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

Area of Science:

• Stem Cell Biology
• Regenerative Medicine
• Gene Therapy

Background:

• Induced pluripotent stem cell (iPSC) therapies hold great promise for regenerative medicine but carry risks of tumor formation from residual undifferentiated cells.
• Suicide gene strategies are being explored to enhance the safety of iPSC-based therapies by enabling targeted elimination of unwanted cells.

Purpose of the Study:

• To evaluate the efficacy of a dimerizable inducible caspase-9 (iCasp9) suicide gene system for mitigating tumor formation in mouse and rhesus iPSCs.
• To assess the impact of different promoters (CMV, EF1α, EOS-C(3+)) on iCasp9 expression and apoptosis induction.
• To investigate the in vivo efficacy of the suicide gene system in preventing or controlling teratoma formation.

Main Methods:

• Lentiviral delivery of the iCasp9 suicide gene into mouse iPSCs (miPSC) and rhesus iPSCs (RhiPSC) under the control of CMV, EF1α, or EOS-C(3+) promoters.
• In vitro induction of apoptosis using the chemical inducer AP1903.
• In vivo assessment of teratoma formation and progression following iPSC transplantation and AP1903 administration.
• Analysis of iCasp9 expression stability and promoter methylation during differentiation.

Main Results:

• The EF1α promoter drove the most effective apoptosis induction in iPSCs upon AP1903 treatment, with varying efficacy for other promoters.
• In vitro teratoma formation was delayed or prevented in EF1α-iCasp9 miPSCs.
• In vivo administration of AP1903 delayed teratoma progression but did not completely eliminate tumors.
• iCasp9 expression, particularly from the EF1α promoter, was downregulated during differentiation due to promoter hypermethylation, which could be reversed by 5-azacytidine.

Conclusions:

• The EF1α promoter-driven iCasp9 system shows potential for iPSC safety but requires optimization for complete tumor ablation.
• The stability and level of suicide gene expression, influenced by promoter methylation, are critical factors for successful iPSC-based regenerative medicine strategies.