Exon-Skipping for a Pathogenic COL6A1 Variant in Ullrich Congenital Muscular Dystrophy

Single nucleotide variants that alter splice sites or splicing regulatory elements can lead to the skipping of exons, retention of introns, or insertion of pseudo-exons (PE) into the mature mRNA transcripts. When translated, these changes can disrupt the function of the synthesized protein. Splice-switching antisense oligonucleotides (ASOs) are synthetic, modified nucleic acids that can correct these aberrant splicing events. They are currently in active clinical development for a number of conditions and have been approved by regulatory agencies for the treatment of neuromuscular disorders such as Duchenne muscular dystrophy and spinal muscular atrophy. We have previously reported that splice-switching ASOs effectively skip a pathogenic PE that causes Ullrich congenital muscular dystrophy (UCMD). This erroneous PE insertion is caused by a deep-intronic variant located within intron 11 of COL6A1 (c.930+189 C>T). Here, we describe the detailed protocols and workflow that our labs have used to assess the efficacy of ASOs to skip this PE in vitro. The protocols include designing ASOs; isolating, culturing, and transfecting fibroblasts; extracting RNA and protein; and validating splicing correction at the mRNA and protein levels using quantitative reverse transcription PCR (qRT-PCR) and western blot assays, respectively.