Gene transfer offers unprecedented potential to modify physiologic phenomena. Unfortunately, insufficient and transient gene expression limit this potential. For example, the investigators have been able to use gene transfer methods to introduce immunosuppressive cytokines (IL-10, TGFb) into cardiac allografts to prolong graft survival, but have been unable to achieve permanent engraftment or tolerance. Although the use of strong viral promoters has improved efficacy somewhat, limited gene expression is still the major barrier to gene therapy. It is increasingly recognized now that specific immune responses to vector components and gene transfer products result in destruction of vectors or infected cells and are a significant impediment to stable expression. Preliminary data now demonstrate that the T lymphocyte derived cytokines IFNg and TNFa limit gene expression, without killing cells, by mechanisms involving negative regulation of transcription, especially from certain viral promoters and enhancers such as HCMVie and RSV-LTR. Since gene therapy is considered for treatment of diseases which are associated with many changes in cytokines (e.g., cancer, AIDS, transplantation, atherosclerosis, autoimmunity, ischemia-reperfusion); it is hypothesized that cytokines inhibit vector gene expression in transduced cells and that determination of the mechanisms involved in cytokine-regulated gene expression will fundamentally alter the design of vectors for gene transfer and gene therapy. The objectives of the proposal are to define how cytokines regulate the expression from gene transfer vectors in order to improve strategies for gene delivery and control and thereby optimize protocols for gene therapy.
The specific aims are: (1) demonstrate that TNFa and IFNg differentially regulate expression from selected viral and cellular promoters; (2) determine how cytokine response elements regulate transcription; (3) map the cytokine-responsive negative regulatory elements; and (4) demonstrate that manipulation of cytokine responses and response elements will improve gene transfer and gene therapy in an in vivo transplantation model. Experiments will entail the manipulation of cytokines and vector promoter-reporter constructs in vitro in primary myoblasts and the C2C12 myoblast cell line to define cytokine-initiated, promoter-dependent regulation of transcription. Promoter constructs will be transfected into murine cardiac allografts to probe cytokine initiated regulation of transferred gene expression in vivo, and to show that this regulation may be manipulated to improve the utility of gene transfer and gene therapy.
