OC 61.1 - Optimization and Delivery of Endogenous mRNA UTRs using LNPs to Control Exogenous Protein Expression in Platelets

Background: Platelets are frequently transfused to control hemorrhage in trauma and surgery. Due to limitations such as variable donor-based hemostatic efficacy and decreased unit efficacy during hemorrhage, platelet transfusions are not always effective. To overcome these issues, we propose manipulating donor platelets through gene therapy. We have developed a method to deliver mRNA into donor platelets ex vivo using lipid nanoparticles (LNP), enabling exogenous protein expression. Despite successful LNP uptake and mRNA translation, exogenous protein synthesis levels remain low. To try and improve protein synthesis, mRNA sequences were modified in the untranslated region (UTR) upstream of the coding sequence. The 5’ UTR possesses structural features that can influence ribosomal binding and control overall protein synthesis.

Aims: To increase exogenous protein synthesis in donor platelets ex vivo, through optimization of the LNP-delivered mRNA sequences to mimic highly expressed platelet proteins (Figure 1A).

Methods: mRNA sequences were designed to contain 5’ UTRs from endogenous platelet proteins, and a coding sequence for the reporter luciferase. Initial mRNA screens were completed using LNP delivery to hepatocyte and megakaryoblast cell lines to determine if differences in protein synthesis were cell specific. mRNAs were then delivered using LNPs to pooled-donor platelets (obtained from Canadian Blood Services). Protein expression in all cells was determined using the luciferase assay.

Results: Delivered mRNAs containing platelet endogenous UTRs showed platelet-specific enhancement of protein expression compared to other cell types. In platelets, the best performing UTR induced a 22.5-fold increase in expression over the lowest expressing UTR, and a 1.8-fold increase in expression over a standard UTR (Figure 1B).

Conclusion(s): These experiments show for the first time the influence of UTRs on protein synthesis in platelet cells. Additionally, through modification of the delivered mRNA UTRs, it is possible to increase protein expression. This study was supported by the US Department of Defense Investigator-Initiated Research Award.