Engineering Living/Synthetic Hybrid Assemblies (LSHAs) as Functional Units for Synthetic Biology

Lead Research Organisation: Imperial College London
Department Name: Dept of Chemistry

Abstract

The overarching vision of the fellowship concerns the following question: how can biological structures (i.e. cells) be coupled with artificially-constructed elements (i.e. vesicle-based synthetic cells functionalised with molecular machinery) for the construction of living/synthetic hybrid assemblies (LSHAs)? Answering this question requires that significant technological hurdles be overcome, and this is the focus of this fellowship.

Synthetic biology concerns itself with the construction of biological systems unlike those seen in nature. Synthetic cells can either be engineered from the top-down, by taking cells and manipulating their genome, or from the bottom-up, by building artificial cells from scratch using simple chemical building-blocks for user-defined functions. A technological bottleneck has meant that these two approaches have existed in isolation from one another, thus hampering the power and potential of the discipline. The LSHAs developed will serve to bridge this divide. This is of strategic importance to synthetic biology: it promises to open up a new research field and deliver applications-from biosynthesis and biosensing, to bio-chemical computing and therapeutic delivery-that would further cement the area as a key emerging technology.

Strategies to construct hybrid cells will centre on using developing droplet-based microfluidics to encapsulate cells within functionalised synthetic lipid vesicles. In this way, the hybrid cells will have access to the complex biochemical pathways inherent in biological systems, yet still contain artificially-constructed elements designed from the bottom up for bespoke functionality. LSHAs can thus be considered as a novel class of 'artificial eukaryote': they will consist of a vesicle host and a cellular symbiont enjoying a mutually beneficial relationship. A set of engineering rules to biochemically interface the synthetic host with the encapsulated cells will be devised and communication routes between the two will be developed.

Next, their use as a suite of cutting-edge applications via a series of proof-of-concept studies will be demonstrated. These include (i) downstream processing of secreted cellular products by chemical components contained within the vesicle, (ii) using LSHAs as a means to achieve 'membrane transplants'-the replacement of a cellular plasma membrane with an artificial membrane of defined size, composition and asymmetry, and (iii) linking up multiple LSHA units to form a large-scale network capable of processing and propagating chemical signals. This fellowship will therefore act a feasibility roadmap outlining which strategies and applications do and do not work, to aid future advances in the field.

This research will be undertaken in the Department of Chemistry at Imperial College. It will fall within the bracket of the Institute of Chemical Biology, and make use of some of its state of the art microfluidic, optical trapping, and microscopy facilities specially tailored to study systems such as these. It also be conducted in partnership with Prof Booth (Kings College London), who will provide key support for the incorporation of smart, responsive behaviours via mechanosensitive pores, and with Prof Yitzhak Mastai (Bar Ilan, Israel) for the addition of chiral molecular elements into the hybrid cell.

Planned Impact

The overall vision behind this proposal is to investigate how biological cells and sub-cellular components can be coupled together with synthetic elements to yield Living/Synthetic Hybrid Assemblies (LSHAs). The field is at an early stage in its development, and requires significant fundamental research to build a solid platform on which applications can be based. Once this is accomplished, it is expected to generate impact across a wide range of sectors in the UK economy and society. Specific areas of research impact include:

(i) Biofuel and chemical production industries. The incorporation of biological cells acting as organelles within vesicle-based artificial cells will allow biologically manufactured products to be processes by synthetic elements in vesicle downstream. LSHAs could therefore prove a game-changing innovation for these industries in the long-term (>15 years), as they provide a method for biological production and chemical modification integrated in a single microreactor.

(ii) Therapeutic delivery sector. Vesicular systems have been widely used by the pharmaceutical industry for decades. In the short-term (< 5 years), functionalization with biological and chemical components will lead to new responsive vesicle systems that allow targeted delivery and tailored drug release kinetics. In the longer-term (> 10 years), LSHAs are expected to produce a new paradigm in therapeutics, by allowing drug and biomolecule synthesis within delivery vehicles, as and when needed, at the target site.

(iii) Patient Care. Artificial cells have been proposed as the next generation of delivery systems. The development of LSHAs will enable the smart behaviours outlined above to be introduced. This will lead to more efficient pharmaceuticals with decreased side-effects. This applies to small-molecule drugs, but also to protein and DNA based therapeutics, which are suited to being encapsulated (and synthesised) in vesicle-based artificial cells.

(iv) Academic and industrial drug discovery programmes. Methods to replace the plasma cell membrane with an artificial membrane of defined size, composition and asymmetry mean systematic probing of ion-channel drug-targets can be conducted in a finely-controlled and simplified environment, in a lab-on-chip setting. Use of microfluidics lends itself to high-throughput approaches.

(v) Improved understanding of the role of membrane lipid composition in cells. The development of plasma-membrane transplants will act as tool to decipher the effect that membrane composition has on embedded protein activity by allowing variables to be precisely defined. In a wider context, this will shed light on how membranes are implicated in diseases.

(vi) Micro-Electro-Mechanical-Systems (MEMS). The methods proposed to interface living and synthetic systems could see use in the MEMS industry, for examples for the incorporation of dedicated bio-components for energy generation, serving as a renewable power source, or 'biochemical computers' for the processing of biochemical as opposed to electrical information.

(vii) UK Cultural and Artistic Spheres. There have already been several landmark examples of synthetic biology being used in these areas (e.g. self-healing biofabrics for high-end fashion). It is expected that living/synthetic hybrid materials will similarly be used in these areas in the long term (15+ years). A consequence of this will be drawing public attention to the cultural value of science, which is crucial given public concerns about biotechnology.

Due to the pioneering nature of the work, there will be also be new unforeseen applications, depending on unexpected breakthroughs made. In summary synthetic biology has proved itself to be of growing importance to the UK's tech-economy as demonstrated by wealth of synthetic biology companies that have emerged in recent years. There is no doubt that the technologies proposed here will add value to this rapidly expanding sector.

Publications

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Description This project was dedicated to the development of a suite of microfluidic and optofluidic tools and technologies to generate vesicle-based synthetic cells, to manipulate them, and to integrate them with biological cells. The key findings and outcomes of this award are as follows:

1. Development of microfluidic techniques to encapsulate cells in lipid vesicles. This allowed us to use living cells as organelle-like modules within vesicles based synthetic cells. Encapsulated cells have been engineered to act as bioreactors and biosensors. Incorporation of a bio-battery functionality is ongoing. This technology is being taken forward for industrial and clinical use through follow-on collaborative projects.
2. Development of light-responsive vesicle microreactor systems, that can initiate chemical synthesis and catalysis using an external trigger. This is now being used to engineer light-mediated communication between living cells and synthetic cells.
3. Development of optical trapping technologies to link up discrete vesicles in networks of defined architectures. These are linked by double-membrane spanning analogues of cellular gap junctions, which are capable of propagating signals using engineered transmembrane protein pore systems. This is forming the basis of generating large-scale tissue-like materials going forward.
4. Development of laser technologies to fuse defined vesicles with one another with spatiotemporal control. This system can be used is a vesicle microreactor context and to fuse cell membranes with one another.
5. Discovery of liquid phase separation mediated content release from vesicles. This has implication for responsive drug delivery. This concept is being taken forward through follow-on projects.
6. Discovery of the ability to manipulate liquid membrane domains using optical tweezers. This is now being used to shed light on fundamental concepts in membrane biophysics.
7. Development of a synthetic biology approach for drug delivery, where content release rates can be tuned using a protein pores and blockers and a cell-mimetic chassis.
Given the above, the objectives of this award have been met and surpassed.
Exploitation Route 1) The developed technologies could be used by the therapeutic delivery sector to engineer more effective drug delivery systems.
2) The developed technologies could be used by pharma in the context of cell models to test drug-membrane interactions and pharmacokinetic profiles in a model environment.
3) The developed technologies could be used by academic to investigate core concepts in cell biology, especially membrane-associated processes, and to contribute to a better understanding of biological cell membranes.
Sectors Chemicals,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description The drug-delivery aspects of the project are being actively explored through follow-on projects with collaborators from pharma. This includes (i) our development of light-responsive microreactors for in situ chemical synthesis (ii) our development of temperature responsive small-molecule release, for targeted delivery (iii) our development of molecular regulators to tune drug release rates from protein-functionalised vesicles. We are working towards more effective drug delivery approaches using 'artificial cell' therapeutic vehicles. Furthermore, our technologies to link up discrete synthetic cells to form tissue-like networks composed of gap-junction mimics is being developed as minimal tissues to explore drug pharmacokinetics in a model environment. Finally, the ethical implications of this research has fed in to the development of the World Economic Forum code-of-ethics for scientists. Efforts to engage with the public are continuously made, to explain the value, potential, and potential controversies that may arise from this research in the long-term. On average, public outreach events were attended every 6 months, mostly in and around London.
First Year Of Impact 2018
Sector Chemicals,Pharmaceuticals and Medical Biotechnology
Impact Types Societal,Economic,Policy & public services