eBook: Cell and Gene Therapy - 17

viral infections, a bottleneck in the production of
AAV-derived gene therapy products is at the point
of infection, replication, and egress of the virus in
the HEK 293 cell line.
The emergence of new, pathogenic viral strains
and the demand for therapies for genetic diseases
contribute to the urgent need to accelerate manufacturing
processes. While there are many points
at which improvements could be made to speed
virus production, optimizing the cell lines used
in the process has the potential to yield the most
benefit with regards to reduction in cost and time.
Some strategies that could be used to optimize
virus-producing cell lines include optimizing virus-producing
cells (VPCs) for viral vector uptake,
adjust- ing the VPC's growth mode from adherent
to suspension or high-density culture, and genetically
engineering VPCs to enhance their viral production
yield.
Of the above mentioned areas of improvement,
ATCC took a step to create high-titer viral production
cell lines through CRISPR/Cas 9 gene editing.
With the design strategy of enhancing viral particle
yield by eliminating the interferon response pathways
and improving the VPC's survival via deleting/
down regulating of pro-apoptotic genes, we employed
two approaches. The first was to exploit the
fact that cells rely on interferon-elicited pathways
as defense against viral infections. A major effector
of interferon signaling is via the STAT1 protein.
Phosphorylation and the resulting self-dimerization
of STAT1 induces this intracellular signaling
protein to translocate to the nucleus, resulting in
a number of antiviral, antiproliferative, and immunoregulatory
responses by the cell. Thus, ablating
STAT1 from this system results in a profound reduction
in the cell's antiviral response and a concomitant
increase in viral yield.1,2
In this regard,
ATCC scientists used CRISPR/Cas9 genome-editing
technology to knock out the STAT1 gene in Vero
(ATCC® CCL-85™) and MDCK (ATCC® CCL-34™) cells
as a strategy to enhance virus production. With the
STAT1 gene disrupted, the STAT1 protein is not synthesized
and thus the inter- feron signaling cascade
is disrupted. Without the cell's defense against the
viral infection, we hypothesized that virus production
would be significantly enhanced in the newly
created Vero.STAT1 KO (ATCC® CCL-81-VHG™) and
MDCK.STAT1 KO (ATCC® CCL-34-VHG™) cell lines as
compared to that of the parental cell lines.
The second approach that was taken involved
knocking out both the BAX and STAT1 genes. BAX
protein is a central activator of apoptosis via the intrinsic
pathway and has functions in virally induced
cell death. Indeed, BAX has been identified by Cuconati
and colleagues in 2002 to limit AAV production
via the apoptotic response.3
Thus, we hypothesized
that deletion of the BAX gene product from
VPCs would increase cell survival and remove another
hurdle to enhanced virus production. As with
the abovementioned modified cell lines, we used
CRISPR/Cas9 genome-editing technology to knock
out both BAX and STAT1 from the AAV-producing
parental HEK 293 cell line (ATCC® CRL-1573™). As
in the other models, without the VPC's antiviral
defense and major apoptotic regulator, we anticipated
that AAV production would be enhanced
in the newly generated 293.STAT1 BAX KO (ATCC®
CRL-1573-VHG™) as compared to that of the parental
cell line.
Overall, we provide evidence indicating that the
ATCC STAT1 and STAT1/BAX knockout cell lines produce
model clinical viruses at titers 10-fold higher
than the corresponding parental cell lines; these
gene-edited cell lines have the potential to significantly
reduce costs and time expenditures that are
associated with virus and viral vaccine production.
Thus, these optimized cell lines are not only good
17
https://www.atcc.org/products/ccl-85 https://www.atcc.org/products/CCL-34 https://www.atcc.org/products/CCL-81-VHG https://www.atcc.org/products/CCL-34-VHG https://www.atcc.org/products/CRL-1573 https://www.atcc.org/products/CRL-1573-VHG
eBook: Cell and Gene Therapy

Table of Contents for the Digital Edition of eBook: Cell and Gene Therapy
