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

Ribozymes are a promising agent for the gene therapy of dominant negative g enetic disorders by allele-specific mRNA suppression. To test allele-specif ic mRNA suppression in cells, we used fibroblasts from a patient with osteo genesis imperfecta (OI), These cells contain a mutation in one alpha1(I) co llagen 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 stu dies in cell culture we generated cell lines stably expressing active (AR) and inactive (IR) ribozymes targeted to mutant alpha1(I) collagen mRNA, Qua ntitative competitive RT-PCR analyses of type I collagen mRNA, normalized t o p-actin expression levels, revealed that the level of mutant alpha1(I) co llagen mRNA was significantly decreased by similar to 50% in cells expressi ng AR. Normal alpha1(I) collagen mRNA showed no significant reduction when AR or IR was expressed from the pH beta APr-1-neo vector and a small (10-20 %) but significant reduction when either ribozyme was expressed from the pC l.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 o f active ribozyme did not affect cell viability, as assessed by growth rate s. 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 repo rt of ribozymes causing specific suppression of an endogenous mutant mRNA i n cells derived from a patient with a dominant negative genetic disorder.