Submitted March 24, 2008
Accepted June 17, 2008
Interfering RNA mediated purine analog resistance for in vitro and in vivo cell selection
Christopher C. Porter and James DeGregori*
Rick Wilson Center for Cancer and Blood Disorders, The Children's Hospital, Aurora, CO, United States
Biochemistry & Molecular Genetics, University of Colorado Denver, School of Medicine, Aurora, CO, United States
* Corresponding author; email: james.degregori{at}uchsc.edu.
The advancement of gene therapy has been slowed, in part, by inefficient transduction of targeted cells and poor long-term engraftment of genetically modified cells. Thus, the ability to select for a desired population of cells within a recipient would be of great benefit for improving gene therapy. Proposed strategies for in vivo cell selection using drug resistance genes have had disappointing outcomes and/or require highly genotoxic medications to be effective. We hypothesized that resistance to purine analogs, a well-tolerated, relatively low-toxicity class of medications, could be provided to cells using interfering RNA against hypoxanthine phosphoribosyl transferase. Using a lentiviral vector, we found that interfering RNA mediated purine analog resistance (iPAR) provided relative resistance to 6-thioguanine (6TG) in murine hematopoietic cells as compared to control- and un- transduced cells. iPAR attenuated 6TG induced G2/M checkpoint activation, cell-cycle arrest and apoptosis. Furthermore, in recipients of transplanted bone marrow cells with iPAR, treatment with 6TG resulted in increased percentages of transduced peripheral blood cells and hematopoietic progenitor cells (HPC) in the bone marrow. Secondary transplants resulted in higher hematopoietic contributions from 6TG treated primary recipients relative to PBS treated recipients. These findings indicate that iPAR/6TG can be used for in vivo HPC selection.
