eBook: Cell and Gene Therapy - 21

Figure 3: Influenza A titer comparison. Immunostained TCID50
of viral supernatants produced by WT parental and MDCK.STAT1 KO cells. Cells
were infected with Influenza A at an MOI of 0.01. Supernatants were collected 48 hours after infection and used to re-infect WT MDCK cells at
the indicated dilution. (A) Green - anti-Influenza A stain, Blue - nuclear stain. Cells were infected with Influenza A at an MOI of 0.01 for 48h, then
(B) calculated TCID50
of Influenza A viral supernatants produced in MDCK.STAT1 KO cells at 48h post infection (n=3). (C) Additionally, RT-PCR
quantification of Influenza A viral shows a 2-fold increase in Influenza A genomes produced in MDCK.STAT1 KO cells compared to the parental
cells at 48h post-infection.
supernatants after 48 hours. Droplet digital PCR™
quantification of AAV5 viral genomes at 72 hours
post-infection demonstrated an increase of about
80% when comparing 293.STAT1 BAX KO cells to
the parental HEK 293 cells.
Conclusion
An increasing number of viral vaccines are manufactured
in large-scale tissue culture systems
using historical cell lines that are approved for
vaccine manufacturing. In this study, we used
CRISPR/Cas9 gene-editing techniques to increase
the viral production efficiency of some of these
historical cell lines.
Vero and MDCK cells were modified by CRISPR/
Cas9 to produce a truncated STAT1 gene product
that is quickly proteolyzed by the cell. Similarly,
HEK 293 cells were genome edited to knock out ex21
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