A novel adenoviral vector which mediates hypoxia-inducible gene expression selectively in neurons

Abstract

Selective gene expression in neurons is still a challenge. We have developed several expression vectors using a combination of neuron restrictive silencer elements (NRSEs), hypoxia responsive elements (HREs) and CMV minimal promoter (CMVmp). These elements were packaged into replication defective adenovirus to target gene expression selectively in neurons in a hypoxia-regulated manner. Neuronal selectivity and responsiveness to hypoxia of these novel constructs were determined empirically in both neural cell lines and primary cerebellar granule neurons (CGNs). The construct p5HRE-3NRSE exhibited not only the highest level of reporter gene expression in neuronal cells but also in an oxygen concentration-dependent manner when compared with all other constructs. As expected, this construct did not elicit reporter gene expression in non-neuronal cells including human HEK293A and HT29 cells, rat NRK cells, mouse 3T6 cells and 3T3 L1 cells. This construct was packaged into a replication defective adenoviral vector (Ad/5HRE-3NRSE) to determine neuron-selective and hypoxia-inducible gene expression in cultured mouse postmitotic primary CGNs and differentiated human NT2 neurons (NT2/Ns). Remarkably, in response to hypoxia, Ad/5HRE-3NRSE showed strong hypoxia-inducible gene expression selectively in neurons (12-fold induction in CGNs and 22-fold in NT2/Ns), but not in glial cells. Taken together, this vector with restricted gene expression to neurons under the regulation of hypoxia will be a useful tool for investigations of mechanisms of neuronal damage caused by ischemic insult.