Genetic-engineered control of the immunogeneic state of vascular composite allografts during preservation

PROJECT ABSTRACT. This is a design driven study to develop engineered VCA grafts by direct genetic modification to test the efficacy in both detecting immune rejection and reducing immunogenicity in a porcine model of graft rejection. A genetic construct with a transcription factor control element will serve as a biosensor for reporting and therapeutically responding to the immunologic state of a graft. This synthetic gene switch simultaneously drives the secretion of a blood-based biomarker and a therapeutic protein to attenuate a rejection response. In our prior work and preliminary results, we have shown engineering of cells with all the required response elements, a mathematical model that predicts in vivo transduction, direct genetic modification of VCA tissue in rat limbs, and the first 24hr ex vivo pig VCA machine perfusion with a successful transplant in a heterotopic porcine hindlimb model. The rationale here is to scale up the techniques based on our recent successes in rodents, and test the proposed smart-graft approach in a clinically relevant graft rejection model in pigs. This application proposes the development of a functional preservation platform for engineering grafts that feature theranostic biosensors for reporting and responding to the state of the graft. To enable the creation of such grafts and their transplant in a clinically viable fashion, we propose to leverage machine perfusion protocol to provide a functional preservation modality that will also enhance the logistics of VCA transplantation by increasing the VCA storage time from the current limit of 6 hours to 24hours. The proposed work is significant in two synergistic aspects: first, smart-graft technology that can sensitively measure local tissue signaling would enable practical monitoring and diagnosis of acute rejection episodes, which remains elusive and critical from a practical perspective to titrate the dosage of immune suppression in patients. Moreover, auto-delivery of anti- inflammatory therapeutic molecules can transform care of transplant patients since compliance issues in medication would be dramatically reduced, and moreover by delivering such therapeutics at a local and time- sensitive manner will likely increase the overall efficacy and therefore reduce graft rejections. Second, from a practical & logistical perspective, engineering VCA grafts will require an organ culture protocol that provides access and time to achieve such manipulations. The proposed machine perfusion protocol provides the perfect platform to achieve such timelines. In the long term, the methods developed here could also enable for tolerance induction in VCA transplants, and therefore allow for their wide-spread use. Even more broadly, genetically modified organs could extended ex vivo storage duration and provide the infrastructure required to extend HLA typing to all grafts for global matching programs and improved transplant outcomes.