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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK058187-03
Application #
6524290
Study Section
Medical Biochemistry Study Section (MEDB)
Program Officer
Mckeon, Catherine T
Project Start
2000-09-01
Project End
2004-07-31
Budget Start
2002-08-01
Budget End
2003-07-31
Support Year
3
Fiscal Year
2002
Total Cost
$273,700
Indirect Cost
Name
Stanford University
Department
Genetics
Type
Schools of Medicine
DUNS #
800771545
City
Stanford
State
CA
Country
United States
Zip Code
94305
Hillman, R Tyler; Calos, Michele P (2012) Site-specific integration with bacteriophage ?C31 integrase. Cold Spring Harb Protoc 2012:
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
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; 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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