661. Efficient SV40-Mediated Gene Transfer to the Brain

Abstract

Top of pageAbstract Gene transfer to the central nervous system has been accomplished with various vectors. SV40 vectors are able to transduce NT2-derived neurons and microglia in vitro. The goal of the present study was to determine the most effective routes of administration of SV40 vectors to the brain and the parameters of the transduction (diffusion, and distribution of the transgene in the brain as well as number and type of the transduced cells). As a marker, we used a rSV40 viral vector carrying HIV Nef protein with a carboxyl-terminal FLAG epitope tag-SV40(Nef-FLAG)- driven with a CMV-IEP promoter. The vector was injected using stereotaxic conditions into the lateral ventricle (LV) or, as an example of an intra-parenchymal inoculation site, the caudate-putamen (CP) of the rat brain. 4, 7, or 14 days later, transgene expression was assessed by immunocytochemistry on serial coronal cryostat sections. After injection of SV40 (Nef-FLAG) into the LV, FLAG expression was mainly observed in cells in the ependyma and choroid plexus. Numerous transgene-positive cells were observed 4, 7, and 14 days after the injection of SV40 (Nef-FLAG) into the CP. Seven days after the injection, transgene-positive cells were observed at least 4 mm longitudinally away from the injection site, and the average number of transgene-positive cells per hemisphere was 1202. In the CP, many transgene-expressing cells stained positively for NeuN, or MAP2, indicating a substantial neuronal transduction, with more than 95% of the transduced cells co-immunostaining with these neuronal markers. Numerous neurons were transduced in the vicinity of the needle track, but also far from the site of injection. There was no detectable expression of FLAG in the substantia nigra (SN), suggesting that retrograde transport of SV40 (Nef-FLAG) or the Nef FLAG protein from the CP to the SN does not play an important role in rSV40 gene delivery to CNS cells. These results show that SV40 vectors can be used to achieve an efficient gene transfer in neuronal cells when injected in the Rat CP, and to a lesser extent in the ependyma when injected into the LV.