Brahim Belbellaa
email: bbelbellaa@adverum.com

Laurence Reutenauer

Nadia Messaddeq

Laurent Monassier

Hélène Puccio
email: helene.puccio@inserm.fr

High levels of frataxin overexpression leads to mitochondrial and cardiac toxicity in mouse models AUTHORS

Friedreich ataxia (FA) is currently an incurable inherited mitochondrial disease caused by reduced levels of frataxin (FXN). Cardiac dysfunction is the main cause of premature death in FA. AAV-mediated gene therapy constitutes a promising approach for FA, as demonstrated in cardiac and neurological mouse models. While the minimal therapeutic level of FXN protein to be restored and biodistribution have recently been defined for the heart, it is unclear if FXN overexpression could be harmful. Indeed, depending on the vector delivery route and dose administrated, the resulting FXN protein level could reach very high levels in the heart, cerebellum, or in off-target organs such as the liver. The present study demonstrates safety of FXN cardiac overexpression up to 9fold the normal endogenous level, but significant toxicity to the mitochondria and heart above 20-fold. We show gradual severity with increasing FXN overexpression, ranging from subclinical cardiotoxicity to left ventricle dysfunction. This appears to be driven by impairment of mitochondria respiratory chain and ultrastructure, which lead to cardiomyocytes subcellular disorganization, cell death and fibrosis. Overall, this study underlines the need, during the development of gene therapy approaches, to consider appropriately vector expression level, long term safety and biomarkers to monitor such events.

INTRODUCTION

Friedreich ataxia (FA) is a rare neurodegenerative disease characterized by spinocerebellar and sensory ataxia associated with hypertrophic cardiomyopathy (HCM). [START_REF] Burk | Friedreich Ataxia: current status and future prospects[END_REF] Cardiac dysfunction is the main cause of premature death in FA patients. [START_REF] Tsou | Mortality in Friedreich ataxia[END_REF] Nighty-five percent of FA patient present a homozygous (GAA) expansion within the first intron of the frataxin gene (FXN). [START_REF] Campuzano | Friedreich's ataxia: autosomal recessive disease caused by an intronic GAA triplet repeat expansion[END_REF] This pathological expansion causes the heterochromatinization of the locus leading to reduced transcription of the FXN gene. [START_REF] Saveliev | DNA triplet repeats mediate heterochromatin-protein-1-sensitive variegated gene silencing[END_REF] FXN is a highly conserved mitochondrial protein regulating the biosynthesis of iron sulfur clusters (Fe-S) through an interaction with the de novo Fe-S complex assembly machinery. [START_REF] Maio | Mammalian iron-sulfur cluster biogenesis: Recent insights into the roles of frataxin, acyl carrier protein and ATPase-mediated transfer to recipient proteins[END_REF] Fe-S are prosthetic groups crucial for many biological functions, including the mitochondrial respiratory chain, heme, lipoid acid and molybdenum biosynthesis, tRNA thiolation and iron metabolism. [START_REF] Sheftel | Iron-sulfur proteins in health and disease[END_REF][START_REF] Mayr | Lipoic acid biosynthesis defects[END_REF][START_REF] Leimkuhler | Shared function and moonlighting proteins in molybdenum cofactor biosynthesis[END_REF][START_REF] Braymer | Iron-sulfur cluster biogenesis and trafficking in mitochondria[END_REF] Frataxin deficiency leads to impaired Fe-S biogenesis, impairment of Fe-S enzymes, mitochondrial dysfunction, iron metabolism dysregulation and eventually to cellular dysfunction and death. [START_REF] Puccio | Mouse models for Friedreich ataxia exhibit cardiomyopathy, sensory nerve defect and Fe-S enzyme deficiency followed by intramitochondrial iron deposits[END_REF][START_REF] Chandran | Inducible and reversible phenotypes in a novel mouse model of Friedreich's Ataxia[END_REF] The therapeutic potential of AAV-mediated in vivo gene therapy in preventing and rescuing mitochondrial dysfunction associated with FXN deficiency, both in cardiac and neurological tissues, has clearly been demonstrated in several mouse studies. [START_REF] Perdomini | Prevention and reversal of severe mitochondrial cardiomyopathy by gene therapy in a mouse model of Friedreich's ataxia[END_REF][START_REF] Gerard | An AAV9 coding for frataxin clearly improved the symptoms and prolonged the life of Friedreich ataxia mouse models[END_REF][START_REF] Piguet | Rapid and Complete Reversal of Sensory Ataxia by Gene Therapy in a Novel Model of Friedreich Ataxia[END_REF] To successfully translate these initial proof-of-concept studies to the clinic, we undertook to define the therapeutic thresholds conditioning the rescue of the cardiac phenotype, as well as the potential toxic effects associated with FXN overexpression. This first point was addressed in a recent publication describing the dose-response studies we have conducted in the cardiac Mck conditional knockout mouse model (or Mck mouse), which recapitulates most features of the FA cardiomyopathy. [START_REF] Puccio | Mouse models for Friedreich ataxia exhibit cardiomyopathy, sensory nerve defect and Fe-S enzyme deficiency followed by intramitochondrial iron deposits[END_REF][START_REF] Belbellaa | Correction of half the cardiomyocytes fully rescue Friedreich ataxia mitochondrial cardiomyopathy through cell-autonomous mechanisms[END_REF] We demonstrated that the therapeutic outcome of AAV-FXN gene therapy is directly and proportionally correlated with the vector biodistribution in the heart. The correction of only half the cardiomyocyte was sufficient to restore fully the cardiac morphology and function. [START_REF] Belbellaa | Correction of half the cardiomyocytes fully rescue Friedreich ataxia mitochondrial cardiomyopathy through cell-autonomous mechanisms[END_REF] Regarding the maximum level of FXN protein expression tolerated, earlier mouse studies have reported that the constitutive overexpression of FXN in muscle and heart up to 6-fold the normal level was innocuous. [START_REF] Pook | Rescue of the Friedreich's ataxia knockout mouse by human YAC transgenesis[END_REF][START_REF] Miranda | Frataxin overexpressing mice[END_REF] In contrast, several recent studies have suggested otherwise when constitutive or induced FXN overexpression reach higher levels, in yeast (>14-fold), [START_REF] Wang | The role of frataxin in fission yeast iron metabolism: implications for Friedreich's ataxia[END_REF] HEK293 inducible cell line (>13fold), [START_REF] Vannocci | Adding a temporal dimension to the study of Friedreich's ataxia: the effect of frataxin overexpression in a human cell model[END_REF] and in transgenic drosophila (>9-fold). [START_REF] Edenharter | Overexpression of Drosophila frataxin triggers cell death in an iron-dependent manner[END_REF][START_REF] Navarro | Overexpression of human and fly frataxins in Drosophila provokes deleterious effects at biochemical, physiological and developmental levels[END_REF] However, the possible toxicity of similar levels of FXN overexpression were never investigated in preclinical relevant animal models, i.e. rodent or large animal models, nor in the context of AAV-mediated gene therapy.

In the present study, we investigated whether in vivo AAV-mediated gene transfer could be detrimental in wildtype mice or frataxin-deficient Mck mice. We evaluated FXN overexpression in vivo and its functional and morphological consequences on mitochondria and the heart. FXN protein expression over 20-fold the endogenous level is toxic for the mitochondria and results in severe impairment of complexes I and II enzymatic activity, alteration of the mitochondria ultrastructure, cardiomyocytes cell death and fibrosis leading to heart dysfunction. This was also associated with an increased mitochondria biomass in cardiomyocytes and the induction of the integrated stress response (ISR). Overexpression of frataxin up to 9-fold the endogenous level in the cardiac tissue was uneventful, suggesting that the maximum safe level of FXN overexpression is between 9 and 20-fold the normal level. Strikingly, liver overexpression of FXN >90-fold the normal level did not result in any detectable toxicity, suggesting organs specific susceptibility to FXN overexpression in mice.

RESULTS

FXN overexpression leads to impairment of mitochondria SDH activity

Previously, we conducted a dose-response study in 5-weeks old Mck mice injected intravenously with decreasing doses of AAVRh.10-CAG-hFXN-HA vector, from 5x10 [START_REF] Gerard | An AAV9 coding for frataxin clearly improved the symptoms and prolonged the life of Friedreich ataxia mouse models[END_REF] down to 1x10 [START_REF] Perdomini | Prevention and reversal of severe mitochondrial cardiomyopathy by gene therapy in a mouse model of Friedreich's ataxia[END_REF] vg/kg. [START_REF] Belbellaa | Correction of half the cardiomyocytes fully rescue Friedreich ataxia mitochondrial cardiomyopathy through cell-autonomous mechanisms[END_REF] Upon sacrifice at 12 weeks of age, the human FXN protein level measured in the heart by ELISA assay, ranged from 2 to 10,927 ng of FXN per mg of total heart protein (ng/mg). In wild-type (WT) 9 weeks old C57/B6J mice (n=6), the mouse FXN protein level measured by ELISA in the heart was 147±42 ng/mg. Despite more than 70-fold overexpression, the cardiac function and morphology of treated Mck mice was rescued. In the present study, we performed additional histological analysis on the heart tissue sections from the same Mck mice to further investigate the relationship between FXN overexpression in the heart and the rescue of mitochondrial metabolism. Unexpectedly, the heart tissue sections from Mck mice treated at the highest dose of vector, i.e 5x10 [START_REF] Gerard | An AAV9 coding for frataxin clearly improved the symptoms and prolonged the life of Friedreich ataxia mouse models[END_REF] vg/kg, displayed regions with very high level of hFXN expression, where SDH enzymatic activity was systematically impaired (Figures 1A andS1).

This was particularly obvious in the mouse with the highest cardiac vector biodistribution (7.68 per diploid genome on average) and protein concentration (10,927 ng/mg or 74-fold the endogenous level) (Fig. 1A, first row). No SDH impairment was observed in the cardiomyocytes expressing much lower hFXN-HA levels or neighboring these hotspots of expression. Quantification of the heart surface positive for SDH enzymatic activity in the Mck mice treated with the highest dose (n=3, 73%±2%) in comparison to NaCl-injected WT mice (n=7, 83%±4%) revealed that less than 12% of the total heart surface was affected. These hotspots were not associated with cardiomyocyte cell death, fibrosis or cell-infiltrations, as assessed by hematoxylin-eosin (HE) and wheat germ agglutinin (WGA) staining of adjacent heart tissue sections, in comparison to untreated Mck mice (Figure 1B). Interestingly, the cardiomyocytes with high hFXN expression and SDH activity impairment, did not show any iron accumulation following Perls-DAB staining of Fe 3+ (Figure 1C), as this would be expected following FXN deficiency and/or Fe-S biosynthesis impairment. [START_REF] Puccio | Mouse models for Friedreich ataxia exhibit cardiomyopathy, sensory nerve defect and Fe-S enzyme deficiency followed by intramitochondrial iron deposits[END_REF][START_REF] Richardson | The ins and outs of mitochondrial iron-loading: the metabolic defect in Friedreich's ataxia[END_REF] Importantly, the left ventricle (LV) function of these mice did not appear substantially impaired at rest at 12 weeks of age (Figure 1D S1). Most likely, this is explained by the relative small proportion of cardiomyocytes and heart surface affected overall (<12%). Indeed, we showed previously that the functional rescue of the cardiac phenotype is achieved with as low as 50-60% of heart surface corrected. [START_REF] Belbellaa | Correction of half the cardiomyocytes fully rescue Friedreich ataxia mitochondrial cardiomyopathy through cell-autonomous mechanisms[END_REF] It is worth noting that these mice did not display significant LV hypertrophy (Figure 1F), but the LV diameters at the end systole (LV-ESD) and diastole (LV-EDD) appeared slightly higher in the mouse treated at 5x10 13 vg/kg and expressing 74-fold the normal level of FXN (Figure S1A-B). Overall, these results suggested that very high level of hFXN-HA protein in cardiomyocytes could lead to impaired mitochondrial SDH enzymatic activity but without notable iron accumulation.

Overexpression of hFXN in WT mice leads to mitochondrial and cardiac toxicity

In order to rule out any potential confounding effects from the Mck mouse cardiac phenotype, we undertook to replicate these experimental conditions in WT C57/B6J mice. WT mice received 5x10 14 vg/kg (n=4) or 5x10 [START_REF] Gerard | An AAV9 coding for frataxin clearly improved the symptoms and prolonged the life of Friedreich ataxia mouse models[END_REF] vg/kg (n=3) of the same AAVRh.10-CAG-hXN-HA vector at 7 weeks of age (Table S1). At the 5x10 [START_REF] Piguet | Rapid and Complete Reversal of Sensory Ataxia by Gene Therapy in a Novel Model of Friedreich Ataxia[END_REF] vg/kg dose, we were able to replicate similar VCN (≥7) and hFXN-HA expression (≥ 10,000 ng/mg) (Figure 2A-C), as reported above in Mck mice treated at 5x10 13 vg/kg. However, one-log higher of vector dose was required in WT mice to achieve the same vector biodistribution and overexpression than in Mck mice. This might suggest a difference of transgene expression between WT and Mck mice. However, no significant difference in hFXN-HA expression when normalized to the vector copy number was observed between treated WT mice (n=7) and Mck mice (n=35) (Figure S2). At the study endpoint, 21 weeks of age, all treated WT mice were alive, with normal body weight growth curve (Figure 2D) and no behavioral sign of stress or suffering. At necropsy, no gross anatomical anomaly was observed at the levels of the heart, lung, liver, kidney, digestive tract, or skeletal muscles.

The consequences of hFXN-HA overexpression on the heart histology were evaluated following HE and WGA staining, performed on adjacent heart frozen tissue sections (Figures 2E andS3). Both staining revealed and confirmed sparse fibrotic patches, but only in the animals treated at 5x10 14 vg/kg and more visibly in the three mice with the highest transgene expression, ranging from 5,206 up to 12,773 ng/mg, i.e. 34 and 85-fold the normal FXN level, respectively (Figure 2C). Heart tissue sections from WT mice treated at 5x10 13 vg/kg and NaClinjected WT mice appeared similar (Figures 2C andS3). While the overall total extend of heart fibrosis was not statistically different between NaCl-injected WT mice and WT mice treated at 5x10 14 or 5x10 [START_REF] Gerard | An AAV9 coding for frataxin clearly improved the symptoms and prolonged the life of Friedreich ataxia mouse models[END_REF] vg/kg (Figure 2F), the RTqPCR analysis of Col1a1, Col3a1 and Tgfβ1 revealed significant increased expression of Col1a1 and Col3a1 in the heart of WT mice treated at 5x10 14 vg/kg, indicating ongoing fibrosis (Figure 2G).

While WT mice treated at 5x10 14 vg/kg displayed significant increased Nppa gene expression (Figure 1H), indicative of volume/pressure overload, none of them developed severe cardiac dysfunction at 21 weeks of age (Figure 1I-K). Nonetheless, the three mice with the highest level of transgene expression in the 5x10 [START_REF] Piguet | Rapid and Complete Reversal of Sensory Ataxia by Gene Therapy in a Novel Model of Friedreich Ataxia[END_REF] vg/kg group displayed meaningful reduction of LV shortening fraction at rest (Figures 2I and Video S1) and increased heart hypertrophy (Figure 2J-K). Importantly, no functional nor morphologic heart alteration was observed in the mice treated with vehicle or at 5x10 13 vg/kg. The inverse correlation between hFXN-HA expression and the LV SF was statistically significant (Figure 2L).

To assess the mitochondria function in relation to the level of hFXN-HA expression, semiquantitative histoenzymatic assays were performed on adjacent heart tissue sections. We probed the activity of the respiratory chain complexes I, II and IV (Figure 3 andS3). As reported in Mck mice (Figure 1A), the co-labelling and colocalization analysis of SDH enzymatic activity and hFXN-HA revealed hotspots of transgene expression where the SDH enzymatic activity was strongly reduced (Figure 3). However, the overall heart surface affected was relatively limited (Figure S3) and this was observed only in mice treated at 5x10 14 vg/kg. SDH enzymatic activity in WT mice treated at 5x10 13 vg/kg appeared similar to NaCl-injected WT mice (Figures 3 andS3). When quantified, the total surface positive for SDH activity was respectively 95.0% ± 2.1% (n=4) and 98.5% ± 0.3% (n=3), in mice treated at 5x10 14 and 5x10 13 vg/kg. Of note, the SDH staining and image acquisition and quantification were performed in batch, with all individual from a given study processed in a single run, including untreated WT and Mck controls. However, there were some variations from one run to another, at different steps and especially during the image quantification which thresholding result lead to bias in the background signal. This explains the difference in WT mice values, between the analysis of this cohort of mice and the one reported earlier. WT mice treated at 5x10 14 vg/kg also displayed sparse impairment of the enzymatic activity of complex I (NADH), contrary to WT mice treated at 5x10 13 vg/kg. In contrast, the enzymatic activity of complex IV (COX) seemed unaffected in both dose-group (Figures 3 andS3). Strikingly, these results were reminiscent of what is observed in the heart of untreated Mck mice, in which FXN deficiency leads to the impairment of Fe-S enzyme including SDH and NADH, but not COX, dependent on heme cofactors and not Fe-S cofactors. [START_REF] Rich | The mitochondrial respiratory chain[END_REF] The consequences of FXN overexpression on the cardiomyocytes ultrastructure were also assessed by transmission electron microscopy (TEM). In contrast to WT mice treated at 5x10 13 vg/kg or NaCl, WT mice treated at 5x10 14 vg/kg displayed a substantial proportion of cardiomyocytes with severe alterations of their subcellular organization, with scattered and disordered myofibrils (Figure 3). Their mitochondria were swollen with sparce cristae, whose stacking is crucial for mitochondria bioenergetic efficiency. [START_REF] Dudkina | Structure and function of mitochondrial supercomplexes[END_REF] The total volume occupied by mitochondria in cardiomyocytes also appeared significantly increased, suggesting altered mitochondrial proliferation or turn-over. We also frequently observed the presence of electron dense bodies, inside the mitochondria matrix. These were not reminiscent of collapsed cristae nor iron deposits, as commonly observed in FA patients and untreated Mck mice cardiomyocytes. [START_REF] Puccio | Mouse models for Friedreich ataxia exhibit cardiomyopathy, sensory nerve defect and Fe-S enzyme deficiency followed by intramitochondrial iron deposits[END_REF][START_REF] Koeppen | The pathogenesis of cardiomyopathy in Friedreich ataxia[END_REF][START_REF] Ward | Longitudinal evaluation of iron concentration and atrophy in the dentate nuclei in friedreich ataxia[END_REF] To rule-out the presence of mitochondrial iron deposits, adjacent ultrathin sections were stained with bismuth subnitrate to label iron complexed with ferritin, in the mitochondria as well as in lysosomes. [START_REF] Koeppen | The pathogenesis of cardiomyopathy in Friedreich ataxia[END_REF][START_REF] Michael | Iron and iron-responsive proteins in the cardiomyopathy of Friedreich's ataxia[END_REF][START_REF] Koeppen | Friedreich's ataxia: pathology, pathogenesis, and molecular genetics[END_REF][START_REF] Arosio | Ferritin, cellular iron storage and regulation[END_REF] All WT mice displayed lysosomal labelling, as expected since this organelle is central to ferritin turn-over, but none showed mitochondrial labelling in contrast to untreated Mck mice (Figure 3). Collectively, these observations rule out iron accumulation as part of the cardioand mitochondrial toxicity mechanism, which is in line with the lack of Perls labelling observed in treated Mck mice (Figure 1C).

To further investigate the mitochondria biomass, heart tissue sections from the same WT mice were colabelled for SDH enzymatic activity and for prohibitin (PHB) (Figure 4A). PHB is a ubiquitous protein predominately located in the mitochondrial inner membrane, [START_REF] Tatsuta | Formation of membrane-bound ring complexes by prohibitins in mitochondria[END_REF] which we used previously to assess mitochondria biomass. [START_REF] Belbellaa | Correction of half the cardiomyocytes fully rescue Friedreich ataxia mitochondrial cardiomyopathy through cell-autonomous mechanisms[END_REF] In line with the above TEM observations, WT mice treated at 5x10 14 vg/kg displayed substantial increase of PHB labelling, but less than untreated Mck mice. In WT mice treated at 5x10 14 vg/kg, we also observed a substantial co-localization between increased PHB labelling and reduction of SDH enzymatic activity. In contrast, WT mice treated at 5x10 13 vg/kg appeared similar to NaCl-injected WT mice. Interestingly, WT mice treated at 5x10 14 vg/kg also displayed increased cardiac gene expression for Asns, Mthfd2 and Ddit3, hallmark genes of the integrated stress response (ISR) (Figure 4C). The expression of Fgf21 and Gdf15, which are downstream targets gene of the ISR, were also strongly increased, although only statistically significant for Fgf21 (Figure 4D-E). Interestingly, Fgf21 and Gdf15 have been proposed as biomarkers of mitochondria and heart dysfunction. [START_REF] Planavila | FGF21 and Cardiac Physiopathology[END_REF][START_REF] Montero | GDF-15 Is Elevated in Children with Mitochondrial Diseases and Is Induced by Mitochondrial Dysfunction[END_REF] Altogether, these results suggest that cardiac overexpression of hFXN-HA ≥20-30-fold the normal endogenous level is toxic for the heart and mitochondria. The mitochondria toxicity is characterized by severe alterations of the mitochondria ultrastructure and bioenergetics, and the induction of ISR.

High level of FXN overexpression results in acute cardiotoxicity and compromised the gene therapy outcome

While the study in WT mice recapitulated and confirmed the mitochondrial toxicity observed initially in

Mck mice, it was unclear if this toxicity could also be explained by the high dose of vector administrated, the resulting high number of vector copies per cell, and/or the HA tag fused to transgene (Figure 5A). To address these questions, we designed an optimized expression cassette and vector, AAVRh.10-hFXN (Figure 5B). The 5'UTR and Kozak sequences were optimized. The human FXN cDNA was not codon-optimized, but the HA tag was removed. The same promoter and polyA sequences were used in both vector (Figure 5A-B). In this third mouse study, Mck mice at 7-weeks old (upon heart failure) received 2.5x10 [START_REF] Gerard | An AAV9 coding for frataxin clearly improved the symptoms and prolonged the life of Friedreich ataxia mouse models[END_REF] vg/kg of the optimized AAVRh.10-hFXN vector (n=8) or 2.5x10 13 vg/kg of the non-optimized AAVRh.10-CAG-hFXN-HA vector (n=6), and then sacrificed at 15 weeks of age (Table S1). For controls, 7 weeks old WT mice received NaCl (n=10) or the optimized AAVRh.10-hFXN vector at doses of 2.5x10 13 (n=2) or 5x10 [START_REF] Perdomini | Prevention and reversal of severe mitochondrial cardiomyopathy by gene therapy in a mouse model of Friedreich's ataxia[END_REF] vg/kg (n=1).

When compared to the non-optimized vector, the optimized vector led to much higher levels of hFXN expression in the heart and liver despite similar VCN (Figures 5C-F, 8A and 8C-E). The resulting level of hFXN protein in the heart were on average 23,376±9,336 ng/mg and 441±276 ng/mg, respectively, corresponding to 156-fold and 2.9-fold the endogenous FXN level (Figure 5D). This large difference in expression was confirmed by western blot analysis (Figure 5E-F). Despite the very high overexpression in Mck mice treated with the optimized vector, no significant accumulation of FXN precursor (23KDa) was observed (Figure 5H). While we observed some accumulation of the FXN intermediary form (19KDa), the vast majority was processed into the mature form (14KDa). These results suggest that the majority of the transgenic hFXN protein was targeted to the mitochondria, without severe saturation or impairment of the mitochondria import and processing capacity. [START_REF] Schmucker | The in vivo mitochondrial twostep maturation of human frataxin[END_REF] Of note, the biodistribution and expression of the optimized AAVRh.10-hFXN vector was also quantified in WT mice treated at 2.5x10 13 (n=2) or 5x10 [START_REF] Perdomini | Prevention and reversal of severe mitochondrial cardiomyopathy by gene therapy in a mouse model of Friedreich's ataxia[END_REF] vg/kg (n=1). The vector biodistribution was similar to Mck mice treated at identical doses, [START_REF] Belbellaa | Correction of half the cardiomyocytes fully rescue Friedreich ataxia mitochondrial cardiomyopathy through cell-autonomous mechanisms[END_REF] with respectively 0.983 and 0.475 VCN at 2.5x10 13 vg/kg and 0.078 VCN at 5x10 12 vg/kg. In contrast, the vector expression was much lower compared to Mck mice treated with the same vector, with respectively 5,963 and 3,526 ng/mg at 2.5x10 [START_REF] Gerard | An AAV9 coding for frataxin clearly improved the symptoms and prolonged the life of Friedreich ataxia mouse models[END_REF] vg/kg and 355ng/mg at 5x10 12 vg/kg. The expression normalized to the VCN of the optimized vector was 35-fold higher than the expression of the non-optimized vector in the heart of Mck mice (Figure S2). As mentioned above, the expression of the nonoptimized AAVRh.10-CAG-hFXN-HA vector was similar in the heart of WT and Mck mice (Figure S2), despite the significant transcriptional and metabolic dysregulations occurring upon FXN depletion. [START_REF] Puccio | Mouse models for Friedreich ataxia exhibit cardiomyopathy, sensory nerve defect and Fe-S enzyme deficiency followed by intramitochondrial iron deposits[END_REF][START_REF] Perdomini | Prevention and reversal of severe mitochondrial cardiomyopathy by gene therapy in a mouse model of Friedreich's ataxia[END_REF][START_REF] Belbellaa | Correction of half the cardiomyocytes fully rescue Friedreich ataxia mitochondrial cardiomyopathy through cell-autonomous mechanisms[END_REF][START_REF] Huang | Molecular and functional alterations in a mouse cardiac model of Friedreich ataxia: activation of the integrated stress response, eIF2alpha phosphorylation, and the induction of downstream targets[END_REF] In contrast, the expression normalized to the VCN of the optimized AAVRh.10-hFXN vector was on average 5-fold higher in the heart of Mck mice than in WT mice (Figure S2). This increased expression is possibly driven by the strong induction of the ISR and Eif2α phosphorylation in Mck mice heart, [START_REF] Huang | Molecular and functional alterations in a mouse cardiac model of Friedreich ataxia: activation of the integrated stress response, eIF2alpha phosphorylation, and the induction of downstream targets[END_REF] which might favorably impact the transcription and translation of the optimized expression cassette, in part through the optimized 5'UTR and Kozak sequence. In line with this hypothesis, both vectors displayed similar VCN and transcription levels in the liver of Mck mice (Figure 8A-B), which is not depleted in FXN and does not present any functional or molecular phenotype. Nonetheless, the optimization of the 5'UTR and Kozak sequences still resulted in a 48-fold increase of the average hFXN protein levels in the liver (Figure 8C-E). Furthermore, the optimized AAVRh.10-hFXN vector lead to much higher hFXN protein expression in the heart than liver of the same Mck mice (Figures 5D and8C).

To assess the consequences of FXN overexpression on the outcome of FA cardiac gene therapy, the cardiac function and morphology of the same Mck mice was assessed by echocardiography (Table S1).

Longitudinal echocardiography analyses were performed from 7 to 15 weeks of age (Figures 5I-L andS4). By 12 weeks of age, Mck mice treated with either vector displayed full correction of the heart function and morphology.

However, mice treated with the optimized AAVRh.10-hFXN vector started deteriorating afterward, while sustained full correction was achieved in mice treated with the non-optimized AAVRh.10-CAG-hFXN-HA vector. This deterioration was characterized by a decrease of the LV SF (Figure 5I) and CO/BW (Figure 5J) and an increase of the LV mass (Figure 5K) and LV ESD (Figure 5L). The LV EDD was also increased but in a delayed manner compared to the other parameters measured (Figure S4A). Overall, this suggest a primary impairment of the contractile and systolic function of the LV, in line with our previous observation in WT mice treated with the non-optimized AAVRh.10-CAG-hFXN-HA (Figure 2I-J and Video S1). The impairment of the cardiac function in Mck mice treated with the optimized vector is supported by the increased gene expression of Nppa and Nppb, indicative of pressure/volume overload (Figure 5M). Histological analysis of the heart fibrosis and cell infiltration were also performed on heart tissue sections (Figure 6A-B). Following HE stain, Mck mice treated with the optimized vector displayed substantial heart fibrosis, distributed throughout the left ventricle and associated with numerous cell infiltrates (Figure 6A). Mck mice treated with the non-optimized vector displayed a few fibrotic patches. Importantly, heart tissue sections from WT mice treated with the optimized vector at 2.5x10 [START_REF] Gerard | An AAV9 coding for frataxin clearly improved the symptoms and prolonged the life of Friedreich ataxia mouse models[END_REF] (n=2) or 5x10 [START_REF] Perdomini | Prevention and reversal of severe mitochondrial cardiomyopathy by gene therapy in a mouse model of Friedreich's ataxia[END_REF] vg/kg (n=1), looked similar to heart tissue section from NaCl-injected WT mice (Figure 6A). This suggest the lack of intrinsic toxicity of the optimized vector. To quantify the accumulation of extracellular matrix associated with fibrosis, adjacent heart tissue sections were stained with WGA (Figure 6B). Mck mice treated with the non-optimized vector displayed more labelling than WT mice treated with NaCl, but much lower level than Mck mice treated with the optimized vector (Figure 6C). Furthermore, these observations were in agreement with the increased gene expression of Col1a1, Col3a1

and Tgfβ which are indicative of on-going fibrosis (Figure 6D), but also of IL1b and IL6 which are indicative of on-going inflammatory response (Figure 6E). To further investigate the nature of these cell infiltrates, adjacent heart tissue sections were immunolabelled for CD14, a maker of monocytes and macrophages (Figures 6F andS5A), CD45, a general marker of leukocytes including lymphocytes T, B and monocytes (Figure S5B) and CD3, a general marker of lymphocytes including regulatory T cells and cytotoxic/NKT cells (Figure S5C). Spleen tissue sections were used as positive labelling controls for all these markers (Figure S5A-C). In line with the inflammatory and fibrotic response measured, Mck mice treated with the optimized showed extensive infiltrations of CD14+ and CD45+ cells, while mice treated with non-optimized vector showed much lower CD14+ and CD45+ labelling. Importantly, WT mice treated with the optimized vector did not show any cell infiltrates, supporting further the lack of intrinsic immunogenicity of the vector or its formulation (Figure S5A-C). In contrast, we were unable to identify CD3+ cell infiltrates in the heart tissue sections from animal treated with either vector, which would be expected in case of cytotoxic immune response (NK cells), as shown previously. [START_REF] Manno | Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response[END_REF][START_REF] Mingozzi | CD8(+) T-cell responses to adenoassociated virus capsid in humans[END_REF] The investigation of the mitochondria function in Mck mice treated with the optimized vector at 2.5x10 [START_REF] Gerard | An AAV9 coding for frataxin clearly improved the symptoms and prolonged the life of Friedreich ataxia mouse models[END_REF] vg/kg (Figures 7A-B andS6A), revealed similar alterations as reported above in the Mck and WT mice treated with the non-optimized vector at higher doses (Figures 1A and3). However, the severity and extend was much higher in mice treated with the optimized vector, in line with the much higher hFXN overexpression across the heart. In Mck mice treated with the optimized vector at 2.5x10 [START_REF] Gerard | An AAV9 coding for frataxin clearly improved the symptoms and prolonged the life of Friedreich ataxia mouse models[END_REF] vg/kg, co-labeling and colocalization analysis revealed numerous cardiomyocytes throughout the heart, with very high level of FXN expression and impaired SDH enzymatic activity (Figure 7A). These hotspots of expression covered most of the heart surface (Figure S6A). In contrast, Mck mice treated with the non-optimized vector were rescued for SDH enzymatic activity throughout the heart, except for a few fibrotic patches (Figure S6B). Interestingly, the protein level of SDH subunit b (SDHb) was significantly decreased in the heart of the Mck mice treated with the optimized vector, when compared with Mck mice treated with the non-optimized vector (Figure 5E andG). Moreover, Mck mice treated with the optimized vector also showed impaired NADH enzymatic activity throughout the heart (Figures 7B andS6A), in line with previous observations in WT mice treated with the non-optimized vector at 5x10 14 vg/kg (Figures 3 andS3). In contrast, the WT mouse treated with the optimized vector at 2.5x10 [START_REF] Gerard | An AAV9 coding for frataxin clearly improved the symptoms and prolonged the life of Friedreich ataxia mouse models[END_REF] To assess the cardiomyocytes and mitochondria ultrastructure following the administration of the optimized vector, sample from the LV myocardium of these same Mck (n=3) and WT (n=1) mice were collected and observed under TEM after negative staining (Figure 7C). Mck mice treated at 2.5x10 [START_REF] Gerard | An AAV9 coding for frataxin clearly improved the symptoms and prolonged the life of Friedreich ataxia mouse models[END_REF] vg/kg displayed severe alterations of cardiomyocytes subcellular organization, with scattered and disordered myofibrils, accumulation of swollen mitochondria, with very few visible cristae (Figure 7C). We also observed electron dense bodies, inside the matrix of several mitochondria. These did not resemble collapsed cristae nor iron deposits, but appeared similar to what we observed in WT mice treated with the non-optimized vector at 5x10 14 vg/kg (Figure 3). The absence of iron accumulation in the heart of these Mck mice was confirmed with DAB-Perl staining (Figure 7D). Finally, we also analyzed the liver of the same Mck mice treated with the optimized vector at 2.5x10 [START_REF] Gerard | An AAV9 coding for frataxin clearly improved the symptoms and prolonged the life of Friedreich ataxia mouse models[END_REF] vg/kg , to assess possible toxic effects beyond the heart. The level of FXN protein in their liver ranged from 1,419 up to 4,890 ng/mg, when quantified by ELISA assay (Figure 8C). When measured in a distinct cohort of 9-weeks old WT C57/B6J mice, the normal level of mouse FXN protein in the liver is 49.6±7.8 ng/mg (n=5).

Thus, Mck mice treated with the optimized vector at 2.5x10 [START_REF] Gerard | An AAV9 coding for frataxin clearly improved the symptoms and prolonged the life of Friedreich ataxia mouse models[END_REF] vg/kg express up to 97-fold the endogenous level in the liver. The high expression was confirmed by Western-Blot analysis (Figure 8D-E). Despite this high level of overexpression ([hFXN]=2,565±1,1123ng/mg; n=8) and the high VCN (31±10; n=8) measured (Figure 8A-C), the liver histology of the three mice evaluated appeared all normal following HE staining, with no obvious sign of fibrosis or cell infiltrations (Figure 8G). The latter would have been expected if an adaptative immune response was raised against the transgene and driving the cardiotoxicity. Moreover, TEM observation of adjacent liver samples collected from the same mice (n=3) did not reveal any obvious alterations of hepatocytes subcellular organization or alterations of mitochondria ultrastructure and/or biomass (Figure 8G). These results support further the lack of intrinsic toxicity of the AAVRh.10-hFXN vector or its formulation, when administrated in Mck or WT mice (Figure 8G). The lack of similar mitochondrial toxicity in the liver was also supported by quantification of SDHb protein level (Figure 8F), which was similar to Mck mice treated with the non-optimized vector and expressing much lower levels of FXN ([hFXN]=53±14ng/mg; n=3). Altogether, these results support the lack of acute liver toxicity, despite very high level of FXN overexpression, which contrast with the observed cardiotoxicity in the same mice.

DISCUSSION

Here, we showed unequivocally the cardiotoxicity of FXN overexpression when expressed >20-fold the endogenous level and its safety when expressed <9-fold. This was demonstrated in three independent studies, using two different animal models and two different AAVRh.10 vector constructs. The severity of this cardiotoxicity appeared to be proportional to the level of FXN overexpression, but also to the proportion and extent of cardiomyocytes affected throughout the heart. This led to more or less severe impairment of LV function and morphology, which then evolved either towards compensated heart hypertrophy or severe impairment of heart function. As demonstrated here, this cardiotoxicity might also compromise the therapeutic outcome of cardiac gene therapy for FA. The identification of this toxic threshold will help define more precisely the safe and efficient range of FXN expression for sustained correction of the FA cardiac phenotype. Our results also suggest that this toxicity might not be generalizable to all organs and/or cell types, as the mouse liver appeared to be tolerant to FXN overexpression up to 90-fold the normal level.

The [hFXN] values and fold-overexpression numbers reported here correspond to average values reflecting unequal expression of FXN among the cardiomyocytes, some with relatively low or moderate level while hotspots with very high levels. As shown previously, [START_REF] Prasad | Robust cardiomyocyte-specific gene expression following systemic injection of AAV: in vivo gene delivery follows a Poisson distribution[END_REF] the inhomogeneous distribution of the FXN expression in the heart reflects most likely the inhomogeneous diffusion of the AAV vectors throughout the cardiac vasculature and then into the parenchyma. This is most likely inherent characteristic of AAV and intravenous administration. Nonetheless, the safe level of FXN overexpression in the heart (≤9-fold the endogenous level) is in line with previous studies conducted in yeast, mammalian cell lines, transgenic drosophila and mice models overexpressing constitutively FXN. Collectively, these studies reported toxicity when FXN overexpression was higher than 6 to 10-fold the endogenous level, [START_REF] Wang | The role of frataxin in fission yeast iron metabolism: implications for Friedreich's ataxia[END_REF][START_REF] Vannocci | Adding a temporal dimension to the study of Friedreich's ataxia: the effect of frataxin overexpression in a human cell model[END_REF][START_REF] Edenharter | Overexpression of Drosophila frataxin triggers cell death in an iron-dependent manner[END_REF][START_REF] Navarro | Overexpression of human and fly frataxins in Drosophila provokes deleterious effects at biochemical, physiological and developmental levels[END_REF][START_REF] Vannocci | A new tool to determine the cellular metabolic landscape: nanotechnology to the study of Friedreich's ataxia[END_REF][START_REF] Llorens | Causative role of oxidative stress in a Drosophila model of Friedreich ataxia[END_REF] but good tolerance for lower levels. [START_REF] Pook | Rescue of the Friedreich's ataxia knockout mouse by human YAC transgenesis[END_REF][START_REF] Miranda | Frataxin overexpressing mice[END_REF][START_REF] Seguin | Overexpression of the yeast frataxin homolog (Yfh1): contrasting effects on iron-sulfur cluster assembly, heme synthesis and resistance to oxidative stress[END_REF][START_REF] Ristow | Frataxin activates mitochondrial energy conversion and oxidative phosphorylation[END_REF][START_REF] Shoichet | Frataxin promotes antioxidant defense in a thiol-dependent manner resulting in diminished malignant transformation in vitro[END_REF][START_REF] Runko | Overexpression of frataxin in the mitochondria increases resistance to oxidative stress and extends lifespan in Drosophila[END_REF] Importantly, the current study did not investigate if the expression of the human FXN in the mouse organism would overestimate or underestimate the toxic threshold to be identified if a similar study were to be conducted with the overexpression of the mouse FXN protein. Indeed, the precursor and mature form of the mouse and human FXN present respectively 70% and 91% identity. Previous studies have shown that the human orthologue can complement the knockout of the mouse Fxn gene, through the genomic insertion and the constitutive expression of the human Fxn gene locus in transgenic mouse models. [START_REF] Pook | Rescue of the Friedreich's ataxia knockout mouse by human YAC transgenesis[END_REF][START_REF] Sarsero | Human BAC-mediated rescue of the Friedreich ataxia knockout mutation in transgenic mice[END_REF] These transgenic mice have normal phenotype and life span, therefore suggesting that the human FXN protein has the same functionalities as its mouse orthologue. However, it remained to be shown whether the human and mouse FXN protein have the same affinity and kinetic of activation on the mouse Fe-S cluster assembly complex. It is also unclear if large excess of FXN could stifle and impair the Fe-S cluster assembly complex, and how potential difference in affinity would be impactful. As non-human primate (NHP) have closer physiology to human and express FXN orthologue with higher identity, we expect toxicology study conducted in NHP to be more predictive of future clinical trial in FA patients. Indeed, the FXN precursor proteins in cynomolgus and rhesus monkey, are 91 and 92% identical to the human FXN, respectively.

Our results do not support the hypothesis of a potential intrinsic toxicity of the vector construct or production lot used in these mouse studies, as WT mice injected with the same vectors did not show signs of toxicity, unless the FXN protein level was beyond the toxic threshold. Several studies have shown cytotoxicity or genotoxicity following the administration of high dose vector, particularly in large animal model. [START_REF] Hinderer | Severe Toxicity in Nonhuman Primates and Piglets Following High-Dose Intravenous Administration of an Adeno-Associated Virus Vector Expressing Human SMN[END_REF][START_REF] Nault | Recurrent AAV2-related insertional mutagenesis in human hepatocellular carcinomas[END_REF] Here, we have partially ruled out this possibility as much lower dose of the optimized AAVRh.10-hFXN vector also induced high level of FXN expression and cardiotoxicity. In addition, previous mice studies have shown that similar dose of vector, but with different DNA cargo, were well tolerated up to 5x10 14 vg/kg, without noticeable cardiotoxicity at long term. [START_REF] Foust | Rescue of the spinal muscular atrophy phenotype in a mouse model by early postnatal delivery of SMN[END_REF][START_REF] Bockor | Repeated AAV-mediated gene transfer by serotype switching enables long-lasting therapeutic levels of hUgt1a1 enzyme in a mouse model of Crigler-Najjar Syndrome Type I[END_REF][START_REF] Xu | rAAVrh74.MCK.GALGT2 Protects against Loss of Hemodynamic Function in the Aging mdx Mouse Heart[END_REF][START_REF] Zygmunt | rAAVrh74.MCK.GALGT2 Demonstrates Safety and Widespread Muscle Glycosylation after Intravenous Delivery in C57BL/6J Mice[END_REF][START_REF] Manso | Systemic AAV9.LAMP2B injection reverses metabolic and physiologic multiorgan dysfunction in a murine model of Danon disease[END_REF] In the current study, we did not investigate the potential involvement of the innate or adaptative immune response against the vector, transgene and/or transduced cells. [START_REF] Manno | Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response[END_REF][START_REF] Mingozzi | CD8(+) T-cell responses to adenoassociated virus capsid in humans[END_REF][START_REF] Zhu | The TLR9-MyD88 pathway is critical for adaptive immune responses to adenoassociated virus gene therapy vectors in mice[END_REF][START_REF] Ashley | TLR9 signaling mediates adaptive immunity following systemic AAV gene therapy[END_REF] It is likely that the treated mice developed an humoral response against the capsid, including neutralizing antibodies, as shown previously in many mouse gene therapy studies. [START_REF] Zygmunt | rAAVrh74.MCK.GALGT2 Demonstrates Safety and Widespread Muscle Glycosylation after Intravenous Delivery in C57BL/6J Mice[END_REF][START_REF] Manso | Systemic AAV9.LAMP2B injection reverses metabolic and physiologic multiorgan dysfunction in a murine model of Danon disease[END_REF] However, the emergence of antibody against the capsid has not been shown to preclude the long-term expression of transgene or to drive cardiac inflammation, but only the re-administration of the same vector. [START_REF] Leborgne | IgG-cleaving endopeptidase enables in vivo gene therapy in the presence of anti-AAV neutralizing antibodies[END_REF] Here, we did not explore the potential presence of antibodies against the transgene and more importantly the development of a potential cytotoxic cell response targeting the transduced cardiomyocytes. The cytotoxic immune response was shown previously to result in loss of transgene expression in animal models and patients. [START_REF] Manno | Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response[END_REF][START_REF] Mingozzi | CD8(+) T-cell responses to adenoassociated virus capsid in humans[END_REF][START_REF] Zhu | The TLR9-MyD88 pathway is critical for adaptive immune responses to adenoassociated virus gene therapy vectors in mice[END_REF][START_REF] Ashley | TLR9 signaling mediates adaptive immunity following systemic AAV gene therapy[END_REF] In the present study, the AAVRh.10 vectors were delivered intravenously, leading to broad biodistribution and strong expression in many organs and muscles. [START_REF] Perdomini | Prevention and reversal of severe mitochondrial cardiomyopathy by gene therapy in a mouse model of Friedreich's ataxia[END_REF][START_REF] Hu | RH10 provides superior transgene expression in mice when compared with natural AAV serotypes for neonatal gene therapy[END_REF] Therefore, we would expect a potential cytotoxic immune response to lead to cell infiltration, not only in the heart, but also in all transduced organs, especially the liver for which the AAVRh.10 capsid has the highest tropism. We will investigate these questions in future studies, as they are critical for future clinical development.

Cardiotoxic FXN overexpression appeared to be driven by cell autonomous impairment of the mitochondria function and structure, most likely followed by cardiomyocytes cell death, heart fibrosis and LV contractile dysfunction. Strikingly, the mitochondria toxicity affected NADH and SDH enzymatic activities but not COX. The severe ultrastructure anomalies of the mitochondria most likely contributed to its functional impairment, [START_REF] Dudkina | Structure and function of mitochondrial supercomplexes[END_REF] which altogether leads to higher mitochondria biomass. These features are reminiscent of the cardiac, mitochondrial and biochemical phenotype observed in untreated Mck mice and are hallmarks of FXN deficiency. [START_REF] Puccio | Mouse models for Friedreich ataxia exhibit cardiomyopathy, sensory nerve defect and Fe-S enzyme deficiency followed by intramitochondrial iron deposits[END_REF][START_REF] Perdomini | Prevention and reversal of severe mitochondrial cardiomyopathy by gene therapy in a mouse model of Friedreich's ataxia[END_REF][START_REF] Belbellaa | Correction of half the cardiomyocytes fully rescue Friedreich ataxia mitochondrial cardiomyopathy through cell-autonomous mechanisms[END_REF][START_REF] Wagner | Friedreich's ataxia reveals a mechanism for coordinate regulation of oxidative metabolism via feedback inhibition of the SIRT3 deacetylase[END_REF] However, it is unclear if FXN toxic overexpression was associated or caused by (partial) impairment of Fe-S biogenesis or handling. Indeed, these two cardiac phenotypes differ from one another by the accumulation of mitochondrial iron upon FXN deficiency, [START_REF] Richardson | The ins and outs of mitochondrial iron-loading: the metabolic defect in Friedreich's ataxia[END_REF] which was consistently not observed upon FXN overexpressing in the three mouse studies conducted here. Indeed, cellular iron metabolism and dysregulation is mediated by IRP1, which is a cytosolic Fe-S protein. [START_REF] Richardson | The ins and outs of mitochondrial iron-loading: the metabolic defect in Friedreich's ataxia[END_REF] These results are in line with previous overexpression studies conducted in yeast, [START_REF] Wang | The role of frataxin in fission yeast iron metabolism: implications for Friedreich's ataxia[END_REF] human cells, [START_REF] Vannocci | Adding a temporal dimension to the study of Friedreich's ataxia: the effect of frataxin overexpression in a human cell model[END_REF] and drosophila [START_REF] Edenharter | Overexpression of Drosophila frataxin triggers cell death in an iron-dependent manner[END_REF] where only modest increase in labile-iron was observed. Future studies will be needed to address the underlying mechanism, through time course analysis of pathological events, including exhaustive biochemical analysis of the Fe-S biosynthesis, handling and Fe-S enzymes.

These findings would likely apply to all in-vivo gene replacement strategy for FXN, independently of the AAV serotype or other type of viral vector used. Besides gene replacement strategies, this would also be of concern for alternative approaches if presenting the same risk of acute FXN tissue concentration, either due to their mechanism of action or their delivery modality. This would include synthetic mRNA, [START_REF] Nabhan | Intrathecal delivery of frataxin mRNA encapsulated in lipid nanoparticles to dorsal root ganglia as a potential therapeutic for Friedreich's ataxia[END_REF] FXN protein replacement, [START_REF] Vyas | A TAT-frataxin fusion protein increases lifespan and cardiac function in a conditional Friedreich's ataxia mouse model[END_REF][START_REF] Britti | Frataxin-deficient neurons and mice models of Friedreich ataxia are improved by TAT-MTScs-FXN treatment[END_REF] and in-vivo gene transfer of strong artificial transcription factors. [START_REF] Chapdelaine | Development of an AAV9 coding for a 3XFLAG-TALEfrat#8-VP64 able to increase in vivo the human frataxin in YG8R mice[END_REF][START_REF] Cherif | Increased Frataxin Expression Induced in Friedreich Ataxia Cells by Platinum TALE-VP64s or Platinum TALE-SunTag[END_REF] While the current study was focused on the heart, this mitochondrial toxicity is likely to affect other organs. The dorsal root ganglia, the spinal cord, the cerebellum and dentate nucleus, are important targets to address the neurological symptoms and would be of particular interest for future studies. The spleen and kidney are major off-target sites for AAV vectors following intravenous delivery and should also be considered carefully. Importantly, the FXN level varies largely among these organs, [START_REF] Campuzano | Friedreich's ataxia: autosomal recessive disease caused by an intronic GAA triplet repeat expansion[END_REF][START_REF] Puccio | Mouse models for Friedreich ataxia exhibit cardiomyopathy, sensory nerve defect and Fe-S enzyme deficiency followed by intramitochondrial iron deposits[END_REF] most likely along cell-type specific metabolism and abundance of mitochondria. In the present and previous studies, [START_REF] Piguet | Rapid and Complete Reversal of Sensory Ataxia by Gene Therapy in a Novel Model of Friedreich Ataxia[END_REF][START_REF] Belbellaa | Correction of half the cardiomyocytes fully rescue Friedreich ataxia mitochondrial cardiomyopathy through cell-autonomous mechanisms[END_REF] we have shown that the normal mouse FXN level is 147ng/mg in the heart, 49ng/mg in the liver, 12-25ng/mg in dorsal root ganglia and 28ng/mg in the cerebellum. Therefore, we can hypothesize different therapeutic index for each one of these organs. To ensure the safety of future clinical trials, AAV vectors will need to be designed to manage appropriately their expression profile across these different tissues. This could be achieved with (1) synthetic weak promoter or the endogenous human frataxin promoter, [START_REF] Puspasari | Long range regulation of human FXN gene expression[END_REF][START_REF] Borlongan | Rescue of the Friedreich Ataxia Knockout Mutation in Transgenic Mice Containing an FXN-EGFP Genomic Reporter[END_REF][START_REF] Li | Defining Transcription Regulatory Elements in the Human Frataxin Gene: Implications for Gene Therapy[END_REF] (2) vector de-targeting strategies, [START_REF] Wang | A Rationally Engineered Capsid Variant of AAV9 for Systemic CNS-Directed and Peripheral Tissue-Detargeted Gene Delivery in Neonates[END_REF][START_REF] Xie | MicroRNA-regulated, Systemically Delivered rAAV9: A Step Closer to CNS-restricted Transgene Expression[END_REF] and/or (3) the design of expression cassette responsive to negative cellular feedback loop. Leveraging our knowledge of the cellular response to toxic FXN overexpression might help achieve the self-regulation of the vector expression and avoid toxic overexpression. For example, the 3'UTR sequence could be engineered to targeted by inhibitory endogenous microRNA, specifically induced by the ISR, [START_REF] Chitnis | miR-211 Is a Prosurvival MicroRNA that Regulates chop Expression in a PERK-Dependent Manner[END_REF][START_REF] Ye | Self-adjusting synthetic gene circuit for correcting insulin resistance[END_REF] or other cellular stress response such as oxidative stress response. [START_REF] Shah | Understanding the role of NRF2regulated miRNAs in human malignancies[END_REF][START_REF] Sun | Nrf2-miR-129-3p-mTOR Axis Controls an miRNA Regulatory Network Involved in HDACi-Induced Autophagy[END_REF] In addition, specific routes of drug delivery might also present risk of high local concentration, such as intracardiac injection, [START_REF] Merentie | Efficacy and safety of myocardial gene transfer of adenovirus, adeno-associated virus and lentivirus vectors in the mouse heart[END_REF] or local delivery in the central nervous system. [START_REF] Hordeaux | Toxicology Study of Intra-Cisterna Magna Adeno-Associated Virus 9 Expressing Iduronate-2-Sulfatase in Rhesus Macaques[END_REF][START_REF] Hordeaux | Toxicology Study of Intra-Cisterna Magna Adeno-Associated Virus 9 Expressing Human Alpha-L-Iduronidase in Rhesus Macaques[END_REF] Due diligence in the design of appropriate pharmacokinetic and toxicity studies, in relevant large animal model, will be crucial for the development of safe clinical therapeutic protocol. Finally, the identification of biomarkers to monitor subclinical toxicity and manage such event, would be very advantageous. In this regard, the current study has identified potential candidates, including FGF21, to be explored in future studies.

In summary, the overexpression of FXN is safe when ≤ 9-fold the normal endogenous level but cardiotoxic when greater than 20-fold. The pathological mechanism was partially elucidated and is most likely primed by the alteration of the mitochondria structure and function. Depending on the percentage of cardiomyocytes affected, this resulted either in subclinical or acute cardiotoxicity. The toxic threshold and potential read-out for toxicity identified here will support the design of robust and meaningful toxicity studies, for safer FA gene therapy clinical trials.

MATERIALS AND METHODS

Adeno-associated viral vector constructs and production.

The AAVRh.10-CAG-hFXN-HA and AAVRh.10-hFXN vectors are described in Figure 5A-B, andencode respectively the human frataxin cDNA, including the mitochondrial targeting sequence, and fused or not in Cterminal to the HA tag. These vectors were produced by triple transfection method in HEK293, [START_REF] Rabinowitz | Cross-packaging of a single adenoassociated virus (AAV) type 2 vector genome into multiple AAV serotypes enables transduction with broad specificity[END_REF] respectively at the Vector Core at the University Hospital of Nantes (France) and the Belfer Gene Therapy Core facility at Weill Cornell Medical College.Their respective concentration was measured as 8.7x10 13 and 1.96x10 [START_REF] Piguet | Rapid and Complete Reversal of Sensory Ataxia by Gene Therapy in a Novel Model of Friedreich Ataxia[END_REF] vector genomes per mL (vg/mL), by qPCR. Their purity was checked with a combination of the following assay: potential protein contamination by SDS-PAGE and Coomassie Blue Staining, endotoxin contamination with Endosafe®-PTSTM LA (< 0.5 EU/m), sterility by growth in liquid and agar based test media (no contamination detected at 48H), and qPCR quantification of residual DNA material from AAV REP or Adenovirus plasmids.

Animal procedures

WT C57/B6J mice were supplied from Charles River laboratory, France. Mck mice were generated in 100% C57BL/6J background, with a conditional deletion of Fxn gene in cardiac and skeletal muscle (Mck-Cre-Fxn L3/L ) and genotyped, as described previously. [START_REF] Puccio | Mouse models for Friedreich ataxia exhibit cardiomyopathy, sensory nerve defect and Fe-S enzyme deficiency followed by intramitochondrial iron deposits[END_REF] Note that Mck mice develop a cardiac dysfunction at 5 weeks of age, progressively worsening into heart failure at 7 weeks of age, eventually causing their death around 10 weeks of age. [START_REF] Puccio | Mouse models for Friedreich ataxia exhibit cardiomyopathy, sensory nerve defect and Fe-S enzyme deficiency followed by intramitochondrial iron deposits[END_REF] Housing animal facility was controlled for temperature and humidity, 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 HP/2012/09/27 and 5344). They were performed in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health). Five-and seven-weeks old animals were anesthetized with isoflurane prior to the retro-orbital injection of vector, in 100µL bolus. Control littermate or WT mice were injected with equivalent volume of NaCl 0.9% solution. Survival and comorbidities were evaluated daily and body weight, weekly. Echocardiography were performed with the Sonos 5500 system (Hewlett Packard) with a 15 MHz linear transducer (15L6) or with the Vevo 2100 system (Fujifilm Visualsonics) with a 25 to 55 MHz transducer, as described previously. [START_REF] Belbellaa | Correction of half the cardiomyocytes fully rescue Friedreich ataxia mitochondrial cardiomyopathy through cell-autonomous mechanisms[END_REF] Briefly, echocardiography was performed under isoflurane anesthesia (0.5-1.5%) and 0.8 mL/min O2, as to maintain the heart rhythm between 450 and 550 bpm. Historical data were used as reference for untreated Mck mice survival, body weight and cardiac function Following intraperitoneal injection of ketamine-xylazine and blood sampling, mice were perfused with cooled NaCl 0.9%. and the heart and liver were retrieved quickly.

Briefly, the apex of the heart was sampled in cryotubes and flash frozen in liquid nitrogen, for subsequent DNA and RNA extraction and analysis, as described previously. [START_REF] Belbellaa | Correction of half the cardiomyocytes fully rescue Friedreich ataxia mitochondrial cardiomyopathy through cell-autonomous mechanisms[END_REF] Two small pieces from the left ventricle's anterior wall (less than 1mm3) were sampled in Karnovsky's fixative and processed for transmitted electron microscopy (TEM) morphological analysis. A thin transversal section (around 2mm thick) was cut at the middle level of the heart for protein analysis. The middle to the basis of the heart was embedded in OCT and frozen in isopentane chilled with liquid nitrogen for histological analysis.

The liver medial left lobe was sampled and fixed in PBS 1x formaldehyde 10%, dehydrated and then embedded in paraffin for histological analysis. Two small pieces from the left lateral lobe (less than 2mm 3 ) were sampled, fixed in Karnovsky's fixative for TEM analysis. The remaining lobes -lateral left, medial right, lateral right and caudate lobes -were sampled in a cryotube and flash frozen in liquid nitrogen for subsequent DNA, RNA and protein analysis.

The spleen was fixed in PBX 1x Formaldehyde 4%, embedded in OCT and frozen in isopentane chilled with liquid nitrogen for histological analysis.

Histochemistry

Frozen sections from the heart and the spleen, and liver paraffine sections, were collected on Superfrost® glass slides. For each staining, the group and number of animals analyzed is reported in the respective figure's legend (1-3 tissue sections stained per animal for each histological analysis). Heart and liver tissue sections were stained with hematoxylin-eosin (HE). In addition, heart tissue sections were stained with wheat germ agglutinin conjugated with Alexa 488nm (WGA), or stained with DAB-enhanced Perls labelling, or used to perform in-situ histoenzymatic activity assay for succinate dehydrogenase or cytochrome C oxidase, as previously described. [START_REF] Belbellaa | Correction of half the cardiomyocytes fully rescue Friedreich ataxia mitochondrial cardiomyopathy through cell-autonomous mechanisms[END_REF] NADH dehydrogenase in-situ histoenzymatic activity was performed on heart tissue section accordingly to Luna et al. [START_REF] Luna | Histopathologic methods and color atlas of special stains and tissue artifacts[END_REF] Immunofluorescent labelling, with or without SDH co-labelling, was performed as described previously, [START_REF] Belbellaa | Correction of half the cardiomyocytes fully rescue Friedreich ataxia mitochondrial cardiomyopathy through cell-autonomous mechanisms[END_REF] with the following antibodies and dilutions: frataxin (anti-FXN, 1/50, IGBMC, FXN935 and anti-HA, 1/100, Abcam, Ab9110), prohibitin (1/300, Ab28172), Sqstm1 (1/300, 2C11, H00008878-M01, Millipore) and acetyl-lysine (1/200, Ab21623, ABCAM). The FXN antibodies recognize both the human and mouse FXN, including the precursor, intermediate and mature forms.

For labelling of CD3, CD14 and CD45 positive cells, tissue sections were fixed in 4% PFA for 5min, permeabilized in PBS1x 0.3% Triton X-100 at RT for 10min and then blocked with PBS, 1% NGS, 5% BSA, 0.2% Tween (PBS-NBT) for 30min at RT. Subsequently, tissues sections were incubated O/N at 4°C with the rabbit antibody against CD3 (1/100, ab16669, ABCAM) or CD14 (1/100, ab183322, ABCAM,) or CD45 (1/100, ab10558, ABCAM) and afterward with alexa fluor-488nm conjugated goat anti-rabbit antibody (1/300, Molecular Probes).

Imaging of the heart, liver and spleen tissue sections were performed with the Hamamatsu NanoZoomer 2.0 slide scanner.

Electron microscopy analysis

The samples were fixed with 2.5% glutaraldehyde and 2.5% paraformaldehyde in cacodylate buffer (0.1 M, pH 7.4), washed 30 minutes in cacodylate buffer, post-fixed with 1% osmium tetroxide in 0.1M cacodylate buffer for 1 hour at 4°C and dehydrated through graded alcohol (50, 70, 90, and 100%) and propylene oxide for 30 minutes each. Samples were oriented and embedded in Epon 812. Semithin sections were cut at 2µm with the Leica Ultracut UCT ultramicrotome and stained with 1% Toluidine blue and 1% sodium borate. Ultrathin sections were cut at 70nm and contrasted with uranyl acetate and lead citrate. Electron microscopy observation and image acquisition were performed at 70kv with the Morgagni 268D electron microscope (FEI Electron Optics, Eindhoven, and the Netherlands) and equipped with the Mega View III camera (Soft Imaging System). The bismuth sodium tartrate staining of ferritin was performed as described previously. [START_REF] Michael | Iron and iron-responsive proteins in the cardiomyopathy of Friedreich's ataxia[END_REF][START_REF] Ainsworth | An ultrastructural staining method for enhancing the size and electron opacity of ferritin in thin sections[END_REF] DNA, RNA and protein analysis DNA, RNA and protein extraction and quantification, as well as cDNA synthesis, were performed as described previously. [START_REF] Belbellaa | Correction of half the cardiomyocytes fully rescue Friedreich ataxia mitochondrial cardiomyopathy through cell-autonomous mechanisms[END_REF] Briefly, absolute quantification of vector biodistribution was performed in triplicate and the quantitative RT-PCR in duplicate, using the Quantitect Sybr® Green CR kit (Qiagen, 1037795) and the Light Cycler 480 II (Roche Biosciences). Primers were used at 0.4µM final concentration and their sequence are reported in Table S3. qPCR amplification program: 1 cycle, 15min 95°C; 50 cycles, 15sec 94°C-30sec 60°C-30s 72°C; 1 cycle for melting curve. Depending on the expression level the target genes quantified, cDNA dilution ranging from 1/50 up to 1/2,000 were performed. As working Ct values ranged between 12 and 34 cycles, 18S and Hprt were used for normalization respectively when the target genes required high and low cDNA dilution for the qPCR assay.

SimpleStep Human Frataxin ELISA Kit (ABCAM, ab176112) and SimpleStep Mouse Frataxin ELISA kit (ABCAM, ab199078) were used to quantify the heart and liver tissue concentration in human and mouse frataxin respectively, following manufacturer instructions.

For Western Blot analysis, between 10 to 30µg of total protein extract were loaded and migrated in 14% or 4-12% SDS PAGE gels (Novex, Nupage 4-12% Bis-Tris Midi Gels 26 wells). Following transfer onto nitrocellulose membranes (Amersham Protran 0.45µm NC), Red Ponceau staining (Sigma Aldrich, P7170-1L), and blocking, the membranes were immunoblotted O/N at 4°C with the following antibodies and dilution: mouse anti-frataxin (1/10,000, 4F9, IGBMC), mouse anti-complex II 30KDa subunit SDHb (1/10,000, 21A11AE7, Novex), mouse anti-glyceraldehyde-3-phosphate dehydrogenase (1/30,000, MAB374, Millipore) and/or mouse anti-beta-tubulin (1/10,000, IGBMC). After incubation for 2 hours at 4°C with goat anti-mouse IgG H+L (1/10,000, 115-035-146, Jackson Immunoresearch), the signal was revealed using the SuperSignal West Dura Extended Duration Substrate (ThermoSienctific, 34075) and imaged with the Amersham Imager 600 (GE Healthcare).

Statistical, correlation and regression analysis

Unless otherwise specified, data are reported as mean ± standard deviation (SD). Statistical analyses were carried out using GraphPad Prism 8.3 (GraphPad Software, La Jolla California USA) and methods are described in the

  -F and S1A-C). Mck mice treated at 5x10 13 vg/kg displayed LV shortening fraction (SF) and cardiac blood output normalized to body weight (CO/BW) corrected to WT levels (Figure 1D-E), similarly to Mck mice treated at 2.5x10 13 vg/kg. Importantly, Mck mice treated at 5x10 13 vg/kg displayed a sustained functional rescue at long term (Figure S1D-H), when evaluated by echocardiography until 25 weeks of age in a separate cohort of mice (n=5; Table

  vg/kg and expressing 9.2-fold the endogenous FXN level, displayed normal SDH and NADH enzymatic activity (Figure 7A-B and S6A). The second WT mouse treated similarly but overexpressing 38.7-fold the endogenous level of FXN, displayed few and sparce cardiomyocytes with reduced SDH and NADH enzymatic activity.

FriedreichFigure 1 .

 1 FIGURES LEGENDS

Figure 2 .

 2 Figure 2. High level of FXN-HA overexpression in the heart of wild-type mice treated with AAVRh.10-

Figure 3 .

 3 Figure 3. High level of FXN-HA overexpression in wild-type mice cardiomyocytes leads to mitochondrial

Figure 4 .

 4 Figure 4. Cardiotoxic overexpression of FXN-HA is associated with increased mitochondria biomass and

Figure 5 .

 5 Figure 5. Cardiotoxicity FXN overexpression is independent of the dose administrated and vector

Figure 6 .

 6 Figure 6. FXN cardiotoxic overexpression is associated with acute heart fibrosis and inflammation. (A)

Figure 7 .

 7 Figure 7. FXN cardiotoxic overexpression is associated with impaired mitochondrial function and

ACKNOWLEDGMENTS

We thank Dr Hugues Jacob, Karim Hnia, Nicolas Charlet-Berguerand and Pr. Jean-Louis Mandel for scientific discussions and Dr Wyatt Delphino and Mehdi Gasmi for critical review of the manuscript. We thank Dr. Ronald G. Crystal (Department of Genetic Medicine, Weill Cornell Medicine, New York) for the production of the optimized construct and vector, Dr. Mustapha Oulad-Abdelghani for providing the 4F9 mouse monoclonal antibodies against FXN. We thank Dr. Tania Sorg, Ghina Bou-About, Mrs Josiane Hergueux, the mouse clinic institute phenotyping platform and Pr. Luc Dupuis for logistical and technical assistance. This work was supported by the Lefoulond Delalande foundation -Institut de France [post-doctoral research fellowships years 2016]; the

arrows indicate non-iron mitochondrial electron dense bodies; myo, myofibrils; mito, mitochondria; nuc, nucleus.

(D) DAB-enhanced Perls labelling of iron deposits.