Abstract
On October 15 2010 the meeting ‘Recombinant Pharmaceutical Manufacturing from Plants – The Future of Molecular Farming’ hosted by EuroScicon was held at BioPark Hertfordshire, Welwyn Garden city, UK. The scientific program of this very eventful meeting was wide ranging and covered diverse aspects of biopharming. The highlights presented included: safety issues in biopharming; coexpression of multiple proteins; steps towards vaccine generation; and engineering of secondary metabolites and medicinal plants. This article summarizes the stimulating scientific presentations and fruitful panel discussions that subsequently arose during and after this event.
In his opening remarks the Chair, Ian Graham, Centre for Novel Agricultural Products (CNAP) Director and Weston Chair of Biochemical Genetics, University of York, UK, started with an overview of recombinant pharmaceuticals. He emphasized biopharming as a vast area with countless possibilities waiting for scientific exploration, specifically areas such as antibodies, recombinant proteins, metabolites, development of platform technologies and the public opinion towards biopharming.
Coexpression of multiple proteins
The opening lecture on ‘development of a virus-derived system for the coexpression of multiple proteins at defined levels in plant cells’ was given by George Lomonossoff from the John Innes Centre, Norwich, UK. He addressed a major challenge in the development of plants as bioreactors: the design of a system that can direct the synthesis of multiple proteins within the same cell at defined and differing levels. Major challenges for stable expression systems exist concerning time consumption, variable levels of expression and difficulties using various screening methods. Transient expression systems offer several advantages, particularly transgene expression in only a few days using mature plants, which has shown demonstrated success for single peptides. Lomonossoff presented a transient expression system based on a deleted version of Cowpea mosaic virus (CPMV) RNA-2, the hyper-translatable CPMV, which permitted an extremely high level and rapid production of proteins without viral replication Citation[1]. The system involved insertion of the gene to be expressed between a modified 5´ UTR and the 3´ UTR from CPMV RNA-2. Alteration of the AUG codon was found to significantly enhance the translation level of the inserted transgene. Retaining the recombinant proteins within the endoplasmic reticulum also helped to increase expression levels. Coexpression of four structural proteins from bluetongue virus to produce virus-like particles (bluetongue virus VLPs) for immunological analysis was also part of the discussion.
Chloroplast-expressed antigen vaccines
Andreas Lössl from the University of Natural Resources and Applied Life Sciences, Vienna, Austria, gave a talk on plastid-derived vaccine antigens. Chloroplast transformation can address the risk of transgenic pollen flow as pollen rarely contains plastids. Furthermore, the plastid genome allows expression of complete operons (e.g., phbA, B and C), and this makes it possible to produce multiple antigens by one successful step of transformation. An additional advantage of this organelle transformation technique is the extremely precise insertion of transgenes and the absence of silencing effects. Additionally, an inducible expression system is available to regulate plastid-derived protein production by ethanol induction. This novel approach allows for a controlled expression of transgenes with pharmaceutical impact, and thus plastid-based systems can pave the way towards affordable pharmaceuticals for developing countries.
A recent development is the expression of human papillomavirus (HPV) L1 capsomeres in tobacco chloroplasts. Various types of HPV are causatively associated with cervical carcinoma, which is the second most common cancer in women worldwide, particularly in developing countries. Capsomeres have recently been demonstrated to be highly immunogenic structures. Compared to VLP-based HPV vaccines they offer a number of advantages as a potential cost-effective alternative. A modified HPV-16 L1 gene was expressed as a protein that retained the ability to assemble to capsomeres in tobacco chloroplasts Citation[2]. Assembly of capsomeres was examined and verified by cesium chloride density gradient centrifugation and sucrose sedimentation analysis. An antigen capture ELISA confirmed the formation of capsomeres by using a conformation-specific monoclonal antibody that recognized the properly assembled L1 proteins. These results present the possible production of a low-cost second-generation vaccine from plastids against cervical cancer.
Genetic engineering of secondary metabolites
The health benefits conferred by numerous secondary metabolites have led to several approaches to elevate their levels in foodstuffs. Two talks focused on health promoting polyphenols and carotenoids. Cathie Martin from the Norwich Research Park, UK, reported the potential of plant science to improve preventative medicine to combat chronic diseases. Epidemiological studies have demonstrated the efficacy of diets enriched with fruits and vegetables to reduce the incidences of chronic disease due to their contribution of important phytonutrients, which serve to promote antioxidant defense mechanisms. Polyphenols are one of the examples that have been shown to decrease the risk of cardiovascular disease and cancer. Such foods could benefit consumers in both developing and developed countries. Flavonoids are a large group of polyphenolic compounds. Based on their core structure they can be grouped into different classes: chalcones, flavonols and anthocyanins. In nature more than 6000 different flavonoids have been identified that are effective hydrophilic antioxidants. The health protective properties of anthocyanins contained in many commonly consumed fruits and vegetables could be increased in order to yield better health benefits. Martin and colleagues succeeded in producing tomato fruits that accumulated anthocyanins at levels considerably higher than attained in earlier studies. Another effect in genetically engineered tomato plants was a threefold boost in the hydrophilic antioxidant capacity of the fruits. In one study, this anthocyanin-containing tomato significantly extended the lifespan of cancer-susceptible mice that were fed with the new tomatoes compared with mice that were fed with normal tomatoes.
Paul Fraser from the Royal Holloway University London, UK, talked about the progress made in the genetic engineering of isoprenoids in solanaceous species, particularly in tomatoes. He explained different modifications of carotenoid biosynthesis in chloroplast and chromoplasts of tomato fruit Citation[3]. Most interesting was the observation that the endogenous carotenoid pathways in higher plants do not seem to react to engineered changes. Often this resistance appears in the form of intrinsic regulatory mechanisms that are ‘silent’ until manipulation of the pathway is initiated. These mechanisms may include feedback inhibition, metabolite channeling, and counteractive metabolic and cellular perturbations.
Molecular breeding of medicinal plants
Malaria is a severely life-threatening infectious disease that affects approximately half of the world’s population with risk of infection. In 2008 alone, 247 million cases of malaria occurred and nearly 1 million deaths were recorded. One of the most effective treatments for malaria is an artemisinin combination therapy. Artemisinin is the active compound of the plant Artemisia, grown in China, Vietnam and East Africa where it has been used for years to treat intestinal parasites. The WHO lauds it as a safe malaria treatment candidate.
In his talk Ian Graham (CNAP Director and Weston Chair of Biochemical Genetics, University of York) presented his ongoing research, the CNAP Artemisia Research Project. This project is aimed to make use of the most recent developments in genetics, bioinformatics and analytical technologies to speed up the breeding of Artemisia annua. A series of specially developed assays were used to screen several of Artemisia progeny rapidly and select the most promising ones for use in plant breeding. Plants were selected for desirable features including increased ratio of leaf to stem tissue, plant bushiness, delayed flowering, and leaf size and shape. Using the new genetic map, plants were selected not just on the basis of observable features, but also according to their genotype profile. Subsequently, plants can be selected if they show positive quantitative trait loci scores for key traits such as leaf area and artemisinin concentration. The CNAP project is well on track to produce molecularly bred, high artemisinin yielding Artemisia varieties by 2012.
Containment strategies in biopharming
Denis Murphy from the University of Glamorgan, UK (also Biotechnology Advisor to United Nations Food and Agriculture Organization), addressed the challenges of segregating biopharmed crops from mainstream crops, particularly those destined for food or feed use. Recently, serious concerns have been raised in the scientific community and also in the public, regarding the usage of major food crops as platforms for production of pharmaceutical compounds. Great emphasis is required for the rigorous separation of food and nonfood varieties of the same crop species via a range of either physical or biological methods. Murphy stressed that very effective methods for biocontainment consist of the use of plastids for transgene expression, inducible and transient expression systems and also physical containment of plants or cell cultures Citation[4]. Containment can be increased by the use of nonfood crops, noncrop plants or in vitro cultures as production platforms instead of food crops. The use of nonfood crops is a very promising option that may obtain approval of the EU for plant-based production of pharmaceuticals. Shifting the research focus from the major food crops of rice and maize to nonfood crops will also contribute to achieve acceptability of genetically modified (GM) plants. The speaker was optimistic about the EU approval of nonfood crops for biopharming within the next decade. It was clear that, in the current atmosphere of heightened concerns about food safety and biosecurity, the future of biopharming will largely be determined by the extent to which the sector is able to maintain public confidence. It requires thoroughly considered approaches to address the issues of containment and to comply with the security regulations for GM crop production systems.
Conclusion
This was a reasonably well organized meeting that enabled efficient interaction and networking between the participants in the biopharming area. The meeting addressed a wide spectrum of topics within molecular farming, ranging from recombinant protein expression to breeding of medicinal plants. Presentations covered numerous technological novelties along with valuable contributions on safety issues and containment strategies.
Information resource
This meeting was organized by Euroscicon (www.euroscicon.com). The next Euroscicon meeting on molecular pharming will take place on September 20 2011: ‘Molecular Pharming – Recent Progress in Manufacturing Medicines and Plants’ (www.regonline.co.uk/molecular2011).
Financial & competing interests disclosure
The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
No writing assistance was utilized in the production of this manuscript.
References
- Sainsbury F, Thuenemann EC, Lomonossoff GP. pEAQ: versatile expression vectors for easy and quick transient expression of heterologous proteins in plants. Plant Biotechnol. J.7, 682–693 (2009).
- Waheed MT, Thönes N, Müller M et al. Transplastomic expression of a modified human papillomavirus L1 protein leading to the assembly of capsomeres in tobacco: a step towards cost effective second generation vaccines. Transgenic Res. DOI: 10.1007/s11248-010-9415-4 (2010) (Epub ahead of print).
- Fraser PD, Enfissi EMA, Bramley PM. Genetic engineering of carotenoid formation in tomato fruit and the potential application of systems and synthetic biology approaches. Arch. Biochem. Biophys.482, 196–204 (2009).
- Murphy DJ. Improving containment strategies in biopharming. Plant Biotechnol. J.5, 555–569 (2007).