A recurrent COL6A1 pseudoexon insertion causes muscular dystrophy and is effectively targeted by splice-correction therapies
Véronique Bolduc, … , Francesco Muntoni, Carsten G. Bönnemann
Véronique Bolduc, … , Francesco Muntoni, Carsten G. Bönnemann
Published March 21, 2019
Citation Information: JCI Insight. 2019;4(6):e124403. https://doi.org/10.1172/jci.insight.124403.
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Research Article Muscle biology Therapeutics

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

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 (COL6-RD), inserts an in-frame pseudoexon into COL6A1 mRNA, encodes a mutant collagen α1(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.

Authors

Véronique Bolduc, A. Reghan Foley, Herimela Solomon-Degefa, Apurva Sarathy, Sandra Donkervoort, Ying Hu, Grace S. Chen, Katherine Sizov, Matthew Nalls, Haiyan Zhou, Sara Aguti, Beryl B. Cummings, Monkol Lek, Taru Tukiainen, Jamie L. Marshall, Oded Regev, Dina Marek-Yagel, Anna Sarkozy, Russell J. Butterfield, Cristina Jou, Cecilia Jimenez-Mallebrera, Yan Li, Corine Gartioux, Kamel Mamchaoui, Valérie Allamand, Francesca Gualandi, Alessandra Ferlini, Eric Hanssen, the COL6A1 Intron 11 Study Group, Steve D. Wilton, Shireen R. Lamandé, Daniel G. MacArthur, Raimund Wagener, Francesco Muntoni, Carsten G. Bönnemann

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Figure 5

Cas9/dual-gRNA targeted genomic excision of the +189C>T mutation corrects the COL6A1 pseudoexon splicing defect.

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Cas9/dual-gRNA targeted genomic excision of the +189C>T mutation corr...
(A) Schematic depicting position of the 2 gRNAs (gRNA upstream 1, U1; and downstream 1, D1) that were designed to excise a 103-nucleotide fragment harboring the pseudoexon-generating mutation from the deep intronic region of COL6A1 intron 11. gRNA-U1 and -D1 were subcloned into a Cas9-GFP plasmid (Cas9+U1D1) harboring 2 gRNA cassettes for dual gRNA expression. (B) Amplification of genomic DNA from HEK293T cells transfected with 5 μg of the Cas9+U1D1 plasmid and GFP-enriched by cell sorting 48 hours after transfection. (C) HEK293T cells cotransfected with 1 μg of each splicing reporter (+189C, WT; +189T, mutant), and 5 μg of the Cas9+U1D1 plasmid, and sorted after 48 hours to enrich for GFP expression, were assessed for pseudoexon splicing by RT-PCR. (D) Amplification of genomic DNA from human patient fibroblast cell line IR1, 48 hours after nucleofection with 5 or 10 μg of the Cas9+U1D1 plasmid (GFP-sorted). (E) IR1 patient fibroblasts were nucleofected with 10 μg of Cas9+U1D1 plasmid, and sorted for GFP expression after 48 hours, before RNA isolation. Expression of the pseudoexon was assessed by endpoint RT-PCR. (F) US8 patient fibroblasts were nucleofected with 10 μg of Cas9+U1D1, followed by GFP enrichment, and cell lysates were harvested for immunoblotting. Membranes were probed with a pseudoexon-specific antibody [Pex11α1(VI)] and a housekeeping control (α-tubulin), or with an antibody assaying total collagen α1 (VI) expression [α1(VI)-C] and α-tubulin.