21EBTA: Engineering plant cell factories for the production of biomedicines and their ingredients. (Acronym; Celfacto)

The COVID-19 pandemic caused by Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2) was impossible to predict, but scientific discoveries have played a major role in our ability to manage the disease state. Although the vaccine scale-up has been highly publicised, underpinning their development is a wide range of scientific tools and resources developed over many years. Our proposed project will develop these tools and resources to ensure, biology can be used to deliver better treatments and preventions in the future. The approaches will not be limited to COVID-19 but will have applicability to many other biomedicines. The project will use a plant-based host as its production platform, by nature this means the system is sustainable and will not use fossil fuel derived precursors and energy, thus improving the environmental credentials of the manufacturing process. New Plant Breeding Techniques (NPBTs) will be used, which implies the genetic manipulations used are more acceptable to the consumer, the procedures will be contained, transient expression will be used, while stably transformed material will only use genes from sexually compatible species. The plant species to be used is Nicotiana benthamiana. This type of tobacco accommodates rapid infection by soil bacterium Agrobacterium, which facilitates the transient expression of heterologous genes. Our valuable production targets will be:
(i) Squalene; this molecule is the leading adjuvant, which means in can stimulate our immune system, less protein (antigen) is required for effective vaccination and it can form nano-structures that better maintain functional protein structures. Presently squalene is extracted from sharks' liver oil and demand is outstripping supply.
(ii) SARS-CoV-2 neutralising antibodies will be produced in the plant based platform. These antibodies represent a treatment for COVID which goes beyond the current preventative vaccination approach that will only work if patients do not have the virus.
(iii) Virus Like Particles (VLPs) of SARS-CoV-2 will be generated in the programme, these are safe structures that offer better vaccine components, as they represent or mimic the structure of the virus more precisely, leading to better immunological responses.
Production yield and quality are major determinants influencing the adoption and widespread use of plant-based manufacturing for biomedicines. Our project will address these aspects by generating several lines with reduced protease activity and altered glycosylation potential. In this way the losses and alterations by degradation of the protein products will be reduced improving competitiveness. In addition, by removing the diverse potential for plant derived glycosylation, the products will be more human-like in nature.
Collectively these outputs have important societal and economic benefits and integrated dialogue will be used to inform diverse stake holders. These activities will include focus groups and workshops performed in the UK and internationally.

Nicotiana benthamiana is a well-established plant-based system that has been adopted by several companies for the production of antibodies, antigens, enzymes and in some cases small molecules. Protein quality and yield from Agrobacterium infiltration of N. benthamiana can be effected by endogenous protein degradation and altered glycosylation patterns.
In the proposed project we will use intragenic components to create a stably transformed N. benthamiana lines producing the valuable adjuvant squalene. Squalene, an isoprenoid molecule, will be produced by orthogonal engineering of farnesyl pyrophosphate (FPP) and squalene synthase into the plastid organelle. Isopentenyl pyrophosphate (IPP) supply in the plastid will be enhanced through the intragenic expression of 1-deoxy-D-xylulose-5-phosphate synthase (DXS), an influential step in the pathway. Transient expression of SARS-CoV-2 neutralising antibodies or Virus Like Particles (VLPs) in the squalene producing background will be carried out to show the potential of using the biomass as a multipurpose feedstock.
Multiplexed genome editing will be used to knock out proteases in N. benthamiana, particularly the subtilase gene family. The potential of these low protease activity lines to produce improved quantity and quality of SARS-CoV-2 (IgG) neutralising antibodies, termed COVA 2_15 will be determined. In a similar manner, the potential of low protease activity lines to yield greater amounts and quality of hybrid IgA neutralising antibodies to SARS-CoV-2 and VLPs will be determined. The production of antibodies and VLPs in plants can result in non-native glycosylation patterns, effecting potency and specificity. Modifications involving xylose and fucose are common in plants but not found in humans. Therefore, using genome editing, a suite of glycosyl transferases will be downregulated. These lines will then be used to produce hybrid IgA neutralising antibodies to SARS-CoV-2 and VLPs.