Recessive dystrophic epidermolysis bullosa (RDEB) is a genetic blistering disorder, caused by mutations in the COL7A1 gene encoding for type VII collagen (C7) protein. These mutations result amongst other complications in fragility of the skin and mucosal membranes. RNA exon-skipping is the modulation of splicing of a pre-mRNA in order to prevent inclusion of a targeted exon into the mRNA. Many RDEB patients harbour a mutation in exon 73 of the COL7A1 gene. We aimed to identify an antisense oligonucleotide (AON) that would exclude the in-frame exon 73 from the COL7A1 mRNA. Exon-skipping in COL7A1 can result in a slightly shortened C7 protein. QR-313, a fully phosphorothioated and 2’O-methylated AON, composed of 21 bases was selected.  This sequence is optimized for length, sequence, lack of immunogenicity and optimal manufacturability. We have tested the effectiveness of QR-313 using multiple cell-types, including primary wild type fibroblasts, (modified) HaCaT cells and dermal fibroblasts compound heterozygous RDEB patient, harbouring a mutation in exon 73. These cells were examined for their ability to induce exon 73 skipping from the COL7A1 mRNA using an in vitro transfection system. Products were analysed using PCR and results demonstrate that QR-313 efficiently excludes exon 73 from the COL7A1 mRNA. Secondly, we have assessed the RNA exon skip potential of QR-313 in human skin equivalents (HSEs). HSEs are composed of a dermal layer, harbouring fibroblasts, with a differentiated epidermal layer on top. Before application of QR-313, HSEs were wounded by partial removal of the epidermis, to mimic the blistered skin of RDEB patients. Topical application of QR-313 in a carbomer-based hydrogel for 24-48 hrs, resulted in the exclusion of exon 73 from the COL7A1 mRNA in dermal fibroblasts and epidermal keratinocytes. QR-313 is a potential novel RNA oligonucleotide that could treat RDEB due to exon 73 mutations by exon-skipping.

In vitro evaluation of QR-313; an anti-sense oligonucleotide designed to skip exon 73 from the COL7A1 mRNA