COLLAGEN RELATED MUSCLE DISEASES: EP.62 Increasing allele selectivity of small interfering RNAs to target a dominant-negative glycine substitution causing a collagen VI-related dystrophy

Abstract

Collagen VI-related dystrophies (COL6-RDs) are a group of frequently severe, congenital-onset muscular dystrophies for which there is no effective treatment. Dominant-negative mutations, in particular glycine substitutions and in-frame exon skips, are common in COL6A1, COL6A2 and COL6A3 genes, and they are capable of incorporating into the hierarchical assembly of the encoded collagen α1, α2 and α3 (VI) chains and as a consequence, produce a dysfunctional collagen VI extracellular matrix. RNA directed therapeutics offer great opportunities to silence dominant-negative mutations if designed to selectively target the mutant allele, as haploinsufficiency for the COL6 genes is not associated with clinical disease. To achieve the potential of RNA directed therapeutics for dominant conditions, the design of highly active, yet allele-specific, antisense oligonucleotides or siRNAs is required. Targeting single missense substitutions proves to be challenging, as the mutant target differs from the normal copy by only one nucleotide. To increase selectivity of siRNA silencing towards a common glycine substitution (G293R) in COL6A1, we deliberately introduced a second mismatch into the siRNA design, to destabilize hybridization to the normal allele while maintaining activity on the mutant transcript. A series of siRNAs in which the location of the second mismatch was located at various positions along the siRNA sequence were first screened in HEK293 cells with fluorescence reporters for both the normal and the mutant allele. We found at least two siRNAs that retain high silencing activity towards the mutant allele as evidenced by fluorescence and immunoblotting quantification, while preserving the expression of the normal allele. Treatment of patient-derived fibroblasts with these siRNAs effectively rescued COL6 matrix deposition. The design strategy that we tested here increases the differential targeting potential of specific mutations that were previously difficult to discriminate.