1086 QR-313, an antisense oligonucleotide, restores expression of functional type VII collagen in DEB patient cells

Abstract

Dystrophic epidermolysis bullosa (DEB) is a blistering disorder caused by mutations in the COL7A1 gene, encoding type VII collagen (C7). Absence of functional C7 leads to blistering due to fragility of the skin and mucous membranes, and disease complications such as scarring and squamous cell carcinoma. Mutations in the in-frame exon 73 are the most common cause of DEB. QR-313 is designed to skip exon 73 from COL7A1 mRNA, thereby excluding mutations in this exon from the transcript, which leads to the formation of a slightly shorter C7 (C7Δ73). In this study, it was assessed whether C7 lacking the amino acids encoded by exon 73 is functional by investigating the thermostability and binding capacity of C7Δ73. In addition restoration of C7 protein was tested in DEB patient cells after QR-313 treatment. To assess the biochemical properties of C7Δ73, wild type (WT) C7 and C7Δ73 were expressed in HEK293T cells and purified. Limited trypsin digestion followed by western blot analysis to test the thermostability of the triple helix, revealed that C7Δ73 is resistant to digestion by trypsin to a similar extent as WT C7. Furthermore, binding of C7Δ73 with two major interaction partners of C7, type IV collagen and laminin-332, was determined using a solid phase binding assay. The results indicated that binding of C7Δ73 to both interaction partners was comparable to that of WT C7. Cells from a patient with recessive DEB that do not produce C7 were treated with QR-313. Protein restoration and functionality of the C7Δ73 produced were tested using various assays. In conclusion, the results of this study show that QR-313-mediated exon 73 skip leads to the formation of functional C7Δ73 protein. These results support the start of a Phase Ib/II clinical trial.