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Leung, Jerry

University of British Columbia

Platelet transfusions are essential for managing bleeding and hemostatic dysfunction, but may become ineffective during certain circumstances such as in trauma. Optimizing systems that can enhance the hemostatic potential of platelets could lead to increased efficacy of transfusions and improve patient outcomes. Modifying transfusable donor platelets to express therapeutic proteins is the most direct method to enhance platelets. However, no appropriate methods exist for genetically modifying platelets collected from blood donors. This thesis showed that platelet-optimized lipid nanoparticles containing mRNA (mRNA-LNP) enable exogenous protein expression in human and rat platelets. Exogenous protein expression did not appear to require, nor correlate with platelet activation. Transfected platelets retained hemostatic function and accumulated in regions of vascular damage after transfusion into rats with hemorrhagic shock. These results suggest that platelets can be genetically modified without impairing hemostasis, where further optimization can expand the therapeutic potential of platelets. Engineering the platelet precursor cells, megakaryocytes (MKs), can also yield modified platelets, but can only currently be achieved through viral vectors. No methods currently exist to genetically modify MKs with non-viral techniques. mRNA-LNP represent a scalable strategy to transfect and modify MKs with a variety of nucleic acid payloads. This thesis showed that the clinically approved mRNA-LNP can transfect cord-blood derived MKs without affecting MK maturation. Expression of the coagulation factor VII (FVII) also decreased clot time in FVII-deficient plasma when assessed through rotational thromboelastometry. This was the first study to demonstrate LNP transfection of in vitro cultured MKs. Optimizing LNP further can create highly potent systems that may enable improved transfection of both platelets and MKs. This thesis showed that formulating LNP in the presence of high concentration pH 4 buffer can induce “bleb” structures and improve the transfection potency both in vitro and in vivo. Induction of bleb structure and improved potency is dependent on the type of pH 4 buffer employed, and the improved transfection potencies can be partially attributed to enhanced integrity of the encapsulated mRNA. These results suggest that enhanced transfection can be achieved by optimizing formulation parameters to improve mRNA stability through formation of bleb structures.

Text

2024-10-31

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/86226

Biochemistry and Molecular Biology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/