Long-Term Transgene Expression in Fetal Rat Suprachiasmatic Nucleus Neurografts Followingex VivoAdenoviral Vector-Mediated Gene Transfer

Abstract

Ex vivogene transfer to fetal suprachiasmatic nucleus (SCN)-containing solid piece neurografts was explored using a first-generation prototype adenoviral vector containing the reporter gene LacZ (Ad–LacZ). Transgene expression was examined at different intervals following grafting in the IIIrd ventricle of rat brain and was compared to that of explant cultures. Large numbers of β-galactosidase-positive cells were observed 8 days postgrafting. The number of stained cells had decreased considerably at 21 days but transduced cells were still present at 70 days.In vitroculturing of infected SCN tissue revealed high expression up to 21 days, indicating that thein vivoandin vitrofates of Ad–LacZ-infected cells were different. The main reason for this difference appeared to be cell loss by necrosis in the initial phase after transplantation, a phenomenon not related to the infection with Ad–LacZ since it similarly occurred in control grafts.In vivoinflammatory responses, observed after immunostaining for macrophages and T-lymphocytes, were also comparable in control and Ad-LacZ-treated transplants, except that cytotoxic T-cells were observed in the Ad–LacZ-treated transplants and not in controls. The recruitment of these cells was, however, minor and primarily observed at 8 days postgrafting, indicating that a major immunological rejection of the transduced graft did not occur. In both control and Ad–LacZ-infected transplants similar survival and intraimplant neuritic growth of SCN cells were visible.Ex vivogene transfer of solid piece fetal SCN grafts with adenoviral vectors therefore appeared to be a nontoxic long-term gene-introducing procedure. This would in principle enable the local production of neurotrophic factors within the transplant and has the potential to improve functional SCN neurografting.