Next Generation of Transgenic Plants: From Farming to Pharming

*Alp Ayan, Sinan Meriç, Tamer Gümü*ş *and Çimen Atak*

## **Abstract**

The number of approaches related to recombinant protein production in plants is increasing rapidly day by day. Plant-based expression offers a safe, cost-effective, scalable, and potentially limitless way to rapidly produce recombinant proteins. Plant systems, which have significant advantages over animal and yeast recombinant protein production systems, are particularly promising for the large-scale production of antibodies and therapeutic proteins. Molecular pharming with transgenic plant systems become prominent among other production systems with its low cost, absence of human or animal pathogen contaminants, and the ability to use post-translational modifications such as glycosylation. The ability to produce recombinant pharmaceutical proteins in plant seeds, plant cells and various plant tissues such as hairy roots and leaves, through the stable transformation of the nuclear genome or transient expression, allows for the establishment of different production strategies. In particular, the rapid production of candidate proteins by transient expression, which eliminates the need for lengthy transformation and regeneration procedures, has made plants an attractive bioreactor for the production of pharmaceutical components. This chapter aimsto exhibit the current plant biotechnology applications and transgenic strategies used for the production of recombinant antibodies, antigens, therapeutic proteins and enzymes, which are used especially in the treatment of various diseases.

**Keywords:** molecular pharming, plant-derived pharmaceutical, therapeutic proteins, transient expression, recombinant proteins

## **1. Introduction**

In the last couple of decades, many initiatives have been carried out in which different disciplines came together for sustainable farming in respect to the increasing food demands of the world population which is expected to reach 9 billion thresholds in 2050. Conventional breeding, mutation breeding and especially transgenic technology have been frequently used to prevent yield losses which are caused by the decreasing amount of arable land due to various reasons such as urbanization, desertification and salinity and drought, various diseases, weeds and insects. With advances in modern biotechnology, many approaches have been used to improve crop varieties, from marker-assisted selection (MAS) to recently developed new plant breeding techniques (NPBTs). In particular, genetic modifications have significantly expanded the genetic pool that has been used by plant breeders since the mid-90s. Thus, the development of new plants with many different agricultural traits has gained great momentum.

After the acceleration of commercialization of transgenic crops, which were initially developed for agronomic purposes such as insect and herbicide tolerance for only producers, especially stacked GM events, in which two or more characteristics are introduced together, emerged. Approximately 30 different transgenic plants with many different characteristics for both the producer and the consumer as disease resistance, abiotic stress tolerance, increase in nutrition and food quality, including fruits and vegetables as *Phaseolus vulgaris* (bean), *Solanum melongen* (eggplant), *Cucumis melo* (melon), *Carica papaya* (papaya), *Prunus domestica* (plum), *Beta vulgaris* (sugar beet), had been approved [1, 2]. Stacked GM crops have a combination of several traits and even four different traits (genetic modification) are possible to be located in one GM event, as in the Widestrike™ Roundup Ready Flex™ cotton and Herculex™ RW Roundup Ready™-2 maize samples [3]. Moreover, in the last decade, new genome editing techniques such as zinc finger nuclease (ZFN) technology, clustered regularly interspaced short palindromic repeat (CRISPR)-associated nuclease systems and transcription activator-like effector nucleases (TALENs), which provides precise genome modifications, enabled the reproduction of plants which does not contain recombinant DNA. The recently adopted cisgenesis/intragenesis, RNA interference and new genome editing techniques have enabled the development of many genetically edited organisms (GEOs) as well.

Along with all these developments, it has been understood after the 2000s that GMOs, which have become increasingly dominant in the agricultural field, can also be utilized effectively in medicine and industrial fields. Especially, the increase in chronic and infectious diseases as a result of overpopulation, and the outbreak of epidemics and pandemics cause demands that exceed the production capacity of molecules used for diagnosis, treatment and prevention. To satisfy these demands, cost-effective systems that can produce high-quality products, and allow to take action quickly are developed. In addition, it is a fact that models that support smallscale production will be needed in recent years, as is the case with personalized medicines. Especially in the last two decades, it has been possible to produce plantbased pharmaceutical proteins that can be used in the diagnosis and treatment of many diseases with the developments in gene transfer and production strategies of recombinant proteins in plants.

Thanks to improvements in basic points such as new transformation strategies (stable or transient), transformation methods, appropriate promoter selection and codon optimization, high-scale and lower cost recombinant proteins in plants can be produced rapidly [4, 5]. Thus, the interest in producing recombinant proteins for direct use as a product without aiming for a specific change in phenotype or metabolism and obtaining them in purified form or crude plant extract has increased. This potential provided by the production of recombinant pharmaceutical proteins in plants for therapeutic applications was realized much later than in bacterial, yeast and animal systems. On the other hand, in the early periods, interest in the production of non-pharmaceutical products such as industrial enzymes, cosmetic ingredients, biosensors or biocatalysts, feed, biofuel in plants due to their short development times, low purification costs and less regulatory burdens was much higher than pharmaceutical products [6].

The first applications of molecular farming were come to fruition in the late 1980s by producing various antibodies and human-specific proteins in transgenic plants. Among all expression systems, plant-based systems have started to be preferred as an attractive alternative with their advantages in low production cost, high level of transgene expression, rapid scalability, the riskless transmission of human and animal pathogens and production of proteins with secondary modification (**Table 1**). It is also possible to produce recombinant proteins in dry tissues such as seeds or grains, which reduces storage and transportation costs and puts plant


#### *Next Generation of Transgenic Plants: From Farming to Pharming DOI: http://dx.doi.org/10.5772/intechopen.102004*

**Table 1.**

*Comparison of different production systems according to their efficiency.*

systems one step ahead. While plants can produce complex proteins similar to other eukaryotic systems, they can modify these proteins post-translationally. However, plants can present enhanced post-translational glycosylation modifications, unlike yeast and insects, which have prokaryotic expression systems or very simple glycosylation patterns. Plant cells have slight differences in their glycosylation patterns compared to mammalian cells, and these differences can be rearranged by genetic engineering [7]. Since different glycan structures affect the stability of glycoproteins, subcellular targeting, immunogenicity, pharmacokinetic behavior and biological activity, it is essential to produce the recombinant protein with the appropriate glycosylation pattern [8, 9]. One of the most significant strategies which are developed to control the glycosylation of recombinant proteins in plants is subcellular targeting that prevent the addition of undesired sugar residues. Another important strategy is glycoengineering that avoids the addition of plant glycans and even replaces them with human proteins [9]. In the following years, studies have accelerated and the term of molecular pharming has become prominent with the increasing demand for the production of recombinant pharmaceutical proteins in many different plant systems [9].

Following the enhanced knowledge of gene transfer to plants and finely-tuned gene transfer methods specific to many species such as *Nicotiana tabacum* and *N. benthamiana* (tobacco plant), *Lactuca sativa* (lettuce), *Glycine max* (soybean), *Oryza sativa* (rice), *Solanum lycopersicum* (tomato), *Medicago sativa* (alfalfa), *Zea mays* (maize), it is possible to produce many recombinant proteins including monoclonal antibodies, enzymes, growth factors, therapeutic proteins and vaccines in various plant tissues or cell. Recombinant protein production can be accomplished in the whole plant, as well as in certain parts such as seeds, leaves and fruits [10]. The selected production strategy must express the protein with high efficiency and fully come up with the requirements of the regulations in terms of safety and production quality. To produce a recombinant protein, optimization of the coding sequences of a gene that is desired to transfer, determination of the gene expression strategy as stable or transient, selection of convenient plants and cost-efficient methods for isolation and purification of target proteins are required. Stable gene transfer which is performed by introducing the target gene into the nuclear or plastid genome by *Agrobacterium* and particle bombardment respectively results in stable expression in plant tissues. Although the methods for plant transformation vary according to target species, the target genome (nuclear or plastid), the structure of the gene to be transferred, there are two common approaches: direct (e.g. biolistic or microparticle bombardment) or indirect (*Agrobacterium tumefaciens* or *Agrobacterium rhizogenes*). It is possible to obtain a whole transgenic plant by using plant tissue culture methods together with mentioned transformation methods [11]. Moreover, high-scale production of recombinant proteins becomes possible for many different plant species by establishing various cultures (e.g., callus culture, hairy root culture or suspension culture) from transformed plant tissues or cells [12].

It is crucial to state that developing transgenic plants by stable transformation is more time-consuming by comparing to transient expression systems. In addition, stable transformation strategies need improvement to increase the level of transgene expression. Especially, due to the random insertion of the transgene into the nuclear genome, the different profiles that emerge as a result of the positional effect should be followed meticulously. Unstable gene expression, as well as transgeneinduced gene silencing, may occur together with multiple insertions [4]. Unlike stable expression, when we focus on recombinant protein production strategies based on transient expression of transgenes carried by bacteria (*A. tumefaciens*) or viral vectors (tobacco mosaic virus (TMV), cauliflower mosaic virus (CMV), alfalfa mosaic virus (AVM)), it is seen obviously that stable integration of transgene

#### *Next Generation of Transgenic Plants: From Farming to Pharming DOI: http://dx.doi.org/10.5772/intechopen.102004*

is not necessary. Thus, it becomes possible to produce recombinant protein rapidly by avoiding time-consuming transformation and regeneration procedures. With transient expression by *Agrobacterium* infiltration or viral vectors, it is possible to ensure rapid expression of the transgene even 3–4 hours after transformation and to produce recombinant protein by reaching the maximum expression level in the range of 18–48 hours [4]. Other important advantages of transient expression are that they can reach higher expression levels compared to stable transformation and maintain gene expression in the range of 10–14 days.

One of the important handicaps in recombinant protein production in plants is the lack of suitable, reliable and inexpensive purification methods for each plant. This deadlock becomes even more difficult when the purification of pharmaceuticals must fulfill the stringent criteria mandated by the "good manufacturing practice (GMP)" standards. It is known that purification practices performed in accordance with legal standards comprise approximately 80% of the total costs [13]. Apart from purification by chromatography, membrane filtration and fusion of various polypeptides, there is a need to develop more efficient and cost-effective purification strategies. On the other hand, with the redesign of plant parts containing the target recombinant protein in a form suitable for an oral route such as edible vaccines, it is possible to get rid of the heavy financial burdens of purification strategies. One of the issues that need to be carefully considered regarding the production of pharmaceutical proteins in plants is regulation and biosafety issues. Especially, economic damages to farmers and the food industry as a result of the co-mingling of food and feed crops with plants designed for pharmaceutical production, the spread of transgenes through pollen or seeds, undesirable exposure of non-target organisms such as insects, birds and horizontal gene transfer are among the emerging risks [14].
