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Abstract

Uncontrolled hemorrhage secondary to trauma is the leading cause of death amongst people from ages 1-46. Platelet transfusions along with whole blood and plasma are the gold-standard for maintaining hemostasis as they detect sites of damage in vasculature and form a clot to stop bleeding. However, in cases of severe bleeding, transfused platelets have limited coagulability making transfusions ineffective, which highlights a critical need for improved platelet products. A promising strategy to overcome current platelet product limitation is to enhance the innate function of platelets by using lipid nanoparticles (LNP) to deliver exogenous cargo. Optimized LNP loaded with mRNA (mRNA-LNP) are able to engineer platelets to express reporter proteins when suspended in a non-clinical crystalloid solution. The modified platelets retained in vitro physiology, and exogenous protein expression was not correlated with activation, and accumulated in sites of damage when tested in a hemorrhagic model in rats. However, for genetically engineered platelets to be established as a viable cell therapy, this technology would have to be developed with clinical compatibility in mind. Platelets are currently stored in plasma or in plasma supplemented with platelet additive solution (PAS) at supraphysiological concentrations at room temperature or at 4 ºC, depending on the indications they will be used for. PAS has been developed to support platelet viability during storage, reduce transfusion related adverse reactions, and standardize storage conditions. This thesis describes mRNA-LNP optimized for transfecting platelets directly in plasma or plasma supplemented with PAS and demonstrates scalability to both physiological and supraphysiological platelet concentrations. The process of transfecting platelets with mRNA-LNP in clinical solutions does not affect in vitro physiology, morphology, and their ability to be stored. Aligning the compatibility of this transfection system could enable future mRNA-LNP-based platelet products and other cellular therapies.