CYTOKINE-FACILITATED TRANSDUCTION LEADS TO LOW-LEVEL ENGRAFTMENT IN NONABLATED HOSTS

Using a murine bone marrow transplantation model, we evaluated the lon g-term engraftment of retrovirally transduced bone marrow cells in non myeloablated hosts. Male bone marrow was stimulated in a cocktail of i nterleukin-3 (IL-3), IL-6, IL-11, and stem cell factor (SCF) for 48 ho urs, then cocultured on the retroviral producer line MDR18.1 for an ad ditional 24 hours. Functional transduction of hematopoietic progenitor s was detected in vitro by reverse transcriptase-polymerase chain reac tion (RT-PCR) amplification of multiple drug resistance 1 (MDR1) mRNA from high proliferative potential-colony forming cell (HPP-CFC) coloni es. After retroviral transduction, male bone marrow cells were injecte d into nonablated female mice. Transplant recipients received three TA XOL (Bristol-Myers, Princeton, NJ) injections (10 mg/kg) over a 14-mon th period. Transplant recipient tissues were analyzed by Southern blot and fluorescence in situ hybridization for Y-chromosome-specific sequ ences and showed donor cell engraftment of approximately 9%. However, polymerase chain reaction amplification of DNAs from bone marrow, sple en, and peripheral blood showed no evidence of the transduced MDR1 gen e. RT-PCR analysis of total bone marrow RNA showed that transcripts fr om the MDR1 gene were present in a fraction of the engrafted donor cel ls. These data show functional transfer of the MDR1 gene into nonmyelo ablated murine hosts. However, the high rates of in vitro transduction into HPP-CFC, coupled with the low in vivo engraftment rate of donor cells containing the MDR1 gene, suggest that the majority of stem cell s that incorporated the retroviral construct did not stably engraft in the host. Based on additional studies that indicate that ex vivo cult ure of bone marrow induces an engraftment defect concomitantly with pr ogression of cells through S phase, we propose that the cell cycle tra nsit required for proviral integration reduces or impairs the ability of transduced cells to stably engraft. (C) 1997 by The American Societ y of Hematology.