710. Stem Cell Gene Therapy: Synergizing Improved Gene Repair/Modification & Targeted Integration of Transgenes to Transient In Vivo Topical Minigene Expression

Abstract

With their self-renewing and homing/differentiative abilities, stem cells are the ideal targets for most gene therapy protocols: they are either long-term carriers of therapeutic genes or mobilized/recruted targets of transient regenerative processes (eg revascularization). Long term gene therapy is thus amenable to synergistic combinations where ex vivo/in vivo genetic engineering of stem cells associates to transient in vivo topical expression of minigenes encoding various homing/regenerative factors. Such a combination of long-term and transient gene therapy protocols stands as a powerful mean to tackle inherited and degenerative/acquired disorders. Indeed, transient magnification of stem cell regenerative potential, in which therapeutic homing is a key feature, should culminate in efficient targeted repopulation dynamics of engineered stem cells and differentiated derivatives in relevant tissues/organs, thereby providing appropriate gene therapeutics and life-long maintenance/replacement of therapeutic engineered cells. This long-term gene therapy strategy is hampered by a major safety concern: random integration of therapeutic DNA. However, promising site-specific integration vectors based on AAV rep or phage phiC31 integrase are at the pre-clinical stage. Unlike these approaches, gene targeting is driven by homologous recombination and has thus target flexibility. It mediates DNA exchanges between chromosomal and transfecting/transducing DNA, thereby providing the means to modify at will the sequence of target chromosomal DNA. Gene targeting stands thus as the ultimate process for gene repair/alteration and targeted (site-specific) transgene integration. It is however overwhelmed by random integration and is highly inefficient unless a double-strand break (DSB) hits target chromosomal DNA.