CORNEAL ENDOTHELIAL CELL REGENERATION

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The goal of this proposal is to circumvent penetrating keratoplasty (PKP) by stimulating the growth of in situ corneal endothelial cells (CEC). The rationale for this strategy is based on the observation that virally introduced SV40 large T-antigen gene can rescue in vitro cultured human CEC from senescent non-proliferation. These immortalized (more appropriately, life-(extended) cells retain morphologic and immunologic features observed in primary cultures of normal adult CEC. Since this immortalizing gene can induce proliferation of corneal endothelial cells in vitro, we propose. to test the hypothesis that an immortalizing protein can induce limited proliferation when introduced into cells. Thus many negative feature associated with gene therapy approaches using transforming genes can be avoided by focusing on specific protein introduction. The half-life of the protein dictates a limited presence to initiate a transient state of proliferation and then as the protein is normally degraded, the cells return to "normal". Therefore, we propose to determine whether the specific introduction of a potent intracellular mitogen (SV40) large T-antigen) can stimulate limited in vivo proliferation of the normally non-dividing corneal endothelial cells thus maintaining cornea function and abrogate the need for many cornea transplants. For this system to work in corneal endothelial cells, we need to address two important questions and to do this, we propose the following specific aims; 1. Test the hypothesis that purified SV40 large T-antigen can be introduced in cultured cells and extend this strategy to human corneal endothelials cells (HCEC). It will be determined whether the introduced SV40 T-antigen localizes to the nucleus and can stimulate DNA synthesis in non-replicating cells. 2. Test the hypothesis that Herpes Simplex Virus (HSV) delta L-particles can be used as an efficient "entry vehicle" for the specific delivery of protein larvae T-antigen into cells and validate its effectiveness in both cultured cells and in an animal model.