(provide by applicant): We have developed new technology for site-specific integration in mammalian cells. The technology involves use of novel phage integrases that mediate efficient integration at compact recognition sites. Sites recognized by the enzymes occur at low frequency in mammalian genomes and can be used to target integration. We have validated this technology in mammalian tissue culture cells, and it is ready for transfer to animals. In vivo experiments are now essential for application of the technology to improve existing methodologies for gene therapy and for construction of transgenic animals. The proposed experiments include the generation of transgenic mice that carry recognition sites for the integrases. These animals will be used for gene therapy studies and to improve basic technology for creation of transgenic mammals. We will carry out gene therapy studies in wild-type, transgenic, and disease model mice by introducing plasmid DNA to the liver by the hydrodynamic tail vein injection method. We will deliver a plasmid carrying the gene for a phage integrase along with a plasmid carrying the therapeutic gene and a recognition site for the integrase. The enzyme will mediate site-specific integration of the therapeutic gene into the genome at sites recognized by the enzyme. We will use this technology to develop effective permanent gene therapy treatments for emphysema and hemophilia B by introducing the a1-antitrypsin and factor IX genes. Success in these experiments will lead to application of this innovative technology to other gene therapy settings and progress toward clinical trials in patients.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL068112-02
Application #
6538077
Study Section
Medical Biochemistry Study Section (MEDB)
Program Officer
Link, Rebecca P
Project Start
2001-07-01
Project End
2004-06-30
Budget Start
2002-07-01
Budget End
2003-06-30
Support Year
2
Fiscal Year
2002
Total Cost
$282,349
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:
Chavez, Christopher L; Keravala, Annahita; Chu, Jacqueline N et al. (2012) Long-term expression of human coagulation factor VIII in a tolerant mouse model using the ?C31 integrase system. Hum Gene Ther 23:390-8
Woodard, Lauren E; Keravala, Annahita; Jung, W Edward et al. (2010) Impact of hydrodynamic injection and phiC31 integrase on tumor latency in a mouse model of MYC-induced hepatocellular carcinoma. PLoS One 5:e11367
Woodard, L E; Hillman, R T; Keravala, A et al. (2010) Effect of nuclear localization and hydrodynamic delivery-induced cell division on phiC31 integrase activity. Gene Ther 17:217-26
Keravala, Annahita; Ormerod, Brandi K; Palmer, Theo D et al. (2008) Long-term transgene expression in mouse neural progenitor cells modified with phiC31 integrase. J Neurosci Methods 173:299-305
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
Calos, Michele P (2006) The phiC31 integrase system for gene therapy. Curr Gene Ther 6:633-45
Portlock, Joylette L; Keravala, Annahita; Bertoni, Carmen et al. (2006) Long-term increase in mVEGF164 in mouse hindlimb muscle mediated by phage phiC31 integrase after nonviral DNA delivery. Hum Gene Ther 17:871-6
Ishikawa, Yoshinori; Tanaka, Nobuyuki; Murakami, Kazuhiro et al. (2006) Phage phiC31 integrase-mediated genomic integration of the common cytokine receptor gamma chain in human T-cell lines. J Gene Med 8:646-53
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

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