Limb perfusion as a route for re-conditioning and genetic engineering: preventing damage and decreasing immunogenicity to support survival after allogeneic transplantation

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Vascularized composite allograft (VCA) transplantation is an established and valid treatment in the management of disfiguring tissue damages, massive tissue loss or amputation. Unfortunately, conventional reconstructive methods often fail in completely restoring function in patients with severe facial or limb injuries. As an alternative, VCA transplantation represents a process by which functional units such as an arm are retrieved from a donor and transplanted into the patient, allowing for the possibility of greater functional recovery. Despite the very encouraging functional outcomes, VCA recipients have been subject to a burden of immunosuppression comparable to that of solid organ transplantation, including cytomegalovirus infection and neoplasia. In addition, they have shown a high incidence of acute rejection episodes with long-term sequelae and chronic rejection. HLA play an important role in the activation of immune responses after allogeneic transplant settings and are the trigger for both acute and chronic rejection. Machine perfusion is a novel approach to decrease preservation injury, improve graft assessment, and increase organ acceptance for transplantation. Recently, we demonstrated the great potential of using ex vivo perfusion strategies to genetically modify organs such as the lung and kidney towards reduction of their immunogenicity and improving graft survival. Therefore, this project aims at using ex vivo limb machine perfusion strategies to prevent tissue damage due to ischemia reperfusion injury and simultaneously allowing for the genetic modification of the limb endothelium to improve graft survival after MHC incompatible transplantation. This proposal is based on a limb transplantation rat model. Rat hind limbs will be ex vivo perfused with lentiviral vectors encoding for shRNAs targeting MHC class I and II transcripts to silence their expression in a stable manner. Our previous studies showed that silencing MHC expression contributes to increase graft survival after transplantation even in the absence of immunosuppression. To assess the effect of ex vivo machine perfusion on the limb endothelium, cytokine signatures and tissue injury markers will be measured in the perfusates. Furthermore, the protocols previously established by our group to genetically modify solid organs will be adapted and improved to achieve an optimal transduction efficiency of the limb tissues. After transplantation experiments, graft survival and the strength of the immune response in absence of immunosuppression will be comprehensively monitored. Importantly, an extensive characterization and evaluation of safety aspects related to the generation and transplantation of genetically modified VCA will be performed. The results achieved by this project will define a new road to ensure the routinely application of VCA transplantation as optimal reconstructive strategy with gain of function and eliminating the burden of immunosuppression.