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 (COLE-RD), inserts an in-frame pseudoexon into COL6A1 mRNA, encodes a mutant collagen alpha 1(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.

A recurrent COL6A1 pseudoexon insertion causes muscular dystrophy and is effectively targeted by splice-correction therapies

Gualandi F.;Ferlini A.;
2019

Abstract

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 (COLE-RD), inserts an in-frame pseudoexon into COL6A1 mRNA, encodes a mutant collagen alpha 1(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.
2019
Bolduc, V.; Reghan Foley, A.; Solomon-Degefa, H.; Sarathy, A.; Donkervoort, S.; Hu, Y.; Chen, G. S.; Sizov, K.; Nalls, M.; Zhou, H.; Aguti, S.; Cummin...espandi
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11392/2408100
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