's abstract) Immunosuppressive gene therapy holds promise as an inductive therapy in transplantation. However, many fundamental aspects of this technology must be addressed before it may be applied to clinical transplantation. These include questions regarding the use of viral vs. non-viral vectors, underlying mechanisms of action, effects on systemic immunity, and the duration of transgene expression. The investigators have developed a model of TGFbeta1 gene transfer into mouse vascularized cardiac allografts to address these issues. Donor hearts are perfused with either DNA-liposome complexes or adenoviral vectors which encode the active form of TGFbeta1. Interestingly, CD4+ cells are readily suppressed by this modality, while CD8+ cells are not. This differential sensitivity is most pronounced when adenoviral vectors are used. The overall hypothesis to be tested is that transient depletion of CD8+ cells will enhance the efficacy of immunosuppressive gene therapy. Hence, the Specific Aims will: 1) Define mechanisms by which TGFbeta1 gene transfer mediates immunosuppressive activities. T cell functional assays will identify critical immune functions which are either turned off or turned on by TGFbeta1 gene transfer, and adoptive transfer studies will determine if regulatory cells are induced by TGFbeta1 gene transfer. 2) Elucidate mechanisms by which adenoviral mediated TGFbeta1 gene transfer stimulates CD8+ cells. The investigators will test the hypothesis that adenoviral vectors, but not DNA-liposome complexes, stimulate production of inflammatory cytokines which over-ride TGFbeta1 suppression of CD8+ cells. 3) Determine the impact of TGFbeta1 gene transfer on systemic immune surveillance. They will assess the effects of TGFbeta1 gene transfer on primary and memory responses to Listeria and on antibody responses to influenza immunization. 4) Determine if TGFbeta1 gene expression may be silenced following initial inductive immunosuppression. Tetracycline regulated promoters will be used to limit the duration of TGFbeta1 expression, and gene transfer of decorin, which binds and neutralizes TGFbeta1, will be employed to neutralize the transgene product. This study will provide insight for optimizing immunosuppressive gene therapy in clinical transplantation.
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