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Comprehensive mRNA-sequencing-based characterization of three HEK-293 cell lines during an rAAV production process for Gene Therapy Applications

ABSTRACT: Human embryonal kidney cells (HEK-293) are the most common host cells used for transient recombinant adeno-associated virus (rAAV) production in pharmaceutical industry. To better cover the expected gene therapy product demands in the future, different traditional strategies such as cell line sub-cloning and/or addition of chemical substances to the fermentation media have been used to maximize titers and improve product quality. A more effective and advanced approach to boost yield can be envisaged by characterizing the transcriptome of different HEK-293 cell line pedigrees with distinct rAAV productivity patterns to subsequently identify potential gene targets for cell engineering. In this work, the mRNA expression profile of three HEK-293 cell lines, resulting in various yields during a fermentation batch process for rAAV production, was investigated to gain basic insight into cell variability and eventually to identify genes that correlate with productivity. Mock runs using only transfection reagents were performed in parallel as a control. We found significant differences in gene regulatory behaviors between the three cell lines at different growth and production stages. The evaluation of these transcriptomics profiles combined with collected in-process control parameters and titers shed some light on potential cell engineering targets to maximize transient production of rAAV in HEK-293 cells Comparison of three HEK-293 suspension cell lines transcriptomics during an AAV production process

ORGANISM(S): Homo sapiens

PROVIDER: GSE226054 | GEO | 2024/05/13

REPOSITORIES: GEO

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Project description:BACKGROUND:Isobutanol is a promising next generation biofuel with demonstrated high yield microbial production, but the toxicity of this molecule reduces fermentation volumetric productivity and final titers. Organic solvent tolerance is a complex, multigenic phenotype that has been recalcitrant to rational engineering approaches. We apply experimental evolution followed by genome resequencing and a gene expression study to elucidate genetic bases on adaptation to exogenous isobutanol stress. RESULTS:The adaptations acquired in our evolved lineages exhibit antagonistic pleiotropy between minimal and rich medium, and appear to be specific to the effects of longer chain alcohols. By examining genotypic adaptation in multiple independent lineages, we find evidence of parallel evolution in hfq, mdh, acrAB, gatYZABCD, and rph genes. Many isobutanol tolerant lineages show reduced rpoS activity, perhaps related to mutations in hfq or acrAB. Consistent with the complex, multigenic nature of solvent tolerance, we observe adaptations in a diversity of cellular processes. Many adaptations appear to involve epistasis between different mutations, implying a rugged fitness landscape for isobutanol tolerance. We observe a trend of evolution targeting post-transcriptional regulation and high centrality nodes of biochemical networks. Collectively, the genotypic adaptations we observe suggest mechanisms of adaptation to isobutanol stress based on remodelling the cell envelope and surprisingly, stress response attenuation. CONCLUSIONS:We have discovered a set of genotypic adaptations that confer increased tolerance to exogenous isobutanol stress. Our results are immediately useful to efforts to engineer more isobutanol tolerant host strains of E. coli for isobutanol production. We suggest that rpoS and post-transcriptional regulators, such as hfq, RNA helicases, and sRNAs may be interesting mutagenesis targets for futurue global phenotype engineering. Two strains (WT strain and G3.2 mutant strain), each with two culture conditions (with and without isobutanol in medium). Three biological replicates for each strain/culture condition. Twelve samples in total.