DIFFERENTIAL NEUROPEPTIDE-Y GENE-EXPRESSION IN POSTMITOTIC VERSUS DIVIDING NEUROBLASTOMA-CELLS DRIVEN BY AN ADENOASSOCIATED VIRUS VECTOR

The ability to express exogenous mammalian genes stably in post-mitoti c cells such as neurons remains an important goal for those attempting to modulate neurotransmission through gene delivery. We therefore inv estigated how differentiation to a post-mitotic state affected the exp ression of an exogenous gene encoding for neuropeptide Y (NPY) followi ng transfection with an adeno-associated virus (AAV) derived vector. T his vector (pJDT95npy) was constructed with rat NPY cDNA (551 bp) inse rted downstream from the indigenous AAV p5, p19 and p40 promoters to c haracterize their relative abilities to drive NPY mRNA expression. Tra nsfection of dividing neuroblastoma CHP126 cells with pJDT95npy result ed in the differential expression of chimeric NPY mRNAs derived from e ach promoter. P40-driven species became dominant after 1 month post-tr ansfection. Vector integration into chromosomal DNA was demonstrated b y Southern blot analyses, indicating at least some region-selective in tegration. In dividing cell extracts, only a low level of pro-NPY immu noreactivity and no mature NPY immunoreactivity was recovered. However , after differentiation of the pJDT95npy-transfected CHP 126 cells to a post-mitotic state, significant levels of pro-NPY and mature NPY wer e recovered in the cells and media. Differentiation also had a time-de pendent effect on mRNA expression: a spike of p5 driven expression on day 3 was followed predominantly by p40-driven expression on day 5. Th is study indicates that AAV-derived vectors using the p40 promoter may be used to express genes in post-mitotic cells such as neurons.