G.P.216: Allele-specific silencing of a dominant-negative mutation using siRNA or LNA antisense oligonucleotides alleviates the phenotype of a cellular model of Ullrich congenital muscular dystrophy

Abstract

Congenital muscular dystrophy type Ullrich (UCMD) is a severe progressive disorder of early childhood onset, presenting with generalized muscle weakness, distal joint hypermobility, proximal joint contractures and respiratory failure as the main features. At present, there are no pharmacological treatment options available for children affected with this disease. In the laboratory we aim at exploring targeted RNAi and antisense approaches as potential therapies for UCMD. Dominant and recessive mutations in the three genes coding for collagen type VI (COL6A1, COL6A2, COL6A3) underlie UCMD, with dominant-negative mutations accounting for the majority of cases. Achieving allele-specific silencing of the mutant collagen VI transcript would convert this dominant-negative state into a clinically asymptomatic haploinsufficient state. We have previously demonstrated the allele-specificity and efficiency of siRNA oligos to downregulate the expression of a mutant COL6A3 transcript in vitro. We have now extended our study to Locked Nucleic Acid (LNA)-containing antisense oligonucleotides, which are short, stable, and RNaseH-competent oligonucleotides that can be delivered without any carrier. We used outcome measures such as unsaturated PCR, quantitative RT-PCR and immunoblot on treated cell lysates, and confocal microscopy on treated fixed cells, and found that both RNAi and RNase-H pathways were comparably effective. This study provides further insights into the comparative allele-specificity of these two pathways to target dominant mutations at the transcript level, with the goal of developing optimal compounds for in vivo application.