In Vitro Investigations on Optimizing and Nebulization of IVT-mRNA Formulations for Potential Pulmonary Based Alpha-1-Antitrypsin Deficiency Treatment

Abstract

Background: In vitro transcribed (IVT) mRNA has come into focus in recent years as a potential therapeutic approach for the treatment of genetic diseases. The pulmonary delivery of IVT-mRNA encoding alpha-1-antitrypsin (A1AT) is a promising strategy for protein replacement therapy of alpha-1-antitrypsin deficiency (AATD). The nebulized A1AT-mRNA formulations would be a highly acceptable and tolerable remedy for the AATD patients in the future. Method: we first optimized parameters that were influencing the transfection efficiency of formulations containing IVT-mRNA and Lipofectamine2000 based on human bronchial epithelial cells transfection. Cell viability was evaluated by performing MTT assay after transfection with different IVT-mRNA lipoplexes. Functional analysis was employed to assess the biological function of A1AT proteins produced from optimized formulations using anti-trypsin assay and anti-elastase assay. Results: Lipoplexes prepared by IVT-mRNA encoding A1AT (A1AT-mRNA) in optimum conditions can successfully transfect human bronchial epithelial cells without significant toxicity. A reduction in transfection efficiency was observed for aerosolized lipoplexes that can be partially overcome by increasing the initial amount of components. A1AT produced from cells transfected by nebulized A1AT-mRNA lipoplexes is functional and can successfully inhibit the enzyme activity of trypsin as well as elastase. Conclusion: Aerosolization of A1AT-mRNA therapeutic constitute a potentially powerful means to transfect airway epithelial cells with the purpose of producing functional A1AT while bringing along the unique advantages of IVT-mRNA.