CLEAVAGE OF COLLAGEN RNA TRANSCRIPTS BY HAMMERHEAD RIBOZYMES IN-VITROIS MUTATION-SPECIFIC AND SHOWS COMPETITIVE-BINDING EFFECTS

We report here the in vitro use of hammerhead ribozymes as an approach to the gene therapy of osteogenesis imperfecta (OI). Our strategy for the treatment of this dominant genetic disorder is based on selective reduction of the level of the mRNA transcripts from the mutant allele . We studied the in vitro cleavage activity of five different hammerhe ad ribozymes targeted against synthetic transcripts of two naturally o ccurring human collagen mutations and against a point mutation introdu ced into a construct containing a portion of the mouse COL1A1 gene, Th is is the first demonstration that ribozyme cleavage is absolutely dep endent on the presence of the ribozyme cleavage site introduced by the disease-causing mutation. Cleavage specificity and activity were unch anged when the cleavage site was located in transcripts of progressive ly longer length. Cleavage efficiency depended directly on the ratio o f ribozyme/substrate, as well as on the time and temperature of incuba tion, We investigated the competitive effects of both total RNA and no rmal synthetic transcripts on ribozyme cleavage activity. The ribozyme was able to localize and cleave its specific target even in the prese nce of a vast excess of total RNA. However, cleavage efficiency was li nearly inhibited by the presence of a non-cleavable competitor substra te which contained a ribozyme binding site identical to the site prese nt in the cleavable target. Although this competition could be elimina ted by introducing a mismatch into one ribozyme binding arm, the prese nce of the mismatch decreased ribozyme cleavage efficiency. The mutati on-specificity of ribozyme cleavage demonstrated in this work provides support for in vivo studies aimed at ribozyme development as a treatm ent for dominant negative genetic disorders.