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- Title
525. Mutants of phiC31 Integrase with Increased Efficiency and Specificity.
- Authors
Keravala, Annahita; Lee, Solomon; Olivares, Eric C.; Thyagarajan, Bhaskar; Farruggio, Alfonso; Gabrovsky, Vanessa E.; Calos, Michele P.
- Abstract
The site-specific integrase from bacteriophage phiC31, catalyzes precise, unidirectional recombination between two ∼30-40 bp sequences, a phage attachment site (attP) and a bacterial attachment site (attB). In mammalian cells, phiC31 integrase can recombine its own sites and can also integrate plasmids with the attB site into native sequences that bear partial identity to attP, referred to as pseudo attP sites. The enzyme recognizes a hierarchy of such sites and, in cell lines, appears to have several hundred potential integration sites, though most integration events occur at a subset of preferred sites. For the purpose of gene therapy, it appears desirable to obtain integration at only one or a small set of safe, well-expressed locations in the genome. In a previous study we demonstrated that by using a genetic screening assay in E. coli, we could create mutant forms of phiC31 integrase with enhanced preference for a pre-existing favored integration site in the human genome. However, this enzyme displayed a low overall integration efficiency. To create phiC31 integrase mutants with enhanced functionality, we devised a screen in mammalian cells to identify mutants with increased recombination efficiency at the attP site. We isolated a collection of such mutants, and combination of several of them led to the identification of an enzyme that mediated the majority of integration events at a single location, at a pre-integrated attP site. This enzyme may be useful for the rapid creation of cell lines with integration at a fixed location. The study also provided proof-of-principle for creation of integrases highly specific for endogenous sequences, a project is underway in the lab. Other mutants from the screen showed an elevated integration frequency at endogenous pseudo attP sites and are being tested in animals as improved gene therapy tools. We have also undertaken the structural characterization of phiC31 integrase. The location of the DNA-binding domain is being investigated by creating deletion mutants and examining them for att site binding with gel mobility shift assays. The increased efficiency and specificity attainable via mutagenesis and protein design will further enhance the utility of phiC31 integrase as a valuable tool in gene therapy.Molecular Therapy (2006) 13, S201–S202; doi: 10.1016/j.ymthe.2006.08.596
- Subjects
GENE therapy; CELL culture; BACTERIOPHAGES; GENETIC engineering; ESCHERICHIA coli; HUMAN gene mapping
- Publication
Molecular Therapy, 2006, Vol 13, pS201
- ISSN
1525-0016
- Publication type
Article
- DOI
10.1016/j.ymthe.2006.08.596