Verbatim): Allogeneic bone marrow transplantation is a curative therapy for hematologic malignancies, marrow failure states, and selected inherited metabolic diseases. Unfortunately, BMT is associated with significant morbidity and mortality related to graft vs. host disease (GvHD). Attempts to mitigate GvHD using T-cell depletion resulted in increased rates of graft failure, post-transplant lymphoproliferative diseases, leukemia relapse and opportunistic infections. We propose to genetically modify T-cells using novel cell surface chimeric suicide genes. We will test the expression and purification of novel fusion suicide genes in vitro and in vivo using instructive transgenic and """"""""knock-in"""""""" murine pre-clinical models. The optimal method and potential role of genetically manipulated T-cells to mitigate GvHD while maintaining engraftment and graft vs. leukemia can only be defined by developing new reagents and novel pre-clinical murine transplant models. We propose to generate important new reagents to study T-cell transduction by suicide genes and to use a number of well-defined pre-clinical models to develop a rational approach and a clear foundation for future clinical trials.
In Aim I, we will design methods to optimize transduction, selection, and expansion of transduced T-cells, using OKT3 and 11-3 and CD3/CD28 magnetic beads. In addition, we will generate novel second-generation suicide genes designed to optimize detection and killing in response to prodrug. We will also test the survival and function of transduced murine and human T cells using in vivo using allogeneic transplant models and several murine SCID models.
Aims II and III, we will use the chimeric suicide genes developed in Aim I to generate informative transgenic and knock-in pre-clinical murine models in which these suicide genes will be expressed in all (Aim II) or in subsets (Aim III) of peripheral T-cells. Allogeneic bone marrow transplantation will be performed using the transgenic and knock-in mice developed in Aims II and III to further develop the optimal method of suicide in vivo, its effect on GvHD and engraftment, and its possible role in clinical trials.
In Aim 1 V, we will utilize three novel murine leukemia models in which the effects of genetically modified T-cells and their in vivo suicide on GvHD, engraftment and graft vs. leukemia can be compared to animals receiving unmanipulated T-cells and T-cell depleted BM. These studies will provide new insights into the pathophysiology of GvHD and its effective treatment. Issues regarding the use of genetically manipulated T-cells to control GvHD can best be investigated through the use of informative animal models and the comprehensive studies described in this proposal.
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