Development of a Targeted Therapy for Charcot Marie Tooth 1A (CMT1A) Neuropathy Based on siRNA PMP22 Squalene Nanoparticles.

Summary Charcot Marie Tooth 1A (CMT1A) is the most common hereditary neuropathy in the world. It is due to a duplication of the PMP22 gene. To date, most of the tested treatments are more palliative than curative. The objective of this project is to develop a new treatment for CMT1A based on the normalization of PMP22 gene overexpression by targeted siRNA therapy. For this purpose, eight PMP22 siRNAs were designed and tested in the MSC-80 cell line. In vitro, siPM22 #7 showed 50% inhibition of the PMP22 gene and corresponding protein expression without affecting cell viability. This siRNA was chosen for further studies. In order to protect the PMP22 siRNA from enzymatic degradation and to improve its cellular internalization, we coupled it to a cholesterol precursor, squalene (SQ), which has the particularity of forming nanoparticles (NP) in water. After bioconjugation, obtaining and characterization of PMP22-SQ siRNA NPs, we show that their lipophilic character remains stable over time and that they remain biologically active. In vitro, in MSC80 cells, the PMP22-SQ siRNA NPs inhibit, like the transfected PMP22 siRNA, the expression of the PMP22 gene at 50%. In vivo, i.v. injected PMP22-SQ siRNA NPs resituate motor activity as well as nerve conduction velocity and muscle action potential, in two CMT1A transgenic mouse models, carrying one extra copy (JP18 B6) or two extra copies (JP18/Y13 B6) of PMP22. After sacrifice of the mice, we show by confocal microscopy that the 2 transcription factors KROX20 and SOX10 as well as neurofilaments are increased in sciatic nerve sections treated with PMP22-SQ siRNA NPs showing myelination and axonal regeneration. In electron microscopy, we observe a normalization of the phenotype which translates into myelin compaction after internalization of PMP22-SQ siRNA NPs in Schwann cells and axons. In conclusion, we show that a targeted treatment with PMP22 siRNA NPs normalizing PMP22 expression could be a future therapy for CMT1A disease. This work is supported by a public funding supervised by the French National Research Agency (ANR) within the framework of the "investment of future" program: Labex NanoSaclay, reference: ANR-10-LABX-0035 and, protected by an international patent No.