ABSTRACT Protein replacement therapy has been a cornerstone in treating genetic diseases (e.g., hemophilia) with loss or reduction of the function of a particular protein, by using recombinant proteins or recombinant engineered proteins. However, most protein therapeutics have short circulation lives, and thus require frequent invasive infusion to maintain their therapeutic efficacy. For example, the most common treatment for hemophilia A caused by a deficiency of blood clotting factor VIII (FVIII) is factor concentrate replacement, which is associated with burdensome frequent intravenous infusion. To address the unmet medical need of hemophilia, the major objective of this project is to develop a non-invasive oral mRNA delivery nanoplatform for durable protein replacement therapy requiring infrequent dosing. Synthetic mRNA has shown enormous potential for biomedical applications, with mRNA vaccines already clinically approved for COVID-19. Various delivery strategies have been developed to improve mRNA translation; however, an ongoing challenge of mRNA therapy is managing the transient efficacy due to its relatively short half-life, and oral mRNA delivery remains elusive. In my previous work, I have identified a unique poly(zwitterion)-lipid-based micelle platform that can cross the intestinal epithelial barrier and lead to a very potent oral bioavailability of biomolecules such as insulin. Recently, I have also discovered a new type of ionizable lipids that can extend the duration of mRNA-mediated protein expression. In this F32 project, I propose to combine the epithelium-crossing poly(zwitterion)-lipids and the unique ionizable lipids to develop an innovative mRNA delivery platform for oral, durable replacement therapy of bleeding disorders. In Aim 1, I will synthesize new poly(zwitterion)-lipids and ionizable lipids and generate a series of new mRNA lipid nanoparticles (LNPs), and systematically investigate their effects on oral transcytosis and the durability of protein expression in vitro and in vivo. In Aim 2, we will select the top-performing LNPs for oral delivery of FVIII mRNA and evaluate the FVIII mRNA nanotherapy in healthy and hemophilia mouse models. With the successful completion of this project, we expect that the oral durable mRNA delivery strategy will provide a more effective and robust therapy for hemophilia and other bleeding disorders.