Characterization of an immuno ‘stealth’ derivative of the herpes simplex virus thymidine-kinase gene

The cellular immune response against transgene-encoded neoantigens is a potential hurdle in gene therapy applications where long-term expression of transgenes is desired. Here a new optimized derivative of the herpes simplex virus 1-thymidine-kinase (HSV1-TK) gene is described. The HSV-TK gene is frequently used in experimental studies on gene-directed enzyme prodrug therapy. In the optimized gene, the HSV-TK coding region is fused with the codons for the Gly-Ala repeat of the Epstein–Barr virus nuclear-antigen 1 to prevent proteasomal degradation of the HSV-TK. To measure the protective effect in vitro, a model cytotoxic T lymphocyte epitope derived from the ovalbumin was inserted in the TK. Cells expressing the GAr-modified TK do not present TK-derived peptides in the major histocompatibility complex. Furthermore, conservative nucleotide substitutions were introduced, which prevent splicing, as well as mutations that render the TK-expressing cells more sensitive to ganciclovir (GCV). The GAr HSV-TK fusion protein is fully functional in vitro. This HSV-TK gene may be especially useful in those gene therapy applications where an immune response against the transgene-encoded product would frustrate the treatment.