Department of Molecular Biology, Dankook University, Yongin, Korea.
J Gene Med. 2011 Feb;13(2):89-100. doi: 10.1002/jgm.1545.
Group I intron-based trans-splicing ribozyme, which can specifically reprogram human telomerase reverse transcriptase (hTERT) RNA, could be a useful tool for tumor-targeted gene therapy. In the present study, the therapeutic feasibility of this ribozyme was investigated by analyzing trans-splicing efficacy in vivo as well as in cells.
We assessed transgene activation, degree of ribozyme expression, targeted hTERT mRNA level, or the level of trans-splicing products in hTERT(+) cells or in human tumor nodules xenografted in animals after ribozyme administration.
The activity and efficacy of the trans-splicing ribozyme in cells was dependent on the amount of endogenous hTERT mRNA and/or the accumulation of ribozyme RNA in cells. Intracellular activity of the ribozyme reached a plateau when no more targetable substrate mRNA was available or the ribozyme RNA level was fully saturated. In addition, the efficacy of ribozyme in xenografted tumor tissues was dependent on the dose of the delivered ribozyme-encoding adenoviral vector, indicating the potential of the ribozyme expression level as a determining factor for the in vivo efficacy of the trans-splicing ribozyme. On the basis of these results, we enhanced the intracellular ribozyme activity by increasing the ribozyme expression level transcriptionally and/or post-transcriptionally.
We analyzed ribozyme efficacy and determined the most influential factors of its trans-splicing reaction in mammalian cell lines as well as in vivo. The present study could provide insights into the optimization of the trans-splicing ribozyme-based RNA replacement approach to cancer treatment.
基于 I 类内含子的反式剪接核酶能够特异性重编程人端粒酶逆转录酶（hTERT）RNA，可作为肿瘤靶向基因治疗的有用工具。本研究通过分析体内和细胞内的反式剪接效果，研究了这种核酶的治疗可行性。
我们评估了转基因激活、核酶表达程度、靶向 hTERT mRNA 水平或在给予核酶后 hTERT(+)细胞或异种移植于动物的人肿瘤结节中反式剪接产物的水平。
反式剪接核酶在细胞中的活性和功效取决于内源性 hTERT mRNA 的量和/或核酶 RNA 在细胞中的积累量。当没有更多可靶向的底物 mRNA 时，或者核酶 RNA 水平完全饱和时，核酶的细胞内活性达到平台期。此外，核酶在异种移植肿瘤组织中的功效取决于递送的核酶编码腺病毒载体的剂量，这表明核酶表达水平有潜力成为反式剪接核酶体内功效的决定因素。基于这些结果，我们通过转录和/或转录后增加核酶表达水平来提高细胞内核酶活性。
我们分析了核酶功效，并确定了其在哺乳动物细胞系和体内反式剪接反应的最具影响力的因素。本研究可为优化基于反式剪接核酶的 RNA 替代方法治疗癌症提供思路。
