Donor chimerism and gene correction with low dose irradiation and retroviral-mediated gene transfer (Rmgt) In murine x-linked chronic granulomatous disease (X-cgd)

Our laboratory has reported the correction of neutrophil NADPH oxidase function by RMGT in murine X-CGD. Few studies, however, have employed nonmyeloablative conditioning with RMGT. We have evaluated the effect of radiation dose on donor chimerism using a congenic C57B1/6, B6.SJL mouse model. At six months post-transplant, blood cells from recipients given 20 × 106 fresh marrow cells and 160 or 300 cGy conditioning were 50.8 ± 14% and 82.8 ± 5.4% donor in origin, respectively. This chimerism has been stable for up to 12 months. The long-term repopulating ability (LTRA) of irradiated marrow was determined using the competitive repopulation assay. Marrow from mice given 160 or 300 cGy competed 22% or 7% as well as fresh competitor cells, respectively. We next mock-transduced marrow cells on a non-virus producing packaging cell line to examine the effects of the 5 day in vitro transduction process. Mock-transduced cells completely repopulated the marrow of lethally irradiated hosts, but recipients given 160 cGy and 20 × 106 mock-transduced cells showed 18.8 ± 6.1% donor chimerism at six months, compared to 50.8 ± 14% for fresh marrow. Finally, X-CGD marrow cells were transduced with MSCV-m91neo, and 20 × 106 transduced cells were transplanted into 160 cGy X-CGD recipients. At 4 months post-transplant, we detected 6.6 ± 5.9% corrected neutrophils by NBT assay; 1100 cGy recipients given 2 × 106 transduced cells had 54.2 ± 6.8% NBT positive neutrophils. These results show that low dose irradiation decreases host marrow LTRA, that ex vivo culture of donor cells for RGMT impairs subsequent engraftment, and that low numbers of gene-corrected cells can be detected following RMGT and nonmyeloablative conditioning.