siRNA-mediated Allele-specific Silencing of a COL6A3 Mutation in a Cellular Model of Dominant Ullrich Muscular Dystrophy
Congenital muscular dystrophy type Ullrich (UCMD) is a severe disorder of early childhood onset for which currently there is no effective treatment. UCMD commonly is caused by dominant-negative mutations in the genes coding for collagen type VI, a major microfibrillar component of the extracellular...
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| Format: | Article |
| Language: | English |
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Elsevier
2014-01-01
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| Series: | Molecular Therapy: Nucleic Acids |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2162253116302876 |
| _version_ | 1811256075539709952 |
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| author | Véronique Bolduc Yaqun Zou Dayoung Ko Carsten G Bönnemann |
| author_facet | Véronique Bolduc Yaqun Zou Dayoung Ko Carsten G Bönnemann |
| author_sort | Véronique Bolduc |
| collection | DOAJ |
| description | Congenital muscular dystrophy type Ullrich (UCMD) is a severe disorder of early childhood onset for which currently there is no effective treatment. UCMD commonly is caused by dominant-negative mutations in the genes coding for collagen type VI, a major microfibrillar component of the extracellular matrix surrounding the muscle fibers. To explore RNA interference (RNAi) as a potential therapy for UCMD, we designed a series of small interfering RNA (siRNA) oligos that specifically target the most common mutations resulting in skipping of exon 16 in the COL6A3 gene and tested them in UCMD-derived dermal fibroblasts. Transcript analysis by semiquantitative and quantitative reverse transcriptase PCR showed that two of these siRNAs were the most allele-specific, i.e., they efficiently knocked down the expression from the mutant allele, without affecting the normal allele. In HEK293T cells, these siRNAs selectively suppressed protein expression from a reporter construct carrying the mutation, with no or minimal suppression of the wild-type (WT) construct, suggesting that collagen VI protein levels are as also reduced in an allele-specific manner. Furthermore, we found that treating UCMD fibroblasts with these siRNAs considerably improved the quantity and quality of the collagen VI matrix, as assessed by confocal microscopy. Our current study establishes RNAi as a promising molecular approach for treating dominant COL6-related dystrophies. |
| first_indexed | 2024-04-12T17:34:37Z |
| format | Article |
| id | doaj.art-26ccc429f7984cf4af94ba4792058041 |
| institution | Directory Open Access Journal |
| issn | 2162-2531 |
| language | English |
| last_indexed | 2024-04-12T17:34:37Z |
| publishDate | 2014-01-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Molecular Therapy: Nucleic Acids |
| spelling | doaj.art-26ccc429f7984cf4af94ba47920580412022-12-22T03:23:01ZengElsevierMolecular Therapy: Nucleic Acids2162-25312014-01-013C10.1038/mtna.2013.74siRNA-mediated Allele-specific Silencing of a COL6A3 Mutation in a Cellular Model of Dominant Ullrich Muscular DystrophyVéronique Bolduc0Yaqun Zou1Dayoung Ko2Carsten G Bönnemann3Neuromuscular and Neurogenetic Disorders of Childhood Section, Neurogenetics Branch, National Institute of Neurological Disorders and Stroke/National Institutes of Health, Bethesda, Maryland, USANeuromuscular and Neurogenetic Disorders of Childhood Section, Neurogenetics Branch, National Institute of Neurological Disorders and Stroke/National Institutes of Health, Bethesda, Maryland, USANeuromuscular and Neurogenetic Disorders of Childhood Section, Neurogenetics Branch, National Institute of Neurological Disorders and Stroke/National Institutes of Health, Bethesda, Maryland, USANeuromuscular and Neurogenetic Disorders of Childhood Section, Neurogenetics Branch, National Institute of Neurological Disorders and Stroke/National Institutes of Health, Bethesda, Maryland, USACongenital muscular dystrophy type Ullrich (UCMD) is a severe disorder of early childhood onset for which currently there is no effective treatment. UCMD commonly is caused by dominant-negative mutations in the genes coding for collagen type VI, a major microfibrillar component of the extracellular matrix surrounding the muscle fibers. To explore RNA interference (RNAi) as a potential therapy for UCMD, we designed a series of small interfering RNA (siRNA) oligos that specifically target the most common mutations resulting in skipping of exon 16 in the COL6A3 gene and tested them in UCMD-derived dermal fibroblasts. Transcript analysis by semiquantitative and quantitative reverse transcriptase PCR showed that two of these siRNAs were the most allele-specific, i.e., they efficiently knocked down the expression from the mutant allele, without affecting the normal allele. In HEK293T cells, these siRNAs selectively suppressed protein expression from a reporter construct carrying the mutation, with no or minimal suppression of the wild-type (WT) construct, suggesting that collagen VI protein levels are as also reduced in an allele-specific manner. Furthermore, we found that treating UCMD fibroblasts with these siRNAs considerably improved the quantity and quality of the collagen VI matrix, as assessed by confocal microscopy. Our current study establishes RNAi as a promising molecular approach for treating dominant COL6-related dystrophies.http://www.sciencedirect.com/science/article/pii/S2162253116302876allele-specific siRNAcollagen type VIdominant negativeUllrich congenital muscular dystrophy |
| spellingShingle | Véronique Bolduc Yaqun Zou Dayoung Ko Carsten G Bönnemann siRNA-mediated Allele-specific Silencing of a COL6A3 Mutation in a Cellular Model of Dominant Ullrich Muscular Dystrophy Molecular Therapy: Nucleic Acids allele-specific siRNA collagen type VI dominant negative Ullrich congenital muscular dystrophy |
| title | siRNA-mediated Allele-specific Silencing of a COL6A3 Mutation in a Cellular Model of Dominant Ullrich Muscular Dystrophy |
| title_full | siRNA-mediated Allele-specific Silencing of a COL6A3 Mutation in a Cellular Model of Dominant Ullrich Muscular Dystrophy |
| title_fullStr | siRNA-mediated Allele-specific Silencing of a COL6A3 Mutation in a Cellular Model of Dominant Ullrich Muscular Dystrophy |
| title_full_unstemmed | siRNA-mediated Allele-specific Silencing of a COL6A3 Mutation in a Cellular Model of Dominant Ullrich Muscular Dystrophy |
| title_short | siRNA-mediated Allele-specific Silencing of a COL6A3 Mutation in a Cellular Model of Dominant Ullrich Muscular Dystrophy |
| title_sort | sirna mediated allele specific silencing of a col6a3 mutation in a cellular model of dominant ullrich muscular dystrophy |
| topic | allele-specific siRNA collagen type VI dominant negative Ullrich congenital muscular dystrophy |
| url | http://www.sciencedirect.com/science/article/pii/S2162253116302876 |
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