297. Sensitivity and Mechanisms of Resistance of Human Induced Pluripotent Stem Cells To the Inducible Caspase-9 Safety Switch

Abstract

Introduction of inducible caspase-9 suicide gene (iC9) is one of the most appealing safety strategies for the cell therapy. iC9-mediated cell death is produced by direct activation of the pro-apoptotic molecule in the downstream portion of the intrinsic apoptotic pathway, so that potent and rapid killing is observed in the presence of the chemical inducer of dimerization (CID). To control the cell fate of human induced pluripotent stem cells (hiPSC) and minimize the possible unwanted effect of therapies using hiPSC, we produced hiPSC that expressed the iC9 safety system. In this system, over 99% of iC9-transduced hiPSC (iC9-iPSC) could be effectively eliminated in less than 24hrs after exposure to CID. There were, however, a small number of resistant cells that subsequently outgrew, and we have now investigated the mechanisms of resistance and devised approaches by which it may be overcome.We first purified a resistant population by culturing iC9-iPSC with CID. iC9-resistant iPSC were not killed even at a 30 fold higher dose of CID (% of live cells after CID exposure = 81.33±2.18%). iC9-and iC9-resistant iPSC had similar iC9 copy number. However, iC9 expression and induction of caspase-9/-3 activation was significantly lower in iC9-resistant iPSC than in iC9-iPSC. The CpG islands of the EF1α promoter sequence used as the iC9 transgene promoter, were more highly methylated in iC9-resistant iPSC, and the HDAC inhibitor, LBH589, could restore iC9 expression and partially enhance the CID sensitivity in iC9-resistant iPSC (% of live cells after LBH589+CID exposure; 45.25±3.4%). iC9 re-transduction in iC9-resistant iPSC could also restore the iC9 expression to similar levels as initially sensitive cells and greatly enhanced their sensitivity to killing by CID (% of live cells 24 hours after CID exposure = 24.16±2.33%). The sensitivity of the re-transduced cells remained lower even after increasing iC9 expression by re-transduction. In these cells, bcl-2 protein was highly expressed compared to the parental iPSC or iC9-iPSC. Moreover, the bcl-2 inhibitor, ABT-737, enhanced expression of activated caspase-9/-3, and augmented sensitivity to cytotoxicity produced by CID (% of live cells after ABT373+CID exposure; 18.31±0.03%).Bcl-2 family proteins regulate the intrinsic apoptotic pathway by blocking the mitochondrial disruption which triggers the activation of caspase-9 singling cascade. Because the caspase-9 signaling cascade also induces mitochondrial disruption through the cleavage of bcl-2 family protein, which thereby amplifies the intrinsic apoptotic pathway, our data suggest that both epigenetic silencing of iC9 transgene and overexpression of bcl-2 protein account for the resistance seen in a small proportion of iC9-iPSC to induction of apoptosis (Figure 1), and indicate means by which the high efficiency of killing mediated by this system can be further increased. View Large Image | Download PowerPoint Slide