Design and validation of cell-based potency assays for frataxin supplementation treatments

Abstract

Friedreich's ataxia (FRDA) is a multisystem, autosomal recessive disorder caused by mutations in the frataxin (FXN) gene. As FRDA is considered a FXN deficiency disorder, numerous therapeutic approaches in development or clinical trials aim to supplement FXN or restore endogenous FXN expression. These include gene therapy, protein supplementation, genome editing or upregulation of FXN transcription. To evaluate efficacy of these therapies, potency assays capable of quantitative determination of FXN biological activity are needed. Herein, we evaluate the suitability of mouse embryonic fibroblasts derived from Fxn G127V knock-in mice (MUT MEFs) as a candidate for cell-based potency assays. We demonstrate that these cells, when immortalized, continue to express minute amounts of Fxn and exhibit a broad range of phenotypes that result from severe Fxn deficiency. Exogenous FXN supplementation reverses these phenotypes. Thus, immortalized MUT MEFs are an excellent tool for developing potency assays to validate novel FRDA therapies. Care needs to be exercised while utilizing these cell lines, as extended passaging results in molecular changes that spontaneously reverse FRDA-like phenotypes without increasing Fxn expression. Based on transcriptome analyses, we identified the Warburg effect as the mechanism allowing cells expressing a minimal level of Fxn to thrive under standard cell culture conditions.