CHARACTERIZATION OF AN AAV-DERIVED CONSTRUCT CONTAINING AN ANTISENSE NMDA-R1 GENE

D.H. Shafron, A.L. Day, B. Jevelli, S. Wang, R.T. Frizzell, E.M. Meyer
University of Florida College of Medicine, Gainesville, FL 32620

NMDA-R1 antisense oligonucleotides recently were shown to down-regulate NMDA-R1 receptor density of in vivo, and to be protective against excitotoxic cell damage in a rat model of ischemia. Similar technology was effectively used to reduce sound-induced seizure frequency in genetically prone mice. Long-term control against glutamate-induced excitotoxic cell injury, however, may require the use of continuous promoter-driven expression of antisense genes in neurons. Adeno-associated virus (AAV) based vectors were developed by our laboratory for post-mitotic gene transfer and stable expression using several neuronal systems. These systems include hypothalamic neuropeptide Y (NPY) gene delivery and expression, increasing hypothalamic NPY immunoreactivity and food intake in rats. We now describe the subcloning of a 2063 base-pair fragment of the NMDA-R1 gene, in an antisense orientation, into an AAV-based gene delivery system in order to effect long-term expression of this antisense gene product. This fragment was isolated and blunt-end ligated into pJDT95dlk, an AAV-based gene delivery plasmid, downstream from the indigenous AAV p5, p19, and p40 promoters. Cultured PC-12 cells were subsequently transfected with this construct utilizing Lipofectamine (Gibco BRL). Transfected cells showed no morphologic changes, and there was no difference in cell viability compared to control cells transfectecd with pJDT95dlk without insert. Revers-transcriptase polymerase chain reaction was utilized to evaluate for AAV vector driven expression of the antisense NMDA-R1 insert 4 days post-transfection. This analysis showed a strong band of appropriate size in the experimental group, which was characterized further with endonuclease digestion. Studies are underway to examine the effects of long-term expression of this antisense product in other neuronal preparations, including brain.