New non-viral gene transfer procedures are needed for human gene therapy in order to achieve long-term maintenance and expression of newly introduced genes. The purpose of this project is to evaluate the Sleeping Beauty (SB) transposon system for its efficacy to catalyze integration of several transgenes into selected tissues in mice and rats. The SB system is binary, consisting of a transposon containing a transgene and a source of transposase enzyme. The SB transposase is able to mediate enhanced integration of marker/reporter genes in several mammalian culture cell lines in vitro as well as in fertilized embryos of zebrafish and Xenopus. Here, we propose to extend these studies in order to determine the efficacy of the SB transposon system as a gene transfer vector. Preliminary results indicate that the transposon can be delivered to livers of adult mice for long-term expression using hydrodynamic pressure. This procedure may not be possible to use in humans. Accordingly, we will evaluate the efficiencies of alternative delivery of the SB system to livers of whole animals affected by single-gene disorders that mimic conditions of human disease. Furthermore, as an additional precaution to curtail potential long-term effects of SB transposase, we will examine two strategies to curtail transposase activity. We will concentrate on gene therapy for mucopolysaccharidosis.
The Specific Aims of this project are to: 1) Construct safer transposon vectors with either insulator elements, immediately inside both ends of the transposon vector to keep the enhancers driving a transgene from activating neighboring genes or that contain a """"""""suicide gene"""""""". 2) Determine the efficiency of SB for gene transfer and expression in the livers of mice; SB will be tested as a vector system for gene transfer into the liver using a transposon engineered to express genes whose activities are deficient in certain human diseases. 3) Determine the efficiency of SB for gene transfer and expression in the livers of mice using purified SB transposase rather than its gene; to assure limitation of transposase activity, the efficacy of using purified SB transposase accompanying transposons will be tested in liver cells. The experiments in this project will provide an assessment of the capacity of SB as a gene transfer vector targeting therapeutically important organs in an in vivo gene therapy protocols. The results will lay the groundwork for optimization of this vector system and its future application to human gene therapy.
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