CMT2A is a rare inherited axonal peripheral neuropathy caused by mutations in the mitofusin 2 (MFN2) gene and characterized by progressive distal muscle weakness, atrophy, sensory loss, and normal or near-normal nerve conduction velocities. MFN2 mutations have been characterized and numerous mechanisms proposed to unravel CMT2A pathophysiology; however, no cure for this devastating disease is available. Thus, it is crucial to identify a treatment to cure or at least mitigate CMT2A progression.

To investigate a novel therapeutic approach for Charchot-Marie-Tooth disease type 2A (CMT2A) using RNAi/gene therapy combination.

Our proposed approach combined RNA interference (RNAi) with gene therapy as therapeutic strategy for CMT2A. In induced pluripotent stem cells (iPSCs) obtained from CMT2A patients we targeted the MFN2 mutant allele with specific short hairpin RNAs (shRNAs) and simultaneously introduced a mutagenized MFN2 gene able to be resistant to shRNA activity and still encoding the native protein. We then differentiated iPSCs into spinal motor neurons (MNs) and analyzed the sub-cellular parameters we previously found to be altered in CMT2A in vitro model (Rizzo et al., 2016) were evaluated to assess the impact of our therapy.

We successfully silenced endogenous MFN2 gene and restored functional MFN2 protein level in CMT2A cells without altering iPSC pluripotency. In addition, MNs differentiated from both treated and untreated CMT2A iPSCs showed typical MN morphology and expressed the main MN markers. Qualitative and quantitative alterations characteristic of CMT2A disease (Rizzo et al., 2016) were restored after our therapy in CMT2A MNs. Specifically, we observed an increase in mitochondria number, an improvement of mitochondrial axonal transport, re-established sensitivity to apoptosis and reduction of authophagy/mytophagy.

Overall, our results led to a significant level of rescue in CMT2A MNs, suggesting RNAi/gene therapy combined approach as a promising therapeutic strategy for CMT2A.