Genetic Modification of Limbs UsingEx VivoMachine 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.