We have previously reported that in vitro cleavage of collagen transcripts from osteogenesis imperfecta (OI) patients by hammerhead ribozymes is allele-specific and is absolutely dependent on the pressence of the disease-causing mutation. This was encouraging for the potential application of hammerhead ribozymes as agents to selectively reduce the level of RNA transcripts from the mutant allele.

We investigated the competitive effects of both total RNA and normal allele transcripts on ribozyme cleavage activity. The ribozyme was able to localize and cleave its specific target even in the presence of a vast excess of total RNA, up to a weight ratio of cleavable RNA/total RNA of 1:500. However, cleavage of the mutant RNA was linearly inhibited by the presence of a non-cleavable competitor substrate which contained a ribozyme binding site identical to the site present in the cleavable target. This is analogous to the intracellular situation, where both normal and mutant allele transcripts are present. This competition could be eliminated by introducing a mismatch either into the sequence complementary to one ribozyme binding arm in the non-cleavable competitor transcript or into one binding arm of the ribozyme. In the later case, the mismatch which eliminated competition also caused a decrease in ribozyme cleavage activity against the cleavable target. We have initiated experiments in cultured fibroblasts from OI patients to determine whether the translational machinery and other RNA binding proteins will cause sufficient cycling of the ribozyme off the normal transcript or whether it will be necessary to introduce mismatches into the binding arms of ribozymes used as therapeutic agents for genetic disease.