Abstract
We are exploring the use of ribozymes and novel dimeric minizymes as gene-inactivating agents for treatment of AIDS and CML desease, respectively. High-level expression under control of the pol III promoter would be advantageous not only for conventional ribozymes but also for minizymes since high-level expression should enhance the likelihood of the dimerization of minizymes. Three kinds of conventional ribozymes were designed, each targets against HIV-1 gene. The RNA polymerase III-based promoter we chose was the promoter of a human gene for tRNAVal which has been used successfully in the suppression of target genes by ribozymes. The designed ribozyme was connected downstream of the tRNAVal portion with different linker sequences, therefore, the efficiencies of the binding to substrate were different for each ribozyme transcript. Expression level of ribozymes was correlated with the half-lives of each ribozyme transcript, and the effect of the ribozyme-mediated repression of HIV-1 gene in living cells were in proportion to the stability of ribozymes. As a result, the most stable ribozyme in vivo could inhibit the proliferation of HIV-1 almost completely. We demonstrated previously that minimized-hammerhead ribozyme with low activity form very active dimeric structures with a common stem II. We are exploring the use of dimeric minizymes as gene-inactivating agents for treatment of CML desease. High-level expression under control of the pol III promoter would be advantageous for minizymes and enhance the likelihood of their dimerization. Therefore, we chose the promoter for a human gene for tRNAVal. Although we feared initially that the tRNAVal portion of the transcript might hinder the dimerization of tRNAVal driven minizymes, the novel tRNA-embedded minizyme had strong activity, To our surprise, the cleavage activity of this minizyme that had been expressed either in vitro or in HeLa cells was almost one order of magnitude higher than that of the tRNAVal-embedded conventional hammerhead ribozyme. Our results indicate that tRNA-emhedded minizymes should be considered as potential candidates for gene-inactivating agents.
