191. Frataxin: A Putative Biomarker for Minimal Effective Dosage of AAV Gene Therapy

Friedrich ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by a triplet repeat expansion in the frataxin gene (FXN), which encodes the mitochondrial protein frataxin. Deficiency in frataxin expression results in severe mitochondrial dysfunction leading to progressive gait abnormality, impaired muscle coordination, muscle weakness, hyporeflexia, dysmetria, dysarthria, and hypertrophic cardiomyopathy. Currently, there are no approved treatments for FRDA, which is the most common occurring autosomal recessive ataxia, affecting 1 in 50,000 people worldwide. Precise quantification of frataxin levels is critical for establishing the effectiveness of potential therapies. Therefore, methods that improve detection of FXN expression will facilitate the implementation of novel therapies into a clinical setting. Our overall objective is to establish an approach for effective gene transfer and quantification of frataxin levels sufficient to restore mitochondrial function. Stable isotope labeling of amino acids in cell culture or in mammals (SILAC/M) is a novel and sensitive mass spectrometry based approach we propose for the quantification of frataxin levels; therefore enabling determination of required levels for correction of FXN deficiency.We have generated a rAAV2/9-FXN vector expressing codon optimized human FXN for use in models of FRDA. Renal epithelial cells (REC) were isolated from control and FRDA patients, and was SILAC used to quantify AAV-mediated FXN expression in-vitro. Induced pluripotent stem cells (IPSC) were generated from REC's, and subsequently differentiated to cardiomyocytes and neurons. AAV-mediated correction of FXN was verified by increased mitochondrial activity including reduced iron deposition and increased aconitase function in FRDA cells. Preliminary results show that SILAC/M are able to accurately measure endogenous and vector derived FXN expression in-vitro and in-vivo. Moreover, improvement in mitochondrial function was correlated with levels of FXN expression in vitro. Future studies will involve systemic dosing of rAAV9-FXN in a novel model of FRDA.