“Ex vivo” regional gene therapy using lentiviral (LV) vectors to over-express bone morphogenetic
protein 2 (BMP-2) is an effective way to enhance bone healing in animal models. Here, we
evaluated two different “ex vivo” approaches using either “same day” rat bone marrow cells (SDRBMCs)
or cultured rat bone marrow cells (C-RBMCs), both transduced with a LV based two-step
transcriptional activation system overexpressing GFP (LV-TSTA-EGFP), to assess the fate of the
transduced cells and the safety of this approach. The transduced cells were implanted in femoral defects of syngeneic rats.
Animals were sacrificed at 4, 14, 28 and 56 days after surgery (n=5 per group). Viral copies were detectable in the defect
site of SD-RBMC group and gradually declined at 8w (5 log decrease compared to 4d). In the C-RBMC animals, there
was a 2-4 log decline in the viral copy numbers at 2w and 4w, but at 8w there was a relative rise (about 100 fold) in the
number of the viral vectors in the defect site of 4 (out of 5) animals compared to the previous time points. For both gene
transfer approaches, the pattern of tissue distribution was non-specific and no histological abnormalities were noted in either
group. In summary, we demonstrated that the LV-TSTA transduced cells remain in the defect site for at least 56 days,
though the numbers decreased over time. There were no consistent findings of viral copies in internal organs which is encouraging
with respect to the development of this strategy for use in humans.