Development of an inducible caspase-9 safety switch for pluripotent stem cell–based therapies

Abstract

Induced pluripotent stem cell (iPSC) therapies offer a promising path for patient-specific regenerative medicine. However, tumor formation from residual undifferentiated iPSC or transformation of iPSC or their derivatives is a risk. Inclusion of a suicide gene is one approach to risk mitigation. We introduced a dimerizable-“inducible caspase-9” (iCasp9) suicide gene into mouse iPSC (miPSC) and rhesus iPSC (RhiPSC) via a lentivirus, driving expression from either a cytomegalovirus (CMV), elongation factor-1 α (EF1α) or pluripotency-specific EOS-C(3+) promoter. Exposure of the iPSC to the synthetic chemical dimerizer, AP1903, in vitro induced effective apoptosis in EF1α-iCasp9-expressing (EF1α)-iPSC, with less effective killing of EOS-C(3+)-iPSC and CMV-iPSC, proportional to transgene expression in these cells. AP1903 treatment of EF1α-iCasp9 miPSC in vitro delayed or prevented teratomas. AP1903 administration following subcutaneous or intravenous delivery of EF1α-iPSC resulted in delayed teratoma progression but did not ablate tumors. EF1α-iCasp9 expression was downregulated during in vitro and in vivo differentiation due to DNA methylation at CpG islands within the promoter, and methylation, and thus decreased expression, could be reversed by 5-azacytidine treatment. The level and stability of suicide gene expression will be important for the development of suicide gene strategies in iPSC regenerative medicine.

Rhesus iPSC Safe Harbor Gene-Editing Platform for Stable Expression of Transgenes in Differentiated Cells of All Germ Layers

“…The rhesus AAVS1-CAGcopGFP donor plasmid was deposited at Addgene (73439). The copGFP marker gene was replaced with hDCD19 6,23 following digestion with BsrGI and MluI. The PCR primers used to produce each insert for Gibson Assembly are listed in Table S2.…”

“…[5][6][7] Genomic safe harbors (GSHs) refer to regions in the genome that permit sufficient expression of newly integrated DNA without adverse effects on the host cell or organism. 8 Although specific loci or general criteria identifying prospective GSHs have been proposed, 8,9 no GSH has yet been fully validated in a large animal model or a clinical setting.…”

Redirecting Specificity of T cells Using the Sleeping Beauty System to Express Chimeric Antigen Receptors by Mix-and-Matching of VL and VH Domains Targeting CD123+ Tumors

“…On the 5’ side of the resulting CAR sequence, there is an in- frame inducible caspase 9 sequence (iCasp9) [50, 51], followed by a Furin element and F2A peptide sequence, which together make an auto-cleavage site within the protein, resulting in two mature proteins from the single polypeptide sequence [52]. The iCasp9 element creates a chemically inducible suicide switch in CAR + cells.…”

Induced Pluripotent Stem Cells for Traumatic Spinal Cord Injury

“…For this reason, efficient techniques to differentiate cells prior to transplantation, and remove potentially harmful cells after transplantation, will be important (Chen et al, 2013b). Several approaches are currently being developed for this purpose including in vitro removal of high proliferation rate cells, antibodies specifically targeting high-risk subpopulations (e.g., potentially teratoma-forming cells), and selective in vivo ablation of transplanted cells using suicide genes (Wu et al, 2014). …”