525. CD8+ T Cell Tolerance To Epitopes Downstream of Non-Sense Mutations

Abstract

Recent success in gene therapy clinical trials have shown great promise towards developing efficacious treatments, however immune responses to the therapeutic protein resulting in transgene expression attenuation or loss still remains a concern. Expression can elicit a CD8+ T cell response against “non-self” regions of the protein as they were not presented during the process of central tolerance. Individuals with genes containing non-sense mutations could be at increased risk of developing a CD8+ T cell response following gene therapy as peptide sequences downstream of the mutation would not have previously been presented to the immune system. The effect of nonsense and missense mutations on the immune response to corrected gene expression was studied using the OTC gene, which is mutated in ornithine transcarbamylase deficiency. The immunodominant epitope SIINFEKL was fused to the C-terminus of OTC to evaluate the effect of mutations on the ability to elicit a transgene-specific immune response, as a surrogate for expression and presentation of SIINFEKL to CD8+ T cells. Using an ex-vivo stimulation assay, CD8+ T cells from OT-1 mice were mixed with fibroblasts transiently transfected with a panel of mutant or wild-type human hOTC-SIINFEKL constructs. T cell activation was determined by IFN-γ ELISPOT assay. Surprisingly, activation of T cells occurred to epitopes in the C-terminal region of genes containing non-sense mutations upstream of the SIINFEKL epitope. The most immunogenic nonsense mutant was selected for further studies in C57BL/6 mice. Packaging of this mutant hOTC-SIINFEKL transgene into highly immunogenic adenovirus 5 (Ad5) elicited an immune response in vivo to the downstream SIINFEKL epitope. To study if expression and presentation of epitopes downstream non-sense mutations to CD8+ T cells could result in tolerance induction all of the non-sense mutant constructs were used to generate AAV8 vectors. C57BL/6 mice were administered the non-immunogenic AAV8 vectors and then subsequently challenged the mice with Ad5 hOTC-SIINFEKL or Ad5 OVA. Mice pre-treated with AAV8 had a drastically reduced CD8+ T cell activation to the SIINFEKL epitope compared to no treatment as measured by flow cytometry and IFN-γ ELISPOT assay. These results suggest that patients with non-sense mutations could have immunological tolerance to the therapeutic protein decreasing the risk of a transgene specific immunologic response.