Allele dependent silencing of collagen type I using small interfering RNAs targeting 3[prime]UTR indels [ndash] a novel therapeutic approach in osteogenesis imperfecta

Abstract

Introduction: Osteogenesis imperfecta (OI) is a heterogenous disease of connective tissue, usually caused by dominant mutations in the genes encoding collagen I, COL1A1 and COL1A2 respectively. Severe forms are lethal, often due to thoracic fractures and intracerebral hemorrhaging. Bisphosphonates are generally the drug of choice, but the effect is neither sufficient nor satisfactory. A promising field is gene silencing through RNA interference, as silencing of the mutated allele would convert a severe OI to a mild OI type I in the case of a COL1A1 mutation and to a phenotypically normal patient in the case of a COL1A2 mutation. However, more than 800 different mutations have been described and creating unique small interfering RNAs (siRNAs), each targeting one mutation would be labour intensive. In this study allele preferential siRNAs were used to target two common 3′ untranslated region insertion/deletions (3′UTR indels) in COL1A1 and COL1A2 in a mutation independent approach. Method: Allele frequencies of the 3′UTR indels were determined in a cohort of 96 healthy individuals and in 96 patients with OI. Four siRNAs targeting each allele of the indels in COL1A1 and COL1A2 were designed and tested in various concentrations in cultures of primary bone derived cells using magnet-assisted transfection (MATRA). RNA was prepared 72 h post transfection and cDNA was created and sequenced. The software PeakPicker was used for calculations of insertion/non-insertion allele ratios. siRNA results were normalized to cell populations treated with negative control siRNAs. Quantitative PCR was used to determine the collagen type I mRNA abundances in treated and negative control treated cell populations. Results: Approximately 50% of individuals genotyped were heterozygous for each of the indels and thus potential candidates for therapy. Allele dependent silencing was observed, with the most promising results for siRNAs complementary to the insertion harbouring alleles. The allele ratio was shifted from 1 to 0.21 and 0.19 for COL1A1 and COL1A2 respectively at the highest concentration used. A slightly lower concentration rendered the ratios 0.21 and 0.34 and an appropriate reduction in mRNA abundance was subsequently demonstrated. This shift represents an increase in normal fibrils from 25 to 69% in COL1A1 and 50 to 75% in COL1A2. In conclusion allele dependent silencing of collagen type I utilizing indels is a promising mutation independent approach for OI.