Dissertation/Thesis Abstract

Safety and utility of phage integrases for gene therapy
by Woodard, Lauren Elizabeth Harkinson, Ph.D., Stanford University, 2009, 155; 3382971
Abstract (Summary)

The &phis;C31 integrase is a phage recombinase that is sequence-specific and is able to mediate integration into endogenous sites in mammalian chromosomes, making it useful for integrating non-viral gene therapy. We have explored the dynamics of &phis;C31 integrase protein expression in mammalian cells and show it to be degraded by the proteasome in a cell-cycle dependent manner. Additionally it is cytoplasmically localized. Fusion of the integrase to a nuclear localization signal did not aid integration efficiency in vitro or for Factor IX gene therapy in mice. To investigate how &phis;C31 integrase may obtain access to the genomic DNA when it is localized to the cytoplasm, we asked if the enzyme required cell division for activity. Using IdU to mark cells that went through S-phase after gene therapy administration to the liver, we determined that &phis;C31 integrase does not require cell division, suggesting that it may be a useful method to integrate into quiescent tissues in the future. These studies also uncovered a large degree of proliferation in the liver that occurred after the hydrodynamic injection to transfect the hepatocytes. Indeed, in safety testing to determine if &phis;C31 integrase-mediated chromosomal rearrangements induce carcinogenesis in the liver, we found that hydrodynamic injection cooperated with the oncogene C-MYC, which is known to be affected by cellular proliferation. Surprisingly, expression of inactive or active &phis;C31 integrase protein reversed this effect and returned the tumor latency to that of untreated transgenic mice. These results indicate that &phis;C31 integrase is a safe gene therapy method, while hydrodynamic injection alone was statistically tumor-inducing in the mouse cancer model. Finally, we tested if &phis;C31 integrase could be combined with a novel muscle stem cell technology to deliver the missing gene to dystrophic muscle, we undertook studies with wild-type mesoangioblasts obtained from two sources. Unfortunately, these cells were not actually muscle stem cells but rather were the feeder layer STO cells, which we discovered formed malignant pleomorphic sarcomas in mice. These cells had a high degree of aneuploidy, but did differentiate in vitro. Therefore, we have found that mesoangioblasts may not be a viable therapeutic strategy for repair of dystrophic muscle. In the future, other stem cell therapies will be tested for this purpose. In conclusion, phage integrases appear to be safe and well-suited to gene therapy applications.

Indexing (document details)
Advisor: Calos, Michele P.
School: Stanford University
School Location: United States -- California
Source: DAI-B 70/10, Dissertation Abstracts International
Subjects: Molecular biology, Genetics, Cellular biology
Keywords: Gene therapy, Hydrodynamic injections, Liver, Phage integrases, Proteasome
Publication Number: 3382971
ISBN: 9781109447545
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