Long-Term Expression of Human Coagulation Factor VIII in a Tolerant Mouse Model Using the u C31 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.

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Available from: Michele Pamela Calos, Oct 07, 2015
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    • "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]. "
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    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.
    Advances in Genetics Research, Volume 13, Edited by Kevin V. Urbano, 01/2014: chapter 1: pages 1-22; Nova Publishers., ISBN: 978-1-63463-091-7
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    ABSTRACT: Site-specific recombinases (SSRs) can perform DNA rearrangements, including deletions, inversions and translocations when their naive target sequences are placed strategically into the genome of an organism. Hence, in order to employ SSRs in heterologous hosts, their target sites have to be introduced into the genome of an organism before the enzyme can be practically employed. Engineered SSRs hold great promise for biotechnology and advanced biomedical applications, as they promise to extend the usefulness of SSRs to allow efficient and specific recombination of pre-existing, natural genomic sequences. However, the generation of enzymes with desired properties remains challenging. Here, we use substrate-linked directed evolution in combination with molecular modeling to rationally engineer an efficient and specific recombinase (sTre) that readily and specifically recombines a sequence present in the HIV-1 genome. We elucidate the role of key residues implicated in the molecular recognition mechanism and we present a rationale for sTre's enhanced specificity. Combining evolutionary and rational approaches should help in accelerating the generation of enzymes with desired properties for use in biotechnology and biomedicine.
    Nucleic Acids Research 12/2012; 41(4). DOI:10.1093/nar/gks1308 · 9.11 Impact Factor
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    ABSTRACT: Phage-encoded serine integrases mediate directionally regulated site-specific recombination between short attP and attB DNA sites without host factor requirements. These features make them attractive for genome engineering and synthetic genetics, although the basis for DNA site selection is poorly understood. Here we show that attP selection is determined through multiple proofreading steps that reject non-attP substrates, and that discrimination of attP and attB involves two critical site features: the outermost 5-6 base pairs of attP that are required for Int binding and recombination but antagonize attB function, and the "discriminators" at positions -15/+15 that determine attB identity but also antagonize attP function. Thus, although the attachment sites differ in length and sequence, only two base changes are needed to convert attP to attL, and just two more from attL to attB. The opposing effect of site identifiers ensures that site schizophrenia with dual identities does not occur.
    PLoS Genetics 05/2013; 9(5):e1003490. DOI:10.1371/journal.pgen.1003490 · 7.53 Impact Factor
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