243. The Use of Thymidine Kinase Mutants as a Safeguard Switch for IPS Cells

Abstract

Induced pluripotent stem (iPS) cells have great potential for a number of applications in gene therapy and regenerative medicine. However, transplanted iPS cells spontaneously form teratoma in vivo. Residual undifferentiated iPS cells in terminally differentiated population hampers accelerated translation to human applications. In addition, forced reprogramming of somatic cells to generate iPS cells and their ability to proliferate indefinitely also raises the concern about their tumorigenicity. Introducing a suicide gene to iPS cells in advance is one of strategies to prevent tumor development. Thymidine kinase of herpes simplex virus (HSV-TK) has been applied to humans in many studies, particularly for cancer treatment. The HSV-TK phosphorylates the prodrug ganciclovir (GCV), an analog of guanosine nucleoside. The phosphorylated GCV is incorporated into host DNA and terminates the elongation of DNA strands, resulting in cell death. The therapeutic range of GCV is narrow and the administration of GCV often induces adverse reactions. Aiming at a more potent and thus less toxic TK, mutations in the HSV-TK had been screened in previous studies. However, no studies have been conducted on the direct comparison of the efficacy of mutant TKs for iPS cells. In the current study, we examined some TK mutants with substitution at the nucleoside binding site that had been reported before (Black, et al., 1996; Black, et al., 2001; Mercer, et al., 2002; Balzarini, et al., 2006). We made iPS cell clones that constitutively express individual mutant TK. Treatment of the TK-iPS clones with GCV revealed that cells with mutant SR11 (IF160 LL+A168Y), SR39 (LIF159IFL+AL168FM), Q7530 (IF160LL+AL168YF), and A168H were 10-fold more sensitive to GCV than wild type TK. Some other mutants that had been shown to have an enhanced killing activity in their original reports did not show an advantage over the wild type. These results suggested that TK mutants were more potent than the wild type and have to be screened and selected for a specific purpose.