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  • Book
    Alfonso Pedro Farruggio.
    The "integrase" enzyme of the bacterial virus phiC31 mediates the unidirectional fusion of the phiC31 and host-bacterium genomes. This recombination reaction occurs between two specific DNA sequences (att-sites), and can be performed by the enzyme without accessory factors in a wide variety of cellular environments, including human cells. Although its primary application to date in eukaryotes has been in organisms where the native phiC31 integrase (C31-int) att-sites have been pre-introduced, C31-int can also function on certain endogenous sequences (pseudosites) in animal genomes, albeit with significantly reduced activity. The ability of C31-int to perform pseudosite integration has made it the focus of several whole-animal and tissue-culture gene-therapy studies over the past decade, as there is currently no means to safely maintain the therapeutic DNA that is delivered for gene-therapy. The focus of my graduate work has been to assist these efforts to apply C31-int for gene-therapy. Specifically, I have sought to increase our basic scientific understanding of DNA recognition by C31-int through deletion and chimera studies (Chapters 2-3), and I have also attempted to improve the implementation of C31-int applications in mammals through my contributions to applied C31-int projects (Chapters 4-6). The results from my deletion and chimera studies have helped to clarify that C31-int employs DNA binding domains that are largely modular and have also helped to lay the groundwork for a potential means to derive serine integrases with novel specificities. The results from my contributions to applied C31-int projects have made it possible to reverse the wild-type reaction in mammalian cells (Chapter 4), to produce high-quality mouse induced-pluripotent-stem cells (iPSC) with C31-int (Chapter 5), and to genetically engineer mouse iPSCs (Chapter 6). Although C31-int will still require significant engineering before it is suitable for use in human gene therapy, and although additional work will be required to fully realize the potential of our iPSC-production and -engineering scheme in human cells, my experience with C31-int and related enzymes that I describe in this dissertation has left me optimistic that these are not unreasonable goals.