A set of gene knockouts as a resource for global lipidomic changes.

Enzyme specificity in lipid metabolic pathways often remains unresolved at the lipid species level, which is needed to link lipidomic molecular phenotypes with their protein counterparts to construct functional pathway maps. We created lipidomic profiles of 23 gene knockouts in a proof-of-concept study based on a CRISPR/Cas9 knockout screen in mammalian cells. This results in a lipidomic resource across 24 lipid classes. We highlight lipid species phenotypes of multiple knockout cell lines compared to a control, created by targeting the human safe-harbor locus AAVS1 using up to 1228 lipid species and subspecies, charting lipid metabolism at the molecular level. Lipid species changes are found in all knockout cell lines, however, some are most apparent on the lipid class level (e.g., SGMS1 and CEPT1), while others are most apparent on the fatty acid level (e.g., DECR2 and ACOT7). We find lipidomic phenotypes to be reproducible across different clones of the same knockout and we observed similar phenotypes when two enzymes that catalyze subsequent steps of the long-chain fatty acid elongation cycle were targeted.

Model-based approach for predicting the impact of genetic modifications on product yield in biopharmaceutical manufacturing-Application to influenza vaccine production.

A large group of biopharmaceuticals is produced in cell lines. The yield of such products can be increased by genetic engineering of the corresponding cell lines. The prediction of promising genetic modifications by mathematical modeling is a valuable tool to facilitate experimental screening. Besides information on the intracellular kinetics and genetic modifications the mathematical model has to account for ubiquitous cell-to-cell variability. In this contribution, we establish a novel model-based methodology for influenza vaccine production in cell lines with overexpressed genes. The manipulation of the expression level of genes coding for host cell factors relevant for virus replication is achieved by lentiviral transduction. Since lentiviral transduction causes increased cell-to-cell variability due to different copy numbers and integration sites of the gene constructs we use a population balance modeling approach to account for this heterogeneity in terms of intracellular viral components and distributed kinetic parameters. The latter are estimated from experimental data of intracellular viral RNA levels and virus titers of infection experiments using cells overexpressing a single host cell gene. For experiments with cells overexpressing multiple host cell genes, only final virus titers were measured and thus, no direct estimation of the parameter distributions was possible. Instead, we evaluate four different computational strategies to infer these from single gene parameter sets. Finally, the best computational strategy is used to predict the most promising candidates for future modifications that show the highest potential for an increased virus yield in a combinatorial study. As expected, there is a trend to higher yields the more modifications are included.

CRISPR/Cas9-based knockout pipeline for reverse genetics in mammalian cell culture.

We present a straightforward protocol for reverse genetics in cultured mammalian cells, using CRISPR/Cas9-mediated homology-dependent repair (HDR) based insertion of a protein trap cassette, resulting in a termination of the endogenous gene expression. Complete loss of function can be achieved with monoallelic trap cassette insertion, as the second allele is frequently disrupted by an error-prone non-homologous end joining (NHEJ) mechanism. The method should be applicable to any expressed gene in most cell lines, including those with low HDR efficiency, as the knockout alleles can be directly selected for.

Model-based analysis of influenza A virus replication in genetically engineered cell lines elucidates the impact of host cell factors on key kinetic parameters of virus growth.

The best measure to limit spread of contagious diseases caused by influenza A viruses (IAVs) is annual vaccination. The growing global demand for low-cost vaccines requires the establishment of high-yield production processes. One possible option to address this challenge is the engineering of novel vaccine producer cell lines by manipulating gene expression of host cell factors relevant for virus replication. To support detailed characterization of engineered cell lines, we fitted an ordinary differential equation (ODE)-based model of intracellular IAV replication previously established by our group to experimental data obtained from infection studies in human A549 cells. Model predictions indicate that steps of viral RNA synthesis, their regulation and particle assembly and virus budding are promising targets for cell line engineering. The importance of these steps was confirmed in four of five single gene overexpression cell lines (SGOs) that showed small, but reproducible changes in early dynamics of RNA synthesis and virus release. Model-based analysis suggests, however, that overexpression of the selected host cell factors negatively influences specific RNA synthesis rates. Still, virus yield was rescued by an increase in the virus release rate. Based on parameter estimations obtained for SGOs, we predicted that there is a potential benefit associated with overexpressing multiple host cell genes in one cell line, which was validated experimentally. Overall, this model-based study on IAV replication in engineered cell lines provides a step forward in the dynamic and quantitative characterization of IAV-host cell interactions. Furthermore, it suggests targets for gene editing and indicates that overexpression of multiple host cell factors may be beneficial for the design of novel producer cell lines.

Influenza virus intracellular replication dynamics, release kinetics, and particle morphology during propagation in MDCK cells.

Influenza viruses are respiratory pathogens and can cause severe disease. The best protection against influenza is provided by annual vaccination. These vaccines are produced in embryonated chicken eggs or using continuous animal cell lines. The latter processes are more flexible and scalable to meet the growing global demand. However, virus production in cell cultures is more expensive. Hence, further research is needed to make these processes more cost-effective and robust. We studied influenza virus replication dynamics to identify factors that limit the virus yield in adherent Madin-Darby canine kidney (MDCK) cells. The cell cycle stage of MDCK cells had no impact during early infection. Yet, our results showed that the influenza virus RNA synthesis levels out already 4 h post infection at a time when viral genome segments are exported from the nucleus. Nevertheless, virus release occurred at a constant rate in the following 16 h. Thereafter, the production of infectious viruses dramatically decreased, but cells continued to produce particles contributing to the hemagglutination (HA) titer. The majority of these particles from the late phase of infection were deformed or broken virus particles as well as large membranous structures decorated with viral surface proteins. These changes in particle characteristics and morphology need to be considered for the optimization of influenza virus production and vaccine purification steps. Moreover, our data suggest that in order to achieve higher cell-specific yields, a prolonged phase of viral RNA synthesis and/or a more efficient release of influenza virus particles is required.

Impaired antiviral response of adenovirus-transformed cell lines supports virus replication.

Activation of the innate immune response represents one of the most important cellular mechanisms to limit virus replication and spread in cell culture. Here, we examined the effect of adenoviral gene expression on the antiviral response in adenovirus-transformed cell lines; HEK293, HEK293SF and AGE1.HN. We demonstrate that the expression of the early region protein 1A in these cell lines impairs their ability to activate antiviral genes by the IFN pathway. This property may help in the isolation of newly emerging viruses and the propagation of interferon-sensitive virus strains.

Impact of defective interfering particles on virus replication and antiviral host response in cell culture-based influenza vaccine production.

During the replication of influenza viruses, defective interfering particles (DIPs) can be generated. These are noninfectious deletion mutants that require coinfection with a wild-type virus but interfere with its helper virus replication. Consequently, coinfected cells mainly produce DIPs. Little is known about how such noninfectious virus particles affect the virus yield of cell culture-based influenza vaccine production. We compared infections of Madin-Darby canine kidney cells with two seed virus preparations of the influenza virus strain A/Puerto Rico/8/34 that contain different amounts of DIPs. A combination of conventional RT-PCR, RT-qPCR, and flow cytometry revealed that DI genomes indeed strongly accumulate in coinfected cells and impede the viral RNA synthesis. Additionally, cells infected at the higher DIP concentration showed a stronger antiviral response characterized by increased interferon-ß expression and apoptosis induction. Furthermore, in the presence of DIPs, a significant fraction of cells did not show any productive accumulation of viral proteins at all. Together, these effects of DIPs significantly reduce the virus yield. Therefore, the accumulation of DIPs should be avoided during influenza vaccine production which can be achieved by quality controls of working seed viruses based on conventional RT-PCR. The strategy for the depletion of DIPs presented here can help to make cell culture-based vaccine production more reliable and robust.