Rapid Production of Nonglycosylated Reteplase for Affordable Thrombolytic Therapy
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Project Summary/Abstract Cardiovascular disease is the leading cause of death in the United States, and acute myocardial infarction (AMI) demands rapid thrombolytic treatment to restore blood flow and reduce mortality. Reteplase, an FDA- approved recombinant tissue plasminogen activator, offers important clinical advantages, including extended plasma half-life and simplified bolus administration. However, commercial reteplase is produced exclusively overseas in E. coli through complex refolding and purification steps, driving costs above $100,000 per gram and restricting access. These barriers are particularly acute in Central Appalachia, where heart disease mortality is 42% higher than the national average. This R15 AREA project will establish a plant-based transient expression platform for producing nonglycosylated, bioactive reteplase at reduced cost. Using a modified BeYDV geminiviral vector in Nicotiana benthamiana, reteplase will be directed to two non-glycosylating compartments: (1) the cytosol, which supports yield and simplifies recovery, and (2) the chloroplast thylakoid lumen, which promotes correct disulfide bond formation and stability. Preliminary undergraduate-led work has already shown that plant-produced apoplast- targeted reteplase dissolves fibrin and human blood clots comparably to the commercial drug, demonstrating feasibility. Specific Aims are to: (1) design and codon-optimize reteplase constructs for cytosolic and thylakoid lumen targeting, (2) express and biochemically validate reteplase in N. benthamiana, and (3) functionally validate plant-derived reteplase in fibrin and whole-blood clot assays while scaling production using vacuum- based agroinfiltration. The expected outcome is a rapid, safe, and cost-effective production platform for high- quality reteplase without post-expression deglycosylation. Beyond thrombolysis, this system is adaptable to other therapeutic proteins and vaccines, advancing a versatile model for decentralized biomanufacturing. At the University of Pikeville—a primarily undergraduate institution where over 59% of students are Pell- eligible—this project integrates biomedical discovery with education. Students will participate in all phases of research, from construct design to protein purification and functional assays, gaining skills in molecular cloning, plant biotechnology, and translational medicine. Over three years, 30–40 undergraduates are expected to co- author presentations and publications, expanding biomedical research capacity and STEM workforce development in Central Appalachia.
