Dr. Federica Rizzo, Dr. Stefania Corti, Prof. Giacomo P. Comi
Centro Dino Ferrari Università degli Studi di Milano

Charcot-Marie-Tooth disease type 2A (CMT2A) is a sensory-motor polyneuropathy, characterized by the involvement of motor and sensitive neurons, resulting in progressive weakness, limb muscle atrophy and loss of sensitivity. Mitofusin2 (MFN2) gene has been identified as causative of the disease. The MFN2 protein,
located in the outer mitochondrial membrane, is involved in the mitochondrial functions. A treatment for CMT2A is not currently available. In our project, we aim to develop an effective therapy, based on the understanding of the disease molecular mechanisms, helpful not only to identify new therapeutic targets, but also to define specific disease hallmarks.
We first generated fibroblasts from CMT2A patients with different MFN2 mutations. The reprogramming of mature somatic cells into induced pluripotent stem cells (iPSCs) provides the derivation of disease-specific cell types, such as motor and sensitive neurons, affected in the disease. Based on this method, we successfully generated human iPSCs from a CMT2A patient and demonstrated their differentiation into motor neurons. In particular, we observed an alteration in mitochondria localization, a reduction in the amount of mitochondrial DNA and a dysfunction of the mitochondrial respiratory chain, identifying specific hallmarks of the disease phenotype. These defects are more evident in neuronal cells compared to fibroblasts, in agreement with neuronal specificity of the disease. In addition in vitro models, the analysis of these aspects has been conducted in the only currently available mouse model of CMT2A (MitoCharc 1) to extend its characterization, searching for biomarkers of disease phenotype.

We aimed to develop a therapeutic approach for this disorder. We silenced endogenous MFN2 gene by short harpin RNA (shRNA) in CMT2A fibroblasts. At the same time, in order to restore correct MFN2 protein levels, we transfected a MFN2 c-DNA modified to be resistant to shRNA-mediated silencing. The results of this strategy were very promising in CMT2A fibroblasts, and preliminary data were also obtained in the CMT2A mouse model.

This study contributed to deepen the knowledge about disease molecular mechanisms, generating an in vitro model of CMT2A by patient-specific iPSCs, and to identify a possible therapeutic strategy for CMT2A.

The future developments of our research project will be:
– to increase the number of iPSC lines obtained from CMT2A patients and to differentiate them into neurons
-to test our RNAi therapeutic strategy in CMT2A neurons
-to apply our strategy in CMT2A mouse model, using adeno-viral type 9, capable of transferring our constructs into neuronal cells.

Recently, we decide to test CRISPR-CAS9 genome editing system to correct the disease MFN2 gene mutation directly at the DNA level. We will establish gene-editing complexes that include a DNA-cutting enzyme called Cas9 bound to a short RNA guide strand that is programmed to bind to a specific genome sequence, guiding Cas9 in its cutting. At the same time, we will also deliver a DNA template strand. Cells repair the damage produced by Cas9 copying from the template and thus introducing new genetic material into the genome, which results in the correction of MFN2 mutation. The CRISPR system is considered a gold standard strategy for genome editing, such as demonstrated by positive results obtained in other pathologies.