Molecular Farming Strategy for the Rapid Production of Protein-Based Reagents for Use in Infectious Disease Diagnostics.

Recombinant proteins are a major breakthrough in biomedical research with a wide range of applications from diagnostics to therapeutics. Strategic construct design, consistent expression platforms, and suitable upstream and downstream techniques are key considerations to produce commercially viable recombinant proteins. The recombinant antigenic protein production for use either as a diagnostic reagent or subunit vaccine formulation is usually carried out in prokaryotic or eukaryotic expression platforms. Microbial and mammalian systems dominate the biopharmaceutical industry for such applications. However, there is no universal expression system that can meet all the requirements for different types of proteins. The adoptability of any expression system is likely based on the quality and quantity of the proteins that can be produced from it. The huge demand of recombinant proteins for different applications requires an inexpensive production platform for rapid development. The molecular farming scientific community has been promoting the plant system for nearly 3 decades as a cost-effective alternative to produce high-quality proteins for research, diagnostic, and therapeutic applications. Here, we discuss how plant biotechnology could offer solutions for the rapid and scalable production of protein antigens as low-cost diagnostic reagents for use in functional assays.

Plant molecular farming-derived epidermal growth factor revolutionizes hydrogels for improving glandular epithelial organoid biofabrication.

Epidermal growth factor (EGF) is a known signaling cue essential towards the development and organoid biofabrication particularly for exocrine glands. This study developed an in vitro EGF delivery platform with Nicotiana benthamiana plant-produced EGF (P-EGF) encapsulated on hyaluronic acid/alginate (HA/Alg) hydrogel to improve the effectiveness of glandular organoid biofabrication in short-term culture systems. Primary submandibular gland epithelial cells were treated with 5 - 20 ng/mL of P-EGF and commercially available bacteria-derived EGF (B-EGF). Cell proliferation and metabolic activity were measured by MTT and luciferase-based ATP assays. P-EGF and B-EGF 5 - 20 ng/mL promoted glandular epithelial cell proliferation during 6 culture days on a comparable fashion. Organoid forming efficiency and cellular viability, ATP-dependent activity and expansion were evaluated using two EGF delivery systems, HA/Alg-based encapsulation and media supplementation. Phosphate buffered saline (PBS) was used as a control vehicle. Epithelial organoids fabricated from PBS-, B-EGF-, and P-EGF-encapsulated hydrogels were characterized genotypically, phenotypically and by functional assays. P-EGF-encapsulated hydrogel enhanced organoid formation efficiency and cellular viability and metabolism relative to P-EGF supplementation. At culture day 3, epithelial organoids developed from P-EGF-encapsulated HA/Alg platform contained functional cell clusters expressing specific glandular epithelial markers such as exocrine pro-acinar (AQP5, NKCC1, CHRM1, CHRM3, Mist1), ductal (K18, Krt19), and myoepithelial (α-SMA, Acta2), and possessed a high mitotic activity (38-62% Ki67 cells) with a large epithelial progenitor population (∼70% K14 cells). The P-EGF encapsulation strikingly upregulated the expression of pro-acinar AQP5 cells through culture time when compared to others (B-EGF, PBS). Thus, the utilization of Nicotiana benthamiana in molecular farming can produce EGF biologicals amenable to encapsulation in HA/Alg-based in vitro platforms, which can effectively and promptly induce the biofabrication of exocrine gland organoids.

Farming for Pharming: Novel Hydroponic Process in Contained Environment for Efficient Pharma-Grade Production of Saffron.

Soilless cultivation of saffron (Crocus sativus) in a controlled environment represents an interesting alternative to field cultivation, in order to obtain a standardized high-quality product and to optimize yields. In particular, pharma-grade saffron is fundamental for therapeutic applications of this spice, whose efficacy has been demonstrated in the treatment of macular diseases, such as Age-related Macular Degeneration (AMD). In this work, a hydroponic cultivation system was developed, specifically designed to meet the needs of C. sativus plant. Various cultivation recipes, different in spectrum and intensity of lighting, temperature, photoperiod and irrigation, have been adopted to study their effect on saffron production. The experimentation involved the cultivation of corms from two subsequent farm years, to identify and validate the optimal conditions, both in terms of quantitative yield and as accumulation of bioactive metabolites, with particular reference to crocins and picrocrocin, which define the 'pharma-grade' quality of saffron. Through HPLC analysis and chromatography it was possible to identify the cultivation parameters suitable for the production of saffron with neuroprotective properties, evaluated by comparison with an ISO standard and the REPRON® procedure. Furthermore, the biochemical characterization was completed through NMR and high-resolution mass spectrometry analyses of saffron extracts. The whole experimental framework allowed to establish an optimized protocol to produce pharma-grade saffron, allowing up to 3.2 g/m2 harvest (i.e., more than three times higher than field production in optimal conditions), which meets the standards of composition for the therapy of AMD.

Regulation of Molecular Farming Products.

The regulation of molecular farming is a complex topic because plants and plant-based systems are relative newcomers among the many production platforms available for recombinant proteins. The regulations specific for different types of product (human/veterinary pharmaceuticals and medical devices, cosmetics, diagnostics, and research reagents) must therefore be overlaid with the regulations governing hitherto unfamiliar production platforms, and this must be achieved in different jurisdictions that handle genetically modified organisms (and genetically modified plants in particular) in very different ways. This chapter uses examples of different product types and production methods in three different jurisdictions (the USA, the EU, and Canada) to demonstrate some of the challenges facing the regulatory authorities.

A Brief Overview of Potential Treatments for Viral Diseases Using Natural Plant Compounds: The Case of SARS-Cov.

The COVID-19 pandemic, as well as the more general global increase in viral diseases, has led researchers to look to the plant kingdom as a potential source for antiviral compounds. Since ancient times, herbal medicines have been extensively applied in the treatment and prevention of various infectious diseases in different traditional systems. The purpose of this review is to highlight the potential antiviral activity of plant compounds as effective and reliable agents against viral infections, especially by viruses from the coronavirus group. Various antiviral mechanisms shown by crude plant extracts and plant-derived bioactive compounds are discussed. The understanding of the action mechanisms of complex plant extract and isolated plant-derived compounds will help pave the way towards the combat of this life-threatening disease. Further, molecular docking studies, in silico analyses of extracted compounds, and future prospects are included. The in vitro production of antiviral chemical compounds from plants using molecular pharming is also considered. Notably, hairy root cultures represent a promising and sustainable way to obtain a range of biologically active compounds that may be applied in the development of novel antiviral agents.

Plants as sources of natural and recombinant anti-cancer agents.

Herbal remedies were the first medicines used by humans due to the many pharmacologically active secondary metabolites produced by plants. Some of these metabolites inhibit cell division and can therefore be used for the treatment of cancer, e.g. the mitostatic drug paclitaxel (Taxol). The ability of plants to produce medicines targeting cancer has expanded due to the advent of genetic engineering, particularly in recent years because of the development of gene editing systems such as the CRISPR/Cas9 platform. These technologies allow the introduction of genetic modifications that facilitate the accumulation of native pharmaceutically-active substances, and even the production heterologous recombinant proteins, including human antibodies, lectins and vaccine candidates. Here we discuss the anti-cancer agents that are produced by plants naturally or following genetic modification, and the potential of these products to supply modern healthcare systems. Special emphasis will be put on proteinaceous anti-cancer agents, which can exhibit an improved selectivity and reduced side effects compared to small molecule-based drugs.

Rapid Transient Production of a Monoclonal Antibody Neutralizing the Porcine Epidemic Diarrhea Virus (PEDV) in Nicotiana benthamiana and Lactuca sativa.

Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea, vomiting, dehydration, weight loss, and high mortality rate in neonatal piglets. Porcine epidemic diarrhea (PED) has been reported in Europe, America, and Asia including Thailand. The disease causes substantial losses to the swine industry in many countries. Presently, there is no effective PEDV vaccine available. In this study, we developed a plant-produced monoclonal antibody (mAb) 2C10 as a prophylactic candidate to prevent the PEDV infection. Recently, plant expression systems have gained interest as an alternative for the production of antibodies because of many advantages, such as low production cost, lack of human and animal pathogen, large scalability, etc. The 2C10 mAb was transiently expressed in Nicotiana benthamiana and lettuce using geminiviral vector. After purification by protein A affinity chromatography, the antibody was tested for the binding and neutralizing activity against PEDV. Our result showed that the plant produced 2C10 mAb can bind to the virus and also inhibit PEDV infection in vitro. These results show excellent potential for a plant-expressed 2C10 as a PEDV prophylaxis and a diagnostic for PEDV infection.

Recombinant biologic products versus nutraceuticals from plants - a regulatory choice?

Biotechnology has transformed the potential for plants to be a manufacturing source of pharmaceutical compounds. Now, with transgenic and transient expression techniques, virtually any biologic, including vaccines and therapeutics, could be manufactured in plants. However, uncertainty over the regulatory path for such new pharmaceuticals has been a deterrent. Consideration has been given to using alternative regulatory paths, including those for nutraceuticals or cosmetic agents. This review will consider these possibilities, and discuss the difficulties in establishing regulatory guidelines for new pharmaceutical manufacturing technologies.

Plant molecular pharming for the treatment of chronic and infectious diseases.

Plant molecular pharming has emerged as a niche technology for the manufacture of pharmaceutical products indicated for chronic and infectious diseases, particularly for products that do not fit into the current industry-favored model of fermenter-based production campaigns. In this review, we explore the areas where molecular pharming can make the greatest impact, including the production of pharmaceuticals that have novel glycan structures or that cannot be produced efficiently in microbes or mammalian cells because they are insoluble or toxic. We also explore the market dynamics that encourage the use of molecular pharming, particularly for pharmaceuticals that are required in small amounts (such as personalized medicines) or large amounts (on a multi-ton scale, such as blood products and microbicides) and those that are needed in response to emergency situations (pandemics and bioterrorism). The impact of molecular pharming will increase as the platforms become standardized and optimized through adoption of good manufacturing practice (GMP) standards for clinical development, offering a new opportunity to produce inexpensive medicines in regional markets that are typically excluded under current business models.

Assessing the bioconfinement potential of a Nicotiana hybrid platform for use in plant molecular farming applications.

BACKGROUND: The introduction of pharmaceutical traits in tobacco for commercial production could benefit from the utilization of a transgene bioconfinement system. It has been observed that interspecific F1Nicotiana hybrids (Nicotiana tabacum × Nicotiana glauca) are sterile and thus proposed that hybrids could be suitable bioconfined hosts for biomanufacturing. We genetically tagged hybrids with green fluorescent protein (GFP), which was used as a visual marker to enable gene flow tracking and quantification for field and greenhouse studies. GFP was used as a useful proxy for pharmaceutical transgenes. RESULTS: Analysis of DNA content revealed significant genomic downsizing of the hybrid relative to that of N. tabacum. Hybrid pollen was capable of germination in vitro, albeit with a very low frequency and with significant differences between plants. In two field experiments, one each in Tennessee and Kentucky, we detected outcrossing at only one location (Tennessee) at 1.4%. Additionally, from 50 hybrid plants at each field site, formation of 84 and 16 seed was observed, respectively. Similar conclusions about hybrid fertility were drawn from greenhouse crosses. In terms of above-ground biomass, the hybrid yield was not significantly different than that of N. tabacum in the field. CONCLUSION: N. tabacum × N. glauca hybrids show potential to contribute to a bioconfinement- and biomanufacturing host system. Hybrids exhibit extremely low fertility with no difference of green biomass yields relative to N. tabacum. In addition, hybrids are morphologically distinguishable from tobacco allowing for identity preservation. This hybrid system for biomanufacturing would optimally be used where N. glauca is not present and in physical isolation of N. tabacum production to provide total bioconfinement.

Oral immunotherapy with transgenic rice seed containing destructed Japanese cedar pollen allergens, Cry j 1 and Cry j 2, against Japanese cedar pollinosis.

Transgenic rice accumulating the modified major Japanese cedar pollen allergens, Cryptomeria japonica 1 (Cry j 1) and Cryptomeria japonica 2 (Cry j 2), which were deconstructed by fragmentation and shuffling, respectively, in the edible part of the seed was generated by transformation of a good-tasting rice variety, 'Koshihikari'. These modified cedar pollen antigens were deposited in ER-derived protein bodies (PB-I), which are suitable for delivery to the mucosal immune system in gut-associated lymphoid tissue when orally administered because antigens bioencapsulated in PB-I are resistant against hydrolysis by intestinal enzymes and harsh environments. Mice fed transgenic seeds daily for three weeks and then challenged with crude cedar pollen allergen showed marked suppression of allergen-specific CD4(+) T-cell proliferation, IgE and IgG levels compared with mice fed nontransgenic rice seeds. As clinical symptoms of pollinosis, sneezing frequency and infiltration of inflammatory cells such as eosinophils and neutrophils were also significantly reduced in the nasal tissue. These results imply that oral administration of transgenic rice seeds containing the structurally disrupted Cry j 1 and Cry j 2 antigens, serving as universal antigens, is a promising approach for specific immunoprophylaxis against Japanese cedar pollinosis.