Author contribution
Acquisition and analysis of data: CC, JA, BL, DS, CP, AG, LB, AC, LN,
JF, JM
Study design, interpretation of data and manuscript drafting: CC, JA,
MJW, FMW, PP
The data that support the findings of this study are available from the
corresponding author upon reasonable request
Introduction
Recombinant Adeno-Associated Viruses (rAAV) have several advantages that
make them vectors of choice for the development of gene therapy. They
are non-pathogenic in humans, are able to infect dividing as well as
non-dividing cells, and different serotypes can target different
specific organs/cells types (Sha et al., 2021; Wang et al., 2019).
Today, the most common way to produce rAAV is by transient transfection
of HEK (Human Embryonic Kidney) 293 cells (Blessing et al., 2019; Sha et
al., 2021; Strobel et al., 2019). In the 1970s, the HEK293 cell line was
generated by transforming human primary embryonic kidney cells with
viral DNA fragments from Adenovirus 5, resulting in the integration ofE1A and E1B sequences into chromosome 19 (Graham et al.,
1977; Lin et al., 2014; Louis et al., 1997). Ten years later, HEK293
were stably transfected with a plasmid carrying the coding sequencesSV40GP6 and SV40GP7 encoding for the Simian Virus (SV) 40
Large and Small T-antigens, respectively (DuBridge et al., 1987; Heinzel
et al., 1988; Rio et al., 1985). The resulting new cell line, known as
HEK293T, is commonly used in research and manufacturing. However, as
reported in several studies, SV40 T-antigen and SV40-transformed cells
are able to induce tumors in hamsters and mice (Eddy et al., 1962;
Girardi et al., 1962; Reddel et al., 1993; Vilchez et al., 2003), which
raises safety issue concerns when the production from HEK293T is
intended for clinical use.
ExcellGene’s proprietary suspension-cultured HEKExpress® cell line in
animal component-free media is derived from HEK293T cells. In order to
reduce the previously-cited risks and provide safer rAAV of high
quality, the T-antigen coding sequence was deleted from HEKExpress®
genome, using the CRISPR-Cas9 strategy as previously described (Bae et
al., 2020). Optimized transfection and cloning protocols lead to a fast,
reliable, and CMC-compliant generation of a high number of
clonally-derived cell populations. T-antigen screening revealed that
100% of the tested clones were T-antigen negative. Cell phenotypic
parameters such as growth, viability, transfectability as well as AAV
productivity were not affected by the deletion. On the contrary, AAV
titers were even higher in some T-antigen-deleted clones than in the
parental HEKExpress®. The resulting HEKzeroT® cell line, also maintained
under suspension growth conditions in animal component-free media,
sustains high growth rates and viabilities and generates high AAV titers
from the mL-scale to the 10s of liters.
Materials & Methods
Plasmid design
The gRNAs targeting the SV40 T-antigen coding sequence were synthetized
and cloned into the pD1401-AD plasmid by ATUM (Menlo Park, CA, USA). The
gRNA sequences are as follow: gRNA_5’ (5’-TATGCTCATCAACCTGACTT-3’) and
gRNA_3’ (5’-CAGCCATATCACATTTGTAG-3’), targeting the beginning and the
end of the T-antigen coding sequence, respectively.
Cell culture
The proprietary cell line HEKExpress® (ExcellGene, Monthey, Switzerland)
was used to generate, develop, and establish the clonally-derived
HEK293T deleted for the T-antigen coding sequence.
Cells were grown in suspension at a cell density of
0.3-0.6×106 cells/mL in chemically-defined medium
supplemented with GlutaMax™ from Gibco (Thermo Fisher Scientific,
Waltham, Massachussets, USA) and shaken in a humidified ISF-1-W orbital
shake incubator (Kuhner, Birsfelden, Switzerland) at 140 rpm, 37°C and
8% CO2.
Single-cell cloning
Clonal cell lines were obtained by image-assisted cell distribution into
single wells of 96-well plates (f.sight, Cytena GmbH, Freiburg,
Germany). Wells containing single cells were identified using the QC
images of single-cell dispenser device and image-based clonality
assessment (Cell Metric®, Solentim, Wimborne, United Kingdom). Obtained
clones were expanded, transferred to TPP® TubeSpin 50 bioreactor tubes,
and frozen in mini-banks.
Generation of T-antigen-negative clonal populations
Optimized ExcellGene transfection and cloning media were used for the
transfection and the culture of HEKExpress® cells. The transfection of
cells was done in animal component-free medium using the vector
pD1401-AD containing the gRNA sequences. Two days post-transfection,
single-cell cloning was performed. Once clonal growth led to a
well-established population, cells were transferred to suspension
culture and further expanded.
Clone screening
DNA of clones was extracted using the DNeasy Blood & Tissue Kit
(Qiagen, Hilden, Germany) according to manufacturer’s instructions.
Clone screening was performed by duplex-qPCR (using two sets of
primers). One primer set targets the large T antigen coding sequence
(Fwd, 5’-TAAAGCATTGCCTGGAACGC-3’, Rev, 5’-AAACTCAGCCACAGGTCTGTAC-3’) and
was used at a final concentration of 500 nM for each primer. The second
primer set targets the human ACTB (ß-Actin) coding sequence (Fwd,
5’-AACACGGCATTGTCACCAAC, Rev, 5’-TCTTTTCACGGTTGGCCTTG-3’) and was used
at a final concentration of 100 nM for each primer. PCR reactions were
performed using iTaq™ Universal SYBR® Green Supermix (BioRad, Hercules,
California, USA) in two steps. After an initial denaturation at 95°C for
5 min, 39 cycles of denaturation (95°C, 15 sec) and annealing-extension
(60°C, 30 sec) were performed. The PCR products from duplex-qPCR were
diluted in 6× Gel Loading Purple Dye (New England BioLabs, Ipswich,
Massachusetts, USA) and 5 or 10 µL were electrophoretically separated on
2% agarose gel pre-stained with SYBR™ Safe DNA Gel Stain (Thermo Fisher
Scientific, Waltham, Massachussets, USA).
TLA analysis
TLA sequencing (de Vree et al., 2014) was performed by Cergentis
(Utrecht, Netherlands) using three sets of primers. Reads were aligned
using the human HG38 genome as host reference genome sequence.
AAV production
Triple transfections were performed on clonal populations for AAV
production in different scale according to ExcellGene’s procedures and
reagents: 10 mL-scale in bioreactor tubes; 25 mL- and 250 mL-scales in
shake flasks, and 1 L-scale in Mobius® 3L Single-use Bioreactor. Cells
were co-transfected with a pHelper plasmid containing AAV helper genes
pAAV-GFP (containing the GFP coding sequence flanked by AAV ITRs) and
pRepCap8 or pRepCap9 (containing the AAV8 or AAV9 rep andcap genes, respectively). Cell cultures were harvested 72 h
later, lysed with NP-40 (Merck KGaA, Darmstadt, Germany) and treated
with benzonase (Merck KGaA, Darmstadt, Germany) for 1 h at 37°C. After
addition of EDTA, samples were cleared either by centrifugation or
filtration and supernatants were analyzed by AAV8 or AAV9 Xpress ELISA
(both by Progen, Heidelberg, Germany) and dPCR (QIAcuity by Qiagen,
Hilden, Germany) to determine AAV capsid titers and AAV genome titers,
respectively. The primers and the probe used for dPCR target the GFP
coding sequence (Fwd, 5’-TGCAAAGACCCCAACGAGAA-3’, Rev,
5’-GGCGGCGGTCACGAA-3’, probe, 5’-CGCGATCACATGGTCCTGCTGG-3’). Primers
were used at a final concentration of 8 µM and the probe at a final
concentration of 4 µM.
Stability study
Cell populations were thawed from RCB and passaged every 3-4 days at a
cell density of 0.3-0.6×106 cells/mL. For two months,
cells were maintained in culture and banked every fourth passages (every
other week) as SS-RCBs (Stability Study-Research Cell Bank).
One vial per condition and SS-RCB were thawed, together with one vial
from the initial RCB as a starting point. After recovery, cells were
transfected and AAV8 titers were determined as described in the previous
section.
Results and Discussion
HEKExpress® genotypic characterization
To characterize the integration sites of the pRTAK plasmid carrying the
T-antigen coding sequence originally integrated in the HEKExpress® cell
line, Targeted Locus Amplification (TLA) sequencing was performed. A
single integration site of the pRTAK plasmid was found in the chromosome
(chr) 3 equivalent, between positions 8,590,507 and 9,140,859 of the
HG38 human reference genome sequence (Figure 1A-B), with a copy number
estimated to be between 1 and 4. In addition, a genomic deletion of
approximately 550 kb was detected between the identified junctions,
represented by the green arrows in Figure 1B. According to the RefSeq,
this region contained the genes CAV3 , RAD18 and the exons
1 and 2 of SRGAP3 . The Figure 1D represents the chr 3 in red with
this deletion and the integration of the plasmid carrying the T-antigen
sequence, with the confirmed junctions in blue. These observations are
consistent with a previous study where the authors characterized their
HEK293T cell line (Bae et al., 2020).
Generation of T-antigen-negative clonal populations
The CRISPR/Cas9 system was used to mediate T-antigen deletion in
HEKExpress®. The design of gRNA sequences targeting the beginning and
the end of the T-antigen coding sequence (Figure 1C) was based on the
work of Bae et al. (2020). In this study, the authors co-transfected the
cells with the plasmid carrying the CAS9 and gRNA sequences and
cloned them by limiting dilution. Here, we improved the strategy using
ExcellGene’s (proprietary) transfection and cloning media and an
automating single-cell dispenser. As shown in Figure 2A, this device is
able to recognize a single cell in the Region Of Interest (ROI) and
dispense it into 96-well plates, together with quality control images to
prove the monoclonality of the colonies. Two days after transfection of
the plasmid encoding for both Cas9 and the gRNA sequences, 1,823 single
cells were dispensed (Figure 2B-C). Approximately 21% of the cells were
able to form a monoclonally-derived cell population in 96-well plates,
and 11.7% were expanded until banking (Figure 3B). The Figure 2B shows
a single cell dispensed at D0 (day of single-cell cloning) and dividing
until forming a colony at D13. From pool generation to clonal population
banking, the whole procedure was achieved within a month. Compared to
the limiting dilution method that is traditionally used for single-cell
isolation in numerous studies (Bae et al., 2020; Levin et al., 2020;
Spidel et al., 2016; Thorne et al., 2009; Yuan et al., 2011), our
procedure does not only enable the high-throughput isolation of cells in
a short period of time, but also provides the required traceability to
work in a CMC-compliant environment.
Analysis of T-antigen-negative clonal populations
Several approaches were used to assess the T-antigen-encoding CDS
deletion in clonal populations. First, a simple qPCR was performed. All
tested clonal populations were lacking the T-antigen sequence (data not
shown). To confirm that the absence of PCR product is due to the
deletion, a duplex-qPCR was performed using two sets of primers, one
targeting the T-antigen coding sequence SV40GP6 , and another
targeting ß-ACTIN . The PCR products were subjected to
agarose-electrophoresis (Figure 3A). The amplification from theSV40GP6 and ß-ACTIN coding sequences should lead to the
presence of a 107 bp fragment and a 141 bp fragment, respectively. As
illustrated in Figure 3A, the ß-ACTIN amplification product was
present in the profile of both HEKExpress® and clones, showing that the
PCR reactions occurred and the ß-ACTIN sequence was, as expected,
present in the DNA extracted from the tested cells. On the contrary, the
T-antigen product was present in HEKExpress® parental cells but not in
the tested clones, demonstrating that the T-antigen coding sequence is
absent in the clones. Altogether, our results showed that the locus
encoding for the Small and Large T antigens was successfully deleted in
100% of the screened clones (Figure 3B), while this percentage reached
only 33% in the work of Bae et al. (Bae et al., 2020). Our strategy
combining improved transfection and cloning protocols thus leads to a
faster yet reliable workflow for monoclonally-derived cell line
generation.
AAV productivity screening
AAV8 productivity was then assessed to screen the clonal populations.
Cells were transfected with the triple-transfection method and harvested
72 h post-transfection. The HEKExpress® cell line was also included as a
reference. As shown in Figure 4, AAV8 titers varied from a clonal
population to another, from 67% to 254% for capsid titers and from
13% to 203% in genome titers, when compared to titers in HEKExpress®.
This suggests that the T-antigen deletion itself does not impact AAV
production and that highly productive clones have been isolated. Based
on this data and on other parameters (transfection efficiency,
productivity and full/empty capsid ratio, data not shown), five top
clones were selected for further analysis: #6, #8, #23, #38 and
#114.
Stability study
The five top clones were evaluated during a stability study (SS) in
which cell parameters and AAV production were assessed. Along with
HEKExpress® as reference, cells were maintained in culture for two
months with regular generation of SS-banks every other week. HEKExpress®
as well as the clonal populations showed a constant high viability and
high VCD up to 10-15.106 cells/mL (Figure 5A).
Furthermore, the doubling time was stable and similar between the
parental cell line and the clones, with an average of 20 h (Figure 5B).
This evidence demonstrates that the T-antigen deletion does not affect
cell growth, in contrast with previous observations by Bae et al.(2020) who noticed that their T-antigen clones were growing slower,
probably due to other, unidentified factors. One vial of each SS-bank
was then thawed to perform triple transfection for AAV8 production. From
P00 (passage 00 corresponding to the initial bank) to P16 (passage 16,
two months later), AAV8 produced in HEKExpress® had similar titers, with
approximately 6×1011 viral capsids/mL and
8×1010 viral genomes copies/mL, showing that AAV
production is stable even after 50-60 generations (Figure 5C-D). On the
contrary, clones #6 and #38 presented variations in titers after
several passages. AAV production in clone #6 decreased with the time,
from 1×1011 to 1×108 viral
capsids/mL, and from 1×1010 to 1×108viral genome copies/mL. It is of importance to note that a copy number
of 108 is the limit of detection for both ELISA and
dPCR, meaning that the lowest measured titers are not accurate and might
be overestimated. Clone #38 was unable to produce AAV at P00 and titers
at other passages show variation of one to two logs. These data indicate
that these clones are not stable for AAV production and were therefore
not chosen for further investigation. Clones #8, #23 and #114
behaviours are similar to the parental line HEKExpress®: stable
production of viral capsids and genome up to 1×1012copies/mL and 3×1011 copies/mL, respectively. These
AAV titers, observed in HEKExpress® and T-antigen negative cells, are in
line with titers obtained with other HEK293 cell lines in the literature
(Chahal et al., 2014; Grieger et al., 2016; Vandenberghe et al., 2010;
Zhao et al., 2020).
Taking into account the data obtained from AAV production screening and
stability study, the clone #8 was chosen to go on with a scale-up
process.
Scalability study
Scalability of AAV production in top clone #8 was then assessed. Cells
were seeded at 2×106 cells/mL and transfected for AAV9
production in 10 mL-, 25 mL-, 250 mL- and 1 L-scale, respectively. Cell
parameters were measured every day and harvest was performed at day 3
post-transfection. AAV9 capsid and genome titers were determined by
ELISA and dPCR. As shown in Figure 6A, cell parameters are very similar
between the different conditions, with a maximum VCD at D3 between
5.5×106 and 6.4×106 cells/mL and
viability above 90%. When grown in 1 L STR (Stirred Tank Reactor), VCD
and viability are slightly lower than in smaller scales (e.g., a
viability of 83% was observed at D3). Interestingly, this small
difference does not have an impact on AAV9 production, since no apparent
difference was observed for capsid as well as for genome titers from 10
mL- to 1 L-scale production (Figure 6B). Thus, clone #8 is able to
produce high AAV titers in a stable way at different scales and was
chosen for the establishment of the HEKzeroT® cell line.
Conclusions
T-antigen negative monoclonally-derived cell populations were
successfully and quickly generated thanks to optimized ExcellGene’s
proprietary transfection and cloning protocols and in a CMC-compliant
environment. The final clone chosen as HEKzeroT® showed higher AVV
titers than the parental cell line HEKExpress® without any disturbance
in cell growth, stability, and scalable AAV production. Altogether, this
makes HEKzeroT® cell line a preferred choice for the safe and
high-throughput manufacturing of rAAV aimed at the development of new
gene therapies.