Article
Biotechnology & Applied Microbiology
Ngoc Tam Tran, Emilie Lecomte, Sylvie Saleun, Suk Namkung, Cecile Robin, Kristina Weber, Eric Devine, Veronique Blouin, Oumeya Adjali, Eduard Ayuso, Guangping Gao, Magalie Penaud-Budloo, Phillip W. L. Tai
Summary: Over the past two decades, significant progress has been made in the manufacturing of adeno-associated virus (AAV) vectors to meet the production demands for preclinical and clinical trials. However, the presence of empty AAV capsids and particles containing inaccurate vector genomes remains a concern. Different methods have been utilized to separate empty capsids from full particles, but no single technique can completely eliminate empty or partial capsids. This study reveals that vectors produced by different manufacturing systems exhibit varying degrees of truncated and unresolved species, and purified empty particles actually contain genomes composed of a single truncated and/or unresolved inverted terminal repeat (ITR). These findings provide valuable insights into the efficacy, safety, and quantification of clinical vectors.
HUMAN GENE THERAPY
(2022)
Article
Multidisciplinary Sciences
Matthew J. Byrne, Matthew G. Iadanza, Marcos Arribas Perez, Daniel P. Maskell, Rachel M. George, Emma L. Hesketh, Paul A. Beales, Marc D. Zack, Colin Berry, Rebecca F. Thompson
Summary: Insect pests are a major cause of crop losses worldwide, amounting to an estimated economic cost of $470 billion annually. Biotechnological tools, such as transgenic plants harboring genes encoding insecticidal proteins, have been introduced to control these pests. The Vip3 family proteins from Bacillus thuringiensis are believed to exert their insecticidal activity through pore formation on the membrane. Researchers have presented cryo-EM structures of a Vip3 family toxin in both inactive and activated forms, demonstrating the pore-forming conformation of activated Vip3Bc1 on the membrane.
NATURE COMMUNICATIONS
(2021)
Article
Genetics & Heredity
Mafalda M. Dias, Joao Vidigal, Daniela P. Sequeira, Paula M. Alves, Ana P. Teixeira, Antonio Roldao
Summary: The study successfully established a stable master clonal Hi5 cell line that can flexibly express single or multiple proteins of interest, using RMCE and FACS technologies.
G3-GENES GENOMES GENETICS
(2021)
Article
Multidisciplinary Sciences
Tomoko Kamasaki, Yumi Miyazaki, Susumu Ishikawa, Kazuya Hoshiba, Keisuke Kuromiya, Nobuyuki Tanimura, Yusuke Mori, Motosuke Tsutsumi, Tomomi Nemoto, Ryota Uehara, Shiro Suetsugu, Toshiki Itoh, Yasuyuki Fujita
Summary: Research has found that characteristic finger-like membrane protrusions are projected from both normal and RasV12 cells at their interface, and FBP17 protein accumulates in RasV12 cells and surrounding normal cells, playing a positive role in the formation of these protrusions and the apical elimination of RasV12 cells. It is also shown that cdc42 acts upstream of these processes, indicating that the cdc42/FBP17 pathway is a crucial trigger of cell competition.
Article
Fisheries
B. S. Anoop, Jayesh Puthumana, Cijo George Vazhappilly, Salini Kombiyil, Rosamma Philip, Anas Abdulaziz, Isaac Sarojini Bright Singh
Summary: The study successfully developed a novel hybrid cell line by fusing shrimp lymphoid organ cells with Sf9 cells, achieving success after numerous attempts. This cell line contains genomic components from both shrimp and Sf9, and is envisioned to serve as a model system for biomedical research in vitro.
FISH & SHELLFISH IMMUNOLOGY
(2021)
Article
Biochemistry & Molecular Biology
Chuan Chen, Zening Wang, Minhyo Kang, Ki Baek Lee, Xin Ge
Summary: A cell cycle arrested recombinase-mediated cassette exchange (aRMCE) technology is developed to construct large monoclonal combinatorial libraries, with great potentials in various applications. The optimized method achieves high efficiency in target gene replacement and successfully demonstrates directed evolution of fragment crystallizable (Fc).
NUCLEIC ACIDS RESEARCH
(2023)
Review
Biotechnology & Applied Microbiology
Marianna Teixeira de Pinho Favaro, Jan Atienza-Garriga, Carlos Martinez-Torro, Eloi Parlade, Esther Vazquez, Jose Luis Corchero, Neus Ferrer-Miralles, Antonio Villaverde
Summary: The recent outbreaks of Ebola, avian influenza, monkeypox, and the COVID-19 pandemic have highlighted the urgent need for effective and cost-effective vaccines against infectious diseases. New DNA or RNA-based vaccination strategies have shown promise, but their long-term protection needs to be evaluated. Subunit vaccines based on recombinant proteins obtained from genetic engineering also have potential for development.
MICROBIAL CELL FACTORIES
(2022)
Article
Genetics & Heredity
Daniel Mariyappa, Arthur Luhur, Danielle Overton, Andrew C. Zelhof
Summary: Generating stable Drosophila cell lines is essential for in vivo experiments and genetic screens, with recent advances utilizing the RMCE process through the attP/PhiC31 integrase system. However, current techniques are limited to a few embryonic cell lines, prompting the need for a more universal method. The combination of CRISPR/Cas9 and the PhiC31 integrase system has shown promising results in efficiently generating stable transgenic cassettes in various existing Drosophila cell lines.
G3-GENES GENOMES GENETICS
(2021)
Article
Biotechnology & Applied Microbiology
Henry de Malmanche, Mazhar Hussain, Esteban Marcellin, Steve Reid, Sassan Asgari
Summary: The Sf9 cell line, widely used for the production of recombinant proteins and virus-like particles, has been found to use RNA interference (RNAi) as an antiviral response to baculovirus infection. Knocking out Dicer-2, a key protein in the RNAi pathway, resulted in increased viral replication in Sf9 cells, indicating a potential role for RNAi in managing chronic viral infections in this cell line.
JOURNAL OF GENERAL VIROLOGY
(2022)
Article
Biochemistry & Molecular Biology
Swagata Roy, Megha Sravani Bondada, Yaoyao Zhang, Katy Moffat, Venugopal Nair, Yongxiu Yao
Summary: This study utilized CRISPR-Cas9 technology to perform targeted editing of the integrated proviral long terminal repeat (LTR) sequence, and found that LTR deletion resulted in significant reduction of gene expression, including c-myc, and induced cell apoptosis, affecting cell proliferation. Additionally, deletion of c-myc had a major effect on cell proliferation, similar to LTR deletion, indicating the significance of c-myc in ALV-induced lymphomagenesis.
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
(2022)
Review
Biotechnology & Applied Microbiology
Evan Tan, Cara Sze Hui Chin, Zhi Feng Sherman Lim, Say Kong Ng
Summary: Animal cell-based expression platforms are capable of producing complex biomolecules, with human cell lines offering the advantage of more human-like post-translational modifications. HEK293 is widely utilized due to its high transfectivity, rapid growth rate, and ability to grow in a serum-free, suspension culture. This review discusses the use of HEK293 cells in biotherapeutics production, comparing their usage against other host cell lines and summarizing factors influencing the choice of host cell lines.
FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY
(2021)
Article
Food Science & Technology
Laura Franz, Klaus Raming, Ralf Nauen
Summary: This study confirmed the importance of mutations in the extracellular loop 4 of the ATP-binding cassette transporter C2 for resistance to Cry1F in the fall armyworm.
Article
Multidisciplinary Sciences
Meenakshi Venkatesan, Cameron Semper, Stig Skrivergaard, Rosa Di Leo, Nathalie Mesa, Martin Kroyer Rasmussen, Jette Feveile Young, Margrethe Therkildsen, Peter J. Stogios, Alexei Savchenko
Summary: This study presents a cost-effective method for producing functionally active GFs in Escherichia coli, which can significantly reduce the cost of cell culture medium and facilitate the production of cell-cultured meat and tissue engineering.
Article
Biotechnology & Applied Microbiology
Bronwyn Jean Chetty, Kentaro Inokuma, Tomohisa Hasunuma, Willem Heber van Zyl, Riaan den Haan
Summary: Enhanced cellulase activity on the cell surface of engineered strains for CBP was achieved through overexpression of cellulase secretion genes. The improved strains showed significant increases in cellulolytic activity and ethanol yields, making them more efficient for 2G biofuel production.
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
(2022)
Article
Biotechnology & Applied Microbiology
Jose Vicente Carratala, Jan Atienza-Garriga, Hector Lopez-Laguna, Esther Vazquez, Antonio Villaverde, Julieta M. Sanchez, Neus Ferrer-Miralles
Summary: To improve the purification yield and purity of difficult proteins, a novel method has been developed using bile salts to supplement crude cell extracts, resulting in a substantial improvement in protein capture by affinity chromatography.
MICROBIAL CELL FACTORIES
(2023)
Article
Biotechnology & Applied Microbiology
Ana P. Teixeira, Pascal Stucheli, Simon Auslander, David Auslander, Pascal Schonenberger, Samuel Hurlemann, Martin Fussenegger
Summary: The researchers have developed an antibiotic-free selection system for generating stable transgenic cell lines, which utilizes cellobiose as the primary energy source. By combining this system with a PiggyBac transposase-based integration strategy, they were able to efficiently generate stable transgenic cell lines that expressed high levels of cargo proteins. This strategy was successfully validated by integrating sequences encoding two biopharmaceutical proteins, erythropoietin and the monoclonal antibody rituximab, and confirmed their efficient production in both cellobiose- and glucose-containing medium.
METABOLIC ENGINEERING
(2022)
Correction
Genetics & Heredity
Mafalda M. Dias, Joao Vidigal, Daniela P. Sequeira, Paula M. Alves, Ana P. Teixeira, Antonio Roldao
G3-GENES GENOMES GENETICS
(2022)
Article
Chemistry, Medicinal
Diana Fontinha, Francisca Arez, Isabella Ramella Gal, Goncalo Nogueira, Diana Moita, Tobias Hyun Ho Baeurle, Catarina Brito, Thomas Spangenberg, Paula M. Alves, Miguel Prudencio
Summary: Combination therapy is effective in reducing drug resistance in malaria. The combination of M5717 and pyronaridine shows potent activity against blood stage Plasmodium infection. Additionally, pyronaridine enhances the activity of M5717 against hepatic infection. These findings support the clinical evaluation of pyronaridine as a partner drug for M5717.
ACS INFECTIOUS DISEASES
(2022)
Article
Chemistry, Multidisciplinary
Ana F. Louro, Marta A. Paiva, Marta R. Oliveira, Katharina A. Kasper, Paula M. Alves, Patricia Gomes-Alves, Margarida Serra
Summary: This study isolated extracellular vesicles (EV) from key stages of human induced pluripotent stem cell-cardiomyocyte differentiation and identified hiPSC as a promising cell biofactory for EV production. The findings demonstrate that hiPSC-EV have pro-angiogenic and pro-proliferative properties, suggesting their potential in cardiac regenerative applications.
Article
Pharmacology & Pharmacy
Barbara Fernandes, Rute Castro, Farien Bhoelan, Denzel Bemelman, Ricardo A. Gomes, Julia Costa, Patricia Gomes-Alves, Toon Stegmann, Mario Amacker, Paula M. Alves, Sylvain Fleury, Antonio Roldao
Summary: This study developed a scalable bioprocess using the insect cells-baculovirus expression vector system (IC-BEVS) to produce high-quality S protein. The insect cell-derived S protein exhibited glycan processing similar to mammalian cells and mid-term stability upon storage. The results demonstrate the potential of using IC-BEVS to produce the highly glycosylated S protein for a virosome-based COVID-19 vaccine candidate.
Article
Biotechnology & Applied Microbiology
Barbara Fernandes, Marcos Sousa, Rute Castro, Anja Schaefer, Julia Hauser, Kai Schulze, Mario Amacker, Marco Tamborrini, Gerd Pluschke, Paula M. Alves, Sylvain Fleury, Antonio Roldao
Summary: This study explores different insect cell lines, culture conditions, and purification strategies to develop a scalable, high-yield, and high-quality Plasmodium falciparum candidate antigen for a malaria vaccine. Results show that adding antioxidants improves PfCyRPA production, and the 4x-His affinity tag has the highest purification recovery and production yield. Additionally, PfCyRPA expressed in insect cells shows no difference in protein quality and stability compared to expression in human cells.
FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY
(2022)
Article
Physiology
Ana F. Louro, Nikolaus Virgolini, Marta A. Paiva, Ines A. Isidro, Paula M. Alves, Patricia Gomes-Alves, Margarida Serra
Summary: Extracellular Vesicles (EV) play a critical role in regulating regenerative processes in wounded tissues through cell-to-cell communication. This study investigated the piRNA profiles of EV derived from different stages of differentiation and maturation, and found significant differences between piRNA and miRNA expression. Only a small number of piRNA were found to be deregulated in EV. These results provide valuable information for further studying the role of piRNA in the bioactivity and therapeutic potential of EV.
FRONTIERS IN PHYSIOLOGY
(2022)
Article
Biotechnology & Applied Microbiology
Tobias Strittmatter, Viktor Haellman, Paul Argast, Peter Buchman, Ana Palma Teixeira, Martin Fussenegger
Summary: Mechanical cues play a role in embryonic development and patterning. The effects of mechanical stress on the differentiation of human induced pluripotent stem cells (hiPSCs) were studied, and it was found that mechanical stress alters lineage commitment and reduces Wnt signaling.
BIOTECHNOLOGY PROGRESS
(2023)
Article
Biochemistry & Molecular Biology
Adrian Bertschi, Bozhidar-Adrian Stefanov, Shuai Xue, Ghislaine Charpin-El Hamri, Ana Palma Teixeira, Martin Fussenegger
Summary: Precise control of therapeutic protein delivery is crucial for gene- and cell-based therapies. A mammalian synthetic gene switch has been developed that regulates transgene expression through exposure to acetoin, a semi-volatile small molecule. This gene switch shows dose-dependent activation of downstream transgenes in response to acetoin, while intake of typical acetoin-containing foods does not activate transgene expression. Delivery of trigger molecules through portable inhalers may enable regular administration of therapeutic proteins for chronic diseases.
NUCLEIC ACIDS RESEARCH
(2023)
Review
Genetics & Heredity
Sofia Fernandes, Joana Diogo, Ana Sofia Coroadinha
Summary: Adeno-associated viruses (AAV) are widely used in gene therapy due to their non-pathogenicity, ability to transduce both dividing and non-dividing cells, and flexibility in targeting different tissues. The success of AAV gene therapy has been demonstrated by the approval of three products. To meet the requirements of high dosage, safety, and reproducibility in clinical trials, production platforms based on stable mammalian cell lines have been proposed as the best strategy, although the methodologies employed may result in different productivities.
CURRENT GENE THERAPY
(2023)
Article
Biochemistry & Molecular Biology
Ana Sofia Moreira, Sandra Bezemer, Tiago Q. Faria, Frank Detmers, Pim Hermans, Laurens Sierkstra, Ana Sofia Coroadinha, Cristina Peixoto
Summary: The use of affinity chromatography can simplify the purification process of viral vectors, resulting in high purity clinical-grade products. This technology contributes to increasing downstream process efficiency, reducing time-to-market.
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
(2023)
Article
Biochemistry & Molecular Biology
Adrian Bertschi, Pengli Wang, Silvia Galvan, Ana Palma Teixeira, Martin Fussenegger
Summary: We constructed a framework for multi-input logic gates based on inducible protein-protein interactions by utilizing the modular structure of bacterial transcription factors. We were able to convert gene switches from OFF-type to more widely applicable ON-type systems and create mammalian gene switches responsive to new inducers. By combining both OFF and ON modes of action, we built a compact and high-performance bandpass filter, and demonstrated cytosolic and extracellular dimerization. Cascading up to five pairwise fusion proteins yielded robust multi-input AND logic gates, and combinations of different pairwise fusion proteins afforded a variety of 4-input 1-output AND and OR logic gate configurations.
NATURE CHEMICAL BIOLOGY
(2023)
Review
Cell Biology
Ana Sofia Coroadinha
Summary: Gene therapy relies on the delivery of genetic material to the patient's cells to provide therapeutic treatment. Lentiviral (LV) and adeno-associated virus (AAV) vectors are two commonly used and efficient delivery systems. However, the therapeutic efficacy of these vectors is hindered by host restriction factors (RFs) that block their transduction. In this review, we discuss the identified RFs that impede LV and AAV vector transduction and limit their therapeutic effectiveness.
Article
Biochemical Research Methods
Tiago A. Vaz, Ana F. Rodrigues, Ana S. Coroadinha
Summary: This study improves the production of lentiviral vectors (LV) for gene therapy by optimizing cell culture conditions and transfection parameters. By adapting 293T cells to serum-free suspension cultures, increasing cell density, adding nutrient boosters, and optimizing transfection conditions, the LV yield was significantly increased.
BIOTECHNOLOGY JOURNAL
(2023)