*Purpose: Classical immunosuppressants lead to systemic immune shutdown, though necessary to mediate transplant rejection, it may lead to various complications. To reduce off-target immunosuppression while retaining increased transplant survival, we propose direct modification of the endothelium of vascular transplants ex vivo to achieve localized immunomodulation. We developed an enzymatic approach to modify the surface of the endothelium at the transplant storage conditions using novel non-toxic immunosuppressive and anti-inflammatory/anti-oxidant polymers. We tested the efficacy of this approach for immunosuppression and reduction in graft rejection.

*Methods: We developed an enzymatic organ engineering method using tissue transglutaminase and polyglycerol polymer conjugates containing immunosuppressive sialic acid or sulfate moieties. In vitro mechanistic studies were performed using EaHy.926 cells to replicate the endothelium. In vivo experiments were done using mice models allograft blood vessel and renal transplantation. The performance of the modification was assessed using serum, and histological examination of tissue sections of excised transplants.

*Results: Enzyme-mediated engineering endothelial surfaces of cultured cells, blood vessels and kidneys using polymers under static cold storage conditions (in UW solution at 4 °C) resulted in uniform modification. In vitro, modified endothelial cells were able to evade immune cell (CAR T Cell and PBMC) induced cytotoxicity, prevented protein leakage and possessed increased scavenging activity of ROS. Modification reduced TNF release in M1-like monocytes. In transplant setting, reduced early inflammation in transplants correlated with reduced medial thickening and pro-inflammatory markers in serum. In turn, this resulted in long-term survival of vessel transplant without rejection in the absence of immune suppressants. Histological analysis of engineered renal grafts revealed less infiltration and mesangial expansion and normal kidney function relating to a healthier graft. Furthermore, de novo generation of donor-specific antibody was reduced in engineered grafts.

*Conclusions: We have developed a novel organ engineering approach that prevents immune-mediated rejection of transplants via improved vascular protection. Ex-vivo engineering using immune cloaking polymers allow for localized immunosuppression, making this an enticing and viable strategy for elimination or reduction in the use of broad-acting immunosuppressants.

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