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

Abstract

The COL7A1 gene encodes homotrimer fibrils essential for anchoring dermal and epidermal layers, and pathogenic mutations in COL7A1 can cause recessive or dominant dystrophic epidermolysis bullosa. As a monogenic disease gene, COL7A1 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 COL7A1: 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 COL7A1 exons 10 and 73 for excision from the mature mRNA, we discovered that antisense oligomers comprised of 2′-O-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 COL7A1 pre-mRNA.

Induction of cryptic pre-mRNA splice-switching by antisense oligonucleotides

“…1 ; Table 2 ). Motifs were considered when one or more motif nucleotides were masked by the targeting AO, as even partially covering a motif by two nucleotides influences splice outcome 38 . The examined AOs were found to consistently mask SRSF1 motifs, with exception of the AO H2D(+10–12) targeting the SRSF2 exon 2 donor site.…”

Antisense RNA Therapeutics: A Brief Overview

“…AONs designed to skip exon 36 restored the reading frame and produced a functional protein able to rescue the cellular phenotype in patientderived cells [12]. Following the same strategy, AON molecules to skip different exons of COL7A1 have been developed for dystrophic epidermolysis bullosa, a skin disease inherited in both dominant and recessive fashion [13][14][15][16].…”

Polyglutamine Ataxias: Our Current Molecular Understanding and What the Future Holds for Antisense Therapies

“…However, our laboratory first recognised the applicability of these oligomers to splice switching in the mid-2000s [167][168][169], and subsequently, a collaboration was established to explore PMOs as agents to treat Duchenne muscular dystrophy (DMD) in association with AVI Pty Ltd, now known as Sarepta Therapeutics, Cambridge, MA. Our laboratory has since evaluated PMOs for translation blockade, exon skipping and exon inclusion, and selection of transcription start sites [170][171][172][173]. The first of our splice-switching applications to gain clinical approval is the PMO Exondys 51, targeting exon 51 of the DMD transcript.…”