- NCCR MSE

Projekte

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Beschreibung [methodology]

The ultimate goal of this project is to create complex networks of reactors/processors as a basis for molecular factories. Over the long term, it is anticipated that the organelles and model cells that we will design and develop will provide a new, theragnostic strategy, very much in demand today in the medical domain. In addition, molecular factories will be specifically designed to support applications in environmental science, food science and technology.

Polymer supramolecular structures in the form of micelles, vesicles, and films are of particular interest as building blocks/templates for molecular factories. If appropriately designed from the point of view of chemical nature and properties, these structures can favour the insertion/encapsulation/attachment of biological molecules that serve as active blocks without dramatically impairing their function.

Polymer vesicles and planar membranes selectively permit encapsulation of a variety of biomolecules, ranging from low molar mass components to functional enzymes and proteins, without hampering their activities. In addition, polymer membranes modified by the insertion of channel proteins allow for the selective exchange of molecular components/reaction products between the inside and outside of a membrane cavity, while the functionalization of their surfaces supports targeted approaches.

The approach differs from others in that, due to its chemical nature, the membrane itself is permeable by inserting channel proteins.

Initially, the variety of amphiphilic copolymers, and later the multifunctionality and responsiveness of biomolecule – polymer assemblies will be extended, by integrating them in complex networks that will support molecular factories.

 

Scientific Highlight

Nanoreactors against malaria (see link to article “Nanomimics of Host Cell Membranes Block Invasion and Expose Invasive Malaria Parasites“ below): Polymer vesicles were designed by the two NCCR-Groups Meier and Palivan to present specific host cell receptors on their surface. Such nanoreactors mimick red blood cells, which are the target of Plasmodium falciparum parasites that cause malaria. When added to a parasite culture, these nanoreactors efficiently interrupt the life cycle of P. falciparum by rapidly binding to the surface of malaria parasites. This inhibits the invasion of uninfected red blood cells, thus efficiently terminating the malaria blood-stage cycle. This new patented strategy offers promising treatments for several severe diseases.

Molecular systems derive their functional breadth from the interplay of multiple elements. The successful cooperation of these elements is often limited to narrow windows of operation, which are often difficult to identify.

We are optimizing complex in vitro systems so they can successfully operate in these windows. For this, we develop cell free systems that allow the synthesis of multiple catalysts and other protein-based elements, and compartmentalize the synthesis in nanoliter or picoliter-sized droplets. This helps us to investigate thousands of system compositions per minute. We use this to develop design rules for multi-membered systems and prepare such droplets for analysis in a classical way (i.e., by fluorescence) and by label-free methods, such as mass-spectrometry. This way we can optimize system function for a variety of objectives, ranging from enzyme evolution to the engineering of smart systems for metabolic diseases.

We engineer synthetic cellular memory platforms that enable the reconstruction of cellular histories and can be applied as living diagnostics.

A fundamental challenge in biology is to understand how cells function and integrate complex molecular information to perform different behaviors. For example, the differentiation of a stem cell into two daughter cells with distinct identities or the transformation of a normal cell into a cancer cell. This challenge has motivated the creation of numerous technologies facilitating detailed intracellular observations at the level of DNA, RNA, protein, and metabolites. Despite the power of these approaches, they generally require destructive methods and therefore observations are limited to a few snapshots in time or select asynchronous cellular processes. One provocative solution to this is to introduce DNA writing and molecular recording platforms within cells that enable the encoding, storage, and retrieval of molecular information.

Towards the goal of continuously recording molecular events within cells, my laboratory is developing and applying Record-seq, a ‘transcriptional recording’ platform that employs CRISPR spacer acquisition from RNA to capture and convert intracellular RNAs into DNA, permanently storing transcriptional information in the DNA of living cells. The newly acquired sequences serve as transcriptional records, which are retrievable via deep sequencing and can be leverage to reconstruct cellular histories. This technology provides an entirely new mode of interrogating dynamic biological and physiological processes and opens up numerous avenues for future work in engineering cellular systems.

Clinical and immunohistochemical studies indicate that the tumor microenvironment, including stromal and immunocompetent components, plays key roles in the control of tumor progression and response to treatment. Moreover, increased recognition is given to the importance of using human cell- based models to predict the outcome of therapeutic strategies.

The goal of the project is to engineer a 3D human organotypic model of colorectal cancer including associated tumor microenvironment components, as test system for new immune-oncology agents. The further inclusion of hepatocytes in the model in a second phase of the project will enable to investigate, along with the antitumor efficacy, also the possible liver toxicity of the therapeutic agents.

This project focuses on the development of an innovative methodology for the assembly of hierarchical architectures on both solid and polymer platforms. The goal is to design multi-functional systems supporting cascade reactions in 2D networks of nano-compartments (polymersomes, liposomes and combination of thereof) by their controlled anchoring and patterning on surfaces. We will develop strategies and proof-of-principle systems with material turn-over based upon either soft-condensed matter supports (polymers) or hard, metal oxide supports. New soft–hard interfaces will be designed to provide a micro- and nanoscale environment which can modulate properties and activity.

Dimethylfumarate is an emergent pharmaceutical compound with annual sales of 4412 Mio $ in 2016. Currently, the main clinical applications are in treating psoriasis and multiple sclerosis. In addition, dimethylfumarate might be therapeutic against asthma, cancer, inflammatory bowl disease, intracerebral hemorrhage, osteoarthritis, chronic pancreatitis, retinal ischemia, and ischemic stroke.

The goal of this project is to engineer a cell-like device for targeted delivery of the electrophilic drug dimethylfumarate. In the first phase of the project we plan to identify a vesicle based enzyme reaction that is triggered by external stimuli to produce and release dimethylfumarate.

An ideal cell-based diabetes therapy in humans should consist of an autonomous core system enabling closed-loop control of D-glucose sensing and insulin release, coupled to a user-defined control interface that allows the metabolically inert L-glucose to monitor the temporal activity of the therapeutic core. In this context, L-glucose functions as a patient-centred adjuvant molecule that is not only free of hepatic side- effects, but also represents an optimal nutritional sugar for diabetic patients.

The focus of this project is the design and construction of an L-glucose-dependent control interface for future cell-based diabetes therapies and the validation of L-glucose polymersome-dependent control of therapeutic transgene activities in mice.

Our research focuses on the deployment of multiple immobilized molecular catalysts on a flow microreactor platform to convert elementary starting materials over telescoped reactions into chemically and structurally complex products. The interactions of different agents in multiple compartments on a solid-state platform result in a molecular factory.

A combination of multiple catalyst permutations and applied reaction conditions enables screening of diverse parameter spaces for the conversion of suitable starting materials. This approach aims at not only synthesizing one catalysis product from one starting material, but multiple products from the same starting material and whole compound libraries. This bio-inspired approach resembles the biosynthetic processes that are taking place in a biological cell, in which multiple metabolites are often produced from a single molecule.

Our research comprises the design and synthesis of complementary catalysts, linkers and starting materials for heterogeneous catalysis with molecular catalyst monolayers, as well as the immobilization of these catalysts in flow microreactors and conducting synthetic operation on these platforms. This project is in close collaboration with the Mayor group and IBM Research - Zurich in Rüschlikon. The silicon-based flow microreactors are being fabricated by microfabrication techniques, are scalable in the number of compartments, and allow various reaction control features such as nanoscale electrode arrays, catalyst-supporting surfaces, externally controllable micro-heaters and nanophotonic sensing sites to be implemented and to be used as reaction feedback controls.

The chemical and physical processes enabling the transformation of matter in living systems is regulated by complex feedback loops. Such tightly cross-regulated processes enable highly complex reaction cascades as well as transport and exchange mechanisms that have not yet been achieved in synthetic systems. The project „Nanopores as Solid-State Approach to Interlinked Reaction Compartments" strives to simulate isolated feedback mechanisms to investigate the underlying regulation mechanisms in detail and to identify the parameters governing the system.

To fabricate such interlinked reaction compartments, we aim at fabricating nanopore devices based on a solid-state approach using top-down fabrication techniques. Self-assembled monolayers (SAMs) of functional molecular building blocks are physically separated but remain addressable by electrical, optical or electrochemical means. The SAMs are highly oriented which enables correlations between chemical structure and electronic as well as ionic transport properties in single-molecular junctions to be studied. 

Preliminary studies will allow us to mimic a biological response and to investigate isolated feedback mechanisms in detail. Additionally, from a materials point of view, the resulting oligomers may be interesting. Their physical properties are length-dependent and, thanks to a feedback mechanism, their length-distribution may become tunable by specific parameters dictated by the system, leading to a new size-control approach with wide potential applications in material science. Modular systems can easily be expanded with additional functionalities. For example redox-­dependent chromophores that will facilitate the investigation of the systems dynamics as it can be investigated by optical microscopy.

The project is not only geared towards investigating feedback mechanisms across vesicle membranes but also towards integrating vesicles as molecular factories. With such vesicles, molecular devices enabling photo-induced charge separation across the vesicle membrane will be studied. In a later stage of the project, we envisage electrochemical interconnecting of different functionalized vesicles to build up gradients of chemical potentials. 

Building autonomous synthetic organelles and cells with a defined function using a repertoire of functional modules (toolkit) and containing inside a minimal metabolism for survival, represents the ultimate goal of this project group.

Such complex processors will open a wide variety of possibilities ranging from environmental to medical applications. One of the most important challenges will be to provide a large repertoire of engineered and modular biomolecular-transport and -energy conversion systems for assembly of nanoreactors with diverse functionalities in lipid bilayers and block copolymers.

Initially, modules will include light-­driven proton pumps and proton-driven solute transporters in the membrane, and metabolizing enzymes inside the container. Next, more complex powering systems will be explored such as combinations of light-­driven proton pumps with sodium/proton antiporters with the objective: to energise sodium-driven solute transporters. This will significantly increase the repertoire and specificity of translocating modules.

The availability of numerous, highly specialized membrane proteins in milligram amounts offers the unique opportunity to use them as building blocks and toolkit to assemble molecular factories in the form of nanoreactors and functional surfaces using bottom-up approaches.

This project group has a strong expertise and knowledge in biochemistry, function and structure of membrane proteins. Furthermore, the group already possesses a significant number of recombinant transport proteins for different solutes such as peptides, sugars, amino acids and antibiotics that can be used as modules for engineering and assembly of nanoreactors.

Artificial Protein Scaffolds for Controlled Assembly of Supramolecular Complexes.

Artificial protein complexes are of high interest for the build-up of molecular factories because they enable assembly of multiple functional subunits in close proximity to one another, providing enhanced catalysis of chemical reactions in series. Assembly of binding proteins (e.g., antibodies) onto scaffolds is also highly desirable for delivery of protein therapeutics because the multi-valent complexes exhibit increased bound lifetime, effectively increasing the apparent affinity of a specific binding molecule to the cell surface.

A major limitation, however, remains the preparation of large scaffolds onto which enzymes and antibodies can assemble. Several strategies have been developed for synthesizing scaffolds, for example, using repeated copies of receptor proteins, or DNA-based scaffolds. These approaches, however, are limited in terms of scaffold size, specificity and/or stability.

The Nash Group synthesizes molecular scaffolds using repetitive protein building blocks called elastin-like polypeptides, and incorporates bioorthogonal functional groups for linkage to proteins and enzymes. The outcomes will be scaffold proteins which provide a versatile platform that is generalizable to many different reaction cascades and binding molecules. This work represents a simplified and scalable process to generate supramolecular complexes with novel functionality.

This project shows true interdisciplinary, transversal research: Clinical tests are conducted with light sensitive, molecular systems in partnership with the Friedrich-Miescher Institute of our industry-partner, Novartis. Should the tests be successful, this project could enable blind people to see in black and white again and, eventually, regain their full color vision.

Retinitis pigmentosa (RP) refers to a diverse group of progressive, hereditary diseases of the retina that lead to incurable blindness and affects 2 million people worldwide. Artificial photoreceptors constructed by gene delivery of light-activated channels or pumps (functional molecular modules) to surviving cell types in the remaining retinal circuit have shown to restore photosensitivity in animal models of RP at the level of the retina and cortex as well as behaviourally.

Simply said, in a degenerated macula the first step is missing: there are no more rods and cones that can detect light and subsequently convert light into neural signal. The visual nerves however are intact. In tests with apes and dogs the genetically delivered molecular factories dock successfully with the visual nerve of the eye and are activated by light, producing certain impulses that enables blind animals to see again.

Protein engineering is the field of discovering novel, synthetic proteins or improving a known protein’s activity (e.g., antibody binding affinity for a particular antigen) by using either rational or directed evolution methods. A new method that combines elements of both rational design and directed evolution is deep mutational scanning, which combines high-throughput screening with next-generation DNA sequencing to assess the functional impact of mutations across the protein sequence.

This project aims to establish deep mutational scanning as a viable approach for engineering complex therapeutic proteins. As a starting point, we will use already existing therapeutic monoclonal antibodies against cancer targets, such as Herceptin, Avastin, Cetuximab, and Rituximab, and improve their binding affinity and expression stability. We will also use sequencing data and combine it with biophysical and structural modelling to determine if we can improve the functional scoring systems generated by deep mutational scanning.

One major objective in synthetic biology is the bottom-up assembly of functional nanocells. These structures consist of lipid or polymer membranes, which serve as architectural scaffolds for functional modules such as membrane and soluble proteins. The former is embedded in the membrane, while the latter is encapsulated inside the container. The correct orientation of membrane proteins in the membrane is essential in order to avoid functional short circuits and obtain fully functional nanocells.

In this project, we propose the use of fusion proteins combined with clipping proteins to functionalize nanocells with more than one type of membrane protein.

Scientific and ethical researchers join to identify ethical issues that involve all projects by debating them in an empirically well-informed and argumentatively sound way, and developing sound policy recommendations on how to deal with them.

Molecular Systems Engineering is a rapidly evolving field cutting across traditional disciplinary boundaries. The large extent to which technological aims and design strategies are applied to the biological systems results in an unprecedented level of interference with living nature and the human body.

The risks, benefits, uncertainties as well as novel ideas and paradigms related to this increased potential raise a large set of interesting ethical, societal and policy questions. Moreover, new technological developments resulting from projects involved might impact significantly on the daily lives of future generations and affect their view on the world.

The NCCR Molecular Systems Engineering opens and welcomes an interdisciplinary discussion of exciting and controversial philosophical issues that relate to all of its projects. This can include the meaning of concepts such as “nature”, “life”, “artificiality” or “design” and the relation of these concepts to human design at nanoscale and into the biological world, as well as the design and production of molecular machines and factories, the engineering of cellular functions, the construction of organic-inorganic hybrids and the use of directed evolution as a “designing aid”.

Another area of relevance is future applications with expected benefits and potential risks that may raise ethical and societal issues, e.g. diagnostic or therapeutic tools for medicine or applications towards the production of energy.

A bioethics training module tailored towards the needs of scientists will be developed and offered. Additionally, there are regular interaction and the exchange of experiences with the ethics and public relations office.

This project uses state-of-the-art molecular and genomic editing platforms to engineer immune cells for applications in biotechnology and cellular immunotherapy.

Our ongoing projects are focused on applying molecular and genome editing tools to engineer various types of immune cells. The precise genomic exchange of highly similar immunogenomic genes has not been demonstrated before using genome-editing tools, thus a series of aims and milestones to advance this goal have been established. For example, in one of aims we are engineering mammalian cellular factories for protein production. In another aim we are using precise genome editing to improve cellular therapeutics for transplantation and cancer. We will combine our efforts with the network of NCCR researchers to push the boundaries of immunological systems engineering. 

Scientific Highlights

  • Plug-and-(dis)play mammalian cells. We have used precise genome editing to develop a platform for rapid generation of stable cell lines capable of surface expression and secretion of recombinant proteins. The simplicity of our plug-and-(dis)play platform is highlighted by the fact that it only requires a single transfection and screening step to generate stable cells. We envision these cell lines can be applied for generating recombinant protein reagents and therapeutics. 
  • Reprogramming MHC-specificity immune cells. We have established a proof-of-concept for MHC-allelic replacement, which could be used in the future for improving the donor-host matching, which is a major challenge in allogeneic cellular transplantations in cancer. We used genome editing methods to precisely exchange the MHC region of immune cells, which were then subsequently verified for functions immune activity. Our methods can be applied for the engineering of other immunogenomic regions, which would have value in cellular immunotherapy.

The objective of this project is to move beyond primary systems to maximize complexity and cumulate emergent properties that are a) significant (conceptual or practical), b) absent in the individual components, and c) inaccessible otherwise.

Ongoing projects focus on the development of orthogonal dynamic covalent bonds for advanced systems interfacing; current emphasis is on boronic esters from bioadhesives.  A second specific objective is the creation of artificial enzymes that operate with interactions that are new-to-nature; current emphasis is on the interfacing of anion-π interactions and streptavidin mutant libraries. A third specific objective focuses on disulfide exchange chemistry on cell surfaces to interface living cells with functional systems such as protein complexes involved in gene editing, artificial metalloenzymes for metabolic engineering, and liposomes or polymersomes as artificial organelles. In this project, emphasis is exclusively on added value from collaborations within the network of this NCCR, i.e., research that could not be realized without this NCCR. 

Scientific Highlights

  • The creation of the first anion-π enzyme: In sharp contrast to the ubiquitous cation-π catalysis in biology, anion-π catalysis, that is the stabilization of anionic transitions states on π-acidic aromatic surfaces, has been just been introduced in chemistry and has so far been unknown in biological systems.The creation of the first artificial enzyme that operates with anion-π interactions became possible by combining expertise from the Ward group on streptavidin mutant libraries and expertise from the Matile group on anion-π catalysis.  The emergent properties obtained from systems interfacing are fully selective catalysis of intrinsically disfavoured but biologically most relevant enolate chemistry, no trace of the intrinsically favoured but irrelevant product, and enantioselectivity near perfection (95% ee).

  • The discovery of the third orthogonal dynamic covalent bond: So far, only disulfide exchange under basic and hydrozane exchange under acidic conditions could be operated independently.  The combination of expertise from the Gademann group on bioadhesives and the Matile group on dynamic covalent surface architectures has lead to the introduction of boronic esters that can exchange independently from disulfides under and hydrozanes.  The availability of a third orthogonal dynamic covalent bond is of fundamental interest for systems interfacing.

In this project we aim to use visible light to drive electrons across a membrane and to accumulate charges in such a way that NAD+ (nicotinamide adenosine dinucleotide) can be converted to NADH. The latter can then fuel enzymatic cascade reactions which ultimately lead to value-added products that are synthesized in the closed compartment of a molecular factory. The use of vesicles made from block copolymer membranes that make up the closed compartment offers the advantage that the enzymatic chemistry can be performed in a protected environment and that oxidation and reduction products resulting from photochemistry can be spatially separated from one another.

The photophysical and photochemical aspects of this project are addressed by the Wenger group while the enzyme chemistry is taken care of by the Ward group. Vesicles built from block copolymers will be made in collaboration with the groups of Meier and Palivan.

In the first phase of the project, strategies for photodriven electron accumulation are explored in suitable model compounds. For example, covalently linked Ru(bpy)32+ (bpy = 2,2’-bipyridine) – naphthalene diimide (NDI) systems are investigated with a view to reducing NDI to NDI- and finally NDI2- in the course of photoirradiation of the Ru(bpy)32+ photosensitizer in presence of sacrificial or non-sacrificial electron donors. Once the challenge of achieving efficient electron accumulation has been tackled, it will be possible to perform light-driven two-electron reduction of suitable small molecule catalysts which are able to reduce NAD+ to NADH.

In parallel, the Wenger group explores the possibility of obtaining other fuels by means of photochemistry, for example formate from CO2. Formate can act as an electron source for various enzymatic reactions hence this molecule can potentially be used to initiate enzyme cascades. Close interaction between the Wenger and Ward groups is key for this purpose. 

Furthermore, strategies for the oriented incorporation of molecular wires into block copolymer membranes are explored jointly between the Palivan and Wenger groups. In order to obtain net electron transfer from the outside to the inside of the molecular factories made from vesicles, it will be essential to orient donor-bridge-acceptor systems properly in the membrane. 

Possible long-term perspectives include the incorporation of proton pumps into the vesicles in order to transport protons across the membranes, in addition to electrons. This avenue will be explored jointly with the group of Fotiadis. An addition possible avenue is the use of artificial metalloenzymes in the enzyme cascades that rely on engineered anion-π catalysis, as implemented jointly be the groups of Matile and Ward.

The project is dedicated to rational design and engineering of mammalian cells augmented with complex functionalities, for applications in non-medical biotechnology.

The two application areas include rational forward design of bioproducing mammalian cell lines, and the development of sentinel (reporter) cells for highly-informative drug discovery assays. In the former, we combine the laters tools of genetic engineering such as synthetic biology and automation, with the recent genome editing approaches and rational in situ control of the bioproduction process on a single cell level, to achieve end-to-end workflow for biomanufacturing cells without the need for screening. In the latter project, we harness the power of biomolecular computing to engineerer reporter cells that detect multiple drug effects in parallel. This dramatically increases the information content of a cell-based drug screening assay without reducing the throughput.

In the design of biological systems (synthetic biology), the aims range from detailed model-based design of novel systems to general concepts for the rational and – potentially automatic – design of large-scale circuits.

Robust systems designed to enable the use of engineered molecular systems in biotechnological and medical applications are of particular interest. This project will address various aspects of methods and model development for Molecular Systems Engineering to enable rational (model-based) design of molecular systems and component features such as kinetic parameters in iterations with experimental (analysis) approaches.

For the rational design of synthetic molecular systems, mathematical models of different types have been developed. While a growing number of computational design tools implements such formal approaches, major challenges remain, for example, in terms of systematic model development and in the rational design of informative experiments for systems characterization.

In the area of model-based analysis and design of genetic circuits, the development of computational models and design methods will enable rational design of parts and circuits. Another focus on the design of engineered metabolic networks will support molecular engineering of complex biosynthetic and energy-producing systems by integration from molecular modules to factories.

Publications

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Projektleitung Mitarbeiter [grouplink]
Wolfgang Meier Jens Gaitzsch
Viktoria Mikhalevich
Cora-Ann Schönenberger
Dimitri Hürlimann

Meier group @UniBas

Sven Panke Daniel Gerngross
Lukas Huber
Markus Jeschek
Eirini Rousounelou
Peter Ruppen
Sanja Tunjic

Panke group @D-BSSE

Randall Platt Mariia Cherepkova
Maria Kuhn
Sushmita Poddar
Antonio Santinha
Florian Schmidt
Niels Weisbach

Platt group @D-BSSE

Ivan Martin
Yaakov Benenson
Manuele Giuseppe Muraro

Martin group @UniBas

Benenson group @D-BSSE

Catherine E. Housecroft
Cornelia G. Palivan
Myrto Kyropoulou
Dalin Wu

Housecroft group @UniBas

Palivan group @UniBas

Florian Seebeck
Petra Dittrich
N.N.

Seebeck group @UniBas

Dittrich group @D-BSSE

Wolfgang Meier
Martin Fussenegger
N.N.

Meier group @UniBas

Fussenegger group @D-BSSE

Christof Sparr Daniel Moser
Felix Raps
Dragan Miladinov
Zlatko Joncev

Sparr group @UniBas

Marcel Mayor Marius Ciobanu
Tim Hohner
Gabriel Puebla-Hellman
David Vogel
Giulia Prone

Mayor group @UniBas

Dimitrios Fotiadis Stephan Hirschi
Mirko Stauffer

Fotiadis group @UniBe

Michael Nash Byeong Seon Yang
Jaime Fernandez de Santaella Sunyer

Nash group @UniBas

Botond Roska Jacek Krol
Magdalena Renner
Tamas Szikra

Roska group @FMI

Bruno Correia
Sai Reddy
Pablo Gainza

Correia group @EPFL

Reddy group @D-BSSE

Dimitrios Fotiadis
Wolfgang Meier
Daniel J. Müller
N.N.

Fotiadis group @UniBe

Meier group @UniBas

Müller group @D-BSSE

Nikola Biller-Andorno Sebastian Wäscher

Biller-Andorno group @UniZH

Sai Reddy Theresa Pesch
Derek Mason
Jakub Kucharczyk

Reddy group @D-BSSE

Stefan Matile Eline Bartolami
Javier López Andarias
Xiang Zhang
Quentin Laurent
Xiaoyu Hao

Matile group @UniGe

Oliver Wenger Mirjam Schreier
Jasmin Anastasia Kübler
Christopher Bryan Larsen

Wenger group @UniBas

Yaakov Benenson Raffaele Altamura

Benenson group @D-BSSE

Jörg Stelling Claude Magnard
Asli Azizoglu
Fabian Rudolf

Stelling group @D-BSSE