The clinical application of advanced next-generation sequencing technologies is increasingly uncovering novel classes of mutations that may serve as potential targets for precision medicine therapeutics. Here, we show that a deep intronic splice defect in the COL6A1 gene, originally discovered by applying muscle RNA sequencing in patients with clinical findings of collagen VI–related dystrophy (COL6-RD), inserts an in-frame pseudoexon into COL6A1 mRNA, encodes a mutant collagen α1(VI) protein that exerts a dominant-negative effect on collagen VI matrix assembly, and provides a unique opportunity for splice-correction approaches aimed at restoring normal gene expression. Using splice-modulating antisense oligomers, we efficiently skipped the pseudoexon in patient-derived fibroblast cultures and restored a wild-type matrix. Similarly, we used CRISPR/Cas9 to precisely delete an intronic sequence containing the pseudoexon and efficiently abolish its inclusion while preserving wild-type splicing. Considering that this splice defect is emerging as one of the single most frequent mutations in COL6-RD, the design of specific and effective splice-correction therapies offers a promising path for clinical translation.
Véronique Bolduc, A. Reghan Foley, Herimela Solomon-Degefa, Apurva Sarathy, Sandra Donkervoort, Ying Hu, Grace S. Chen, Katherine Sizov, Matthew Nalls, Haiyan Zhou, Sara Aguti, Beryl B. Cummings, Monkol Lek, Taru Tukiainen, Jamie L. Marshall, Oded Regev, Dina Marek-Yagel, Anna Sarkozy, Russell J. Butterfield, Cristina Jou, Cecilia Jimenez-Mallebrera, Yan Li, Corine Gartioux, Kamel Mamchaoui, Valérie Allamand, Francesca Gualandi, Alessandra Ferlini, Eric Hanssen, the COL6A1 Intron 11 Study Group, Steve D. Wilton, Shireen R. Lamandé, Daniel G. MacArthur, Raimund Wagener, Francesco Muntoni, Carsten G. Bönnemann
Cas9/dual-gRNA targeted genomic excision of the +189C>T mutation corrects the