eBook: Cell and Gene Therapy - 18

candidates for enhancing virus titers for vaccine
development, they also provide useful tools to
package lentiviral, adenoviral, and retroviral vectors
for the delivery of DNA sequences into target
cells for various applications such as gene therapy
and cancer immunotherapy.
Results and Discussion
STAT1 and STAT1/BAX Knockout Strategy
and Execution
We employed the CRISPR/Cas9 genome-editing
platform to generate the desired, targeted STAT1
knockout (Vero.STAT1 KO and MDCK. STAT1 KO)
or STAT1 and BAX double knockout (293.STAT1
BAX KO) VPCs. Our strategy was to generate outof-frame
insertions and deletions near the start of
STAT1 and BAX genes, which would result in a functional
protein knockout. Because of the transient
nature of the Cas9 construct transfection strategy,
the constructs themselves were not integrated into
the gene-edited clones. To execute on this strategy,
single guide RNAs (sgRNAs) were designed and
built to guide Cas9 to bind and cut the desired intronic
regions in the STAT1 and BAX gene targets.
We then transiently transfected Cas9 and either the
STAT1 gRNA or STAT1 and BAX gRNA constructs into
the viral production cells. Once expressed within
the cells, CRISPR/Cas9 created a double-strand
break (DSB) in the appropriate gene. The cells'
non-homologous end joining (NHEJ) repair mechanisms
then created small insertions and deletions
at the repair site. After gene editing was complete,
the clones were subjected to a battery of tests.
Sanger sequencing was used to confirm that the
correct frame-shift mutation occurred, and western
blot was used to confirm that expression of the
target genes was absent (Figure 1). Additionally,
the VPCs were evaluated for off-target editing, stability,
morphology, and cell growth. We found that
the targeted gene knockout was very stable, and
there was no off-target editing in similar sequences
throughout the whole genome. Additionally,
the VPCs displayed very similar morphology and
equiv- alent growth rate as their parental cell lines.
Biofunctional Evaluation of VPCS: Viruses
for Vaccine Development
Once the Vero.STAT1 KO, MDCK.STAT1 KO, and 293.
STAT1 BAX KO cell lines were created and quality
control analyses were performed, we evaluated
their virus production capacity as compared to that
of their respective parental cell lines. Generally, the
strategy for evaluating virus production was the
same for all of the cell lines that we tested.
Vero.STAT1 KO and Vero cells were infected with
Dengue virus type 2 strain New Guinea C (ATCC®
VR-1584™); after seven days post-infection, the
supernatants were harvested, serially diluted, and
used to re-infect fresh cell cultures. Viruses within
the cells were then subjected to immunofluorescence
staining. High-content microscopy was used
to capture fluorescence images of each well for the
eval- uation of infectious viral titer by TCID50
(Figure
2A and B). When compared to the virus production
capacity of Vero cells, a 10-fold increase in Dengue
virus-infected cells was observed in the Vero.STAT1
KO cells. Additionally, RT-PCR was performed in the
STAT1 KO and parental cell lines (Figure 2C), where
we saw a 30-fold increase in Dengue viral genomes
in the KO cell line.
We performed similar experiments in MDCK.STAT1
KO and the MDCK parental cell line. In this set of
experiments, we infected both cell lines with Influenza
A virus (H1N1; ATCC® VR-1736™) and harvested
supernatants 48 hours post-transduction. Serial
dilutions of the viral supernatants were again
18
https://www.atcc.org/products/vr-1584 https://www.atcc.org/products/vr-1736
eBook: Cell and Gene Therapy

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