1
Targeted biallelic integration of an inducible Caspase 9 suicide gene
for safer cellular therapies prevents development of drug-resistant
escapees in human iPSCs
Stephanie Wunderlich
1,2
; Alexandra Haase
1,2
; Sylvia Merkert
1,2
; Kirsten Jahn
3
, Maximillian
Deest
3
, Silke Glage
4,2
; Wilhelm Korte
1,2
; Andreas Martens
1,2
; Andreas Kirschning
5,2
; Andre
Zeug
6,2
; Evgeni Ponimaskin
6,2
; Gudrun Goehring
7,2
; Mania Ackermann
8,2
, Nico Lachmann
8,2
;
Thomas Moritz
9,10,2
; Robert Zweigerdt
1,2
; Ulrich Martin
1,2
1
Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO),
Department of Cardiothoracic, Transplantation and Vascular Surgery, Hannover Medical
School, Hannover, Germany;
2
REBIRTH - Research Center for Translational Regenerative Medicine;
3
Department of Psychiatry, Social Psychiatry, and Psychotherapy, Hannover Medical School,
Hannover, Germany;
4
Institute for Laboratory Animal Science, Hannover Medical School, Hannover, Germany;
5
Institute for Organic Chemistry, Leibniz University Hannover, Hannover, Germany;
56
Department of Cellular Neurophysiology; Hannover Medical School, Hannover, Germany;
7
Department of Human Genetics, Hannover Medical School, Hannover, Germany;
8
Department of Pediatric Pneumology, Allergology and Neonatology, Hannover Medical
School, Hannover, Germany;
9
RG Reprogramming and Gene Therapy, Hannover Medical School, Hannover, Germany;
10
Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany;
11
JRG Translational Hematology of Congenital Diseases, Hannover Medical School,
Hannover, Germany;
*Corresponding author: Prof. Dr. Ulrich Martin
Carl-Neuberg-Straße 1
30625 Hannover, Germany
Martin.ulrich@mh-hannover.de
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2
Abstract
Drug-inducible suicide systems may help to minimize risks of cellular therapies due to the
tumor forming potential of human induced pluripotent stem cells (hiPSCs). Recent research
challenged the usefulness of such systems since rare drug-resistant subclones were observed
that showed elimination or silencing of the transgene.
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. In mice,
drug treatment generally led to rapid elimination of teratomas, but individual 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
.
Transgene elimination, presumably via Loss of Heterozygosity (LoH), or methylation of the
CAG promoter but not methylation of the
PPP1R12C locus were identified as underlying
mechanisms. In contrast, we never observed any escapees from biallelic iCASP9 hiPSCs,
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 cell number in tumors of a size that is readily detectable
during routine screening procedures.
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3
Introduction
Pluripotent stem cell (PSC) technologies come out of age and a number of clinical trials
applying embryonic stem cell (ESC) or induced pluripotent stem cell (iPSC)-based cell
products are ongoing or in preparation. The tumorigenic potential of PSC-derived cell products,
however, either in terms of contaminating undifferentiated cells that form teratoma or due to
malignant transformation caused by mutations acquired and enriched during reprogramming
1
or culture expansion
2
is considered as a major safety concern.
While improved protocols for targeted differentiation of PSCs into the therapeutic derivatives
of interest substantially reduced the risk for teratoma formation, introduction of synthetic fail-
safe systems with drug-inducible suicide genes have been proposed to further decrease
tumour risks.
Such fail-safe systems include the inducible expression of the herpes simplex virus-thymidine
kinase (HSV-TK), which has been clinically applied already more than 25 years ago
3
, and the
inducible Caspase9 (iCASP9) safety switch system
4
that has been clinically applied more
recently in T cells using a retroviral approach
5
.
In a first study that attempted to quantitatively define the safety level of PSC transplants with
integrated fail-safe systems, ESCs carrying an HSV-TK system controlled by one allele of the
endogenous CDK1 locus were applied
6
. Despite general functionality of the HSV-TK suicide
system concerning elimination of cycling ESCs, rare proliferating subclones of mouse ESCs
were observed in vivo and in vitro with an average frequency of 6.6x10
-8
that became resistant
to ganciclovir. Remarkably, not only transgene silencing but also loss of heterozygosity (LoH)
was identified as underlying mechanism for failure of the integrated drug-inducible suicide
system in mouse ESCs. Finally, the authors demonstrated that in case of ESCs with biallelic
(homozygous) integration of HSV-TK in the CDK1 locus, no resistant clones appeared among
a total of 1.2x10
8
cells pointing to a substantially higher safety level.
One potential disadvantage of the applied HSV-TK system is that HSV-TK as viral transgene
may be immunologically recognized in vivo leading to killing of HSV-TK expressing cells even
without application of ganciclovir. It should be stressed that placing a HSV-TK transgene under
control of a cell cycle-dependent locus such as CDK1 (cyclin-dependent kinase 1)
6
, indeed
restricts the targeted elimination to cycling cells such as tumour cells. This concept, though, is
probably not applicable if the iPSC-derived graft has to proliferate in order to fulfil its therapeutic
function, because all cycling cells that express the HSV-TK gene may become target of the
host’s immune response. In addition, the HSV-TK fail-safe system is especially effective in
fast-dividing cells and may fail to eliminate populations of slowly dividing tumour cells. Also,
the use of the prodrug ganciclovir as potent antiviral drug e.g. for treatment of serious herpes
virus infections may not be possible in affected patients.
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4
Because of these limitations and the lack of data for human iPSCs (hiPSCs), we aimed to
quantitatively define the safety level of hiPSC transplants with another integrated drug-
inducible suicide system, the iCASP9 safety switch. In this system, the Caspase activation and
recruitment domain of human Caspase 9, a gene expressed in fetal and adult tissue that is
upregulated during induction of apoptosis, has been replaced by a modified dimerizer-binding
domain of the FK506-binding protein 12
5
. This system requires caspase dimerization after
application of an otherwise bioinert small-molecule (Chemical Inducer of Dimerization, CID) for
activation of intracytoplasmic caspase-3 /7 and induction of apoptosis
4,7
(Figure 1A). Because
Caspase 9 is an endogenous gene, it is considered non-immunogenic.
In order to ensure safe application of the safety switch and efficient cell killing also in slowly
dividing hiPSC derivatives we have integrated the iCASP9 construct into the the
PPP1R12C /
AAVS1 (Adeno-Associated-Virus Site 1) safe harbour locus
8
. iCASP9 was placed under
control of a synthetic promoter consisting of the cytomegalovirus early enhancer element and
chicken beta-actin (CAG) promoter, known for robust expression in undiffentiated hiPSCs and
their differentiated progeny
9,10
(Figure 1B). In consideration of the results of Liang et al.
6
we
have generated hiPSC clones either heterozygous or homozygous for the integrated iCASP9
safety switch. Potential emergence of cell clones escaping the induced suicide was monitored
in vivo and in vitro among therapeutically relevant cell numbers.
Results
Generation of Human iPSC Lines with heterozygous or homozygous (mono- or biallelic)
integration of an iCASP9 suicide gene coupled to a Tomato fluorescent reporter. In order
to enable inducible induction of apoptosis, we applied transcription activator-like effector
nucleases (TALEN)-mediated gene editing to integrate an iCASP9 safety switch into the
AAVS1 safe harbor locus. iCASP9 gene was place under control of the CAG promoter. A
dTomato-fluorescent protein fused to a nuclear membrane localization signal (dTomato
nucmem
)
was utilized to facilitate indirect monitoring of iCASP9 expression and discrimination from
autofluorescence, and was coupled to the iCASP9 gene via a 2A site (Figure 1B). Allele-
specific PCR analysis confirmed site-specific integration into one or both alleles of the AAVS1
locus of Phoenix
11
and iCBPSC2
12
lines (Figure S1A&B). For all further procedures and
analyses, one correctly targeted monoallelic (heterozygous) and biallelic (homozygous) clone
per cell line was chosen. These clones showed considerable levels of dTomato
nucmem
expression (Figure 1C), absence of chromosomal aberrations as demonstrated by karyotyping
(Figure 1D) expressed typical pluripotency markers, differentiated into derivatives of all three
germ layers and formed teratoma upon injection into NODSCID mice (Figure S2). For clarity,
all selected iCASP9 cell lines that were applied thereafter are abbreviated according to their
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5
origin and genotype: monoallelic iCASP9 Phoenix, biallelic iCASP9 Phoenix, monoallelic
iCASP9 hCBiPS2, biallelic iCASP9 hCBiPS2.
Chemical inducers of dimerization (CIDs) efficiently induce apoptosis in monoallelic
and biallelic iCASP9 iPSC clones. Undifferentiated cells of monoallelic and biallelic iCASP9
Phoenix and iCASP9 hCBiPS2 clones cultivated as monolayer on geltrex were treated for 24h
with different concentration of two CID variants AP1903 and AP20187 before staining of viable
cells with Calcein staining. Intercalating DNA-binding live-dead dyes such as 7-AAD or
propidium iodide that were used in previous studies
10,13
were found not to be useful for this
purpose apparently because dead cells after DNA degradation are detected by flow cytometry
as false negative “viable” cells. We were never been able to confirm such seemingly viable
cells after reseeding in cell culture (data not shown). In contrast, Calcein provided much more
reliable results. The the non-fluorescent acetomethoxy derivate of calcein is transported
through the cellular membrane into live cells. Intracellular esterases that are lacking in dead
cells remove the acetomethoxy group, the molecule gets trapped inside and gives out strong
green fluorescence. Determination of the proportion of Calcein
pos
cells by FACS revealed very
efficient killing of cells of all four mono- / bi-allelic cell lines with both CID variants at
concentration ≥ 0,1nM (Figure 2).
In vivo CID treatment of preformed teratoma after injection of monoallelic iCASP9
hiPSCs eliminates human cells and teratoma in most but not all mice. In vivo experiments
in NODSCID mice were conducted to confirm the above results in vivo (Figure 3A). All mice
after injections of iPSCs under the kidney capsule showed massive increase of girth as typical
sign for teratoma formation after 8 weeks. Mice that received non transgenic Phoenix wildtype
cells and subsequent CID treatment, as well as mice after injection of monoallelic iCASP9
Phoenix iPSCs followed by vehicle-treatment all developed teratoma that stained positive for
human nuclear antigen (Figure 3B). In contrast, in three out of five mice injection of transgenic
monoallelic iCASP9 Phoenix iPSCs followed by treatment with CID resulted in almost complete
elimination of frequently cystic teratoma structures (Table 1 and Figure 3B). In these mice,
only some abnormal puffy mouse tissue with eosinophilic infiltration around the kidney could
be detected (upper middle panel), probably representing fibrotic mouse tissue that formed in
response to the massive CID-induced cell death and the resulting infiltration of phagocytes and
granulocytes followed by pro-fibrotic cytokine release. In the remaining two out of five mice
also extensive elimination of frequently cystic teratoma structures was observed. However, in
these mice also tumor like tissue was detected around the kidney that stained positive with an
anti-human nucleoli antibody, suggesting that despite killing of the vast majority of engrafted
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