896. Long Term Transgene Expression in the Brain Delivered by SV40-Derived Vectors

Abstract

Top of pageAbstract Gene transfer to the central nervous system (CNS) has been studied using various vectors. Recombinant, Tag-deleted SV40 vectors provide long-term transgene expression in vitro and in vivo in multiple non-CNS cell types, and can be used in vitro to transduce both NT2-derived and primary human neurons, and primary human brain microglia. We previously reported administering a rSV40 vector into the caudate-putamen (CP) of the rat brain using stereotaxic conditions. This vector carries HIV-1 Nef protein with a carboxyl-terminal FLAG epitope tag driven with a CMV-IEP promoter, SV(Nef-FLAG). Transgene expression was assessed 4, 7 or 14 days later by immunostaining of serial coronal cryostat sections. Numerous transgene-positive cells were seen at those times, at least several mm longitudinally away from the injection site. The present study aimed to determine whether these vectors could also deliver long term expression of a transgene, up to 3 months after unilateral injection into the CP, hippocampus, and cerebellum (lobe 6). The uninjected side, or saline injection, were used as controls. No cells on the uninjected side were immunopositive for FLAG.Sections incubated with non-immune primary antibody (immunostain negative controls) were also negative. The number of transgene-positive cells counted 1, 2 and 3 months after injection of the vector in the CP was higher than the one at d14, and showed a wider anatomic distribution. Transgene-positive cells were seen not only in the CP, but also in different brain structures along the needle track. The longitudinal extent of the transduced area was similar along the whole CP for time points greater than 4 days. 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. Astrocytes generally did not express the transgene. Long term transgene expression was also seen in the hippocampus (dentate gyrus) and in the cerebellum. More transgene-positive cells were observed when 10 microl of vector were given than with an injection of 1 microl. In the cerebellum, transgene-positive cells colocalized with calbindin, a marker for Purkinje cells. Previous studies of rSV40 gene delivery did not identify inflammatory or immunologic responses to the vector. In these studies, we also looked for evidence of inflammatory or immune reaction to the transduction process, using sections stained with neutral red (NR): no inflammation was seen at any time point in brains injected in any of the different structures. Likewise, no CD8-positive T cells were identified in sections of brains injected with SV(Nef-FLAG). These results show that SV40-derived vectors can be used to achieve long term and efficient CNS gene transfer without evidence of inflammatory or immune reaction, preferentially to neurons and to a lesser extent, microglial cells, when injected in different parenchymal structures of the rat brain.