790. Evaluation of Immune Responses to Dystrophin in Humanized mdx HLA-A|[ast]|0201 Dystrophic Mi Application in Duchenne Muscular Dystrophy Gene Therapy after U7 snRNA-Mediated Exon Skipping

Cellular immune responses may compromise long-term expression in Dystrophin-based gene therapy treatments. To predict cytotoxic T-cell responses mediated by dystrophin (DYST), we designed a new H-2-negative HLA-A*0201 transgenic mouse model breaded on mdx background bearing a stop mutation in DYST exon 23. After DYST plasmid injection we identified a shared mouse/human specific DYST epitope located on exon 24, which elicits HLA-A*0201-restricted cytotoxic T cell activities in all mice. We found however, that rescue of dystrophic muscle by a single administration of an AAV vector expressing antisense sequences linked to a modified U7 small nuclear RNA (Goyenvalle A, Vulin A, Fougerousse F, Leturcq F, Kaplan JC, Garcia L, Danos O., Science. 2004, 306 : 1796-9) restores long term expression of DYST in muscle fibres and does not induce CTL activity against DYST in mdx HLA-A*0201 mice. This persistent exon skipping removes exon 23 and restores DYST exon 24 on the dystrophin messenger mRNA of the mdx HLA-A*0201 mouse. Several procedures were then attempted to induce muscle inflammation secondary to exon skipping. They failed to trigger effector CTL responses and failed to trigger immune rejection of rescued muscles fibres. We conclude that in contrast to delivery of DYST plasmid, rescuing dystrophic muscle through U7 snRNA-mediated exon skipping does not induce a primary immune response directed by exon 24 DYST sequence. The knowledge of HLA-A*0201-restricted human DYST peptides will be of importance to assay the occurrence of immune responses in HLA-A*0201 positive humans enrolled in future clinical studies.