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\begin{document}

\title{Increased recombinant AAV production by HEK293 cells using small
molecule chemical additives}



\author[1]{Joseph Scarrott}%
\author[1]{Yusuf B. Johari}%
\author[1]{Thilo Pohle}%
\author[2]{Ping Liu}%
\author[2]{Ayda Mayer}%
\author[1]{David James}%
\affil[1]{The University of Sheffield Department of Chemical and Biological Engineering}%
\affil[2]{REGENXBIO Inc}%


\vspace{-1em}



  
  \date{May 22, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Recombinant adeno-associated virus (rAAV) has established itself as a
highly efficacious gene delivery vector with a well characterised safety
profile allowing broad clinical application. Recent successes in
rAAV-mediated gene therapy clinical trials will continue to drive demand
for improved rAAV production processes to reduce costs. Here we
demonstrate that small molecule bioactive chemical additives can
significantly increase recombinant AAV vector production by HEK cells up
to 3-fold. Nocodazole (an anti-mitotic agent) and M344 (a selective
histone deacetylase inhibitor) were identified as positive regulators of
rAAV8 genome titre in a microplate screening assay. Addition of
nocodazole to triple-transfected HEK293 suspension cells producing rAAV
arrested cells in G2/M phase, increased average cell volume, and reduced
viable cell density relative to untreated rAAV producing cells at
harvest.. Final crude genome vector titre from nocodazole treated
cultures was \textgreater{}2-fold higher compared to non-treated
cultures.. Further investigation showed nocodazole addition to cultures
to be time critical.Genome titre improvement was found to be scalable
and serotype independent across two distinct rAAV serotypes, rAAV8 and
rAAV9. Furthermore, a combination of M344 and nocodazole produced a
positive additive effect on rAAV8 genome titre, resulting in a 3-fold
increase in genome titre compared to untreated cells.%
\end{abstract}%



\sloppy


\textbf{Increased recombinant AAV production by HEK293 cells using small
molecule chemical additives}

Joseph M. Scarrott\textsuperscript{1}, Yusuf B.
Johari\textsuperscript{1}, Thilo H. Pohle\textsuperscript{1}, Ping
Liu\textsuperscript{2}, Ayda Mayer\textsuperscript{2}, David C.
James\textsuperscript{1}*

1. Department of Chemical and Biological Engineering, University of
Sheffield, Mappin St., Sheffield S1 3JD, U.K.

2. Cell Line Development, REGENXBIO Inc., Rockville, MD 20850, U.S.A.

* to whom correspondence should be addressed.

Tel: +44 114 222 7505; E-mail: d.c.james@sheffield.ac.uk

\textbf{Abstract}

Recombinant adeno-associated virus (rAAV) has established itself as a
highly efficacious gene delivery vector with a well characterised safety
profile allowing broad clinical application. Recent successes in
rAAV-mediated gene therapy clinical trials will continue to drive demand
for improved rAAV production processes to reduce costs. Here we
demonstrate that small molecule bioactive chemical additives can
significantly increase recombinant AAV vector production by HEK cells up
to 3-fold.

Nocodazole (an anti-mitotic agent) and M344 (a selective histone
deacetylase inhibitor) were identified as positive regulators of rAAV8
genome titre in a microplate screening assay. Addition of nocodazole to
triple-transfected HEK293 suspension cells producing rAAV arrested cells
in G2/M phase, increased average cell volume, and reduced viable cell
density relative to untreated rAAV producing cells at harvest.. Final
crude genome vector titre from nocodazole treated cultures was
\textgreater{}2-fold higher compared to non-treated cultures.. Further
investigation showed nocodazole addition to cultures to be time
critical.Genome titre improvement was found to be scalable and serotype
independent across two distinct rAAV serotypes, rAAV8 and rAAV9.
Furthermore, a combination of M344 and nocodazole produced a positive
additive effect on rAAV8 genome titre, resulting in a 3-fold increase in
genome titre compared to untreated cells.



\textbf{Introduction}

Recent regulatory approvals for recombinant adeno-associated virus
(rAAV) mediated gene therapy products (Luxturna\selectlanguage{ngerman}® in 2017 and Zolgensma®
in 2019) - together with a significant increase in the number of rAAV
gene therapies in clinical trials\textsuperscript{{[}1{]},{[}2{]}} has
highlighted the need for improved production process technology with
respect to titre, product quality and cost. Indeed, the high cost of
manufacturing rAAV gene therapies to support the typically high
therapeutic dosages used (e.g. 6 x 10\textsuperscript{13} -- 2 x
10\textsuperscript{14} vector genomes/kg\textsuperscript{{[}3{]}}) has
limited the economic viability of AAV gene therapies despite evidence of
promising clinical efficacy. Therefore there is an urgent need for
robust, intensified process technology to support production of Good
Manufacturing Practice (GMP) quality material. As nearly all current
rAAV production processes rely on co-transfection of three plasmids
encoding the necessary AAV and helper genes into HEK293 cell hosts for
transient production of rAAV within 3-5 days\textsuperscript{{[}4{]}},
efficient use of plasmid DNA (itself produced using a costly
GMP-approved process) is also of paramount importance.

rAAV is a complex macromolecular product requiring a diverse network of
cellular processes and molecular interactions to enable coordinated
cellular synthesis - host-cell proteins, transiently expressed viral
helper, capsid and replicase genes as well as the single stranded
therapeutic viral DNA payload itself \textsuperscript{{[}5{]}--{[}7{]}}.
Small molecule enhancers of recombinant protein production have
demonstrated efficacy in a wide variety of mammalian cell
lines\textsuperscript{{[}3,10,11{]}} and the addition of chemicals as
diverse as sodium chloride, sodium butyrate, and soy peptones have been
shown in previous studies to improve rAAV production
yields\textsuperscript{{[}11{]}--{[}13{]}}. Targeting of discrete
pathways involved in the replication, packaging, and trafficking of
viral particles by bioactive small molecule cell culture additives
therefore offers a simple and cost-effective way of increasing viral
titre and reducing overall production costs.

Here we show that microplate plate-based screening of chemical additives
can be used to rapidly identify positive effectors of rAAV synthesis in
HEK cells and that significant improvements in viral genome titre can be
obtained by subsequent optimisation of dosing regimen. Furthermore, we
also demonstrate that a combination of two functionally distinct small
molecule enhancers may act additively, resulting in a 3-fold increase in
viral vector titre (both increased genome titre and increased capsid
titre) to substantially improve the efficient use of transfected plasmid
DNA.

\textbf{Materials and Methods}

\textbf{Cell culture}

Proprietary suspension adapted Human Embryonic Kidney 293 (HEK293) cells
were provided by REGENXBIO (Rockville, MD). Cells were cultured in a
serum free medium supplemented with L-glutamine (Thermo Scientific,
Waltham, MA). Cells were maintained in an orbital shaking incubator
(Infors, Bottmingen, Switzerland) at 30 mL culture volumes in 125 mL
Erlenmeyer flasks (Corning) at 37°C, 5\% CO\textsubscript{2} and 85\%
humidity, with agitation at 140 rpm. Smaller scale cultures were grown
in shallow-well 24-well plates (0.7 mL culture volume/well, 240 rpm
shaking) (Corning, Acton, MA) using the Deutz
system\textsuperscript{{[}14{]}}. Routine cell density and cell
viability measurements were performed on a ViCell automated cell counter
(Beckman-Coulter, Brea, CA). Measurements of density and viability and
determination of mean cell volume during rAAV production runs were
performed using the Norma HT system (Iprasense) utilising 3 \selectlanguage{greek}µ\selectlanguage{english}l total
cell culture and 20 \selectlanguage{greek}µ\selectlanguage{english}m slide chambers. Mean cell volume (\emph{V} ) was
calculated as \(V=\frac{4}{3}\pi r^{3}\) where \emph{r} = measured cell
diameter/2.

\textbf{rAAV vector production in suspension adapted HEK293 cells}

Proprietary ITR, Helper and Rep/Cap plasmid vectors used for rAAV8 and
rAAV9 production were supplied by REGENXBIO using GFP as a
reporter/transgene. Plasmids were transfected using polyethylenimine
(PEI) as a gene delivery vehicle at optimised PEI:DNA and DNA:cell
ratios. For rAAV production, plasmids and PEI were separately diluted in
a serum free medium before being combined and incubated at RT. After
incubation, the DNA:PEI polyplex transfection mixture was added to
cells. For analysis, 450 \selectlanguage{greek}µ\selectlanguage{english}L of total cell culture was added to 50 \selectlanguage{greek}µ\selectlanguage{english}L 10x
cell lysis buffer containing 1x cOmplete, EDTA-free Protease Inhibitor
Cocktail (Roche) and incubated for 1 h at 37 \selectlanguage{ngerman}°C with gentle agitation.
Samples were briefly centrifuged at 12,000 RPM to remove cell debris and
the resulting supernatant used to determine viral genome and capsid
titre.

\textbf{Measurement of rAAV genome titre}

Genome titre was quantified by digital droplet PCR (ddPCR). Aliquots of
post-lysis supernatant (5 \selectlanguage{greek}µ\selectlanguage{english}L) from total cell culture were treated with
DNAse I (Roche, Basel, Switzerland) to remove residual plasmid DNA.
DNAse I-treated samples were diluted 1000- or 10,000-fold in PCR
dilution buffer (GeneAmp PCR Buffer I (Thermo Scientific), 0.02\%
UltraPure Salmon Sperm DNA Solution (Invitrogen, Waltham, MA), 0.1\%
Pluronic F-68 non-ionic surfactant). Droplet formation and subsequent
post-PCR droplet analysis was performed using the QX200 system (Bio-Rad,
Hercules, CA), with absolute quantification of AAV genome copies/\selectlanguage{greek}µ\selectlanguage{english}l
determined using the Quantasoft analysis software (Bio-Rad). Genome
detection was achieved using primers and a FAM-labelled probe targeting
the PolyA sequence of the pAAV-CAG-GFP plasmid. Capsid titre
quantification was performed using the AAV8 titration ELISA (Progen,
Heidelberg, Germany) from total cell lysis supernatant diluted in 1x
ASSB assay buffer (Progen).

\textbf{Chemical additive screening in microplates}

Chemical additives for screening were diluted in either sterile water or
dimethyl sulfoxide (DMSO, 100\% v/v) where appropriate. Low, medium and
high concentrations for each chemical were based on available literature
describing their observed effects on recombinant protein
expression\emph{in vitro} (listed in Table S1). Cells were initially
grown to a density of 4 x 10\textsuperscript{6} viable cells/mL in
Erlenmeyer flasks and mid-exponential phase cells were transfected with
AAV-encoding plasmids prior to transferring to 24-shallow-well
microplates (Corning) at 2.8x10\textsuperscript{6} cells per well, 20
min post-transfection. Chemical additives were added to cells 24 hours
post-transfection (HPT). Cell cultures were harvested at 72 HPT for
viral genome titre analysis by ddPCR.

\textbf{HEK cell DNA content analysis by flow cytometry}

Cells (1 x 10\textsuperscript{6}) were harvested 72 HPT and fixed in
70\% (v/v) EtOH at 4\selectlanguage{ngerman}°C for 30 min, with gentle vortexing to prevent
clumping. EtOH was removed and the cells washed twice in 1x PBS. Fixed
cells were treated with 100 \selectlanguage{greek}µ\selectlanguage{english}l RNAse A (100 \selectlanguage{greek}µ\selectlanguage{english}g/mL in PBS; Qiagen) for 5
min at RT and the DNA stained with the subsequent addition of 400 \selectlanguage{greek}µ\selectlanguage{english}l
propidium iodide (PI) (50 \selectlanguage{greek}µ\selectlanguage{english}g/mL in PBS; Thermo Scientific) at room
temperature for a minimum of 30 min. PI-stained cells were analysed by
flow cytometry using an LSRII instrument (BD Biosciences). Gated single
cell populations were detected based on PI signal and the resulting
histograms analysed using FlowJo software to determine the relative
distribution of cells within the cell cycle (G1/S/G2-M) phases.

\textbf{Immunocytochemistry}

rAAV producing cells were harvested 24 hours after nocodazole addition.
1 x 10\textsuperscript{6} cells were spun down at 250xg for 5 min,
culture media was removed, and cells resuspended in 1 mL PBS.
Resuspended cells were incubated with no agitation for 30 min at RT and
allowed to adhere by gravity sedimentation to lysine-coated glass
coverslips in 24-well plates. After 30 min, PBS and unadhered cells were
removed by gentle aspiration. Adhered cells were fixed by 10min
incubation at RT with 0.5 mL 4\% (v/v) paraformaldehyde in PBS. Fixed
cells were washed with PBS before permeabilization with 0.5 mL 0.5\%
(v/v) Triton-X100 for 10 min. Permeabilised cells were blocked by 10\%
normal goat serum (Life Technologies, Carlsbad, CA; \#016201) in PBS for
30 min. Cells were then incubated with anti-Fibrillarin antibody (Abcam,
Cambridge, UK; ab5821, 0.1 \selectlanguage{greek}µ\selectlanguage{english}g/mL in blocking buffer) for 1 h at RT
before incubation with goat anti-Rabbit Alexa-594 conjugated secondary
antibody (Abcam; ab150080, 1:1000 dilution in blocking buffer) for 45
minutes at room temperature. Coverslips were transferred to a glass
slide and mounted with Fluoroshield mounting media containing DAPI
(Sigma-Aldrich; F6507) for visualisation of nuclei. Slides were imaged
using a Nikon Eclipse Ti fluorescent microscope and images adjusted for
brightness and contrast using ImageJ software.

\textbf{Results}

\textbf{Initial screening of chemical additives to increase AAV genome
titre}

To identify novel small molecule enhancers of rAAV production, a panel
of 15 small molecule culture additives was chosen based on either their
reported propensity to increase recombinant protein expression in
mammalian cell systems, or their reported positive effects on rAAV
transduction efficiency. Due to the diverse mechanisms involved in AAV
vector production, the panel featured chemicals that exert their
influence on recombinant protein expression via a broad range of
functional mechanisms. Specifically, we tested chemicals reported to be
``chemical chaperones'' (TUDCA, TMAO,
betaine)\textsuperscript{{[}15{]}--{[}17{]}}, cell cycle modulators
(nocodazole, BI-2536)\textsuperscript{{[}18{]},{[}19{]}}, caspase
inhibitors (z-VAD-fmk)\textsuperscript{{[}20{]}}, histone deacetylase
inhibitors (NaBu, VPA, M344,
apicidin)\textsuperscript{{[}10{]},{[}21{]}--{[}23{]}}, insulin-mimetics
(lithium chloride and zinc sulphate)\textsuperscript{{[}24{]}}, and
proteasome inhibitors (ONX0912, MLN9708,
MG132)\textsuperscript{{[}25{]}--{[}27{]}}.

To enable multi-parallel screening of small molecule additives, we
employed pre-optimized small-scale cultures (700 \selectlanguage{greek}μ\selectlanguage{english}L culture volume) in
24-well microplates that exhibited a similar rAAV production profile to
shake flask cultures (data not shown). Triple transfection was performed
using PEI in a serum-free medium, and chemical additives were introduced
to rAAV8-producing suspension HEK293 cells at 24 HPT. Each additive was
used at three different concentrations (Low, medium, high -- values
based on previous literature, see Table S1). Total cell culture was
harvested at 72 HPT, cells were chemically lysed and the rAAV8 genome
titre in the crude supernatant was measured by ddPCR. Crude genome titre
in cells containing chemical additives was compared to that of untreated
rAAV8 producing cells to determine changes to titre mediated by the
small molecules. Several chemicals displayed a clear
concentration-dependent impact on genome titre, both positively
(z-VAD-fmk) and negatively (LiCl, ONX0912) (Fig. 1A). Several compounds
with similar mechanistic properties showed a marked reduction in rAAV
titre at the tested concentrations -- most notably inhibitors of
proteasomal function (ONX0912, MLN9708, MG132) and the reported chemical
chaperones (TUDCA, TMAO, betaine). Notably, the screening showed that
high relative dose nocodazole (4 \selectlanguage{greek}µ\selectlanguage{english}M) added to culture 24 HPT resulted in
a 1.34-fold increase in rAAV8 titer compared to untreated control cells
(p = 0.62, n = 2), while M344 appeared to be effective at low dose (2.5
\selectlanguage{greek}µ\selectlanguage{english}M) with a 1.36-fold increase in rAAV8 titer (p = 0.64, n = 2). Of note,
nocodazole treatment increased mean cell volume at all three
concentrations, an attribute that appears to correlate positively with
measured genome titre (r\textsuperscript{2} = 0.79, p \textless{}
0.0001) (Fig. 1B) and has been shown in previous reports to be a major
cellular determinant of recombinant protein productivity in Chinese
hamster ovary (CHO) cells \textsuperscript{{[}28{]}--{[}30{]}}.
Nocodazole treated cells were found to be up to 27\% larger than control
cells. Nevertheless, this phenotypic variation was not observed with
M344, in which the increase in titer was not accompanied by an increase
in cell volume. It may therefore be inferred that these molecules
modulate rAAV expression via distinct mechanisms independent of each
other and that increased control of rAAV production may be achieved by
the application of specific combinations of small molecule effectors.

\textbf{Early addition of nocodazole enhances rAAV8 production in HEK293
cells}

A recent study showed that \emph{cap} gene expression, translation and
assembly of Cap proteins into empty AAV particles is a kinetically rapid
process\textsuperscript{{[}31{]}}. \textasciitilde{}80\% of capsids are
assembled within the first 24 HPT whereas Rep mediated replication of
rAAV genome from the \emph{cis} -ITR plasmid is slower, peaking after
capsid protein levels have plateaued due to the inhibitory action of Rep
binding to the packaging plasmid and inhibiting transcription or by
translational repression of \emph{cap} mRNA. As capsid secretion from
the nucleus into the cytoplasm is independent of genome loading,
pre-assembled empty capsids can be secreted from the site of rAAV genome
loading, thus depleting the pool of empty capsids available to newly
replicated rAAV genome \textsuperscript{{[}31{]},{[}32{]}}. Therefore,
we hypothesized that the effect of a given chemical effector may be
positive (or further enhanced), neutral, or negative with respect to the
timing of its deployment,

To further investigate the impact of nocodazole in enhancing rAAV
production, we deployed high dose nocodazole (4 \selectlanguage{greek}µ\selectlanguage{english}M) between 0 -- 48 HPT
(Figure 2). Addition of 4 \selectlanguage{greek}µ\selectlanguage{english}M nocodazole immediately (0 HPT) and 4 HPT
resulted in a considerable reduction in VCD at 72 HPT of 74\% and 64\%
respectively, compared to rAAV8 producing cells without nocodazole
(Ctrl) (Fig. 2A). Also apparent at early addition timepoints was a
slight reduction in cell viability (p \textgreater{} 0.05; Fig. 2B) and
a significantly higher mean cell volume compared to cultures not treated
with nocodazole (up to 23\%; Fig. 2C), consistent with previous work by
Tait et al., (2004) who observed increased cell size and decreased
viability in nocodazole treated CHO cells producing a recombinant
monoclonal antibody. Most strikingly, addition of nocodazole at 4 HPT
resulted in a 2.2-fold increase in rAAV8 genome titre at harvest
compared to control cells (Fig. 2D). Addition at the earlier timepoint
of 0 HPT was sub-optimal, with a 10\% reduction in rAAV8 titer compared
to the titer of cells treated at 4 HPT (Figure 2D). We therefore
conclude that early addition (\textasciitilde{}4 HPT) of nocodazole to
rAAV producing cells is an effective positive mediator of rAAV
production in a small-scale, transient expression, suspension HEK cell
system.

\textbf{Nocodazole improves rAAV genome titre in two different
serotypes}

To investigate the effect of nocodazole addition in a system more
representative of large scale rAAV production, culture volume was scaled
up from the initial microplate culture volume of 0.7 mL to 30 mL culture
volume in shake flasks. To rule out a serotype-dependent effect of
nocodazole addition, plasmids for both rAAV8 and rAAV9 serotypes were
separately transfected in HEK293 cells and cultured with and without the
addition of 4 \selectlanguage{greek}µ\selectlanguage{english}M nocodazole. rAAV genome titre was measured from 24 --
72 HPT. All measured parameters of nocodazole induced cell cycle arrest
(reduced VCD, decreased viability, and increased mean cell volume) and
increased rAAV genome titre were consistent with those seen at smaller
culture volumes (Fig. 3A \& B). Comparable to microplate-based cultures,
mean viral genome titre was increased by up to 2.5-fold in
nocodazole-treated cultures compared to untreated cultures (Fig. 3C).
Importantly there were no observed significant differences in the
above-described measurements between cultures producing rAAV8 or rAAV9,
suggesting a broad applicability for nocodazole addition in rAAV
manufacturing.

\textbf{rAAV producing cultures treated with nocodazole have an
increased proportion of cells in G2/M phase}

Nocodazole is an anti-mitotic agent, used as both a chemotherapeutic and
as a common agent of cell cycle
synchronisation\textsuperscript{{[}18{]},{[}33{]}--{[}35{]}}. Nocodazole
exerts its effect by reversibly inhibiting the polymerisation of
\selectlanguage{greek}β-\selectlanguage{english}tubulin, destabilising microtubules and preventing the formation of
mitotic spindles, thus arresting cells within the G2/M phase of the cell
cycle. Cells treated with nocodazole typically enter mitosis but are
unable to progress through cytokinesis, leading to either apoptosis in
cells remaining in arrested mitosis for an extended period of time, or
subsequent ``mitotic slippage'' into G0/G1 phase followed by
apoptosis\textsuperscript{{[}35{]}--{[}38{]}}. In addition to its use as
a cell synchronisation agent, nocodazole has previously been shown to
increase transient recombinant protein expression in a mammalian cell
system\textsuperscript{{[}39{]}}.

Flow cytometry was carried out to determine the cell cycle status of
rAAV8-producing cultures treated with nocodazole. Untreated rAAV8
producing cells were found to be almost entirely in either G1 or S phase
at 72 HPT (Fig. 4A). Cells treated with 4 \selectlanguage{greek}μ\selectlanguage{english}M nocodazole at the point of
transfection (0 HPT) resulted in a very significant proportion (38\%)
arresting in G2/M phase (Fig. 3B). The ratio of cells in G2/M : G1 phase
was found to decrease the later nocodazole was added (Fig. 4C). Of note
is the observation that a significant number (26\%) of cells remain
arrested in G2/M phase at 72 HPT when nocodazole is added as late as 24
HPT, yet the positive effect on rAAV8 genome titre is substantially
lessened compared to earlier treatment. This would suggest a critical
temporal component to the addition of nocodazole, such that later
additions to cell culture are sub-optimal in terms of producing high
rAAV titres, likely due to the rapid assembly process of cap proteins
that largely occurred within 24 HPT\textsuperscript{{[}31{]}}.

The nucleolus is a dynamic compartment within the nucleus which
undergoes extensive remodelling during cell cycle progression,
particularly during mitosis\textsuperscript{{[}40{]}}. Previous
immunocytochemical examination of nucleolar localisation throughout
normal cell cycle progression shows a distinctive dispersal of nucleolar
protein staining throughout the cytoplasm during prometaphase and
metaphase -- concomitant with the breakdown of the nuclear membrane
during mitosis \textsuperscript{{[}41{]}--{[}43{]}}. Nucleolar proteins
in cells in interphase and prophase are typically found located within
the nucleus itself. Of note, the nucleolus is considered to be a likely
site for AAV capsid assembly and Rep-mediated loading of AAV genome into
capsids \textsuperscript{{[}44{]}--{[}47{]}}, as well as being linked
more generally to viral replication in other human
viruses\textsuperscript{{[}48{]}}.

Cells from rAAV8 producing cultures - both with and without nocodazole
addition 4 HPT - were harvested and fixed at 24 HPT. Cells were stained
with a nucleolar marker (fibrillarin - a protein component of the
nucleolus associated with ribosomal RNA
processing\textsuperscript{{[}42{]}}), and DAPI (a nuclear DNA stain).
Widefield fluorescent microscopy was used to visualise the phenotypic
changes induced by nocodazole addition. Untreated cells displayed highly
localised or punctate staining of fibrillarin within the area of nuclear
DNA staining (Fig. 4D), indicative of cells in interphase or prophase.
In contrast, cells treated with nocodazole exhibited a proportion
displaying a disorganised nuclear phenotype - with fibrillarin
distributed broadly throughout the cytoplasm, and with condensed nuclear
DNA (Fig. 4E) --indicative of cells either progressing through mitosis
or arrested within G2/M phase.

\textbf{Small molecule additives can be combined to further enhance
rAAV8 production}

In order to evaluate whether specific combinations of effectors could
act synergistically to further increase rAAV production, we utilized
nocodazole in combination with either z-VAD-fmk or M344 (see Figure 1).
M344 is a synthetic analogue of the anti-fungal drug Trichostatin A, an
inhibitor of Class I and IIB histone deacetylases, and recently used as
an enhancer of recombinant protein expression in mammalian cell
culture\textsuperscript{{[}10{]}}. Z-VAD-fmk is a pan-caspase
inhibitor\textsuperscript{{[}20{]}}. Each chemical was added to rAAV8
producing HEK293 cultures together with nocodazole at 4 HPT in 24-well
microplates. The inclusion of z-VAD-fmk in the initial screening
experiment was predicated on its anti-caspase activity, as we
hypothesised caspase mediated apoptosis - linked to rAAV production and
expression of AAV2 Rep proteins - would negatively affect final crude
titre\textsuperscript{{[}49{]},{[}50{]}}. Additionally, caspase-mediated
apoptosis induced by nocodazole-mediated cell cycle dysregulation would
further increase cell death within the production cultures. Increased
cell viability and VCD in cultures treated with nocodazole/z-VAD-fmk
relative to untreated, nocodazole treated or nocodazole/M344 treated
cells suggests that apoptosis was reduced (Fig. 5A \& B) but with an
unexpected reduction in crude genome titre (Fig. 5D). The observed
reduction may be a consequence of off-target induction of autophagy via
inactivation of \emph{n} -glycanase 1 (NGLY1) that has been shown to
occur in HEK293 cells treated with z-VAD-fmk\textsuperscript{{[}51{]}}.
While the role of autophagy in rAAV production is undetermined,
inhibition of autophagy has been shown to increase recombinant protein
expression in CHO cells\textsuperscript{{[}52{]},{[}53{]}} and
unintentional upregulation of autophagy may result in a reduction in
viral component proteins necessary for production of high viral titres.
Interestingly, the highest mean cell volume, which appeared to correlate
positively with genome titre, was measured in cells treated with both
nocodazole and z-VAD-fmk (Fig. 5C). This may be a result of reduced
apoptotic cell death allowing cell size to increase but with the caveat
that any benefit gained from this from a production perspective is
attenuated by the potential negative off-target effects of z-VAD-fmk.

We observed that the combination of 2.5 \selectlanguage{greek}µ\selectlanguage{english}M M344 and 4 \selectlanguage{greek}µ\selectlanguage{english}M nocodazole
produced an additive effect, increasing crude rAAV genome titre 2.6-fold
compared to untreated cultures, an improvement on nocodazole alone
(2-fold increase compared to untreated) (Fig 5D). This additive effect
was also observed in 30 mL shake flask cultures, whereby measurements of
VCD and viability closely replicated those observed in the screening
assay (Fig. 6A), together with an improvement in genome titre with
nocodazole/M344 from a 2.6-fold increase in the screening assay to a
3-fold increase compared to untreated cells in larger volume cultures
(Fig. 6B). AAV8 intact capsid-specific ELISA analysis of total capsid
titre showed a significant increase in intact capsids after addition of
nocodazole (4.3-fold increase) and nocodazole/M344 (11.3-fold increase)
compared to untreated controls (data not shown). We anticipate that rAAV
vector development could complement the process engineering strategy to
further maximize rAAV titer (e.g. high intact capsid levels are likely
to benefit from hybrid Rep with improved genome packaging
efficiency)\textsuperscript{{[}54{]}} while combinatorial empirical
modeling will enable systematic determination of optimal chemical dosage
and timing.

Taken together, these data show that nocodazole, either alone or in
combination with select small molecules, can reproducibly boost both
genome and total viral particle titre in a transient suspension HEK293
rAAV production system and that it is both scalable and applicable to
production of rAAVs derived from two phylogenetically distinct and
clinically translatable pseudotyped capsids.

\textbf{Discussion}

Improving the yield of intact, genome-containing rAAV particles during
viral vector production is a critical step to reducing overall
production costs. Here we describe a simple and robust method by which
viral vector titre, a fundamental critical quality attribute of viral
vector production, may be quickly improved in an established and
previously optimised suspension culture system. Small molecule enhancers
of recombinant protein expression have been extensively used across a
wide range of mammalian cell systems to improve transient production
performance\textsuperscript{{[}8{]}--{[}10{]},{[}22{]},{[}39{]},{[}55{]},{[}56{]}}.
The ease of use and low cost of small molecule enhancers (particularly
for chemicals with pronounced biological activity at low dosages) makes
them an attractive solution to improving rAAV yield.

Investigation of the cellular mechanisms underpinning increased viral
genome titre point to the arrest of rAAV producing cells in mitosis soon
after PEI-mediated transient transfection of rAAV producing plasmids.
Prior studies have shown a strong preference for wildtype AAV
replication within the G2/M phase of the cell
cycle\textsuperscript{{[}57{]},{[}58{]}}. Nocodazole has been shown to
significantly improve transient transfection efficiency in CHO
cells\textsuperscript{{[}39{]}} which may stem from increased nuclear
permissibility of transfection complexes due to the breakdown of the
nuclear membrane during mitosis. A reduction in cell proliferation
caused by nocodazole addition may also benefit viral production by
reducing plasmid copy number dilution and maintaining mRNA transcript
levels \textsuperscript{{[}9{]}}. A recent study utilising a
CRISPR-mediated genome wide screening strategy identified two target
genes (ITPRIP and SKA2) that when modulated in cells increased rAAV
genome titre and improved full/empty capsid ratios, with both target
genes (strongly, in the case of SKA2) associated with cell cycle
modulation \textsuperscript{{[}59{]}}. Further to this, a proteomic
study of HEK cells during AAV5 production highlighted a number of
proteins involved in cell cycle and proliferation as being strongly
downregulated during production \textsuperscript{{[}60{]}}. The
molecular effects of cell cycle arrest within the G2/M phase on rAAV
production are unknown, but there is a potential correlation between the
loss of essential nucleolar functions and the nucleolar localization of
viral proteins, while the volume increase could possibly minimize
crowding effects due to accumulation of viral
proteins\textsuperscript{{[}61{]}}. This apparent link between the cell
cycle and AAV production, and the abundance of cell cycle modulating
molecules, necessitates further investigation into the use of cell cycle
modulators for both improving rAAV production yields and ultimately
improving our understanding of the underlying biological processes
governing rAAV production.

The apparent important temporal aspect of cell cycle regulation within
the production process may also provide avenues for non-chemical
interventions to improve vector yield as this relationship becomes
better understood. Whilst we have not investigated the mechanism behind
M344-mediated titre enhancement, HDAC6 (of which M344 is a selective
inhibitor) has been shown to bind to, and regulate clearance of,
ubiquitinated proteins via induction of the heat-shock cellular
response\textsuperscript{{[}62{]}}. AAV capsid proteins are a known
target of ubiquitination post-viral entry\textsuperscript{{[}63{]}}, and
the ubiquitin-proteasome pathway (UPP) has been suggested to play an
active role in AAV capsid monomer degradation\textsuperscript{{[}64{]}},
therefore we posit a link between M344-mediated UPP dysregulation and
increased crude viral titre. Due to the robustness of the results
between small-scale plate-based cultures and larger scale shake flasks,
we believe that the screening process shown here could be further scaled
down and automated to increase throughput, due to the availability of
instrumentation that can rapidly and accurately dispense very small
volumes of drugs into culture. As minimal volumes are required for ddPCR
analysis of genome titre, identification of novel enhancers of rAAV
could be rapidly incorporated into existing rAAV production platforms
with minimal changes to existing protocols.

In summary, we show that the use of readily available small molecule
enhancers can significantly improve rAAV production yield. We show that
small molecule enhancers of rAAV production are amenable to optimisation
in an existing suspension HEK293 cell system and that positive hits from
initial small-scale screening of enhancer molecules can translate to
larger scale production platforms. We also show that increased titre
resulting from nocodazole treatment is consistent across two different
serotypes, suggesting broad applicability in rAAV manufacturing.

\textbf{Acknowledgments}

This study was supported by REGENXBIO Inc., U.S.A. The authors thank
Susan Clark (University of Sheffield) for technical assistance in flow
cytometry analysis and Lynne Baxter (University of Sheffield) for access
to widefield fluorescence microscopy equipment.

\textbf{Conflicts of interest}

The authors declare no conflicts of interest.

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\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1} -- Initial screen of small molecule culture additives
identifies nocodazole, M344 and z-VAD-fmk as novel putative enhancers of
rAAV genome titre. Cells were analysed 72 hours post transfection (HPT)
for rAAV8 genome titre, shown here as the fold change relative to rAAV8
producing cells containing no small molecule enhancers (Ctrl).
Nocodazole, M344, and z-VAD-fmk are highlighted (dashed boxes)
(\textbf{A} ). Mean cell volume vs genome titre fold change is shown in
(\textbf{B} ), dashed lines indicate control values.
R\textsuperscript{2} and p-value calculated from Pearson correlation
coefficient of mean cell volume vs genome titre. Data shown in
\textbf{A} are mean \selectlanguage{ngerman}± SEM for two independent experiments carried out in
technical duplicate, with the exception of TUDCA, TMAO, Betaine and LiCl
which are mean only data from one independent experiment carried out in
technical duplicate (data from these experiments is omitted in
\textbf{B} ). NaBu - Sodium butyrate; VPA -- Valproic acid; TUDCA -
Tauroursodeoxycholic acid; TMAO - Trimethylamine N-oxide; LiCl --
Lithium chloride\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 2 --} Addition of nocodazole to rAAV8 producing cells to
increase genome titre is time-dependent. rAAV8 producing cells were
cultured in 24-well microplates and 4 \selectlanguage{greek}µ\selectlanguage{english}M nocodazole was added at 0-, 4-,
24-, or 48-HPT or left untreated (Ctrl). At 72 HPT cells were harvested
and viable cell density (VCD) (\textbf{A} ), viability (\textbf{B} ) and
mean cell volume (\textbf{C} ) was measured. Final crude genome titre
expressed as a fold change relative to Ctrl is shown in (\textbf{D} ).
Data shown for VCD, viability and mean cell diameter are the mean \selectlanguage{ngerman}±
S.E.M. n \textgreater{} 3 independent biological replicates. Data shown
for genome titre fold change are the mean ± S.E.M. n \selectlanguage{english}[?] 3 independent
biological replicates. Data were analysed by one-way ANOVA followed by
post-hoc Holm-\selectlanguage{polish}Š\selectlanguage{ngerman}ídák multiple comparisons test with respect to Ctrl. * p
\textless{} 0.05, ** p \textless{} 0.01, *** p \textless{} 0.001, **** p
\textless{} 0.0001.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 3} -- Increased genome titre in nocodazole treated cells
is maintained in larger scale cultures and across two separate
serotypes. 30 mL shake flask cultures were transfected with rAAV8 or
rAAV9 producing plasmids and cultured for 72 HPT in the presence (rAAV8:
; rAAV9: ) or absence (rAAV8: ; rAAV9: #) of 4 \selectlanguage{greek}µ\selectlanguage{english}M nocodazole added 4
hours post-transfection. Cultures were measured daily for viable cell
density (VCD) (\textbf{A --} solid lines), viability (\textbf{A --}
dashed lines) and mean cell volume (\textbf{B} ). Fold change analysis
of genome titre for both serotypes in the presence (+) or absence (--)
of 4 \selectlanguage{greek}µ\selectlanguage{english}M nocodazole at 72 HPT is shown in (\textbf{C} ). Data shown are
the mean \selectlanguage{ngerman}± S.E.M. n = 3 independent biological replicates. Fold change
between nocodazole treated and untreated cultures was analysed for each
serotype separately by Student's unpaired two-tailed t-test with respect
to untreated cultures. ** p \textless{} 0.01, *** p \textless{} 0.001.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 4 --} rAAV8 producing cells treated with nocodazole
arrest in G2/M phase. Flow cytometry histograms triggered against
propidium iodide signal for cells show the relative abundance of cells
in either G1 (blue), S (orange) or G2/M (green) phases of the cell cycle
in the absence (\textbf{A} ) or presence (\textbf{B} ) of 4 \selectlanguage{greek}µ\selectlanguage{english}M
nocodazole addition at 0 HPT. Ratio of cells in G2/M phase to G1 phase
at 72 HPT with 4 \selectlanguage{greek}µ\selectlanguage{english}M nocodazole added at 0, 4, 24, 48 HPT or untreated
(Ctrl) (\textbf{C} ). Representative fluorescent composite images of
rAAV-producing cells fixed 24 HPT show staining of the fibrillar
component of the nucleolus with anti-fibrillarin (red) and nuclear DNA
with DAPI (blue). Cells displaying disorganised nucleolar morphology are
indicated by white arrows in non-treated cells (\textbf{D} ) and cells
treated 4 hours post-transfection with 4 \selectlanguage{greek}µ\selectlanguage{english}M nocodazole (\textbf{E} ).
Images taken using 100 x oil objective. Scale bar 10 \selectlanguage{greek}µ\selectlanguage{english}m. Data shown in
(\textbf{C} ) are mean \selectlanguage{ngerman}± SEM for three biological replicates.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Figure 5 --}Combinatorial use of small molecule enhancers has an
additive effect on genome titre. rAAV8 producing cells were treated 4
HPT with small molecule enhancers of genome titre or left untreated.
Cells were harvested 72 HPT and measurements taken of viable cell
density (VCD) (\textbf{A} ), viability (\textbf{B} ), mean cell volume
(\textbf{C} ), and rAAV8 genome titre as a fold change with respect to
untreated cells (-/-/-) (\textbf{D} ). Data shown are the mean \selectlanguage{ngerman}± S.E.M.
n = 3 independent biological replicates. Data were analysed by one-way
ANOVA followed by post-hoc Holm-\selectlanguage{polish}Š\selectlanguage{ngerman}ídák multiple comparisons test with
respect to untreated cells (-/-/-). * p \textless{} 0.05, ** p
\textless{} 0.01, *** p \textless{} 0.001, **** p \textless{} 0.0001.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

\textbf{Figure 6 --} Genome titre improvements are retained in larger
scale cultures. 30mL shake flask cultures were transfected with rAAV8
producing plasmids with no chemical additives (#), with the addition of
4\selectlanguage{greek}µ\selectlanguage{english}M nocodazole at 4 hours post-transfection (), or with the addition of
4 \selectlanguage{greek}µ\selectlanguage{english}M nocodazole and 2.5 \selectlanguage{greek}µ\selectlanguage{english}M M344 at 4 hours post-transfection ().
Cultures were measured daily for VCD (\textbf{A} -- solid lines) and
viability (\textbf{A} -- dashed lines). Genome titre was measured 72
hours post-transfection and is shown as fold change with respect to
untreated cultures (\textbf{B} ). Data shown are the mean \selectlanguage{ngerman}± S.E.M. n = 3
independent biological replicates. Data were analysed by one-way ANOVA
followed by post-hoc Holm-\selectlanguage{polish}Š\selectlanguage{ngerman}ídák multiple comparisons test with respect
to untreated cells (-/-). * p \textless{} 0.05, ** p \textless{} 0.01

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