mRNA loaded Lipid Nanocapsule Formulation: a promising therapeutic strategy to treat Pseudoxanthoma elasticum
Résumé
Pseudoxanthoma elasticum (PXE) is a rare genetic disorder characterized by progressive ectopic calcification, which is caused by mutations in the ABCC6 gene. PXE predominantly affects the elastic fibers of the skin, Bruch’s membrane in the retina and the cardiovascular system. Unfortunately, no curative treatment exists. To compensate the deficiency of the protein normally expressed in hepatocytes, the delivery of the corresponding mRNA has emerged as a promising therapeutic strategy. However, this approach requires a nanocarrier to cross the cell membrane, protect the mRNA, and ensure delivery to the targeted tissues. During the Covid-19 pandemic, lipid nanoparticles (LNP) highlighted the huge potential of nanomedicines: they successfully demonstrated their ability to protect and deliver mRNA. Among LNP, lipid nanocapsules (LNC) were designed and patented in 2014 by the MINT laboratory (patent FR 3026009 (A1)) with the goal of protecting genetic material such as DNA, siRNA, and miRNA. The objective of this project is to formulate and characterize mRNA loaded LNC, assessing their in vitro toxicity on the hepatocyte cell lines HepG2 and SNU-387 and to validate the therapeutic strategy in Abcc6−/− mice. As a model to validate the formulation, mRNA encoding Enhanced Green Fluorescent Protein (EGFP) was initially used. In parallel, a total of 948.2 µg of ABCC6 mRNA was produced in Prof. Pichon’s laboratory. Both ABCC6 and EGFP mRNA-loaded LNC were fully characterized. Subsequently, dose-dependent cytotoxicity was assessed using MTT and LDH assays, resulting in the determination of an IC50 that will be utilized in future experiments. Cell internalization studies using flow cytometry revealed an efficient uptake of DiD-labeled EGFP mRNA loaded LNC over time with a 100% internalization after 24 hours. Finally, in vivo studies were conducted in healthy mice by intravenous injection of DiD-labeled LNC. The results showed a significant accumulation of LNC in the liver 24 hours post-injection: ABCC6 being predominantly expressed in the liver, this biodistribution is an important result. Encouragingly, hepatic mRNA expression was also observed 6h post-injection, offering promising prospects for future therapeutic applications in PXE.