e.g., hTafazzin) under a ubiquitous promoter-enhancer (e.g., CB7 hybrid promoter). Vector efficacy was evaluated in the tetracycline-inducible, short hairpin / shRNA-mediated TAZ knockdown mouse model (TAZ KD); a long-tenn 90-day safety and pharmacology study was also conducted in cynomolgus macaques that received 2 x 1013GC / kg IV.

[0280] In tire TAZ KD mouse model, IV vector administration resulted in robust expression of human TAZ (as determined by in situ hybridization) and improved survival, cardiac function, and levels of the cardiac biomarker GDF-15. No safety concerns were observed during tire 90-day study in nonhuman primates that received IV TAZ vector. Wc observed that one of our engineered capsids led to the highest cardiac expression in macaques (~ 10-40-fold increase in human TAZ transcripts within left ventricular regions of the heart compared to AAVhu68).

[0281] EXAMPLE 1. Production of rAAV comprising Tafazzin. In the studies herein, an engineered human Tafazzin (also referred to as hTafazzin, huTafazzin. hTAZ, huTAZ) sequence and rAAV comprising hTafazzin were generated and comparative studies were performed.

[0282] The rAAV are generated using triple transfection techniques, utilizing (1) a trans plasmid encoding AAV2 rep proteins and the AAVhu68 VP1 cap gene, (2) a plasmid comprising adenovirus helper genes not provided by the packaging cell line which expresses adenovirus El a, and (3) a cis plasmid containing the vector genome for packaging in the AAV capsid. See, e.g., US 2020 / 0056159. The cis plasmid is designed to contain the vector genome comprising hTafazzin. The rAAV comprising an AAV9 capsid, and a mutant AAV9 capsid are generated using similar triple transfection technique. The mutant AAV9 capsid are referred to throughout as AAV9- IIRGDPA, which comprises 'TIRGDPA"’ (SEQ ID NO: 24) amino acid insert. AAV9- AVIRGDV, which comprises an ‘‘AVIRGDV’’ (SEQ ID NO: 25) amino acid insert, AAV9- RGDYREV, which comprises an ‘RGDYREV” (SEQ ID NO: 42) amino acid insert, and AAV 9- RGDYHQV, which comprises an “RGDYHQV” (SEQ ID NO: 44) amino acid insert, AAV9- RGDLHGY, which comprises an “RGDLHGY” (SEQ ID NO: 27) amino acid insert, or AAV9- PYQRGDH, which comprises an "PYQRGDI I" (SEQ ID NO: 29) amino acid insert located between residues 588 and 589 of the capsid, as numbered based on the full-length VP1 of AAV9 (SEQ ID NO: 23).

[0283] The nucleic acid molecule comprising the vector genome packaged in the capsid contains an AAV2 - 5’ inverted terminal repeat (ITR) and an AAV2 - 3’ ITR at the extreme 5’ and 3’ end of the vector genome, respectively. The vector genome further comprises between the ITRs, the expression cassette packaged into the AAV capsid w hich have sequences encoding a hTafazzin. The expression cassette further comprises regulatory’ sequences operably linked to the engineered coding sequences, the regulatory control sequence of which includes an optional enhancer, a promoter, an optional intron (e.g.. a hybrid CB7 hybrid promoter comprising a cytomegalovirus immediate-early (CMV IE) enhancer and the chicken P-actin promoter, optionally with spacer sequence, optionally with a chimeric intron comprising chicken beta actin intron and further comprising a chicken beta-actin splicing donor (including the exon sequence, chicken beta actin intron) and rabbit beta-globin splicing acceptor), wherein the expression cassette further includes a rabbit beta-globin (RBG) polyA. SEQ ID NO: 6 refers to expression cassette of ITR.CB7.CI.TAZ.RBG.ITR, and SEQ ID NO: 5 refers to vector genome of rAAV. CB7.CI.TAZ.RBG, also referred to below as rAAV. CB7.CI.TAZ.rBG, rAAV.hTaz, rAAV. Tafazzin, or rAAV.hTafazzin. Productivity and recovery summary of the production (e.g., using iCellis 200m2with downstream chromatography purification) of rAAV comprising Tafazzin is summarized in Table 1 (recovery summary), 2 (productivity), and 3 (enrichment summary) below.

[0284] Table 1.

[0285] Table 2.

[0286] Table 3.

[0287] These results show that productivity and enrichment values were comparable among the

[0288] AAVhu68, AAV9- IIRGDPA and AAV9- AVIRGDV capsid in the yield and manufacturability assessment.

[0289] Additionally, we performed mass spectrometry analysis (N=l) of deamidation of the rAAV capsid comprising the AAV9 mutant AVIRGDV. These results are shown in Table 4, below-.

[0290] Table 4.

[0291] As can be seen, in this early study, the AAV9-AVIRGDV mutant retains tire deamidation pattern of tire AAV9 [sec, US 2020 / 0407750 Al, incorporated herein by reference], in that it is highly deamidated (at least 50% deamidation) at positions N57, N329. N452 and N512.

[0292] EXAMPLE 2. Evaluation of rAAV comprising Tafazzin in mice.

[0293] Initially, for this study, we evaluated a mouse model of Barth Syndrome using tetracycline induced shRNA silencing / TAZKD mice. Briefly, TAZ RD vs. TAZ WT mice are reported in literature to reproduce / mimic key phenotypes similar to that of Barth syndrome in humans. Barth syndrome is X linked multisystem disorder characterized by cardiomyopathy, skeletal myopathy, neutropenia, and growth retardation. Prenatal death or morbidity can occur from fetal cardiomyopathy. Tafazzin deficiency causes severe deficiency and altered molecular species of the phospholipid cardiolipin which is important for membrane integrity and respiratory function of the mitochondria. The resultant cardiomyopathy is presented as dilated or hypertrophic cardiomyopathy, endomyocardial fibroelastosis, and / or a developmental cardiomyopathy known as myocardial (left ventricular) noncompaction or hypertrabeculation (“spongy myocardium'’), which is hallmark of Barth syndrome. TAZ KD mice mimic above phenotypes developmentally and into adulthood, except for neutropenia.

[0294] The TAZKD (B6.Cg-Gt(ROSA)26Sortm37(Hl / tetO-RNAi:Tafazzin)Arte / ZkhuJ) mouse line was donated by the Zaza Khuchua laboratory at the Children's Hospital Medical Center to the Jackson Laboratory and maintained on a C57BL / 6J background. This knock-in mutation (also referred to as a tafazzin knock-down or TAZKD) strain is a tetracycline inducible shRNA- mediated TAZ knock-down mouse model of Barth syndrome. In-house breeding to obtain WT Control and TAZKD is perfonned by breeding WT females to HET males. WT C57BL / 6J female breeders may be ordered directly from tire Jackson Laboratory. Female mice are placed on 625mg / kg doxycycline chow 1 week prior to mating and are maintained on only this chow until after pups are weaned. No other food source is offered to the female breeders or the associated litters during this period. Doxycycline chow moistened with water may be offered if needed. WT female mice and HET male mice are placed into a cage together with 625mg / kg doxycycline chow for up to 1 week. Female mice are checked twice daily for evidence that copulation has occurred (plug checks). Each female mouse is housed once they are confinned to have a copulatory plug. The resulting litters are dosed after weaning according to the protocol. All mice from resulting litters are then maintained on only 625mg / kg doxycycline chow throughout the duration of their entire life. See also, jax_org / strain / 014648#; Soustek, M.S., et al., Characterization of a Transgenic Short Hairpin RNA-Induced Murine Model of Tafazzin Deficiency, Hum Gene Ther. 2011 Jul; 22(7): 865-871; Redelsperger, I. M., et al., (2016), Stability' of Doxycycline in Feed and Water and Minimal Effective Doses in Tctracyclinc- Inducible Systems, Journal of the American Association for Laboratory Animal Science, 55(4), 467-474; and Phoon CK, et al., Tafazzin knockdown in mice leads to a developmental cardiomyopathy with early diastolic dysfunction preceding myocardial noncompaction, J Am Heart Assoc, 2012 Apr;l(2):jah3-e000455, Epub 2012 Apr 24. which are incorporated herein by reference in their entireties.

[0295] FIG. 9A shows heart / body weight ratio, plotted as percent wild-type, in wild-type and TAZKD mice. FIG. 9B shows tafazzin / viculin levels ratio, plotted as percent wild-ty pe, as measured by western blot in heart tissue in wild-type and TAZKD mice. FIG. 9C shows levels of cardiolipin, plotted as percent wild-type, as measured in heart in wild-type and TAZKD mice. FIG. 9D shows levels of serum biomarker GDF-15, plotted as concentration (pg / mL), in wildtype and TAZKD mice. These data confirm that TAZKD mice display cardiac hypertrophy, reduced Tafazzin / haploinsufficiency at protein level in heart, increased immature cardiolipin (un-esterified: esterified mature cardiolipin) using mass spectrometry, and elevated serum GDF- 15, a biomarker for cardiac mitochondrial damage / injury (vs. WT littermates, when fed Doxycycline (625 ppm doxycycline hyclate) in diet).

[0296] Next, we examined pharmacological efficacy of AAV9-AVIRGDV.hTafazzin (or AAV- AVIRGDV, or AVIRGDV), AAV9-IIRGDPA.hTafazzin (or AAV-IIRGDPA, or IIRGDPA) in TAZKD mouse model on Day 110 following administration with lei 3 (1 x 1013) GC / mice AAV. FIG. 10A shows baseline serum levels of GDF-15 (before treatment). FIG. 10B shows serum levels of GDF-15 in mice on day 110 following administration of AAV in TAZKD mice. FIG. 11 A shows percent ISH (in situ hybridization)-positive cells (hTafazzin) as measured in heart tissue on day 110 following AAV administration. FIG. 1 IB shows expression levels of tafazzin (tafazzin / vinculin), as measured by western blot, and plotted as percent PBS. Vinculin protein is a housekeeping protein used as a loading control in the western blot analysis. See also, blog_cellsignal.com / choosing-a-westem-blot-loading-control-cst-blog and abcam com / primary- antibodies / loading-control-guide. Additionally, we performed in vivo, noninvasive echocardiography on Day 90, post AAV administration.

[0297] FIG. 12A shows interventricular septum thickness (IVSd) of tire left ventricular wall, plotted as mm. FIG. 12B shows end-diastolic left ventricular posterior wall thickness (LVPWd) of the left ventricular wall, plotted as mm. FIG. 12C shows left ventricular internal end diastolic diameter (LVIDd; i.e., left ventricular lumen size), plotted as mm. FIG. 12D shows left ventricular internal diameter end systole (LVIDs; i.e., left ventricular lumen size), plotted as mm. FIG. 12E shows left ventricular mass (LVM) as estimated by Echo, plotted as mg. FIG. 12F show s left ventricular mass index (LVMI) as estimated by Echo, plotted as AU. FIG. 12G show's relative wall thickness (RWT; i.e., systolic function), plotted as AU. FIG. 12H shows fractional shortening (FS; i.e., systolic function), plotted as percent. These data confirm that AAV- AVIRGDV was shown to be effective in reducing relative w all thickness, increasing left ventricular lumen diameters / reducing hypertrophy, and restored cardiac function to normal (vs. pseudo-augmented cardiac function due to hypertrophic and hyperdynamic hearts in KO PBS mice)

[0298] Next, we examined in vivo potency in WT-C57B6J treated with 2.5E13 (2.5 x 1013GC) of AAV following 1 w eek. FIG. 13A show s representative microscopic images of ISH analysis of cardiac tissue (left ventricle). FIG. 13B shows quantified results of ISH analysis, plotted as percent ISH-positive cells, in mice following AAV administration.

[0299] Next, in this study we examined efficacy of a gene therapy strategy using a cardiotropic capsid, a systemic route of administration, ubiquitous promoter connected to an engineered tafazzin sequence. We performed a pilot efficacy study to evaluate rAAV.hTafazzin (rAAV.CB7.CI.TAZ.rBG) in tafazzin knock down (TAZKD) mice. Heterologous doxycycline induced shRNA-mediated TAZKD murine model of Barth Syndrome (BTHS) was used. Briefly, mice were administered intravenously, at 1 month of age during which there is a baseline cardiac hypertrophy present, with rAAV.hTafazzin at a dose of 1 x 1011GC per mice (approximately 5 x 1012GC / kg). Functional echo-cardiogram, LC / MS analysis of cardiac lipids (heart apex used for mass spectrometry analysis), GDF-15 cardiac oxidative stress fluid biomarker analysis were performed at around 8 months of age. Necropsy was performed in Day 210 (about 8 months of age).

[0300] FIG. 2A shows analysis of cardiac function plotted as percent of fractional shortening in hearts in mice following administration with rAAV.hTafazzin or PBS (control) in TAZKD and wild-type (WT) mice. FIG. 2B shows analysis of cardiac function plotted as relative wall thickness (mm) of hearts in mice following administration with rAAV.hTafazzin or PBS (control) in TAZKD and wild-type (WT) mice. FIG. 2C shows results of the LC / MS (mass spectrometry) analysis of measured levels of monolysocardiolipin (MLCL) in ratio to cardiolipin (CL) (MLCL:CL) plotted as percent ratio standardized against MCLC:CL levels as measured in WT- PBS mice. FIG. 2C shows measured levels of GDF-15 plotted as percent standardized against GDF-15 levels as measured in WT-PBS mice.

[0301] The results of the pilot efficacy study show that a reversal of hypertrophic cardiomyopathy -related cardiac function was sustained over time, as seen from echocardiogram analysis as soon as 1 -month post-injection. Additionally, normalization of substrate to mature cardiolipin ratio and reduction of GDF-15 levels (i.e., reduction in oxidative stress) was observed.

[0302] The studies in TAZ knock-down (TAZKD) mice demonstrated rapid amelioration of cardiac function by echocardiography as soon as 1 -month post-administration of rAAV, which was sustained over time (total duration of the study 8 months). We also demonstrated normalization of substrate to mature cardiolipin ratio in heart tissue showing TAZ gene transfer produced a functional rescue in cardiomyocytes and a reduction of circulating levels of GDF-15 (indicating correction of oxidative stress). Together our data suggest that AAV mediated gene transfer to heart was successful, restored cardiolipin levels in heart, and ameliorated mitochondrial functional leading to decreased oxidative stress.

[0303] Next, we performed a potency study to evaluate potency of vector lots for use in NHP studies. Briefly, in this study we administered mice via intravenous injection with 2 x 1012GC / kg of rAAVhu68.hTafazzin (AAVhu68 capsid), rAAV9-AVIRGDV.hTafazzin (mutated AAV9 capsid (cardiotropic)), rAAV9-IIRGDPA.hTafazzin (n = 3 per group (male, C57B6 / J, 4-6weeks old, of about 22g)). Necropsy was performed on Day 7 post-rAAV administration. Tissues were collected for In Situ Hybridization (ISH) and biodistribution analysis. FIG. 1 A shows representative images of ISH analysis in heart and liver tissue collected from mice. Transduction was observed in all AAV-treated mice. The results of ISH analysis show higher transduction in mice administered with rAAV9- IIRGDPA capsid, in comparison to AAVhu68 capsid. Additionally, the results of the ISH analysis show lower transduction levels in liver tissues in mice administered with rAAV9- IIRGDPA. FIG. IB shows results of ISH analysis plotted as percent positive cardiomyocytes following rAAV administration in mice.

[0304] EXAMPLE 3. Evaluation of rAAV comprising Tafazzin in Non-human Primates (NHPs).

[0305] In this study, we evaluated pharmacology and safety of rAAV comprising a cardio tropic AAV capsid (AAVhu68, AAV9- IIRGDPA, AAV9-AVIRGDV) and administered intravenously in Cynomolgus Macaques at low Dose (2 x 1013or 2el3 GC / kg: n=2). FIG. 3 shows a study schematic. During days DO to D28 monitoring of potential acute toxicity was performed (e.g., liver injury, thrombotic microangiopathy (TMA)). Additionally, EKG and nerve conduction studies were performed on DO, D28, D60, and D90. On D90, necropsy was performed, and tissues were collected for analysis, including DNA and RNA biodistribution, ISH, whole slide morphometry, H&E pathology.

[0306] FIG. 4A shows TAZ DNA levels in left ventricle, plotted as GC / diploid cell, following AAVhu68.hTafazzin. AAV9- IIRGDPA.hTafazzin. AAV9-AVIRGDV.hTafazzin administration. FIG. 4B shows TAZ RNA levels in left ventricle, plotted as vector GC / 100 ng total RNA (as normalized to U6), following AAVhu68.hTafazzin, AAV9- IIRGDPA.hTafazzin, AAV9- AVIRGDV.hTafazzin administration. FIG. 4C shows TAZ DNA levels in septum, plotted as GC / diploid cell, following AAVhu68.hTafazzin, AAV9- IIRGDPA.hTafazzin, AAV9- AVIRGDV.hTafazzin administration. FIG. 4D shows TAZ RNA levels in septum, plotted as vector GC / 100 ng total RNA (as normalized to U6), following AAVhu68.hTafazzin. AAV9- IIRGDPA.hTafazzin, AAV9- AVIRGDV.hTafazzin administration. FIG. 4E shows TAZ DNA levels in left atrium, plotted as GC / diploid cell, following AAVhu68.hTafazzin, AAV9- lIRGDPA.hTafazzin, AAV9- AVIRGDV.hTafazzin administration. FIG. 4F shows TAZ RNA levels in left atrium, plotted as vector GC / 100 ng total RNA (as normalized to U6), following AAVhu68.hTafazzin, AAV9- IIRGDPA.hTafazzin, AAV9- AVIRGDV.hTafazzin administration. FIG. 4G shows TAZ DNA levels in liver, plotted as GC / diploid cell, following AAVhu68.hTafazzin, AAV9- IIRGDPA.hTafazzin, AAV9- AVIRGDV.hTafazzin administration. FIG. 4H shows TAZ RNA levels in liver, plotted as vector GC / 100 ng total RNA (as normalized to U6), following AAVhu68.hTafazzin, AAV9- IIRGDPA.hTafazzin, AAV9- AVIRGDV.hTafazzin administration. FIG. 41 shows TAZ DNA levels in diaphragm, plotted as GC / diploid cell, following AAVhu68.hTafazzin, AAV9- IIRGDPA.hTafazzin, AAV9- AVIRGDV.hTafazzin administration. FIG. 4J shows TAZ RNA levels in diaphragm, plotted as vector GC / 100 ng total RNA (as normalized to U6), following AAVhu68.hTafazzin. AAV9- IIRGDPA.hTafazzin, AAV9- AVIRGDV.hTafazzin administration. FIG. 4K shows TAZ DNA levels in quadriceps, plotted as GC / diploid cell, following AAVhu68.hTafazzin, AAV9- IIRGDPA.hTafazzin, AAV9- AVIRGDV.hTafazzin administration. FIG. 4L shows TAZ RNA levels in quadriceps, plotted as vector GC / 100 ng total RNA (as normalized to U6), following AAVhu68.hTafazzin, AAV9- IIRGDPA.hTafazzin, AAV9- AVIRGDV.hTafazzin administration. These results show that a 30-50-fold increase in cardiac expression of TAZ was observed in heart tissue, and 50-fold increase in diaphragm transduction post administering with rAAV9- AVIRGDV.hTafazzin.

[0307] FIG. 14A shows DNA biodistribution in various tissues on day 90 following AAV administration, plotted as vector genome copies (GC) / diploid cell. FIG. 14B shows DNA distribution on day 90 following AAV administration, plotted as fold over hu68. DNA biodistribution is summarized in Table 5, below. These results confirm that AAV -AVIRGDV and AAV-IIRGDPA show improved (vs. AAVhu68) AAV transduction in various organs. Data represents multiple sections / regions of the target tissues / animal used for DNA extraction.

[0308] Table 5.

[0309] FIG. 14C shows RNA biodistribution in various tissues on day 90 following AAV administration, plotted as vector RNA copies / 100 ng total RNA (normalized to U6 delta CT). FIG. 14D shows RNA distribution on day 90 following AAV administration, plotted as fold over hu68. RNA biodistribution is summarized in Table 6, below. These results confirm that AAV- AVIRGDV and AAV-IIRGDPA show improved (vs. AAVhu68) AAV mRNA expression in various organs. Data represents multiple sections / regions of tire target tissues / animal used for RNA extraction. Table 6.

[0310] FIG. 5 A shows results of ISH analysis, plotted as percent ISH-positive cells in tissue of left ventricle of the heart. FIG. 5B shows results of ISH analysis, plotted as percent ISH-positive cells in tissue of intraventricular septum of the heart. FIG. 5C shows results of ISH analysis, plotted as percent ISH-positive cells in tissue of right ventricle of the heart. FIG. 5D shows results of ISH analysis, plotted as percent ISH-positive cells in diaphragm tissue. FIG. 5E shows results of ISH analysis, plotted as percent ISH-positive cells in quadricep tissue. These results confirm widespread and robust cardiac transduction in cardiac and diaphragm tissue, and mild transduction in quadricep tissue following administration with rAAV9-AVIRGDV.hTafazzin. These results confinn robust cardiac transduction at low IV dose (2xl013GC / kg), and over 2-fold higher than transduction levels required to achieve efficacy in the mouse model. No safety concerns in 90-day study were observed. Transduction efficiency as maintained throughout the heart (right ventricle, interventricular septum, atria). These data further show that rAAV transduction according to ISH and vector biodistribution in various cardiac and non-cardiac tissues indicate rAAV9-AVIRGDV.hTafazzin exhibited a 30 - 50x enrichment compared to AAVhu68 (n=2 Cynos / group: 2E13 GC / kg; 90 days pi. Biodistribution data plotted as average fold expression vs. AAVhu68).

[0311] FIG. 6 shows representative microscopy images of hepatocytes at day 90 following AAVhu68.hTafazzin, AAV9-IIRGDPA.hTafazzin, AAV9-AVIRGDV.hTafazzin administration. These results show a more transcriptionally active hepatocytes at day 90 following rAA V - AV IRGD V . h Tafazzin admini strat ion .

[0312] FIG. 7A shows measured levels of aspartate aminotransferase (AST) in blood samples on DO to D90 following AAVhu68.hTafazzin, AAV9- IIRGDPA.hTafazzin, AAV9- AVIRGDV.hTafazzin administration. FIG. 7B show s measured levels of alanine aminotransferase (ALT) in blood samples on DO to D90 following AAVhu68.hTafazzin, AAV9- IIRGDPA.hTafazzin, AAV9- AVIRGDV.hTafazzin administration. FIG. 7C shows measured levels of platelet count in blood samples on DO to D90 following AAVhu68.hTafazzin, AAV9— IIRGDPA.hTafazzin, AAV9- AVIRGDV.hTafazzin administration. FIG. 7D show s measured levels of d dimer in blood samples on DO to D90 following AAVhu68.hTafazzin, AAV9- IIRGDPA.hTafazzin, AAV9- AVIRGDV.hTafazzin administration. FIG. 7E show s measured levels of troponin I in blood samples on DO to D90 follow ing AAVhu68.hTafazzin, AAV9- IIRGDPA.hTafazzin, AAV9- AVIRGDV.hTafazzin administration.

[0313] FIG. 8 show s representative microscopy images of H&E pathology analysis of liver, heart and nervous tissues. These results show no safety concerns following administration AAVhu68.hTafazzin. AAV9- IIRGDPA.hTafazzin. AAV9- AVIRGDV.hTafazzin. These results confirm good safety profile of AAVs in NHPs, as clinical observations, body weight, echo cardio, ECG, nerve conduction and sensory nerve action potential amplitudes, liver, heart, DRG histology w ere normal / unchanged.

[0314] Example 4: Further Evaluation of rAAV comprising Tafazzin in Non-human Primates (NHPs).

[0315] In this study, we evaluated pharmacology and safety of rAAV comprising a cardio tropic AAV capsid (AAVhu68, AAV9-RGDYREV, AAV9-RGDYHQV, AAV9-RGDLHGY, AAV9- PYQRGDH) and administered intravenously in Cynomolgus Macaques at low- Dose (2 x 1013or 2el3 GC / kg; n=2). FIG. 15 shows a study schematic. During days DO to D30 monitoring of potential acute toxicity was performed (e.g., liver injury, thrombotic microangiopathy (TMA)). Additionally, EKG and nen e conduction studies were performed on DO, D30, D60, and D90. Biopsies were perfomred on Day 60 and Day 90. On D90. necropsy was performed, and tissues were collected for analysis, including DNA and RNA biodistribution, ISH, whole slide morphometry, H&E pathology.

[0316] FIG. 16 shows RNA levels, plotted as transcript copies per / 100 ng RNA, as examined in brain (frontal cortex), heart, liver, diaphragm, skeletal muscle tissues. These results show 4-23- fold increase in heart tissues, and 9-229-fold increase in skeletal muscle transduction w ith AAV9- RGDYHQV, AAV9-RGDLHGY, AAV9-PYQRGDH. For heart and some skeletal muscles, several samples w ere harvested across tire tissues to ensure distribution of the vector transduction. The individual data points presented here are due to this increased sampling. FIG. 17 shows RNA levels, plotted as transcript copies per / 100 ng RNA, as examined in diaphragm, biceps brachii, biceps femoris, deltoid, gastrocnemius, gluteus maximus, soleus, and vastus lateralis tissues. These results show increase in transduction across all skeletal muscles evaluated. For heart and some skeletal muscles, several samples were harvested across the tissues to ensure distribution of the vector transduction. The individual data points presented here are due to this increased sampling. FIG. 18 shows RNA levels, plotted as transcript copies per / 100 ng RNA. as examined in gastrocnemius muscle tissues following biopsies collected on days 0, 30, 60, and 90. D30. D60 one biopsy per muscle was performed: D90 necropsy, whole muscle grid sampling was performed to ensure whole muscle distribution of the vector transduction. The individual data points presented here are due to this increased sampling.

[0317] FIG. 19A shows representative images of the in situ hybridization (ISH) analysis of gastrocnemius tissue as examined on day 30 and day 60. FIG. 19B shows results of in situ hybridization (ISH) analysis in gastrocnemius tissue, plotted as percent hTafazzin-positive fibers as normalized to an AAVhu68 control. These results show widespread and robust skeletal muscle transduction in gastrocnemius tissue from biopsies performed on day 30 and day 60. Additionally, these results show over 17-fold increase in signal with AAV-RGDYHQV, over 19- fold increase in signal with AAV-RGDLHGY, and over 13 -fold increase in signal with AAV- PYQRGDH over AAVhu68 by image quantification.

[0318] FIG. 20A shows representative images of the in situ hybridization (ISH) analysis of gastrocnemius, vastus lateralis, pectoralis. heart, soleus, and diaphragm tissues. FIG. 30B shows results of ISH analysis, plotted as percent ISH-positive myofibers in tissue from biceps barchii, deltoid, diaphragm, gastrocnemius, pectoralis. soleus, and vastus lateralis. These results confirm the widespread and robust cardiac and skeletal muscle transduction in tissues collected from necropsy on day 90 following rAAV administration.

[0319] FIG. 21 show s results of ISH analysis, plotted as percent ISH-positive cells in tissue collected from necropsy on day 90 following rAAV administration (left ventricle, septum, biceps brachii, deltoid, diaphragm, gastrocnemius, soleus, vastus, pectoralis, and liver). These results confirm tire expression of Tafazzin in various muscle tissues.

[0320] We further evaluated pharmacology and safety of rAAV comprising a cardio tropic AAV capsid (AAV clade F, AAV-RGDYREV) and administered intravenously in non-human primates (NHP) at a dose (3 x 1013or 3el3 GC / kg). At 3 months following administration, necropsy was performed, and tissues were collected for analysis (i.e., reverse transcription polymerase chain reaction (RT-PCR)). FIG. 22 show s results of RT-PCR, plotted as copies / 100 ng input RNA in tissue collected at 3 months following AAV administration (IV) 3xl013GC / kg (3E13 / Kg). These results show about 20-1000-fold (e.g.. about 50-fold, about 200-fold, about 5000-fold) increase in signal with AAV-RGDYREV by RT-PCR. Example 5: Further Evaluation of rAAV comprising Tafazzin in mice (MED) and Non-human Primates (toxicology).

[0321] Barth syndrome is a pediatric-onset. X-linked mitochondrial cardiomyopathy caused by loss of function of tafazzin (TAZ), a protein highly expressed in cardiac and skeletal muscles. TAZ is critical to maintaining the mitochondrial inner membrane structure, and thus cellular energy levels, through its role in cardiolipin metabolism. Heterozygous females are generally asymptomatic, but affected boys develop cardiomyopathy during infancy, a mild proximal myopathy, and neutropenia. Many patients do not respond to conventional heart failure treatments and 12% require heart transplantation at a median age of 1.7 years. There is no FDA- approved treatment.

[0322] The aim of the study is to restore physiological levels of tafazzin in cardiac and skeletal muscle through AAV-mediated gene transfer using modified rAAV-AVIRGDV, a novel clade F engineered capsid with an embedded (i.e., XXXRGDX) motif in HVRVIII conferring enhanced cardiac and skeletal muscle tropism. A further aim of the study is to demonstrate efficacy in a tafazzin-deficient mouse model and establish the minimum effective dose (MED) as well as potential translational biomarkers supporting a plausible mechanism of action for accelerated approval readiness. A further aim of the study is to confirm pharmacology and safety in healthy nonhuman primates (NHP) in a Good Laboratory Practice (GLP)-compliant toxicology study to support a phase 1 / 11 clinical trial.

[0323] Above-described data demonstrates a durable heart over muscle transduction in nonhuman primates and confirms the expression of the tafazzin in L ventricle, septum, diaphragm and quadricep tissues when adult macaques were administered with rAAV (Clade F and rAAV- AVIRGDV, comprising engineered tafazzin coding sequence under strong ubiquitous strong promoter (i.e., CB7)) at a dose of 2xl013GC / kg with, wherein higher expression shown in rAAV- AVIRGDV lOOLead Cardiotropic Capsid.

[0324] We also examined minimum effective dose (MED) in a Tafazzin Knockdown (KD; TAZKD) mouse model. The study was performed in a tetracycline-induced shRNA TAZ KD mouse model, wherein mice were administered IV (tail) with 2xl012to IxlO14GC / kg of rAAV (rAAV- AVIRGDV.hTaz; 2xl012GC / kg; 6xl012GC / kg; 2xl013GC / kg; IxlO14GC / kg; n=16 males / group). The duration of the study was 110 days. Various readouts were examined: bodyweight (weekly); survival; serum GDF-15 ELISA (baseline, D25, D55, & D85); echocardiography (baseline, D60, D90); antechGLP bloodwork and serum chemistry; LC / MS for cardiac ratio of MonolysocardiolipimCardiolipin; cardiac ISH for tafazzin; H&E for toxicology Western Blot for cardiac tafazzin and vinculin. Animal death only observed in group 4 (G4), High Dose ( 1 e 14 GC / kg). Most deaths occurred 2 to 4 weeks after treatment. Average HET body weight assumed for each cohort at treatment. Surviving mice received slightly less than lel4 GC / kg.

[0325] We observed that GDF-15 was elevated in TAZKD mouse relative to WT (wild-type). Additionally, we observed a dose-dependent response to treatment via ELISA. High Dose group levels were elevated at D25 compared to KD-PBS group. FIG. 31A shows measured serum GDF- 15 concentration, plotted as pg / mmol, on days 25, 55, and 85. FIG. 3 IB shows measured serum GDF-15 concentration, plotted as pg / mmol, on day 85. TAZKD animals displayed significantly elevated GDF-15 levels in all groups at baseline timepoint. Mice Group 1 (Gl), dose 2el2 GC / kg; G2, dose 6el2 GC / kg; G3, dose 2el3 GC / kg; G4 dose lel4 GC / kg; G5, KD-PBS; G6, WT PBS.

[0326] FIG. 32A shows a plot of measured body weights of mice administered with rAAV at various doses. FIG. 32B shows as plot of measured body weights of mice on day 91 post rAAV administration. Mice Group 1 (Gl), dose 2el2 GC / kg; G2, dose 6el2 GC / kg; G3, dose 2el3 GC / kg; G4 dose lel4 GC / kg; G5, KD-PBS; G6, WT PBS.

[0327] FIG. 33A shows body weight at scheduled necropsy date, plotted as grams (g). FIG. 33B shows a plot of heart weight to brain weight ratio, plotted as mg / mg. FIG. 33C shows a plot of heart weight to necropsy body weight, plotted as mg / g. Mice Group 1 (Gl), dose 2el2 GC / kg; G2. dose 6el2 GC / kg; G3, dose 2el3 GC / kg; G4 dose le 14 GC / kg; G5, KD-PBS: G6. WT PBS.

[0328] FIG. 34A shows measured ALP (alkaline phosphatase) levels, plotted as 1U / L, in mice at various doses of administered rAAV. FIG. 34B shows measured ALT (alanine aminotransferase) levels, plotted as IU / L, in mice at various doses of administered rAAV. FIG. 34C shows measured ALB (albumin) levels, plotted as g / dL, in mice at various doses of administered rAAV. FIG. 34D shows measured GLOB (globulin) levels, plotted as g / dL, in mice at various doses of administered rAAV. FIG. 34E shows measured MG levels, plotted as mg / dL, in mice at various doses of administered rAAV. FIG. 34F shows measured WBC (white blood cells) levels, plotted as 103 / pL, in mice at various doses of administered rAAV. FIG. 34G shows measured HB (hemoglobin) levels, plotted as g / dL, in mice at various doses of administered rAAV. FIG. 34H shows measured PLT (platelet count) levels, plotted as 103 / pL, in mice at various doses of administered rAAV. FIG. 341 shows measured MON (monocytes) levels, plotted as 103 / pL, in mice at various doses of administered rAAV. FIG. 34J shows measured EOS (eosinophil) levels, plotted as l O’ / pL. in mice at various doses of administered rAAV. Mice Group 1 (Gl), dose 2el2 GC / kg; G2, dose 6el2 GC / kg; G3, dose 2el3 GC / kg; G4 dose le 14 GC / kg; G5, KD-PBS; G6, WT PBS.

[0329] FIG. 23 A shows dose-dependent normalization of the cardiolipin content in heart 100 days post administration of rAAV in mice, plotted as percent wild type (WT)-PBS. FIG. 23B shows a dose-dependent reduction of GDF-15 in serum, a mitochondrial function biomarker following administration of rAAV in mice, plotted as pg / mL of GDF-15. FIGs. 24A to 24E show representative microscopy images showing a dose-dependent increase in human tafazzin expression, in heart according to in situ hybridization (ISH). These results show support for MED at a dose of 2xl012GC / kg, and for optimal dose at 6xl012GC / kg to 2xl013GC / kg.

[0330] Next, we examined an optimal heart transduction in NHP at 6x10'2GC / kg and absence of dose-limiting toxicity in NHP. The study (i.e., GLP (Good Laboratory Practice (GLP) toxicology)) w as performed in male juvenile macaque, w herein animals were administered 2xl012GC / kg to 2xl013GC / kg of rAAV (vehicle, 2xlO12GC / kg, 6xl012GC / kg, 2xl013GC / kg; n=2 per group), and necropsy was performed at 90- and 180-day post administration. The results of tire study showed no bloodwork change at 2xl012and 6x 102GC / kg. Furthermore, the results showed transient liver enzyme elevation at 2xl013GC / kg on Day 3 without repercussion on liver function or clinical presentation. Furthermore, tire results of the study showed normal echocardiogram and cardiac ultrasound throughout the study.

[0331] FIG. 25A show s measured AST (aspartate transaminase) levels, plotted as IU / L, in NHP on Day -5 to Day 90 at various doses. FIG. 25B shows measured AST levels, plotted as IU / L, in NHP on Day -5 to Day 90 post rAAV administration at 6x10’2GC / kg. FIG. 26A and FIG. 26B show representative microscopy images showing huma tafazzin mRNA levels in heart tissue (gray against black background; according to in situ hybridization (ISH), 4x magnification), post administration of rAAV at 6xl012GC / kg and 12xl013GC / kg. FIG. 26C shows tafazzin RNA in various tissues (left ventricle, right ventricle, septum, quadricep, bicep, liver), plotted as copies / lOOng RNA, as measured by RTqPCR. These results show that human tafazzin expression in heart is optimal at 6x1012GC / kg.

[0332] FIG. 27A shows vector DNA biodistribution in various tissues on day 90 post rAAV administration. FIG. 27B shows RNA biodistribution in various tissues on day 90 post rAAV administration. FIG. 28A shows RNA biodistribution in heart tissue (left ventricle) on day 90 and day 180 post administrator with rAAV at various doses (vehicle, 2xl012GC / kg, 6xl012GC / kg, 2xl013GC / kg). FIG. 28B shows RNA biodistribution in liver on day 90 and day 180 post administrator with rAAV at various doses (vehicle, 2xlO12GC / kg, 6xl012GC / kg, 2xl013GC / kg). FIG. 29 shows results of the ISH analysis (tafazzin In situ hybridization VisioPharm quantification), plotted as percent ISH+ (ISH positive) cells in various tissues (left ventricle, septum, quadricep, bicep, lover and pectoralis) post at day 90 and day 180 post rAAV administration at various doses (from left to right for each tissue; vehicle, 2xlO12GC / kg, 6xl012GC / kg, 2xl013GC / kg). Mice Group 1 (Gl). dose 2el2 GC / kg; G2, dose 6el2 GC / kg; G3. dose 2el3 GC / kg (at DI 80); G4 dose 2el3 GC / kg. FIG. 30A shows measured ALP (alkaline phosphatase) levels, plotted as IU / L, in NHP on Day -6 to Day 90 at various doses of administered rAAV. FIG. 30B shows measured BUN (blood urea nitrogen) levels, plotted as mg / dL, in NHP on Day -6 to Day 90 at various doses of administered rAAV. FIG. 30C shows measured GGT (gamma-glutamyl transferase) levels, plotted as IU / L, in NHP on Day -6 to Day 90 at various doses of administered rAAV. FIG. 30D shows measured platelets levels, plotted as 103 / pL, in NHP on Day -6 to Day 90 at various doses of administered rAAV. FIG. 30E shows measured WBC (white blood cell) levels, plotted as 103 / pL, in NHP on Day -6 to Day 90 at various doses of administered rAAV. FIG. 30F shows measured DDimer levels, plotted as ng / niL, in NHP on Day -6 to Day 90 at various doses of administered rAAV. FIG. 30G shows measured PT (prothrombin time) levels, plotted as seconds (sec), in NHP on Day -6 to Day 90 at various doses of administered rAAV. FIG. 3 OH shows measured aPTT (activated partial thromboplastin time) levels, plotted as seconds (sec), in NHP on Day -6 to Day 90 at various doses of administered rAAV. FIG. 301 shows measured ALT (alanine aminotransferase) levels, plotted as IU / L, in NHP on Day -6 to Day 90 at various doses of administered rAAV.

[0333] Overall, we observed that cardiac transduction in nonhuman primates is optimal at 6xl012GC / kg, (using rAAV-AVIRGD.hTAZ) dose, which is 10- to 20-fold lower than what is currently employed in clinical trials using clade F vectors. There was no dose-limiting toxicity that was identified in a GLP-compliant NHP study testing doses up to 2xl013GC / kg. We demonstrated robust efficacy in a tafazzin-deficient mouse model and established a minimum effective dose (MED) of 2xl012GC / kg. Mouse and NHP pharmacology and toxicology studies demonstrated the potential for efficacy and a wide safety margin that support progression of this gene therapy to a phase I / II clinical trial in Barth syndrome. Additionally, we observ ed that translational biomarkers such as MLCL / CL ratio can be used as secondary efficacy endpoints and could be leveraged for accelerated approval in the case of positive results.

[0334] All documents cited in this specification are incorporated herein by reference are incorporated by reference. US Provisional Patent Application No. 63 / 597,905, filed November 10, 2024, US Provisional Patent Application No. 63 / 661,136, filed June 18, 2024, and International Patent Application No. PCT / US24 / 55346, filed November 11, 2024, US Provisional Patent Application No. 63 / 728,468, filed December 5, 2024, US Provisional Patent Application No. 63 / 804.233, filed May 12, 2025 are incorporated herein by reference in its entirety. While the invention has been described with reference to particular embodiments, it will be appreciated that modifications can be made without departing from the spirit of the invention. Such modifications are intended to fall within the scope of the appended claims.

Claims

1. UPN-25-1101I.PCTCLAIMS:

1. A composition comprising a recombinant adeno-associated virus (rAAV) comprising an adeno-associated virus (AAV) capsid and a nucleic acid molecule comprising an expression cassette in the capsid, wherein the capsid is a clade F capsid comprising an AVIRGDV (SEQ ID NO: 25) peptide inserted between amino acid position 588 and 589 with reference to AAV9 (SEQ ID NO: 23), and further wherein the expression cassette is a nucleic sequence comprising a cDNA sequence comprising SEQ ID NO: 1 encoding a human Tafazzin (hTafazzin) operably linked to at least one regulatory control sequence, wherein the composition comprises rAAV which is for use at a dose of 2 x 1012GC / kg to 2 x 1013GC / kg.

2. An improved method of delivering human Tafazzin to a target tissue to afford expression levels at least 100 times to 5000 times higher than a reference unmodified clade F capsid, the method comprising delivering a composition comprising 2 x 1012GC / kg to 2 x 1013GC / kg of a recombinant adeno-associated virus (rAAV) comprising an adeno-associated virus (AAV) capsid and a nucleic acid molecule comprising an expression cassette in the capsid, wherein the capsid is a clade F capsid comprising an AVIRGDV (SEQ ID NO: 25) peptide inserted amino acid between position 588 and 589 with reference to AAV9 (SEQ ID NO: 23), and further wherein the expression cassette is a nucleic sequence comprising a cDNA sequence comprising SEQ ID NO: 1 encoding a human Tafazzin (hTafazzin) operably linked to at least one regulator}' control sequence.

3. The composition according to claim 1 or the method according to claim 2, wherein the at least one regulatory sequence comprises a CB7 hybrid promoter comprising a cytomegalovirus immediate-early (CMV IE) enhancer and tire chicken -actin promoter, optionally with spacer sequence, optionally with a chimeric intron comprising chicken beta actin intron and further comprising a chicken beta-actin splicing donor including the exon sequence, chicken beta actin intron, and rabbit beta-globin splicing acceptor.

4. The composition according to claim 1 or claim 3, or the method according to claim 2 or claim 3, wherein the at least one regulatory sequence comprises rabbit beta-globin polyadenylation (poly A) signal sequence.

5. The composition according to any one of claim 1, 3, 4 or the method according to any one of claims 2, 3, 4, wherein the capsid is produced by a sequence encoding SEQ ID NO: 38.

6. The composition according to any one of claims 1, 3-5 , wherein the rAAV is for use in the treatment of Barth Syndrome or a disease associated with a mutation in a Tafazzin (TAZ) gene7. The composition according to any one of claims 1. 3-6, wherein the rAAV is formulated for use at a dose at 6 x 1012GC / kg.

8. The composition according to any one of claims 1, 3-7, wherein the rAAV is formulated for intravenous (IV) injection.

9. A method of treating Barth Syndrome or a disease associated with a mutation in Tafazzin (TAZ) gene in a subject in a need thereof, said method comprising administering to the subject the composition according to any one of claims 1, 3-9.

10. A composition according to any one of claims 1, 3-9 for use in preparing a medicament for treatment of Barth Syndrome or a disease associated with a mutation in Tafazzin (TAZ) gene.

11. Use of a rAAV vector for treating a condition associated with dysfunctional Tafazzin gene comprising delivering a recombinant adeno-associated virus (rAAV) comprising an adeno-associated virus (AAV) capsid and a nucleic acid molecule comprising an expression cassette in the capsid, wherein the capsid is a clade F capsid comprising an AVIRGDV (SEQ ID NO: 25) peptide inserted between amino acid position 588 and 589 with reference to AAV9 (SEQ ID NO: 23), and further wherein the expression cassette is a nucleic sequence comprising a cDNA sequence comprising SEQ ID NO: 1 encoding a human Tafazzin (hTafazzin) operably linked to at least one regulatory control sequence, wherein a dose of rAAV is 2 x 1012GC / kg to 5 x 1013GC / kg.

12. Use of a recombinant adeno-associated virus (rAAV) in the manufacture of a medicament for treatment of Barth Syndrome or a disease associated with a mutation in Tafazzin (TAZ) gene, wherein the rAAV comprises an adeno-associated virus (AAV) capsid and a nucleicacid molecule comprising an expression cassete in the capsid, wherein the capsid is a clade F capsid comprising an AVIRGDV (SEQ ID NO: 25) peptide inserted between amino acid position 588 and 589 with reference to AAV9 (SEQ ID NO: 23). and further wherein the expression cassete is a nucleic sequence comprising a cDNA sequence comprising SEQ ID NO: 1 encoding a human Tafazzin (hTafazzin) operably linked to at least one regulatory control sequence, wherein a dose of the rAAV is 2 x 1012GC / kg to 5 x 1013GC / kg.

13. Use of a composition according to any one of claims 1, 3-9 in the manufacture of a medicament for treatment of Barth Syndrome or a disease associated with a mutation in Tafazzin (TAZ) gene.

14. Use of a composition according to any one of claims 1, 3-9 in the manufacture of a medicament for improved delivery of human Tafazzin to a target tissue to afford expression levels at least 100 times to 5000 times higher than a reference unmodified clade F capsid.

Images