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\begin{document}

\title{A new T-antigen negative HEK293 cell line with improved AAV productivity}



\author[1]{Paco Pino}%
\author[1]{Coralie Croissant}%
\author[1]{Joshua Armitano}%
\author[1]{Bertrand Lazuech}%
\author[1]{Danijel Švec}%
\author[1]{Cyril Pugin}%
\author[1]{Anaïs Guesdon}%
\author[1]{Louise Bryan}%
\author[1]{Antonio Castro}%
\author[1]{Léa Neuhaus}%
\author[1]{Giulia Fonti}%
\author[1]{Jacopo Martinis}%
\author[1]{Maria Wurm}%
\author[1]{Florian M. Wurm}%
\affil[1]{ExcellGene SA}%


\vspace{-1em}



  
  \date{May 26, 2024}


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\selectlanguage{english}
\begin{abstract}
Viral vectors for gene therapy, such as recombinant Adeno-Associated
Viruses (rAAV), are produced in Human Embryonic Kidney (HEK) 293 cells.
However, the presence of the SV40 T-antigen-encoding CDS \emph{SV40GP6}
and \emph{SV40GP7} in the HEK293T genome raises safety issues when these
cells are used in manufacturing for clinical purposes. We developed a
new T-antigen-negative HEK cell line from ExcellGene's proprietary
HEKExpress\selectlanguage{ngerman}®, using the CRISPR-Cas9 strategy. We obtained a high number
of clonally-derived cell populations and all of them were demonstrated
T-antigen negative. Stability study and AAV production evaluation showed
that the deletion of the T-antigen-encoding locus did not impact neither
cell growth nor viability nor productivity. The resulting CMC-compliant
cell line, named HEKzeroT®, is able to produce high AAV titers, from
small to large scale.%
\end{abstract}\selectlanguage{ngerman}%



\sloppy


\textbf{A new T-antigen negative HEK293 cell line with improved AAV
productivity}

Coralie Croissant, Joshua Armitano, Bertrand Lazuech, Danijel \selectlanguage{polish}Š\selectlanguage{english}vec,
Cyril Pugin, Ana\selectlanguage{ngerman}ïs Guesdon, Louise Bryan, Antonio Castro, Léa Neuhaus,
Giulia Fonti, Jacopo Martinis, Maria J. Wurm, Florian M. Wurm, Paco Pino

ExcellGene SA, Route de l'Ile-Au-Bois 1A, CH-1870 Monthey, Switzerland

Corresponding author:\emph{paco.pino@excellgene.com}, +41 24 471 96 60

Running Title: \textbf{A T-antigen negative cell line for AAV
manufacturing}

\textbf{Abstract}

Viral vectors for gene therapy, such as recombinant Adeno-Associated
Viruses (rAAV), are produced in Human Embryonic Kidney (HEK) 293 cells.
However, the presence of the SV40 T-antigen-encoding CDS
\emph{SV40GP6}and \emph{SV40GP7} in the HEK293T genome raises safety
issues when these cells are used in manufacturing for clinical purposes.
We developed a new T-antigen-negative HEK cell line from ExcellGene's
proprietary HEKExpress®, using the CRISPR-Cas9 strategy. We obtained a
high number of clonally-derived cell populations and all of them were
demonstrated T-antigen negative. Stability study and AAV production
evaluation showed that the deletion of the T-antigen-encoding locus did
not impact neither cell growth nor viability nor productivity. The
resulting CMC-compliant cell line, named HEKzeroT®, is able to produce
high AAV titers, from small to large scale.

\textbf{Keywords}

AAV; Gene therapy; HEK293; Manufacturing; T-antigen

\textbf{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

\textbf{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
of\emph{E1A} and \emph{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
sequences\emph{SV40GP6} and \emph{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.

\textbf{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
0.3-0.6×10\textsuperscript{6} cells/mL in chemically-defined medium
supplemented with GlutaMax\selectlanguage{english} 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, 37degC and
8\% CO\textsubscript{2}.

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(r), Solentim, Wimborne, United Kingdom). Obtained
clones were expanded, transferred to TPP(r) 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(r) 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 \emph{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\selectlanguage{english} Universal SYBR(r) Green Supermix (BioRad, Hercules,
California, USA) in two steps. After an initial denaturation at 95degC for
5 min, 39 cycles of denaturation (95degC, 15 sec) and annealing-extension
(60degC, 30 sec) were performed. The PCR products from duplex-qPCR were
diluted in 6x Gel Loading Purple Dye (New England BioLabs, Ipswich,
Massachusetts, USA) and 5 or 10 \selectlanguage{greek}µ\selectlanguage{english}L 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\selectlanguage{ngerman}® 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 \emph{rep}
and\emph{cap} 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 \selectlanguage{greek}µ\selectlanguage{english}M and the probe at a final
concentration of 4 \selectlanguage{greek}µ\selectlanguage{english}M.

Stability study

Cell populations were thawed from RCB and passaged every 3-4 days at a
cell density of 0.3-0.6\selectlanguage{ngerman}×10\textsuperscript{6} 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 \emph{CAV3} , \emph{RAD18} and the exons
1 and 2 of \emph{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 \emph{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 \emph{SV40GP6} , and another
targeting \emph{ß-ACTIN} . The PCR products were subjected to
agarose-electrophoresis (Figure 3A). The amplification from
the\emph{SV40GP6} and \emph{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 \emph{ß-ACTIN} amplification product was
present in the profile of both HEKExpress® and clones, showing that the
PCR reactions occurred and the \emph{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 10-15.10\textsuperscript{6} 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 \emph{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 6×10\textsuperscript{11} viral capsids/mL and
8×10\textsuperscript{10} 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 1×10\textsuperscript{11} to 1×10\textsuperscript{8} viral
capsids/mL, and from 1×10\textsuperscript{10} to
1×10\textsuperscript{8}viral genome copies/mL. It is of importance to
note that a copy number of 10\textsuperscript{8} 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 1×10\textsuperscript{12}copies/mL and 3×10\textsuperscript{11}
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 2×10\textsuperscript{6} 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
5.5×10\textsuperscript{6} and 6.4×10\textsuperscript{6} 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.

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\textbf{Figure 1: Genotypic characterization of HEKExpress® cells} . (A)
TLA sequence coverage across HEKExpress® genome using the primers
targeting the T-antigen coding sequence (set 1). The plot show that the
integration site of the plasmid is located on the chromosome 3
equivalent (chr3). (B) TLA sequence coverage across the integration
locus in human chr3 using the primers targeting the T-antigen coding
sequence (set 1) or chr3 (set 3 and 4). The coverage of set 1 shows a
deletion of 550 kb in HEKExpress® genome (green arrows) compared to the
human hg38 genome. The coverage of sets 3 and 4 confirm the junctions of
the integrated plamid pRTAK. (C) Map of pRTAK plasmid originally
integrated in HEKExpress® cell line. The large and small T-antigen
sequences are indicated in dark purple and gRNAs (gRNA\_beginning and
gRNA\_end) targeting the gene in orange. (D) Map of chr3 equivalent (in
red) with the 550 kb deletion and the integration of the pRTAK plasmid
containing the T-antigen sequence. Plasmid-chromosome junctions
confirmed by TLA are indicated in blue.

\textbf{Figure 2: CMC compliant cloning of HEKEpress® cells after
CRISPR/Cas9.} (A) Images from the single-cell dispenser showing a single
cell to dispense. The first four images show the cell coming in the ROI
(blue circle). The last image is taken after the dispensing and show
that the cell was successfully dispensed. Scale bar: 50 \selectlanguage{greek}µ\selectlanguage{english}m. (B) Scan of
a 96-well plate at day 13 post-seeding. Wells surrounded in green
indicate monoclonally-derived cell populations ready to be transferred
for expansion and represent approximatively 21\% of the total printed
cells. (C) Images from the clone imager device taken at D0 (day 0), D1,
D2 and D13 after single-cell cloning. The image at D0 confirms the
dispensing of a single cell, that divides into two cells at D1 until
forming a single-cell derived colony at D13. Scale bar: 200 \selectlanguage{greek}µ\selectlanguage{english}m.

\textbf{Figure 3: Analysis of T-antigen removal on clonally derived cell
populations} . (A) A duplex-qPCR was performed on genomic DNA extracts,
using two sets of primers targeting the T-antigen and the \selectlanguage{ngerman}ß-ACTIN coding
sequences. PCR products (5 or 10 \selectlanguage{greek}µ\selectlanguage{english}L) were then electrophoretically
separated on agarose gel. The PCR products sizes from the T-antigen and
\selectlanguage{ngerman}ß-ACTIN targeting primers pairs are 107 bp and 141 bp, respectively. (B)
Summary of single-cell cloning and KO colonies.

\textbf{Figure 4: AAV productivity of T-antigen-negative clonally
derived cell populations} . Triple transfection was performed on cells
and AAVs were harvested 72h later. Viral capsid (A) and genome (B)
titers were determined by ELISA and dPCR, respectively. Data were
normalized with titers from HEKExpress® as reference. Error bars
represent means ± standard deviation of two technical replicates.

\textbf{Figure 5: T-antigen negative cell performance and stability} .
Cells were maintained in culture for two months, with regular banking
every other week. ``PXX'' represents the passage number of the cell
culture after a bank was generated. Viable Cell Densities (VCD) and
viabilities were measured (A) and doubling times calculated (B). The
data corresponding to clone \#8 in (B) are representative of other top
clones. (C-D) One vial of each bank was thawed and AAV8 virions were
produced as described in Figure 4. Viral capsid (C) and genome (D)
titers were determined by ELISA and dPCR, respectively. Error bars
represent means ± standard deviation of two technical replicates.

\textbf{Figure 6: T-antigen negative cell performance in different scale
bioreactors} . Clone \#8 was seeded in different working volumes from 10
mL to 1 L and transfected for AAV9 production as described in Figure 4.
(A) Viable Cell Density (VCD) and viability were assessed every day. (B)
Viral capsid and genome titers were determined by ELISA and dPCR,
respectively.\selectlanguage{english}
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