Recombinant adeno-associated virus (rAAV) is one of the most widely used viral vectors for gene therapy. It is used in very high doses for the treatment of many diseases, making large-scale production for clinical applications challenging. We have established a synthetic biology-based platform to construct stable production cell lines, which can be induced to produce rAAV2.
View Article and Find Full Text PDFRecombinant adeno-associated virus (rAAV) is a widely used viral vector in gene therapy. To meet the growing clinical demand, a scalable production technology which can efficiently produce high-quality products is required. We have developed a synthetic biology strategy to generate HEK293-based cell lines which have integrated essential AAV and adenoviral helper genes and are capable of producing rAAV upon induction.
View Article and Find Full Text PDFAppl Microbiol Biotechnol
June 2024
Recombinant adeno-associated virus (rAAV) is a major gene delivery vehicle. We have constructed a stable rAAV producer cell line by integrating essential rAAV genome, viral and helper genes into the genome of HEK293 cell under the control of inducible promoters. Upon induction, the cell line produces transducing rAAV.
View Article and Find Full Text PDFBiotechnol Bioeng
September 2024
Mammalian cells are commonly used as hosts in cell culture for biologics production in the pharmaceutical industry. Structured mechanistic models of metabolism have been used to capture complex cellular mechanisms that contribute to varying metabolic shifts in different cell lines. However, little research has focused on the impact of temporal changes in enzyme abundance and activity on the modeling of cell metabolism.
View Article and Find Full Text PDFRecombinant adeno-associated virus (rAAV) is widely used as an in vivo delivery vector for gene therapy. It is used in a very large dose, and the large quantities required for broad applications present manufacturing challenges. We have developed a synthetic biology platform of constructing cell lines integrated with essential viral genes which can be induced to produce rAAV without plasmid transfection or virus transduction.
View Article and Find Full Text PDFRecombinant adeno-associated virus (rAAV) is among the most commonly used vectors for gene therapy. It is commonly produced by transfection of HEK293 cells with three plasmids each containing the vector genome including gene of interest (GOI), helper functions, and rep and cap genes for genome replication and capsid formation. To meet the potential clinical needs, the productivity of the production system needs to be enhanced.
View Article and Find Full Text PDFInfluenza A virus (IAV) is a negative-sense RNA virus that causes seasonal infections and periodic pandemics, inflicting huge economic and human costs on society. The current production of influenza virus for vaccines is initiated by generating a seed virus through the transfection of multiple plasmids in HEK293 cells followed by the infection of seed viruses into embryonated chicken eggs or cultured mammalian cells. We took a system design and synthetic biology approach to engineer cell lines that can be induced to produce all viral components except hemagglutinin (HA) and neuraminidase (NA), which are the antigens that specify the variants of IAV.
View Article and Find Full Text PDFRecombinant adeno-associated virus (rAAV) is among the most commonly used in vivo gene delivery vehicles and has seen a number of successes in clinical application. Current manufacturing processes of rAAV employ multiple plasmid transfection or rely on virus infection and face challenges in scale-up. A synthetic biology approach was taken to generate stable cell lines with integrated genetic modules, which produced rAAV upon induction albeit at a low productivity.
View Article and Find Full Text PDFCell therapy offers the potential for curative treatment of cancers. Although T cells have been the predominantly used cell type, natural killer (NK) cells have attracted great attention owing to their ability to kill cancer cells and because they are naturally suitable for allogeneic applications. Upon stimulation by cytokines or activation by a target cell, NK cells proliferate and expand their population.
View Article and Find Full Text PDFAn 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 (Ad), which requires helper virus preparation and introduces additional variability. A simple method that has high sensitivity and removes the need for helper virus would improve assay consistency and facilitate high-throughput applications such as rAAV producer cell line development.
View Article and Find Full Text PDFRecombinant 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.
View Article and Find Full Text PDFOver 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.
View Article and Find Full Text PDFInfluenza A virus (IAV) infection causes acute respiratory disease with potential severe and deadly complications. Viral pathogenesis is not only due to the direct cytopathic effect of viral infections but also to the exacerbated host inflammatory responses. Influenza viral infection can activate various host signaling pathways that function to activate or inhibit viral replication.
View Article and Find Full Text PDFis considered a leading cause of hospital-acquired infections. Treatment of these infections has become a major challenge for clinicians because some strains are resistant to multiple clinically used antibiotics. Moreover, the presence of biofilms can make infections with more difficult to eradicate with current antibiotic therapies.
View Article and Find Full Text PDFThe 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.
View Article and Find Full Text PDFEnterococcus faecalis, a member of the commensal flora in the human gastrointestinal tract, has become a threatening nosocomial pathogen because it has developed resistance to many known antibiotics. More concerningly, resistance gene-carrying E. faecalis cells may transfer antibiotic resistance to resistance-free E.
View Article and Find Full Text PDFIn 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.
View Article and Find Full Text PDFInfluenza 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; mRNA, cRNA, and 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 has been limited by the ability to detect all three different RNA species in a scalable manner.
View Article and Find Full Text PDFBiotechnol Bioeng
May 2021
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.
View Article and Find Full Text PDFCell 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.
View Article and Find Full Text PDFMammalian 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.
View Article and Find Full Text PDFPredicting drug-induced liver injury in a preclinical setting remains challenging, as cultured primary human hepatocytes (PHHs), pluripotent stem cell-derived hepatocyte-like cells (HLCs), and hepatoma cells exhibit poor drug biotransformation capacity. We here demonstrate that hepatic functionality depends more on cellular metabolism and extracellular nutrients than on developmental regulators. Specifically, we demonstrate that increasing extracellular amino acids beyond the nutritional need of HLCs and HepG2 cells induces glucose independence, mitochondrial function, and the acquisition of a transcriptional profile that is closer to PHHs.
View Article and Find Full Text PDFFor high-frequency transfer of pCF10 between cells, induced expression of the pCF10 genes encoding conjugative machinery from the operon is required. This process is initiated by the cCF10 (C) inducer peptide produced by potential recipient cells. The expression timing of an "early" gene just downstream of the inducible promoter, has been studied extensively in single cells.
View Article and Find Full Text PDFDuring the development of cell lines for therapeutic protein production, a vector harboring a product transgene is integrated into the genome. To ensure production stability and consistent product quality, single-cell cloning is then performed. Since cells derived from the same parental clone have the same transgene integration locus, the identity of the integration site can also be used to verify the clonality of a production cell line.
View Article and Find Full Text PDFChinese hamster ovary (CHO) cells are used for industrial production of protein-based therapeutics (i.e., "biologics").
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