Novel alternative to human red blood cell using pig red blood cells expressing human transgenes

Introduction: Pig red blood cells (pRBCs) are potentially useful to solve the worldwide shortage of human blood. As an alternative to human red blood cells (hRBCs), pRBCs have similarities to human RBCs and could overcome the transfusion immune response through a gene editing system. In particular, triple-knockout (TKO) pig (lacking Gal, Neu5Gc and Sda) RBCs are known to be free of aggregation and lysis in vitro. However, pRBCs expressing human transgenes, which are essential for prolonging the life-span of transfused pRBCs in vivo, have not yet been developed. So in vivo studies on pRBCs have been quite limited. For significant advanced xenotransfusion study, the development and comparative study of transgenic pRBCs are essential. Method: Production of transgenic pRBCs (hCD55, hCD39 expressed) conducted through knock-in system by the GGTA1 endogenous promoter. pRBCs were prepared (WT, wild type; GTKO, aGal knockout; GTKO/hCD55/hCD39, GTKO expressing hCD55, hCD39; TKO, aGal/Neu5Gc/Sda knockout; TKO/hCD55/hCD39, TKO expressing hCD55, hCD39). To access aGal, Neu5Gc, Sda, hCD55 and hCD39 expression on RBCs, flow cytometry (FACS) and confocal analysis were performed. Glycosylation alteration of pRBC membranes was characterized by NanoLC-Q-TOF-MS system. In vitro comparative analysis of pRBCs was conducted with pooled human and NHP serum. IgG or IgM binding to pRBCs was accessed by FACS and total antibody binding was characterized by hemagglutination. Complement-mediated damage was measured by cytotoxicity and C3 deposition. Phagocytosis to pRBCs was assayed by FACS. Results: Knocked-in transgenes (hCD55, hCD39) were expressed on pRBCs in GTKO/CD55/CD39 and TKO/CD55/CD39. aGal, Neu5Gc and Sda were all not detected on TKO pRBCs. The MS results showed that TKO pRBCs distribution has completely changed from pig N-glycan to neutral and human like N-glycan (NeuAc complex). Human and NHP IgG, IgM binding, hemagglutination decreased in TKO pRBCs to a similar level to human or NHP RBCs. The human, NHP complement mediated-cytotoxicity and C3 deposition of GTKO/CD55/CD39 pRBCs significantly decreased compared to GTKO pRBCs. C3 deposition of TKO/CD55/CD39 pRBCs also was lower than TKO pRBCs. In addition, phagocytosis of GTKO/CD55/CD39 pRBCs was also reduced compared with GTKO pRBCs by inhibition of C3 convertase formation, but TKO based pRBCs phagocytosis more reduced compared GTKO/CD55/CD39 pRBCs phagocytosis. Conclusion: GGTA1 endogenous promoter-mediated expression system caused expression of both hCD55, hCD39 on pRBCs. Not only that, but our data support that expression of hCD55, hCD39 on pRBCs prevents an effective immunological reaction. We also confirmed the distribution of N-glycans and the similarity with hRBCs in TKO pRBCs. Our results and technique will serve as an excellent resource to produce TKO based multitransgenic pRBCs for xenotransfusion. This research was financially supported by the Institute of Civil Military Technology Cooperation funded by the Defense Acquisition Program Administration and Ministry of Trade, Industry and of Korean government under grant No. 22-CM-18.