Inducible caspase-9 suicide gene under control of endogenous oct4 to safeguard mouse and human pluripotent stem cell therapy.

Pluripotent stem cells (PSCs) are promising in regenerative medicine. A major challenge of PSC therapy is the risk of teratoma formation because of the contamination of undifferentiated stem cells. Constitutive promoters or endogenous SOX2 promoters have been used to drive inducible caspase-9 (iCasp9) gene expression but cannot specifically eradicate undifferentiated PSCs. Here, we inserted iCasp9 gene into the endogenous OCT4 locus of human and mouse PSCs without affecting their pluripotency. A chemical inducer of dimerization (CID), AP1903, induced iCasp9 activation, which led to the apoptosis of specific undifferentiated PSCs in vitro and in vivo. Differentiated cell lineages survived because of the silence of the endogenous OCT4 gene. Human and mouse PSCs were controllable when CID was administrated within 2 weeks after PSC injection in immunodeficient mice. However, an interval longer than 2 weeks caused teratoma formation and mouse death because a mass of somatic cells already differentiated from the PSCs. In conclusion, we have developed a specific and efficient PSC suicide system that will be of value in the clinical applications of PSC-based therapy.

Increased Susceptibility of the CD57- NK Cells Expressing KIR2DL2/3 and NKG2C to iCasp9 Gene Retroviral Transduction and the Relationships with Proliferative Potential, Activation Degree, and Death Induction Response.

Nowadays, the use of genetically modified NK cells is a promising strategy for cancer immunotherapy. The additional insertion of genes capable of inducing cell suicide allows for the timely elimination of the modified NK cells. Different subsets of the heterogenic NK cell population may differ in proliferative potential, in susceptibility to genetic viral transduction, and to the subsequent induction of cell death. The CD57-NKG2C+ NK cells are of special interest as potential candidates for therapeutic usage due to their high proliferative potential and certain features of adaptive NK cells. In this study, CD57- NK cell subsets differing in KIR2DL2/3 and NKG2C expression were transduced with the iCasp9 suicide gene. The highest transduction efficacy was observed in the KIR2DL2/3+NKG2C+ NK cell subset, which demonstrated an increased proliferative potential with prolonged cultivation. The increased transduction efficiency of the cell cultures was associated with the higher expression level of the HLA-DR activation marker. Among the iCasp9-transduced subsets, KIR2DL2/3+ cells had the weakest response to the apoptosis induction by the chemical inductor of dimerization (CID). Thus, KIR2DL2/3+NKG2C+ NK cells showed an increased susceptibility to the iCasp9 retroviral transduction, which was associated with higher proliferative potential and activation status. However, the complete elimination of these cells with CID is impeded.