Specificity of ribozymes against the bcr-abl mRNAs in vitro.

Chronic myelogenous leukaemia (CML) is a disease characterized by the presence of the Philadelphia chromosome and the k r d l fusion gene, which result from a reciprocal translocation between chromosomes 9 and 22. Two forms of bcrabl mRNA exist, b3a2 and b2a2, depending upon whether the translocation includes bcr exon 3 or not. The p2 lP-*’ protein, a deregulated tyrosine kinase, is implicated in progression of CML. We are investigating the use of ribozymes to down-regulate the k r d l gene expression. Ribozymes are catalytic RNA molecules that can be designed as specific endoribonucleases [I]. We have made three hammerhead nbozymes, two designed to cleave the b3a2 mRNA at two distinct sites 12 nucleotides (nt) apart and the third ribozyme designed to cleave the b2a2 mRNA. The cleavage sites were chosen close to the k r d l fusion point to encourage the specificity of the ribozymes for the fusion mRNA relative to the wild-type abl and bcr mRNAs. cDNAs for each ribozyme were cloned into the pGEM-3Z plasmid so that in vim transcription with SP6 RNA polymerase produced the active ribozymes. In vim ribozyme cleavage reactions were performed in conditions previously described [ I ] using in vim genaated transcripts of cDNA fragments of abl, hcr, and k r d l mRNAs as substrates. We found that ribozymes designed to cleave either b3a2 or b2a2 mRNA 9nts 3’ of the fusion point are non-specific; they cleave both h c r d l and abl RNAs. However the third ribozyme, designed to cleave 3nts 5’ of the fusion point of b3a2 is specific for its substrate [2]. Under none of the expenmental conditions med was complete cleavage of the substrate RNA observed, including when the three ribozymes were used together. It was recently noted by Tabler et d., [3] that the shortening of one flanking arm of the hammerhead ribozyme can increase the ribozyme’s specificity. Shortening the arm of the non-specific b3a2 ribozyme, from lOnt to 4nt capable of basepairing with the substrate, resulted in an increase in specificity; the ribozyme no longer cleaved the wild-type ubl RNA [2]. Using this asymmetric ribozyme design we had a DNA-RNA hybrid ribozyme made for studies with cell cultures (Figure I ) . Flanking arms made of DNA, whilst the catalyhc core of the ribozyme remained RNA, have been shown by us (unpublished results) and others [4] to a m f a increased resistance to nuclease degradation. Using this hybrid ribozyme in virro cleavage exwments have been carried out. Reliminary results suggest that the DRD-l ribozyme is more specific for its substrate than the equivalent all RNA ribozyme. At 50’C the all RNA ribozyme is the more active, however at 37’C the hybrid and RNA ribozymes appear to have similar activities. Using the cationic liposome LipfedAMINE” the hybrid ribozyme has been transiently transfected into a bcrdl expressing