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An important quality attribute of a recombinant Adeno-Associated Virus (rAAV) as a therapeutic vector is its infectivity. Current assays to quantify infectious rAAV rely on coinfection with a helper virus such as adenovirus, which requires helper virus preparation and introduces additional variability. Here we describe a stable assay cell line that was generated by integrating the coding sequences for AAV Rep68 and adenovirus E4orf6 and DNA Binding Protein (DBP) under the control of inducible promoters. The Rep68 protein expression was further modulated by a ligand-responsive destabilization domain. In several benchmarks, the cell line gave comparable titers to those obtained using a classical adenovirus coinfection method. The cell line was also used to titer vectors of multiple AAV serotypes. This cell line has the potential to serve as an effective and robust tool for product quality evaluation.

Recombinant adeno-associated viruses (rAAV) are important gene delivery vehicles for gene therapy applications. Their production relies on plasmid transfection or virus infection of producer cells, which pose a challenge in process scale-up. Here, we describe a template for a transfection-free, helper virus-free rAAV producer cell line using a synthetic biology approach. Three modules were integrated into HEK293 cells including an rAAV genome and multiple inducible promoters controlling the expression of AAV Rep, Cap, and helper coding sequences. The synthetic cell line generated infectious rAAV vectors upon induction. Independent control over replication and packaging activities allowed for manipulation of the fraction of capsid particles containing viral genomes, affirming the feasibility of tuning gene expression profiles in a synthetic cell line for enhancing the quality of the viral vector produced. The synthetic biology approach for rAAV production presented in this study can be exploited for scalable biomanufacturing.

Over the last two decades, model-based metabolic pathway optimization tools have been developed for the design of microorganisms to produce desired metabolites. However, few have considered more complex cellular systems such as mammalian cells, which requires the use of nonlinear kinetic models to capture the effects of concentration changes and cross-regulatory interactions. In this study, we develop a new two-stage pathway optimization framework based on kinetic models that incorporate detailed kinetics and regulation information. In Stage 1, a set of optimization problems are solved to identify and rank the enzymes that contribute the most to achieving the metabolic objective. Stage 2 then determines the optimal enzyme interventions for specified desired numbers of enzyme adjustments. It also incorporates multi-scenario optimization, which allows the simultaneous consideration of multiple physiological conditions. We apply the proposed framework to find enzyme adjustments that enable a reverse glucose flow in cultured mammalian cells, thereby eliminating the need for glucose feed in the late culture stage and enhancing process robustness. The computational results demonstrate the efficacy of the proposed approach; it not only captures the important regulations and key enzymes for reverse glycolysis but also identifies differences and commonalities in the metabolic requirements for different carbon sources.

Different regions of a mammalian genome have different accessibilities to transcriptional machinery. The integration site of a transgene affects how actively it is transcribed. Highly accessible genomic regions called super-enhancers have been recently described as strong regulatory elements that shape cell identity. Super-enhancers have been identified in Chinese hamster ovary (CHO) cells using the Assay for Transposase-Accessible Chromatin Sequencing (ATAC-seq). Genes near super-enhancer regions had high transcript levels and were enriched for oncogenic signaling and proliferation functions, consistent with an immortalized phenotype. Inaccessible regions in the genome with low ATAC signal also had low transcriptional activity. Genes in inaccessible regions were enriched for remote tissue functions such as taste, smell, and neuronal activation. A lentiviral reporter integration assay showed integration into super-enhancer regions conferred higher reporter expression than insertion into inaccessible regions. Targeted integration of an IgG vector into the Plec super-enhancer region yielded clones that expressed the immunoglobulin light chain gene mostly in the top 20% of all transcripts with the majority in the top 5%. The results suggest the epigenomic landscape of CHO cells can guide the selection of integration sites in the development of cell lines for therapeutic protein production.

Mammalian cells are the main tool for the production of therapeutic proteins, viruses for gene therapy, and cells for cell therapy. In production processes cell metabolism is the main driver that causes changes in the growth environment and affects productivity and product quality. Of all nutrients, glucose has the most prominent impact on bioprocesses. We summarize recent findings on the regulation of glucose and energy metabolism in cultured cells. Local allosteric regulations and post-translational modifications of enzymes in metabolic networks interplay with global signaling and transcriptional regulation. These regulatory networks sustain homeostasis across the cytosolic and mitochondrial compartments. Understanding the regulation of glucose metabolism and metabolic state is crucial for enhancing process productivity and product quality.

The Madin–Darby Canine Kidney (MDCK) cell line is among the most commonly used cell lines for the production of influenza virus vaccines. As cell culture-based manufacturing is poised to replace egg-based processes, increasing virus production is of paramount importance. To shed light on factors affecting virus productivity, we isolated a subline, H1, which had twice the influenza virus A (IAV) productivity of the parent (P) through cell cloning, and characterized H1 and P in detail on both physical and molecular levels. Transcriptome analysis revealed that within a few hours after IAV infection, viral mRNAs constituted over one fifth of total mRNA, with several viral genes more highly expressed in H1 than P. Functional analysis of the transcriptome dynamics showed that H1 and P responded similarly to IAV infection, and were both subjected to host shutoff and inflammatory responses. Importantly, H1 was more active in translation and RNA processing intrinsically and after infection. Furthermore, H1 had more subdued inflammatory and antiviral responses. Taken together, we postulate that the high productivity of IAV hinges on the balance between suppression of host functions to divert cellular resources and the sustaining of sufficient activities for virus replication. Mechanistic insights into virus productivity can facilitate the process optimization and cell line engineering for advancing influenza vaccine manufacturing.

Blastocyst complementation combined with gene editing is an emerging approach in the field of regenerative medicine that could potentially solve the worldwide problem of organ shortages for transplantation. In theory, blastocyst complementation can generate fully functional human organs or tissues, grown within genetically engineered livestock animals. Targeted deletion of a specific gene(s) using gene editing to cause deficiencies in organ development can open a niche for human stem cells to occupy, thus generating human tissues. Within this review, we will focus on the pancreas, liver, heart, kidney, lung, and skeletal muscle, as well as cells of the immune and nervous systems. Within each of these organ systems, we identify and discuss (i) the common causes of organ failure; (ii) the current state of regenerative therapies; and (iii) the candidate genes to knockout and enable specific exogenous organ development via the use of blastocyst complementation. We also highlight some of the current barriers limiting the success of blastocyst complementation.

Enterococcal pheromone responsive conjugative plasmids like pCF10 promote horizontal spread of antibiotic resistance genes following induction of plasmid-containing cells by potential recipients. Transcription of conjugation genes from promoter P(Q) is inhibited by the master regulator PrgX, further repressed when PrgX is in complex with the inhibitory I peptide, and allowed when PrgX is in complex with the C inducing peptide. Single cell analysis has shown that heterogeneity in the pheromone response is prevalent. Here, we systematically varied levels of regulatory molecules to better understand why some individual cells have increased propensity for induction. In this study, PrgX was confirmed to repress P(Q) in the absence of exogenous peptides in vivo, but cells with increased levels of PrgX were shown to be more prone to induction. Further, ablation of endogenous I reduced PrgX levels, resulting in reduced basal repression and loss of inducibility. Reduction of both endogenous peptides by washing increased the inducibility of cells. Together, these results show that endogenous PrgX, C, and I levels can impact the induction potential of a cell and establish the importance of basal I for regulation. These results also suggest that PrgX/C complexes may directly activate prgQ transcription, contrary to a long-standing working model.

Mucin-type O-glycans have profound effects on the structure and stability of glycoproteins. O-Glycans on the cell surface proteins also modulate the cell's interactions with the surrounding environments and other cells. The synthetic pathway of O-glycans involves a large number of enzymes with diverse substrate specificity. The expression pattern of these enzymes is cell and tissue-specific, thus making the pathway highly diverse. To facilitate pathway analysis in a cell and tissue-specific fashion, we developed an integrated platform of RING (Rule Input Network Generator) and O-GlycoVis. RING uses an English-like reaction language to describe the substrate specificity of enzymes and additional constraints on the formation of the glycan products. Using this information, the RING generates a list of possible glycans, which is used as input into O-Glycovis. O-GlycoVis displays the glycan distribution in the pathway and potential reaction paths leading to each glycan. With the input glycan data, O-GlycoVis also traces all possible reaction paths leading to each glycan and outputs pathway maps with the relative abundance levels of glycans overlaid. O-Glycan profiles from two breast cancer cell lines, MCF7 and T47d, human umbilical vascular endothelium cells, Chinese Hamster Ovary cells were generated based on transcriptional data and compared with experimentally observed O-glycans. This RING-based program allows rules to be added or subtracted for network generation and visualization of networks of O-glycosylation network of different tissues and species.

Influenza A virus (IAV) is a respiratory pathogen that has caused significant mortality throughout history and remains a global threat to human health. Although much is known about IAV replication, the regulation of IAV replication dynamics is not completely understood., Influenza A virus (IAV) is a segmented negative-sense RNA virus and is the cause of major epidemics and pandemics. The replication of IAV is complex, involving the production of three distinct RNA species, namely mRNA, cRNA, and viral RNA (vRNA), for all eight genome segments. While understanding IAV replication kinetics is important for drug development and improving vaccine production, current methods for studying IAV kinetics have been limited by the ability to detect all three different RNA species in a scalable manner. Here, we report the development of a novel pipeline using total stranded RNA sequencing (RNA-Seq), which we named influenza virus enumerator of RNA transcripts (InVERT), that allows for the simultaneous quantification of all three RNA species produced by IAV. Using InVERT, we provide a full landscape of the IAV replication kinetics and found that different groups of viral genes follow different kinetics. The segments coding for RNA-dependent RNA polymerase (RdRP) produced more vRNA than mRNA, while some other segments (NP, NS, and hemagglutinin [HA]) consistently made more mRNA than vRNA. vRNA expression levels did not correlate with cRNA expression, suggesting complex regulation of vRNA synthesis. Furthermore, by studying the kinetics of a virus lacking the capacity to generate new polymerase complexes, we found evidence that further supports a model in which cRNA synthesis requires newly synthesized RdRP and that incoming RdRP can only generate mRNA. Overall, InVERT is a powerful tool for quantifying IAV RNA species to elucidate key features of IAV replication. IMPORTANCE Influenza A virus (IAV) is a respiratory pathogen that has caused significant mortality throughout history and remains a global threat to human health. Although much is known about IAV replication, the regulation of IAV replication dynamics is not completely understood. This is due in part to both technical limitations and the complicated replication of the virus, which has a segmented genome and produces three distinct RNA species for each gene segment. We developed a new approach that allows the methodical study of IAV replication kinetics, shedding light on many interesting features of IAV replication biology. This study advances our understanding of the kinetics of IAV replication and will help to facilitate future research in the field.

Additional file of Genome-wide inference of regulatory networks in Streptomyces coelicolor

Authors’ original file for figure 4

Additional file 8: Additional network modules enriched and with consensus sequence. Network modules 20, 45, 636, and 691 enriched in a protein class and a GO term and containing a consensus sequence in all of its members (see Table 2and additional file7). (PDF 250 KB)

Cell culture processes are used to produce the vast majority of protein therapeutics, valued at over US$180 billion per annum worldwide. For more than a decade now, these processes have become highly productive. To further enhance capital efficiency, there has been an increase in the adoption of disposable apparatus and continuous processing, as well as a greater exploration of in-line sensing, various -omic tools, and cell engineering to enhance process controllability and product quality consistency. These feats in cell culture processing for protein biologics will help accelerate the bioprocess advancements for virus and cell therapy applications.

In cell culture processes cell growth and metabolism drive changes in the chemical environment of the culture. These environmental changes elicit reactor control actions, cell growth response, and are sensed by cell signaling pathways that influence metabolism. The interplay of these forces shapes the culture dynamics through different stages of cell cultivation and the outcome greatly affects process productivity, product quality, and robustness. Developing a systems model that describes the interactions of those major players in the cell culture system can lead to better process understanding and enhance process robustness. Here we report the construction of a hybrid mechanistic-empirical bioprocess model which integrates a mechanistic metabolic model with subcomponent models for cell growth, signaling regulation, and the bioreactor environment for in silico exploration of process scenarios. Model parameters were optimized by fitting to a dataset of cell culture manufacturing process which exhibits variability in metabolism and productivity. The model fitting process was broken into multiple steps to mitigate the substantial numerical challenges related to the first-principles model components. The optimized model captured the dynamics of metabolism and the variability of the process runs with different kinetic profiles and productivity. The variability of the process was attributed in part to the metabolic state of cell inoculum. The model was then used to identify potential mitigation strategies to reduce process variability by altering the initial process conditions as well as to explore the effect of changing CO2 removal capacity in different bioreactor scales on process performance. By incorporating a mechanistic model of cell metabolism and appropriately fitting it to a large dataset, the hybrid model can describe the different metabolic phases in culture and the variability in manufacturing runs. This approach of employing a hybrid model has the potential to greatly facilitate process development and reactor scaling.

For high-frequency transfer of pCF10 between E. faecalis cells, induced expression of the pCF10 genes encoding conjugative machinery from the prgQ operon is required. This process is initiated by the cCF10 (C) inducer peptide produced by potential recipient cells. The expression timing of prgB, an “early” gene just downstream of the inducible promoter, has been studied extensively in single cells. However, several previous studies suggest that only 1 to 10% of donors induced for early prgQ gene expression actually transfer plasmids to recipients, even at a very high recipient population density. One possible explanation for this is that only a minority of pheromone-induced donors actually transcribe the entire prgQ operon. Such cells would not be able to functionally conjugate but might play another role in the group behavior of donors. Here, we sought to (i) simultaneously assess the presence of RNAs produced from the proximal (early induced transcripts [early Q]) and distal (late Q) portions of the prgQ operon in individual cells, (ii) investigate the prevalence of heterogeneity in induced transcript length, and (iii) evaluate the temporality of induced transcript expression. Using fluorescent in situ hybridization chain reaction (HCR) transcript labeling and single-cell microscopic analysis, we observed that most cells expressing early transcripts (Q(L), prgB, and prgA) also expressed late transcripts (prgJ, pcfC, and pcfG). These data support the conclusion that, after induction is initiated, transcription likely extends through the end of the conjugation machinery operon for most, if not all, induced cells. IMPORTANCE In Enterococcus faecalis, conjugative plasmids like pCF10 often carry antibiotic resistance genes. With antibiotic treatment, bacteria benefit from plasmid carriage; however, without antibiotic treatment, plasmid gene expression may have a fitness cost. Transfer of pCF10 is mediated by cell-to-cell signaling, which activates the expression of conjugation genes and leads to efficient plasmid transfer. Yet, not all donor cells in induced populations transfer the plasmid. We examined whether induced cells might not be able to functionally conjugate due to premature induced transcript termination. Single-cell analysis showed that most induced cells do, in fact, express all of the genes required for conjugation, suggesting that premature transcription termination within the prgQ operon does not account for failure of induced donor cell gene transfer.

The vast majority of cells isolated from tissues of mammals are anchorage-dependent, meaning that they require surface adhesion in order to multiply. Cells that are subject to senescence thus appear to “count” their number of doublings. Senescence is regulated by cellular events and is often thought to be related to aging. Cells isolated from normal or cancerous differentiated tissues often retain their differentiated properties to different extents depending on the chemical and physical environment. Stem cells must be capable of self-renewal and differentiation into their target cells. The cells commonly used for the production of biologics are derived from different tissues of different species. Cells isolated from different species differ in their chromosome number and genome sequence. The cellular membrane is in a dynamic state; membrane constituents are continuously being added and removed. This is not only for membrane expansion and cell growth, but also for turnover and vesicle trafficking.

Cultured mammalian cells are the workhorse for the manufacturing of the class of pharmaceuticals known as biologics, including viral vaccines and many protein medicines. Critical to cell culture advances was the arrival of membrane-filtration-based medium sterilization, first by ultrafiltration with a nitrocellulose membrane and later by microfiltration. The arrival of recombinant DNA technology in the 1970s made the production of various protein reagents a simpler task. It expanded the repertoire of biochemical reagents and greatly increased our ability to culture cells that require growth factors and cytokines. The most effective way to fight viral diseases has been widespread immunization by vaccines. The majority of viral vaccines are produced in cell culture. Proteins derived from blood, tissues, and cell cultures were used for therapeutic purposes long before the arrival of recombinant DNA technology. The basic process flowsheet for cell culture manufacturing is almost identical to that for microbial fermentation.

This chapter provides succinct descriptions of the roles of key medium components in supporting cell growth. Early in the industrialization of cell culture, media design efforts focused on enhancing cell proliferation and on eliminating animal serum or other animal-derived media components. The use of serum-free and animal component-free media has been extended to the cell line development stage to minimize the exposure of cells to adventitious agents throughout the entire cell banking and manufacturing process. Many medium components that have been traditionally used in cell culture are a complex mixture of different components, such as animal serum, plant hydrolysate, soybean phospholipids, and yeast extract. To streamline the process development of a new product, the medium that gives rise to high productivity for a given product is adopted as a platform medium for future media development for cell lines. Some nonessential amino acids may be secreted by cells and accumulate in culture medium, including alanine, asparagine, glutamic acid, and proline.

This chapter discusses the scaling up of a bioreactor based on geometrical similarity. The stirred tank bioreactor is the standard vessel for growing mammalian cells in industrial processes. Impellers can generally be classified into two types according to the flow pattern they generate: axial flow and radial flow. The purpose of agitation is to provide a homogenous chemical environment. Spatial heterogeneity of nutrients may subject cells to an oscillating environment as they traverse the reactor, leading to inferior growth or production performance. Cells in a stirred bioreactor may encounter shear stress caused by fluid flow, or be subjected to direct impact by moving mechanical parts such as the impeller. The design objective in scaling up a process is to produce product in quantities proportional to the scale. To meet that goal, all nutrients are supplied proportionally to the scale. In scale translation, the relationship of geometry-related physical parameters cannot all be kept constant.

The initial focus, which was on chemical drugs, has extended to protein biologics. The drive was prompted by the success of continuous processes in the chemical industry. Many enzymatic biotransformation, waste treatment, and biodegradation processes have long been operated continuously. When cell culture was first adopted as the production vehicle for biopharmaceuticals, continuous operation was explored as an industrial process. The material balance equations for cells and the rate-limiting substrate reflect the balance of the amount of cell mass or substrate carried into and out of the reactor by the fluid flow. The development of continuous cultures for the manufacturing of protein biologics was driven by the production of recombinant proteins that are labile and produced at low levels. Centrifugation is a standard unit operation in many downstream recovery processes. However, most centrifuges are not designed for long-term continuous and aseptic operations. In the early stages of perfusion culture development, in-line centrifuges were used for intermittent cell recycling.

The cells used in cell culture processes are all derived from animals. Their genetic and epigenetic control circuits and their physiology have evolved to carry out the physiological functions of the tissue from which they were derived, not to sustain themselves and proliferate in in vitro culture. Cell metabolism generates waste metabolites, some of which may become toxic if allowed to accumulate to high levels. A drastic difference between a bioreactor and a human body is the absence of a metabolite-balancing mechanism. The main limiting factor for productivity in a fed-batch operation is the accumulation of metabolites and culmination of other growth inhibitory factors. A key factor in increasing the productivity of a fed-batch culture process is providing a balanced chemical environment that minimizes the production of metabolites and prolongs the time it takes for them to accumulate to inhibitory levels.