IN VIVO AND IN VITRO EVALUATION OF THE COMBINATION OF RNA INTERFERING AND GENE THERAPY FOR TREATING MITOFUSIN2-RELATED DISEASES

Background: Mitofusin-2 (MFN2) is an outer mitochondrial membrane protein essential for mitochondrial networking in most cells. Autosomal dominant mutations in the MFN2 gene cause Charcot-Marie-Tooth type 2A disease (CMT2A), a severe and disabling sensory-motor neuropathy. Here, we propose a novel therapeutic strategy tailored to the correction of the root genetic defect of CMT2A, based on the combination of RNA- interference (RNAi) and gene therapy. Methods: We devised a strategy whereby both the mutant and wild-type MFN2 mRNA are inhibited by a short hairpin RNA (shRNA), while the wild-type protein is restored by overexpressing a cDNA encoding functional MFN2 modified to be resistant to RNAi. We tested this strategy in vitro, in CMT2A patient-specific human induced pluripotent stem cell (iPSC)-differentiated motor neurons (MNs), transfecting them with two constructs, one encoding a shRNA directed against endogenous MFN2 sequence, the gene responsible for CMT2A, and one encoding an exogenous MFN2 sequence designed to be resistant to silencing. We also delivered the constructs in vivo in newborn MitoCharc1 CMT2A and Thy1.2-MFN2R94Q transgenic mice via cerebrospinal fluid (CSF) delivery using adeno-associated virus 9 (AAV9). Results: We demonstrated the correct silencing of endogenous MFN2 and replacement with an exogenous copy of the functional wild-type gene in cells. This approach significantly rescues the CMT2A MN phenotype in vitro, stabilizing the altered axonal mitochondrial distribution and correcting abnormal mitophagic processes. After delivery of an AAV9 encoding either the shRNA alone or the combination of shRNA/MFN2 cDNA, we observed a correct MFN2 molecular correction in the MitoCharc1 and Thy1.2-MFN2R94Q mice, but the treatment proved toxic after one month for Thy1.2- MFN2R94Q animals treated with AAV9-encoded shRNA and after two months of life for mice treated with AAV9-encoded shRNA/MFN2 cDNA. Conclusions: Overall, the combined RNAi and gene therapy strategy proved able to effectively silence mutant MFN2 and restore wild-type protein levels. In addition, it was able to correct alterations in mitochondrial distribution and mitophagic processes in CMT2A MNs. In vivo, this therapeutic strategy achieved molecular efficacy but proved toxic for treated animals. It will therefore be necessary to investigate further the reasons for this toxicity in order to find a safe and effective treatment for CMT2A patients.