TRIPLE RIBOZYME-MEDIATED DOWN-REGULATION OF THE RETINOBLASTOMA GENE

We have been developing triple ribozyme (TRz) constructs which consist of two cis-acting ribozymes flanking an internal trans-acting ribozym e, which is targeted to a cellular RNA. Actions of the two cis-acting ribozymes efficiently liberate the internal ribozyme with minimal nons pecific flanking sequences, The liberated internal targeted ribozyme s hows substantially greater catalytic activity than TRz preparations, c onstructs which cannot undergo self-liberation or than single ribozyme s with flanking vector sequences. Here we construct a TRz which was ta rgeted to retinoblastoma gene (Rb) mRNA, which cleaved Rb target RNA i n vitro as expected. A number of tetracycline-regulatable clones stabl y transfected with the Rb-targeted TRz were developed and analyzed. Th e internal targeted ribozymes were efficiently liberated in vivo and t he stably transfected clones showed varied reductions in Rb mRNA, whic h were contingent upon ribozyme expression and catalytic activity. The two clones showing major reductions in Rb mRNA (and pRb) levels (>70% reduction) showed abnormal morphology, loss of contact inhibition and the ability to grow in soft agar, as well as altered compartmentation of repetitive B2 transcripts, a phenomenon previously associated with immortalization and/or transformation. TRz constructs coupled with ti ssue-specific promoters should allow development of irt vivo models in which Rb function is markedly reduced in a tissue-specific manner.