GENETICALLY MODIFIED PRIMARY ASTROCYTES AS CELLULAR VEHICLES FOR GENE THERAPY IN THE BRAIN

Combining genetic engineering and cell transplantation has been proposed as one way to overcome the limited availability of donor tissue that may restrict the application of graft therapy to neurological diseases. Important issues in this approach concern the choice of a suitable cellular vehicle, and the method of gene insertion. In this regard, we have investigated the use of brain-derived primary astrocytes as cellular vehicles for gene therapy, because they can be transfected, divide in culture, are brain-region specific, possess a secretory mechanism, and may migrate several mm from the transplant site. To address the issue of gene insertion, we have generated stably transfected primary rat astrocytes using the nonviral calcium phosphate method to co-transfect a reporter construct (RSV-chloramphenicol acetyltransferase (CAT), or human enkephalin promoter CAT, plus a neomycin resistance plasmid (pRSVNeo). Modified astrocytes were then propagated by transfer to selective media containing G418 (300 μg/mL) for 3 wk. The presence of the reporter gene product (CAT) was demonstrated by immunocytochemistry, and by biochemical assay of CAT enzyme catalytic activity. These genetically modified astrocytes were followed for up to 3 wk after transplantation into the rat striatum. Criteria used to distinguish transplanted astrocytes included histological evidence of abundant nuclei interrupting the normal cytoarchitecture of the striatum, astrocyte morphology, and the presence of CAT enzyme activity. Our data indicates that genetically modified astrocytes are an important candidate vehicle for use in transplantation therapy in neurological diseases. We suggest that genetically modified astrocytes can also be used for studying the human enkephalin promoter, other promoters, and expressed proteins using this paradigm.