Mitochondria are cell organelles that provide the energetic requirements of the body. The majority of cellular ATP is produced by the membrane protein ATP synthase through a proton gradient across the mitochondrial inner membrane. Alterations in ATP synthase biogenesis can result in severe mitochondrial diseases affecting tissues with high energy requirements as brain and muscles. Mitochondrial diseases affect approximately 20 million people in the EU, causing 35 % of deaths during the first year of life of newborns. However, the available therapeutic approaches, are still extremely limited and there is no specific treatment for ATP synthase deficiencies. To improve the treatments currently available for mitochondrial diseases, the project will focus on the realization of artificial mitochondria (AM). Based on artificial lipid vesicles, AM will be fabricated by means of microfluidics methods, a powerful tool able to produce identical replicas of a given bio-inspired membrane-object. ATP synthase will be expressed and assembled within the lipid bilayer by encapsulating cell-free protein expression systems. To test the ability of AM as in-situ energy fabrication systems, targeting-AM will be endocytosed inside cultured cells and ATP synthesis will be triggered by taking advantage of the proton gradient provided by endosomes. Finally, by enclosing other plasmids encoding for diverse proteins, AM can be used as energy-factoring pockets to elicit protein expression just when internalized within cells. This novel approach may constitute an advanced new concept in gene therapy to more effectively create breakthroughs in improving human health.