Tissue Factor Knockdown in Porcine Islets: An Effective Approach to Suppressing the Instant Blood-Mediated Inflammatory Reaction

Abstract

Tissue factor (TF) expression on islets has been shown to trigger instant blood-mediated inflammatory reaction (IBMIR), leading to rapid islet loss in portal vein islet transplantation. This study investigated whether antisense RNA-mediated TF gene knockdown in islets could suppress IBMIR as a strategy to overcome IBMIR. Neonatal porcine islet cell clusters (NICCs) were transfected with or without TF-specific antisense RNA or a nonspecific RNA by a lipid-based method. Expression of both TF gene and protein in NICCs was analyzed after transfection by real-time PCR, Western blot, and FACS, respectively. The impact of antisense RNA transfection on NICC viability and in vitro function was examined by FACS and insulin release test, respectively. The effect of TF knockdown in NICCs on IBMIR was assessed with an in vitro tubing loop assay using human blood. A significant reduction in TF gene and protein expression was achieved in TF antisense RNA but not control RNA transfected NICCs, which did not affect NICCs' viability or their insulin secreting capacity. Incubation of TF antisense RNA transfected with human blood resulted in a considerable reduction in blood clot formation, platelet consumption, and complement and coagulation activation compared to that observed in the loops containing human blood and untreated or control RNA transfected NICCs. Consistent with these findings, infiltrating neutrophils in the blood clots with entrapped TF antisense RNA transfected NICCs was also reduced substantially compared to that seen in the clots containing untreated or control RNA transfected NICCs. This study presents a nontoxic TF antisense RNA-mediated TF knockdown in porcine islets that leads to an effective suppression of IBMIR, suggesting a potentially new strategy to improve islet transplantation outcomes.

Gene Editing, Gene Therapy, and Cell Xenotransplantation: Cell Transplantation Across Species

“…The impact of tissue factor knockdown in neonatal porcine islets on IBMIR was demonstrated in vitro by treating islets with tissue factor-specific antisense RNA and exposing them to human blood. Reduced clot formation, complement, and coagulation activation were observed with modified islets compared to control islets [36]. In mice, expression of human endothelial protein C receptor (hEPCR) on islets improved graft survival and function, reduced inflammation and coagulation, and allowed diabetes correction using less islets than required when wild-type mice were used as donors [37].…”

“…Encapsulated islets implanted in poorly vascularized sites might escape this reaction at least in the early stages of engraftment but IBMIR causes a quick loss of up to 60% of islets implanted in the portal vein [32]. Transgenic expression of human CD46 [33•, 34], CD55, and CD59 [35] and knockdown of tissue factor gene [36] were shown to avoid this early damage to the graft by decreasing IBMIR both in vivo and in vitro. hCD46 expression in porcine islets increased graft survival to more than 3 months compared to only 46 days in the case of wild-type islets transplanted to immunosuppressed macaques [34].…”

Xenotransplantation for Islets from Clinical Side

“…The quality parameters of islet products involved sterility, purity, viability and activity, cell population, and functionality. It has been suggested that transplantation of poor quality islet product would cause the inconsistencies of the ability of islet transplants to reverse diabetes [25,26], so islet quality control is critical to both determining the suitability of islets for transplantation as well as to maintain a long-term functional graft in recipients posttransplantation.…”

“…Insulin capacity was obtained by the ratio of the insulin content to the DNA content in microgram in 1000 NICC IEQ [25].…”

Islet cell transplantation for the treatment of type 1 diabetes: recent advances and future challenges

“…The direct impact of IBMIR on early loss of islet function and mass has yet to be fully characterized. However, given that platelet activation is one of the primary contributing factors in the generation of an inflammatory response, IBMIR is most likely one of the key processes that elicits an early immune response 55–59. In a study conducted by the Uppsala Group, it was demonstrated that IBMIR is initiated upon intraportal infusion 60.…”