Delivery of a hammerhead ribozyme specifically downregulates mutant type Icollagen mRNA in a murine model of osteogenesis imperfecta

Osteogenesis imperfecta (OI) is a systemic heritable disorder of connective tissue, caused by a mutation in one of the genes for type I collagen, whos e cardinal manifestation is bone fragility. Several studies have identified two molecular mechanisms of collagen type I defects. In chain exclusion, t he mutant chain is not incorporated into the collagen triple helix, whereas in chain nonexclusion, it is. The dominant-negative effect of nonexcluded mutations must be taken into account in all strategies aimed at correcting the collagen defects in individuals affected with moderate or several OI. H erein, we describe the application of hammerhead ribozymes to selectively t arget the mutant minigene transcript expressed in a murine calvarial osteob last cell line. Active and control inactive ribozymes were tested in vitro on both mutant and normal targets and in the minigene-expressing cell line. Active ribozyme cleaved its target with high efficiency and specificity in both a time-dependent and dose-dependent manner. After delivery of a riboz yme expression construct, intracellular ribozyme was detected, along with a relative reduction in mutant transcript level.