Gene therapy using adenovirus

Abstract

Glial cell line-derived neurotrophic factor (GDNF) has a strong neuroprotective property. It prevents neuronal cell death in various situations, and intracerebroventricular injection of this molecule actually reduced ischemic brain injury in vivo. However, GDNF needs to be given intracerebroventricularly because the blood-brain barrier inhibits its entry into the brain parenchyma from intravascular space. In order to get over this problem, gene transfer could be a promising method. Adenovirus is an appropriate vector to transfer genes into the brain, because this virus successfully infects non-dividing cells as neurons. In the ischemic brain, however, initiation of protein synthesis is inhibited, and thus, whether transferred gene becomes translated into protein remained uncertain. In an attempt to clarify this issue, we injected adenovirus containing lacZ gene into the brain after transient or permanent middle cerebral artery (MCA) occlusion. In the brain after transient MCA occlusion, lacZ activity was not or only minimally observed in the reperfused brain until 2 days. However, the activity dramatically increased at 7 days of reperfusion at a level similar to that of the control, which diminished by 21 days. In the brain with permanent MCA occlusion, lacZ activity was increased from 8 h to 2 days in the MCA territory, which was less stronger than that of the control. Based on the finding of the successful expression of transferred lacZ gene, we injected adenovirus containing GDNF gene into the brain followed by making transient MCA occlusion. The infarction volume was measured with 2,3,5-triphenyltetrazolium chloride staining method, and it was revealed that GDNF-containing adenovirus reduced the infarction volume at 1 day of reperfusion. The numbers of TUNEL, immunoreactive caspase-3, and cytochrome c-positive neurons were markedly reduced in the GDNF gene transferred group, suggesting that amelioration of brain damage was caused by reduction of apoptotic signals activation. As the next step, we investigated therapeutic time window of GDNF gene-containing adenovirus. In the group of adenovirus injection at just before or just after the MCA occlusion, the infarction volume was significantly smaller than the control group. On the other hand, adnenovirus injection at 1 h after the reperfusion failed to reduce the infarction volume. This means that the GDNF gene transfer with use of adenovirus could be effective for treating stroke, if the vector was applied at an early time after the ischemic insult.