• 미생물학회지
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• 제35권3호
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• pp.211-217
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• 1999

표적 RNA 의 구조가 Tetrahymena thermophila 의 group I intron 에 의한 trans-splicing 반응에 미치는 영향을 분석하기 위해 강력한 stem-loop 형태의 안정된 구조를 갖고 있는 표적 RNA mapping 분석 방법을 이용한 결과 in vitro 뿐만 아니라 in vivo 에서도 stem 부위의 염기들에 반해 loop 부위의 염기들이 ribozyme 에 의해 잘 인지되었으며 이러한 결과는 그러한 부위들을 인지할 수 있는 ribozyme 들에 의한 trans-cleavage 그리고 trans-splicing 반응을 수행함으로써 검증하였다. 또한 이러한 trans-splicing 반응은 정확하게 일어남을 반응 산물의 염기서열 결정을 통해 확인하였다. 따라서 표적 RNA 의 구조가 in vitro 및 in vivo 에서의 ribozyme 활성에 매우 중요한 요인임을 확인하였다.

• Journal of Microbiology and Biotechnology
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• 제15권6호
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• pp.1408-1413
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• 2005

Elevated expression of carcinoembryonic antigen (CEA) has been implicated in various biological aspects of neoplasia such as tumor cell adhesion, metastasis, blocking of cellular immune mechanisms, and antiapoptosis function. Thus, the CEA could be an important target for anticancer therapy. In this study, we developed Tetrahymena group 1 intron-based trans-splicing ribozymes that can specifically target and replace CEA RNA. To this end, we first determined which regions of the CEA RNA were accessible to ribozymes by employing an RNA mapping strategy that was based on a trans-splicing ribozyme library. Next, we assessed the ribozyme activities by comparing the trans-splicing activities of several ribozymes that targeted different regions of the CEA RNA, and then the ribozyme that could target the most accessible site was observed to be the most active with high fidelity in vitro. Moreover, the specific trans-splicing ribozyme was found to react with and altered the target CEA transcripts in mammalian cells with high fidelity. These results suggest that the Tetrahymena ribozyme can be utilized to replace CEA RNAs in tumors with a new RNA-harboring anticancer activity, thereby hopefully reverting the malignant phenotype.

• Genomics & Informatics
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• 제3권4호
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• pp.172-174
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• 2005

Recent anti-cancer approaches have been based to target tumor-specifically associated and/or causative molecules such as RNAs or proteins. As this specifically targeted anti-cancer modulator, we have previously described a novel human cancer gene therapeutic agent that is Tetrahymena group I intron-based trans-splicing ribozyme which can reprogram and replace human telomerase reverse transcriptase (hTERT) RNA to selectively induce tumor-specific cytotoxicity in cancer cells expressing the target RNA. Moreover, the specific ribozyme has been shown to efficiently retard tumor tissues in xenograft mice which had been inoculated with hTERT-expressing human cancer cells. In this study, we assessed specificity of trans-splicing reaction in cells to evaluate the therapeutic feasibility of the specific ribozyme. In order to analyze the trans-spliced products by the specific ribozyme in hTERT-positive cells, RT, 5'-end RACE-PCR, and sequencing reactions of the spliced RNAs were employed. Then, whole analyzed products resulted from reactions only with the hTERT RNA. This study suggested that the developed ribozyme perform highly specific RNA replacement of the target RNA in cells, hence trans-splicing ribozyme will be one of specific agents for genetic approach to revert cancer.

• Journal of Microbiology and Biotechnology
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• 제22권3호
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• pp.431-435
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• 2012

In this study, we describe a novel approach to achieve replicative selectivity of conditionally replicative adenovirus that is based upon trans-splicing ribozyme-mediated replacement of cancer-specific RNAs. We developed a specific ribozyme that can reprogram human telomerase reverse transcriptase (hTERT) RNA to induce adenoviral E1A gene expression selectively in cancer cells that express the RNA. Western blot analysis showed that the ribozyme highly selectively triggered E1A expression in hTERT-expressing cancer cells. RT-PCR and sequencing analysis indicated that the ribozyme-mediated E1A induction was caused via a high fidelity trans-splicing reaction with the targeted residue in the hTERT-expressing cells. Moreover, reporter activity under the control of an E1A-dependent E3 promoter was highly transactivated in hTERT-expressing cancer cells. Therefore, adenovirus containing the hTERT RNA-targeting trans-splicing ribozyme would be a promising anticancer agent through selective replication in cancer cells and thus specific destruction of the infected cells.

• Journal of Microbiology
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• 제41권4호
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• pp.340-344
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• 2003

The group I intron from Tetrahymena thermophila has been demonstrated to employ splicing reactions with its substrate RNA in the trans configuration. Moreover, we have recently shown that the transsplicing group I ribozyme can replace HCV-specific transcripts with a new RNA that exerts anti-viral activity. In this study, we explored the potential use of RNA replacement for cancer treatment by developing trans-splicing group I ribozymes, which could replace tumor-associated RNAs with the RNA sequence attached to the 3' end of the ribozymes. Thymidine phosphorylase (TP) RNA was chosen as a target RNA because it is known as a valid cancer prognostic factor. By performing an RNA mapping strategy that is based on a trans-splicing ribozyme library, we first determined which regions of the TP RNA are accessible to ribozymes, and found that the leader sequences upstream of the AUG start codon appeared to be particularly accessible. Next, we assessed the ribozyme activities by comparing trans-splicing activities of several ribozymes that targeted different regions of the TP RNA. This assessment was performed to verify if the target site predicted to be accessible is truly the most accessible. The ribozyme that could target the most accessible site, identified by mapping studies, was the most active with high fidelity in vitro. Moreover, the specific trans-splicing ribozyme reacted with and altered the TP transcripts by transferring an intended 3' exon tag sequence onto the targeted TP RNA in mammalian cells with high fidelity. These results suggest that the Tetrahymena ribozyme can be utilized to replace TP RNAs in tumors with a new RNA harboring anti-cancer activity, which would revert the malignant phenotype.

• BMB Reports
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• 제36권6호
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• pp.538-544
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• 2003

The hepatitis C virus (HCV) is a major causative agent of chronic hepatitis and hepatocellular carcinoma. The development of alternative antiviral therapies is warranted because current treatments for the HCV infection affect only a limited number of patients and lead to significant toxicities. The HCV genome is exclusively present in the RNA form; therefore, ribozyme strategies to target certain HCV sequences have been proposed as anti-HCV treatments. In this study, we determined which regions of the internal ribosome entry site (IRES) of HCV are accessible to ribozymes by employing an RNA mapping strategy that is based on a trans-splicing ribozyme library. We then discovered that the loop regions of the domain IIIb of HCV IRES appeared to be particularly accessible. Moreover, to verify if the target sites that were predicted to be accessible are truly the most accessible, we assessed the ribozyme activities by comparing not only the trans-splicing activities in vitro but also the trans-cleavage activities in cells of several ribozymes that targeted different sites. The ribozyme that could target the most accessible site identified by mapping studies was then the most active with high fidelity in cells as well as in vitro. These results demonstrate that the RNA mapping strategy represents an effective method to determine the accessible regions of target RNAs and have important implications for the development of various antiviral therapies which are based on RNA such as ribozyme, antisense, or siRNA.

• 미생물학회지
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• 제44권3호
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• pp.186-192
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• 2008

C형 간염바이러스(hepatitis C virus; HCV) 복제를 효과적이며 특이적으로 제어할 수 있는 유전산물을 개발하기 위하여 특정 리간드 존재에 의해 allosteric하게 그 활성이 조절될 수 있는 HCV 유전체 표적 trans-splicing 리보자임(trans-splicing aptazyme)을 발굴하였다. 이러한 trans-splicing aptazyme은 특정 리간드와 특이적으로 결합하는 RNA aptamer 부위, aptamer와 리간드와의 결합에 의해 리보자임 활성을 유도할 수 있도록 구조적 변이를 전달할 수 있는 communication module부위 및 HCV IRES의 +199 nt를 인지하는trans-splicing리보자임 등으로 구성되도록 설계하였다. 특히 trans-splicing 리보자임의 catalytic core의 P6과 P8 부위에 aptamer와 communication module을 삽입하였을 때 가장 allosteric하게 리보자임 활성이 유도되었다. 이러한 리보자임은 리간드가 없거나 대조 리간드가 존재할 때에는 trans-splicing 반응을 유도하지 못하였으나 특정 리간드가 존재할 때에만 효과적이며 특이적으로 trans-splicing 반응을 유도하여 표적 RNA를 변형시킬 수 있음을 관찰하였다. 이러한 aptazyme은 HCV 증식에 대해 특이적이며 효과적인 억제를 위한 선도물질로 이용 가능할 뿐 아니라 HCV 치료선도 물질의 스크리닝용 도구로서도 활용될 수 있을 것이다.

• Journal of Microbiology
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• 제42권4호
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• pp.361-364
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• 2004

Internal ribosome entry site (IRES) of the hepatitis C virus (HCV) is known to be essential for HCV replication and most conserved among HCV variants. Hence, IRES RNA is a good therapeutic target for RNA-based inhibitors, such as ribozymes. We previously proposed a new anti-HCV modulation strategy based on trans-splicing ribozymes, which can selectively replace HCV transcripts with a new RNA that exerts anti-HCV activity. To explore this procedure, sites which are accessible to ribozymes in HCV IRES were previously determined by employing an RNA mapping method in vitro. In this study, we evaluate the intracellular accessibility of the ribozymes by comparing the trans-splicing activ­ities in cells of several ribozymes targeting different sites of the HCV IRES RNA. We assessed the intra­cellular activities of the ribozymes by monitoring their target-specific induction degree of both reporter gene activity and cytotoxin expression. The ribozyme capable of targeting the most accessible site iden­tified by the mapping studies then harbored the most active trans-splicing activity in cells. These results suggest that the target sites predicted to be accessible are truly the most accessible in the cells, and thus, could be applied to the development of various RNA-based anti-HCV therapies.

• Genomics & Informatics
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• 제2권1호
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• pp.45-52
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• 2004

The self-splicing group I intron from Tetrahymena thermophila has been demonstrated to perform splicing reaction with its substrate RNA in the trans configuration. In this study, we explored the potential use of the trans-splicing group I ribozymes to replace a specific RNA with a new RNA that exerts any new function we want to introduce. We have chosen thymidine phosphorylase (TP) RNA as a target RNA that is known as a valid cancer prognostic factor. Cancer-specific expression of TP RNA was first evaluated with RT-PCR analysis of RNA from patients with gastric cancer. We determined next which regions of the TP RNA are accessible to ribozymes by employing an RNA mapping strategy, and found that the leader sequences upstream of the AUG start codon appeared to be particularly accessible. A specific ribozyme recognizing the most accessible sequence in the TP RNA with firefly luciferase transcript as a 3' exon was then developed. The specific trans-splicing ribozyme transferred an intended 3' exon tag sequence onto the targeted TP transcripts, resulting in a more than two fold induction of the reporter activity in the presence of TP RNA in mammalian cells, compared to the absence of the target RNA. These results suggest that the Tetrahymena ribozyme can be a potent anti-cancer agent to modify TP RNAs in tumors with a new RNA harboring anti-cancer activity.

• Genomics & Informatics
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• 제7권4호
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• pp.181-186
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• 2009

Myotonic dystrophy type 1 (DM1), which is a dominantly inherited neurodegenerative disorder, results from a CTG trinucleotide repeat expansion in the 3'-untranslated region (3'-UTR) of the myotonic dystrophy protein kinase (DMPK) gene. Retention of mutant DMPK (mDMPK) transcripts in the nuclei of affected cells has been known to be the main cause of pathogenesis of the disease. Thus, reducing the RNA toxicity through elimination of the mutant RNA has been suggested as one therapeutic strategy against DM1. In this study, we suggested RNA replacement with a trans -splicing ribozyme as an alternate genetic therapeutic approach for amelioration of DM1. To this end, we identified the regions of mDMPK 3'-UTR RNA that were accessible to ribozymes by using an RNA mapping strategy based on a trans-splicing ribozyme library. We found that particularly accessible sites were present not only upstream but also downstream of the expanded repeat sequence. Repair or replacement of the mDMPK transcript with the specific ribozyme will be useful for DM1 treatment through reduction of toxic mutant transcripts and simultaneously restore wild-type DMPK or release nucleus-entrapped mDMPK transcripts to the cytoplasm.