Scalable mRNA Synthesis for advanced therapeutic applications

Muscle atrophy is a condition characterized by progressive loss of muscle mass and function, with limited therapeutic approaches currently available. Gene therapy, based on the delivery of nucleic acids, represents a promising strategy, but faces several challenges such as instability in physiological fluids and poor cellular uptake. Nowadays, lipid nanoparticles (LNPs) have emerged as efficient carriers to overcome these limitations. This thesis aims to develop and optimize a reproducible protocol for the in vitro synthesis of high-quality messenger RNA (mRNA) in high yields, defining a scalable and cost-effective workflow that could potentially support future applications of mRNA-based gene therapy. The Mitochondrial Calcium Uniporter (MCU) was selected as a potential therapeutic gene for this study. This choice was based on previous research showing that increased expression of MCU may help reduce muscle atrophy. Additionally, Luciferase was used as a reporter gene and control construct in the experiments. The study first focused on the design of optimized DNA templates for in vitro transcription (IVT), then compared different transcription systems, and critical steps such as capping, polyadenylation, and RNA purification were refined to minimize gene products loss and enhance overall efficiency. The resulting mRNAs were validated in cell culture via Lipofectamine and LNPs transfection, confirming correct protein expression and functionality through Western blot and reporter luminescence or mtCa2+ analyses.