Ex Vivo Nanoparticle Drug Delivery Targeted to Human Allograft Endothelium

7. 项目总结/文摘

7. Project Summary/Abstract Peri-transplantation inflammation of solid organ allografts exacerbates acute cell-mediated rejection and increases late graft loss, primarily caused by chronic rejection. The two most common causes of perioperative inflammation are ischemia reperfusion injury (IR) and, in sensitized patients, pre-formed donor specific antibodies, both of which deposit antibody and complement membrane attack complexes (MACs) on graft endothelial cells (ECs). MAC deposition is linked to increased rejection by inducing expression of IL-15/IL- 15Rα complexes on the EC surface where they can be trans-presented to host alloreactive lymphocytes, intensifying T cell responses. In a human immune system mouse model, this results in increased rejection of human arterial interposition grafts. We hypothesize that preventing this specific response of ECs to MACs by treating the graft rather than the recipient will reduce early rejection episodes and late term graft loss without increasing systemic immunosuppression. In aim 1, we will develop and optimize new antibody-targeted, degradable polymer nanoparticles (NPs) for delivery of therapeutic RNA selectively to ECs that can prevent IL- 15/IL-15Rα complexes. We will evaluate efficacy and duration of effects in both cultured cells and perfused human vessel segments. Agents to be tested include siRNAs that, due to sustained release from the NP, will produce a sustained knockdown of target proteins, as well as mRNAs encoding Cas enzymes and guide strands that can produce permanent gene disruption or epigenetic silencing. Access and transduction of ECs in human organs is more challenging than transduction of isolated cells or vessel segments. In Aim 2, we will further develop approaches to optimize delivery of the antibody-targeted NPs to ECs of kidneys and hearts that have been declined for transplantation using established approaches of ex vivo normothermic machine perfusion. These experiments will exploit advances already made by our team, such as fibrinolytic clearing of fibrinogen/erythrocyte occlusions of graft vasculature to increase access to the whole vasculature and improved coupling of targeting antibodies using monobody adapters that greatly enhance binding to ECs. In aim three, we will directly test the hypothesis that prevention of IL-15 trans-presentation can prevent the consequences of peri-operative injuries using both our well established model of human artery segment interposition grafts in human immune system mouse recipients and in a new model of heterotopic transplants of mouse hearts following ex vivo perfusion. Regardless of the validity of our IL-15 hypothesis, the technologies developed in all three aims can be readily adapted for use against other EC targets to either complement or in lieu of targeting IL-15 trans-presentation.