1) Evaluation of Adeno-Associated Virus (AAV-2) as a Vector for Gene Transfer into Cells of Glial Origin: AAV-2 is single-stranded DNA parvovirus which has recently emerged as potentially useful tool for gene transfer, both ex vivo and in vivo. To determine whether AAV-2 could be used for gene transfer into glial cells, both primary glial cells (100% GFAP positive) and a glial cell line (SVG) were transduced with a Lac-z expressing AAV-2 recombinant. Using an MOI of between 1-10%, 95% of the primary glial cells were transduced and 99% of the glial cell line was transduced. To determine whether a functional protein could be effectively transferred, the SVG cell line was transduced with a human CD4 expressing AAV-2 recombinant. A stable CD4 expressing SVG cell line was easily established as determined by immuno-histochemistry and Western blotting for CD4. To determine whether this CD4 is functional, the SVG- CD4 cells were infected with HIV-1 (IIb strain). Viral replication and the number of infected cells was ten-fold higher in the SVG-CD4 cells than in he parental SVG cell line, consistent with the expression of functional CD4 protein. 2) Development of Chimeric JCV Vector for Gene Transfer into Cells of Glial Origin: JC virus is a human polyoma virus which has a glial-specific promoter. As such, a recombinant JCV could also be a useful tool for glial specific gene transfer. We have already constructed two different viral chimeras in which the early portion of the viral genome (the large T protein gene) has been replaced with a cDNA marker gene (either tyrosine hydroxylase or green fluorescent protein). In addition constructs have been made which have one of three different JCV promoters (Mad1, Mad4, and Mad8). When the chimeric DNA is transfected into cells which do not express T protein, these constructs have been found to express the marker gene, but fail to replicate. However, when these constructs were transfected into a cell line (SVG) which complements the chimera by producing T protein, the SVGs act as a packaging cell line and produce high titer recombinant JCV. This recombinant virus has been sued to infect primary human fetal glial cells and has been found to efficiently express the marker gene TH. Southern blots of DNA extracted from the infected cultures confirmed that the viral genome was the chimeric construct. Thus, a recombinant JCV particle has been packaged, and a novel packaging cell system has been established.
