We propose to apply methods and insights of bioengineering and human immunology to a surgical therapy, namely organ transplantation. Our goal is to produce safe, efficient, selective and sustained knock down of immunostimulatory proteins within human graft endothelial cells (EC) by developing ex vivo targeted nanoparticle transfection of siRNA so as to reduce allograft rejection in humanized pre-clinical models. Rejection remains an important cause of graft loss and current regimens of host immunosuppression produce significant complications. Our novel approach will reduce rejection instead by modifying the alloantigenicity of the graft. By focusing on human-based models, we address two fundamental limitations of most rodent transplant models. First, adult humans, but not experimental rodents, have circulating effector memory T cells capable of directly recognizing non-self-major histocompatibility complex (MHC) molecules and, upon activation, causing graft rejection. The high frequency of alloreactive memory cells is thought to account for the failure in humans of many therapies successful in rodents. Second, human endothelial cells (ECs), unlike rodent ECs, express and directly present non-self-class II MHC molecules to circulating effector memory T cells, initiating rejection and bypassing the need for graft dendriti cells (""""""""passenger leukocytes"""""""") to activate na?ve host T cells seen in rodent models. Our experiments with cultured human ECs and with humanized mouse models of allograft rejection have revealed crucial roles for EC-expressed co-stimulators and EC- derived cytokines as well as EC-expressed MHC molecules in T cell activation. Furthermore, human effector memory T cells are still somewhat plastic and can be irreversibly directed along different pathways by their initial contact with graft ECs. In other words, changes in the expression of immune stimulatory or regulatory molecules by ECs in the perioperative period can have lasting effects on graft outcomes. In current clinical practice, the graft vasculature is flushed with an organ preservation solution so that ECs throughout the graft come in contact with the perfusate. We will optimize conditions for ex vivo delivery of siRNAs using biodegradable polymer nanoparticles engineered to efficiently transfect ECs lining human blood vessels and to produce a more sustained change in the EC phenotype achieved by current transfection approaches (specific aim 1);we will use this approach to knock down specific immunomodulatory molecules, examples being CIITA, LFA-3, raptor and/or IL-1a, in cultured human ECs and assess effects on the activation of allogeneic memory T cells in vitro, compared to conventional EC transfections (specific aim 2);and we will use nanoparticle- mediated transfection to knock down molecules identified as important in aim 2 in the ECs lining human artery segments ex vivo prior to implantation into mice reconstituted with a human immune system allogeneic to the artery donor, assessing the effect on acute and subacute graft rejection (specific aim 3). These pre-clinical studies will provide proof of concept for our novel approach to improve the outcome of allotransplantation.
Immune-mediated rejection by the recipient of a transplanted organ is still a major cause of transplantation failure and commonly used drugs that suppress the host's immune system to control rejection can cause serious complications, limiting the doses that can be administered to patients. We propose to develop and apply an alternative approach to reduce rejection by modifying the ability of a human graft to activate the host's immune system through the administration of bioengineered nanoparticles that deliver short interfering RNA molecules to the endothelial cells that line the blood vessels of the graft. We anticipate that modifying human graft endothelial cells for a short period at the time of transplantation will permanently reduce the host's immune response to the graft, improving graft outcome, reducing the need for immunosuppressive agents, and potentially improving health outcomes for millions of transplant recipients.
|Cheng, Christopher J; Tietjen, Gregory T; Saucier-Sawyer, Jennifer K et al. (2015) A holistic approach to targeting disease with polymeric nanoparticles. Nat Rev Drug Discov 14:239-47|