Cancer-type organic anion transporting polypeptide 1B3 is a promising target for spliceosome-mediated RNA trans-splicing based suicide gene therapy

抄録

[Background] An RNA trans-splicing molecule (RTM) is a promising tool for cancer gene therapy. Once introduced intracellularly, RTM leads to trans-splicing between a target pre-mRNA and a suicide gene (e.g. herpes simplex virus 1 thymidine kinase, HSV-tk) to generate the suicide protein. Meanwhile, we previously identified the cancer-type organic anion transporting polypeptide1B3 (Ct-OATP1B3) that is expressed in various cancer tissues in a cancer-specific manner in humans. Therefore, Ct-OATP1B3 has an excellent expression profile as a target of RTM-mediated gene therapy. In this study, we aimed to clarify the feasibility that Ct-OATP1B3 can be a target for a RTM-mediated suicide gene therapy.

[Methods] RTM44, which contains the specific domain binding to Ct-OATP1B3 pre-mRNA, was designed utilizing a fluorescence-based system. mutRTM44, which lacks trans-splicing activity, was also designed as a negative control. RTM44 functionality was examined by Western blotting analysis, in vitro cytotoxic assays, along with in vivo studies utilizing xenograft mice carrying human colorectal cancer LS180 cells stably expressing RTM44 (RTM/LS) or mutRTM44 (mutRTM/LS), where ganciclovir (GCV) was used as a HSV-tk activity-dependent cytotoxic drug.

[Results] The results of Western blotting analysis showed that the fusion protein of Ct-OATP1B3 and HSV-tk was successfully produced in RTM/LS cells, but not in mutRTM/LS cells, indicating that RTM44 can precisely facilitate trans-splicing between two genes. Consistently, in the cytotoxic assay, GCV (100 µM) treatment significantly decreased the RTM/LS180 cell viability to 16.4 ± 4.8% of that of the control cells, while the treatment did not affect the mutRTM/LS cell viability. Furthermore, the results of in vivo study showed that the growth of RTM/LS-tumors was significantly inhibited by GCV treatment, which was indicated by the smaller mean tumor weight, compared to that of the mutRTM/LS-tumors (0.15 g vs. 0.4 g, respectively).

[Conclusion] Our results demonstrate that Ct-OATP1B3 is a promising target for a RTM-mediated suicide gene therapy. In light with its expression characteristics, the Ct-OATP1B3-targeted therapy will be expected to provide an effective way to fight against various cancer types.