Empty virus-like particles (eVLPs) as bio-compatible targeted drug-delivery vehicles

A major challenge in pharmacology is to devise methods whereby drugs can be delivered specifically to target tissues. This is a particular issue in the case of anti-cancer drugs which usually discriminate between cancerous and normal cells by the fact that the cancer cells are dividing more rapidly. However anti-cancer drugs are toxic to all cells and thus often have severe side-effects. To avoid this, it would clearly be desirable to target the drug molecule specifically to the cancerous tissue. A potential means of achieving this would be to package or encapsulate the drug molecules inside a particle which is designed to bind solely to the cancerous tissue. Such encapsulation would have the additional advantage of protecting the drug from breakdown in blood plasma. For this to become a reality it will be necessary to develop particles which can be modified on their outer surface to achieve the desired targeting and which can contain drug molecules. The particles need to be small enough to be able to move in the bloodstream, are not toxic and to be able to enter cells. One type of particle which has all these characteristics is the plant virus, cowpea mosaic virus (CPMV), which can be produced in large quantities by infecting plants. Previous research has shown that it is possible to 'stick' molecules on the surface of CPMV particles which enable them to be targeted to specific cells. Despite these advantages, CPMV particles have not, to date, been exploited as a drug-delivery vehicle. The reason for this is that particles produced by the infection of plants are already filled with the virus' own genetic material. This means that there is little or no room to put anything else, such as drug molecules, inside the particles. Even if such molecules could somehow be squeezed in, there would still be concerns about administering particles containing viral genetic material, even though it is from a plant virus that cannot infect animals. This project addresses the issue of the genetic material within the virus particles by exploiting the recent discovery at the John Innes Centre of a method of producing large quantities of pure empty (lacking genetic material) virus-like particles (eVLPs) of CPMV in plants. The method involves simultaneously expressing genes coding for a precursor of the viral coat proteins and the enzyme used to process it using a recently developed highly efficient plant transient expression system. Applying this approach we will produce particles which are modified so that they will specifically bind to proteins expressed on the surface of cancerous, but not normal, cells. This modification will be done either chemically or by making genetic fusions to the virus coat protein. We will investigate the best way of loading the targeted particles with the anti-cancer drug, gemcitabine. To do this we will make use of pores in the virus particles which allow small molecules to enter under certain conditions. The ability of the targeted particles to deliver the drug to cancer cells will be investigated by testing their ability to bind to and kill cancers in culture. This will be the first important step in potentially developing brand new therapeutic agents to tackle human cancer.

This project is aimed at developing empty (RNA-free) virus-like particles (eVLPs) of cowpea mosaic virus (CPMV) as a targeted drug delivery system. CPMV particles have already been shown to be well tolerated in mice and have been investigated as imaging agents. However, despite these properties, the presence of RNA within particles isolated from infected plants has, to date, precluded the use of CPMV for drug delivery. The project will exploit the recent success at JIC in the production of empty capsids using the CPMV-HT transient expression system and the applicants' experience in the genetic and chemical modification of CPMV particles. Although the ultimate aim will be the development of a generic targeted drug delivery system, the project will focus on the delivery of the anti-cancer drug, gemcitabine, to cells expressing a cancer-specific integrin. This receptor is absent from most healthy adult tissues but is over-expressed in a range of tumours including oral squamous cell carcinomas and lung and breast carcinomas. Thus, it is a tumour-specific target which is of particular interest to our industrial partner Aura Biosciences Inc.. To produce eVLPs which target to this sequence, we will express either a peptide, A20FMDV2, with known integrin-binding properties or a single-chain antibody (scFv) on the eVLP surface using established chemical and genetic methods. To load the eVLPs with gemcitabine, we will make use of pores which occur at the 5-fold particle axes. These allow small molecules to enter the eVLPs in a pH-dependent manner and thus access to the interior of the VLPs can be controlled. We will optimise methods for loading the drug into the VLPs including mutagenesis of amino acids which surround the pore and line the interior of the capsid. The ability of the targeted particles to deliver the drug to cancer cells will be investigated by testing their ability to bind to and kill cells expressing the integrin in culture.

The approach taken by the project, the development of a targeted drug delivery system based on empty virus-like particles (eVLPs) from cowpea mosaic virus (CPMV), is designed to solve a critical issue in drug delivery by encapsulating therapies within a protein nanoparticle engineered for precise targeting, immune system evasion, and efficient cellular uptake. The use of a virus-derived nanoparticle also offers the prospect of scalable manufacturing. The initial focus of the project is on applying the eVLP technology to the treatment of cancer. Such nanoparticle-enabled chemotherapy is designed to be more effective and better tolerated than current therapies, allowing more patients to be treated and to help improve survival of this devastating disease. This would clearly have a major impact in the treatment of cancer. However, the technology for targeted drug delivery could be applied to many other diseases and thus would have a general impact in the field of medicine. To ensure the impact of this research is fully exploited, we will consider intellectual property (IP) issues at an early stage. Clearly patents directly arising from the research will be filed in collaboration with our Industrial collaborator, Aura Biosciences Inc. The applicants already have extensive experience of commercialising their research. Prof. Lomonossoff has filed several patents on the expression of foreign proteins in plants and, in particular, the use of chimaeric virus particles; commercial applications of the technology are being pursued. Dr. Evans and Prof. Lomonossoff have recently filed a patent, in collaboration with Aura Biosciences Inc, on the use of plant virus particles for delivery of drugs and other biological reagents. The JIC has staff dedicated to and experienced in the protection and exploitation of intellectual property and has an excellent working relationship with, Plant Biosciences Ltd (PBL) an on-site independent technology transfer company. PBL have already entered into discussion with Aura Biosciences regarding an option agreement on the eVLP technology. In addition to considering IP issues, it will be necessary to engage with various users, beneficiaries and stakeholders.These will include commercial organisations, the public sector and the wider general public. In addition, the peptide A20FMDV2 idntified and characterised by the group led by John Marshall is protected internationallly by patents held by Cancer Research Technologies. Aura Biosciences has recently ben granted a license for the commercial deve,opment of this peptide. Though the focus of project is the use of CPMV eVLPs for targeted drug delivery, the results of the research are likely to have an impact far beyond this immediate area. Because of it's attractive properties, CPMV has already been widely investigated as a nanoparticle for applications in field as diverse as bio-medicine and material science. However, to date all these applications have used virus particles containing RNA with concomitant problems of biosafety and lack of loadability. These have hampered the development and exploitation of CPMV-based technologies. The technology for producing eVLPs, which will be refined during the project, overcomes these limitations and will therefore greatly assist the development of CPMV-based applications. As well as having a direct impact on the development of CPMV-based technologies, the results will have a general impact on the general development of nanoparticle-based technologies. Thus the information gained about targeting and loading CPMV will be applicable to a wide-range of other nanoparticles of viral or other origin. The hope is that these targeted particles will selectively deliver therapy and thus allow higher doses of drug to accumulate in the target organ. The molecular target od A20FMDV2, integrin alpha-v beta-6, is upregulated on many different forms of cancer and thus makes our expected success valuable for improving UK health.