Current status on: Biopharming: crops for the production of Therapeutic & Pharmaceutical proteins Biopharming, also known as molecular farming, is the production of pharmacologically active substances, either induced or increased through the application of genetic engineering. The first instance of artificial gene expression in an organism to produce a pharmaceutical product was the synthesis of insulin in the bacterium E. coli (Goeddel et al. , 1979).
This type of biotechnology has since moved from microbial cell cultures to applications in eukaryotic organisms, such as plants and animals. The first genetically modified (GM) plants to be used for biopharming were tobacco and tomato plants which produced human serum albumin (Sijmons et al. , 1990). Since those early breakthroughs, a wide range of plant-derived pharmaceutical and therapeutic proteins have been produced, including antibodies, subunit vaccines, human blood products, hormones and growth regulators (Twyman et al. , 2003).
There have been advances in methodology also; plant cell cultures and plants as ‘bioreactors’ have been used as techniques to more efficiently extract the protein product and modification of genes controlling storage and secretion shows potential for more effective extraction. Biopharming has a very valid use as a humanitarian tool, with Golden Rice having the potential to save millions of people at risk of blindness due to lack of dietary vitamin A and the concept of vaccines produced in food crops that can simply be consumed directly.
Many products have been developed without ever reaching the market and numerous companies have gone bankrupt in pursuit of economical biopharming, for example, Biolex Therapeutics (Bagley, 2012)and Large Scale Biology Corp (Bloomburg, n. d. ). As GM technology becomes increasingly prevalent throughout the world and pressures to reduce the use of conventional chemical-based methods in agriculture and other forms of industry, it’s applicability to the production of pharmaceutical & therapeutic proteins is bound to increase.
However, as with all emerging GM based technologies, there are groups opposed to biopharming who hold considerable power in determining how widespread the concept could potentially become. Certain strains of the bacterium Vibrio cholera cause cholera, an infection in the small intestines. A study in 2010 (Renuga et al. , 2010) attempted to produce an edible cholera vaccine through recombinant genes inserted into banana callus cultures. The gene encoding cholera toxin subunit B (CT-B) was cloned using Escherichia coli-derived vector PRK2013.
Following amplification using PCR, the CT-B gene was inserted into the plant transformation vector (plasmid) PGA 643 to create the plasmid PCAMBIA. This plasmid was inserted into the bacterium Agrobacterium tumefaciens and from here, into a banana callus culture. The callus was maintained and differentiated into plantlets, thus producing bananas expressing recombinant CT-B antigens. Using bananas as a medium for delivering vaccines was selected because the fruit is edible in its raw form and can the plant can be grown in a number of developing countries where cholera is prevalent like Haiti and regions of East Africa (WHO, 2012).
There are plant-derived vaccines that are in the stages of clinical trials. Non-Hodgkin’s lymphoma is a variety of blood disease that occurs when B or T lymphocytes become cancerous. Tobacco plants have been genetically engineered to produce single-chain variable region (scFv) vaccines to treat lymphoma. Conventional methods of lymphoma vaccine production involve creating hybrid cell lines with a patient’s tumour cells and screening for tumour-specific immunoglobulins to then produce antibodies which are coupled to various proteins before they are administered to the patient.
This method is difficult and time consuming and a more efficient and cheaper plant-based technique was developed in the late nineties (McCormick et al. , 1999) and was shown to protect lab mice from a lethal tumour dose from genetically identical individuals suffering with lymphoma. However, this method has its own shortcomings; complex engineering of scFvs is required and purification cannot be conducted via standard methods. A phase 1 clinical study (McCormick et al. 2008) evaluated safety, immune response and clinical outcome of 16 patients who were given doses of scFv lymphoma vaccines derived from GM plants. No adverse effects reported were attributed to administration of the vaccine, but rather due to coadministration with lymphocyte growth factors. Over half of the group were characterised as having specific immune responses, however the small sample size means these results could be negligible. Further developments have been made in regard to plant-produced lymphoma vaccines. Entire immunoglobulins, rather than cFvs, have been constructed in tobacco plants, without the negative aspects associated with the previous technique (McCormick et al. , 1999). (Bendandi et al. , 2010) achieved a manufacturing process of less than 14 weeks, with expression and purification of the antigen taking only 2 weeks, offering a level of protection equal to vaccines produced from hybrid cell lines. Perhaps the most well-known utilisation of GM plants to synthesise compounds concerned with health, whilst not strictly categorised as biopharming, is the Golden Rice project.
Millions of people in developing countries have rice as their primary source of nutrition. To prevent rotting when in storage, the aleurone layer is removed, which contains carotenoids vital for vitamin A production which are absent in the main body of the endosperm. If the body is lacking these vitamin A precursors then immunological responses are diminished and blindness is common, with young children and pregnant women being the worst affected. In 2004 an estimated 670,000 children under the age of five died from a deficiency in the vitamin (Black et al. 2008). A biosynthetic pathway to formulate ? -carotene (a precursor to Vitamin A) has been developed (Ye et al. , 2000) and subsequently improved (Paine et al. , 2005) to produce more ? -carotene. The first creation used the insertion of two genes into the rice genome and association with an endosperm-specific promoter to restrict expression to the edible part of the plant; psy from daffodil and crtI from the bacterium Erwinia uredovora. Only 1. 6 µg/g (dry) of ? -carotene was produced in greenhouse conditions using this variety (Beyer et al. 2002), however subsequent field tests yielded four times that amount on average (Al-Babili and Beyer, 2005). A second variety, developed in 2005, used the psy gene extracted from maize after proving the limiting factor to carotenoid accumulation was the daffodil psy gene (Paine et al. , 2005). Golden Rice was expected to be in the hands of Asian farmers by 2002 (Potrykus, 2010) but despite extensive research and development, strict regulations and other factors have hampered field tests and thus delayed bringing Golden Rice to the people who potentially need it to save their lives.
GM-based regulations call for extensive regulatory screening procedures and trials, culminating to 17 years (Potrykus, 2010). The regulatory dossier alone requires a whole team of specialists to conduct expensive studies concerning molecular genetics, biochemistry etc. for four years. Prior to this it took six months to simply acquire the legal rights to develop what would become Golden Rice. Anti-GM groups as well as the media have played a part in delaying production and even more so, giving the public a biased image of the risks and benefits associated with biopharming.
Greenpeace China claimed a Golden Rice trial that used Chinese schoolchildren in the study had violated governmental plans, leading the Chinese media to liken it to Japanese biological war crimes that used Chinese civilians in the second world war (Hvistendahl and Enserink, 2012). Earthjustice have also released pamphlets and articles on the internet condemning biopharming in general stating a number of risks, for example, that biopharming may create “a public epidemic of disease, and allergic reactions”.
They attribute scientific reviews as the source of their information, yet provide no references to any material (Earthjustice, n. d. ). Actions of anti-GM groups hinder the public’s understanding of biopharming and the vast array of potential benefits it has to bring. A survey assessing the public’s perceptions to biopharming (Nevitt et al. , 2006) found 56%, 47% and 28% of respondents were concerned with adverse health effects, adverse environmental effects and moral issues, respectively. There are genuine concerns over potential risks and disadvantages amongst the scientific community as well.
The phase of testing required to ensure genetic stability and biochemical activity of the product is longer than in microbial and animal systems (Twyman et al. , 2003). As with all GM technology, there is risk of cross-contamination. A biopharming-related incident occurred in 2002 which resulted in the ordered destruction of 500,000 bushels of soybean that might have been contaminated with the protein avidin as the farmers had not left the land fallow for a year to allow complete removal of the biopharmed crops (Fox, 2003). Outweighing the concerns and disadvantages of biopharming, are the positive aspects.
Firstly, production costs of plant-based molecular pharming are relatively low. The average production costs per gram of pharmaceutical using transgenic plants is $10-20, versus $50-100 and $500-5000 for yeast and mammalian cell cultures, respectively (Elbehri, 2005). Plants as a pharmaceutical production system are far greater in terms of safety than mammalian or microbial cultures because they lack human pathogens and endotoxins (Twyman et al. , 2003). Storage and secretory pathways can potentially be exploited in plants to make attainting the protein product easier.
Proteins lacking specific trafficking signals are secreted into the cell wall and can accumulate in great quantities if gene expression is high. Furthermore, proteins can be secreted via the roots into a medium from which they can be extrapolated (Vitale and Pedrazzini, 2005). High-level transcription is vital for high yields and it can be regulated with further genetic modification. The promoter and polyadenylation sites are the two most important elements determining the level of transcription and are often derived from the 19S and 35S transcripts of the cauliflower mosaic virus.
Plants have many advantages when it comes to producing pharmaceutical and therapeutic proteins. As technology advances, it is fair to say that the role of plants as pharmaceutical producers will only increase as the associated costs become lower and yields and number of proteins able to be synthesised become higher. Plant biopharming has the potential to save countless lives, both in developing nations, where basic vaccines are hard to access and nutritional deficiencies are prevalent, and developed regions, which invest huge amounts of money to control diseases, such as cancer.
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