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

使用锤头核酶针对子宫内膜癌中的端粒酶成分进行基因治疗。

基本信息

批准号：
10671528
负责人：
YOKOYAMA Yasuhiro
金额：
$ 1.15万
依托单位：
Gifu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10671528/
关键词：
Telomerase Endometrial carcinoma Hammerhead ribozyme RNA component of human telomerase Catalytic subunit of human telomerase Gene therapy Expression vector テロラメーゼRNA成分テロラメーゼ触媒成分リボザイム

项目摘要

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.

研究了锤头状核酶抑制端粒酶活性用于癌症治疗的可能用途。在人端粒酶的组分中，选择RNA组分hTR和蛋白组分的催化亚基hTERT的mRNA作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3种单价核酶（36-RZ、180-RZ和315-RZ）和1种二价核酶（36-51-RZ），对hTR模拟底物RNA进行体外切割，结果表明3种核酶对hTR模拟底物RNA的切割活性相当。当它们被导入石川细胞时，只有靶位点位于模板区域周围的36-RZ和36-51-RZ表现出抑制活性。它们抑制端粒酶活性至少96小时，尽管36-RZ比36-51-RZ有效得多。接下来，我们使用pHbAPr-1-neo/36-RZ、质粒载体和重组逆转录病毒载体将36-RZ导入细胞。pHbAPr-1-neo/36 RZ对石川细胞有很强的毒性，但对AN 3CA细胞的生长有很强的抑制作用。转导的36-RZ在AN 3CA细胞中有效地抑制端粒酶活性。然而，逆转录病毒转导的36-RZ到石川细胞没有引起有效的抑制端粒酶。其中，靶向hTERTmRNA 5 '端和3'端的两种核酶（14-RZ和3951-RZ）在RNA转染实验中表现出抑制活性。然后，我们将14-RZ亚克隆到pHbAPr-1-neo质粒载体中，并导入石川细胞。G418抗性克隆端粒酶活性减弱，核酶表达明显。因此，我们认为靶向hTR模板区的36-RZ和靶向hTERTmRNA 5 '端的14-RZ是靶向端粒酶的肿瘤基因治疗的候选靶点。