CRISPR/Cas9-based approaches for Frataxin gene reactivation in Friedreich’s ataxia

Friedreich’s ataxia (FA) is an autosomal-recessive neurodegenerative disorder caused by the silencing of the Frataxin (FXN) gene, due to the expansion of GAA trinucleotide repeats in its first intron. Transcriptional activators constitute an innovative approach to overcome the repression of endogenous FXN. Moreover, newly available CRISPR/Cas9-associated systems represent a powerful editing platform for genome engineering. Intriguingly, the chance to selectively excise the FA pathological GAA expansion represents an intriguing opportunity for a permanent restoration of FXN expression.

To promote FXN gene expression reactivation we compared two complementary CRISPR/Cas9 approaches based on either stimulate FXN expression or to excise the GAA pathological tract. For the former approach, FA patient fibroblasts were transduced with a lentiviral vector expressing the dCas9-VP160, containing the VP16 transactivator. Multiple sgRNAs targeting the FXN gene promoter were used to enhance the initiation of gene transcription. For the latter approach, we explored the compact and high-fidelity Neisseria meningitidis Cas9 (Nme2Cas9) in order to selectively remove the GAA pathological expansion. We produced an all-in-one AAV vector expressing Nme2Cas9 with two sgRNAs targeting the regions flanking the GAA triplet expansion.

We demonstrated that the transcriptional activator, when combined with one or more selected sgRNAs, stimulate FXN expression by 2 to 4 fold, restoring RNA and protein levels comparable to the control condition. Simultaneously, we assessed the efficiency of the AAV-Nme2Cas9 system with two selected sgRNAs in exciding the GAA expansion in patient fibroblasts.

Our findings indicate that dCas9-transcriptional activators can be effectively employed as tools for FXN gene reactivation in patient fibroblasts. Moreover, we confirmed that the NmeCas9 is an efficient nuclease that, giving its reduced size, is suitable for gene therapy application. These results pave the way for testing these approaches on FA mouse models to evaluate the efficiency of symptomatic rescue.