97. Translational Studies toward Optimizing In Vivo Bone Marrow Stem Cell Gene Transfer Using Lentiviral Vector.

In situ stem cell gene transfer, mediated by intra-bone marrow (iBM) injection of a lentiviral vector (LV), presents a novel approach that could potentially take full advantage of any stem cells in bone cavity and overcome many difficulties encountered in ex vivo gene transfer. Our recent studies have demonstrated significant gene transfer into hematopoietic stem cells (HSC) and mesenchymal stem/ progenitor cells (MSC) after intrafemoral injection of LV in adult mice without any pre-conditioning. In this study, we sought to determine if HSC and MSC gene transfer can be enhanced by pretreatment with 5-fluorouracil (5-FU) in combination with in vivo selection. An advanced 3rd-generation LV was constructed expressing a variant of O6-methylguanine DNA methyltransferase (MGMTP140K) and GFP under regulation of elongation factor 1- alpha promoter. VSVG-pseudotyped vector (7 × 107 TU/ml) was injected into femurs of adult C57/Bl6 mice that were pretreated with a sublethal dose of 5-FU either 2- (n=6) or 5- (n=6) days before injection, in comparison with non-conditioning mice (n=7) or buffer-injected controls (n=6). Two days post injection, BM of femurs from injected side of some mice (2-3 per group) were harvested and transplanted into lethally irradiated mice. Only one (out of 4) BMT recipient derived from 2-D treatment group demonstrated significant level of GFP+ PBL (9.7%) two months post transplantation, although successful engraftment was indicated in recipients from all LV-injected groups (n=13). To monitor gene transfer in progenitor cells, CFC assay was also performed using BM of femurs from the short-term mice. Similar levels of GFPcontaining colonies were observed in both 2-D pretreated (mean of 8.1%) and un-conditioned (mean of 8.2%) mice, while less GFP colonies (mean of 3.6%) were detected in 5-D pretreated mice. To evaluate stem cell gene transfer and monitor potential hematologic toxicity, we performed complete blood count and FACS analysis periodically in primary injected mice. All blood parameters were normal except that mild monocytosis and leukocytosis were detected in most of LV-injected (8 of 12) and all buffer-injected (3 of 3) mice up to two month post injection, suggesting acute inflammation related to iBM injection procedure. Four-weeks post injection, various levels of GFP-expressing cells were detected in lymphoid and myeloid subpopulations of all 2D-pretreated (mean of 1.9% and 2.5% respectively), all 5D-pretreated (mean of 1% and 0.5%) and two (out of 4) un-conditioned mice. However, 9-weeks after injection, the highest GFP labeling was observed in one of the un-conditioned mice (7% and 11.7%). Further studies are underway to charaterize gene transfer in HSC and MSC, and enhance transgene frequency by MGMTP140K-mediated in vivo selection. Our preliminary data suggested that timing of preconditioning is critical for improving LV-mediated in vivo gene transfer. This study will provide valuable information to optimize in situ BM stem cell gene transfer which might lead toward a novel approach for treatment of human diseases.Molecular Therapy (2006) 13, S40–S40; doi: 10.1016/j.ymthe.2006.08.116 [ABSTRACT FROM AUTHOR]