While functional correction of genodermatoses has been demonstrated using retroviral mediated ex vivo gene therapy, the cost and traumatic features of this procedure has led to the search for additional non-viral technologies for gene therapy. Non-viral vector gene transfer vectors have been plagued by the inability to get both therapeutic and persistent levels of gene expression in vivo. They have recently described a non-viral vector system based on DNA transposons that integrates an expression cassette into the chromosomal DNA of hepatocytes in vivo. Stable gene transfer is achieved by transient expression from the transposase gene resulting in the integration of transposon DNA flanking an expression cassette. As a result, life-long and therapeutic concentrations of human factor IX have been achieved in hemophilia B mice. Moreover, preliminary studies suggest there is no toxicity and secondary transposition if it occurs is extremely rare. The two major limitations of this system are the limited gene transfer capacity (5.0 kb), and the efficiency of integration, which is 5% of transfected hepatocytes in vivo and varies between different cell types in culture. While the effectiveness of this transposon has been demonstrated for diseases like hemophilia, there are other diseases in which the efficiency and size of the DNA insert will need to be increased. Thus, they plan to use random mutagenesis and DNA shuffling to develop a transposon-based vector that can integrate more efficiently and larger pieces of DNA. They will begin to identify cellular factors involved in transposition. This may give an idea of the rate limiting factors required for transposition and help to explain why there is variation in transposition efficiencies within different cell types. Finally, whey propose to address a safety issue related to determining the true efficacy of secondary transposition events in vitro and in vivo. While the new vectors will first be tested in liver, the biological system where the vector has been most well studied, they plan to collaborate with the other projects herein and pursue the use of the first generation transposon as well as the improved versions for skin-related gene transfer studies.
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