Expression of exogenous proteins in donor platelets treated with lipid nanoparticles encapsulating mRNA

ABSTRACT

Background: Platelets are transfused therapeutically for hemostasis, and are an integral part of hemorrhage management. However, transfusions can be ineffective in the most severe cases of hemorrhage. Platelets are also a potential cell therapy in other applications, but development has been hindered by inadequate methods to control which proteins are expressed by platelets. Currently, there are no methods to express exogenous proteins in transfusable platelets, which would expand their use to help treat the diseases they modulate. A method is therefore needed to modify transfusable platelets, and thus enhance their protein composition for specific applications. Aim(s) To produce engineered, transfusable platelets to enhance their natural coagulability and functional repertoire by directly transfecting donor-derived platelets with mRNA via lipid nanoparticle (LNP)-mediated delivery. The recent advances through the COVID-19 mRNA vaccines demonstrates the clinical safety and efficacy of LNP-mediated gene therapy, and thus offers a promising strategy to effectively engineer modified platelets. Method(s) Donor-derived platelets were washed and subsequently incubated with a systematic array of LNPs encapsulating Cy5-labeled mRNA encoding for nanoluciferase in comparison to commercial transfection reagents. LNP uptake and platelet activation via CD62p levels was assessed following 4 hours by flow cytometry, while luciferase expression was assessed by normalizing the luminescence intensity to the total protein content. Result(s) Platelets took up the mRNA through all conditions tested;nanoluciferase was only expressed, however, in platelets treated with LNPs and not commercial reagents. Systematically optimizing LNPs increased nanoluciferase expression nine-fold relative to pre-optimized LNPs. Exogenous protein expression did not appear to correlate with mRNA uptake nor platelet activation. Conclusion(s) Platelets transfected with LNPs can express exogenous protein. Further optimization can eventually lead to the creation of a platform technology that in the long-term will allow platelets to deliver therapeutic proteins and yield more effective platelet products.