Transcriptional activators rescue Frataxin expression and downstream pathways in Friedreich’s ataxia diseased cell types

Friedreich’s ataxia (FA) is an autosomal recessive neurodegenerative disorder caused by the epigenetic silencing of the Frataxin (FXN) gene, resulting from the pathological expansion of GAA·TTC trinucleotide repeats in its first intron. Given the strong correlation between FXN silencing and repressive chromatin states, we explored targeted epigenetic reactivation strategies using engineered transcriptional activators. We employed the CRISPR activation (CRISPRa) system based on a lentiviral vector encoding dCas9 fused to the potent VP160 activation domain, along with multiple sgRNAs targeting FXN promoter. This system successfully restored FXN expression in primary fibroblasts, iPSC-derived neural progenitor cells (NPCs), cortical neurons, and sensory neurons derived from a mildly affected patient (PTS, 330/300 GAA repeats) and a more severely affected patient (PTL, 530/1000 GAA repeats). Rescue of disease-associated pathways was also observed, upon CRISPRa treatment. Additionally, we tested zinc-finger activators (ZFAs), consisting of zinc-finger proteins targeting FXN promoter and fused to the VP64 activation domain. Given their compact size and human origin, ZFAs offer great promise for translational applications. Notably, we showed that a specific combination of three ZFAs effectively reactivated gene expression in PTS-derived fibroblasts, NPCs and sensory neurons. However, ZFAs were not effective on PTL-derived cells. Overall, our findings demonstrated that targeted epigenetic reactivation of the endogenous FXN gene holds strong therapeutic potential. Remarkably, our comparative analysis of CRISPRa and ZFAs highlighted CRISPRa as the most powerful and reliable tool for restoring endogenous FXN expression in the context of FA.