Without argument, the most important goal of gene therapy is to fix the somatic endogenous gene in a gene defect. This goal circumvents most major problems associated with current strategies of gene therapy mediated by viruses or proviruses, antisense oligonucleotides, or ribozymes. Our intent is to repair a gene much like the normal cellular process of nucleotide excision repair or mismatch repair, which occurs as a perfectly normal everyday occurrence in human cellular physiology.
Our aims are to employ the normal DNA repair enzymes of the eye and to supply a suitable repair substrate to the nucleus of the cell. Having provided this substrate, we will rely on the normal DNA repair enzymes of the cell to recognize a single nucleotide mismatch between the substrate and the endogenous gene and replace the defective base with the wildtype nucleotide. This process is now known as genoplasty. Our principal model systems will be the commonly available animal models of human oculocutaneous tyrosinase-negative albinism, white mice and New Zealand white rabbits. Using genoplasty, we plan to repair precisely known lesions in the tyrosinase gene somatically in RPE cells. Thus, this research will have high impact on the future directions of gene therapy in the eye. It is novel, no one else is doing this in the eye, and it is innovative because it uses the endogenous gene and normal DNA repair enzymes to allow the cell to fix itself. Our guiding hypothesis is that genoplasty works by mass action. The more substrate put into the cell, the more likely that gene repair will occur. We further hypothesize that virtually every cell has the right DNA repair enzymes and ancillary proteins. Thus, to achieve gene repair, all we need to do is get an adequate amount of substrate into the nucleus, and nature will take its course.
Our aims are: 1) to develop better assays for detecting gene repair of the tyrosinase gene in somatic RPE cells, 2) to achieve high levels of transfection and nuclear localization in ocular tissues, and 3) to design better substrates.
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