The central hypothesis in this proposal is as follows: Infection of liver grafts with genes expressing factors capable of inhibition of the alloreactive immune response will influence regional immunity. Several basic prerequisites must be met to test this hypothesis. First, a system of efficient gene delivery to cells and organs of interest must be refined, and gene expression of functional protein must be assured. Finally, the protein products of inserted genes must be capable of inhibiting the immune response.
The first aim, then, is to define efficient methods of viral mediated gene transfer into rat livers. We will perform a systematic qualitative and quantitative analysis of the methods, efficiency, infected cell subtypes, and stability of gene transfer in liver grafts infected with adenoviral vectors. Our preliminary results demonstrate that adenoviral vectors, when compared to retroviral vectors, are more versatile and fulfill the necessary prerequisites for efficient gene delivery.
The second aim i s the quantification of protein production in the liver after gene transfer under various conditions, to confirm biologic activity of expressed protein, and to examine the regulatory role of promoters in protein production. Thus, the range of gene expression for recombinant proteins will be defined.
The third aim i s to study the regional hepatic immune response after transplantation of grafts infected with TGF-beta. TGF-beta will be used as a representative potent immune regulatory protein. In vitro and in vivo assays will be used to determine whether excessive local production of this inhibitory factor modifies the response of alloreactive T cells, and patterns of allograft rejection. In summary, this proposal explores methods of using gene therapy techniques to address mechanistic issues of liver transplantation immunobiology. The ultimate goal is to define biological principles by which gene therapy efforts may be applied to experimental and clinical liver transplantation.
