| dc.description.abstract | Small-molecule drugs have dominated the pharmaceutical industry and physician's prescription
pads since the beginning of modern medicine. The most recent decades, however,
are most aptly characterized as the era of biologics. Indeed, 7 of the top 10 revenue generating
therapeutics in 2020 were biologics, and although they are yet to be counted
among the most commonly prescribed drugs, the drum beat of new product approvals
and demand for biologic therapeutics is intensifying. Despite the potential of biologics as
therapeutics, one of the factors that currently hinder their use is exorbitant cost of production.
In preceding years, important developments in the optimization of media, feeding
strategies, and downstream processing have led to signifi cant improvement in the yield and
decreased production cost of recombinant therapeutics. The most recent decade, however,
has witnessed a revolution in the field akin to the systems biology approach toward rational
design practiced for improving microbial production hosts; enabled by advancements in
whole-genome sequencing, genome-scale models, and development of sophisticated genetic
engineering tools, a new wave of engineered production hosts and platforms with improved
or completely novel biochemical properties are being developed, leading to improved product
yield and quality, and decreased production costs.
The baculovirus expression vector system (BEVS) is an established platform for the
manufacture of recombinant proteins, viral vaccines, and gene therapy vectors. Despite
the fi rst recombinant protein being produced in the BEVS in the early 1980s, much of the
intervening years has seen it utilized predominantly as a research tool in academic laboratories
rather than as a commercial manufacturing platform. Consequently, relatively little
attention has been devoted to its improvement as a production platform. Nevertheless,
several BEVS-manufactured vaccines and therapeutics have recently been licensed for use
in animals and humans, signifying that it may yet nd mainstream use as a commercial
manufacturing platform.
Although the BEVS boasts many features that make it attractive as a manufacturing
platform, to realize its full potential, intrinsic limitations must be addressed: the lytic infection
cycle and resulting short bioprocess duration can limit overall yield of recombinant
proteins, and large amounts of progeny virus, cellular proteins, and debris from lysed cells
are contaminants that necessitate extensive purifi cation steps to achieve product purity.
Additionally, genome instability remains a major barrier to scalability due to rapid loss
of foreign gene expression. Although periodic reports in the literature describe strategies
aimed at reducing contaminant progeny virions or improving yield and/or quality of the
recombinant protein product by targeted deletion or addition of endogenous and heterologous
genes in the baculovirus genome, genomes available commercially remain virtually
unmodifi ed. This thesis seeks to address these issues through the development of genetic
tools aimed at optimization of the baculovirus genome.
We initiated this work by developing a platform for efficient scrutiny of baculovirus
genes through targeted gene disruption and transcriptional repression using CRISPR-Cas9 technology.
Using cell lines that were developed for constitutive expression of the Cas9 or
dCas9 proteins, sequence-specifi c disruption or downregulation of target genes was achieved
with efficiencies of up to 99%. The key factors affecting efficiency were choice of promoter
for sgRNA expression and spacer sequence selection for gene targeting. CRISPR-mediated
gene disruption was more effective than transcriptional repression in all cases. As a result
of these fi ndings, we confi rmed sequence-specifi c disruption of the AcMNPV GP64 and
VP80 structural proteins for recombinant protein production with reduced baculovirus
contamination. Targeting these genes resulted in greater than 94% reduction in budded
virus release. Importantly, production of the model cytoplasmic protein GFP and a model
virus-like particle based on the HIV-1 Gag protein was not signi ficantly affected by gene
disruption, indicating that our approach could be more efficient than previously reported
strategies.
Next, a microplate-based assay was developed to allow for efficient scrutiny of several
baculovirus genes in parallel. The assay involved transfection of Sf9 cells constitutively
expressing the Cas9 protein with a plasmid encoding a sgRNA, followed by infection with
a recombinant BEV. Expression of a gfp reporter gene and analysis of infectious virus titer
in cell culture supernatants were used as analogs for late gene expression and progeny
budded virus release, thus providing insight toward the effect of various gene disruptions
on the virus infection cycle. The critical factors established in the development of the
assay included viable cell density, choice of transfection reagent, the amount of plasmid
DNA transfected, the ratio of transfection reagent:plasmid DNA, the time interval between
transfection and infection, virus multiplicity of infection, and the time interval between infection
and harvest/analysis. The assay was used to scrutinize the effect of disrupting 13
AcMNPV genes, and the results agreed with those previously reported in all cases. Importantly,
results could be realized in less than 2 weeks, which represented an improvement in
efficiency of up to several months.
Finally, bioinformatics was used to select and evaluate baculovirus promoters with different
expression characteristics than those routinely employed for foreign gene expression.
We assesed the selected promoters by expressing model cytoplasmic and secreted proteins,
and provided experimental evidence of the importance of promoter selection for foreign
gene expression. We also examined sequence determinants that may be important for late
gene transcription and translation initiation on a genome-wide scale. | en |
