In Vivo Target RNA Specificity of Trans-Splicing Phenomena by the Group I Intron

The Tetrahymena group I intron has been shown to employ a trans-splicing reaction and has been modified to specifically target and replace human telomerase reverse transcriptase (hTERT) RNA with a suicide gene transcript, resulting in the induction of selective cytotoxicity in cancer cells that express the target RNA, in animal models as well as in cell cultures. In this study, we evaluated the target RNA specificity of trans -splicing phenomena by the group I intron in mice that were intraperitoneally inoculated with hTERT-expressing human cancer cells to validate the anti-cancer therapeutic applicability of the group I intron. To this end, an adenoviral vector that encoded for the hTERT-targeting group I intron was constructed and systemically injected into the animal. 5'-end RACE-PCR and sequencing analyses of the trans-spliced cDNA clones revealed that all of the analyzed products in the tumor tissue of the virus-infected mice resulted from reactions that were generated only with the targeted hTERT RNA. This study implies the in vivo target specificity of the trans - splicing group I intron and hence suggests that RNA replacement via a trans -splicing reaction by the group I intron is a potent anti-cancer genetic approach.

Specificity of Intracellular Trans-Splicing Reaction by hTERT-Targeting Group I Intron

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.

Construction of a trans-splicing ribozyme for restoring EGFP truncation mutation / 生物工程学报

Special designed group I intron ribozymes can specifically splice objective RNA, repair the mutant gene in RNA level. The specificity of ribozyme is determined by nucleotides specific internal guide sequence (IGS) introduced to the enzyme. In this study, fragment sequence containing Tetrahymena thermophilia intron I of 26S rRNA gene was cloned and cis-splicing activity of this ribozyme was confirmed by in vitro transcription. For evaluating the trans-splicing activity of this ribozyme, a truncated mutant Green Fluorescence Protein (GFP) vector, XYQ5/XYQ10- pEGFP-C2, was constructed. This vector deleted the 3' end 564bp fragment of EGFP coding sequence, led to the lost the activity of emitting green fluorescence. Trans-splicing ribozyme plasmids ptrans-rib-CMV2 for remedy of the truncated mutant EGFP was constructed by PCR and molecular cloning techniques. This vector utilizing cloned 26S rRNA intron 1 as core enzyme; selecting T-G site at 194bp of EGFP coding sequence as splicing receptor, designed an IGS which is inversely complement to the 188-193nt of EGFP mRNA; the 195-890bp fragment of EGFP coding sequence was ligated to the 3'-end of ribozyme core. The fragment containing these components was inserted to a eukayotic expression vector pRC-CMV2. Using linearized XYQ5/XYQ10- pEGFP-C2 and ptrans-rib-CMV2 as templates, truncated EGFP mRNA and the constructed ribozyme vector were transcribed and mixed to evaluate the trans-splicing activity. Analysis of in vitro transcription products mix by RT-PCR verified the existence of wild type EGFP mRNA molecule. Co-transfection of XYQ5/XYQ10- pEGFP-C2 with ptrans-rib-CMV2 to Hela cells proved this ribozyme restored green fluorescence within cell, but the efficiency was low.

Functional Modification of a Specific RNA with Targeted Trans-Splicing

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.