Hammerhead ribozymes selectively suppress mutant type I collagen mRNA in osteogenesis imperfecta fibroblasts.

TL;DR: This is the first report of ribozymes causing specific suppression of an endogenous mutant mRNA in cells derived from a patient with a dominant negative genetic disorder.

Abstract: Ribozymes are a promising agent for the gene therapy of dominant negative genetic disorders by allele-specific mRNA suppression. To test allele-specific mRNA suppression in cells, we used fibroblasts from a patient with osteogenesis imperfecta (OI). These cells contain a mutation in one alpha1(I) collagen allele which both causes the skeletal disorder and generates a novel ribozyme cleavage site. In a preliminary in vitro assay, ribozymes cleaved mutant RNA substrate whereas normal substrate was left intact. For the studies in cell culture we generated cell lines stably expressing active (AR) and inactive (IR) ribozymes targeted to mutant alpha1(I) collagen mRNA. Quantitative competitive RT-PCR analyses of type I collagen mRNA, normalized to beta-actin expression levels, revealed that the level of mutant alpha1(I) collagen mRNA was significantly decreased by approximately 50% in cells expressing AR. Normal alpha1(I) collagen mRNA showed no significant reduction when AR or IR was expressed from the pHbetaAPr-1-neo vector and a small (10-20%) but significant reduction when either ribozyme was expressed from the pCI.neo vector. In clonal lines derived from cells expressing AR the level of ribozyme expression correlated with the extent of reduction in the mutant:normal alpha1(I) mRNA ratio, ranging from 0.33 to 0.96. Stable expression of active ribozyme did not affect cell viability, as assessed by growth rates. Ribozyme cleavage of mutant mRNA results in a reduction in mutant type I collagen protein, as demonstrated by SDS-urea-PAGE. This is the first report of ribozymes causing specific suppression of an endogenous mutant mRNA in cells derived from a patient with a dominant negative genetic disorder.