The generation of a herpes simplex virus vector to target motor neurons
Herpes simplex virus (HSV) is a neurovirulent virus that in the course of natural infection of man predominantly infects sensory neurons. The aim of this project was to develop a safe, nonvirulent HSV, capable of expressing exogenous genes which altered the binding characteristics of the virus so that tropism was directed predominantly to motor nerves. It was envisaged that these viruses could then act as prototypes for gene therapy vectors targeted to the treatment of motor nerve diseases. To achieve this, two mutant viruses were created, RFa and RFb. These contained deletions of the main HSV glycoprotein involved in cellular binding (glycoprotein C). Gene fusions were created of truncated portions of gC (amino acids 377-511(RFa) and amino acids 477-511 (RFb)) to E. coli heat-labile enterotoxin B-subunit (LTB). The gene fusions were inserted in the RL1 gene thereby abolishing expression of the virulence factor ICP34.5. LTB is a ligand which binds to several gangliosides, including GM1 and GM2 which are motor neuron markers. It was hoped that by deletion of the main viral protein involved in adsorption to cells and replacing it with an LTB-containing fusion protein, the tropism of the mutant viruses could be altered to promote an increase in motor neuron infection. RFb was constructed. RFa constructed but could not be purified to homogeneity. This was thought to be due to poor adsorption/penetration or cell-to-cell spread, brought about by expression of the LTB fusion protein. RFb was analysed to determine the effect of expression of the novel LTB fusion protein within the context of the HSV genome. Western blot analysis using antibodies directed against LTB failed to detect expression of the LTB-gC fusion protein. In vitro replication studies showed that the RFb was non-virulent as demonstrated by its inability to replicate in growth arrested 3T6 cells, a phenotype characteristic of HSV which fails to produce ICP34.5. However no marked difference in virus replication kinetics was seen between RFb and wild type HSV (17+) on two motor neuron-like cell lines (NSC-19 and NSC-34).