The therapeutic potential of ΦC31 integrase as a gene therapy system.

Stanford University School of Medicine, Department of Genetics, Stanford, CA 94305-5120, USA.
Expert opinion on biological therapy (Impact Factor: 3.74). 07/2011; 11(10):1287-96. DOI: 10.1517/14712598.2011.601293
Source: PubMed


INTRODUCTION: The φC31 integrase system is a phage-derived system that offers the ability to integrate plasmid DNA into the chromosomes at a subset of endogenous preferred locations associated with robust gene expression. Recent progress highlights the unique advantages of this system for in vivo gene therapy and for use in stem cells. AREAS COVERED: The φC31 integrase system has been under development for ten years and has been demonstrated to be effective for integration of plasmids in a variety of tissues and organs for gene therapy in animal systems, as well as in isolated human cells. We focus on work with the φC31 integrase system during the past 12-18 months. This work has centered on a series of papers involving in vivo delivery of the integrase system to the liver and a variety of studies demonstrating the utility of the integrase system in stem cells. EXPERT OPINION: We conclude that the φC31 integrase system has significant potential for liver gene therapy, if effective DNA delivery methods for large mammals become available. The φC31 integrase system displays an outstanding fit for use in pluripotent stem cells, and this area is expected to be the subject of intense development.

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Available from: Michele Pamela Calos
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    • "PhiC31 integrase recognizes relatively short but moderately specific sequences in mammalian genomes7. Thus, a phiC31 integrase system exhibits several characteristics, such as site-specificity and unidirectional recombination, and allow the successful use of integrase in various fields of study, including gene delivery in vitro5 or in vivo8, gene therapy9, and production of transgenic animals10. "
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    ABSTRACT: The Streptomyces phage phiC31 integrase was tested for its feasibility in excising transgenes from the barley genome through site-specific recombination. We produced transgenic barley plants expressing an active phiC31 integrase and crossed them with transgenic barley plants carrying a target locus for recombination. The target sequence involves a reporter gene encoding green fluorescent protein (GFP), which is flanked by the attB and attP recognition sites for the phiC31 integrase. This sequence disruptively separates a gusA coding sequence from an upstream rice actin promoter. We succeeded in producing site-specific recombination events in the hybrid progeny of 11 independent barley plants carrying the above target sequence after crossing with plants carrying a phiC31 expression cassette. Some of the hybrids displayed fully executed recombination. Excision of the GFP gene fostered activation of the gusA gene, as visualized in tissue of hybrid plants by histochemical staining. The recombinant loci were detected in progeny of selfed F(1), even in individuals lacking the phiC31 transgene, which provides evidence of stability and generative transmission of the recombination events. In several plants that displayed incomplete recombination, extrachromosomal excision circles were identified. Besides the technical advance achieved in this study, the generated phiC31 integrase-expressing barley plants provide foundational stock material for use in future approaches to barley genetic improvement, such as the production of marker-free transgenic plants or switching transgene activity.
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