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\begin{document}

\title{Advanced Cell Technologies: Making Protein, Cell, and Gene Therapies a
Reality}



\author[1]{Paula Alves}%
\author[2]{Isabelle Geada}%
\author[1]{Antonio Roldao}%
\author[2]{Michael Betenbaugh}%
\affil[1]{Instituto de Biologia Experimental e Tecnologica}%
\affil[2]{Johns Hopkins University Whiting School of Engineering}%


\vspace{-1em}



  
  \date{May 22, 2024}


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\selectlanguage{english}
\begin{abstract}
Researchers and engineers came together in Lisbon at the 27
\textsuperscript{th} Meeting of the European Society for Animal Cell
Technology (ESACT 2022), to discuss the latest advances in technologies
associated with protein-based biologics production, new modalities and
cell, gene and tissue therapies. Main contributions focused on how the
capabilities of production platforms can be enhanced, and how to
leverage them to generate new products. Some of the advances that were
presented are discussed below, including those related with cell line
development, metabolic engineering, analytics, CHO and insect cells
platforms engineering, vesicle and viral vector production, and gene and
cell therapy, along with some concluding remarks on the future of this
important field.%
\end{abstract}%



\sloppy


\textbf{Editorial}

Advanced Cell Technologies: Making Protein, Cell, and Gene Therapies a
Reality

Isabelle Geada\textsuperscript{1}, Antonio Rold\selectlanguage{ngerman}ão\textsuperscript{2};
Michael Betenbaugh\textsuperscript{1*} and Paula
Alves\textsuperscript{2,3*}

1 - Johns Hopkins University, Whiting School of Engineering, Baltimore,
USA

2 -~iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12,
2780-901~Oeiras, Portugal

3 -~Instituto de Tecnologia Química e Biológica António Xavier,
Universidade Nova de Lisboa, Av. da República, 2780-157~Oeiras, Portugal

*Special Issue Guest Editors

Researchers and engineers came together in Lisbon at the
27\textsuperscript{th} Meeting of the European Society for Animal Cell
Technology (ESACT 2022), to discuss the latest advances in technologies
associated with protein-based biologics production, new modalities and
cell, gene and tissue therapies. Main contributions focused on how the
capabilities of production platforms can be enhanced, and how to
leverage them to generate new products. Some of the advances that were
presented are discussed below, including those related with cell line
development, metabolic engineering, analytics, CHO and insect cells
platforms engineering, vesicle and viral vector production, and gene and
cell therapy, along with some concluding remarks on the future of this
important field.

\textbf{Improving CHO cell line performance through cell and bioprocess
engineering}

A continuing goal in mammalian biomanufacturing processes is to
evaluate, modify, and ultimately enhance the performance of CHO cells in
culture. Indeed, one of the major challenges in CHO cell culture is long
term cell line stability. Torres \emph{et al} . took a deeper look into
this issue, examining changes in cell culture performance, gene
expression, and metabolism over time for two cell lines. Especially
interesting was the upregulation of genes involved in cell proliferation
and survival concomitantly with changes in metabolites' uptake and
production rates. Acknowledging the impact that culture time has on the
cell, researchers are now exploring ways to improve cell line stability,
aiming to design more predictable, consistent, and productive expression
system. One of such approaches is targeted integration; however,
identifying a CHO cell genomic loci capable of supporting high-level
protein expression is still a bottleneck. To address this need,
Lee\emph{et al} ., implemented the ``Thousands of Reporters Integrated
in Parallel'' (TRIP) high-throughput screening method, identifying
several hotspot candidates in the CHO genome exhibiting high transgene
mRNA expression. In another study, Marx \emph{et al.} presented a fast
and robust method -the nanopore Cas9-targeted sequencing (nCats)
pipeline - to characterize cell clones and isolate the most promising
ones. This method was able to identify integration sites, the
composition of the integrated sequence, and the DNA methylation status
in CHO cells in a single sequencing run. Building up on CRISPR/Cas9
technology for mammalian cells, Lee \emph{et al} . developed an all-in
one reporter system to quantify gene disruption and site-specific
integration (SSI) in CHO cells. Using this system, it was possible to
identify specific molecules (inhibitors of DNA repair pathways) that
enhance SSI efficiency and thus accelerate cell engineering. Another
approach to further improve mammalian cell factories is to ameliorate
cell's capacity to handle proteotoxic stress, which can result in
cellular apoptosis. In Segatori \emph{et al.} , the challenges and
opportunities in synthetic biology for improving these programmable cell
factories is detailed. An important contribution to the field of cell
line and metabolic engineering is provided by Kontoravdi \emph{et al.}
with a review on the current state of CHO genome-scale metabolic models
(GEM), their inability to model intracellular metabolism and capture
extracellular phenotypes. In addition, Kontoravdi \emph{et al} .
presented an improved GEM, iCHO2441, as well as two cell line specific
GEMs for CHO-S and CHO-K1 that may serve as foundations for better
design and assess next-generation flux analysis techniques. Jimenez del
Val \emph{et al} . propose a more compact network model, CHOmpact, that
can provide improved interpretations from simulations, including
identification of shifts in key metabolic behaviours. This model could
also serve as a platform for dynamic models used for process control and
optimization. The advancement in process analytical techniques (PAT) and
artificial intelligence (AI) has enabled the generation of enormous
culture datasets from biomanufacturing processes. AI-based data-driven
models permit the correlation of biological and process conditions and
cell culture states. This approach was exploited by Lee \emph{et
al.}that describe data-driven prediction models for forecasting
multi-step ahead profiles of mAbs produced in CHO towards bioprocess
digital twins.

A complementary approach to cell line engineering is the development of
improved biomanufacturing processes. To address that need, Ben
Yahia\emph{et al.} worked on intensified processes by optimizing the
feeding strategy and specific power input (P/V) in a high-cell-density
(HCD) seed bioreactor operated in fed-batch mode towards improved
monoclonal antibodies (mAb) expression in the production bioreactor.
Interestingly they report a positive impact of cellular ``organized
stress'' in the seed bioreactor on the production performance. Chotteau
\emph{et al} . implemented a Design of Experiment (DoE) approach to
design an optimal CHO culture medium capable of supporting the operation
of microbioreactors in perfusion at HCD and low specific perfusion rates
while maintaining constant specific product quality attributes such as
N-glycosylation profile of the produced antibody. Using a similar
statistical design methodology, Ladiwala \emph{et al} . fine-tuned
specific amino acids levels in reference basal and feed media in order
to limit production of inhibitory metabolites, ultimately enhancing peak
viable cell densities and product titers. Alternatively, Naik \emph{et
al.} looked at adding glycolysis inhibitors to limit the production of
lactate as metabolic by-product in CHO cell cultures. They found that
specific glucose analogs could lower peak lactate concentrations while
also providing an increase in the final titers, although there was some
changes in glycosylation patterns indicating an effect on product
quality.

\textbf{Viral vectors, extracellular vesicle, and vaccines}

With the rise of new modalities such as viral vectors, extracellular
vesicles and vaccines, there is a pressing need to continue improving
biomanufacturing capabilities of these biologics. Several presentations
at ESACT 2022 were devoted to addressing this topic, some of which being
reflected in this special issue as a way of recognizing the enormous
efforts of the scientific community to date in this area which are
likely to further expand in the future as these modalities become an
increasingly important part of the biomanufacturing landscape.

Scalable and cost-effective bioprocesses for production of viral vectors
for gene and cell therapy are currently in high demand. Thus, developing
new, innovative technologies for production of these products is an
emerging thrust in biomanufacturing. One of the most used vectors in
gene therapy is the recombinant Adeno-Associated Virus, or rAAV,
produced in a variety of cell types. As an alternative to traditional
transient transfection systems using HEK93 cell lines, Escandell\emph{et
al} . developed a scalable system for rAAV production using a stable
HeLa cell line; it proved to be time-efficient and easy to scale-up
while capable of generating high rAAV yields and full to empty capsid
ratios. Another increasingly important platform for rAAV production is
the insect cells-baculovirus expression vector system (IC-BEVS). To
understand the variability in insect populations and its impact on rAAV2
titers, Isidro \emph{et al} . analyzed the Sf9 insect cell transcriptome
using single-cell RNA-seq. While transcriptional heterogeneity in Sf9
insect cells prior to infection exists, mainly associated with cell
cycle, that is exacerbated upon infection with the differential
expression of baculovirus genes and rAAV transgenes. The genes and
pathways identified will inform the path forward for cell and process
engineering towards improved rAAV2 production. Finally, Yoon\emph{et
al.} reviews the advances made in rAAV bioproduction as well as the
challenges ahead in making these therapeutics accessible. Lentivirus
(LV) are another important viral vector used in gene and cell therapies
for which large scale production remains a major challenge. Aiming to
solve this issue, Klimpel \emph{et al} . developed suspension-adapted
stable packaging cell lines in a scalable and serum-free production
process, and compared alternative methods to remove doxycycline in order
to initiate LV generation. Oncolytic viruses, another emerging modality,
shares the same bioprocess limitations as rAAV and LV. Genzel \emph{et
al} . developed an optimized perfusion process for large-scale
production of recombinant vesicular stomatitis virus-based fusogenic
oncolytic virus (rVSV-OV). Three cell lines (AGE1.CR, BHK-21, and
HEK293SF) were evaluated in HCD cultures, with a 15-30-fold increase in
volumetric productivities being observed compared to batch process,
establishing perfusion as a viable process for large-scale production of
rVSV-OV.

In the area of vaccine development, Palomares \emph{et al.} applied
phage display technology to produce epitopes (or mimotopes) that when
attached to AAV virus-like particles (VLP) can produce an antibody
response to recognize and potentially protect against Zika and Dengue
viruses\emph{.} In another study, Lorenzo \emph{et al.} described a
process to purify HIV-1 Gag VLPs from contaminating host extracellular
vesicles using a series of downstream isolation stages including
multiple filtration and chromatography steps.

Another emerging modality for delivery of therapeutics are extracellular
vesicles produced by mammalian production hosts, and a number of studies
explored methods to further improve their capabilities. In one study,
Estes \emph{et al} . identified specific metabolic pathways in HEK293
and CHO-S cell lines capable of improving extracellular vesicle titers
using high throughput siRNA screens. Cholesterol biosynthesis was one of
those pathways, results showing that the addition of statins to cell
culture increased vesicle productivity up to 9-fold. In addition,
Belliveau\emph{et al} . investigated the impact of osmotic and ammonia
stress on the microRNA (miRs) within extracellular vesicles (EVs) of CHO
cells. While normal culture conditions included higher levels of mir-92a
and mir-23a in EVs, stress condition resulted in the enrichment of
let-7a, let-7b, and let-7c miRs, which regulate core oncogenes and may
alter the balance between cell cycle and apoptosis.

\textbf{Stem cells and cell therapies}

Other important emerging modalities reviewed at ESACT 2022 were stem
cells and immune cells; there were several presentations examining the
development of these cell-based products, some of which are included in
this special issue. For example, Vives \emph{et al} . evaluated the
impact of 3D printing to preserve critical quality attributes in bone
tissue regeneration using natural hydrogel scaffolds. Interestingly,
hyaluronic acid, gelatin, and fibrin applied as bioinks enabled optimal
recovery of mesenchymal stromal cells while also preserving
proliferation and osteogenic capacity by reducing shear stress and
providing structural support. In addition, Costa \emph{et al} .
described an integrated bioreactor and downstream process enabling the
scalability of mesenchymal stromal cells production of extracellular
vesicles, which yielded considerably higher yields than those achieved
with static cultures while preserving the quality of the EVs and their
ability to stimulate angiogenesis. Fed-batch alone or in combination
with perfusion were described by Fernandes-Platzummer \emph{et al.} as a
process to generate bone marrow-derived MsCs and MSC-EV in stirred tank
bioreactors. To complement these experimental investigations,
Lewis\emph{et al} . proposes an \emph{in silico} approach (using
mathematical models) to describe cell-to-cell communications important
to immune cells cell functions and ultimately immunotherapies.

An overriding topic at the conference (and the field of
biomanufacturing) was the challenges and opportunities of introducing
new therapeutic platforms. This special issue starts with a publication
by Schaefer \emph{et al} . that discuss the barriers, including
technology, business, regulatory and people-driven concerns, that arise
when introducing new therapeutic technologies and present enablers for
reducing the gaps between advances and innovation in biopharmaceutical
manufacturing and final therapy delivery to patients.

\textbf{References}

1 Torres, Mauro \emph{et al,} ``Long term culture promotes changes to
growth, gene expression and metabolism in CHO cells that are independent
of production stability''

2 Lee, Kelvin \emph{et al,} ``A compendium of stable hotspots in the CHO
genome''

3 Marx, Nicolas \emph{et al,} ``Nanopore Cas9-targeted sequencing
enables accurate and simultaneous identification of transgene
integration sites, their structure and epigenetic status in recombinant
Chinese hamster ovary cells''

4 Lee, Kelvin \emph{et al,} ``High-efficiency and multi-locus targeted
integration in CHO cells using CRISPR-mediated donor nicking and DNA
repair inhibitors.''

5 Segatori, Laura \emph{et al,} ``Cell factory engineering: challenges
and opportunities for synthetic biology applications''

6 Kontoravdi, Cleo \emph{et al,} ``How reliable are Chinese Hamster
Ovary (CHO) cell genome-scale metabolic models?''

7 Jimenez del Val, Ioscani \emph{et al,} ``CHOmpact: a reduced metabolic
model of Chinese hamster ovary cells with enhanced interpretability''

8 Lee, Dong-Yup \emph{et al,} ``Data-driven prediction models for
forecasting multi-step ahead profiles of mammalian cell culture towards
bioprocess digital twins``

9 Ben Yahia, Bassem \emph{et al,} ``Organized stress'' for robust
scale-up of intensified production process with fed-batch seed
bioreactor``

10 Chotteau, Veronique \emph{et al,} ``Optimization of perfusion medium
with perfusion microbioreactors for high density CHO cell cultures at
very low perfusion rate aided by Design of Experiments``

11 Ladiwala, Pranay \emph{et al,} ``Addressing amino acid-derived
inhibitory metabolites and enhancing CHO cell culture performance
through DOE-guided media modifications``

12 Naik, Harnish Mukesh \emph{et al,} ``Chemical inhibitors of
hexokinase-2 enzyme reduce lactate accumulation, alter glycosylation
processing, and produce altered glycoforms in CHO cell cultures``

13 Escandell, José \emph{et al} , ``Towards a scalable bioprocess for
rAAV production using a HeLa stable cell line''

14 Isidro, Inês \emph{et al} , ``Impact of dual baculovirus infection on
the Sf9 insect cell transcriptome during AAV production using
single-cell RNA-seq''

15 Yoon, Seongkyu \emph{et al} , ``Critical Challenges and Advances in
Recombinant Adeno-Associated Virus (rAAV) Biomanufacturing

16 Klimpel, Maximilian \emph{et al} , ``Development of a perfusion
process for continuous lentivirus production using stable suspension
producer cell lines

17 Genzel, Y \emph{et al} , ``Process intensification strategies towards
cell culture-based high-yield production of a fusogenic oncolytic virus

18 Palomares, Laura A. \emph{et al} , ``Mimotope discovery as a tool to
design a vaccine against zika and dengue viruses''

19 Lorenzo, Elianet \emph{et al} , ``Downstream process design for Gag
HIV-1 based virus-like particles''

20 Estes, Scott \emph{et al} , ``Therapeutic extracellular vesicle
production is substantially increased by inhibition of cellular
cholesterol biosynthesis''

21 Belliveau, Jessica \emph{et al} , `` The microRNomes of Chinese
Hamster Ovary (CHO) cells and their extracellular vesicles, and how they
respond to osmotic and ammonia stress''

22 Vives, Joaquim \emph{et al} , ``Preservation of critical quality
attributes of mesenchymal stromal cells in 3d bioprinted structures by
using natural hydrogel scaffolds''

23 H G, Marta Costa \emph{et al} , ``Enhanced bioprocess control to
advance the manufacture of mesenchymal stromal cell-derived
extracellular vesicles in stirred-tank bioreactors''

24 Fernandes-Platzgummer, Ana \emph{et al} , ``Optimized operation of a
controlled stirred tank reactor system for the production of mesenchymal
stromal cells and their extracellular vesicles''

25 Lewis, Nathan E. \emph{et al} , ``Reconstructing the Cell-Cell
Interaction Network Among Mouse Immune Cells''

26 Schaefer, Gene \emph{et al,} ``Driving adoption of new technologies
in biopharmaceutical manufacturing''

\selectlanguage{english}
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