Abstract
Collagen VI-related congenital muscular dystrophies (COL6-CMDs) are the second most common form of congenital muscular dystrophy. Currently, there is no effective treatment available. COL6-CMDs are caused by recessive or dominant mutations in one of the three genes encoding for the α chains of collagen type VI (COL6A1, COL6A2, and COL6A3). One of the most common mutations in COL6-CMD patients is a de novo deep intronic c.930+189C > T mutation in COL6A1 gene. This mutation creates a cryptic donor splice site and induces incorporation of a novel in-frame pseudo-exon in the mature transcripts. In this study, we systematically evaluated the splice switching approach using antisense oligonucleotides (ASOs) to correct this mutation. Fifteen ASOs were designed using the RNA-tiling approach to target the misspliced pseudo-exon and its flanking sequences. The efficiency of ASOs was evaluated at RNA, protein, and structural levels in skin fibroblasts established from four patients carrying the c.930+189C > T mutation. We identified two additional lead ASO candidates that efficiently induce pseudo-exon exclusion from the mature transcripts, thus allowing for the restoration of a functional collagen VI microfibrillar matrix. Our findings provide further evidence for ASO exon skipping as a therapeutic approach for COL6-CMD patients carrying this common intronic mutation.
Highlights
Congenital muscular dystrophy (CMD) is a term coined in 1908 that is used to identify a group of heterogeneous muscle wasting diseases with onset at birth or infancy.[1]
Our results showed two regions of the pseudo-exon sequence, targeted by antisense oligonucleotides (ASOs)-3 to ASO-6 and ASO-9 to ASO-12, respectively, where ASOs successfully skipped the mutant transcripts with efficiencies over 90%, including 91.3% (±4.6) after ASO of 25-mer long (ASO-5) treatment and 97.5% (±2.2) after ASO-6 treatment (Figure 2C)
Efficacy of ASO to Restore Collagen VI Protein Expression in the extracellular matrix (ECM) In our recently reported study using Phosphorodiamidate morpholino oligomer (PMO), we identified an effective sequence region targeted by ASO-9, ASO-10, ASO-11, and ASO-12.32 in this study, we focused on an additional region targeted by ASO-3, ASO-4, ASO-5, and ASO-6, evaluating their efficacy at protein level by immunofluorescence staining (Figure 3)
Summary
Congenital muscular dystrophy (CMD) is a term coined in 1908 that is used to identify a group of heterogeneous muscle wasting diseases with onset at birth or infancy.[1]. The a3 chain is encoded by COL6A3 (NM_004369.4) located on chromosome 2q27.5 The three a chains share the N-terminal and C-terminal globular domains connected by a short triple-helical domain consisting of Gly-X-Y repeating sequences, where X is often proline and Y is often hydroxyproline or hydroxylysine.[6] Together, the three a chains assemble to form the large higher-order collagen VI protein that is an important component of the extracellular matrix (ECM). Tetramers undergo post-translational modifications and are secreted into the extracellular compartment, where they align in an interlinking end-to-end association to form a network of beaded microfibrils.[8,9,10,11] Collagen VI microfibrils are ubiquitously distributed throughout connective tissues, anchoring components of the basal lamina to the surrounding ECM.[12] This function is crucial for signal transduction and cell integrity, in skeletal muscle that continuously undergoes contraction-induced mechanical stress.[9] In skeletal muscles, collagen VI is synthesized by the interstitial muscle fibroblasts and represents one of the major components of the ECM.[13]
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