The goal of this proposal is to continue advancing this innovative site-specific integration technology toward utility in gene therapy through studies in animals. The technology uses the phage phiC31 integrase to provide safe and efficient site-specific integration of incoming gene therapy vectors at preferred locations in the genome. PhiC31-mediated integration provides robust, long-term expression of the integrated therapeutic gene, without the risk of random integration. During the two years of the grant to date, we demonstrated that the phiC31 integrase technology was effective in providing long-term, high level liver expression of factor IX in mice. We also demonstrated therapeutic expression of collagen VII and laminin B3 in human skin grafted on to mouse models and expression of human alpha1 antitrypsin in mouse liver. In addition, we used the technology to create transgenic animals. ? ? We now wish to continue development of the integrase technology for long-term gene therapy by moving to larger animal models in two important tissues, liver and muscle. We have already demonstrated effective DNA delivery methods for these tissues. In both liver and muscle we observed more robust and longer-lived gene expression when phiC31 integrase technology was used, compared to unintegrated plasmid DNA. We will employ hydrodynamic delivery of the phiC31 integrase system to express therapeutic levels of factor IX from the livers of rats, rabbits, and dogs. We will also explore alternative methods to deliver the integrase system to liver. In addition, we will use the phiC31 integrase technology, delivered to muscle by DNA injection and electroporation, for integration and long-term expression of plasmids bearing VEGF and other angiogenic factors to correct ischemia in mice, rats, and rabbits. By scaling up to animals more similar to humans, these experiments will move the site-specific integrase technology closer to the clinic. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
2R01HL068112-04
Application #
6822697
Study Section
Special Emphasis Panel (ZRG1-GTIE (90))
Program Officer
Link, Rebecca P
Project Start
2001-07-01
Project End
2007-06-30
Budget Start
2004-07-01
Budget End
2005-06-30
Support Year
4
Fiscal Year
2004
Total Cost
$324,515
Indirect Cost
Name
Stanford University
Department
Genetics
Type
Schools of Medicine
DUNS #
009214214
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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