Despite careful donor selection and extensive laboratory testing, a small risk of virus transmission by transfusion still exists. Additional efforts in donor selection and infectious disease testing may not substantially reduce this risk. However, the development of virus inactivation methods should provide an additional level of safety. The goal of this research is to develop practical methods to photoinactivate intracellular and extracellular viruses in red cell (RBC) suspensions of transfusable hematocrit under conditions that will neither harm the recipient nor the in vitro/in vivo properties of red cells. More specifically, the photosensitizer 1,9-dimethylmethylene blue (DMMB) and red light will be used to rapidly inactivate (within 10-20 second timeframe) virus in thin films of oxygenated RBC suspensions. Based on preliminary studies, virus inactivation will be enhanced and photoinduced RBC damage will be diminished by the addition of a single substance that acts as a competitive inhibitor to prevent DMMB binding to RBCs. The competitive inhibitor will be safe to transfuse to recipients and shares several structural features with DMMB, yet it will not absorb red light nor produce reactive oxygen species upon red light illumination.
The specific aims of the research are to: 1) Develop a simple and reproducible method for oxygenating full units of RBCs in a closed system; 2) Design a flow cell system suitable for rapid photoinactivation of 1-2 mm thin films of 45-60% hematocrit RBC of 1-2 mm thin films of 45-60% hematocrit RBS suspensions with minimal alterations of RBC properties during 42 day 1-6 degrees C storage; 3) Identify an analytical procedure to quantitate levels of DMMB, its photoproducts, and the competitive inhibitor in blood; 4) Develop a device to remove DMMB, photoproducts and the competitive inhibitor from blood that is compatible with 42 day 1-6 degrees C storage; 5) Perform in vitro and in-vivo genotoxicity and acute toxicity testing for DMMB and phototreatment in an animal (baboon) model, and finally, 7) Investigate safety and RBC recovery and lifespan following virucidal phototreatment in a Phase 1 clinical trial conducted during the final year of the study.
