Targeted skipping of a single exon harboring a premature termination codon mutation by antisense oligoribonucleotide for dystrophic epidermolysis bullosa

The transfer of normal genes into somatic cells is one strategy to treat patients with genetic diseases. However, this strategy has still encounters technical problems including effi cacy of gene transfer rate and practical clinical safety. Thus, other strategies including pharmacological therapy or gene correction, are receiving increasing attention. Dystrophic epidermolysis bullosa (DEB) is caused by mutations in the COL7A1 encoding type VII collagen. This study examined the feasibility of antisense oligoribonucleotide (AON) therapy for DEB. AON was designed to induce skipping of a targeted exon containing a premature termination codon mutation, resulting in restoration of the open reading frame. We targeted exon 70 of COL7A1 since a recurrent mutation 5818delC was localized to exon 70 in Japanese DEB patients. We initially designed and synthesized two AONs to modulate splicing of exon 70 and found that one AON induced effective skipping of normal exon 70 containing the 16 amino acids. Attachment and migration analyses showed that recombinant collagen without contribution of exon 70 had similar effect to normal type VII collagen. Next, we synthesized mutation-specifi c AON by deleting cytosine at 5818. Introduction of this AON into DEB keratinocytes harboring 5818delC without expression of type VII collagen showed that the AON induced skipping of exon 70 in the abnormal 5818delC allele. Furthermore, 6.2 % of the DEB keratinocytes started to express type VII collagen in vitro after application of the mutation-specifi c AON. Injection of the AON into rat model grafted the DEB keratinocytes and fi broblasts induced detectable of type VII collagen expression at the basement membrane zone. We conclude that skipping of targeted exons using mutation-specifi c AON may show potential for future gene therapy for DEB patients