RAPID METHOD FOR CONSTRUCTION OF RECOMBINANT HSV GENE-TRANSFER VECTORS

Herpes simplex virus type 1 (HSV-1) is a neurotrophic human pathogen t hat naturally persists in neurons in a latent state and carries a larg e number of viral functions which can be replaced by foreign genes to create a vector for gene therapy applications. in this report we descr ibe a two-step method for insertion/deletion mutagenesis of HSV genes and the efficient insertion of transgenes into these locations in the viral genome. The first step is the insertion of a reporter gene (lacZ ) cassette flanked by Pacl restriction enzyme sites not otherwise foun d in the viral genome, using standard marker transfer procedures to in terrupt a portion of the target HSV gene. The second step is substitut ion of the reporter gene with other foreign cDNAs by digestion of the vector DNA with Pacl to remove the lacZ gene and subsequent repair of the vector genome by homologous recombination with a transgene express ion plasmid. Potential recombinants indentified by a 'clear plaque' ph enotype after X-gal staining arose at high frequency (80-100%). Of the se, recombinants containing the transgene in place of the lacZ gene ra nged from 19-65%, Insertion of the transgene expression construct into the viral genome eliminates the Pacl sites allowing this method to be used repeatedly for the sequential deletion of multiple HSV genes whi le inserting multiple transgenes. This procedure was repeated in succe ssion to produce a vector carrying two independent expression cassette s at distinct viral loci.