phiC31 integrase for non-viral gene therapy in the mammalian retina.

摘要

The integrase from bacteriophage &phis;C31 normally recombines attB and attP to integrate the phage genome into the Streptomyces genome. Used out of context, it can also catalyze recombination between attB and endogenous sequences in mammalian genomes, termed pseudo attP sites, resulting in efficient, site-specific integration of plasmid DNA. &phis;C31 integrase has been studied as a useful gene therapy vector because it confers site-specific integration and long-term transgene expression following non-viral gene transfer.; Here we describe the integration specificity of &phis;C31 integrase in the human genome, and show that 60% of observed integrations occur in the 20 most frequently observed genomic sites or repeat elements. We also define a consensus for &phis;C31 integrase mediated integration and discuss the safety of &phis;C31 integrase as a gene therapy vector.; In subsequent chapters, we describe the utility of &phis;C31 integrase as a gene therapy vector in vivo. In mouse liver, we observe long-term transgene expression and site-specific genomic integration with &phis;C31 integrase. We then demonstrate gene transfer to rat retinal pigment epithelium by using electroporation and long-term transgene expression in the presence of &phis;C31 integrase. In experiments in rabbit retina, we introduce a new technology for non-viral gene transfer called electron-avalanche transfection. Electron-avalanche transfection uses microelectrodes to produce a high electric field with synchronized tensile stress to achieve much higher transfection efficiency than either method alone. We evaluate the safety of electron-avalanche transfection and show that it is less damaging than conventional electroporation and that it does not damage rabbit retina. Finally we use electron-avalanche transfection in combination with &phis;C31 integrase to demonstrate stable gene transfer to rabbit retina.