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.

Keywords: Collagens; Extracellular matrix; Muscle Biology; Neuromuscular disease; Therapeutics.

Figure 1. A deep intronic COL6A1 mutation creates a donor splice site, prompting the insertion of a pseudoexonic sequence.

(A) Immunofluorescence images of muscle biopsy sections of patient US1 and one unaffected control costained for collagen VI (red), basement membrane marker laminin (green), and with nuclear stain DAPI (blue). Scale bars: 50 μm. (B) Sashimi plots comparing skeletal muscle biopsy (top) and cultured dermal fibroblast (bottom) RNA sequencing reads from patient UK1, at the COL6A1 intron 11 locus. (C) Genomic DNA chromatograms for the COL6A1 +189C>T mutation testing in family US2. (D) Schematics of the COL6A1 +189C>T mutation locus depicting the pseudoexon sequence boundaries, and splicing outcomes. Splice-modulating antisense oligomers targeting the pseudoexon pre-mRNA sequence are used to promote its skipping as a therapeutic strategy. (E) Splicing reporter (minigene) constructs were prepared by subcloning various segments of COL6A1 genomic DNA (intervening sequence 11 [IVS-11], exons 11 to 13 [Ex-11-13], or exons 10 to 13 [Ex-10-13]) into the pET01 vector (top). Minigenes were transfected into HEK293T cells, and RNA was isolated 24 hours later. Electrophoretic gel image (bottom) represents reverse transcription PCR (RT-PCR) products, amplified using pET01 primers (arrows). Composition of the amplicons is exemplified for Ex-10-13 (right). L = molecular weight ladder. Gel representative of 3 transfections. (F) The pseudoexon percent spliced in (PSI) index was calculated from the RNA sequencing data of 4 muscle specimens and 1 dermis-derived cultured fibroblast specimen, and indicates the percentage of COL6A1 transcript reads that include the pseudoexon. (G and H) Detection of the 72-nucleotide pseudoexon expression in 3 cultured dermal fibroblasts and their corresponding muscle biopsy specimens (G), or in patient F1’s muscle biopsy, cultured dermis- and muscle-derived fibroblasts (H), using RT-PCR. Primers spanning COL6A1 exons 10 to 20 (G), or exons 10 to 15 (H) were used for amplification. RT-PCR gel images were quantified by densitometry (ImageJ) to determine the percentage pseudoexon expression (% p-exon, indicated below each lane and representing the average of 3 to 4 gel quantifications). (I) Location of the single-nucleotide polymorphism rs1980982 relative to the +189C>T mutation site. cDNA from US14 fibroblasts, in whom the “T” allele of rs1980982 is located in cis to the +189T mutation (below), was amplified with primers located in exons 10 and 19 (arrows) for subsequent shotgun cloning.

Figure 2. The COL6A1 pseudoexon is translated and exerts a dominant-negative effect on collagen VI matrix assembly in cultured dermal fibroblasts.

(A) Schematic representing the exon composition of the triple helical domains’ N-terminal ends for each of the 3 α chains of collagen VI, including the predicted pseudoexon peptide sequence (shown in red) inserted between exons 11 and 12 of the α1 chain. The positions of the cysteine residues critical for dimer and tetramer stabilization are indicated for each chain. (B) A polyclonal antibody [Pex11-α1(VI)] was raised against the pseudoexon peptide, and used to probe a denaturing and reducing immunoblot of cell lysate (cellular, C) and media (secreted, S) protein extracts from cultured dermal fibroblasts of 2 patients and 1 unaffected individual. Probing with the preimmune sera is shown (right). (C) An immunoblot prepared as described in B was probed with the Pex11-α1(VI) antibody (left), then stripped and probed with an antibody detecting α1(VI) collagen [α1(VI)-C] (right). (D) Dermal fibroblasts were cultured in the presence of L-ascorbic acid, and medium was collected to measure collagen VI microfibrillar length using rotary shadowing electron microscopy (EM). Representative negative-staining EM images showing isolated collagen VI microfibrils (13 to 44 microfibrils were imaged per sample). Scale bars: 200 nm. The collagen VI microfibrillar length (number of tetramers per microfibril) is reported as a dot plot (below), where lines represent mean ± standard deviation. *P < 0.001 by Mann-Whitney U test, Bonferroni corrected for 2 comparisons. (E) Dermal fibroblasts from 1 unaffected individual, 1 patient carrying the glycine substitution COL6A1 G290R, and 2 +189C>T patients were cultured for 3 days in the presence of L-ascorbic acid, and then cells were fixed and immunostained for matrix-deposited collagen VI (green) and with the nuclear stain DAPI (blue). Scale bars: 25 μm.

Figure 3. PMOs efficiently skip the COL6A1 pseudoexon.

(A) Location of the phosphorodiamidate morpholino antisense oligomers (PMOs) targeting the splice acceptor (SA), the splice donor (SD), or sequences within the pseudoexon (PEX). Asterisks in PMO-SA, PMO-PEX3, and PMO-PEX4 denote the presence of a mismatch between the oligomer sequence and the target sequence. (B) HEK293T cells expressing the minigene construct Ex-11-13 were transfected with each PMO oligomer at the indicated concentrations, and RNA was isolated 48 hours later. Electrophoretic gels of RT-PCR products amplified as in Figure 1E are shown (top), representative of 3 transfection replicates. PCR fragment densities were quantified from the gel images using ImageJ, to determine the ratio of pseudoexon over normal (pseudoexon/WT) products for each lane. Graph (bottom) reports the pseudoexon/WT ratios from 3 replicate treatments. Lines represent the average of the technical replicates ± standard deviation. PMO-NT = nontargeting PMO. Mock = transfection reagent only. (C) RT-PCR detection of the pseudoexon expression in patient IR1 cultured dermal fibroblasts following a 48-hour treatment with selected PMOs is shown as an example. (D–F) Relative gene expression in patient-derived cultured dermal fibroblasts treated for 48 hours with PMOs at the indicated concentrations, measured by quantitative RT-PCR assays specific for the pseudoexon (D and F) or for total COL6A1 (E). Expression levels were normalized to the housekeeping gene PGK1 and measured as relative to the corresponding mock-treated fibroblasts. Each data point represents the average of 2 to 3 treatments on 1 biological replicate, and lines represent the average of the 3 biological replicates (patients R1, IR1, and CA1). Repeated-measures 2-way ANOVA with Bonferroni’s multiple comparisons test was applied. ^#P < 0.05 for all concentrations on the graph; *P < 0.05 for the indicated concentration.

Figure 4. Skipping of the COL6A1 pseudoexon mitigates the dominant-negative effect in cultured dermal fibroblasts.

Cultured dermal fibroblasts from controls and patients were treated with the nontargeting control PMO (PMO-NT), or with PMO-PEX1, or a combination of PMO-PEX2 and PMO-SD, at a total PMO concentration of 30 μM for each condition, in the presence of L-ascorbic acid, for 4 days. (A) Immunoblots of the medium (secreted) or cell lysate (cellular) fractions were probed with the pseudoexon-specific antibody [Pex11-α1(VI)], and then blots were stripped and probed with an antibody detecting collagen α1(VI). Tubulin was probed as control. Mock = transfection reagent only. (B) Fibroblast cultures were fixed and stained for matrix-deposited collagen VI (green). Nuclei were stained with DAPI (blue). Scale bars: 25 μm. (C) Microfibrillar length (number of tetramers per collagen VI microfibril) was measured as described in Figure 2D, and is reported as a dot plot. Lines represent mean ± standard deviation. *P < 0.05, **P < 0.0002 by Mann-Whitney U test, Bonferroni corrected for 2 comparisons.

Figure 5. Cas9/dual-gRNA targeted genomic excision of the +189C>T mutation corrects the COL6A1 pseudoexon splicing defect.

(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.