Plant-made pharmaceuticals

Abstract

Plant-made pharmaceuticals (PMP) have great potential in terms of production costs, scalability, safety, environmental protection, and consumer acceptability. The first PMP were antibodies and antigens produced in stably transformed transgenic plants in the around 90s. Even though the effort using stable transgenic plants is still going on, the mainstream of PMP production has shifted to transient expression in Nicotiana benthamiana. This system involves the expression vectors by Agrobacterium, and its efficiency has been improved by the development of new vector systems and host engineering. The COVID-19 outbreak accelerated this trend through efforts to produce vaccines in plants. Transient expression systems have been improved and diversified by the development of plant virus vectors, which can be classified as full and deconstructed vectors. Full virus vectors spread systemically, allowing for protein production in the entire plant. Compared with conventional agroinfiltration vectors, excellent virus vectors result in higher protein production. Engineering of host plants has included knocking out gene-silencing systems to increase protein production, and the introduction of glycan modification enzymes so that plant-made proteins more resemble animal-made proteins. Hydroponic cultivation systems in plant factories and environmental controls have contributed to efficient protein production in plants. Considering their advantages and small environmental impact, PMP should be more widely adopted for pharmaceuticals' production. However, the initial investment and running costs of plant factories are higher than open filed cultivation. The next objectives are to develop next-generation low-cost plant factories that use renewable energy and recycle materials based on the idea of circular economy.

Keywords: plant molecular farming; plant-made pharmaceutical.

Figure 1. Summary of PMP research papers related to COVID-19/SARS-CoV2 (as of March 31, 2024). (A) Changes in the number of PMP research papers related to COVID-19/SARS-CoV2. (B) Countries that authors are affiliated with which target COVID-19/SARS-CoV2 in PMP research and the number of published papers. (C) Breakdown of COVID-19/SARS-CoV2 related substances targeted for plant expression. (D) Platform plants and plant species used for expression of COVID-19 related substances. “Higher plant cell” includes Nicotiana tabacum BY-2 cell and Medicago truncatula A17 cell. “Unicellular green algae” includes Chlorella and Chlamydomonas reinhardtii. (E) Plant expression systems used for expression of COVID-19 related substances.

Figure 2. Overview of plant expression systems for PMP production.

Figure 3. Plant virus vectors used for expression of recombinant proteins. (A) Full virus vectors: tobacco mosaic virus (TMV, Pogue et al. 2010); cowpea mosaic virus (CPMV, Gopinath et al. 2000); potato virus X (PVX, Baulcombe et al. 1995). (B) Deconstructed virus vectors: TMV/turnip vein-clearing virus (TVCV) (Marillonnet et al. 2005); TMV (Lindbo 2007); CPMV (Cañizares et al. 2006); cucumber mosaic virus (CMV, Fukuzawa et al. 2018); bean yellow dwarf virus (BeYDV, Diamos et al. 2020). *CMV RNA1 is supplied from transgene integrated into the host genome. (C) Transgenic plants harboring viral replicons: TMV/TVCV (Werner et al. 2011), tobacco yellow dwarf virus (TYDV, Dugdale et al. 2013). Abbreviations: GOI, gene-of-interest; MP, movement protein; CP, coat protein; TGB1–3, triple gene block 1–3; Co-Pro, proteinase co-factor; NTB, NTP-binding proteins; VPg, viral protein genome-linked; Pro, proteinase; LCP, SCP, large coat protein, small coat protein; LIR, long intergenic region; SIR, short intergenic region; AlcR, alcohol receptor gene; T7p, T7 bacteriophage RNA promoter; 35Sp, cauliflower mosaic virus 35S promoter; Act2p, A. thaliana ACT2 promoter; AlcAp, AlcA promoter from Aspergillus nidulans; PsaK2T 5′, truncated 5′ UTR of N. benthamiana psaK gene; Ext 3′, intronless tobacco extensin terminator; NbACT 3′, 3′ UTR of N. benthamiana ACT3 gene; Rb7 MAR, tobacco Rb7 matrix attachment region; NOSt, terminator of the agrobacterium nopaline synthase gene; OCSt, terminator of the agrobacterium octopine synthase gene; CaMVt, cauliflower mosaic virus terminator. The length shown in the diagram does not reflect the size of each element in virus vectors.

Figure 4. Variety of glycans in mammals and plants. (A and B) Plant N-glycans mainly differ from mammalian N-glycans by the followings: absence of sialic acid, presence of α(1,3) linked fucose residues on the reducing terminal GlcNAc instead of α(1,6)-linked fucose residues in mammalian N-glycans, and presence of β(1,2) linked xylose residues on the mannose residues of the core oligosaccharide, common moiety between plant and mammalian N-glycans. The α(1,3) linked fucose and β(1,2) linked xylose residues are also known as the plant-specific sugar residues. Interestingly, plant N-glycans frequently have Lewis A antigen, one of human blood group, at their non-reducing end. (C and D) In many cases, N-acetylgalactosamine is firstly linked to serine/threonine residues of proteins in mammalian O-glycans. On the other hand, in plant O-glycans, arabinose and galactose are linked to hydroxyproline and serine residues of proteins. Sialic acid is also absence for plant O-glycans. (E) Glycosaminoglycans are the major mammalian sugar chains and known as sugar parts of proteoglycans. Glycosaminoglycans are a group of anionic polysaccharides composed of sulfated repeating disaccharide building blocks, consist of uronic acids and amino sugars, and are synthesized through common core oligosaccharides on serine residues of core. Glycosaminoglycans include such as heparin, heparan sulfate, and keratan sulfate. Only major glycans are shown in this figure, and glycans show further diversity in various species. Abbreviations: Ara, arabinose; Asn, asparagine; Fuc, fucose; GalNAc, N-acetylgalactosamine; GlcA, glucuronic acid; GlcNAc, N-acetylglucosamine; Gal, galactose; Hyp, hydroxyproline; Man, mannose; Ser, serine; Thr, threonine; Xyl, xylose.