Therapeutic applications of the ΦC31 integrase system

Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305-5120, USA.
Current Gene Therapy (Impact Factor: 2.54). 08/2011; 11(5):375-81.
Source: PubMed


The potential use of the ΦC31 integrase system in gene therapy opens up the possibilities of new treatments for old diseases. ΦC31 integrase mediates the integration of plasmid DNA into the chromsomes of mammalian cells in a sequence-specific manner, resulting in robust, long-term transgene expression. In this article, we review how ΦC31 integrase mediates transgene integration into the genomes of target cells and summarize the recent preclinical applications of the system to gene therapy. These applications encompass in vivo studies in liver and lung, as well as increasing ex vivo uses of the system, including in neural and muscle stem cells, in cord-lining epithelial cells, and for the production of induced pluripotent stem cells. The safety of the ΦC31 integrase system for gene therapy is evaluated, and its ability to provide treatments for hemophilia is discussed. We conclude that gene therapy strategies utilizing ΦC31 integrase offer great promise for the development of treatments in the future.

Download full-text


Available from: Michele Pamela Calos
  • Source
    • "A number of recombinase systems have been developed, and are being optimized or fine-tuned for safe use in human genetic therapy. These include C31, cre/lox, Sleeping Beauty, PiggyBac, and zinc finger nucleases [44] [45] [46] [47] [48] [49] [50] [51] [52]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: A bewildering variety of plasmids have been developed for a wide array of purposes in biotechnology and biomedical research. These purposes include transient and stable protein expression, mutation, recombination-mediated nuclear gene knockout, promoter analysis, RNA stability, targeted RNA degradation, and gene therapy. Each plasmid has been optimized for only one of these functions, and consequentially, convenient restriction enzyme sites surround only one genetic element. However, an optimal expression system often requires the use of different genetic elements that are rarely found on the same plasmid (such as differing promoters or eukaryotic antibiotic resistance), and exchange of these elements between plasmids is difficult. We retrofitted pcDNA3 (an archaic but well-validated and useful plasmid from Stratagene, Inc.) to facilitate the exchange of various genetic elements by inserting useful restriction endonuclease sites at the borders of these elements. With the retrofitted plasmid, called pc3.5, we can exchange not only the ectopically expressed gene of interest but also promoters or polyadenylation sites of the gene of interest, as well as the eukaryotic antibiotic resistance gene, its promoter or polyadenylation sequences. We have used derivatives of these plasmids for transient and stable gene expression, promoter analysis and induction, and mesenchymal stem cell engineering. Retrofitting plasmids is a simple process than can be applied to a wide variety of plasmid or viral vector systems to diversify and optimize their utility.
    Full-text · Chapter · Jan 2014
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Viruses are ubiquitous and can infect any of the three existing cellular lineages (Archaea, Bacteria and Eukarya). Despite the persisting negative public perception of these entities, scientists learnt how to domesticate some of them. The study of molecular mechanisms essential to the completion of viral cycles has greatly contributed to deciphering fundamental processes in biology. Nowadays, viruses have entered the biotechnological era and numerous applications have already been developed. Viral-derived tools are used to manipulate genetic information, detect, diagnose, control and cure infectious diseases, or even design new structural assemblies. With the recent advances in the field of metagenomics, an overwhelming amount of information on novel viruses has become available. As current tools have been historically developed from a limited number of viruses, the potential of discoveries from new archaeal, bacterial and eukaryotic viruses may be limited only by our understanding of the multiple facets of viral cycles.
    Full-text · Article · Oct 2012 · Virology
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Starting in 1991, the advance of Tyr-recombinases Flp and Cre enabled superior strategies for the predictable insertion of transgenes into compatible target sites of mammalian cells. Early approaches suffered from the reversibility of integration routes and the fact that co-introduction of prokaryotic vector parts triggered uncontrolled heterochromatization. Shortcomings of this kind were overcome when Flp-Recombinase Mediated Cassette Exchange entered the field in 1994. RMCE enables enhanced tag-and-exchange strategies by precisely replacing a genomic target cassette by a compatible donor construct. After "gene swapping" the donor cassette is safely locked in, but can nevertheless be re-mobilized in case other compatible donor cassettes are provided ("serial RMCE"). These features considerably expand the options for systematic, stepwise genome modifications. The first decade was dominated by the systematic generation of cell lines for biotechnological purposes. Based on the reproducible expression capacity of the resulting strains, a comprehensive toolbox emerged to serve a multitude of purposes, which constitute the first part of this review. The concept per se did not, however, provide access to high-producer strains able to outcompete industrial multiple-copy cell lines. This fact gave rise to systematic improvements, among these certain accumulative site-specific integration pathways. The exceptional value of RMCE emerged after its entry into the stem cell field, where it started to contribute to the generation of induced pluripotent stem (iPS-) cells and their subsequent differentiation yielding a variety of cell types for diagnostic and therapeutic purposes. This topic firmly relies on the strategies developed in the first decade and can be seen as the major ambition of the present article. In this context an unanticipated, potent property of serial Flp-RMCE setups concerns the potential to re-open loci that have served to establish the iPS status before the site underwent the obligatory silencing process. Other relevant options relate to the introduction of composite Flp-recognition target sites ("heterospecific FRT-doublets"), into the LTRs of lentiviral vectors. These "twin sites" enhance the safety of iPS re-programming and -differentiation as they enable the subsequent quantitative excision of a transgene, leaving behind a single "FRT-twin". Such a strategy combines the established expression potential of the common retro-and lentiviral systems with options to terminate the process at will. The remaining genomic tag serves to identify and characterize the insertion site with the goal to identify genomic "safe harbors" (GOIs) for re-use. This is enabled by the capacity of "FRT-twins" to accommodate any incoming RMCE-donor cassette with a compatible design.
    Full-text · Article · Nov 2012 · Gene
Show more