Nonsequential Splicing Events Alter Antisense-Mediated Exon Skipping Outcome in COL7A1

Abstract

The <i>COL7A1</i> gene encodes homotrimer fibrils essential for anchoring dermal and epidermal layers, and pathogenic mutations in <i>COL7A1</i> can cause recessive or dominant dystrophic epidermolysis bullosa. As a monogenic disease gene, <i>COL7A1</i> constitutes a potential target for antisense oligomer-mediated exon skipping, a therapy applicable to a growing number of other genetic disorders. However, certain characteristics of <i>COL7A1</i>: many exons, low average intron size, and repetitive and guanine-cytosine rich coding sequence, present challenges to the design of specific and effective antisense oligomers. While targeting <i>COL7A1</i> exons 10 and 73 for excision from the mature mRNA, we discovered that antisense oligomers comprised of 2'-<i>O</i>-methyl modified bases on a phosphorothioate backbone and phosphorodiamidate morpholino oligomers produced similar, but distinctive, splicing patterns including excision of adjacent nontargeted exons and/or retention of nearby introns in some transcripts. We found that the nonsequential splicing of certain introns may alter pre-mRNA processing during antisense oligomer-mediated exon skipping and, therefore, additional studies are required to determine if the order of intron removal influences multiexon skipping and/or intron retention in processing of the <i>COL7A1</i> pre-mRNA.