Antisense hammerhead ribozymes have the capability to cleave complementary RNA in a sequence-dependent manner. In osteogenesis imperfecta, a genetic disorder of connective tissue, mutant collagen type I has been shown to participate in but not sustain formation of the triple helix. Selective ablation of mutant collagen gene transcript could potentially remove the mutant gene product and reverse the dominant-negative effect exerted by the abnormal protein. In earlier studies we showed that the hammerhead ribozyme Col1A1Rz547 selectively cleaved a mutant Col1A1 gene transcript in a murine calvarial osteoblast cell line. In order to test the possible therapeutic efficacy of this approach, a dramatic downregulation of the mutant transcript must be achieved, a function directly related to high steady-state level of intracellular ribozyme. We report significantly enhanced expression of Col1A1Rz547 by vaccinia T7 polymerase following infection with an attenuated T7-pol vaccinia virus as shown both by the intracellular level of the ribozyme and the cleavage of the mutant Col1A1 gene transcript. We also describe the engineering of a multimeric ribozyme construct comprising eight subunits, which can self-cleave to monomers. These studies suggest the potential use of multimeric ribozymes expressed by a vaccinia-based system in the therapy of a variety of disorders.