Gene therapy with hammerhead ribozymes targeting telomerase components in the endometrial carcinoma.

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A possible utility of hammerhead ribozymes to suppress telomerase activity for a cancer therapy was studied. Among the components of human telomerase, the hTR, an RNA component and the mRNA of hTERT, a catalytic subunit of protein components, were chosen as substrates of the hammerhead ribozymes. A number of ribozymes were designed, and their cleavage activity and inhibitory activity to telomerase were studied. Three kinds of monovalent ribozyme (36-RZ, 180-RZ and 315 RZ) and a divalent ribozyme (36-51-RZ ) were designed to cleave hTR.All the ribozymes showed potent but equivalent cleavage activity against hTR mimic substrate RNA in vitro. When they were introduced into Ishikawa cells, only the 36-RZ and 36-51-RZ, of which the target sites were localized around the template region, exhibited inhibitory activity. They suppressed telomerase activity for at least 96 hours, though the 36-RZ is much more potent a than 36-51-RZ.Next we introduced the the 36-RZ into cells using pHbAPr-1-neo/36RZ, a plasmid vector and a recombinant retroviral vector. The pHbAPr-1-neo/36RZ was very toxic to Ishikawa cells, but was very inhibitory to the growth of AN3CA cells. Transduced 36-RZ worked well in AN3CA cells to suppress telomerase activity. However, the retroviral transduction of the 36-RZ into Ishikawa cells did not provoke a potent inhibition to telomerase.Against hTERT mRNA, we designed 7 kinds of hammerhead ribozymes. Among them, two ribozymes (14-RZ and 3951-RZ) targeting the 5'end end 3' end of hTERT mRNA showed inhibitory activity in RNA transfection study. Next we subcloned the 14-RZ into pHbAPr-1-neo plasmid vector and introduced into Ishikawa cells. The clones resistant to G418 showed attenuated telomerase activity with the apparent expression of ribozyme. Taken together, we concluded that 36-RZ targeting the template region of hTR and 14-RZ targeting 5'end of hTERT mRNA would be candidates for cancer gene therapy targeting telomerase.