A multi-mutant herpes simplex virus vector has minimal cytotoxic effects on the distribution of filamentous actin, alpha-actinin 2 and a glutamate receptor in differentiated PC12 cells

To develop effective gene therapy techniques that target populations of neu rons in the spinal cord, suitable vectors must be developed that will under go efficient, retrograde transport from an appropriate peripheral site and will not be cytotoxic. Our previous work (LeVatte ef al, 1998a) has demonst rated that a replication defective herpes simplex virus vector 14H Delta 3v hsZ, that has been substantially detoxified, is retrogradely transported fr om peripheral sites and can infect large numbers of the targeted spinal neu rons. We plan to develop targeted gene therapy approaches designed to modul ate the excitatory glutamatergic methyl-D-aspartate (NMDA) receptor in spin al cord neurons as a means of ameliorating a form of episodic high blood pr essure that occurs after spinal cord injury. In this report, we demonstrate that, in differentiated PC12 cells, a neuronal-like cell line, the virus v ector does not appear to alter aspects of the cytoskeletal architecture imp ortant to the proper distribution of the NMDA receptor. In turn, the distri bution of endogenous NMDA receptor 1 subunit protein (NMDAR1) or a transfec ted NMDAR1-green fluorescent fusion protein was also found to be unaltered after vector infection. However, whereas endogenous NMDAR1 distribution was maintained, vector infection did tend to reduce the level of its expressio n. This drop in endogenous NMDAR1 expression coincided with the expression of the HSV immediate early genes ICP0 and ICP27 over the first 24-48 h. The se results indicate that the 14H Delta 3vhsZ herpes simplex virus vector is suitable to use in future strategies to alter the level of gene expression in targeted populations of spinal cord neurons.