Genetic Modification of Limbs Using Ex Vivo Machine Perfusion.

Genetic engineering is a promising tool to repair genetic disorders, improve graft function, or reduce immune responses toward allografts. Ex vivo organ perfusion systems have the potential to mitigate ischemic-reperfusion injury, prolong preservation time, or even rescue organ function. We aim at combining both technologies to develop a modular platform allowing the genetic modification of vascularized composite (VC) allografts. Rat hind limbs were perfused ex vivo under subnormothermic conditions with lentiviral vectors. Specific perfusion conditions such as controlled pressure, temperature, and flow rates were optimized to support the genetic modification of the limbs. Genetic modification was detected in vascular, muscular, and dermal limb tissues. Remarkably, skin follicular and interfollicular keratinocytes, as well as endothelial cells showed stable transgene expression. Furthermore, levels of injury markers such as lactate, myoglobin, and lactate dehydrogenase, as well as histological analyses showed that ex vivo limb perfusion with lentiviral vectors did not cause tissue damage and limb cytokine secretion signatures were not significantly affected. The use of ex vivo VC perfusion in combination with lentiviral vectors allows an efficient and stable genetic modification representing a robust platform to genetically engineer limbs toward increasing graft survival after transplantation.

Genetic Engineering of the Kidney to Permanently Silence MHC Transcripts During ex vivo Organ Perfusion.

Organ gene therapy represents a promising tool to correct diseases or improve graft survival after transplantation. Polymorphic variation of the major histocompatibility complex (MHC) antigens remains a major obstacle to long-term graft survival after transplantation. Previously, we demonstrated that MHC-silenced cells are protected against allogeneic immune responses. We also showed the feasibility to silence MHC in the lung. Here, we aimed at the genetic engineering of the kidney toward permanent silencing of MHC antigens in a rat model. We constructed a sub-normothermic ex vivo perfusion system to deliver lentiviral vectors encoding shRNAs targeting ß2-microglobulin and the class II transactivator to the kidney. In addition, the vector contained the sequence for a secreted nanoluciferase. After kidney transplantation (ktx), we detected bioluminescence in the plasma and urine of recipients of an engineered kidney during the 6 weeks of post-transplant monitoring, indicating a stable transgene expression. Remarkably, transcript levels of ß2-microglobulin and the class II transactivator were decreased by 70% in kidneys expressing specific shRNAs. Kidney genetic modification did not cause additional cell death compared to control kidneys after machine perfusion. Nevertheless, cytokine secretion signatures were altered during perfusion with lentiviral vectors as revealed by an increase in the secretion of IL-10, MIP-1α, MIP-2, IP-10, and EGF and a decrease in the levels of IL-12, IL-17, MCP-1, and IFN-γ. Biodistribution assays indicate that the localization of the vector was restricted to the graft. This study shows the potential to generate immunologically invisible kidneys showing great promise to support graft survival after transplantation and may contribute to reduce the burden of immunosuppression.