Effects of ex vivo transduction of mesencephalic reaggregates with bcl-2 on grafted dopamine neuron survival

Abstract

Survival rates of dopamine (DA) neurons grafted to the denervated striatum are extremely poor (5–20%). Gene transfer of survival promoting factors, such as the anti-apoptotic protein bcl-2, to mesencephalic DA neurons prior to transplantation (ex vivo transduction) offers a novel approach to increase graft survival. However, specific criteria to assess the efficacy of various vectors must be adhered to in order to reasonably predict successful gene transfer with appropriate timing and levels of protein expression. Cell culture results utilizing three different herpes simplex virus (HSV) vectors to deliver the reporter β-galactosidase gene (lacZ) indicate that transduction of mesencephalic cells with a helper virus-free HSV amplicon (HF HSV-TH9lac) that harbors the 9-kb tyrosine hydroxylase (TH) promoter to drive lacZ gene expression elicits the transduction of the highest percentage (≈50%) of TH-immunoreactive (THir) neurons without significant cytotoxic effects. This transduction efficiency and limited cytotoxicity was superior to that observed following transduction with helper virus-containing HSV (HC HSVlac) and helper virus-free HSV amplicons (HF HSVlac) expressing lacZ under the transcriptional control of the HSV immediate-early 4/5 gene promoter. Subsequently, we assessed the ability of HSV-TH9lac and the bcl-2 expressing HSV-TH9bcl-2 amplicon to transduce mesencephalic reaggregates. Although an increase in bcl-2 and β-galactosidase protein was induced by transduction, amplicon-mediated overexpression of bcl-2 did not lead to an increase in grafted THir neuron number. Even with highly efficient viral vector-mediated transduction, our results demonstrate that ex vivo gene transfer of bcl-2 to mesencephalic reaggregates is ineffective in increasing grafted DA neuron survival.