Current treatment options for neuroblastoma fail to eradicate the disease in the majority of high-risk patients, clearly mandating development of innovative therapeutic strategies. Gene therapy represents a promising approach for reversing the neoplastic phenotype or driving tumor cells to self-destruction. We presently studied the effects of adenovirus-mediated gene transfer of human polynucleotide phosphorylase (hPNPase(old-35)), a 3',5'-exoribonuclease with growth-inhibitory properties, in neuroblastoma cells. Transgene expression was driven by either the cytomegalovirus (CMV) promoter or by a tumor-selective promoter derived from progression elevated gene-3 (PEG-3). Our data demonstrate that efficient adenoviral transduction of neuroblastoma cells and robust transgene expression are feasible objectives, that the PEG-3 promoter is capable of selectively targeting gene expression in the majority of neuroblastoma cells, and that hPNPase(old-35) induces profound growth suppression and apoptosis of malignant neuroblastoma cells, while exerting limited effects on normal neural crest-derived melanocytes. These findings support future applications of hPNPase(old-35) for targeted gene-based therapy of neuroblastoma and suggest that combination with the PEG-3 promoter holds promise for creating a potent and selective neuroblastoma therapeutic. J. Cell. Physiol. 219: 707-715, 2009. (c) 2009 Wiley-Liss, Inc.