261. Immune Evasion in Gene Therapy

Abstract

A problem associated with the gene-therapy concept is the T cell-mediated immune response, which is elicited by synthesis and presentation of transgene-derived and vector-derived peptides and which leads to rapid clearance of the transduced cells and loss of transgene expression. One way to avoid the immune response is by blocking the ubiquitin/proteasome pathway. Interference in this pathway is widely applied by viruses, for instance by Epstein-Barr virus (EBV) in its nuclear antigen 1 (EBNA-1). EBNA-1 is required for the maintenance of the viral episomes in the infected cells. Although EBNA-1 specific CTL have been described, they cannot recognize EBV-infected cells. The failure to recognize endogenously expressed EBNA-1 has previously been attributed to a glycine-alanine rich (GAr) stretch in the EBNA-1 sequence that protects the protein from proteasomal degradation. We have shown previously that inclusion of the full length EBNA-1 derived GAr domain could be exploited to abrogate CTL-induced killing of cells expressing the transgene (ref 1). Firstly we demonstrated that inclusion of the EBNA-1 derived GAr domain does not interfere with enzyme function. We tested three different enzymes that all retained their activity. In addition, a recombinant adenovirus expressing GAr-containing β-galactosidase was able to deliver functional enzyme in vivo. However, expression of the GAr-β-gal as well as the control β-gal resulted in the induction of a strong β-galactosidase-specific CTL response. This can be attributed to cross-priming, as vaccination of BALB/c mice (H-2d) with C57Bl/6 (H-2b) fibroblasts expressing GAr-β-galactosidase resulted in the induction of β-gal-specific CTLs. Nonetheless, LacZ-specific CTL did not recognize target cells expressing GAr-LacZ in vitro. These results showed that the GAr can be exploited to create 'stealth' genes by hiding specific foreign antigens from CTL-mediated immune-attack. Currently, we are testing the hypothesis that fusion with the GAr will lead to a prolonged expression of transgenes in vivo. Preliminary evidence indicates that the modification leads to prolonged expression of β-gal in vivo after adenovirus-mediated intra-muscular gene transfer. In addition, we generated stable B16 mouse melanoma cell lines expressing either thymidine kinase (TK) or GAr-TK. The tumor take and survival of these cells in C57Bl/6 mice are being studied either in the presence or in the absence of antigen-specific CTL.