Targeted biallelic integration of an inducible Caspase 9 suicide gene in iPSCs for safer therapies

Stephanie Wunderlich; Alexandra Haase; Sylvia Merkert; Kirsten Jahn; Maximillian Deest; Helge Frieling; Silke Glage; Wilhelm Korte; Andreas Martens; Andreas Kirschning; Andre Zeug; Evgeni Ponimaskin; Gudrun Göhring; Mania Ackermann; Nico Lachmann; Thomas Moritz; Robert Zweigerdt; Ulrich Martin

doi:10.1016/j.omtm.2022.05.011

Targeted biallelic integration of an inducible Caspase 9 suicide gene in iPSCs for safer therapies

Mol Ther Methods Clin Dev. 2022 May 31:26:84-94. doi: 10.1016/j.omtm.2022.05.011. eCollection 2022 Sep 8.

Authors

Stephanie Wunderlich^{1

2}, Alexandra Haase^{1

2

3}, Sylvia Merkert^{1

2

3}, Kirsten Jahn⁴, Maximillian Deest⁴, Helge Frieling⁴, Silke Glage^{2

5}, Wilhelm Korte^{1

2}, Andreas Martens^{1

2}, Andreas Kirschning^{2

6}, Andre Zeug^{2

7}, Evgeni Ponimaskin^{2

7}, Gudrun Göhring^{2

8}, Mania Ackermann^{9

10}, Nico Lachmann^{2

3

9

10

11}, Thomas Moritz^{2

12

10}, Robert Zweigerdt^{1

2}, Ulrich Martin^{1

2

3}

Affiliations

¹ Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery, Hannover Medical School, 30625 Hannover, Germany.
² REBIRTH - Research Center for Translational Regenerative Medicine, 30625 Hannover, Germany.
³ Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Germany.
⁴ Laboratory for Molecular Neuroscience, Department of Psychiatry, Social Psychiatry and Psychotherapy, Hannover Medical School, 30625 Hannover, Germany.
⁵ Institute for Laboratory Animal Science, Hannover Medical School, 30625 Hannover, Germany.
⁶ Institute for Organic Chemistry, Leibniz University Hannover, 30167 Hannover, Germany.
⁷ Department of Cellular Neurophysiology; Hannover Medical School, 30625 Hannover, Germany.
⁸ Department of Human Genetics, Hannover Medical School, 30625 Hannover, Germany.
⁹ Department of Pediatric Pneumology, Allergology and Neonatology, Hannover Medical School, 30625 Hannover, Germany.
¹⁰ Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany.
¹¹ Cluster of Excellence RESIST (EXC 2155), Hannover Medical School, 30625 Hannover, Germany.
¹² RG Reprogramming and Gene Therapy, Hannover Medical School, 30625 Hannover, Germany.

Abstract

Drug-inducible suicide systems may help to minimize risks of human induced pluripotent stem cell (hiPSC) therapies. Recent research challenged the usefulness of such systems since rare drug-resistant subclones were observed. We have introduced a drug-inducible Caspase 9 suicide system (iCASP9) into the AAVS1 safe-harbor locus of hiPSCs. In these cells, apoptosis could be efficiently induced in vitro. After transplantation into mice, drug treatment generally led to rapid elimination of teratomas, but single animals subsequently formed tumor tissue from monoallelic iCASP9 hiPSCs. Very rare drug-resistant subclones of monoallelic iCASP9 hiPSCs appeared in vitro with frequencies of ∼ 3 × 10^-8. Besides transgene elimination, presumably via loss of heterozygosity (LoH), silencing via aberrant promoter methylation was identified as a major underlying mechanism. In contrast to monoallelic iCASP9 hiPSCs, no escapees from biallelic iCASP9 cells were observed after treatment of up to 0.8 billion hiPSCs. The highly increased safety level provided by biallelic integration of the iCASP9 system may substantially contribute to the safety level of iPSC-based therapies.

Keywords: Caspase 9; gene editing; induced pluripotent stem cells; loss of heterozygosity; silencing; suicide gene.