**2.2 Platforms based on transplastomic plants**

Transplastomic plants are a valuable alternative to transgenic plants for the production of recombinant pharmaceutical proteins. Transplastomic plants are obtained by the insertion of expression constructs into the plastid genome by particle bombardment. Since the *Agrobacterium* T-DNA (transfer DNA) complex is targeted to the nucleus, it is unsuitable for gene transfer to chloroplasts [24, 52]. Following the transformation procedure, the bombarded leaf explants are regenerated, and transplastomic plants with homoplastomic transformation (in which every chloroplast carries the transgene) are finally selected, recurring to a selection medium containing spectinomycin or in combination with streptomycin [53].

Plastid transformation can result in high yields of heterologous proteins because multiple copies of the genome are present in each plastid, and photosynthetic cells may contain hundreds or thousands of plastids [54]. As an example, the expression of a proteinaceous antibiotic in tobacco chloroplasts has achieved up to 70% of the total soluble proteins, which is the highest recombinant protein accumulation accomplished so far in plants [55]. Furthermore, chloroplasts provide a natural biocontainment of transgene flow since genes in chloroplast genomes are maternally inherited and consequently not transmitted through pollen, thereby avoiding unwanted escape into the environment. Other advantages of chloroplast engineering include the ability to express several genes as operons, and the accumulation of recombinant proteins in the chloroplast, thus reducing toxicity to the host plant [24].

Finally, transplastomic production platforms offer the possibility of oral delivery [54, 56]. In fact, it has been demonstrated that chloroplast-derived therapeutic proteins, delivered orally via plant cells, are protected from degradation in the stomach, probably due to the bioencapsulation of the therapeutic protein by the plant cell wall. They are subsequently released into the gut lumen by microbes that digest the plant cell wall, where the large mucosal intestine area offers an ideal system for oral drug delivery [57].

A shortcoming of expressing proteins via the chloroplast genome is that routine plastid engineering is still limited to tobacco, a crop that is not edible and thus unsuitable for oral delivery of therapeutic proteins. In addition, the synthesis of glycoproteins is not possible in the chloroplast system, as plastids do not carry out glycosylation [24]. Nevertheless, the expression of human somatotropin [58] in tobacco established that chloroplasts are capable of properly folding human proteins with disulfide bonds. In another study, the production of native cholera toxin B subunit

*Genetic Engineering - A Glimpse of Techniques and Applications*

transformation procedures [38].

rhagic disease virus [45].

II clinical trial [34]. Rice has also been widely used as host for peptide expression, especially for the production of allergen peptides (e.g., pollen and mite allergies) [35, 36]. Recent studies report that rice has the potential to offer an oral delivery system for vaccine antigens and therapeutic proteins and peptides [25, 35, 37].

Barley seeds have also been developed as commercial platforms. In comparison to other cereal crops, barley is less widely grown. However, this fact added to the self-pollinating nature of barley can be viewed as an advantage since the risk of contamination and outcrossing with non-transgenic crops is minimized. Considering this benefit, an Iceland-based company, ORF Genetics (https://orfgenetics.com/), has targeted barley grain as the expression host for several human cytokines and growth factors [19]. Other molecular pharming companies, such as Ventria Bioscience and Maltagen, have also been developing barley-based production platforms. Although barley is still recognized for its recalcitrance to transformation, over the last decade some progress has been made in the development of reliable

The use of legume seeds, such as soybean and pea, for the production of recombinant pharmaceutical proteins, has been less explored than cereal-based platforms, with platforms based on legume seeds having yet to achieve commercial success. However, the fact that legume seeds have exceptionally high protein content (20–40%) can be exploited to achieve high yields of recombinant protein [39]. Soybean seeds have been used to express recombinant growth factors [40, 41], coagulation factors [42], and vaccine peptides [43]. Transgenic pea seeds have been previously used to produce a single-chain Fv fragment (scFV) antibody used in cancer diagnosis and therapy [44]. In another study, pea seeds were used to produce a vaccine that showed high immunogenicity and protection against rabbit hemor-

Safflower and rapeseed seeds are rich in oil and are, thus, referred as oilseeds.

Oilseeds can provide useful recombinant pharmaceutical protein production systems. SemBioSys (http://www.sembiosys.ca/), with its oleosin-fusion platform, has been a pioneer in that field. Oleosins are the principal membrane proteins of oil bodies; oleosins confer peculiar structural properties to the oil bodies that offer simple extraction and purification procedures [46]. In the oleosin-fusion platform the recombinant protein is fused with oleosin and consequently targeted to the oil bodies. The fusion protein is then recovered through simple purification of the oil bodies and separated from oleosin by endoprotease digestion. Commercial production of hirudin in safflower by SemBioSys constituted the first report of an oilseedderived protein [47]. The company has been focusing on safflower as its primary host ever since, with safflower-derived insulin being in phase I clinical trial [32]. Finally, fruit and vegetable crops can also be employed for molecular pharming. A major advantage of protein expression in fruit and vegetable crops is that edible organs can be consumed uncooked, unprocessed, or partially processed, making them particularly suitable for the production of recombinant subunit vaccines, nutraceuticals, and antibodies designed for topical application [29]. The oral delivery of recombinant therapeutics is one of the differentiating factor of molecular pharming in comparison to mainstream biopharmaceutical production systems, with several pharmaceutical products being produced in tomato fruits, potato tubers, and lettuce leaves for this purpose [3]. Tomato fruits are particularly useful for protein expression because the fruits are palatable as raw tissue but can also be lyophilized and stored for a long time [25]. Recently, human coagulation factor IX (hFIX) was expressed specifically in tomato fruits, constituting the first report on the expression of hFIX in plant [48]. Another study described the expression in tomato fruits of a thymosin α1 concatemer [49], an immune booster that plays an important role in the maturation, differentiation, and function of T cells.

**54**

[59] demonstrated the capacity of chloroplasts to fold and assemble oligomeric proteins correctly. Other therapeutic proteins expressed in tobacco chloroplasts include interferons alpha-2a and alpha-2b [60, 61] and anti-cancer therapeutic agents such as human soluble tumor necrosis factor (TNF) [62] and azurin [63]. Recently, chloroplast transformation of lettuce has also been developed [64, 65] to provide oral delivery transplastomic systems [66, 67]. Several therapeutic proteins were produced in lettuce chloroplast, namely, proinsulin [66, 67], tuberculosis vaccine antigens [68], and human thioredoxin 1 protein [69]. The chloroplast production platform has yet to achieve commercial success, though the referred developments in this field augur a promising future for therapeutic protein production in chloroplasts.
