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Graduate Student Supervision
Doctoral Student Supervision (Jan 2008 - May 2019)
Platelets are small, anucleate cells that circulate in the blood stream and mediate hemostasis, inflammation, and angiogenesis. Platelet transfusions are used to treat active bleeding as well as to prevent bleeding during thrombocytopenia or prior to surgery. Yet there are situations where transfusions do not adequately stop bleeding, such as during trauma, which is associated with platelet dysfunction. A method for genetically modifying platelets might enhance their efficacy and lead to new therapeutic uses for platelets. As platelets are anucleate, directly modifying platelets requires messenger RNA (mRNA). Attempts to transfect platelets with mRNA have not been successful, and it is unknown whether lipid-based materials could be used as mRNA transfection agents for platelets.Lipid nanoparticles (LNPs) have been used for nucleic acid delivery in vitro and in vivo. In this thesis, the ability of four different classes of LNPs to deliver mRNA to platelets was compared. These classes consisted of LNPs containing cationic lipids (cLNPs) that are highly effective in vitro, LNPs containing ionizable cationic lipids (icLNPs) developed for in vivo use, LNPs without a cationic lipid commonly used to encapsulate proteins or small molecules, and a commercially available agent previously used for short interfering RNA delivery to platelets. To identify ideal conditions for transfection with mRNA, uptake under various storage conditions and the ability of the LNPs to alter platelet activation was quantified. Finally, the ability of platelets to translate and release the mRNA was assessed. Two approaches were taken for mRNA delivery. In one approach, mRNA was synthesized inside of liposomes, indicating proteins, DNA, and small molecules can be delivered to platelets using LNPs. In the second approach, in vitro transcribed mRNA was directly delivered to platelets using icLNPs and cLNPs, and mRNA delivered to platelets using cLNPs was released in microparticles. These were the first examples of direct delivery of mRNA to platelets, and the first step towards creating genetically modified platelets. While protein synthesis in LNP-treated platelets was not detected, optimizing the LNP formulations used here may lead to a transfection agent for platelets that allows for de novo synthesis of exogenous proteins in the future.
No abstract available.
Platelets are small, anucleate blood cells that are important mediators of many physiological and pathological processes. These include hemostasis, thrombosis, wound healing, inflammation, immunity, and malignancy. There are currently several uses for platelet therapy in the clinic, such as to increase platelet counts for the prevention of spontaneous bleeding, and to stop uncontrolled bleeding during trauma and surgery. Although platelet transfusions are an efficacious component in preventing and stopping bleeding in most cases, they are still insufficient to stop the most severe cases of surgical and traumatic bleeding. Traumatic bleeding is further complicated by trauma-induced coagulopathy, which often presents with platelet dysfunction and is not corrected by transfusions of normal platelets. Strategies to enhance the endogenous function of platelets to increase the efficacy of platelet transfusions has not been rigorously explored, especially during active bleeding in trauma-induced coagulopathy.When activated by specific stimuli, platelets locally secrete a variety of biologically active molecules in order to contribute to many physiological and pathophysiological processes. For example, platelets can recognize areas of vascular damage and respond by locally adhering, aggregating, and activating to initiate primary hemostasis. Platelets also release procoagulant molecules and mediate the formation of active coagulation factor complexes to ultimately form an insoluble fibrin clot and seal the wound. Taken together, developing strategies to modify the endogenous function of platelets may be a first step towards a platform system that could target many diseases. Moreover, strategies to load platelets with biomolecules could allow for the local delivery of therapeutics to disease sites using endogenous platelet machinery. This provides significant motivation to test our overarching hypothesis, that the endogenous function of platelets can be modified ex vivo through the delivery of liposome-encapsulated enzymes.The objectives of this thesis were to: i) develop a platform approach to deliver biomolecules to platelets, ii) engineer anucleate platelets to transcribe RNA, and iii) increase the coagulability of transfusable platelets. The results shown here demonstrate proof-of-concept that endogenous platelet function can be extended through the delivery of lipid-encapsulated enzymes, and provides new approaches to potentially enhancing current platelet transfusions.