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.
Highlights
Dystrophic epidermolysis bullosa (DEB) is an inherited disease caused by mutations in COL7A1 that compromise expression of functional gene products
We speculate that since COL7A1 exons are separated by small introns, targeting a single exon may impact on the splicing of adjacent exons, with considerable cross-talk between these exons. Some of these multiexon skipping and intron retention events would not be detected with reverse transcription polymerase chain reaction (RT-PCR) assays designed to detect excision of the targeted exon, highlighting the need to include several flanking exons in any RT-PCR assay designed to monitor changes in pre-mRNA processing. For both exons 10 and 73 we show that the phosphorodiamidate morpholino oligomer (PMO) chemistry was able to enhance exon skipping in comparison to that induced by a corresponding oligonucleotide composed of 2 -O-methyl modified bases on a phosphorothioate backbone (2 -OMe) using the methods tested
In Duchenne muscular dystrophy (MD), mutations that lead to premature termination of translation are associated with severe disease, whereas most in-frame deletions are associated with the milder allelic condition, Becker MD
Summary
Dystrophic epidermolysis bullosa (DEB) is an inherited disease caused by mutations in COL7A1 that compromise expression of functional gene products. Mutations that result in premature termination codons, affecting both alleles, and resulting in absent or markedly reduced type VII collagen protein (C7), are characteristic of RDEB, termed generalized severe [1]. These mutations can result in reduced COL7A1 mRNA through nonsense-mediated decay, and absent or incomplete polypeptides, leading to lack of anchoring fibril deposition in the epidermal basement membrane layers. A milder recessive form, RDEB, termed generalized intermediate, is commonly caused by compound heterozygous mutations whereby one COL7A1 allele leads to premature termination of translation and the other carries a missense mutation, frequently a glycine substitution. There is an urgent need to develop effective therapies for this devastating disorder
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