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

研究了锤头状核酶抑制端粒酶活性用于癌症治疗的可能性。在人类端粒酶的组成成分中，hTR(RNA成分)和hTERT(蛋白质成分的催化亚单位)的mRNA被选为锤头状核酶的底物。设计了多种核酶，并研究了它们的切割活性和对端粒酶的抑制活性。设计了三种单价核酶(36-RZ、180-RZ和315 RZ)和一种二价核酶(36-51-RZ)来切割hTR.所有核酶在体外对hTR模拟底物RNA都有很强的切割活性.当它们被导入Ishikawa细胞时，只有靶点位于模板区域周围的36-RZ和36-51-RZ显示出抑制活性。他们抑制端粒酶活性至少96小时，尽管36-RZ比36-51-RZ更有效。接下来，我们将36-RZ导入细胞，使用的是pHbAPr-1-neo/36RZ，一个质粒载体和一个重组逆转录病毒载体。PHbAPr-1-neo/36RZ对Ishikawa细胞有很强的毒性，但对AN3CA细胞的生长有很强的抑制作用。转导的36-RZ在AN3CA细胞中抑制端粒酶活性效果良好。然而，将36-RZ逆转录病毒转导入Ishikawa细胞并没有引起对端粒酶的强烈抑制。针对hTERT mRNA，我们设计了7种锤头状核酶。其中，针对hTERT基因5‘端3’端的两种核酶(14-RZ和3951-RZ)在RNA转染研究中显示出抑制活性。接下来，我们将14-RZ亚克隆到pHbAPr-1-neo载体中，并导入Ishikawa细胞。G418抗性株系端粒酶活性降低，核酶表达明显增强。综上所述，我们得出结论：靶向hTERT模板区域的36-RZ和靶向hTERT mRNA 5‘端的14-RZ有望成为以端粒酶为靶点的肿瘤基因治疗的候选药物。

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.