T.P.55 Allele-specific knockdown to mutant mRNA retrieves cellular function in fibroblasts with point-mutated Ullrich CMD

Abstract

Abstract Ullrich congenital muscular dystrophy (UCMD) is an inherited muscle disorder characterized clinically by muscle weakness, distal joint hyperlaxity, and proximal joint contractures. Sporadic/dominant and recessive mutations in the three collagen VI genes, COL6A1 , COL6A2 , and COL6A3 , are reported to be causative. In the sporadic/dominant form, heterozygous point mutations causing glycine substitution in the triple helical domain have been identified. In this study, we examined the effects of mutant allele mRNA-specific knockdown using siRNAs that target point mutations in COL6A1 on the phenotype of UCMD fibroblasts. We designed several siRNAs for COL6A1 point mutation with/without a mismatched base and measured gene-knockdown effect and specificity of those siRNAs on murine cell-lines transfected with normal and mutant human COL6A1 cDNA. The selected siRNAs were introduced into patient’s fibroblasts, and then the cell-associated collagen VI expression and the ability for cell–substrate attachment were evaluated. Two siRNAs were selected out in transfection experiments. Both worked well on the recovery of extracellular localization of collagen VI surrounding fibroblasts, while on cell attachment assay, one of them showed remarkable recovery of cell-attachment. These results suggest that allele-specific knockdown of the mutant mRNA can potentially be considered a therapeutic strategy in UCMD due to COL6A1 point mutations.