TY - JOUR

T1 - Targeted biallelic integration of an inducible Caspase 9 suicide gene in iPSCs for safer therapies

AU - Wunderlich, Stephanie

AU - Haase, Alexandra

AU - Merkert, Sylvia

AU - Jahn, Kirsten

AU - Deest, Maximillian

AU - Frieling, Helge

AU - Glage, Silke

AU - Korte, Wilhelm

AU - Martens, Andreas

AU - Kirschning, Andreas

AU - Zeug, Andre

AU - Ponimaskin, Evgeni

AU - Göhring, Gudrun

AU - Ackermann, Mania

AU - Lachmann, Nico

AU - Moritz, Thomas

AU - Zweigerdt, Robert

AU - Martin, Ulrich

N1 - Funding Information: The authors thank Jennifer Beier, Janina Zöllner, Nicole Cleve, Annika Franke, Alexandra Lipus, and Viktor Lutscher for technical assistance and helpful discussion and Wladimir Solodenko for chemical synthesis of AP1903. We are also thankful to Ian Shum for proofreading. Illustrations in Figures 1, 3, and 5 were created with BioRender.com. The plasmid SFG.iCasp9.2A.ΔCD19 was kindly provided by G. Dotti (Baylor College of Medicine). This work received funding from the German Research Foundation (DFG; grants: Cluster of Excellence REBIRTH EXC 62/2, 62/3, and 62/4), “Förderung aus Mitteln des Niedersächsischen Vorab” (grant: ZN3340) supported by the REBIRTH - Research Center for Translational Regenerative Medicine (until 2019: REBIRTH - Cluster of Excellence), and from the German Center for Lung Research (DZL, BREATH 82DZL002A1). S.W. designed, performed, and analyzed experiments. S.M. and A.H. was involved in the cloning procedure of the donor and design of guide RNAs. K.J. M.D. and H.F. designed, performed, and analyzed Nanopore sequencing. S.G. analyzed the H&E stainings and provided expertise and feedback. W.K. and A.M. performed and analyzed the in vivo experiments (teratoma assay). A.K. provided Y-27632 and AP1903. A.Z. and E.P. were involved in analyzing of remaining human cells and editing the resulting pictures (confocal microscopy). G.G. performed and analyzed the karyotype analysis. M.A. provided expertise and feedback. N.L. helped with data interpretation. T.M. contributed to experimental design. R.Z. contributed expertise and helpful discussion. U.M. S.W. and A.H. wrote the manuscript. The authors have no commercial, proprietary, or financial interest in the products or companies described in this article. Funding Information: The authors thank Jennifer Beier, Janina Zöllner, Nicole Cleve, Annika Franke, Alexandra Lipus, and Viktor Lutscher for technical assistance and helpful discussion and Wladimir Solodenko for chemical synthesis of AP1903. We are also thankful to Ian Shum for proofreading. Illustrations in Figures 1 , 3 , and 5 were created with BioRender.com . The plasmid SFG.iCasp9.2A.ΔCD19 was kindly provided by G. Dotti (Baylor College of Medicine). This work received funding from the German Research Foundation (DFG; grants: Cluster of Excellence REBIRTH EXC 62/2 , 62/3 , and 62/4 ), “Förderung aus Mitteln des Niedersächsischen Vorab” (grant: ZN3340 ) supported by the REBIRTH - Research Center for Translational Regenerative Medicine (until 2019: REBIRTH - Cluster of Excellence), and from the German Center for Lung Research (DZL, BREATH 82DZL002A1 ).

PY - 2022/9/8

Y1 - 2022/9/8

N2 - 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.

AB - 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.

KW - Caspase 9

KW - gene editing

KW - induced pluripotent stem cells

KW - loss of heterozygosity

KW - silencing

KW - suicide gene

UR - http://www.scopus.com/inward/record.url?scp=85132344637&partnerID=8YFLogxK

U2 - 10.1016/j.omtm.2022.05.011

DO - 10.1016/j.omtm.2022.05.011

M3 - Article

AN - SCOPUS:85132344637

VL - 26

SP - 84

EP - 94

JO - Molecular Therapy - Methods and Clinical Development

JF - Molecular Therapy - Methods and Clinical Development

SN - 2329-0501

ER -