Details
| Original language | English |
|---|---|
| Pages (from-to) | 84-94 |
| Number of pages | 11 |
| Journal | Molecular Therapy - Methods and Clinical Development |
| Volume | 26 |
| Early online date | 31 May 2022 |
| Publication status | Published - 8 Sept 2022 |
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
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- Molecular Medicine
- Biochemistry, Genetics and Molecular Biology(all)
- Molecular Biology
- Biochemistry, Genetics and Molecular Biology(all)
- Genetics
Sustainable Development Goals
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In: Molecular Therapy - Methods and Clinical Development, Vol. 26, 08.09.2022, p. 84-94.
Research output: Contribution to journal › Article › Research › peer review
}
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 -