Optimized allele-specific silencing of the dominant-negative COL6A1 G293R substitution causing collagen VI-related dystrophy
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Astrid Brull1, Apurva Sarathy1, Véronique Bolduc1
1Neurogenetics and Neuromuscular Disorders of Childhood Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA.
Abstract:
Collagen VI-related dystrophies (COL6-RDs) are a group of severe, congenital-onset muscular dystrophies for which there is no effective causative treatment. Dominant-negative mutations are common in COL6A1, COL6A2, and COL6A3 genes, encoding the collagen α1, α2, and α3 (VI) chains. They act by incorporating into the hierarchical assembly of the three α (VI) chains and consequently produce a dysfunctional collagen VI extracellular matrix, while haploinsufficiency for any of the COL6 genes is not associated with disease. Hence, allele-specific transcript inactivation is a valid therapeutic strategy, although selectively targeting a pathogenic single nucleotide variant is challenging. Here, we develop a small interfering RNA (siRNA) that robustly, and in an allele-specific manner, silences a common glycine substitution (G293R) caused by a single nucleotide change in COL6A1 gene. By intentionally introducing an additional mismatch into the siRNA design, we achieved enhanced specificity toward the mutant allele. Treatment of patient-derived fibroblasts effectively reduced the levels of mutant transcripts while maintaining unaltered wild-type transcript levels, rescuing the secretion and assembly of collagen VI matrix by reducing the dominant-negative effect of mutant chains. Our findings establish a promising treatment approach for patients with the recurrent dominantly negative acting G293R glycine substitution.
Insights
A new small interfering RNA (siRNA) therapy specifically targets dominant-negative mutations in Collagen VI-related dystrophies (COL6-RDs). This approach silences mutant COL6A1 transcripts, restoring normal collagen VI matrix assembly and offering a promising treatment for this severe muscular dystrophy.
Area of Science:
- Biochemistry
- Genetics
- Molecular Biology
Background:
- Collagen VI-related dystrophies (COL6-RDs) are severe congenital muscular dystrophies with no effective treatments.
- Dominant-negative mutations in COL6A1, COL6A2, and COL6A3 genes cause COL6-RDs by disrupting collagen VI matrix assembly.
- Haploinsufficiency of COL6 genes does not cause disease, making allele-specific targeting a viable therapeutic strategy.
Purpose of the Study:
- To develop an allele-specific therapeutic strategy for COL6-RDs by targeting dominant-negative mutations.
- To design and validate a small interfering RNA (siRNA) capable of selectively silencing mutant COL6A1 transcripts.
Main Methods:
- Development of a novel siRNA with an intentionally introduced mismatch for enhanced allele specificity.
- Testing the siRNA in patient-derived fibroblasts to assess its efficacy in reducing mutant transcripts.
- Evaluating the impact of siRNA treatment on wild-type transcript levels, collagen VI secretion, and matrix assembly.
Main Results:
- The developed siRNA demonstrated robust and allele-specific silencing of the common G293R glycine substitution mutation in the COL6A1 gene.
- Patient-derived fibroblasts treated with the siRNA showed reduced mutant transcript levels without affecting wild-type transcripts.
- The treatment rescued collagen VI matrix secretion and assembly by mitigating the dominant-negative effect of the mutant chains.
Conclusions:
- Allele-specific transcript inactivation using siRNA is a promising therapeutic strategy for COL6-RDs caused by dominant-negative mutations.
- The developed siRNA offers a targeted approach for patients with the G293R glycine substitution in COL6A1.
- This study establishes a foundation for developing effective treatments for specific genetic forms of muscular dystrophy.
