Publications by authors named "Martin Fussenegger"

259 Publications

Synthetic Biology: Emerging Concepts to Design and Advance Adeno-Associated Viral Vectors for Gene Therapy.

Adv Sci (Weinh) 2021 05 26;8(9):2004018. Epub 2021 Feb 26.

Department of Biosystems Science and Engineering ETH Zurich Mattenstrasse 26 Basel 4058 Switzerland.

Three recent approvals and over 100 ongoing clinical trials make adeno-associated virus (AAV)-based vectors the leading gene delivery vehicles in gene therapy. Pharmaceutical companies are investing in this small and nonpathogenic gene shuttle to increase the therapeutic portfolios within the coming years. This prospect of marking a new era in gene therapy has fostered both investigations of the fundamental AAV biology as well as engineering studies to enhance delivery vehicles. Driven by the high clinical potential, a new generation of synthetic-biologically engineered AAV vectors is on the rise. Concepts from synthetic biology enable the control and fine-tuning of vector function at different stages of cellular transduction and gene expression. It is anticipated that the emerging field of synthetic-biologically engineered AAV vectors can shape future gene therapeutic approaches and thus the design of tomorrow's gene delivery vectors. This review describes and discusses the recent trends in capsid and vector genome engineering, with particular emphasis on synthetic-biological approaches.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/advs.202004018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8097373PMC
May 2021

A versatile plasmid architecture for mammalian synthetic biology (VAMSyB).

Metab Eng 2021 Apr 18;66:41-50. Epub 2021 Apr 18.

Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, CH-4058, Basel, Switzerland; Faculty of Science, University of Basel, Mattenstrasse 26, CH-4058, Basel, Switzerland. Electronic address:

Current molecular cloning strategies generally lack inter-compatibility, are not strictly modular, or are not applicable to engineer multi-gene expression vectors for transient and stable integration. A standardized molecular cloning platform would advance research, for example, by promoting exchange of vectors between groups. Here, we present a versatile plasmid architecture for mammalian synthetic biology, which we designate VAMSyB, consisting of a three-tier vector family. Tier-1 is designed for easy engineering of fusion constructs, as well as easy swapping of genes and modules to tune the functionality of the vector. Tier-2 is designed for transient multi-gene expression, and is constructed by directly transferring the engineered expression cassettes from tier-1 vectors. Tier-3 enables stable integration into a mammalian host cell through viral transduction, transposons, or homology-directed recombination via CRISPR. This VAMSyB architecture is expected to have broad applicability in the field of mammalian synthetic biology. The VAMSyB collection of plasmids will be available through Addgene.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ymben.2021.04.003DOI Listing
April 2021

5-Fluorouracil blocks quorum-sensing of biofilm-embedded methicillin-resistant Staphylococcus aureus in mice.

Nucleic Acids Res 2021 Apr 15. Epub 2021 Apr 15.

Department of Biosystems Science and Engineering, ETH Zürich, Mattenstrasse 26, CH-4058 Basel, Switzerland.

Antibiotic-resistant pathogens often escape antimicrobial treatment by forming protective biofilms in response to quorum-sensing communication via diffusible autoinducers. Biofilm formation by the nosocomial pathogen methicillin-resistant Staphylococcus aureus (MRSA) is triggered by the quorum-sensor autoinducer-2 (AI-2), whose biosynthesis is mediated by methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) and S-ribosylhomocysteine lyase (LuxS). Here, we present a high-throughput screening platform for small-molecular inhibitors of either enzyme. This platform employs a cell-based assay to report non-toxic, bioavailable and cell-penetrating inhibitors of AI-2 production, utilizing engineered human cells programmed to constitutively secrete AI-2 by tapping into the endogenous methylation cycle via ectopic expression of codon-optimized MTAN and LuxS. Screening of a library of over 5000 commercial compounds yielded 66 hits, including the FDA-licensed cytostatic anti-cancer drug 5-fluorouracil (5-FU). Secondary screening and validation studies showed that 5-FU is a potent quorum-quencher, inhibiting AI-2 production and release by MRSA, Staphylococcus epidermidis, Escherichia coli and Vibrio harveyi. 5-FU efficiently reduced adherence and blocked biofilm formation of MRSA in vitro at an order-of-magnitude-lower concentration than that clinically relevant for anti-cancer therapy. Furthermore, 5-FU reestablished antibiotic susceptibility and enabled daptomycin-mediated prevention and clearance of MRSA infection in a mouse model of human implant-associated infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkab251DOI Listing
April 2021

Engineering precision therapies: lessons and motivations from the clinic.

Synth Biol (Oxf) 2021 24;6(1):ysaa024. Epub 2020 Nov 24.

Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland.

In the past decade, gene- and cell-based therapies have been at the forefront of the biomedical revolution. Synthetic biology, the engineering discipline of building sophisticated 'genetic software' to enable precise regulation of gene activities in living cells, has been a decisive success factor of these new therapies. Here, we discuss the core technologies and treatment strategies that have already gained approval for therapeutic applications in humans. We also review promising preclinical work that could either enhance the efficacy of existing treatment strategies or pave the way for new precision medicines to treat currently intractable human conditions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/synbio/ysaa024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7998714PMC
November 2020

Rational design and optimization of synthetic gene switches for controlling cell-fate decisions in pluripotent stem cells.

Metab Eng 2021 05 17;65:99-110. Epub 2021 Mar 17.

Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, CH, 4058, Basel, Switzerland; Faculty of Science, University of Basel, Mattenstrasse 26, CH, 4058, Basel, Switzerland. Electronic address:

Advances in synthetic biology have enabled robust control of cell behavior by using tunable genetic circuits to regulate gene expression in a ligand-dependent manner. Such circuits can be used to direct the differentiation of pluripotent stem cells (PSCs) towards desired cell types, but rational design of synthetic gene circuits in PSCs is challenging due to the variable intracellular environment. Here, we provide a framework for implementing synthetic gene switches in PSCs based on combinations of tunable transcriptional, structural, and posttranslational elements that can be engineered as required, using the vanillic acid-controlled transcriptional activator (VanA) as a model system. We further show that the VanA system can be multiplexed with the well-established reverse tetracycline-controlled transcriptional activator (rtTA) system to enable independent control of the expression of different transcription factors in human induced PSCs in order to enhance lineage specification towards early pancreatic progenitors. This work represents a first step towards standardizing the design and construction of synthetic gene switches for building robust gene-regulatory networks to guide stem cell differentiation towards a desired cell fate.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ymben.2021.03.009DOI Listing
May 2021

Gene switch for l-glucose-induced biopharmaceutical production in mammalian cells.

Biotechnol Bioeng 2021 Feb 25. Epub 2021 Feb 25.

Department of Biosystems, Science and Engineering, ETH Zurich, Basel, Switzerland.

In this study, we designed and built a gene switch that employs metabolically inert l-glucose to regulate transgene expression in mammalian cells via d-idonate-mediated control of the bacterial regulator LgnR. To this end, we engineered a metabolic cascade in mammalian cells to produce the inducer molecule d-idonate from its precursor l-glucose by ectopically expressing the Paracoccus species 43P-derived catabolic enzymes LgdA, LgnH, and LgnI. To obtain ON- and OFF-switches, we fused LgnR to the human transcriptional silencer domain Krüppel associated box (KRAB) and the viral trans-activator domain VP16, respectively. Thus, these artificial transcription factors KRAB-LgnR or VP16-LgnR modulated cognate promoters containing LgnR-specific binding sites in a d-idonate-dependent manner as a direct result of l-glucose metabolism. In a proof-of-concept experiment, we show that the switches can control production of the model biopharmaceutical rituximab in both transiently and stably transfected HEK-293T cells, as well as CHO-K1 cells. Rituximab production reached 5.9 µg/ml in stably transfected HEK-293T cells and 3.3 µg/ml in stably transfected CHO-K1 cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/bit.27730DOI Listing
February 2021

Shedding Light on Extracellular Vesicle Biogenesis and Bioengineering.

Adv Sci (Weinh) 2020 Jan 27;8(1):2003505. Epub 2020 Nov 27.

Department of Biosystems Science and Engineering ETH Zurich Mattenstrasse 26 Basel CH-4058 Switzerland.

Extracellular vesicles (EVs) are biocompatible, nano-sized secreted vesicles containing many types of biomolecules, including proteins, RNAs, DNAs, lipids, and metabolites. Their low immunogenicity and ability to functionally modify recipient cells by transferring diverse bioactive constituents make them an excellent candidate for a next-generation drug delivery system. Here, the recent advances in EV biology and emerging strategies of EV bioengineering are summarized, and the prospects for clinical translation of bioengineered EVs and the challenges to be overcome are discussed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/advs.202003505DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7788585PMC
January 2020

Bottom-up de novo design of functional proteins with complex structural features.

Nat Chem Biol 2021 04 4;17(4):492-500. Epub 2021 Jan 4.

Institute of Bioengineering, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.

De novo protein design has enabled the creation of new protein structures. However, the design of functional proteins has proved challenging, in part due to the difficulty of transplanting structurally complex functional sites to available protein structures. Here, we used a bottom-up approach to build de novo proteins tailored to accommodate structurally complex functional motifs. We applied the bottom-up strategy to successfully design five folds for four distinct binding motifs, including a bifunctionalized protein with two motifs. Crystal structures confirmed the atomic-level accuracy of the computational designs. These de novo proteins were functional as components of biosensors to monitor antibody responses and as orthogonal ligands to modulate synthetic signaling receptors in engineered mammalian cells. Our work demonstrates the potential of bottom-up approaches to accommodate complex structural motifs, which will be essential to endow de novo proteins with elaborate biochemical functions, such as molecular recognition or catalysis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41589-020-00699-xDOI Listing
April 2021

Design of Multipartite Transcription Factors for Multiplexed Logic Genome Integration Control in Mammalian Cells.

ACS Synth Biol 2020 11 14;9(11):2964-2970. Epub 2020 Oct 14.

Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, CH-4058 Basel, Switzerland.

Synthetic biology relies on rapid and efficient methods to stably integrate DNA payloads encoding for synthetic biological systems into the genome of living cells. The size of designed biological systems increases with their complexity, and novel methods are needed that enable efficient and simultaneous integration of multiple payloads into single cells. By assembling natural and synthetic protein-protein dimerization domains, we have engineered a set of multipartite transcription factors for driving heterologous target gene expression. With the distribution of single parts of multipartite transcription factors on piggyback transposon-based donor plasmids, we have created a logic genome integration control (LOGIC) system that allows for efficient one-step selection of stable mammalian cell lines with up to three plasmids. LOGIC significantly enhances the efficiency of multiplexed payload integration in mammalian cells compared to traditional cotransfection and may advance cell line engineering in synthetic biology and biotechnology.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acssynbio.0c00413DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7684658PMC
November 2020

Role of YAP/TAZ in Cell Lineage Fate Determination and Related Signaling Pathways.

Front Cell Dev Biol 2020 30;8:735. Epub 2020 Jul 30.

National Engineering Laboratory for Digital and Material Technology of Stomatology, NMPA Key Laboratory for Dental Materials, Beijing Laboratory of Biomedical Materials, Peking University School and Hospital of Stomatology, Beijing, China.

The penultimate effectors of the Hippo signaling pathways YAP and TAZ, are transcriptional co-activator proteins that play key roles in many diverse biological processes, ranging from cell proliferation, tumorigenesis, mechanosensing and cell lineage fate determination, to wound healing and regeneration. In this review, we discuss the regulatory mechanisms by which YAP/TAZ control stem/progenitor cell differentiation into the various major lineages that are of interest to tissue engineering and regenerative medicine applications. Of particular interest is the key role of YAP/TAZ in maintaining the delicate balance between quiescence, self-renewal, proliferation and differentiation of endogenous adult stem cells within various tissues/organs during early development, normal homeostasis and regeneration/healing. Finally, we will consider how increasing knowledge of YAP/TAZ signaling might influence the trajectory of future progress in regenerative medicine.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fcell.2020.00735DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7406690PMC
July 2020

An overview of signaling pathways regulating YAP/TAZ activity.

Cell Mol Life Sci 2021 Jan 3;78(2):497-512. Epub 2020 Aug 3.

Department of Geriatric Dentistry, Peking University School and Hospital of Stomatology, Beijing, 100081, People's Republic of China.

YAP and TAZ are ubiquitously expressed homologous proteins originally identified as penultimate effectors of the Hippo signaling pathway, which plays a key role in maintaining mammalian tissue/organ size. Presently, it is known that YAP/TAZ also interact with various non-Hippo signaling pathways, and have diverse roles in multiple biological processes, including cell proliferation, tissue regeneration, cell lineage fate determination, tumorigenesis, and mechanosensing. In this review, we first examine the various microenvironmental cues and signaling pathways that regulate YAP/TAZ activation, through the Hippo and non-Hippo signaling pathways. This is followed by a brief summary of the interactions of YAP/TAZ with TEAD1-4 and a diverse array of other non-TEAD transcription factors. Finally, we offer a critical perspective on how increasing knowledge of the regulatory mechanisms of YAP/TAZ signaling might open the door to novel therapeutic applications in the interrelated fields of biomaterials, tissue engineering, regenerative medicine and synthetic biology.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00018-020-03579-8DOI Listing
January 2021

Construction of a Multiwell Light-Induction Platform for Traceless Control of Gene Expression in Mammalian Cells.

Methods Mol Biol 2020 ;2173:189-199

Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland.

Mammalian cells can be engineered to incorporate light-responsive elements that reliably sense stimulation by light and activate endogenous pathways, such as the cAMP or Ca pathway, to control gene expression. Light-inducible gene expression systems offer high spatiotemporal resolution, and are also traceless, reversible, tunable, and inexpensive. Melanopsin, a well-known representative of the animal opsins, is a G-protein-coupled receptor that triggers a Gαq-dependent signaling cascade upon activation with blue light (≈470 nm). Here, we describe how to rewire melanopsin activation by blue light to transgene expression in mammalian cells, with detailed instructions for constructing a 96-LED array platform with multiple tunable parameters for illumination of the engineered cells in multiwell plates.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-0716-0755-8_13DOI Listing
March 2021

Phosphoregulated orthogonal signal transduction in mammalian cells.

Nat Commun 2020 06 18;11(1):3085. Epub 2020 Jun 18.

Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, CH-4058, Basel, Switzerland.

Orthogonal tools for controlling protein function by post-translational modifications open up new possibilities for protein circuit engineering in synthetic biology. Phosphoregulation is a key mechanism of signal processing in all kingdoms of life, but tools to control the involved processes are very limited. Here, we repurpose components of bacterial two-component systems (TCSs) for chemically induced phosphotransfer in mammalian cells. TCSs are the most abundant multi-component signal-processing units in bacteria, but are not found in the animal kingdom. The presented phosphoregulated orthogonal signal transduction (POST) system uses induced nanobody dimerization to regulate the trans-autophosphorylation activity of engineered histidine kinases. Engineered response regulators use the phosphohistidine residue as a substrate to autophosphorylate an aspartate residue, inducing their own homodimerization. We verify this approach by demonstrating control of gene expression with engineered, dimerization-dependent transcription factors and propose a phosphoregulated relay system of protein dimerization as a basic building block for next-generation protein circuits.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-020-16895-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7303213PMC
June 2020

Electrogenetic cellular insulin release for real-time glycemic control in type 1 diabetic mice.

Science 2020 05;368(6494):993-1001

Department of Biosystems Science and Engineering, ETH Zurich, CH-4058 Basel, Switzerland.

Sophisticated devices for remote-controlled medical interventions require an electrogenetic interface that uses digital electronic input to directly program cellular behavior. We present a cofactor-free bioelectronic interface that directly links wireless-powered electrical stimulation of human cells to either synthetic promoter-driven transgene expression or rapid secretion of constitutively expressed protein therapeutics from vesicular stores. Electrogenetic control was achieved by coupling ectopic expression of the L-type voltage-gated channel Ca1.2 and the inwardly rectifying potassium channel K2.1 to the desired output through endogenous calcium signaling. Focusing on type 1 diabetes, we engineered electrosensitive human β cells (β cells). Wireless electrical stimulation of β cells inside a custom-built bioelectronic device provided real-time control of vesicular insulin release; insulin levels peaked within 10 minutes. When subcutaneously implanted, this electrotriggered vesicular release system restored normoglycemia in type 1 diabetic mice.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.aau7187DOI Listing
May 2020

Ethical and societal implications of cellular health-monitoring devices.

Sci Transl Med 2020 05;12(545)

Anna Deplazes-Zemp is a senior researcher at the Ethics Research Institute at the University of Zürich, CH-8006 Zürich, Switzerland. Email:

As cell engineering is used to develop new types of implanted health-monitoring devices, this may raise ethical issues for individuals and society.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/scitranslmed.aax6924DOI Listing
May 2020

Genetically encoded betaxanthin-based small-molecular fluorescent reporter for mammalian cells.

Nucleic Acids Res 2020 07;48(12):e67

Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, CH-4058 Basel, Switzerland.

We designed and engineered a dye production cassette encoding a heterologous pathway, including human tyrosine hydroxylase and Amanita muscaria 4,5-DOPA dioxygenase, for the biosynthesis of the betaxanthin family of plant and fungal pigments in mammalian cells. The system does not impair cell viability, and can be used as a non-protein reporter system to directly visualize the dynamics of gene expression by profiling absorbance or fluorescence in the supernatant of cell cultures, as well as for fluorescence labeling of individual cells. Pigment profiling can also be multiplexed with reporter proteins such as mCherry or the human model glycoprotein SEAP (secreted alkaline phosphatase). Furthermore, absorbance measurement with a smartphone camera using standard application software enables inexpensive, low-tech reporter quantification.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkaa342DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7337513PMC
July 2020

Building sophisticated sensors of extracellular cues that enable mammalian cells to work as "doctors" in the body.

Cell Mol Life Sci 2020 Sep 17;77(18):3567-3581. Epub 2020 Mar 17.

Department of Biosystems Science and Engineering (D-BSSE), ETH Zurich, Mattenstrasse 26, 4058, Basel, Switzerland.

Mammalian cells are inherently capable of sensing extracellular environmental signals and activating complex biological functions on demand. Advances in synthetic biology have made it possible to install additional capabilities, which can allow cells to sense the presence of custom biological molecules and provide defined outputs on demand. When implanted/infused in patients, such engineered cells can work as intrabody "doctors" that diagnose disease states and produce and deliver therapeutic molecules when and where necessary. The key to construction of such theranostic cells is the development of a range of sensor systems for detecting various extracellular environmental cues that can be rewired to custom outputs. In this review, we introduce the state-of-art engineering principles utilized in the design of sensor systems to detect soluble factors and also to detect specific cell contact, and we discuss their potential role in treating intractable diseases by delivering appropriate therapeutic functions on demand. We also discuss the challenges facing these emerging technologies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00018-020-03486-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7452942PMC
September 2020

Segregated Nanocompartments Containing Therapeutic Enzymes and Imaging Compounds within DNA-Zipped Polymersome Clusters for Advanced Nanotheranostic Platform.

Small 2020 07 4;16(27):e1906492. Epub 2020 Mar 4.

Department of Chemistry, University of Basel, Mattenstrasse 24a, Basel, 4002, Switzerland.

Nanotheranostics is an emerging field that brings together nanoscale-engineered materials with biological systems providing a combination of therapeutic and diagnostic strategies. However, current theranostic nanoplatforms have serious limitations, mainly due to a mismatch between the physical properties of the selected nanomaterials and their functionalization ease, loading ability, or overall compatibility with bioactive molecules. Herein, a nanotheranostic system is proposed based on nanocompartment clusters composed of two different polymersomes linked together by DNA. Careful design and procedure optimization result in clusters segregating the therapeutic enzyme human Dopa decarboxylase (DDC) and fluorescent probes for the detection unit in distinct but colocalized nanocompartments. The diagnostic compartment provides a twofold function: trackability via dye loading as the imaging component and the ability to attach the cluster construct to the surface of cells. The therapeutic compartment, loaded with active DDC, triggers the cellular expression of a secreted reporter enzyme via production of dopamine and activation of dopaminergic receptors implicated in atherosclerosis. This two-compartment nanotheranostic platform is expected to provide the basis of a new treatment strategy for atherosclerosis, to expand versatility and diversify the types of utilizable active molecules, and thus by extension expand the breadth of attainable applications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/smll.201906492DOI Listing
July 2020

Rewiring of endogenous signaling pathways to genomic targets for therapeutic cell reprogramming.

Nat Commun 2020 01 30;11(1):608. Epub 2020 Jan 30.

Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, CH-4058, Basel, Switzerland.

Rewiring cellular sensors to trigger non-natural responses is fundamental for therapeutic cell engineering. Current designs rely on engineered receptors that are limited to single inputs, and often suffer from high leakiness and low fold induction. Here, we present Generalized Engineered Activation Regulators (GEARs) that overcome these limitations by being pathway-specific rather than input-specific. GEARs consist of the MS2 bacteriophage coat protein fused to regulatory or transactivation domains, and work by rerouting activation of the NFAT, NFκB, MAPK or SMAD pathways to dCas9-directed gene expression from genomic loci. This system enables membrane depolarization-induced activation of insulin expression in β-mimetic cells and IL-12 expression in activated Jurkat cells, as well as IL-12 production in response to the immunomodulatory cytokines TGFβ and TNFα in HEK293T cells. Engineered cells with the ability to reinterpret the extracellular milieu have potential for applications in immunotherapy and in the treatment of metabolic diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-020-14397-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6992713PMC
January 2020

Neurons differentiate magnitude and location of mechanical stimuli.

Proc Natl Acad Sci U S A 2020 01 27;117(2):848-856. Epub 2019 Dec 27.

Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule Zurich, 4058 Basel, Switzerland;

Neuronal activity can be modulated by mechanical stimuli. To study this phenomenon quantitatively, we mechanically stimulated rat cortical neurons by shear stress and local indentation. Neurons show 2 distinct responses, classified as transient and sustained. Transient responses display fast kinetics, similar to spontaneous neuronal activity, whereas sustained responses last several minutes before returning to baseline. Local soma stimulations with micrometer-sized beads evoke transient responses at low forces of ∼220 nN and pressures of ∼5.6 kPa and sustained responses at higher forces of ∼360 nN and pressures of ∼9.2 kPa. Among the neuronal compartments, axons are highly susceptible to mechanical stimulation and predominantly show sustained responses, whereas the less susceptible dendrites predominantly respond transiently. Chemical perturbation experiments suggest that mechanically evoked responses require the influx of extracellular calcium through ion channels. We propose that subtraumatic forces/pressures applied to neurons evoke neuronal responses via nonspecific gating of ion channels.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1909933117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6969492PMC
January 2020

The Role of Protein Engineering in Biomedical Applications of Mammalian Synthetic Biology.

Small 2020 07 7;16(27):e1903093. Epub 2019 Oct 7.

ETH Zurich, Department of Biosystems Science and Engineering, Faculty of Life Science, University of Basel, Mattenstrasse 26, CH-4058, Basel, Switzerland.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/smll.201903093DOI Listing
July 2020

A fully human transgene switch to regulate therapeutic protein production by cooling sensation.

Nat Med 2019 08 8;25(8):1266-1273. Epub 2019 Jul 8.

Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland.

The ability to safely control transgene expression with simple synthetic gene switches is critical for effective gene- and cell-based therapies. In the present study, the signaling pathway controlled by human transient receptor potential (TRP) melastatin 8 (hTRPM8), a TRP channel family member, is harnessed to control transgene expression. Human TRPM8 signaling is stimulated by menthol, an innocuous, natural, cooling compound, or by exposure to a cool environment (15-18 °C). By functionally linking hTRPM8-induced signaling to a synthetic promoter containing elements that bind nuclear factor of activated T cells, a synthetic gene circuit was designed that can be adjusted by exposure to either a cool environment or menthol. It was shown that this gene switch is functional in various cell types and human primary cells, as well as in mice implanted with engineered cells. In response to transdermal delivery of menthol, microencapsulated cell implants harboring this gene circuit, coupled to expression of either of two therapeutic proteins, insulin or a modified, activin type IIB, receptor ligand trap protein (mActRIIB-hFc), could alleviate hyperglycemia in alloxan-treated mice (a model of type 1 diabetes) or reverse muscle atrophy in dexamethasone-treated mice (a model of muscle wasting), respectively. This fully human-derived orthogonal transgene switch should be amenable to a wide range of clinical applications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41591-019-0501-8DOI Listing
August 2019

A modular degron library for synthetic circuits in mammalian cells.

Nat Commun 2019 05 1;10(1):2013. Epub 2019 May 1.

Department of Biosystems Science and Engineering, ETH Zürich, Mattenstrasse 26, CH-4058, Basel, Switzerland.

Tight control over protein degradation is a fundamental requirement for cells to respond rapidly to various stimuli and adapt to a fluctuating environment. Here we develop a versatile, easy-to-handle library of destabilizing tags (degrons) for the precise regulation of protein expression profiles in mammalian cells by modulating target protein half-lives in a predictable manner. Using the well-established tetracycline gene-regulation system as a model, we show that the dynamics of protein expression can be tuned by fusing appropriate degron tags to gene regulators. Next, we apply this degron library to tune a synthetic pulse-generating circuit in mammalian cells. With this toolbox we establish a set of pulse generators with tailored pulse lengths and magnitudes of protein expression. This methodology will prove useful in the functional roles of essential proteins, fine-tuning of gene-expression systems, and enabling a higher complexity in the design of synthetic biological systems in mammalian cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-019-09974-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6494899PMC
May 2019

Synthetic gene circuits for the detection, elimination and prevention of disease.

Nat Biomed Eng 2018 06 11;2(6):399-415. Epub 2018 Jun 11.

Department of Biosystems Science and Engineering, ETH Zürich, Basel, Switzerland.

In living organisms, naturally evolved sensors that constantly monitor and process environmental cues trigger corrective actions that enable the organisms to cope with changing conditions. Such natural processes have inspired biologists to construct synthetic living sensors and signalling pathways, by repurposing naturally occurring proteins and by designing molecular building blocks de novo, for customized diagnostics and therapeutics. In particular, designer cells that employ user-defined synthetic gene circuits to survey disease biomarkers and to autonomously re-adjust unbalanced pathological states can coordinate the production of therapeutics, with controlled timing and dosage. Furthermore, tailored genetic networks operating in bacterial or human cells have led to cancer remission in experimental animal models, owing to the network's unprecedented specificity. Other applications of designer cells in infectious, metabolic and autoimmune diseases are also being explored. In this Review, we describe the biomedical applications of synthetic gene circuits in major disease areas, and discuss how the first genetically engineered devices developed on the basis of synthetic-biology principles made the leap from the laboratory to the clinic.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41551-018-0215-0DOI Listing
June 2018

From synthetic biology to human therapy: engineered mammalian cells.

Curr Opin Biotechnol 2019 08 30;58:108-116. Epub 2019 Mar 30.

Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, CH-4058, Basel, Switzerland; University of Basel, Faculty of Science, Mattenstrasse 26, CH-4058, Basel, Switzerland. Electronic address:

Mammalian synthetic biology has evolved to become a key driver of biomedical innovation in the area of cell therapy. Advances in receptor engineering, immunotherapy and cell implants promise new treatment options for complex diseases. Synthetic receptors have already found applications in cellular immunotherapy for cancer treatment, and are being introduced into the field of cell implants. Here, we discuss prospects for the next generation of engineered mammalian cells for human therapy, highlighting selected recent studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.copbio.2019.02.023DOI Listing
August 2019

A CRISPR/Cas9-based central processing unit to program complex logic computation in human cells.

Proc Natl Acad Sci U S A 2019 04 28;116(15):7214-7219. Epub 2019 Mar 28.

Department of Biosystems Science and Engineering, ETH Zürich, CH-4058 Basel, Switzerland;

Controlling gene expression with sophisticated logic gates has been and remains one of the central aims of synthetic biology. However, conventional implementations of biocomputers use central processing units (CPUs) assembled from multiple protein-based gene switches, limiting the programming flexibility and complexity that can be achieved within single cells. Here, we introduce a CRISPR/Cas9-based core processor that enables different sets of user-defined guide RNA inputs to program a single transcriptional regulator (dCas9-KRAB) to perform a wide range of bitwise computations, from simple Boolean logic gates to arithmetic operations such as the half adder. Furthermore, we built a dual-core CPU combining two orthogonal core processors in a single cell. In principle, human cells integrating multiple orthogonal CRISPR/Cas9-based core processors could offer enormous computational capacity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1821740116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6462112PMC
April 2019

Genetic circuitry for personalized human cell therapy.

Curr Opin Biotechnol 2019 10 7;59:31-38. Epub 2019 Mar 7.

Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, CH-4058 Basel, Switzerland; Faculty of Science, University of Basel, Mattenstrasse 26, CH-4058 Basel, Switzerland. Electronic address:

Synthetic biology uses engineering principles to design and assemble biological components and systems for a variety of applications. On the basis of genetic engineering, synthetic gene switches can be interconnected to construct complex gene circuits, capable of sensing and integrating diverse input signals for precise spatiotemporal control of target gene expression in living cells. Designer cells can be equipped with advanced gene circuitry enabling them to react precisely to pre-programmed combinations of conditions, automatically triggering a specified response, such as therapeutic protein production. Such cells are promising therapeutic modalities for applications where traditional medical treatments have limitations. Herein, we highlight selected recent examples of designer cells with engineered gene circuits targeted toward applications in personalized human medicine.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.copbio.2019.02.003DOI Listing
October 2019

Light-Controlled Mammalian Cells and Their Therapeutic Applications in Synthetic Biology.

Adv Sci (Weinh) 2019 Jan 30;6(1):1800952. Epub 2018 Sep 30.

Department of Biosystems Science and Engineering ETH Zurich Mattenstrasse 26 CH-4058 Basel Switzerland.

The ability to remote control the expression of therapeutic genes in mammalian cells in order to treat disease is a central goal of synthetic biology-inspired therapeutic strategies. Furthermore, optogenetics, a combination of light and genetic sciences, provides an unprecedented ability to use light for precise control of various cellular activities with high spatiotemporal resolution. Recent work to combine optogenetics and therapeutic synthetic biology has led to the engineering of light-controllable designer cells, whose behavior can be regulated precisely and noninvasively. This Review focuses mainly on non-neural optogenetic systems, which are often used in synthetic biology, and their applications in genetic programing of mammalian cells. Here, a brief overview of the optogenetic tool kit that is available to build light-sensitive mammalian cells is provided. Then, recently developed strategies for the control of designer cells with specific biological functions are summarized. Recent translational applications of optogenetically engineered cells are also highlighted, ranging from in vitro basic research to in vivo light-controlled gene therapy. Finally, current bottlenecks, possible solutions, and future prospects for optogenetics in synthetic biology are discussed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/advs.201800952DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6325585PMC
January 2019

Synthetic Biology: Engineering Mammalian Cells To Control Cell-to-Cell Communication at Will.

Chembiochem 2019 04 21;20(8):994-1002. Epub 2019 Mar 21.

Department of Biosystems Science and Engineering (D-BSSE), ETH Zurich, Mattenstrasse 26, 4058, Basel, Switzerland.

Cell-to-cell communication plays a key role in the regulation of many natural biological processes. Recent advances in mammalian synthetic biology are making it possible to rationally engineer cell-to-cell communication for therapeutic and other purposes. Here, we review state-of-the-art engineering principles to control cell-to-cell communication, focusing on communication between mammalian cells with diffusible factors (e.g., small molecules or exosomes) or direct cell contact, and on interkingdom communication between mammalian cells and bacteria. Potential applications include construction of artificial tissues able to perform complex computations, sophisticated cell-based cancer therapies, use of mammalian cells as a new class of cargo delivery modality, development of design principles to control pattern formation of cell populations, and treatment of infectious diseases. We also discuss the challenges facing practical applications, and possible enabling technologies to overcome them.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cbic.201800682DOI Listing
April 2019

Purity by design: Reducing impurities in bioproduction by stimulus-controlled global translational downregulation of non-product proteins.

Metab Eng 2019 03 20;52:110-123. Epub 2018 Nov 20.

ETH Zurich, Department of Biosystems Science and Engineering, Mattenstrasse 26, 4058 Basel, Switzerland; Faculty of Life Science, University of Basel, Mattenstrasse 26, CH-4058 Basel, Switzerland. Electronic address:

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ymben.2018.11.007DOI Listing
March 2019