According to the Department of Health and Human Services, more than 25,000 patients undergo solid organ transplantation every year in the U.S. Approximately 100,000 patients are currently waiting for compatible transplants. The current drugs used in controlling transplant rejection are non-specific, thereby subjecting patients to high risks of infectious diseases and malignancies. This project is motivated by the unique opportunity in transplantation in that isolated organs can be treated using ex vivo methods. Exogenous anti-inflammatory agents can be introduced into transplants prior to implantation. Interleukin-10 is a powerful immunosuppressive cytokine that has been shown to attenuate acute rejection of allogeneic transplant (or """"""""allografts""""""""), but the non-specific effects of IL-10 protein limit its clinical use. The goal of this project is to develop methods to introduce the IL-10 gene ex vivo into donor dendritic cells (DCs) reside within transplant tissues. The phenotype of donor DCs dictates the fate of transplants in recipients. While donor DCs in allografts become inhibitory when forced to express IL-10 in vitro, the outstanding question is whether the donor DCs will remain inhibitory in vivo. Skin grafting between two genetically distinct mouse strains will be used to test the ability of the ex vivo IL-10 gene treatment. The mouse (and human) skin contains high density of DCs, therefore a good representation of commonly transplanted organs (e.g. kidney and lung) in which a significant number of the same cells resides. IL-10 gene particles will be applied to skin allografts ex vivo (prior to transplantation) using polymeric particles as a DNA carrier. The system entails using the low molecular weight polycation O10H6 as a DNA condensing agent on the surface of nickel-displaying PLGA particles. The nickel/his-tag interaction provides a mechanism for adsorbing O10H6, freeing the protonated ornithine amines to maximize DNA capture. By calibrating the particles'surface charge loading of different doses of plasmid DNA can be optimized. Upon completion of the first phase of the project (specific aim 1), we expect to find that allografts treated ex vivo with the IL-10 gene particles to release in vivo donor DCs with inhibitory phenotypes. Rejection will be assessed using florine-19 magnetic resonance imaging to measure in allografts localized inflammation, an early sign of rejection. The key milestone of this aim is to pinpoint the phenotype of donor DCs in vivo associated with a delay in acute rejection. In the second phase (specific aim 2), we expect to document changes in the recipient mice's T cells in respond to IL-10 modified allografts. Key milestones of this aim are to determine 1) the nature and scope of the T cell response, and 2) unwanted side effects associated with the ex vivo treatment. The proposed experiments will be carried out in collaborations with scientists at the Pittsburgh NMR Center for Biomedical Research and Children's Hospital of Pittsburgh. In summary, we expect the research to provide strong rationale for testing ex vivo IL-10 gene therapy in transplantation of DC-rich organs to achieve specific immunosuppression.
This project emphasizes the development of an ex vivo gene therapy approach to attenuate acute transplant rejection.
It aims to test if the longevity of transplants can be extended by forcing white blood cells associated with transplant organs to produce interleukin-10. A polymeric system with low inflammatory potential will be used in ex vivo IL-10 gene transfection of skin transplants in mice. The research entails characterization of the donor's white blood cells in the recipient, how the recipient's immune system responds toward the modified transplants, and the extent to which the transplant remains viable in recipient mice. Successful completion of the study will lead to improved health outcome of a significant number of patients by decreasing the need for indefinite use of non-specific immunosuppressants in transplant recipients.
