Toward Optimization of the Baculovirus Expression Vector System - Development of Genetic Tools to Improve Biologics Production
Year: 2021
Type: dissertation
Abstract: Small-molecule drugs have dominated the pharmaceutical industry and physician's prescription \npads since the beginning of modern medicine. The most recent decades, however, \nare most aptly characterized as the era of biologics. Indeed, 7 of the top 10 revenue generating \ntherapeutics in 2020 were biologics, and although they are yet to be counted \namong the most commonly prescribed drugs, the drum beat of new product approvals \nand demand for biologic therapeutics is intensifying. Despite the potential of biologics as \ntherapeutics, one of the factors that currently hinder their use is exorbitant cost of production. \nIn preceding years, important developments in the optimization of media, feeding \nstrategies, and downstream processing have led to signifi cant improvement in the yield and \ndecreased production cost of recombinant therapeutics. The most recent decade, however, \nhas witnessed a revolution in the field akin to the systems biology approach toward rational \ndesign practiced for improving microbial production hosts; enabled by advancements in \nwhole-genome sequencing, genome-scale models, and development of sophisticated genetic \nengineering tools, a new wave of engineered production hosts and platforms with improved \nor completely novel biochemical properties are being developed, leading to improved product \nyield and quality, and decreased production costs. \n \nThe baculovirus expression vector system (BEVS) is an established platform for the \nmanufacture of recombinant proteins, viral vaccines, and gene therapy vectors. Despite \nthe fi rst recombinant protein being produced in the BEVS in the early 1980s, much of the \nintervening years has seen it utilized predominantly as a research tool in academic laboratories \nrather than as a commercial manufacturing platform. Consequently, relatively little \nattention has been devoted to its improvement as a production platform. Nevertheless, \nseveral BEVS-manufactured vaccines and therapeutics have recently been licensed for use \nin animals and humans, signifying that it may yet nd mainstream use as a commercial \nmanufacturing platform. \n \nAlthough the BEVS boasts many features that make it attractive as a manufacturing \nplatform, to realize its full potential, intrinsic limitations must be addressed: the lytic infection \ncycle and resulting short bioprocess duration can limit overall yield of recombinant \nproteins, and large amounts of progeny virus, cellular proteins, and debris from lysed cells \nare contaminants that necessitate extensive purifi cation steps to achieve product purity. \nAdditionally, genome instability remains a major barrier to scalability due to rapid loss \nof foreign gene expression. Although periodic reports in the literature describe strategies \naimed at reducing contaminant progeny virions or improving yield and/or quality of the \nrecombinant protein product by targeted deletion or addition of endogenous and heterologous \ngenes in the baculovirus genome, genomes available commercially remain virtually \nunmodifi ed. This thesis seeks to address these issues through the development of genetic \ntools aimed at optimization of the baculovirus genome. \n \nWe initiated this work by developing a platform for efficient scrutiny of baculovirus \ngenes through targeted gene disruption and transcriptional repression using CRISPR-Cas9 technology. \nUsing cell lines that were developed for constitutive expression of the Cas9 or \ndCas9 proteins, sequence-specifi c disruption or downregulation of target genes was achieved \nwith efficiencies of up to 99%. The key factors affecting efficiency were choice of promoter \nfor sgRNA expression and spacer sequence selection for gene targeting. CRISPR-mediated \ngene disruption was more effective than transcriptional repression in all cases. As a result \nof these fi ndings, we confi rmed sequence-specifi c disruption of the AcMNPV GP64 and \nVP80 structural proteins for recombinant protein production with reduced baculovirus \ncontamination. Targeting these genes resulted in greater than 94% reduction in budded \nvirus release. Importantly, production of the model cytoplasmic protein GFP and a model \nvirus-like particle based on the HIV-1 Gag protein was not signi ficantly affected by gene \ndisruption, indicating that our approach could be more efficient than previously reported \nstrategies. \n \nNext, a microplate-based assay was developed to allow for efficient scrutiny of several \nbaculovirus genes in parallel. The assay involved transfection of Sf9 cells constitutively \nexpressing the Cas9 protein with a plasmid encoding a sgRNA, followed by infection with \na recombinant BEV. Expression of a gfp reporter gene and analysis of infectious virus titer \nin cell culture supernatants were used as analogs for late gene expression and progeny \nbudded virus release, thus providing insight toward the effect of various gene disruptions \non the virus infection cycle. The critical factors established in the development of the \nassay included viable cell density, choice of transfection reagent, the amount of plasmid \nDNA transfected, the ratio of transfection reagent:plasmid DNA, the time interval between \ntransfection and infection, virus multiplicity of infection, and the time interval between infection \nand harvest/analysis. The assay was used to scrutinize the effect of disrupting 13 \nAcMNPV genes, and the results agreed with those previously reported in all cases. Importantly, \nresults could be realized in less than 2 weeks, which represented an improvement in \nefficiency of up to several months. \n \nFinally, bioinformatics was used to select and evaluate baculovirus promoters with different \nexpression characteristics than those routinely employed for foreign gene expression. \nWe assesed the selected promoters by expressing model cytoplasmic and secreted proteins, \nand provided experimental evidence of the importance of promoter selection for foreign \ngene expression. We also examined sequence determinants that may be important for late \ngene transcription and translation initiation on a genome-wide scale.
Source: UWSpace (University of Waterloo)
Author: Mark Bruder
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W3194590161