Abstract : Among well described animal models of Duchenne muscular dystrophy, the GRMDdog is likely the best model of DMD patients in terms of size and pathological expression of the disease. GRMD is caused by a 3? splice-site point mutation in intron 6, which induces the skipping of exon 7 and thus results in a frame shift that prematurely aborts dystrophin synthesis. By forced exclusion of two exons (6 and 8), it is possible to restore an open-reading frame. Antisense oligonucleotides (AO) allow specifically to target and inhibit individual genes for the treatment of the disorder. However, since the AO are not self-renewed, they cannot achieve long term correction. To overcome this limitation, we have introduced antisense sequences into small nuclear RNAs (snRNA) and vectorized them in AAV vectors. To determine efficient AO, we tested in particular exon splicing enhancer (ESE) motifs, which target internal exon sequences, crucial to pre-mRNA splicing. We designed different 2?O-methyl oligoribonucleotides which were tested first in vitro. Then we have designed AAV vector harboring chimeric U7 snRNA carrying efficient antisense sequences against exons 6 and 8 of the dogdystrophin gene, which were injected in GRMD muscles. Among different antisense oligonucleotides tested in GRMD, the combination of 2 AONs targeting, respectively, ESE of exon 6 and 8 induced delta5–9 in-frame skipping. In this study, we show the efficacy of these ESE sequences for exon-skipping in culture myotubes and in vivo. We also detected an almost complete restoration of dystrophin expression after intramuscular injections and systemic delivery of AAV-[U7-ESE6/U7-ESE8] vectors in GRMD. In this study, we provide evidence that efficient skipping of two exons can be achieved in GRMD through U7snRNA shuttles. Theoretically over 75% of Duchenne patients could benefit from skipping of a single exon and multi-exonskipping would significantly extend this percentage to most DMD mutations.