Correction of half the cardiomyocytes fully rescue Friedreich ataxia mitochondrial cardiomyopathy through cell-autonomous mechanisms.

Friedreich ataxia (FA) is currently an incurable inherited mitochondrial neurodegenerative disease caused by reduced levels of frataxin. Cardiac failure constitutes the main cause of premature death in FA. While adeno-associated virus-mediated cardiac gene therapy was shown to fully reverse the cardiac and mitochondrial phenotype in mouse models, this was achieved at high dose of vector resulting in the transduction of almost all cardiomyocytes, a dose and biodistribution that is unlikely to be replicated in clinic. The purpose of this study was to define the minimum vector biodistribution corresponding to the therapeutic threshold, at different stages of the disease progression. Correlative analysis of vector cardiac biodistribution, survival, cardiac function and biochemical hallmarks of the disease revealed that full rescue of the cardiac function was achieved when only half of the cardiomyocytes were transduced. In addition, meaningful therapeutic effect was achieved with as little as 30% transduction coverage. This therapeutic effect was mediated through cell-autonomous mechanisms for mitochondria homeostasis, although a significant increase in survival of uncorrected neighboring cells was observed. Overall, this study identifies the biodistribution thresholds and the underlying mechanisms conditioning the success of cardiac gene therapy in Friedreich ataxia and provides guidelines for the development of the clinical administration paradigm.

Structure comparison of the chimeric AAV2.7m8 vector with parental AAV2.

The AAV2.7m8 vector is an engineered capsid with a 10-amino acid insertion in adeno-associated virus (AAV) surface variable region VIII (VR-VIII) resulting in the alteration of an antigenic region of AAV2 and the ability to efficiently transduce retina cells following intravitreal administration. Directed evolution and in vivo screening in the mouse retina isolated this vector. In the present study, we sought to identify the structural differences between a recombinant AAV2.7m8 (rAAV2.7m8) vector packaging a GFP genome and its parental serotype, AAV2, by cryo-electron microscopy (cryo-EM) and image reconstruction. The structures of rAAV2.7m8 and AAV2 were determined to 2.91 and 3.02 Å resolution, respectively. The rAAV2.7m8 amino acid side-chains for residues 219-745 (the last C-terminal residue) were interpretable in the density map with the exception of the 10 inserted amino acids. While observable in a low sigma threshold density, side-chains were only resolved at the base of the insertion, likely due to flexibility at the top of the loop. A comparison to parental AAV2 (ordered from residues 217-735) showed the structures to be similar, except at some side-chains that had different orientations and, in VR-VIII containing the 10 amino acid insertion. VR-VIII is part of an AAV2 antigenic epitope, and the difference is consistent with rAAV2.7m8's escape from a known AAV2 monoclonal antibody, C37-B. The observations provide valuable insight into the configuration of inserted surface peptides on the AAV capsid and structural differences to be leveraged for future AAV vector rational design, especially for retargeted tropism and antibody escape.

Neonatal systemic delivery of scAAV9 in rodents and large animals results in gene transfer to RPE cells in the retina.

Systemic delivery of recombinant adeno-associated virus (rAAV) vectors has recently been shown to cross the blood brain barrier in rodents and large animals and to efficiently target cells of the central nervous system. Such approach could be particularly interesting to treat lysosomal storage diseases or neurodegenerative disorders characterized by multiple organs injuries especially neuronal and retinal dysfunctions. However, the ability of rAAV vector to cross the blood retina barrier and to transduce retinal cells after systemic injection has not been precisely determined. In this study, gene transfer was investigated in the retina of neonatal and adult rats after intravenous injection of self-complementary (sc) rAAV serotype 1, 5, 6, 8, and 9 carrying a CMV-driven green fluorescent protein (GFP), by fluorescence fundus photography and histological examination. Neonatal rats injected with scAAV2/9 vector displayed the strongest GFP expression in the retina, within the retinal pigment epithelium (RPE) cells. Retinal tropism of scAAV2/9 vector was further assessed after systemic delivery in large animal models, i.e., dogs and cats. Interestingly, efficient gene transfer was observed in the RPE cells of these two large animal models following neonatal intravenous injection of the vector. The ability of scAAV2/9 to transduce simultaneously neurons in the central nervous system, and RPE cells in the retina, after neonatal systemic delivery, makes this approach potentially interesting for the treatment of infantile neurodegenerative diseases characterized by both neuronal and retinal damages.

Detection of intact rAAV particles up to 6 years after successful gene transfer in the retina of dogs and primates.

Gene transfer to the retina using recombinant adeno-associated viral (rAAV) vectors has proven to be an effective option for the treatment of retinal degenerative diseases in several animal models and has recently advanced into clinical trials in humans. To date, intracellular trafficking of AAV vectors and subsequent capsid degradation has been studied only in vitro, but the fate of AAV particles in transduced cells following subretinal injection has yet to be elucidated. Using electron microscopy and western blot, we analyzed retinas of one primate and four dogs that had been subretinally injected with AAV2/4, -2/5, or -2/2 serotypes and that displayed efficient gene transfer over several years. We show that intact AAV particles are still present in retinal cells, for up to 6 years after successful gene transfer in these large animals. The persistence of intact vector particles in the target organ, several years postadministration, is totally unexpected and, therefore, represents a new and unanticipated safety issue to consider at a time when gene therapy clinical trials raise new immunological concerns.

High Levels of Frataxin Overexpression Lead to Mitochondrial and Cardiac Toxicity in Mouse Models.

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. Adeno-associated virus (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 administered, the resulting FXN protein level could reach very high levels in the heart, cerebellum, or off-target organs such as the liver. The present study demonstrates safety of FXN cardiac overexpression up to 9-fold 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 the mitochondria respiratory chain and ultrastructure, which leads to cardiomyocyte subcellular disorganization, cell death, and fibrosis. Overall, this study underlines the need, during the development of gene therapy approaches, to consider appropriate vector expression level, long-term safety, and biomarkers to monitor such events.

Prevention and reversal of severe mitochondrial cardiomyopathy by gene therapy in a mouse model of Friedreich's ataxia.

Cardiac failure is the most common cause of mortality in Friedreich's ataxia (FRDA), a mitochondrial disease characterized by neurodegeneration, hypertrophic cardiomyopathy and diabetes. FRDA is caused by reduced levels of frataxin (FXN), an essential mitochondrial protein involved in the biosynthesis of iron-sulfur (Fe-S) clusters. Impaired mitochondrial oxidative phosphorylation, bioenergetics imbalance, deficit of Fe-S cluster enzymes and mitochondrial iron overload occur in the myocardium of individuals with FRDA. No treatment exists as yet for FRDA cardiomyopathy. A conditional mouse model with complete frataxin deletion in cardiac and skeletal muscle (Mck-Cre-Fxn(L3/L-) mice) recapitulates most features of FRDA cardiomyopathy, albeit with a more rapid and severe course. Here we show that adeno-associated virus rh10 vector expressing human FXN injected intravenously in these mice fully prevented the onset of cardiac disease. Moreover, later administration of the frataxin-expressing vector, after the onset of heart failure, was able to completely reverse the cardiomyopathy of these mice at the functional, cellular and molecular levels within a few days. Our results demonstrate that cardiomyocytes with severe energy failure and ultrastructure disorganization can be rapidly rescued and remodeled by gene therapy and establish the preclinical proof of concept for the potential of gene therapy in treating FRDA cardiomyopathy.

In vivo gene regulation using tetracycline-regulatable systems.

Numerous preclinical studies have demonstrated the efficacy of viral gene delivery vectors, and recent clinical trials have shown promising results. However, the tight control of transgene expression is likely to be required for therapeutic applications and in some instances, for safety reasons. For this purpose, several ligand-dependent transcription regulatory systems have been developed. Among these, the tetracycline-regulatable system is by far the most frequently used and the most advanced towards gene therapy trials. This review will focus on this system and will describe the most recent progress in the regulation of transgene expression in various organs, including the muscle, the retina and the brain. Since the development of an immune response to the transactivator was observed following gene transfer in the muscle of nonhuman primate, focus will be therefore, given on the immune response to transgene products of the tetracycline inducible promoter.