Abstract:

Ullrich congenital muscular dystrophy (UCMD) is an inherited muscle disorder characterized clinically by muscle weakness, distal joint hyperlaxity, and proximal joint contractures. Sporadic and recessive mutations in the three collagen VI genes, COL6A1, COL6A2, and COL6A3, are reported to be causative. In the sporadic forms, a heterozygous point mutation causing glycine substitution in the triple helical domain has been identified in higher rate. In this study, we examined the efficacy of siRNAs, which target point mutation site, on specific knockdown toward transcripts from mutant allele and evaluated consequent cellular phenotype of UCMD fibroblasts. We evaluated the effect of siRNAs targeted to silence-specific COL6A1 alleles in UCMD fibroblasts, where simultaneous expression of both wild-type and mutant collagen VI resulted in defective collagen localization. Addition of mutant-specific siRNAs allowed normal extracellular localization of collagen VI surrounding fibroblasts, suggesting selective inhibition of mutant collagen VI. Targeting the single-nucleotide COL6A1 c.850G>A (p.G284R) mutation responsible a sporadic autosomal dominant form of UCMD can potently and selectively block expression of mutant collagen VI. These results suggest that allele-specific knockdown of the mutant mRNA can potentially be considered as a therapeutic procedure in UCMD due to COL6A1 point mutations.

日本語要旨:

COL6A1遺伝子の点変異により引き起こされる優性変異型ウルリッヒ病に対するsiRNAによる治療の可能性を解析した。c.850G>A変異を標的とした、数種のsiRNAをデザインした。株化細胞での検索により、変異遺伝子に特異的なジーンサイレンシングを引き起こす２種類のsiRNAを選択した。患者細胞へのこれらsiRNAの導入により、変異COL6A1遺伝子が特異的に抑制され、コラーゲンVIを患者細胞周囲に発現させることに成功した。これらに結果は優性変異型ウルリッヒ病に対する変異遺伝子特異的siRNAの有効性を示すものである。