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Abstract

In transfusion therapy, unintentional mismatching of blood groups remains one of the most common causes of serious and sometimes fatal adverse reactions. In addition, shortages of blood supply in rare blood transfusions and development of alloimmunization in patients undergoing chronic blood transfusions still remain as major challenges in current transfusion medicine. To address these unmet clinical needs, substantial efforts have been made in the past to produce universal blood via cell surface engineering (CSE). These approaches, however, had limitations and induced immune responses. In this body of work, we present new techniques for engineering the red blood cell (RBC) surface to produce universal blood donor cells. Utilizing a significantly more efficient novel two-enzyme system (N-Acetylgalactosamine deacetylase and Galactosaminidase from Flavonifractor plautii) than previously reported glycosidases, we produced enzyme-converted O (ECO) RBCs from both A1 and A2 type RBCs. These ECO RBCs showed similar characteristics as native O RBCs, and the modification reached a clinically significant level of immune compatibility when assessed by the monocyte monolayer assay. Also, a full blood unit conversion from group A to O was achieved using the enzyme system. Next, we developed a CSE technique that can immunocamouflage the non-ABO antigens. For this, two approaches were investigated to covalently attach polymers to the RBC surface: 1) utilization of mammalian and microbial transglutaminase (TGase) in concert with Q-tagged polymers, and 2) utilization of polysialic acids, a biopolymer as an alternative to synthetic polymers, with direct enzymatic polysialylation using polysialyltransferase (PST-109). Both mammalian and microbial TGases showed low reactivity on RBC surfaces, resulting in insufficient polymer grafting and masking of RhD antigen. In contrast, PST-109 mediated polysialylation of RBCs provided excellent camouflage for non-ABO antigens, including RhD, and showed excellent immune compatibility. Finally, we presented a novel CSE technique to produce universal blood. For this, we combined the aforementioned novel two-enzyme system to cleave A antigens and PST-109 mediated polysialylation to camouflage the non-ABO antigens. Using this method, ‘true’ universal donor cells that are both antigen A and RhD stealth are generated, which may significantly improve the blood supply and decrease the transfusion-associated risks.