The goal of this work is to create a system that will produce site-specific integration of gene therapy vectors into the chromosomes. This feature is currently lacking in all cases. The innovative system developed here will provide long-term expression of introduced genes, for the lifetime of the target cells. This outcome is desirable in most gene therapy strategies. In order to achieve efficient and site-specific integration, Dr. Calos will use prokaryotic recombinase enzymes that work in mammalian cells with no added co-factors. These enzymes have long recognition sites that are not expected to occur in mammalian genomes. However, the principal investigator has shown that enzymes also recognize a small number of native sequences, called pseudo sites, with homology to the native recognition site. These pseudo sites can be used for site-specific integration into mammalian chromosomes. The principal investigator will locate such sites for three recombinase enzymes, using integration efficiency and good gene expression in a chromosomal context. Protein engineering will be carried out on the recombinases to improve efficiency and alter DNA recognition specificity. Gene therapy experiments will be initiated with the best enzyme/pseudo site candidates by using introduction of plasmid DNA. The novel site-specific integration strategy developed here can be used to add value to most current gene therapy vectors.
| Hillman, R Tyler; Calos, Michele P (2012) Site-specific integration with bacteriophage ?C31 integrase. Cold Spring Harb Protoc 2012: |
| Keravala, Annahita; Groth, Amy C; Jarrahian, Sohail et al. (2006) A diversity of serine phage integrases mediate site-specific recombination in mammalian cells. Mol Genet Genomics 276:135-46 |
| Chalberg, Thomas W; Portlock, Joylette L; Olivares, Eric C et al. (2006) Integration specificity of phage phiC31 integrase in the human genome. J Mol Biol 357:28-48 |
| Chalberg, Thomas W; Genise, Hilary L; Vollrath, Douglas et al. (2005) phiC31 integrase confers genomic integration and long-term transgene expression in rat retina. Invest Ophthalmol Vis Sci 46:2140-6 |
| Ginsburg, Daniel S; Calos, Michele P (2005) Site-specific integration with phiC31 integrase for prolonged expression of therapeutic genes. Adv Genet 54:179-87 |
| Groth, Amy C; Calos, Michele P (2004) Phage integrases: biology and applications. J Mol Biol 335:667-78 |
| Portlock, Joylette L; Calos, Michele P (2003) Site-specific genomic strategies for gene therapy. Curr Opin Mol Ther 5:376-82 |
| Hollis, Roger P; Stoll, Stephanie M; Sclimenti, Christopher R et al. (2003) Phage integrases for the construction and manipulation of transgenic mammals. Reprod Biol Endocrinol 1:79 |
| Stoll, Stephanie M; Ginsburg, Daniel S; Calos, Michele P (2002) Phage TP901-1 site-specific integrase functions in human cells. J Bacteriol 184:3657-63 |
| Olivares, E C; Hollis, R P; Calos, M P (2001) Phage R4 integrase mediates site-specific integration in human cells. Gene 278:167-76 |
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