P24
Targeted biallelic integration of an inducible Caspase 9 suicide gene in human iPSCs for safer therapies
S Wunderlich² ³ A Haase² ³ ⁴ S Merkert² ³ ⁴ K Jahn⁵ M Deest⁵ H Frieling⁵ S Glage³ ⁶ W Korte² ³ A Martens² ³ A Kirschning³ ⁷ A Zeug³ ⁸ E Ponimaskin³ ⁸ G Göhring³ ⁹ M Ackermann³ ¹⁰ N Lachmann³ ¹⁰ T Moritz³ ¹¹ ¹² R Zweigerdt² ³ U Martin² ³ ⁴
1:Hannover Medical School; 2:Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery, Hannover Medical School, Hannover, 30625, Germany; 3:REBIRTH - Research Center for Translational Regenerative Medicine; 4:Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH); 5:Laboratory for Molecular Neuroscience, Department of Psychiatry, Social Psychiatry, and Psychotherapy, Hannover Medical School, Hannover, 30625, Germany; 6:Institute for Laboratory Animal Science, Hannover Medical School, Hannover, 30625, Germany; 7:Institute for Organic Chemistry, Leibniz University Hannover, Hannover, 30625, Germany; 8:Department of Cellular Neurophysiology; Hannover Medical School, Hannover, 30625 Germany; 9:Department of Human Genetics, Hannover Medical School, Hannover, 30625, Germany; 10:Department of Pediatric Pneumology, Allergology and Neonatology, Hannover Medical School, Hannover, 30625, Germany; 11:RG Reprogramming and Gene Therapy, Hannover Medical School, Hannover, 30625, Germany; 12:Institute of Experimental Hematology, Hannover Medical School, Hannover, 30625, 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 Caspase9 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 ~ 3x10⁻⁸. Besides transgene elimination, presumably via Loss of Heterozygosity (LoH), also silencing via aberrant promoter methylation was identified as a major underlying mechanism. In contrast to monoallelic iCASP9 hiPSCs, we never observed any escapees from biallelic iCASP9 cells, even after treatment of up to 0.8 billion hiPSCs.
In conclusion, biallelic integration of an iCASP9 system in the AAVS1 locus may substantially contribute to the safety level of iPSC-based therapies, which should be calculated by relating clonal escapee frequencies to the number of tumor or leukemia cells that is readily detectable during routine screening procedures.