Development of an Inducible Caspase-9 Safety Switch for Pluripotent Stem Cell Based Therapies

Abstract 2047

Induced pluripotent stem cell (iPSC) based therapies offer a promising path for patient-specific regenerative medicine. However, residual undifferentiated iPSCs within the graft present a potential risk of tumor development. Including a suicide gene system in the transplanted cells would allow the elimination of the transplant in case of an adverse event. For this purpose we propose the use of an inducible caspase 9 (iCasp9) suicide gene system (Di Stasi et al., 2011), consisting of human caspase 9 fused to a modified human FK-binding protein. After conditional iCasp9 dimerization by a synthetic small molecule chemical inducer (AP1903) the marked cells undergo rapid apoptosis (Clackson et al., 1998). To explore the system in the context of iPSC, we studied in vitro and in vivo purging strategies as well as therapeutically modalities using lentiviral constructs expressing iCasp9 either from a constitutional promoters or a pluripotency-specific promoter in a teratoma mouse model.

We designed lentiviral vectors consisting of the iCasp9 gene linked via a 2A peptide to human CD19, driven by either CMV, EF1α, or embryonic stem cell-specific EOS (3+) promoters (Hotta et al., 2009). The iCasp9 lentiviral constructs were introduced into either into mouse iPSCs (C57Bl/6) or rhesus monkey fibroblasts that were subsequently used to derive monkey iCasp9-iPSCs for future nonhuman primate model. In mature rhesus monkey fibroblasts, cells expressing iCasp9/CD19 driven by either CMV or EF1α promoters underwent rapid and complete apoptosis with exposure to the AP1903 dimerizer (0.5μM to 10 μM), whereas the EOS (3+) promoter drove minimal iCasp9-CD19 expression in fibroblasts, as expected. Murine iPSCs transduced with the CMV-iCasp9 construct demonstrated silencing of the transgenes and no significant apoptosis induction with exposure to AP1903 (10 μM). In contrast, both the EF1α and the EOS (3+) promoters highly expressed iCasp9, allowing for successful induction of apoptosis in iPSCs following AP1903 treatment, with dosages ranging from 0.5μM to 10 μM in vitro, cell apoptosis were analyzed by Annexin V/7AAD using flow cytometry.

We next tested the iCasp9 suicide gene system in an in vivo NSG mouse teratoma assay. For this we chose three different approaches: 1) In vitro treatment with AP1903(10 μM) of the iPSCs 4 hours prior to cells injection to NSG mouse (in vitro purging iCasp9-CD19 expressing cells); 2) Immediate I.V. injection of AP1903 (1mg/kg, single does) after injection of the iPSCs into NSG mouse (in vivo purging) ; and 3) Treatment with AP1903(1mg/kg) one week after iPSC injection (in vivo purging after initial teratoma formation). A non-iCasp9 mouse iPSC clone was used as a control in the assay. The results showed that mice injected with iPSCs expressing EOS (3+) or EF1α iCasp9 that underwent in vitro purging with 10 μM AP1903 did not show teratoma formation until 2 months, whereas the non-iCasp9 mouse iPSCs control mice reached the end point size of the teratoma after two weeks(1.494±0.29 g, mean±SD). Mice treated with a single dose of 1mg/kg AP1903 post iPSC cell injection demonstrated markedly delayed teratoma formation. Mice treated one week post iPSC injection with a daily AP1903 regimen (1mg/kg) for four days demonstrated inhibition of further tumor growth, but did not fully ablate existing tumors.

In conclusion, we have shown that the iCasp9/AP1903 system is effective in eliminating iPSCs in vitro and in vivo. Furthermore, we show that efficient transgene expression and subsequent elimination of pluripotent cells depends on the internal promoter of the viral construct. Currently, we are investigating why the induction of the suicide gene failed to ablate existing tumors efficiently (insufficient dosage, silencing of transgenes in vivo).