Pre-clinical evaluation of the Sleeping Beauty transposon system for gene therapy

Nonviral gene transfer has been explored as a potential ex vivo and in vivo gene therapy. The Sleeping Beauty (SB) transposon system provides a nonviral means to achieve chromosomal integration. For SB-mediated gene therapy, a plasmid encoding a SB transposon and its therapeutic cargo would be delivered into a target cell or tissue with a source of SB transposase. Still, persistent expression of the therapeutic gene product remains a consistent obstacle. Much effort has been placed upon defining the factors that would make possible life-long, therapeutic levels of gene expression. Bioluminescence imaging provides an immediate measure of reporter gene expression that is localized at the region where expression occurs. I used bioluminescence imaging to (i) provide the most detailed time-course of plasmid-based expression reported to date, (ii) characterize expression for RNA-encoded sequences in living animals, (iii) monitor gene therapy in pre-clinical animal models, and (iv) demonstrate maintenance of gene expression following stem cell differentiation. One concern in the therapeutic application of the SB system is that persistent expression of transposase could result in transposon instability and genotoxicity. I developed delivery conditions for in vitro transcribed RNA to attain SB transposase levels comparable to those achieved using promoters known to provide levels of transposase protein effective for mediating transposition. Pre-clinical testing of transposase RNA for SB-mediated gene transfer was conducted in fumarylacetoacetate hydrolase (FAH) deficient mice and demonstrated complete phenotypic correction. The SB transposon system was tested in human embryonic stem (ES) cells and multipotent adult progenitor cells (MAPC), important biological and potentially therapeutic cellular systems. Human ES cells engineered with SB exhibited stable gene expression after differentiation into hematopoietic cells or teratoma tumor tissue. Gene transfer studies in MAPC were performed using an internally controlled plasmid to deliver both components of the PhiC31 integrase (&phis;C31) and SIB transposase, providing a direct comparison of these two nonviral integrating vectors. Each system mediated stable gene transfer without affecting the capacity of MAPC to differentiate into endothelium and liver. Combined, these results contribute important developments for the use of nonviral integrating vectors in human gene therapy.