SAGEs: Self-assembled peptide-based cages for the presentation, encapsulation and delivery of bioactive molecules to cells in culture

CONTEXT OF THE RESEARCH: An ability to deliver biologically active molecules (drugs, DNA and proteins) to specified cells either in the lab or the body would impact on many branches of biology and medicine. Imagine being able to selectively find and destroy diseased cells; or "simply" to test the effectiveness of a new drug inside a range of cells before animal and human trials? Unfortunately, there is no general solution to this problem of delivering bioactive molecules within cells, and even bespoke true solutions are few and far between. The problem is not straightforward, and is best illustrated by how biology has evolved viruses to do this.

Viruses are astonishing natural nanoscale packages, usually termed virions. Though they come in many types, all virions perform three functions: (1) they recognise often specific cell types, which they do by presenting molecules on their surfaces to recognize molecules on the target cells; (2) they penetrate the outer barriers of the cell; and (3) they deliver a payload, which is the genetic information to make more virus in the host cell. Functions (1) and (2) are performed by the viral coats, or capsids. Not surprisingly, many people have tried to mimic these structures to deliver payloads other than RNA and DNA.

AIMS, OBJECTIVES AND ASPIRATIONS OF THE RESEARCH: The overall ambition of the proposed work is to produce hollow, cage-like particles that have the diameter of about one hundredth the width of a human hair, so-called SAGE particles. We will do this in a modular way, using small versions of proteins called peptides. Each peptide module will have a specific function to mimic one the properties of virions: one set will be made to recognize specific cell types; another will be used to construct the casing of the particles; and the third set will carry the biologically active payloads. On their own, these modules would not be useful at all. However, if combined correctly they could assemble into virus-like particles, but without the (deadly) RNA and DNA cargo, instead they would contain drugs or useful proteins.

To do this we will build on a multidisciplinary team of chemists, biochemists, cell biologists and molecular modellers that has delivered the SAGE particles. The physical scientists will work together to design and make the assemblies of molecules, and then work with the biologists to test and visualise how they interact with cells and deliver their payloads.

POTENTIAL APPLICATIONS AND BENEFITS: Throughout the research, we will work with a company, Syntaxin, interested in targeting and killing particular diseased cell types in the body. As well as providing reagents and know-how, this partnership will encourage real-life applications, and thus clear and practical end points for our research. In this way, we will explore both the fundamentals of SAGE assembly and engineering, and potential applications of functional SAGEs in cell biology and medicine.

Broadly speaking, this modular and systematic approach to constructing complex biological molecules, assemblies and systems is called "synthetic biology". The aim and spirit of synthetic biology is to make the engineering of biological systems easier (that is, systematic, quick, and predictable) and ultimately to make useful functions and products. For example, synthetic biology is being recognised as increasingly important to generate new medicines, biofuels and fine chemicals. It is being invested in by Government and Research Councils, with the aim of developing the field sufficiently to be of direct benefit to the UK (biotech) industry and economy. One of the key aspects of our proposal is that it fits with this spirit and these aspirations: we aim to make a toolkit of different modules for each of the above three properties; in this way, modules could be combined rapidly, reliably and with predictable outcomes to generate different particles for targeting and tackling different cells and diseases.

We have assembled a multidisciplinary Bristol-based team covering peptide design and synthetic biology, biomolecular modelling, cell biology and microscopy to deliver the following:

1a DESIGN OF NEW SAGE CONSTRUCTS. We will use rational peptide design and molecular modelling to generate and test new peptide modules and SAGE variants that differ in size, stability and function. Our existing modelling gives confidence that this approach will allow the meaningful and rapid evaluation of new designs ahead of more time-consuming experimental work.

1b SAGE CONSTRUCTION AND CHARACTERISATION. We will make and characterise the peptides from 1a using peptide synthesis, biophysics and electron and atomic force microscopy. Successful designs will then be made via recombinant expression of synthetic genes in E. coli, allowing routes to larger SAGE constructs and functional SAGE polypeptides.

2 DECORATING THE SAGES WITH CELL-RECOGNITION MOTIFS. We will design fusion peptides in which cell-binding and internalisation motifs (RGDS, TAT etc) are appended to the outside of the SAGEs. These will then be tested for cell binding and internalisation using light and electron microscopy and different cell types.

3 ENCAPSULATION AND CELLULAR DELIVERY OF BIOACTIVE PEPTIDES AND PROTEINS WITHIN THE SAGE PARTICLES. We will make synthetic and recombinant fusion polypeptides incorporating both cell-binding/internalisation motifs and modules to encapsulate bioactive proteins (e.g. GFP and proteases). These will be tested for internalisation into cells and inhibition of specific cellular functions in different cell types using light and electron microscopy.

As part of the latter, we will work with Syntaxin to incorporate the LC domain (not cytotoxic in itself) from Botulinum neurotoxins into SAGEs. We will smuggle these into cells in culture using the above functionalisations, where we will test for LC's known function of cleaving SNARE proteins and inhibiting vesicular fusion.

During the course of a grant, we would engage with audiences beyond academia. We envisage two broad groups of beneficiaries, and propose to engage them and foster relationships as follows.

1. UK AND INTERNATIONAL BIOTECHNOLOGY INDUSTRY, AND THE UK ECONOMY MORE GENERALLY
If we succeed in making functionalised SAGE particles that interact with and internalise into cells, we envisage potential applications in the delivery of all manner of bioactive molecules to cells in culture and in vivo. This would be extremely exciting, opening up new routes to the delivery of drugs and biologicals in the lab and in medicine.

Though some way off realisation, initially we will explore possibilities for the SAGEs in this domain with Syntaxin, a biotech company based in Abingdon, UK. Hopefully, this will lead to a natural partnership for exploiting and developing the work towards real-life applications. We anticipate that seeing any functional and active SAGEs through to such applications would take 3 - 8 years.

We have filed a patent on the SAGE concept, the first-generation designs, and potential applications areas. If the work outlined in this proposal is successful, we envisage further IP and patent applications. If correct, there is potential for broader applications of the SAGE concept than "just" in cell delivery; it is possibly a platform technology with applications as scaffolds for vaccine development (antigen presentation) and enzyme encapsulation. Therefore, we see potential for a University of Bristol spin-out company, or licensing agreements to develop the SAGE concept in other directions; both of which we will actively explore. These would contribute to both the local Bristol and UK economies. We see a longer timeframe for these applications, 5 - 15 years.

2. UK PUBLIC DEBATE AROUND SYNTHETIC BIOLOGY
Synthetic Biology is an emerging area of research that combines engineering and biology. By applying engineering principles to biological systems, we may be able to re-design, or create from scratch, biological systems that perform new functions. Generally, research in this area can raise societal concerns in terms of safety, security, regulation, ownership, and also how to deliver maximum benefit of any emerging technologies. It is important that the public is aware of developments in this exciting new field.

Both the Royal Academy of Engineering and the BBSRC have commissioned work to explore public attitudes to synthetic biology and its applications. Although public opinion is largely positive about the potential benefits of this work, it is imperative that researchers continue to talk about their research, its likely impacts and limitations, and to hear the concerns and interests of members of the public.

As outlined in our 'Pathways to Impact' statement, we have been very active in public-engagement activities associated with synthetic biology over the past 5 years; and we will continue to organise/participate in open discussions about synthetic biology with a variety of audiences. Specifically, we will engage secondary school pupils, teachers and adult members of the public in discussion and debate about this exciting area of research. We hope that through these interactions, they will be more positively disposed to research in this area, and will have increased trust in the scientists that carry it out.

A second audience that will benefit from the public-engagement activities are the early career researchers in the Woolfson, Banting, Verkade and collaborating groups in Bristol. DNW and JMF in particular will be involved in the planning and delivery of public-engagement activities, receiving dedicated training from qualified professionals as required, as well as experiential training opportunities at the events themselves.

Many of these aspects of our Impact Plan are already in place, and we plan to continue public-engagement activities certainly for the next 3 - 4 years, and probably beyond