Interfering RNA-mediated purine analog resistance for in vitro and in vivo cell selection.
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 compared with control- and untransduced cells. iPAR attenuated 6TG-induced G(2)/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 in the bone marrow. Secondary transplantations resulted in higher hematopoietic contributions from 6TG-treated primary recipients relative to phosphate-buffered saline-treated recipients. These findings indicate that iPAR/6TG can be used for in vivo hematopoietic progenitor cell selection.
Porter
Christopher C
CC
Rick Wilson Center for Cancer and Blood Disorders, The Children's Hospital, Aurora, CO, USA. James.DeGregori@UCHSC.edu
DeGregori
James
J
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D002453:Cell Cycle / Q000187:drug effects / Q000235:genetics; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D004351:Drug Resistance / Q000187:drug effects* / Q000235:genetics*; D055785:Gene Knockdown Techniques / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics; D006412:Hematopoietic Stem Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D016086:Lentivirus / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D011687:Purines / Q000494:pharmacology*; D034741:RNA, Small Interfering / Q000235:genetics / Q000494:pharmacology*; D013866:Thioguanine / Q000494:pharmacology
D011687:Purines; D034741:RNA, Small Interfering; D007041:Hypoxanthine Phosphoribosyltransferase; D013866:Thioguanine