METHODS AND PHARMACEUTICAL COMPOSITION FOR THE TREATMENT AND THE PREVENTION OF CARDIOMYOPATHY DUE TO ENERGY FAILURE

Abstract

The present invention relates to a method for preventing or treating cardiomyopathy due to energy failure in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a vector which comprises a nucleic acid sequence of a gene that can restore energy failure. More particularly, the invention relates to a method for preventing or treating a cardiomyopathy associated with Friedreich ataxia in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a vector which comprises a frataxin (FXN) encoding nucleic acid.

Claims

1. A method for preventing or treating cardiomyopathy due to energy failure in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a vector which comprises a nucleic acid sequence of a gene that can restore energy failure.

2. The method according to claim 1, wherein the cardiomyopathy due to energy failure is a cardiomyopathy associated with Friedreich ataxia and the gene that can restore energy failure is the frataxin (FXN) encoding nucleic acid.

3. The method according to claim 1, wherein said FXN encoding nucleic acid encodes for the amino acid sequence SEQ ID NO:2.

4. The method according to claim 1, wherein the vector comprises the nucleic acid sequence SEQ ID NO:1.

5. The method according to claim 1, wherein the vector is selected from the group consisting of adenovirus, retrovirus, herpesvirus and Adeno-Associated Virus (AAV) vectors.

6. The method according to claim 5, wherein the vector is an AAV vector.

7. The method according to claim 6, wherein the AAV vector is an AAV1, AAV2, AAV3, AAV4, AAS, AAV6, AAV7, AAV8, AAV9, AAVrh10 vector or any AAV derived vector.

8. The method according to claim 7, wherein the AA V vector is an AA Vrh10 vector.

9. The method according to claim 1, wherein the vector is administered intracoronary or directly into the myocardium of the subject.

10. The method according to claim 1, wherein the vector is administered by intravenous injection.

11. (canceled)

12. (canceled)

13. A method for reversing symptoms of cardiomyopathy associated with Friedreich ataxia in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a vector which comprises a frataxin (FXN) encoding nucleic acid.

14. A vector which comprises a nucleic acid sequence of a gene that can restore energy failure for use in treatment or prevention of cardiomyopathy due to energy failure in a subject in need thereof.

15. A vector according to claim 14, wherein the vector comprises a frataxin (FXN) encoding nucleic acid.

16. A vector according to claim 14, wherein the cardiomyopathy is a cardiomyopathy associated with Friedreich ataxia.

17. A vector according to claim 14, wherein the vector is an AAVrh10 vector.

18. (canceled)

19. (canceled)

20. (canceled)

Description

FIGURES

[0109] FIGS. 1A-1C. Administration of AAVrh10.CAG-hXN vector at 3 weeks of age prevents the onset of cardiac failure and rescues survival in pre-symptomatic MCK mice at a dose of 5×10.sup.13 vg/kg. (FIG. 1A) Survival rates of wild-type (black solid line), treated (grey dotted line) and untreated (black dotted line) MCK mice. n=9-10 for each group. (FIGS. 1B-1C) Relative quantification of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and sarcoplasmic reticulum Ca2+ ATPase (Serca2a) mRNA expressions in heart at 8 and 35 weeks for wild-type (white), treated (grey) and untreated (black) MCK mice; n=3-5 per group (*P<0.05; ***P<0.001). mRNA expression was normalized to 18S ribosomal RNA and is presented as a fold change relative to wild-type values. Data are represented as means±SD.

[0110] FIGS. 2A-2F. Administration of AAVrh10.CAG-hXN vector, at a dose of 5×10.sup.13 vg/kg, at 7 weeks of age in symptomatic MCK mice with severe cardiac failure reverses the cardiac contractile dysfunction, Fe—S cluster proteins, and cardiomyocyte and mitochondrial ultrastructure disorganization. (FIGS. 2A-2B) Longitudinal echocardiographic assessment of the left ventricle mass (LVM, left) and the shortening fraction (SF, right) for wild-type (black circles) mice, treated (light grey squares) and untreated (grey triangle) MCK mice. Data are represented as means±SD. n=9-11 for each group. (FIG. 2C) Survival rates of wild-type mice (black solid line), treated (grey dotted line) and untreated (black dotted line) MCK mice. n=9-11 for each group. (FIGS. 2D-2E) Relative quantification of atrial natriuretic peptide (ANP), and brain natriuretic peptide (BNP) and sarcoplasmic reticulum Ca.sup.2+ ATPase (Serca2a) mRNA expressions in heart at 8, 15 and 22 weeks for wild-type (white), treated (grey) and untreated (black) MCK mice; n=3-5 per group and per age (*P<0.05; **P<0.01; ns: statistically non-significant). mRNA expression was normalized to 18S ribosomal RNA and is presented as a fold change relative to wild-type values. Data are represented as means±SD. (FIG. 2F) Biochemical measurements of combined cytosolic and mitochondrial aconitases (Aco) and succinate dehydrogenase (SDH, complex II) activities in heart from wild-type (white) mice, treated (grey) and untreated (black) MCK mice at 8, 15 and 22 weeks of age; n=3-6 per group and per age (**P<0.01). Isocitrate dehydrogenase (IDH) activity was used to normalize SDH and aconitase activites for total mitochondrial content.

[0111] FIGS. 3A-3G. Treatment of MCK mice at 5 weeks with 10 fold decreased dose of vector delays the onset of cardiac failure. (FIGS. 3A-3D) Echocardiographic assessment of the left ventricle shortening fraction (FIG. 3A), ejection fraction (FIG. 3B), cardiac output (FIG. 3C) and left ventricle mass reported to body weight (FIG. 3D) for MCK mice treated with a dose of 5×10.sup.13 vg/kg (□, n=3) or 5×10.sup.12 vg/kg (⋄, n=3), as well as for control wild-type (*, n=4) and untreated MCK (◯, n=10) mice. (FIGS. 3E-3G) Kinetic of individual echocardiographic assessment of left ventricular shortening fraction for MCK mice treated with 5×10.sup.13 vg/kg (FIG. 3E) or 5×10.sup.12 vg/kg (FIGS. 3F-3G).

TABLE-US-00002 TABLE 1 Echocardiographic parameters, percentage of SDH positive cardiomyocytes and vector genome copy per individual mice. Echocardiographic Heart Vector parameters surface copies Dose of Shortening Cardiac LV SDH per vector Fraction Output Mass positive diploid (vg/Kg) Mice (%) (mL/min) (10.sup.−5) (%) genome 5 × 10.sup.13 1 35 100 192 89 11.1 2 32 100 219 77 21.3 3 20 97 265 82 11.5 2.5 × 10.sup.13   1 31 67 198 85 2.9 2 25 91 278 79 7.9 1 × 10.sup.13 1 28 52 239 53 0.6 2 30 80 213 55 N.D. 3 33 116 210 88 3.6 5 × 10.sup.12 1 17 28 410 44 0.1 2 21 39 454 48 0.2 3 33 85 223 58 0.7 WT mice 1 36 102 191 87 0.0 2 36 124 227 86 0.0 3 34 137 195 94 0.0 Abbreviations: vg/kg, vector genome per kilogram; LV, left ventricle; SDH, Succinate dehydrogenase; N.D., not determined. Values are presented as mean of experimental replicate of individual measure.

EXAMPLES

Example 1

[0112] Material & Methods

[0113] Adeno-associated viral vector construction and production Human frataxin (hFXN) cDNA, including the mitochondrial targeting sequence, fused to a HA tag was subcloned in a pAAV2-CAG plasmid (Sondhi, Hackett et al. 2007) to produce pAAV2-CAG-hFXN that included the viral inverted terminal repeat (ITR) from AAV2; the cytomegalovirus/β-actin hybrid promoter, consisting of the enhancer from the cytomegalo-virus immediate-early gene, the promoter, splice donor, and intron from the chicken β-actin gene, and the splice acceptor from rabbit β-globin. The AAVrh10.CAG-hFXN-HA vector was produced as described earlier (Rabinowitz, Rolling et al. 2002) in the Vector Core at the University Hospital of Nantes (http://www.vectors.nantes.inserm.fr). The final titers of the two batches used were 5.4×10.sup.12 vg/ml and 2.15×10.sup.13 vg/ml, respectively.

[0114] Animal Procedures

[0115] Mice with a specific deletion of Fxn gene in cardiac and skeletal muscle (MCK-Cre-FxnL3/L-) (MCK mice) in 100% C57BL/6J background were generated and genotyped as previously described (Puccio, Simon et al. 2001). Mice were maintained in a temperature and humidity controlled animal facility, with a 12 hours light/dark cycle and free access to water and a standard rodent chow (D03, SAFE, Villemoisson-sur-Orge, France). All animal procedures and experiments were approved by the local ethical committee for Animal Care and Use (Com′Eth 2011-07), and were performed in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health). For biodistribution studies, three weeks old wild-type mice were anesthetized by intraperitoneal injection of ketamine/xylazine (75/10 mg/kg) to allow intravenous administration by retro-orbital injection of AAVrh10.CAG-FXN at a dose of 5×10.sup.13 vg/kg, and sacrificed at 7 weeks of age (4 weeks post-injection). For gene therapy studies, three or seven weeks old MCK mice were anesthetized by intraperitoneal injection of ketamine/xylazine (75/10 mg/kg or 60/8 mg/kg, respectively) to allow intravenous administration by retro-orbital injection of AAVrh10.CAG-FXN at a dose of 5×10.sup.13 vg/kg. Untreated MCK and WT mice littermates were injected with equivalent volume of saline solution. Survival was evaluated daily and mice weight weekly. The mice cardiac function was evaluated under isofluorane anesthesia (1-2%) by echocardiography by an experimenter blinded to mice genotype and treatment regimen, as previously described (Seznec, Simon et al. 2004). Animals were killed by CO2 inhalation at 8, 15, 22 or 35 weeks, and tissues samples for biochemical and molecular analysis were immediately frozen in liquid nitrogen. For histological analysis, mice were anesthetized by intraperitoneal injection of ketamine/xylazine and perfused with cooled saline solution. For histological analysis of dorsal root ganglia, spinal cord and cardiac tissue was embedded in OCT Tissue Tek (Sakura Finetechnical, Torrance, Calif.) and snap-frozen in isopentane chilled in liquid nitrogen. Samples of skeletal muscles were directly snap-frozen in isopentane chilled in liquid nitrogen. For electron microscopy analysis, small samples from the middle of left ventricle and its apex were collected, then fixed and embedded in Epon as previously described (Puccio, Simon et al. 2001).

[0116] Histopathology, Enzyme Histochemistry and Electron Microscopy

[0117] For histochemical analysis, 10 μm cryosections were stained either with hematoxylin and eosin (H&E), Sirius red and Fast green to label extracellular collagen, or DAB enhanced Perls to label iron (Fe.sup.3+) deposits (Puccio, Simon et al. 2001).

[0118] Sirius red and fast green staining: Tissue sections were fixed with 10% paraformaldehyde in 0.1 M phosphate buffer (PBS), pH 7.4 for 10 min and then incubated with a saturated solution of picric acid containing 0.1% Direct red 80 (Sigma) for 2 min, washed with 0.5% glacial acetic acid solution followed by deionized water, and subsequently incubated in 0.05% Fast Green solution for 5 min, and then washed with 0.5% glacial acetic acid solution. Finally, sections were dehydrated in graded alcohols, cleared in Histosol Plus (Shandom) for 5 min and mounted using Pertex mounting medium (Histolab Products AB).

[0119] DAB-enhanced Perls iron staining: Tissue sections were fixed with 10% paraformaldehyde in 0.1 M phosphate buffer (PBS), pH 7.4 for 20 min and incubated in Perls solution (1% HCl, 1% Potassium Ferrocyanide) for 30 min. Staining was enhanced by incubation in 0.025% 3′-3′-diaminobenzidine tetrahydrochloride (Sigma-Aldrich), 0.005% H.sub.2O.sub.2 in PBS buffer for 30 min, and then developed in the same buffer. Finally, sections were dehydrated in graded alcohols, cleared in Histosol Plus (Shandom) for 5 min and mounted using Pertex mounting medium (Histolab Products AB).

[0120] Enzyme histochemical analyses: Succinate dehydrogenase (SDH) and Cytochrome C Oxydase (COX) activities were performed on 10 μm cryostat sections of tissues, as previously described (Puccio, Simon et al. 2001).

[0121] Electron microscopy analysis: Ultrathin sections (70 nm) of cardiac tissue were contrasted with uranyl acetate and lead citrate and examined with a Morgagni 268D electron microscope, as described previously (Puccio, Simon et al. 2001).

[0122] Immunofluorescence and Image Acquisition

[0123] Cardiac and spinal cord tissue cryosections were fixed in 4% PFA for 10 min, washed and then permeabilized in methanol at −20° C. for 20 min. Sections were blocked and permeabilized at the same time with PBS, 1% NGS, 5% BSA, 0.3% Triton X-100 for 1 h at room temperature (RT) and then washed in PBS, 0.2% Tween 1% BSA 1% NGS (PBS-TBN). Subsequently, tissues were incubated overnight (0/N) at 4° C. with the rabbit polyclonal antibody against frataxin (FXN935)(1/250) diluted in PBS-TBN (Puccio, Simon et al. 2001). The Alexa fluor-594 goat anti-rabbit antibody (1/500) (Molecular Probes) was incubated for 2 h at RT. Sections were stained with Hoechst and mounted using Aqua-Polymount mounting medium (Polysciences, Inc.). For co-immunolabelling of HA-tag and prohibitin, the tissue section were washed in PBS, 0.05% Tween and then blocked 0/N at 4° C. in M.O.M.™ Mouse Ig Blocking Reagent (Vector Laboratories). Section were then incubated 0/N at 4° C. with the mouse monoclonal antibody to HA tag (1/150) (Covence) diluted in M.O.M.™ diluent (Vector Laboratories). After washing, sections were incubated for 1 h at RT with the goat anti-mouse antibody conjugated to Alexa Fluor-594 nm (1/500) (Molecular Probes) diluted in M.O.M.™ diluent. Subsequently, sections were washed and blocked in PBS, 0.3% Triton, 2% NGS for 1 h30 at RT, washed and incubated for 2 h at RT with the rabbit polyclonal antibody to prohibitin (1/150) (Abcam) diluted in PBS-BTN. The Alexa Fluor-488 nm goat anti-rabbit antibody (1/500) (Molecular Probes) was incubated 1 h30 at RT with the goat anti-rabbit antibody conjugated to Alexa Fluor-488 nm (Molecular Probes) diluted at 1/500 in PBS-BTN. Sections were stained with Hoechst and mounted using Aqua-Polymount mounting medium (Polysciences, Inc).

[0124] Confocal analysis was performed on a Leica TCS SP2 upright confocal microsystem with a Plan Apo CS (numerical aperture 1.4) 63× objective. Observation of whole cardiac cryosections was performed on a Leica Z16 APO A microsystem fitted with a QuanteM-S125C camera and combined with a 2× objective (39 mm working distance).

[0125] Quantitative Real-Time PCR Total

[0126] Total RNA was extracted from frozen heart pulverized with the Precellys24 homogeniser (Bertin Technologies) and using TRI Reagent (MRC) according to the manufacturer's protocol and was treated with DNAse I treatment (Roche Biosciences). cDNA was generated by reverse transcription using the Transcriptor first strand cDNA synthesis kit (Roche biosciences). Quantitative RT-PCR was performed using the SYBR Green I Master (Roche biosciences) and light Cycler 480 (Roche biosciences) with primers described in Supplementary Table S3. 18S ribosomal RNA was used as internal standard.

[0127] Enzyme Activities

[0128] Tissues were immediately frozen in liquid nitrogen. The activities of the respiratory chain enzyme SDH (complex II), the citric acid cycle enzymes isocitrate dehydrogenase, and mitochondrial and cytosolic aconitases were determined as described (Puccio, Simon et al. 2001).

[0129] Immunoblot Analysis

[0130] Extracts of tissues were frozen in liquid nitrogen, and then homogenized in lysis buffer containing Tris-HCl (280 mM, pH 6.8), 10% SDS, 50% glycerol. Total protein extract (10 μg or 50 μg) was analyzed on SDS-glycine polyacrylamide gels. Proteins were transferred to nitrocellulose membranes blocked with 5% non-fat milk and then incubated with the different primary antibodies, polyclonal anti-frataxin (R1250 purified sera IGBMC, 1/1,000), anti-HA (Covance, 1/500), anti-mitochondrial aconitase (R2377 purified sera IGBMC, 1/20,000), anti-Ndufs3 (Invitrogen, 1/4,000), anti-SDH (Invitrogen, 1/4,000), anti-Rieske (Abcam, 1/5,000), anti-lipoic acid (Calbiochem, 1/5,000), anti-GAPDH (Millipore, 1/10,000) and monoclonal anti-beta-tubulin (2A2, IGBMC 1/1,000). Secondary antibody (goat anti-rabbit or anti-mouse IgG, respectively) coupled to peroxidase was diluted at 1/5,000 and used for detection of the reaction with Supersignal Substrate Western blotting (Pierce), according to the manufacturer's instructions.

[0131] Statistical Analysis

[0132] All data are presented as mean±standard deviation of the mean (SD). Statistical analysis was carried out using Statview software (SAS Institute Inc). For statistical comparison of three experimental groups, one-way ANOVA followed by Scheffé's post-hoc test was used. A value of P<0.05 was considered significant. For statistical comparison of two experimental groups, the bilateral Student's t-test was used. P<0.05 was considered significant.

[0133] Quantitative PCR on Genomic DNA

[0134] Genomic DNA was extracted from heart by using a phenol-chloroform method. AAVrh10.CAG-FXN vector genome copy numbers were measured by quantitative PCR using the SYBR Green I Master (Roche Biosciences) and light Cycler 480 (Roche Biosciences). The vector genome copy number per cell (VGC) was evaluated as described (Piguet, Sondhi et al. 2012). The mouse genomic Adck3 sequence was used as internal control.

[0135] Results

[0136] Three week-old MCK mice that do not exhibit yet any clinical, echocardiographic nor biochemical signs of cardiac disease, received a single intravenous injection of AAVrh10-CAG-hFXN at the dose of 5.4×10.sup.13 vg/kg (n=9). Serial echocardiographic measurements identified that the treatment efficiently prevented the development of the cardiac disease associated with frataxin deficiency. While untreated MCK mice developed a rapidly progressing left ventricle hypertrophy associated with a massive geometric remodeling characterized by increased left-ventricular diastolic diameter, the treated MCK mice were indistinguishable from wild-type (WT) littermate animals (data not shown). In parallel, systolic function evaluated by the left-ventricular shortening fraction (SF) and the cardiac output gradually decreased in untreated mice, while the treated MCK mice showed no sign of altered ventricular contractility (data not shown). The absence of echocardiographic phenotype in the treated MCK mice lead to normal growth (data not shown) and survival (35 weeks with no sign of disease), in contrast to untreated mice which die at 65±10 days (FIG. 1A). To assess the cellular and molecular state of the cardiomyocytes, treated MCK mice were sacrificed at 35 weeks of age i.e. more than triple lifespan of untreated mice. Consistent with the evolution towards heart failure, the expression of atrial natriuretic peptide (ANP) and the brain natriuretic peptide (BNP), two markers of pathology-induced stress program induced by hemodynamic overload was markedly increased in the heart from untreated mice at 8 weeks compared to WT (19 and 7 times, respectively, p<0.001) (FIG. 1B). In contrast, no difference could be detected in the expression level of these two markers between the treated MCK mice and the WT littermates, supporting the absence of pathology-induced stress programme due to hemodynamic overload (FIG. 1B). Furthermore, while the expression of sarcoplasmic reticulum Ca2+ ATPase (Serca2a), a critical determinant of cardiac relaxation responsible for diastolic Ca2+ reuptake from cytosol was reduced in untreated mice (3.3 fold, p<0.01), treated MCK mice had normal Serca2a levels (FIG. 1C). Histological analysis confirmed a preserved overall heart organization in 35 week-old treated MCK mice, compared to the myocardial degeneration with cytoplasmic vacuolization in the necrotic cardiomyocytes observed in untreated mice at 8 weeks of age (data not shown). Furthermore, Sirius-red staining (data not shown) and collagen type I and III mRNA expression (data not shown) indicated the absence of myocardial post-necrotic fibrosis in treated animals, in comparison to the massive interstitial fibrosis present in untreated MCK mice at 8 weeks (data not shown).

[0137] Intravenous injection of AAVrh10-FXN led to robust viral transduction of the heart (20.85±6.3 vg/cell) and liver, but also of skeletal muscle and dorsal root ganglia (data not shown). Western blot analysis using an anti-FXN antibody, which equally detects human and mouse frataxins, demonstrated a significant overexpression (>10 fold) of AAVrh10-encoded frataxin compared to endogenous frataxin of WT mice (data not shown). Sustained expression of the AAVrh10-encoded frataxin was seen over 35 weeks (data not shown). Mitochondrial import and maturation of frataxin was complete and non-saturated, as only the cleaved mature form of human frataxin was detected (data not shown). Immunohistochemistry analysis using both anti-FXN and anti-HA antibodies showed a broad expression of human frataxin throughout the heart of the AAV treated MCK mice, with close to 100% of transduced cardiomyocytes in the LV, RV and septum, with some cardiomyocytes expressing higher levels (data not shown). Co-localization with prohibitin demonstrated the expected mitochondrial localization of human frataxin (data not shown).

[0138] In line with the essential function of frataxin in regulating cellular Fe—S cluster biogenesis, it is now commonly accepted that frataxin deficiency leads to a primary Fe—S cluster deficit followed by secondary mitochondrial iron accumulation. Indeed, while untreated MCK mice showed a strong deficit in the Fe—S mitochondrial aconitase (mAco) and succinate dehydrogenase (SDH) (41.3% and 79.8%, respectively) (data not shown), treated mice presented levels of activities similar to WT littermates. Consistent with the widespread expression of hFXN in the heart after AAVrh10-CAG-hFXN injection, colorimetric staining of SDH activity confirmed the correction of Fe—S biogenesis in over 95% of cardiomyocytes (data not shown). While a substantial decrease in the levels of all analysed mitochondrial Fe—S proteins, was detected in untreated mice, as a result of the instability of the respective Fe—S apo-proteins, treated mutants had levels equivalent to WT (data not shown). Similarly, expression of human frataxin prevented the decrease in activity of the Fe—S enzyme lipoic acid synthase, indirectly demonstrated by normal levels of lipoic acid bound α-ketoglutarate dehydrogenase (KGDH) and pyruvate dehydrogenase (PDH) in treated animals in comparison to untreated animals (data not shown). Consistent with the absence of Fe—S cluster deficit, no cellular iron accumulation was observed in the cardiac tissue of treated mice (data not shown). Furthermore, we did not detect any sign of cellular iron homeostasis perturbation in treated animals (data not shown). Finally, electron microscopy analysis demonstrated a normal sarcomere organization of the cardiomyocytes and mitochondria ultrastructure in treated mice. Untreated animals showed sparse atrophied myofibrils and massive mitochondrial proliferation with abnormal collapsed or swollen cristae and iron accumulation (data not shown). All together, these data indicate that human frataxin gene transfer using AAVrh10 in pre-symptomatic MCK mice prevented the development of the mitochondrial FRDA cardiomyopathy at the molecular, cellular and physiological level.

[0139] While preventing the onset of the cardiomyopathy is an important step, at a clinical point of view it appears crucial to determine the therapeutic potential of this gene therapy approach when cardiac dysfunction is already present. Mutant MCK mice were intravenously injected with AAVrh10-CAG-hFXN at the dose of 5.4×10.sup.13 vg/kg (n=9) at 7 weeks, when the ventricular remodeling and left ventricular systolic dysfunction are established, with a major decrease in cardiac output (60±9% versus control values), attesting of cardiac failure. One week after injection at 8 weeks of age, the LV function was already significantly improved, with a 49±5% ejection fraction and a decrease in LV hypertrophy and dilation in the treated mutant mice, whereas untreated animals presented typical signs of heart failure (FIGS. 2A-2B). Echocardiographic parameters regarding cardiac function progressively improved to reach WT values at 11-12 weeks of age, demonstrating a complete recovery of the ventricular systolic function and anatomy. The survival of the mice was significantly prolonged until at least 18 weeks of age (FIG. 2C). In accordance with the rapid reversion observed by echocardiography, human FXN was already strongly expressed one week after injection in heart of treated mutant mice and sustained over 22 weeks, with a mitochondrial localization (data not shown). Similarly, the pathology-induced stress program induced by hemodynamic overload, reflected by the expression of ANP and BNP, was significantly decreased one week after injection (8 weeks) in treated mice (FIG. 2D). By 22 weeks, the expression level of ANP and BNP of treated MCK mice was close to the expression level of WT animals, suggesting a normalization of the hemodynamic load. Furthermore, the expression of Serca2a progressively increased in treated mice between 8 and 22 weeks, indicating that diastolic Ca2+ transport was likely restored (FIG. 2E). The reversal and correction of the cardiac phenotype correlated with a progressive increased in Fe—S proteins activities, mAco and SDH, in levels of the Fe—S proteins Ndufs3, SDH, Rieske, as well as in the lipoic acid bound PDH and KGDH (FIG. 2F). At 22 weeks, some rare patches with low SDH activity were detected in the cardiac tissue of treated mice (data not shown), corresponding to fibrotic scar probably already present at the time of treated. Interestingly, collagen staining and expression (type I and III) showed that interstitial cardiac fibrosis stopped one week post injection (data not shown). Strikingly, a rapid correction of the ultrastructure of the cardiac muscle was also observed one week after injection, with normal sarcomere organization and with a massive decrease in mitochondria (data not shown). In correlation with a still incomplete recovery of the biochemical phenotype one week after treatment, the mitochondria in the treated animals showed some signs of pathology, with the presence of some swollen mitochondria presenting parallel stacks of cristae membranes (data not shown). However, by 22 weeks, sarcomeres and mitochondria organizations completely recovered with no sign of pathological change. All together, these data indicate that AAVrh10.CAG-hFXN treatment in symptomatic MCK mice resulted in a rapid clinical, echocardiographic and biochemical improvement with a complete correction of the FRDA cardiomyopathy.

CONCLUSION

[0140] Our data demonstrates that AAVrh10-mediated transfer of hFXN gene in the myocardium of a mouse model of severe FRDA cardiomyopathy not only prevents the onset of the disease for a sustained period, but also can reverse heart failure and cardiac remodelling. The correction is extremely rapid and efficient, with a striking reversal of the mitochondrial abnormalities and biochemical Fe—S proteins deficit one week after treatment. Despite the severity of cardiac insufficiency at the time of treatment, the cardiac recovery is rapidly progressive, reaching normality within 4-5 weeks of treatment.

[0141] Indeed, the correction of mitochondrial dysfunction in the mouse was associated with a progressive increase of sarcoplasmic reticulum Ca2+-ATPase (Serca2a) gene expression involved in sarcoplasmic reticulum calcium uptake from cytosol. Interestingly, decrease in the expression and activity of Serca2a has been identified in cardiomyocytes from failing human hearts. A rapid correction of the ultrastructure of the cardiac muscle was also observed and the interstitial cardiac fibrosis was stopped one week after treatment, preventing the dilation and massive remodelling of the cardiac tissue. Fibrosis is an early manifestation of FRDA cardiomyopathy and its importance in organ pathology and dysfunction is relevant to a wide variety of diseases, including heart diseases.

[0142] In conclusion, delivery of a vector encoding hFXN in a mammalian model of FRDA cardiomyopathy resulted in i) prevention of the development of disease symptoms in asymptomatic individuals and ii) reversal of disease symptoms in individuals who already exhibited cardiomyopathy, biochemical Fe—S cluster impairment, mitochondrial dysfunction and interstitial cardiac fibrosis.

[0143] Thus, the use of a gene that can restore energy failure may be useful for the treatment and the prevention of a cardiomyopathy due to energy failure (like the use of FXN gene in the case of cardiomyopathy associated with Friedreich ataxia as explained in the examples).

Example 2: Determination of the Minimal Dose of AAVrh10.CAG-FXN Vector Injected Inducing a Detectable Level of Cardiac Transgene Expression

[0144] Material & Methods

[0145] The dose-response evaluation was conducted in 5 weeks-old MCK mice with early-stage systolic dysfunction using the same experimental design as described above. All animal procedures and experiments were approved by the local ethical committee for Animal Care and Use (Com′Eth 2012-016). Briefly, mice were anesthetized and then injected retro-orbitaly with 1004 of AAVrh10.CAG-FXN vector solution diluted in saline solution, for a final injected-dose of 5×10.sup.13, 2.5×10.sup.13, 1×10.sup.13 and 5×10.sup.12 vg/kg, respectively (Table 1). Age matched WT mice were injected with 1004 of saline solution, as control. Survival, growth and cardiac function were evaluated weekly up to 12 weeks of age (FIGS. 3A-3D). All mice were sacrificed at 12 weeks of age. Heart was sampled and dissected transversally in order to snap freeze a small transversal section of 1-2 mm thick from the middle of the left-ventricle and to process separately the apex and the middle-to-base of the heart which were embedded in OCT Tissue Tek and snap-frozen in isopentane chilled in liquid nitrogen. The OCT-included heart samples were cut alternatively at 5 and 100 μm from the apex to the base of the heart. H&E staining as well as SDH and COX activities were performed on 5 μm heart sections, at the level of the apex, middle and base of the left ventricle. Imaging of whole heart sections was performed at high magnification with the Hamamatsu NanoZoomer 2.0 slide scanner and analysis were performed using ImageJ following the color threshold method. RNA and DNA were extracted from 100 μm thick cryosection following the trizol-based protocol as described above. The apex, middle and base of the left-ventricle were analyzed separately. Vector copy per diploid genome and relative expression of transgene were quantified as described above.

[0146] Results

[0147] The dose of 5×10.sup.13 vg/kg used in the original study allows transduction of near 100% of the cardiomyocytes, a transduction efficiency that will very unlikely be achieved in humans. Therefore, to determine the lower therapeutic threshold that is required to rescue the cardiomyopathy associated with frataxin deficiency in the MCK mice, a dose-response study was performed. MCK mice with early stage systolic dysfunction were intravenously injected with AAVrh10-CAG-hFXN at a dose of 5×10.sup.13, 2.5×10.sup.13, 1×10.sup.13, and 5×10.sup.12 vg/kg (n=2-3 per dose), respectively, at 5-weeks of age. Serial echocardiographic measurements were performed weekly up to 12 weeks of age. Although some variability was observed, MCK mice injected at the highest doses (5×10.sup.13, 2.5×10.sup.13 and 1×10.sup.13 vg/kg) did not show any hemodynamic nor morphological anomaly up to 12 weeks of age (FIGS. 3A-3D, Table 1), demonstrating efficient correction of the early stage systolic dysfunction and prevention of cardiac failure onset associated with FXN deficiency. At the lower dose, 5×10.sup.12 vg/kg, while the hemodynamic and morphological measurements are stable and consistent with a correction of the early stage systolic dysfunction and prevention of cardiac failure onset up to 10 weeks of age, a systolic dysfunction (FIGS. 3A-3B) accompanied by a left ventricular remodeling (FIG. 3D) was observed starting 10 weeks of age leading to a rapid declined of the cardiac output (FIG. 3C).

[0148] All animals in each dose tested were sacrificed at 12 weeks of age to determine the transduction efficiency by measuring the vector genome copy per diploid genome and the percentage of corrected cardiomyocytes by SDH staining (Table 1). While variability was observed in the number of vector genome copy per cell, in particular at lower doses, we find a correlation between the vector genome copy, the percentage of SDH positive cells (which correlates very well with the percentage of frataxin expressing cells) and cardiac function (Table 1). Together, these results suggest that at 5 weeks of age, the therapeutic threshold appears to be around 2-3 vg/cell, with 50-60% of SDH positive cardiomyocytes.

Example 3: Efficient Transduction of Monkey and Pig Cardiomyocytes after Intramyocardial Injection of AAVrh10 Vector

[0149] Material & Methods

[0150] AAVrh10-CAG-GFP vector was administered using an identical cardiac surgery procedure in monkey and pig. Following general anesthesia, a median sternotomy was performed. The AAVrh10-CAG-GFP vector was administered directly to the myocardium of the left ventricle as subepicardial injections. In monkey, 9 subepicardial injections were performed, with 3 subepicardial injections 0.5 cm apart in 3 spots (1.66×10.sup.10 vg of vector per injection, 40 μl/injection). The total dose of injected vector per spot was 5×10.sup.10 vg. In pig, 18 subepicardial injections were performed with 3 subepicardial injections in 6 different spots (3 vector injections per spot). In one spot, 3 vector injections were made 0.5 cm apart (as in monkey) and 1.66×10.sup.10 vg (as in monkey) of vector diluted in 40 μl of PBS was injected at each site. In the 5 other spots, vector injection was made 1 cm apart with 8.3×10.sup.10 vg/injection in 3 spots and 1.25×10.sup.11 vg/injection in 2 spots. The total dose of injected vector per spot was therefore 5×10.sup.10 vg (1 spot), 2.5×10.sup.11 vg (3 spots) and 3.75×10.sup.11 vg (2 spots). The cardiac surgery procedure and vector injection were associated with no adverse event, even minimal. In pig, cardiac echography performed 10 days after vector injection was normal, showing no changes in respect to cardiac parameters observed immediately prior to cardiac surgery.

[0151] Monkey and pig were euthanized 3 weeks after vector injection. The left ventricle was dissected from the remaining part of the heart and trimmed in 4 (monkey) or 10 (pig) separate pieces along antero-longitudinal axis. Each piece of left ventricle was then cut in 20 mm-thick slices and one out of two slices were processed for directed visualization of GFP expression under fluorescence microscopy. The other slices were processed for DNA extraction to measure vector copy number. Percentage of GFP-positive cardiomyocytes was assessed using an in-house software. Vector copy number was assessed using quantitative PCR with primers targeting the ITR2 of AAVrh10-CAG-GFP vector and the endogenous albumin gene. Vector copy number was assessed using the cycle (Ct) threshold to detect albumin or ITR2 sequences. At autopsy, there was no macroscopic lesion of left ventricle in monkey and pig. In monkey, there was a mild inflammatory reaction in the myocardium around 2 injection sites in which GFP expression was observed. There was no inflammatory reaction in the myocardium of vector-injected pig.

[0152] Results

[0153] Intravenous injection of AAVrh10-CAG-hFXN vector will likely not be feasible in FRDA patients because it would require the injection of a very high dose of vector that could result in significant adverse events, mostly related to an immune reaction against the AAVrh10 capsid in the periphery. Direct intra-myocardial injection of gene therapy vector through subepicardial injections following mini-thoracotomy has proven to be safe over an average of nearly 12-year follow-up period (Rosengart T K et al, Hum Gene Ther, 2:203-208, 2013). The transduction efficacy of AAV of a given serotype in a given tissue can be species specific. At last efficacy of hFXN gene transfer in cardiomyocytes must be evaluated in a large animal whose heart size is similar to human. To answer to these important pre-clinical steps towards a gene therapy trial in patients with FRDA cardiomyopathy, AAVrh10 vector expressing the reporter green fluorescent protein (GFP) under the control of CAG promoter was injected directly into the myocardium of one monkey (Macaca fascicularis) and one pig. Monkey is the best animal model to anticipate that the observed transduction efficacy of an AAV vector of a given serotype in a given tissue will be similar in human patients. Two-three year-old pigs of 20-30 kg have a size of the heart, in particular of the left ventricle, which is similar to humans.

[0154] AAVrh10-CAG-GFP vector was directly delivered in the myocardium of a monkey through epicardial injections. Three separate spots of myocardium were injected with 3 vector injections per spot. At a non-optimized dose of 5×10.sup.10 vector genome (vg) per spot, GFP expression was observed in 60% of cardiomyocytes (data not shown). The expression of GFP was similar in the 3 spots. The mean volume in each spot in which GFP was expressed was 200 mm3. The mean vector copy number/cell was 1.2.

[0155] AAVrh10-CAG-GFP vector was then directly delivered in the myocardium of a pig through epicardial injections. Six separate spots of myocardium were injected with 3 vector injections per spot. The spots were injected with different doses of vector: 5×10.sup.10 vg (spot#1), 2.5×10.sup.11 vg (spots#2, 3 and 4) and 3.75×10.sup.11 vg (spots#5 and 6). GFP expression was observed in 10% of cardiomyocytes in spot#1 whereas GFP expression was observed in 50 to 95% of cardiomyocytes in spots#2, 3 and 4. Injection of higher dose of AAVrh10-CAG-GFP vector in spots#5 and 6 did not result in significant better transduction of cardiomyocytes. The mean volume in spots#2 to 6 in which GFP was expressed was 330 mm.sup.3. The mean vector copy number/cell in spots #2 to 4 was 1.8.

[0156] Altogether, these results demonstrate that cardiomyocytes of monkey and pig are very well transduced by AAVrh10 vector following the direct administration of the vector to the myocardium. Results obtained in monkey indicates that 30 injections of higher dose of AAVrh10 vector should be sufficient to transduce >50% of cardiomyocytes of the left ventricle in human. Thirty epicardial viral vector injections following mini-thoracotomy has proven to be feasible (1-hour surgical procedure) and safe in humans (Rosengart T K et al, Hum Gene Ther, 2:203-208, 2013). Results of transduction efficacy with AAVrh10 in pig cardiomyocytes indicates that this porcine animal model will be suitable to evaluate the total dose of AAVrh10-CAG-hFXN vector that it will be needed to inject in the left ventricle myocardium of FRDA patients to express frataxin in >50% of their cardiomyocytes.

REFERENCES

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