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

Abstract

Abstract The Tetrahymena group I intron has been shown to em-ploy a trans-splicing reaction and has been modified to specifically target and replace human telomerase re-verse transcriptase (hTERT) RNA with a suicide gene transcript, resulting in the induction of selective cytotox-icity in cancer cells that express the target RNA, in ani-mal 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 in-traperitoneally inoculated with hTERT-expressing human cancer cells to validate the anti-cancer therapeutic ap-plicability of the group I intron. To this end, an ad-enoviral 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 re-placement via a trans-splicing reaction by the group I intron is a potent anti-cancer genetic approach.Keywords: cancer gene therapy, group I intron, hTERT, RACE PCR, RNA replacement, trans-splicing The Tetrahymena group I intron has been shown to per-form replacement of a specific disease-causative or -associated RNA with a transgene transcript that exerts a therapeutic effect through a trans-splicing reaction, specifically in cells that express the target RNA (Drude et al., 2007). Therefore, the trans-splicing group I introns have been developed as gene therapeutic tools to repair mutant transcripts in genetic or malignant diseases or to reprogram transcripts that are involved in virus-infected diseases to induce new gene activity in the viral RNA- expressing cells (Lan et al., 1998; Phylactou et al., 1998; Rogers et al., 2002; Ryu et al., 2003; Kastanos et al., 2004; Shin et al., 2004). Moreover, we recently have developed a specific trans-splicing group I intron that can target and replace human telomerase reverse transcriptase (hTERT) RNA to induce transgene activity specifically in cancer cells that express the RNA (Kwon et al., 2005). The group I intron has been demonstrated to specifically mark tumor cells that express hTERT and furthermore to specifically and effectively mediate regression of tumor cells not only in vitro but in mice that have been xenografted with hu-man tumors (Hong et al., 2008; Jeong et al., 2008). These data indicate that the hTERT RNA-targeting trans-splicing group I intron can be a potent gene ther-apeutic agent against human cancer. A major concern in applying the trans-splicing group I intron into clinical settings as a gene therapeutic agent for cancer is the in vivo specificity of cancer cell regres-sion. For such a specificity, target RNA-independent and nonspecific transgene induction by the group I intron should be avoided. In other words, mis-spliced products should not be generated by the group I intron. In this study, in order to evaluate the therapeutic feasibility of the hTERT-specific group I intron, we assessed the tar-get RNA specificity of the trans-splicing phenomenon by the intron in mice that have been intraperitoneally xeno-grafted with human cancer cells.