Epigenetic regulation of lentiviral and retroviral vectors in murine embryonic stem (ES) cells and genetic modification of ES cells with lentiviral vectors for application in regenerative medicine

Abstract

Embryonic stem cells are characterized by their self-renewal capability and pluripotency. They represent an unlimited cell source for regenerative medicine. In order to genetically modify ES cells for gene therapy, self-inactivating lentiviral vectors (SIN-LVs) have been proposed as versatile tools for the gene transfer <em>in vitro, ex vivo </em>or<em> in vivo</em> leading to stable transgene expression (Matrai et al, 2010; Pfeifer et al, 2002). Although retroviral vectors (RVs) were also used for the genetic modifications of ES cells, a major limitation in using RVs is the epigenetic silencing (Jahner et al, 1982). However, the performance of SIN-RVs as well as SIN-LVs has been reported in HSCs (Schambach et al, 2006a) but not in ES cells. Therefore, in the first part of this work the focus lied on the comparison between SIN-LV and SIN-RV transduction of ES cells. The data obtained showed a similar transgene expression level of both SIN-LVs and SIN-RVs transduced ES cells under undifferentiation conditions. Under differentiation conditions transgene silencing in SIN-LVs as well as SIN-RVs transduced ES cells was observed that mainly was due to CpG methylation. For both vectors the contribution of CpG methylation to transgene silencing was of the same extent. For a more accurate analysis of SIN-LV- and SIN-RV-mediated transgene expression, transduced ES cell clones with either high or low expression level were picked for further analysis. In case of SIN-RV ES cell clones, both clones (low and high expressing) showed strong transgene silencing under differentiation conditions due to CpG methylation. In contrast, only the low expressing SIN-LV ES cell clone showed high CpG methylation level under differentiation conditions. Importantly, the high expressing LV ES cell clone revealed a lower CpG methylation level and transgene expression was still observed under differentiation conditions. Moreover, using subzonal injection for the generation of transgenic mice, injection of SIN-LVs in preimplantation embryos resulted in around 50 % transgenic mice of born pups whereas SIN-RVs subzonal injections led to less than 1 %. This data indicated the successful and efficient usage of SIN-LV transduction of murine embryos whereas SIN-RVs showed very low efficient transduction. Taken together, SIN-LVs seem to be the proper vehicle for gene transfer either in ES cells or in embryos for the generation of transgenic mice.<br /> Using pluripotent stem cell derived cells for regenerative medicine carries the potential risk of tumor formation due to the contamination with residual undifferentiated cells (Blum & Benvenisty, 2008; Nussbaum et al, 2007; Wakitani et al, 2003). Therefore, the specific elimination of pluripotent stem cells was investigated in the second part of this work. Pluripotent stem cells including ES and iPS cells were transduced with LVs carrying thymidine kinase (TK) from Herpes simplex virus under control of promoters of pluripotency genes. TK expressing cells are in turn sensitive to ganciclovir (GCV) as TK converts GCV into a toxic metabolite leading to cell death. Treatment of TK transduced pluripotent cells in not further selected (mixed) cell populations with GCV led indeed to a high elimination of undifferentiated cells. Although picked single LV-transduced ES cell clones showed a higher killing efficiency upon GCV treatment complete elimination of undifferentiated cells was not observed. However, raising TK expression by using the stronger EOS promoters in mixed ES cell populations did also not result in a significant reduction of undifferentiated cells. In order to obtain complete transduced cell populations, a pre-selection strategy with hygromycin resistance gene was thus applied. These STPH (LV-EOS-S4-TK-PGK-Hygromycin)-transduced ES cell populations carrying only low copy number of LVs demonstrated a complete elimination of undifferentiated cells after Hygromycin pre-selection upon GCV administration <em>in vitro</em>. More importantly, no teratoma was observed when pre-selected cells were injected into mice that were treated with GCV. These <em>in vivo</em> data confirmed the <em>in vitro</em> findings and suggested that STPH-transduced ES cells with pre-selection seemed to be completely eliminated upon GCV treatment if they did not undergo differentiation. This gives rise to possible promising applications in future clinical approaches.