Meeting EBTNA 9 ottobre 2015

Oscar Vicente Institute of Plant Molecular and Cellular Biology (IBMCP, UPV-CSIC),

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Oscar Vicente¹* and Monica Boscaiu²

¹Institute of Plant Molecular and Cellular Biology (IBMCP, UPV-CSIC),

²Mediterranean Agroforestal Institute (IAM, UPV). Universitat Politècnica de Valencia

'Green/red’ biotech: GM plants as biofactories of pharmaceutical proteins

Since the production of human insulin in E. coli – the first commercial therapeutic protein provided by recombinant DNA technology – several GMO systems have been used as "bio-factories" for the synthesis of a wide range of proteins with pharmacological activities, by expression of the corresponding cloned genes: hormones, growth factors, blood clotting factors, enzymes, vaccines, antibodies... in general, any protein with application in the diagnosis, prevention or treatment of human (or animal) diseases. Bacterial cultures have several advantages for the production of recombinant proteins, being reliable and relatively cheap systems, easy to establish and maintain, and providing high protein production levels. Nevertheless they have also serious drawbacks, especially for the expression of complex and multimeric proteins, which in many cases are not produced in an active form in the cells, since they do not fold or are not assembled properly. However, their most important limitation is that the bacteria do not possess the machinery responsible for post-translational modification of proteins; most human proteins are modified by phosphorylation, acetylation, glycosylation, etc., and the presence of these groups (especially the correct glycosylation) is essential for their biological activity. Mammalian cells cultures are also very robust and reliable systems, there is a long experience in their industrial use and approval by the competent regulatory authorities, and a whole body of ‘good manufacturing practices’ (GMP) have been developed over the years. Unlike bacteria, however, they ensure (in general) the synthesis of pharmacologically active products, since post-translational modifications of the recombinant protein are the same than that of the native human protein in vivo. That is why animal cell cultures – for example, of Chinese Hamster Ovary (CHO) cells, the 'golden standard' in the industry – are currently the system of choice for the production of biopharmaceuticals, despite having several drawbacks and limitations: high costs of up-front investment, development and maintenance, slow growth of the cells and limited productivity, the difficulty (technical and economic) to scale up or down production, or the risk of contamination by human pathogens (e.g., viruses or prions). Production of recombinant proteins in the so-called “3^rd generation” of transgenic plants provides an alternative, or rather a complement, to systems based on GM-microorganisms or animal cells in in vitro cultures. This is what is known as 'molecular farming' (... or 'molecular pharming' if we refer specifically to pharmacological proteins), and has – at least theoretically – a number of advantages over other commercial production platforms:

i) the methods of plant genetic transformation and regeneration of transgenic plants are relatively cheap and simple, as compared for example to the generation of transgenic animals

ii) the production systems can be established with low up-front investment and maintained cheaply, since they are based on common techniques used for centuries in agriculture (low-tech, low-cost)

iii)the production can be scaled (up or down) easily and cheaply, to adapt to market demands (in principle, simply by increasing or decreasing the cultivation area)

iv) in general, proteins are synthesized in a pharmacologically active form, since the systems of post-translational modification (e.g. glycosylation) in plants are very similar to those of mammalian cells

v) the synthesis of the recombinant protein can be directed to specific organs (and organelles) by using tissue-specific promoters and proper subcellular localization signals. Thus, the protein can be 'encapsulated' in natural plant structures, for example in the endosperm of seeds, facilitating in this way the storage of the protein in an active form, without requiring special conditions such as refrigeration

vi) there is the possibility of developing simple and efficient purification methods

vii) there is no risk of contamination with human pathogens

Another specific application of “pharma-crops” is the production of edible vaccines: ingesting plant material containing a suitable recombinant antigen will activate the immune system at the level of the intestinal mucosa. This approach would eliminate many of the problems associated with the production and application of traditional vaccines, such as high cost, transport and distribution issues (mostly in developing countries): no need to maintain the 'cold chain', avoiding risk of transmitting infections by the use of non-sterile syringes, etc.

Besides stable genetic transformation, protocols have been developed for transient expression of recombinant proteins in plant tissues (tobacco or alfalfa leaves are the commonest), using Agrobacterium tumefaciens, plant viruses or hybrid constructs as vectors. These systems allow the rapid production of substantial quantities of biopharmaceuticals, which would be needed to treat large numbers of individuals in a short span of time in case, for example, of epidemics or bioterrorist attacks. For over 20 years a large number of recombinant proteins, many with possible clinical applications, have been produced in transgenic plants, proving the aforementioned advantages of these platforms; in most cases this work has been limited to academic or 'proof-of-concept' studies. Several recombinant proteins are currently produced in plants, marketed as reagents for research or used in various industries: cosmetics, detergents, food, etc. However, commercial production of biopharmaceuticals in GM plants is lagging far behind with regard to the system of cultured mammalian cells. This is due more to regulatory and technical issues, rather than to purely scientific advances. There is no clear specific rules applicable to the production of pharmaceuticals in plants, and it is very difficult to adapt those existing at present, established for such different biological systems as cells in in vitro cultures (similar regulatory problems exist in the case of the production of therapeutic proteins in transgenic animals). In addition, worldwide, there are only a few facilities authorised for the production of recombinant proteins in plants according to 'good manufacturing practice' (GMP). In recent years, nevertheless, there has been a substantial boost in commercial development and potential applications of molecular pharming, and several specific products are currently undergoing clinical trials, at different phases. Some of the specific milestones that mark this development are: i) the approval by the FDA in May 2012, of 'Elelyso' (recombinant glucocerebrosidase), enzyme used to treat Gaucher's disease (a lysosomal storage disorder), produced by Protalix Biotherapeuticals (Israel) in a carrot cell culture; ii) production by transient expression in Nicotiana benthamiana of the experimental drug ZMapp, which has proven effective against Ebola virus in primates, and contain a combination of three humanized monoclonal antibodies, which recognize a surface glycoprotein of the virus; iii) production in transgenic tobacco of a neutralizing anti-HIV monoclonal antibody, which is used as a topical prophylactic to prevent virus infection (by vaginal application prior to sexual intercourse); the relevant authorities approved a phase I clinical trial, which demonstrated the safety of its use. Although ‘molecular pharming’ will probably never replace the systems of animal cell cultures, much more developed (and where the industry has made major investments), the advantages of using transgenic plants as bio-factories for the production of biopharmaceuticals provide a very interesting market niche for specific products. For example, as mentioned before, when large amounts of protein are required in a short time, which cannot be produced in vitro culture systems. Moreover, technical simplicity, the low initial investment and the also limited running costs, make ‘molecular pharming’ (or, more generally 'molecular farming') an affordable technology to less developed countries. Further developments on scientific, technical and regulatory issues regarding these plant-based platforms for production of recombinant proteins are, therefore, expected in the coming years.

Further reading:

The following recent reviews (and the references therein) provide a complete and up-to-date overview of the issues discussed above:

  -Eva Stoger, Rainer Fischer, Maurice Moloney and Julian K.-C. Ma (2014) Plant Molecular Pharming for the Treatment of Chronic and Infectious Diseases Annu. Rev. Plant Biol. 65: 743-768.

-Shah Fahad et al. (2015). Recent developments in therapeutic protein expression technologies in plants Biotechnol. Lett.  37:265–279