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Trending Papers in biotechnology

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305
Published: Jun 2018
Published: Jun 2018
  • The goal of this synthetic circuit is to fine-tune therapeutic transgene expression in response to the routine intake of beverages, such as tea and coffee without supplementation of any additional chemicals.
  • This paper describes the design of a caffeine-inducible gene switch
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Daniel Bojar
14
From Paper: A monogenic and fast-responding Light-Inducible Cre recombinase as a novel optogenetic switch
From Paper: A monogenic and fast-responding Light-Inducible Cre recombinase as a novel optogenetic switch
  • Compared to other light-inducible Cre recombinases, LiCre displayed faster and stronger activation by light as well as a lower residual activity in the dark
  • This paper reports the development of LiCre, a novel light-inducible Cre recombinase - that allows for toggleable gene expression only in tissues actively exposed to blue light
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Patrick Joyce
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Authors: Daniel Bojar, Martin Fussenegger
Published: Jan 2020
Authors: Daniel Bojar, Martin Fussenegger
Published: Jan 2020
Engineered proteins with enhanced or altered functionality, generated for example by mutation or domain fusion, are at the core of nearly all synthetic biology endeavors in the context of precision medicine, also known as personalized medicine. From designer receptors sensing elevated blood markers to effectors rerouting signaling pathways to synthetic transcription factors and the customized therapeutics they regulate, engineered proteins play a crucial role at every step of novel therapeutic approaches using synthetic biology. Here, recent developments in protein engineering aided by advances in directed evolution, de novo design, and machine learning are discussed. Building on clinical successes already achieved with chimeric antigen receptor (CAR-) T cells and other cell-based therapies, these developments are expected to further enhance the capabilities of mammalian synthetic biology in biomedical and other applications.
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Daniel Bojar
2
Authors: Daniel Bojar, Martin Fussenegger
Published: Jan 2018
Authors: Daniel Bojar, Martin Fussenegger
Published: Jan 2018
Precise control of gene expression with small-molecular or physical inducers has been a central aim of synthetic biology in recent decades, and has led, for example, to dramatic improvements in the production of protein therapeutics. However, the number of inputs for inducible promoters and gene circuits is still limited, and new orthogonal inducers are needed to support the construction of more complex programmable systems in the fields of bioengineering and biocomputing. Here, two new gene switches inducible by the antibiotic simocyclinone D8 (SD8) and the flavonoid luteolin in mammalian cells are presented. Streptomyces antibioticus Tü 6040 has evolved the DNA gyrase inhibitor SD8, which, through the bacterial TetR-like transcriptional repressor SimR, also regulates the multidrug efflux pump SimX. Taking advantage of SimR, as well as the luteolin-binding TetR-like transcriptional repressor EmrR from Sinorhizobium meliloti, we optimized the amounts of transfected genes and DNA operator sites to engineer highly effective, orthogonal transcriptional OFF- as well as ON-switches triggered by SD8 and/or luteolin. We confirmed that SD8 and luteolin are not cytotoxic at the concentrations required for switching, and demonstrated the functionality of these gene switches in a range of biotechnologically relevant cell lines. These switches were combined to generate OR and AND Boolean logic gates, and we confirmed their modularity by the addition of the vanillic acid-responsive transcriptional repressor VanR to generate a three-input AND gate. These additions to the panoply of inducers available for synthetic biology are expected to facilitate advances in the fields of biocomputing, biopharmaceutical manufacturing, and biomedicine. © 2018 The Authors. AIChE Journal published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers. AIChE J, 64: 4237–4246, 2018
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Daniel Bojar
2
Authors: Leo Scheller, Tobias Strittmatter, David Fuchs, Daniel Bojar, Martin Fussenegger
Published: Jul 2018
Authors: Leo Scheller, Tobias Strittmatter, David Fuchs, Daniel Bojar, Martin Fussenegger
Published: Jul 2018
Strategies for expanding the sensor space of designer receptors are urgently needed to tailor cell-based therapies to respond to any type of medically relevant molecules. Here, we describe a universal approach to designing receptor scaffolds that enables antibody-specific molecular input to activate JAK/STAT, MAPK, PLCG or PI3K/Akt signaling rewired to transgene expression driven by synthetic promoters. To demonstrate its scope, we equipped the GEMS (generalized extracellular molecule sensor) platform with antibody fragments targeting a synthetic azo dye, nicotine, a peptide tag and the PSA (prostate-specific antigen) biomarker, thereby covering inputs ranging from small molecules to proteins. These four GEMS devices provided robust signaling and transgene expression with high signal-to-noise ratios in response to their specific ligands. The sensitivity of the nicotine- and PSA-specific GEMS devices matched the clinically relevant concentration ranges, and PSA-specific GEMS were able to detect pathological PSA levels in the serum of patients diagnosed with prostate cancer.
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Daniel Bojar
2
Authors: Pratik Saxena, Daniel Bojar, Henryk Zulewski, Martin Fussenegger
Published: Oct 2017
Authors: Pratik Saxena, Daniel Bojar, Henryk Zulewski, Martin Fussenegger
Published: Oct 2017
We previously reported novel technology to differentiate induced pluripotent stem cells (IPSCs) into glucose-sensitive insulin-secreting beta-like cells by engineering a synthetic lineage-control network regulated by the licensed food additive vanillic acid. This genetic network was able to program intricate expression dynamics of the key transcription factors Ngn3 (neurogenin 3, OFF-ON-OFF), Pdx1 (pancreatic and duodenal homeobox 1, ON-OFF-ON) and MafA (V-maf musculoaponeurotic fibrosarcoma oncogene homologue A, OFF-ON) to guide the differentiation of IPSC-derived pancreatic progenitor cells to beta-like cells. In the present study, we show for the first time that this network can also program the expression dynamics of Ngn3, Pdx1 and MafA in human embryonic stem cell (hESC)-derived pancreatic progenitor cells and drive differentiation of these cells into glucose-sensitive insulin-secreting beta-like cells. Therefore, synthetic lineage-control networks appear to be a robust methodology for differentiating pluripotent stem cells into somatic cell types for basic research and regenerative medicine.
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Daniel Bojar
2
Authors: Daniel Bojar, Martin Fussenegger
Published: Oct 2016
Authors: Daniel Bojar, Martin Fussenegger
Published: Oct 2016
Synthetic biology has revolutionized the field of biology in the last two decades. By taking apart natural systems and recombining engineered parts in novel constellations, it has not only unlocked a staggering variety of biological control mechanisms but it has also created a panoply of biomedical achievements, such as innovative diagnostics and therapies. The most common mode of action in the field of synthetic biology is mediated by synthetic gene circuits assembled in a systematic and rational manner. This review covers the most recent therapeutic gene circuits implemented in mammalian and bacterial cells designed for the diagnosis and therapy of an extensive array of diseases. Highlighting new tools for therapeutic gene circuits, we describe a future that holds a plethora of potentialities for the medicine of tomorrow.
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Daniel Bojar
2
Authors: Daniel Bojar, Tobias Fuhrer, Martin Fussenegger
Published: Mar 2019
Authors: Daniel Bojar, Tobias Fuhrer, Martin Fussenegger
Published: Mar 2019
Capitalizing on the ability of mammalian cells to conduct complex post-translational modifications, most protein therapeutics are currently produced in cell culture systems. Addition of a signal peptide to the product protein enables its accumulation in the cell culture supernatant, but separation of the product from endogenously secreted proteins remains costly and labor-intensive. We considered that global downregulation of translation of non-product proteins would be an efficient strategy to minimize downstream processing requirements. Therefore, taking advantage of the ability of mammalian protein kinase R (PKR) to switch off most cellular translation processes in response to infection by viruses, we fused a caffeine-inducible dimerization domain to the catalytic domain of PKR. Addition of caffeine to this construct results in homodimerization and activation of PKR, effectively rewiring rapid global translational downregulation to the addition of the stimulus in a dose-dependent manner. Then, to protect translation of the target therapeutic, we screened viral and cellular internal ribosomal entry sites (IRESes) known or suspected to be resistant to PKR-induced translational stress. After choosing the best-in-class Seneca valley virus (SVV) IRES, we additionally screened for IRES transactivation factors (ITAFs) as well as for supplementary small molecules to further boost the production titer of the product protein under conditions of global translational downregulation. Importantly, the residual global translation activity of roughly 10% under maximal downregulation is sufficient to maintain cellular viability during a production timeframe of at least five days. Standard industrially used adherent as well as suspension-adapted cell lines transfected with this synthetic biology-inspired Protein Kinase R-Enhanced Protein Production (PREPP) system could produce several medicinally relevant protein therapeutics, such as the blockbuster drug rituximab, in substantial quantities and with significantly higher purity than previous culture technologies. We believe incorporation of such purity-by-design technology in the production process will alleviate downstream processing bottlenecks in future biopharmaceutical manufacturing.
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Daniel Bojar
2
Authors: Kojima, Ryosuke, et al
Published: Apr 2018
Authors: Kojima, Ryosuke, et al
Published: Apr 2018
Exosomes are cell-derived nanovesicles (50–150 nm), which mediate intercellular communication, and are candidate therapeutic agents. However, inefficiency of exosomal message transfer, such as mRNA, and lack of methods to create designer exosomes have hampered their development into therapeutic interventions. Here, we report a set of EXOsomal transfer into cells (EXOtic) devices that enable efficient, customizable production of designer exosomes in engineered mammalian cells. These genetically encoded devices in exosome producer cells enhance exosome production, specific mRNA packaging, and delivery of the mRNA into the cytosol of target cells, enabling efficient cell-to-cell communication without the need to concentrate exosomes. Further, engineered producer cells implanted in living mice could consistently deliver cargo mRNA to the brain. Therapeutic catalase mRNA delivery by designer exosomes attenuated neurotoxicity and neuroinflammation in in vitro and in vivo models of Parkinson’s disease, indicating the potential usefulness of the EXOtic devices for RNA delivery-based therapeutic applications.
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Daniel Bojar
1
Published: Jan 2017
Published: Jan 2017
Synthetic biology, the synthesis of engineering and biology, has rapidly matured and has dramatically increased the complexity of artificial gene circuits in recent years. The deployment of intricate synthetic gene circuits in mammalian cells requires the establishment of very precise and orthogonal control of transgene expression. In this chapter, we describe methods of modulating the expression of transgenes at the transcriptional level. Using cAMP-response element-binding protein (CREB)-dependent promoters as examples, a tool for the precise tuning of gene expression by using different core promoters and by varying the binding affinity of transcription factor operator sites is described.
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Daniel Bojar
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