Characterization of the Sleeping Beauty transposon system for gene therapy applications /

University of Minnesota Ph.D. dissertation. May 2008. Major: Molecular, Cellular, Developmental Biology and Genetics. Advisor: Dr. R. Scott McIvor. 1 computer file (PDF); viii, 164 pages.Science and medicine are merging in development of gene therapy techniques that will likely become a recognized method for the treatment of human disease within our lifetime. The basic goal of gene therapy is to deliver therapeutic genes to target cell populations, allowing the cells themselves to generate a therapeutic gene product, for example to provide a gene product which is missing in the patient. Most of the current gene therapy trials have been based on the use of viral vectors to deliver therapeutic genes. These viral vectors are being used because they have already evolved mechanisms to efficiently enter cells. Because of the complications inherent in the use of viruses for delivery, a nonviral approach would have several advantages if it could achieve similar levels of efficiency. To this end, I set forth to characterize the Sleeping Beauty (SB) transposon system as a potential tool for effective nonviral gene transfer and its eventual use in clinical gene therapy protocols. The SB transposon system consists of a DNA cargo, usually plasmid based, which in the presence of the transposase integrates into chromosomal DNA. One of the key concerns for any gene delivery system is its ability to function in cells that are not dividing, as many cells in the body that are potential targets for gene therapy are non-dividing. In a series of in vitro experiments utilizing various techniques to halt cell division, I determined that cell division is likely not necessary for SB-mediated integration and expression to occur. Secondly, when tracking expression in vivo, it is not possible to distinguish the amount of gene product produced from integrated vs. nonintegrated transposons. Differentiating between these two sources of transgene expression soon after delivery, will allow insight into transposition efficiency in vivo that can relate to its clinical use. Using LoxP recombination sites, a Cre recombinase inducible mouse strain and transposons carrying a murine erythropoietin gene (Epo), I was able to silence expression from nonintegrated transposons and quantify in vivo gene expression specifically from transposed sequences. Over-expression of erythropoietin in the murine model became an unexpected problem due to subsequent erythrocytosis. Delivery of plasmid DNA to the livers of mice results in an initial spike of transgene expression and when coupled with the subsequent ubiquitous expression of the Epo transgene, circulating Epo levels remained greatly elevated, leading to serious health complications and death. To circumvent the initial spike of EPO expression, an inducible promoter was constructed that responds to hypoxic conditions. In this way, expression of erythropoietin should be regulated to prevent over-expression. Insights gained from these studies will contribute to our understanding of the capabilities of the SB system and its potential application to the treatment of human disease in the future