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Ding S, Alexander E, Liang H, Kulchar RJ, Singh R, Herzog RW, Daniell H, Leong KW. Synthetic and Biogenic Materials for Oral Delivery of Biologics: From Bench to Bedside. Chem Rev 2025. [PMID: 40168474 DOI: 10.1021/acs.chemrev.4c00482] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 04/03/2025]
Abstract
The development of nucleic acid and protein drugs for oral delivery has lagged behind their production for conventional nonoral routes. Over the past decade, the evolution of DNA- and RNA-based technologies combined with the innovation of state-of-the-art delivery vehicles for nucleic acids has brought rapid advancements to the biopharmaceutical field. Nucleic acid therapies have the potential to achieve long-lasting effects, or even cures, by inhibiting or editing genes, which is not possible with conventional small-molecule drugs. However, challenges and limitations must be addressed before these therapies can provide cures for chronic conditions and rare diseases, rather than only offering temporary relief. Nucleic acids and proteins face premature degradation in the acidic, enzyme-rich stomach environment and are rapidly cleared by the liver. To overcome these challenges, various delivery vehicles have been developed to transport therapeutic compounds to the intestines, where the active compounds are released and gut microbiota and mucosal immune system also play an important role. This review provides a comprehensive overview of the promises and pitfalls associated with the oral route of administration of biologics, current delivery systems, applications of orally delivered therapeutics, and the challenges and considerations for translation of nucleic acid and protein therapeutics into clinical practice.
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Affiliation(s)
- Suwan Ding
- Department of Biomedical Engineering, Columbia University, 500 West 120th Street, New York, New York 10027, United States
| | - Elena Alexander
- Department of Biomedical Engineering, Columbia University, 500 West 120th Street, New York, New York 10027, United States
| | - Huiyi Liang
- Department of Biomedical Engineering, Columbia University, 500 West 120th Street, New York, New York 10027, United States
| | - Rachel J Kulchar
- Department of Basic and Translational Sciences, School of Dental Medicine, University of Pennsylvania, 240 South 40th Street, Philadelphia, Pennsylvania 19104, United States
| | - Rahul Singh
- Department of Basic and Translational Sciences, School of Dental Medicine, University of Pennsylvania, 240 South 40th Street, Philadelphia, Pennsylvania 19104, United States
| | - Roland W Herzog
- Department of Pediatrics, Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, Indiana 46202, United States
| | - Henry Daniell
- Department of Basic and Translational Sciences, School of Dental Medicine, University of Pennsylvania, 240 South 40th Street, Philadelphia, Pennsylvania 19104, United States
| | - Kam W Leong
- Department of Biomedical Engineering, Columbia University, 500 West 120th Street, New York, New York 10027, United States
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Bharathi JK, Suresh P, Prakash MAS, Muneer S. Exploring recent progress of molecular farming for therapeutic and recombinant molecules in plant systems. Heliyon 2024; 10:e37634. [PMID: 39309966 PMCID: PMC11416299 DOI: 10.1016/j.heliyon.2024.e37634] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2024] [Revised: 08/10/2024] [Accepted: 09/06/2024] [Indexed: 09/25/2024] Open
Abstract
An excellent technique for producing pharmaceuticals called "molecular farming" enables the industrial mass production of useful recombinant proteins in genetically modified organisms. Protein-based pharmaceuticals are rising in significance because of a variety of factors, including their bioreactivity, precision, safety, and efficacy rate. Heterologous expression methods for the manufacturing of pharmaceutical products have been previously employed using yeast, bacteria, and animal cells. However, the high cost of mammalian cell system, and production, the chance for product complexity, and contamination, and the hurdles of scaling up to commercial production are the limitations of these traditional expression methods. Plants have been raised as a hopeful replacement system for the expression of biopharmaceutical products due to their potential benefits, which include low production costs, simplicity in scaling up to commercial manufacturing levels, and a lower threat of mammalian toxin contaminations and virus infections. Since plants are widely utilized as a source of therapeutic chemicals, molecular farming offers a unique way to produce molecular medicines such as recombinant antibodies, enzymes, growth factors, plasma proteins, and vaccines whose molecular basis for use in therapy is well established. Biopharming provides more economical and extensive pharmaceutical drug supplies, including vaccines for contagious diseases and pharmaceutical proteins for the treatment of conditions like heart disease and cancer. To assess its technical viability and the efficacy resulting from the adoption of molecular farming products, the following review explores the various methods and methodologies that are currently employed to create commercially valuable molecules in plant systems.
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Affiliation(s)
- Jothi Kanmani Bharathi
- Department of Genetics and Plant Breeding, Faculty of Agriculture, Annamalai University, Annamalai Nagar, 608002, Tamil Nadu, India
| | - Preethika Suresh
- School of Bioscience and Biotechnology, Vellore Institute of Technology, Vellore, Tamil-Nadu, India
- Department of Horticulture and Food Science, School of Agricultural Innovations and Advanced Learning, Vellore Institute of Technology, Vellore, Tamil-Nadu, India
| | - Muthu Arjuna Samy Prakash
- Department of Genetics and Plant Breeding, Faculty of Agriculture, Annamalai University, Annamalai Nagar, 608002, Tamil Nadu, India
| | - Sowbiya Muneer
- Department of Horticulture and Food Science, School of Agricultural Innovations and Advanced Learning, Vellore Institute of Technology, Vellore, Tamil-Nadu, India
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Kotwal SB, Orekondey N, Saradadevi GP, Priyadarshini N, Puppala NV, Bhushan M, Motamarry S, Kumar R, Mohannath G, Dey RJ. Multidimensional futuristic approaches to address the pandemics beyond COVID-19. Heliyon 2023; 9:e17148. [PMID: 37325452 PMCID: PMC10257889 DOI: 10.1016/j.heliyon.2023.e17148] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2022] [Revised: 06/01/2023] [Accepted: 06/08/2023] [Indexed: 06/17/2023] Open
Abstract
Globally, the impact of the coronavirus disease 2019 (COVID-19) pandemic has been enormous and unrelenting with ∼6.9 million deaths and ∼765 million infections. This review mainly focuses on the recent advances and potentially novel molecular tools for viral diagnostics and therapeutics with far-reaching implications in managing the future pandemics. In addition to briefly highlighting the existing and recent methods of viral diagnostics, we propose a couple of potentially novel non-PCR-based methods for rapid, cost-effective, and single-step detection of nucleic acids of viruses using RNA mimics of green fluorescent protein (GFP) and nuclease-based approaches. We also highlight key innovations in miniaturized Lab-on-Chip (LoC) devices, which in combination with cyber-physical systems, could serve as ideal futuristic platforms for viral diagnosis and disease management. We also discuss underexplored and underutilized antiviral strategies, including ribozyme-mediated RNA-cleaving tools for targeting viral RNA, and recent advances in plant-based platforms for rapid, low-cost, and large-scale production and oral delivery of antiviral agents/vaccines. Lastly, we propose repurposing of the existing vaccines for newer applications with a major emphasis on Bacillus Calmette-Guérin (BCG)-based vaccine engineering.
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Affiliation(s)
- Shifa Bushra Kotwal
- Department of Biological Sciences, BITS Pilani, Hyderabad Campus, Telangana 500078, India
| | - Nidhi Orekondey
- Department of Biological Sciences, BITS Pilani, Hyderabad Campus, Telangana 500078, India
| | | | - Neha Priyadarshini
- Department of Biological Sciences, BITS Pilani, Hyderabad Campus, Telangana 500078, India
| | - Navinchandra V Puppala
- Department of Biological Sciences, BITS Pilani, Hyderabad Campus, Telangana 500078, India
| | - Mahak Bhushan
- Department of Biological Sciences, Indian Institute of Science Education and Research (IISER), Kolkata, West Bengal 741246, India
| | - Snehasri Motamarry
- Department of Biological Sciences, BITS Pilani, Hyderabad Campus, Telangana 500078, India
| | - Rahul Kumar
- Department of Biological Sciences, BITS Pilani, Hyderabad Campus, Telangana 500078, India
| | - Gireesha Mohannath
- Department of Biological Sciences, BITS Pilani, Hyderabad Campus, Telangana 500078, India
| | - Ruchi Jain Dey
- Department of Biological Sciences, BITS Pilani, Hyderabad Campus, Telangana 500078, India
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Akter S, Afrin S, Kim J, Kang J, Razzak MA, Berggren PO, Hwang I. Production of active Exendin-4 in Nicotiana benthamiana and its application in treatment of type-2 diabetics. FRONTIERS IN PLANT SCIENCE 2022; 13:1062658. [PMID: 36618620 PMCID: PMC9812950 DOI: 10.3389/fpls.2022.1062658] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 10/06/2022] [Accepted: 12/05/2022] [Indexed: 06/17/2023]
Abstract
GLP-1 (Glucagon-like peptide-1) is a peptide that stimulates insulin secretion from the β-cell for glycemic control of the plasma blood glucose level. Its mimetic exenatide (synthetic Exendin-4) with a longer half-life of approximately 3.3-4 h is widely used in clinical application to treat diabetes. Currently, exenatide is chemically synthesized. In this study, we report that the GLP-1 analogue recombinant Exendin-4 (Exdn-4) can be produced at a high level in Nicotiana benthamiana, with an estimated yield of 50.0 µg/g fresh biomass. For high-level expression, we generated a recombinant gene, B:GB1:ddCBD1m:8xHis : Exendin-4 (BGC : Exdn-4), for the production of Exendin-4 using various domains such as the BiP signal peptide, the GB1 domain (B1 domain of streptococcal G protein), a double cellulose binding domain 1 (CBD1), and 8 His residues (8xHis) to the N-terminus of Exendin-4. GB1 was used to increase the expression, whereas double CBD1 and 8xHis were included as affinity tags for easy purification using MCC beads and Ni2+-NTA resin, respectively. BGC : Exdn-4 was purified by single-step purification to near homogeneity using both Ni2+-NTA resin and microcrystalline cellulose (MCC) beads. Moreover, Exdn-4 without any extra residues was produced from BGC : Exdn-4 bound onto MCC beads by treating with enterokinase. Plant-produced Exdn-4 (Exendin-4) was as effective as chemically synthesized Exendin-4 in glucose-induced insulin secretion (GIIS) from mouse MIN6m9 cells a pancreatic beta cell line.
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Affiliation(s)
- Shammi Akter
- Department of Life Sciences, Pohang University of Science and Technology, Pohang, South Korea
| | - Shajia Afrin
- Department of Research and Development, BioN Inc., Pohang, South Korea
| | - Jaeyoon Kim
- Department of Research and Development, BioN Inc., Pohang, South Korea
| | - Joohyun Kang
- Department of Life Sciences, Pohang University of Science and Technology, Pohang, South Korea
| | - Md Abdur Razzak
- Department of Life Sciences, Pohang University of Science and Technology, Pohang, South Korea
| | - Per-Olof Berggren
- The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet, Stockholm, Sweden
| | - Inhwan Hwang
- Department of Life Sciences, Pohang University of Science and Technology, Pohang, South Korea
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Mathew M, Thomas J. Tobacco-Based Vaccines, Hopes, and Concerns: A Systematic Review. Mol Biotechnol 2022:10.1007/s12033-022-00627-5. [PMID: 36528727 PMCID: PMC9759281 DOI: 10.1007/s12033-022-00627-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2022] [Accepted: 11/26/2022] [Indexed: 12/23/2022]
Abstract
Emerging infectious diseases have vigorously devastated the global economy and health sector; cost-effective plant-based vaccines (PBV) can be the potential solution to withstand the current health economic crisis. The prominent role of tobacco as an efficient expression system for PBV has been well-established for decades, through this review we highlight the importance of tobacco-based vaccines (TBV) against evolving infectious diseases in humans. Studies focusing on the use of TBV for human infectious diseases were searched in PubMed, Google Scholar, and science direct from 1995 to 2021 using the keywords Tobacco-based vaccines OR transgenic tobacco OR Nicotiana benthamiana vaccines AND Infectious diseases or communicable diseases. We carried out a critical review of the articles and studies that fulfilled the eligibility criteria and were included in this review. Of 976 studies identified, only 63 studies fulfilling the eligibility criteria were included, which focused on either the in vitro, in vivo, or clinical studies on TBV for human infectious diseases. Around 43 in vitro studies of 23 different infectious pathogens expressed in tobacco-based systems were identified and 23 in vivo analysis studies were recognized to check the immunogenicity of vaccine candidates while only 10 of these were subjected to clinical trials. Viral infectious pathogens were studied more than bacterial pathogens. From our review, it was evident that TBV can be an effective health strategy to combat the emerging viral infectious diseases which are very difficult to manage with the current health facilities. The timely administration of cost-effective TBV can prevent the outburst of viral infections, thereby can protect the global healthcare system to a greater extent.
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Affiliation(s)
- Mintu Mathew
- Department of Pharmacology, Amrita School of Pharmacy, Kochi, Kerala India
| | - Jaya Thomas
- Department of Pharmacology, Amrita School of Pharmacy, Kochi, Kerala India
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Rana J, Muñoz MM, Biswas M. Oral tolerance to prevent anti-drug antibody formation in protein replacement therapies. Cell Immunol 2022; 382:104641. [PMID: 36402002 PMCID: PMC9730862 DOI: 10.1016/j.cellimm.2022.104641] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2022] [Revised: 11/07/2022] [Accepted: 11/09/2022] [Indexed: 11/16/2022]
Abstract
Protein based therapeutics have successfully improved the quality of life for patients of monogenic disorders like hemophilia, Pompe and Fabry disease. However, a significant proportion of patients develop immune responses towards intravenously infused therapeutic protein, which can complicate or neutralize treatment and compromise patient safety. Strategies aimed at circumventing immune responses following therapeutic protein infusion can greatly improve therapeutic efficacy. In recent years, antigen-based oral tolerance induction has shown promising results in the prevention and treatment of autoimmune diseases, food allergies and can prevent anti-drug antibody formation to protein replacement therapies. Oral tolerance exploits regulatory mechanisms that are initiated in the gut associated lymphoid tissue (GALT) to promote active suppression of orally ingested antigen. In this review, we outline general perceptions and current knowledge about the mechanisms of oral tolerance, including tissue specific sites of tolerance induction and the cells involved, with emphasis on antigen presenting cells and regulatory T cells. We define several factors, such as cytokines and metabolites that impact the stability and expansion potential of these immune modulatory cells. We highlight preclinical studies that have been performed to induce oral tolerance to therapeutic proteins or enzymes for single gene disorders, such as hemophilia or Pompe disease. These studies mainly utilize a transgenic plant-based system for oral delivery of antigen in conjugation with fusion protein technology that favors the prevention of antigen degradation in the stomach while enhancing uptake in the small intestine by antigen presenting cells and regulatory T cell induction, thereby promoting antigen specific systemic tolerance.
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Affiliation(s)
- Jyoti Rana
- Herman B Wells Center for Pediatric Research, Department of Pediatrics, Indiana University School of Medicine, Indianapolis, IN, USA
| | - Maite Melero Muñoz
- Herman B Wells Center for Pediatric Research, Department of Pediatrics, Indiana University School of Medicine, Indianapolis, IN, USA
| | - Moanaro Biswas
- Herman B Wells Center for Pediatric Research, Department of Pediatrics, Indiana University School of Medicine, Indianapolis, IN, USA.
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Ehsasatvatan M, Kohnehrouz BB, Gholizadeh A, Ofoghi H, Shanehbandi D. The production of the first functional antibody mimetic in higher plants: the chloroplast makes the DARPin G3 for HER2 imaging in oncology. Biol Res 2022; 55:32. [PMID: 36274167 PMCID: PMC9590205 DOI: 10.1186/s40659-022-00400-7] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2022] [Accepted: 10/12/2022] [Indexed: 12/05/2022] Open
Abstract
Background Designed mimetic molecules are attractive tools in biopharmaceuticals and synthetic biology. They require mass and functional production for the assessment of upcoming challenges in the near future. The DARPin family is considered a mimetic pharmaceutical peptide group with high affinity binding to specific targets. DARPin G3 is designed to bind to the HER2 (human epidermal growth factor receptor 2) tyrosine kinase receptor. Overexpression of HER2 is common in some cancers, including breast cancer, and can be used as a prognostic and predictive tool for cancer. The chloroplasts are cost-effective alternatives, equal to, and sometimes better than, bacterial, yeast, or mammalian expression systems. This research examined the possibility of the production of the first antibody mimetic, DARPin G3, in tobacco chloroplasts for HER2 imaging in oncology. Results The chloroplast specific DARPin G3 expression cassette was constructed and transformed into N. tabacum chloroplasts. PCR and Southern blot analysis confirmed integration of transgenes as well as chloroplastic and cellular homoplasmy. The Western blot analysis and ELISA confirmed the production of DARPin G3 at the commercial scale and high dose with the rate of 20.2% in leaf TSP and 33.7% in chloroplast TSP. The functional analysis by ELISA confirmed the binding of IMAC purified chloroplast-made DARPin G3 to the extracellular domain of the HER2 receptor with highly effective picomolar affinities. The carcinoma cellular studies by flow cytometry and immunofluorescence microscopy confirmed the correct functioning by the specific binding of the chloroplast-made DARPin G3 to the HER2 receptor on the surface of HER2-positive cancer cell lines. Conclusion The efficient functional bioactive production of DARPin G3 in chloroplasts led us to introduce plant chloroplasts as the site of efficient production of the first antibody mimetic molecules. This report, as the first case of the cost-effective production of mimetic molecules, enables researchers in pharmaceuticals, synthetic biology, and bio-molecular engineering to develop tool boxes by producing new molecular substitutes for diverse purposes.
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Dunaliella salina as a Potential Biofactory for Antigens and Vehicle for Mucosal Application. Processes (Basel) 2022. [DOI: 10.3390/pr10091776] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022] Open
Abstract
The demand for effective, low-cost vaccines increases research in next-generation biomanufacturing platforms and the study of new vaccine delivery systems (e.g., mucosal vaccines). Applied biotechnology in antigen production guides research toward developing genetic modification techniques in different biological models to achieve the expression of heterologous proteins. These studies are based on various transformation protocols, applied in prokaryotic systems such as Escherichia coli to eukaryotic models such as yeasts, insect cell cultures, animals, and plants, including a particular type of photosynthetic organisms: microalgae, demonstrating the feasibility of recombinant protein expression in these biological models. Microalgae are one of the recombinant protein expression models with the most significant potential and studies in the last decade. Unicellular photosynthetic organisms are widely diverse with biological and growth-specific characteristics. Some examples of the species with commercial interest are Chlamydomonas, Botryococcus, Chlorella, Dunaliella, Haematococcus, and Spirulina. The production of microalgae species at an industrial level through specialized equipment for this purpose allows for proposing microalgae as a basis for producing recombinant proteins at a commercial level. A specie with a particular interest in biotechnology application due to growth characteristics, composition, and protein production capacity is D. salina, which can be cultivated under industrial standards to obtain βcarotene of high interest to humans. D saline currently has advantages over other microalgae species, such as its growth in culture media with a high salt concentration which reduces the risk of contamination, rapid growth, generally considered safe (GRAS), recombinant protein biofactory, and a possible delivery vehicle for mucosal application. This review discusses the status of microalgae D. salina as a platform of expression of recombinant production for its potential mucosal application as a vaccine delivery system, taking an advance on the technology for its production and cultivation at an industrial level.
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Kandoi D, Ruhil K, Govindjee G, Tripathy BC. Overexpression of cytoplasmic C 4 Flaveria bidentis carbonic anhydrase in C 3 Arabidopsis thaliana increases amino acids, photosynthetic potential, and biomass. PLANT BIOTECHNOLOGY JOURNAL 2022; 20:1518-1532. [PMID: 35467074 PMCID: PMC9342616 DOI: 10.1111/pbi.13830] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/09/2021] [Revised: 04/15/2022] [Accepted: 04/18/2022] [Indexed: 05/20/2023]
Abstract
An important method to improve photosynthesis in C3 crops, such as rice and wheat, is to transfer efficient C4 characters to them. Here, cytosolic carbonic anhydrase (CA: βCA3) of the C4 Flaveria bidentis (Fb) was overexpressed under the control of 35 S promoter in Arabidopsis thaliana, a C3 plant, to enhance its photosynthetic efficiency. Overexpression of CA resulted in a better supply of the substrate HCO3- for the endogenous phosphoenolpyruvate carboxylase in the cytosol of the overexpressers, and increased its activity for generating malate that feeds into the tricarboxylic acid cycle. This provided additional carbon skeleton for increased synthesis of amino acids aspartate, asparagine, glutamate, and glutamine. Increased amino acids contributed to higher protein content in the transgenics. Furthermore, expression of FbβCA3 in Arabidopsis led to a better growth due to expression of several genes leading to higher chlorophyll content, electron transport, and photosynthetic carbon assimilation in the transformants. Enhanced CO2 assimilation resulted in increased sugar and starch content, and plant dry weight. In addition, transgenic plants had lower stomatal conductance, reduced transpiration rate, and higher water-use efficiency. These results, taken together, show that expression of C4 CA in the cytosol of a C3 plant can indeed improve its photosynthetic capacity with enhanced water-use efficiency.
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Affiliation(s)
- Deepika Kandoi
- School of Life SciencesJawaharlal Nehru UniversityNew DelhiIndia
| | - Kamal Ruhil
- School of Life SciencesJawaharlal Nehru UniversityNew DelhiIndia
| | - Govindjee Govindjee
- Department of Plant BiologyDepartment of Biochemistry, and Center of Biophysics & Quantitative BiologyUniversity of Illinois at Urbana‐ChampaignUrbanaILUSA
| | - Baishnab C. Tripathy
- School of Life SciencesJawaharlal Nehru UniversityNew DelhiIndia
- Department of BiotechnologySharda UniversityGreater NoidaUPIndia
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Singh VK, Phanindra MLV, Nain V, Gothandapani S, Dhandapani G, Rao KRSS, Kumar A, Kumar PA. Targeting delta-endotoxin (Cry1Ac) of Bacillus thuringiensis to subcellular compartments increases the protein expression, stability, and biological activity. Int J Biol Macromol 2022; 205:185-192. [PMID: 35182560 DOI: 10.1016/j.ijbiomac.2022.02.083] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2022] [Revised: 02/11/2022] [Accepted: 02/14/2022] [Indexed: 11/26/2022]
Abstract
Evolving insect resistance to delta-endotoxins can be delayed by using a few strategies like high dosage, refugia, and gene stacking which require the expression of delta-endotoxins at sufficiently high levels to kill the resistant insects. In this study, we comparatively analyzed the efficacy of targeting truncated cry1Ac protein to the cytoplasm, endoplasmic reticulum (ER), and chloroplast to obtain high protein expression. mRNA and protein profiling of cry1Ac showed that both ER and chloroplast are efficient targets for expressing high levels of truncated cry1Ac. A maximum of 0.8, 1.6, and 2.0% cry1Ac of total soluble protein were obtained when the truncated cry1Ac was expressed in the cytoplasm, routed through ER, and targeted to the chloroplast. We further showed that not only the protein content but also the biological activity of truncated cry1Ac increases by sub-cellular targeting and the biological activity is slightly greater in the ER routed transgenic lines by conducting different bioassays on Helicoverpa armigera. Using native Western analysis, we demonstrated that the truncated cry1Ac protein could exist as oligomers in plant cells and this oligomerization capability is low in the cytoplasm. In conclusion, routing of delta endotoxins through ER is the first choice to obtain high protein expression and bioactivity.
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Affiliation(s)
- Vivek Kumar Singh
- ICAR-National Institute for Plant Biotechnology (Formerly, National Research Centre on Plant Biotechnology), New Delhi, India; Department of Biotechnology, National Institute of Technology, Raipur, India
| | | | - Vikrant Nain
- School of Biotechnology, Gautam Buddha University, Greater Noida, India
| | - Sellamuthu Gothandapani
- ICAR-National Institute for Plant Biotechnology (Formerly, National Research Centre on Plant Biotechnology), New Delhi, India
| | - Gurusamy Dhandapani
- ICAR-National Institute for Plant Biotechnology (Formerly, National Research Centre on Plant Biotechnology), New Delhi, India
| | | | - Awanish Kumar
- Department of Biotechnology, National Institute of Technology, Raipur, India.
| | - Polumetla Ananda Kumar
- ICAR-National Institute for Plant Biotechnology (Formerly, National Research Centre on Plant Biotechnology), New Delhi, India.
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Overexpression of the recombinant human interferon-beta ( rhIFN-β) gene in tobacco chloroplasts. BIOTECHNOLOGIA 2021; 102:367-376. [PMID: 36605601 PMCID: PMC9642931 DOI: 10.5114/bta.2021.111094] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2021] [Revised: 06/26/2021] [Accepted: 07/02/2021] [Indexed: 01/09/2023] Open
Abstract
Chloroplast genetic engineering is a convenient method for the production of recombinant proteins by increasing the expression level of transgenes. Interferon-beta (IFN-β) is a member of type I interferons that possess some pharmaceutical properties. The present study aimed to investigate the overexpression and production of the recombinant human IFN-β gene (rhIFN-β) in the tobacco chloroplast genome. For this purpose, a codon-optimized rhIFN-β was transferred to the pVSR326 plastid vector containing the aadA gene as a selectable marker. The rhIFN-β gene was then successfully introduced into the tobacco chloroplast genome by using a gene gun. The integration of the rhIFN-β gene into the chloroplast genome and the homoplasmy of the T1 progeny were confirmed by PCR and Southern blot analysis, respectively. RT-PCR and western blot analyses confirmed the transcription and translation of the rhIFN-β gene, respectively. An enzyme-linked immunosorbent assay (ELISA) showed that the rhIFN-β protein in transplastomic plants comprised approximately 2.4% of total soluble protein (TSPs). The bioassay confirmed that the rhIFN-β protein expressed in the tobacco chloroplast had a relatively high biological activity (2.9 × 104 IU/ml) and protected human amnionic cells against the vesicular stomatitis virus (VSV). On the basis of these findings, it can be concluded that plastid transformation can serve as an operative method for the production of pharmaceutical recombinant proteins.
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He W, Baysal C, Lobato Gómez M, Huang X, Alvarez D, Zhu C, Armario‐Najera V, Blanco Perera A, Cerda Bennaser P, Saba‐Mayoral A, Sobrino‐Mengual G, Vargheese A, Abranches R, Alexandra Abreu I, Balamurugan S, Bock R, Buyel JF, da Cunha NB, Daniell H, Faller R, Folgado A, Gowtham I, Häkkinen ST, Kumar S, Sathish Kumar R, Lacorte C, Lomonossoff GP, Luís IM, K.‐C. Ma J, McDonald KA, Murad A, Nandi S, O’Keef B, Parthiban S, Paul MJ, Ponndorf D, Rech E, Rodrigues JC, Ruf S, Schillberg S, Schwestka J, Shah PS, Singh R, Stoger E, Twyman RM, Varghese IP, Vianna GR, Webster G, Wilbers RHP, Christou P, Oksman‐Caldentey K, Capell T. Contributions of the international plant science community to the fight against infectious diseases in humans-part 2: Affordable drugs in edible plants for endemic and re-emerging diseases. PLANT BIOTECHNOLOGY JOURNAL 2021; 19:1921-1936. [PMID: 34181810 PMCID: PMC8486237 DOI: 10.1111/pbi.13658] [Citation(s) in RCA: 32] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/30/2021] [Revised: 06/10/2021] [Accepted: 06/22/2021] [Indexed: 05/05/2023]
Abstract
The fight against infectious diseases often focuses on epidemics and pandemics, which demand urgent resources and command attention from the health authorities and media. However, the vast majority of deaths caused by infectious diseases occur in endemic zones, particularly in developing countries, placing a disproportionate burden on underfunded health systems and often requiring international interventions. The provision of vaccines and other biologics is hampered not only by the high cost and limited scalability of traditional manufacturing platforms based on microbial and animal cells, but also by challenges caused by distribution and storage, particularly in regions without a complete cold chain. In this review article, we consider the potential of molecular farming to address the challenges of endemic and re-emerging diseases, focusing on edible plants for the development of oral drugs. Key recent developments in this field include successful clinical trials based on orally delivered dried leaves of Artemisia annua against malarial parasite strains resistant to artemisinin combination therapy, the ability to produce clinical-grade protein drugs in leaves to treat infectious diseases and the long-term storage of protein drugs in dried leaves at ambient temperatures. Recent FDA approval of the first orally delivered protein drug encapsulated in plant cells to treat peanut allergy has opened the door for the development of affordable oral drugs that can be manufactured and distributed in remote areas without cold storage infrastructure and that eliminate the need for expensive purification steps and sterile delivery by injection.
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Affiliation(s)
- Wenshu He
- Department of Crop and Forest SciencesUniversity of Lleida‐Agrotecnio CERCA CenterLleidaSpain
| | - Can Baysal
- Department of Crop and Forest SciencesUniversity of Lleida‐Agrotecnio CERCA CenterLleidaSpain
| | - Maria Lobato Gómez
- Department of Crop and Forest SciencesUniversity of Lleida‐Agrotecnio CERCA CenterLleidaSpain
| | - Xin Huang
- Department of Crop and Forest SciencesUniversity of Lleida‐Agrotecnio CERCA CenterLleidaSpain
| | - Derry Alvarez
- Department of Crop and Forest SciencesUniversity of Lleida‐Agrotecnio CERCA CenterLleidaSpain
| | - Changfu Zhu
- Department of Crop and Forest SciencesUniversity of Lleida‐Agrotecnio CERCA CenterLleidaSpain
| | - Victoria Armario‐Najera
- Department of Crop and Forest SciencesUniversity of Lleida‐Agrotecnio CERCA CenterLleidaSpain
| | - Aamaya Blanco Perera
- Department of Crop and Forest SciencesUniversity of Lleida‐Agrotecnio CERCA CenterLleidaSpain
| | - Pedro Cerda Bennaser
- Department of Crop and Forest SciencesUniversity of Lleida‐Agrotecnio CERCA CenterLleidaSpain
| | - Andrea Saba‐Mayoral
- Department of Crop and Forest SciencesUniversity of Lleida‐Agrotecnio CERCA CenterLleidaSpain
| | | | - Ashwin Vargheese
- Department of Crop and Forest SciencesUniversity of Lleida‐Agrotecnio CERCA CenterLleidaSpain
| | - Rita Abranches
- Instituto de Tecnologia Química e Biológica António XavierUniversidade Nova de LisboaOeirasPortugal
| | - Isabel Alexandra Abreu
- Instituto de Tecnologia Química e Biológica António XavierUniversidade Nova de LisboaOeirasPortugal
| | - Shanmugaraj Balamurugan
- Plant Genetic Engineering LaboratoryDepartment of BiotechnologyBharathiar UniversityTamil NaduIndia
| | - Ralph Bock
- Max Planck Institute of Molecular Plant PhysiologyPotsdam‐GolmGermany
| | - Johannes F. Buyel
- Fraunhofer Institute for Molecular Biology and Applied Ecology IMEAachenGermany
- Institute for Molecular BiotechnologyRWTH Aachen UniversityAachenGermany
| | - Nicolau B. da Cunha
- Centro de Análise Proteômicas e Bioquímicas de BrasíliaUniversidade Católica de BrasíliaBrasíliaBrazil
| | - Henry Daniell
- School of Dental MedicineUniversity of PennsylvaniaPhiladelphiaPAUSA
| | - Roland Faller
- Department of Chemical EngineeringUniversity of California, DavisDavisCAUSA
| | - André Folgado
- Instituto de Tecnologia Química e Biológica António XavierUniversidade Nova de LisboaOeirasPortugal
| | - Iyappan Gowtham
- Plant Genetic Engineering LaboratoryDepartment of BiotechnologyBharathiar UniversityTamil NaduIndia
| | - Suvi T. Häkkinen
- Industrial Biotechnology and Food SolutionsVTT Technical Research Centre of Finland LtdEspooFinland
| | - Shashi Kumar
- International Centre for Genetic Engineering and BiotechnologyNew DelhiIndia
| | - Ramalingam Sathish Kumar
- Plant Genetic Engineering LaboratoryDepartment of BiotechnologyBharathiar UniversityTamil NaduIndia
| | - Cristiano Lacorte
- Brazilian Agriculture Research CorporationEmbrapa Genetic Resources and Biotechnology and National Institute of Science and Technology Synthetic in Biology, Parque Estação BiológicaBrasiliaBrazil
| | | | - Ines M. Luís
- Instituto de Tecnologia Química e Biológica António XavierUniversidade Nova de LisboaOeirasPortugal
| | - Julian K.‐C. Ma
- Institute for Infection and ImmunitySt. George’s University of LondonLondonUK
| | - Karen A. McDonald
- Department of Chemical EngineeringUniversity of California, DavisDavisCAUSA
- Global HealthShare InitiativeUniversity of California, DavisDavisCAUSA
| | - Andre Murad
- Brazilian Agriculture Research CorporationEmbrapa Genetic Resources and Biotechnology and National Institute of Science and Technology Synthetic in Biology, Parque Estação BiológicaBrasiliaBrazil
| | - Somen Nandi
- Department of Chemical EngineeringUniversity of California, DavisDavisCAUSA
- Global HealthShare InitiativeUniversity of California, DavisDavisCAUSA
| | - Barry O’Keef
- Division of Cancer Treatment and DiagnosisMolecular Targets ProgramCenter for Cancer ResearchNational Cancer Institute, and Natural Products Branch, Developmental Therapeutics ProgramNational Cancer Institute, NIHFrederickMDUSA
| | - Subramanian Parthiban
- Plant Genetic Engineering LaboratoryDepartment of BiotechnologyBharathiar UniversityTamil NaduIndia
| | - Mathew J. Paul
- Institute for Infection and ImmunitySt. George’s University of LondonLondonUK
| | - Daniel Ponndorf
- Department of Biological ChemistryJohn Innes CentreNorwich Research Park, NorwichUK
| | - Elibio Rech
- Brazilian Agriculture Research CorporationEmbrapa Genetic Resources and Biotechnology and National Institute of Science and Technology Synthetic in Biology, Parque Estação BiológicaBrasiliaBrazil
| | - Julio C.M. Rodrigues
- Brazilian Agriculture Research CorporationEmbrapa Genetic Resources and Biotechnology and National Institute of Science and Technology Synthetic in Biology, Parque Estação BiológicaBrasiliaBrazil
| | - Stephanie Ruf
- Max Planck Institute of Molecular Plant PhysiologyPotsdam‐GolmGermany
| | - Stefan Schillberg
- Fraunhofer Institute for Molecular Biology and Applied Ecology IMEAachenGermany
- Institute for PhytopathologyJustus‐Liebig‐University GiessenGiessenGermany
| | - Jennifer Schwestka
- Institute of Plant Biotechnology and Cell BiologyUniversity of Natural Resources and Life SciencesViennaAustria
| | - Priya S. Shah
- Department of Chemical EngineeringUniversity of California, DavisDavisCAUSA
- Department of Microbiology and Molecular GeneticsUniversity of California, DavisDavisCAUSA
| | - Rahul Singh
- School of Dental MedicineUniversity of PennsylvaniaPhiladelphiaPAUSA
| | - Eva Stoger
- Institute of Plant Biotechnology and Cell BiologyUniversity of Natural Resources and Life SciencesViennaAustria
| | | | - Inchakalody P. Varghese
- Plant Genetic Engineering LaboratoryDepartment of BiotechnologyBharathiar UniversityTamil NaduIndia
| | - Giovanni R. Vianna
- Brazilian Agriculture Research CorporationEmbrapa Genetic Resources and Biotechnology and National Institute of Science and Technology Synthetic in Biology, Parque Estação BiológicaBrasiliaBrazil
| | - Gina Webster
- Institute for Infection and ImmunitySt. George’s University of LondonLondonUK
| | - Ruud H. P. Wilbers
- Laboratory of NematologyPlant Sciences GroupWageningen University and ResearchWageningenThe Netherlands
| | - Paul Christou
- Department of Crop and Forest SciencesUniversity of Lleida‐Agrotecnio CERCA CenterLleidaSpain
- ICREACatalan Institute for Research and Advanced StudiesBarcelonaSpain
| | | | - Teresa Capell
- Department of Crop and Forest SciencesUniversity of Lleida‐Agrotecnio CERCA CenterLleidaSpain
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13
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Chin-Fatt A, Menassa R. A V HH-Fc Fusion Targeted to the Chloroplast Thylakoid Lumen Assembles and Neutralizes Enterohemorrhagic E. coli O157:H7. FRONTIERS IN PLANT SCIENCE 2021; 12:686421. [PMID: 34122494 PMCID: PMC8193579 DOI: 10.3389/fpls.2021.686421] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 03/26/2021] [Accepted: 04/26/2021] [Indexed: 06/12/2023]
Abstract
Chimeric fusion proteins comprising a single domain antibody (VHH) fused to a crystallizable fragment (Fc) of an immunoglobulin are modular glycoproteins that are becoming increasingly in demand because of their value as diagnostics, research reagents and passive immunization therapeutics. Because ER-associated degradation and misfolding may potentially be limiting factors in the oxidative folding of VHH-Fc fusion proteins in the ER, we sought to explore oxidative folding in an alternative sub-compartment, the chloroplast thylakoid lumen, and determine its viability in a molecular farming context. We developed a set of in-house expression vectors for transient transformation of Nicotiana benthamiana leaves that target a VHH-Fc to the thylakoid lumen via either secretory (Sec) or twin-arginine translocation (Tat) import pathways. Compared to stromal [6.63 ± 3.41 mg/kg fresh weight (FW)], cytoplasmic (undetectable) and Tat-import pathways (5.43 ± 2.41 mg/kg FW), the Sec-targeted VHH-Fc showed superior accumulation (30.56 ± 5.19 mg/kg FW), but was less than that of the ER (51.16 ± 9.11 mg/kg FW). Additionally, the introduction of a rationally designed de novo disulfide bond enhances in planta accumulation when introduced into the Sec-targeted Fc fusion protein from 50.24 ± 4.08 mg/kg FW to 110.90 ± 6.46 mg/kg FW. In vitro immunofluorescent labeling assays on VHH-Fc purified from Sec, Tat, and stromal pathways demonstrate that the antibody still retains VHH functionality in binding Escherichia coli O157:H7 and neutralizing its intimate adherence to human epithelial type 2 cells. These results overall provide a proof of concept that the oxidative folding environment of the thylakoid lumen may be a viable compartment for stably folding disulfide-containing recombinant VHH-Fc proteins.
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Affiliation(s)
- Adam Chin-Fatt
- Agriculture and Agri-Food Canada, London Research and Development Centre, London, ON, Canada
- Department of Biology, University of Western Ontario, London, ON, Canada
| | - Rima Menassa
- Agriculture and Agri-Food Canada, London Research and Development Centre, London, ON, Canada
- Department of Biology, University of Western Ontario, London, ON, Canada
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14
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Rosales-Mendoza S, Cervantes-Rincón T, Romero-Maldonado A, Monreal-Escalante E, Nieto-Gómez R. Transgenic plants expressing a Clostridium difficile spore antigen as an approach to develop low-cost oral vaccines. Biotechnol Prog 2021; 37:e3141. [PMID: 33666366 DOI: 10.1002/btpr.3141] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/15/2020] [Revised: 01/18/2021] [Accepted: 02/04/2021] [Indexed: 01/05/2023]
Abstract
Gastrointestinal infections caused by Clostridium difficile lead to significant impact in terms of morbidity and mortality, causing from mild symptoms, such as a low-grade fever, watery stools, and minor abdominal cramping as well as more severe symptoms such as bloody diarrhea, pseudomembrane colitis, and toxic megacolon. Vaccination is a viable approach to fight against C. difficile and several efforts in this direction are ongoing. Plants are promising vaccine biofactories offering low cost, enhanced safety, and allow for the formulation of oral vaccines. Herein, the CdeM protein, which is a spore antigen associated with immunoprotection against C. difficile, was selected to begin the development of plant-based vaccine candidates. The vaccine antigen is based in a fusion protein (LTB-CdeM), carrying the CdeM antigen, fused to the carboxi-terminus of the B subunit of the Escherichia coli heat-labile enterotoxin (LTB) as a mucosal immunogenic carrier. LTB-CdeM was produced in plants using a synthetic optimized gene according codon usage and mRNA stability criteria. The obtained transformed tobacco lines produced the LTB-CdeM antigen in the range of 52-90 μg/g dry weight leaf tissues. The antigenicity of the plant-made LTB-CdeM antigen was evidenced by GM1-ELISA and immunogenicity assessment performed in test mice revealed that the LTB-CdeM antigen is orally immunogenic inducing humoral responses against CdeM epitopes. This report constitutes the first step in the development of plant-based vaccines against C. difficile infection.
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MESH Headings
- Administration, Oral
- Animals
- Antibodies, Bacterial/blood
- Antigens, Bacterial/genetics
- Antigens, Bacterial/metabolism
- Clostridioides difficile/genetics
- Enterotoxins/genetics
- Escherichia coli Proteins/genetics
- Immunoglobulin G/blood
- Mice
- Mice, Inbred BALB C
- Molecular Farming
- Plants, Genetically Modified/genetics
- Plants, Genetically Modified/metabolism
- Spores, Bacterial/genetics
- Nicotiana/genetics
- Nicotiana/metabolism
- Vaccines, Edible/genetics
- Vaccines, Edible/immunology
- Vaccines, Edible/metabolism
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Affiliation(s)
- Sergio Rosales-Mendoza
- Laboratorio de Biofarmacéuticos Recombinantes, Facultad de Ciencias Químicas, Universidad Autónoma de San Luis Potosí, San Luis Potosí, Mexico
- Sección de Biotecnología, Centro de Investigación en Ciencias de la Salud y Biomedicina, Universidad Autónoma de San Luis Potosí, San Luis Potosí, Mexico
| | - Tomás Cervantes-Rincón
- Laboratorio de Biofarmacéuticos Recombinantes, Facultad de Ciencias Químicas, Universidad Autónoma de San Luis Potosí, San Luis Potosí, Mexico
- Sección de Biotecnología, Centro de Investigación en Ciencias de la Salud y Biomedicina, Universidad Autónoma de San Luis Potosí, San Luis Potosí, Mexico
| | - Andrea Romero-Maldonado
- Laboratorio de Biofarmacéuticos Recombinantes, Facultad de Ciencias Químicas, Universidad Autónoma de San Luis Potosí, San Luis Potosí, Mexico
- Sección de Biotecnología, Centro de Investigación en Ciencias de la Salud y Biomedicina, Universidad Autónoma de San Luis Potosí, San Luis Potosí, Mexico
| | - Elizabeth Monreal-Escalante
- Laboratorio de Biofarmacéuticos Recombinantes, Facultad de Ciencias Químicas, Universidad Autónoma de San Luis Potosí, San Luis Potosí, Mexico
- Sección de Biotecnología, Centro de Investigación en Ciencias de la Salud y Biomedicina, Universidad Autónoma de San Luis Potosí, San Luis Potosí, Mexico
| | - Ricardo Nieto-Gómez
- Laboratorio de Biofarmacéuticos Recombinantes, Facultad de Ciencias Químicas, Universidad Autónoma de San Luis Potosí, San Luis Potosí, Mexico
- Sección de Biotecnología, Centro de Investigación en Ciencias de la Salud y Biomedicina, Universidad Autónoma de San Luis Potosí, San Luis Potosí, Mexico
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15
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Rascón-Cruz Q, González-Barriga CD, Iglesias-Figueroa BF, Trejo-Muñoz JC, Siqueiros-Cendón T, Sinagawa-García SR, Arévalo-Gallegos S, Espinoza-Sánchez EA. Plastid transformation: Advances and challenges for its implementation in agricultural crops. ELECTRON J BIOTECHN 2021. [DOI: 10.1016/j.ejbt.2021.03.005] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
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16
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Daniell H, Jin S, Zhu X, Gitzendanner MA, Soltis DE, Soltis PS. Green giant-a tiny chloroplast genome with mighty power to produce high-value proteins: history and phylogeny. PLANT BIOTECHNOLOGY JOURNAL 2021; 19:430-447. [PMID: 33484606 PMCID: PMC7955891 DOI: 10.1111/pbi.13556] [Citation(s) in RCA: 93] [Impact Index Per Article: 23.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/14/2020] [Revised: 01/11/2021] [Accepted: 01/16/2021] [Indexed: 05/04/2023]
Abstract
Free-living cyanobacteria were entrapped by eukaryotic cells ~2 billion years ago, ultimately giving rise to chloroplasts. After a century of debate, the presence of chloroplast DNA was demonstrated in the 1960s. The first chloroplast genomes were sequenced in the 1980s, followed by ~100 vegetable, fruit, cereal, beverage, oil and starch/sugar crop chloroplast genomes in the past three decades. Foreign genes were expressed in isolated chloroplasts or intact plant cells in the late 1980s and stably integrated into chloroplast genomes, with typically maternal inheritance shown in the 1990s. Since then, chloroplast genomes conferred the highest reported levels of tolerance or resistance to biotic or abiotic stress. Although launching products with agronomic traits in important crops using this concept has been elusive, commercial products developed include enzymes used in everyday life from processing fruit juice, to enhancing water absorption of cotton fibre or removal of stains as laundry detergents and in dye removal in the textile industry. Plastid genome sequences have revealed the framework of green plant phylogeny as well as the intricate history of plastid genome transfer events to other eukaryotes. Discordant historical signals among plastid genes suggest possible variable constraints across the plastome and further understanding and mitigation of these constraints may yield new opportunities for bioengineering. In this review, we trace the evolutionary history of chloroplasts, status of autonomy and recent advances in products developed for everyday use or those advanced to the clinic, including treatment of COVID-19 patients and SARS-CoV-2 vaccine.
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Affiliation(s)
- Henry Daniell
- Department of Basic and Translational SciencesSchool of Dental MedicineUniversity of PennsylvaniaPhiladelphiaPAUSA
| | - Shuangxia Jin
- National Key Laboratory of Crop Genetic ImprovementHuazhong Agricultural UniversityWuhanChina
| | - Xin‐Guang Zhu
- State Key Laboratory for Plant Molecular Genetics and Center of Excellence for Molecular Plant SciencesChinese Academy of SciencesShanghaiChina
| | | | - Douglas E. Soltis
- Florida Museum of Natural History and Department of BiologyUniversity of FloridaGainesvilleFLUSA
- Florida Museum of Natural HistoryUniversity of FloridaGainesvilleFLUSA
| | - Pamela S. Soltis
- Florida Museum of Natural HistoryUniversity of FloridaGainesvilleFLUSA
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17
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Jareonsin S, Pumas C. Advantages of Heterotrophic Microalgae as a Host for Phytochemicals Production. Front Bioeng Biotechnol 2021; 9:628597. [PMID: 33644020 PMCID: PMC7907617 DOI: 10.3389/fbioe.2021.628597] [Citation(s) in RCA: 26] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2020] [Accepted: 01/19/2021] [Indexed: 12/17/2022] Open
Abstract
Currently, most commercial recombinant technologies rely on host systems. However, each host has their own benefits and drawbacks, depending on the target products. Prokaryote host is lack of post-transcriptional and post-translational mechanisms, making them unsuitable for eukaryotic productions like phytochemicals. Even there are other eukaryote hosts (e.g., transgenic animals, mammalian cell, and transgenic plants), but those hosts have some limitations, such as low yield, high cost, time consuming, virus contamination, and so on. Thus, flexible platforms and efficient methods that can produced phytochemicals are required. The use of heterotrophic microalgae as a host system is interesting because it possibly overcome those obstacles. This paper presents a comprehensive review of heterotrophic microalgal expression host including advantages of heterotrophic microalgae as a host, genetic engineering of microalgae, genetic transformation of microalgae, microalgal engineering for phytochemicals production, challenges of microalgal hosts, key market trends, and future view. Finally, this review might be a directions of the alternative microalgae host for high-value phytochemicals production in the next few years.
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Affiliation(s)
- Surumpa Jareonsin
- Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, Thailand
| | - Chayakorn Pumas
- Research Center in Bioresources for Agriculture, Industry and Medicine, Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, Thailand
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18
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Ghag SB, Adki VS, Ganapathi TR, Bapat VA. Plant Platforms for Efficient Heterologous Protein Production. BIOTECHNOL BIOPROC E 2021; 26:546-567. [PMID: 34393545 PMCID: PMC8346785 DOI: 10.1007/s12257-020-0374-1] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2020] [Revised: 01/14/2021] [Accepted: 01/16/2021] [Indexed: 02/07/2023]
Abstract
Production of recombinant proteins is primarily established in cultures of mammalian, insect and bacterial cells. Concurrently, concept of using plants to produce high-value pharmaceuticals such as vaccines, antibodies, and dietary proteins have received worldwide attention. Newer technologies for plant transformation such as plastid engineering, agroinfiltration, magnifection, and deconstructed viral vectors have been used to enhance the protein production in plants along with the inherent advantage of speed, scale, and cost of production in plant systems. Production of therapeutic proteins in plants has now a more pragmatic approach when several plant-produced vaccines and antibodies successfully completed Phase I clinical trials in humans and were further scheduled for regulatory approvals to manufacture clinical grade products on a large scale which are safe, efficacious, and meet the quality standards. The main thrust of this review is to summarize the data accumulated over the last two decades and recent development and achievements of the plant derived therapeutics. It also attempts to discuss different strategies employed to increase the production so as to make plants more competitive with the established production systems in this industry.
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Affiliation(s)
- Siddhesh B. Ghag
- School of Biological Sciences, UM-DAE Centre for Excellence in Basic Sciences, University of Mumbai campus, Kalina, Santacruz, Mumbai, 400098 India
| | - Vinayak S. Adki
- V. G. Shivdare College of Arts, Commerce and Science, Solapur, Maharashtra 413004 India
| | - Thumballi R. Ganapathi
- Plant Cell Culture Technology Section, Nuclear Agriculture & Biotechnology Division, Bhabha Atomic Research Centre, Trombay, Mumbai, 400085 India
| | - Vishwas A. Bapat
- Department of Biotechnology, Shivaji University, Vidyanagar, Kolhapur, Maharashtra 416004 India
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19
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Daniell H. From conception to COVID-19: an arduous journey of tribulations of racism and triumphs. PLANT BIOTECHNOLOGY JOURNAL 2020; 18:2147-2154. [PMID: 32799416 PMCID: PMC7460971 DOI: 10.1111/pbi.13468] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/29/2020] [Revised: 07/06/2020] [Accepted: 07/08/2020] [Indexed: 05/07/2023]
Abstract
Growing up in a densely wooded tropical forest enhanced my curiosity in plants and reading biography of Marie Curie profoundly influenced pursuit of my research career. Early in my career, I developed in vitro functional chloroplasts, capable of expressing foreign genes and this laid the foundation for the chloroplast genetic engineering field. Four decades of research has advanced chloroplast bioreactors for production of industrial enzymes or biopharmaceuticals by small or large companies. Because I experienced firsthand horrors of expensive vaccines or medicines, I devoted most of my career to develop affordable therapeutics. During this long journey, I suffered institutional racial discrimination but was rescued by several guardian angels. This biography gives readers a glimpse of tribulations and triumphs of my journey and recognizes important contributions made by my mentees.
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Affiliation(s)
- Henry Daniell
- School of Dental MedicineUniversity of PennsylvaniaPhiladelphiaPAUSA
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20
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Yarra R. Plastome engineering in vegetable crops: current status and future prospects. Mol Biol Rep 2020; 47:8061-8074. [PMID: 32880066 DOI: 10.1007/s11033-020-05770-3] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2020] [Accepted: 08/28/2020] [Indexed: 01/12/2023]
Abstract
Plastome (plastid genome) engineering has grown up and got smarter for the transgene expression. Plastid transformation has profound benefits over nuclear transformation, includes a higher level of transgene expression, integration via homologous recombination, transgene containment, lack of gene silencing, and position effect. Substantial and fruitful progress has been achieved in plastome engineering of vegetable crops through the use of improved regeneration/selection procedures, plastid transformation vectors with efficient promoters, and 3/, 5/regulatory sequences. Plastid transformation technology developed for vegetable crops being used as a platform for the production of industrially important proteins and some of the genes of agronomic importance has been stably integrated and expressed in plastome. Although great progress has been accomplished in the plastid transformation of vegetable crops, still it is restricted to few species because of the unavailability of whole plastome sequencing. In this review, the author focus on the technology, progress, and advancements in plastid transformation of vegetable plants such as lettuce, tomato, potato, cabbage, cauliflower, eggplant, carrot, soybean, and bitter melon are reviewed. The conclusions, future prospects, and expansion of plastid transformation technology to other vegetable crops for genetic improvement and production of edible vaccines are proposed.
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Affiliation(s)
- Rajesh Yarra
- Department of Agronomy, University of Florida, IFAS, Gainesville, FL, 32611, USA.
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21
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Kumar SRP, Wang X, Avuthu N, Bertolini TB, Terhorst C, Guda C, Daniell H, Herzog RW. Role of Small Intestine and Gut Microbiome in Plant-Based Oral Tolerance for Hemophilia. Front Immunol 2020; 11:844. [PMID: 32508814 PMCID: PMC7251037 DOI: 10.3389/fimmu.2020.00844] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/04/2019] [Accepted: 04/14/2020] [Indexed: 01/03/2023] Open
Abstract
Fusion proteins, which consist of factor VIII or factor IX and the transmucosal carrier cholera toxin subunit B, expressed in chloroplasts and bioencapsulated within plant cells, initiate tolerogenic immune responses in the intestine when administered orally. This approach induces regulatory T cells (Treg), which suppress inhibitory antibody formation directed at hemophilia proteins induced by intravenous replacement therapy in hemophilia A and B mice. Further analyses of Treg CD4+ lymphocyte sub-populations in hemophilia B mice reveal a marked increase in the frequency of CD4+CD25-FoxP3-LAP+ T cells (but not of CD4+CD25+FoxP3+ T cells) in the lamina propria of the small but not large intestine. The adoptive transfer of very small numbers of CD4+CD25-LAP+ Treg isolated from the spleen of tolerized mice was superior in suppression of antibodies directed against FIX when compared to CD4+CD25+ T cells. Thus, tolerance induction by oral delivery of antigens bioencapsulated in plant cells occurs via the unique immune system of the small intestine, and suppression of antibody formation is primarily carried out by induced latency-associated peptide (LAP) expressing Treg that likely migrate to the spleen. Tolerogenic antigen presentation in the small intestine requires partial enzymatic degradation of plant cell wall by commensal bacteria in order to release the antigen. Microbiome analysis of hemophilia B mice showed marked differences between small and large intestine. Remarkably, bacterial species known to produce a broad spectrum of enzymes involved in degradation of plant cell wall components were found in the small intestine, in particular in the duodenum. These were highly distinct from populations of cell wall degrading bacteria found in the large intestine. Therefore, FIX antigen presentation and Treg induction by the immune system of the small intestine relies on activity of a distinct microbiome that can potentially be augmented to further enhance this approach.
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Affiliation(s)
- Sandeep R. P. Kumar
- Herman B Wells Center for Pediatric Research, IAPUI, Indianapolis, IN, United States
| | - Xiaomei Wang
- Department of Pediatrics, University of Florida, Gainesville, FL, United States
| | - Nagavardhini Avuthu
- Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, NE, United States
| | - Thais B. Bertolini
- Herman B Wells Center for Pediatric Research, IAPUI, Indianapolis, IN, United States
| | - Cox Terhorst
- Division of Immunology, Beth Israel Deaconess Medical Center (BIDMC), Harvard Medical School, Boston, MA, United States
| | - Chittibabu Guda
- Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, NE, United States
| | - Henry Daniell
- Department of Basic and Translational Sciences, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA, United States
| | - Roland W. Herzog
- Herman B Wells Center for Pediatric Research, IAPUI, Indianapolis, IN, United States
- Department of Pediatrics, University of Florida, Gainesville, FL, United States
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22
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Betterle N, Hidalgo Martinez D, Melis A. Cyanobacterial Production of Biopharmaceutical and Biotherapeutic Proteins. FRONTIERS IN PLANT SCIENCE 2020; 11:237. [PMID: 32194609 PMCID: PMC7062967 DOI: 10.3389/fpls.2020.00237] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 10/24/2019] [Accepted: 02/14/2020] [Indexed: 06/10/2023]
Abstract
Efforts to express human therapeutic proteins in photosynthetic organisms have been described in the literature. Regarding microalgae, most of the research entailed a heterologous transformation of the chloroplast, but transformant cells failed to accumulate the desired recombinant proteins in high quantity. The present work provides methods and DNA construct formulations for over-expressing in photosynthetic cyanobacteria, at the protein level, human-origin bio-pharmaceutical and bio-therapeutic proteins. Proof-of-concept evidence is provided for the design and reduction to practice of "fusion constructs as protein overexpression vectors" for the generation of the bio-therapeutic protein interferon alpha-2 (IFN). IFN is a member of the Type I interferon cytokine family, well-known for its antiviral and anti-proliferative functions. Fusion construct formulations enabled accumulation of IFN up to 12% of total cellular protein in soluble form. In addition, the work reports on the isolation and purification of the fusion IFN protein and preliminary verification of its antiviral activity. Combining the expression and purification protocols developed here, it is possible to produce fairly large quantities of interferon in these photosynthetic microorganisms, generated from sunlight, CO2, and H2O.
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Park J, Yan G, Kwon KC, Liu M, Gonnella PA, Yang S, Daniell H. Oral delivery of novel human IGF-1 bioencapsulated in lettuce cells promotes musculoskeletal cell proliferation, differentiation and diabetic fracture healing. Biomaterials 2020; 233:119591. [PMID: 31870566 PMCID: PMC6990632 DOI: 10.1016/j.biomaterials.2019.119591] [Citation(s) in RCA: 47] [Impact Index Per Article: 9.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/13/2019] [Revised: 10/16/2019] [Accepted: 10/30/2019] [Indexed: 12/16/2022]
Abstract
Human insulin-like growth factor-1 (IGF-1) plays important roles in development and regeneration of skeletal muscles and bones but requires daily injections or surgical implantation. Current clinical IGF-1 lacks e-peptide and is glycosylated, reducing functional efficacy. In this study, codon-optimized Pro-IGF-1 with e-peptide (fused to GM1 receptor binding protein CTB or cell penetrating peptide PTD) was expressed in lettuce chloroplasts to facilitate oral delivery. Pro-IGF-1 was expressed at high levels in the absence of the antibiotic resistance gene in lettuce chloroplasts and was maintained in subsequent generations. In lyophilized plant cells, Pro-IGF-1 maintained folding, assembly, stability and functionality up to 31 months, when stored at ambient temperature. CTB-Pro-IGF-1 stimulated proliferation of human oral keratinocytes, gingiva-derived mesenchymal stromal cells and mouse osteoblasts in a dose-dependent manner and promoted osteoblast differentiation through upregulation of ALP, OSX and RUNX2 genes. Mice orally gavaged with the lyophilized plant cells significantly increased IGF-1 levels in sera, skeletal muscles and was stable for several hours. When bioencapsulated CTB-Pro-IGF-1 was gavaged to femoral fractured diabetic mice, bone regeneration was significantly promoted with increase in bone volume, density and area. This novel delivery system should increase affordability and patient compliance, especially for treatment of musculoskeletal diseases.
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Affiliation(s)
- J Park
- Department of Basic and Translational Sciences, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - G Yan
- Department of Basic and Translational Sciences, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - K-C Kwon
- Department of Basic and Translational Sciences, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - M Liu
- Department of Basic and Translational Sciences, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - P A Gonnella
- Department of Basic and Translational Sciences, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - S Yang
- Department of Basic and Translational Sciences, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; The Penn Center for Musculoskeletal Disorders, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
| | - H Daniell
- Department of Basic and Translational Sciences, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
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24
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Moon KB, Park JS, Park YI, Song IJ, Lee HJ, Cho HS, Jeon JH, Kim HS. Development of Systems for the Production of Plant-Derived Biopharmaceuticals. PLANTS 2019; 9:plants9010030. [PMID: 31878277 PMCID: PMC7020158 DOI: 10.3390/plants9010030] [Citation(s) in RCA: 46] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 11/13/2019] [Revised: 12/20/2019] [Accepted: 12/23/2019] [Indexed: 12/21/2022]
Abstract
Over the last several decades, plants have been developed as a platform for the production of useful recombinant proteins due to a number of advantages, including rapid production and scalability, the ability to produce unique glycoforms, and the intrinsic safety of food crops. The expression methods used to produce target proteins are divided into stable and transient systems depending on applications that use whole plants or minimally processed forms. In the early stages of research, stable expression systems were mostly used; however, in recent years, transient expression systems have been preferred. The production of the plant itself, which produces recombinant proteins, is currently divided into two major approaches, open-field cultivation and closed-indoor systems. The latter encompasses such regimes as greenhouses, vertical farming units, cell bioreactors, and hydroponic systems. Various aspects of each system will be discussed in this review, which focuses mainly on practical examples and commercially feasible approaches.
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Affiliation(s)
- Ki-Beom Moon
- Plant Systems Engineering Research Center, Korea Research Institute of Bioscience and Biotechnology, 125 Gwahak-ro, Yuseong-gu, Daejeon 34141, Korea; (K.-B.M.); (J.-S.P.); (H.-J.L.); (H.S.C.); (J.-H.J.)
- Department of Biological Sciences, Chungnam National University, 99 Deahank-ro, Yuseong-gu, Daejeon 34134, Korea
| | - Ji-Sun Park
- Plant Systems Engineering Research Center, Korea Research Institute of Bioscience and Biotechnology, 125 Gwahak-ro, Yuseong-gu, Daejeon 34141, Korea; (K.-B.M.); (J.-S.P.); (H.-J.L.); (H.S.C.); (J.-H.J.)
| | - Youn-Il Park
- Department of Biological Sciences, Chungnam National University, 99 Deahank-ro, Yuseong-gu, Daejeon 34134, Korea
| | - In-Ja Song
- National Research Safety Headquarters, Korea Research Institute of Bioscience and Biotechnology, 30 Yeongudanji-ro, Ochang, Chungbuk-do 28116, Korea;
| | - Hyo-Jun Lee
- Plant Systems Engineering Research Center, Korea Research Institute of Bioscience and Biotechnology, 125 Gwahak-ro, Yuseong-gu, Daejeon 34141, Korea; (K.-B.M.); (J.-S.P.); (H.-J.L.); (H.S.C.); (J.-H.J.)
| | - Hye Sun Cho
- Plant Systems Engineering Research Center, Korea Research Institute of Bioscience and Biotechnology, 125 Gwahak-ro, Yuseong-gu, Daejeon 34141, Korea; (K.-B.M.); (J.-S.P.); (H.-J.L.); (H.S.C.); (J.-H.J.)
| | - Jae-Heung Jeon
- Plant Systems Engineering Research Center, Korea Research Institute of Bioscience and Biotechnology, 125 Gwahak-ro, Yuseong-gu, Daejeon 34141, Korea; (K.-B.M.); (J.-S.P.); (H.-J.L.); (H.S.C.); (J.-H.J.)
| | - Hyun-Soon Kim
- Plant Systems Engineering Research Center, Korea Research Institute of Bioscience and Biotechnology, 125 Gwahak-ro, Yuseong-gu, Daejeon 34141, Korea; (K.-B.M.); (J.-S.P.); (H.-J.L.); (H.S.C.); (J.-H.J.)
- Correspondence: ; Tel.: +82-42-860-4493
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Lin B, Cui Y, Yan M, Wang Y, Gao Z, Meng C, Qin S. Construction of astaxanthin metabolic pathway in the green microalga Dunaliella viridis. ALGAL RES 2019. [DOI: 10.1016/j.algal.2019.101697] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
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26
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van Eerde A, Gottschamel J, Bock R, Hansen KEA, Munang'andu HM, Daniell H, Liu Clarke J. Production of tetravalent dengue virus envelope protein domain III based antigens in lettuce chloroplasts and immunologic analysis for future oral vaccine development. PLANT BIOTECHNOLOGY JOURNAL 2019; 17:1408-1417. [PMID: 30578710 PMCID: PMC6576073 DOI: 10.1111/pbi.13065] [Citation(s) in RCA: 29] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/02/2018] [Revised: 12/07/2018] [Accepted: 12/12/2018] [Indexed: 05/19/2023]
Abstract
Dengue fever is a mosquito (Aedes aegypti) -transmitted viral disease that is endemic in more than 125 countries around the world. There are four serotypes of the dengue virus (DENV 1-4) and a safe and effective dengue vaccine must provide protection against all four serotypes. To date, the first vaccine, Dengvaxia (CYD-TDV), is available after many decades' efforts, but only has moderate efficacy. More effective and affordable vaccines are hence required. Plants offer promising vaccine production platforms and food crops offer additional advantages for the production of edible human and animal vaccines, thus eliminating the need for expensive fermentation, purification, cold storage and sterile delivery. Oral vaccines can elicit humoural and cellular immunity via both the mucosal and humoral immune systems. Here, we report the production of tetravalent EDIII antigen (EDIII-1-4) in stably transformed lettuce chloroplasts. Transplastomic EDIII-1-4-expressing lettuce lines were obtained and homoplasmy was verified by Southern blot analysis. Expression of EDIII-1-4 antigens was demonstrated by immunoblotting, with the EDIII-1-4 antigen accumulating to 3.45% of the total protein content. Immunological assays in rabbits showed immunogenicity of EDIII-1-4. Our in vitro gastrointestinal digestion analysis revealed that EDIII-1-4 antigens are well protected when passing through the oral and gastric digestion phases but underwent degradation during the intestinal phase. Our results demonstrate that lettuce chloroplast engineering is a promising approach for future production of an affordable oral dengue vaccine.
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Affiliation(s)
- André van Eerde
- NIBIO – Norwegian Institute of Bioeconomy ResearchDivision of Biotechnology and Plant HealthÅsNorway
| | - Johanna Gottschamel
- NIBIO – Norwegian Institute of Bioeconomy ResearchDivision of Biotechnology and Plant HealthÅsNorway
| | - Ralph Bock
- Max Planck Institute of Molecular Plant PhysiologyPotsdam‐GolmGermany
| | | | | | - Henry Daniell
- Department of BiochemistrySchool of Dental MedicineUniversity of PennsylvaniaPhiladelphiaPAUSA
| | - Jihong Liu Clarke
- NIBIO – Norwegian Institute of Bioeconomy ResearchDivision of Biotechnology and Plant HealthÅsNorway
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Daniell H, Kulis M, Herzog RW. Plant cell-made protein antigens for induction of Oral tolerance. Biotechnol Adv 2019; 37:107413. [PMID: 31251968 PMCID: PMC6842683 DOI: 10.1016/j.biotechadv.2019.06.012] [Citation(s) in RCA: 41] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2018] [Revised: 05/21/2019] [Accepted: 06/24/2019] [Indexed: 12/15/2022]
Abstract
The gut associated lymphoid tissue has effective mechanisms in place to maintain tolerance to food antigens. These can be exploited to induce antigen-specific tolerance for the prevention and treatment of autoimmune diseases and severe allergies and to prevent serious immune responses in protein replacement therapies for genetic diseases. An oral tolerance approach for the prevention of peanut allergy in infants proved highly efficacious and advances in treatment of peanut allergy have brought forth an oral immunotherapy drug that is currently awaiting FDA approval. Several other protein antigens made in plant cells are in clinical development. Plant cell-made proteins are protected in the stomach from acids and enzymes after their oral delivery because of bioencapsulation within plant cell wall, but are released to the immune system upon digestion by gut microbes. Utilization of fusion protein technologies facilitates their delivery to the immune system, oral tolerance induction at low antigen doses, resulting in efficient induction of FoxP3+ and latency-associated peptide (LAP)+ regulatory T cells that express immune suppressive cytokines such as IL-10. LAP and IL-10 expression represent potential biomarkers for plant-based oral tolerance. Efficacy studies in hemophilia dogs support clinical development of oral delivery of bioencapsulated antigens to prevent anti-drug antibody formation. Production of clinical grade materials in cGMP facilities, stability of antigens in lyophilized plant cells for several years when stored at ambient temperature, efficacy of oral delivery of human doses in large animal models and lack of toxicity augur well for clinical advancement of this novel drug delivery concept.
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Affiliation(s)
- Henry Daniell
- Department of Biochemistry, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
| | - Michael Kulis
- Department of Pediatrics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Roland W Herzog
- Department of Pediatrics, Indiana University, Indianapolis, IN 46202, USA.
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28
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Dubey KK, Luke GA, Knox C, Kumar P, Pletschke BI, Singh PK, Shukla P. Vaccine and antibody production in plants: developments and computational tools. Brief Funct Genomics 2019; 17:295-307. [PMID: 29982427 DOI: 10.1093/bfgp/ely020] [Citation(s) in RCA: 37] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/14/2022] Open
Abstract
Plants as bioreactors have been widely used to express efficient vaccine antigens against viral, bacterial and protozoan infections. To date, many different plant-based expression systems have been analyzed, with a growing preference for transient expression systems. Antibody expression in diverse plant species for therapeutic applications is well known, and this review provides an overview of various aspects of plant-based biopharmaceutical production. Here, we highlight conventional and gene expression technologies in plants along with some illustrative examples. In addition, the portfolio of products that are being produced and how they relate to the success of this field are discussed. Stable and transient gene expression in plants, agrofiltration and virus infection vectors are also reviewed. Further, the present report draws attention to antibody epitope prediction using computational tools, one of the crucial steps of vaccine design. Finally, regulatory issues, biosafety and public perception of this technology are also discussed.
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Affiliation(s)
- Kashyap Kumar Dubey
- Department of Biotechnology, Central University of Haryana, Jant-Pali Mahendergarh, Haryana, India.,Microbial Process Development Laboratory, University Institute of Engineering and Technology, Maharshi Dayanand University, Rohtak, Haryana, India
| | - Garry A Luke
- Centre for Biomolecular Sciences, School of Biology, University of St Andrews, North Haugh, St Andrews, Fife KY16 9ST, Scotland
| | - Caroline Knox
- Department of Biochemistry and Microbiology, Rhodes University, Grahamstown, Eastern Cape, South Africa
| | - Punit Kumar
- Microbial Process Development Laboratory, University Institute of Engineering and Technology, Maharshi Dayanand University, Rohtak, Haryana, India
| | - Brett I Pletschke
- Department of Biochemistry and Microbiology, Rhodes University, Grahamstown, Eastern Cape, South Africa
| | - Puneet Kumar Singh
- Enzyme Technology and Protein Bioinformatics Laboratory, Department of Microbiology, Maharshi Dayanand University, Rohtak, Haryana, India
| | - Pratyoosh Shukla
- Enzyme Technology and Protein Bioinformatics Laboratory, Department of Microbiology, Maharshi Dayanand University, Rohtak, Haryana, India
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29
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Tien NQD, Huy NX, Kim MY. Improved expression of porcine epidemic diarrhea antigen by fusion with cholera toxin B subunit and chloroplast transformation in Nicotiana tabacum. PLANT CELL, TISSUE AND ORGAN CULTURE 2019; 137:213-223. [PMID: 32214566 PMCID: PMC7089040 DOI: 10.1007/s11240-019-01562-1] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/23/2018] [Accepted: 01/21/2019] [Indexed: 05/24/2023]
Abstract
The porcine epidemic diarrhea virus (PEDV) belongs to the coronavirus family, which causes acute diarrhea in pigs with higher mortality in piglets less than 2 weeks old. The PEDV is one of the major concerns of the pig industry around the world, including Asian countries and Noth America since first identified in Europe. Currently, there is no PEDV licensed vaccine to effectively prevent this disease. This study was performed for the development of a mucosal PEDV vaccine and B subunit of cholera toxin (CTB) as a carrier was employed to surpass the tolerogenic nature of GALT and induce potent immune responses against the target antigen fused to CTB. An epitope (S1D) alone or conjugated with CTB was constructed into the tobacco chloroplasts expression vector which is controlled under the chloroplast rRNA operon promoter with T7g10 5' UTR and the psbA 3'UTR as a terminator. The homoplastomic lines were obtained by third round screening via organogenesis from the leaf tissues which were verified by PCR with antigen and chloroplast specific primers and then confirmed by Southern blot analysis. While the expression level of the S1D alone as detected by Western blotting was approximately 0.07% of total soluble protein, the CTB-S1D fusion protein was expressed up to 1.4%. The fusion protein showed binding to the intestinal membrane GM1-ganglioside receptor, demonstrating its functionality. The result shows that the highest expression of S1D could be achieved by fusion with a stable CTB protein and chloroplast transformation. Furthermore, the CTB-S1D expressed in chloroplasts of Nicotiana tabacum cv. Maryland could be assembled to pentameric form which increases the possibility to develop a mucosal vaccine against PEDV.
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Affiliation(s)
- Nguyen-Quang-Duc Tien
- Bioactive Material Science, Chonbuk National University, Jeonju, South Korea
- College of Sciences, Hue University, Hue City, Vietnam
| | - Nguyen-Xuan Huy
- Department of Molecular Biology, Chonbuk National University, Jeonju, South Korea
- College of Education, Hue University, Hue City, Vietnam
| | - Mi-Young Kim
- Department of Molecular Biology, Chonbuk National University, Jeonju, South Korea
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Mirzaee M, Jalali-Javaran M, Moieni A, Zeinali S, Behdani M. Expression of VGRNb-PE immunotoxin in transplastomic lettuce (Lactuca sativa L.). PLANT MOLECULAR BIOLOGY 2018; 97:103-112. [PMID: 29633168 DOI: 10.1007/s11103-018-0726-9] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/13/2017] [Accepted: 04/03/2018] [Indexed: 05/03/2023]
Abstract
KEY MESSAGE This research has shown, for the first time, that plant chloroplasts are a suitable compartment for synthesizing recombinant immunotoxins and the transgenic immunotoxin efficiently causes the inhibition of VEGFR2 overexpression, cell growth and proliferation. Angiogenesis refers to the formation of new blood vessels, which resulted in the growth, invasion and metastasis of cancer. The vascular endothelial growth factor receptor 2 (VEGFR2) plays a major role in angiogenesis and blocking of its signaling inhibits neovascularization and tumor metastasis. Immunotoxins are promising therapeutics for targeted cancer therapy. They consist of an antibody linked to a protein toxin and are designed to specifically kill the tumor cells. In our previous study, VGRNb-PE immunotoxin protein containing anti-VEGFR2 nanobody fused to the truncated form of Pseudomonas exotoxin A has been established. Here, we expressed this immunotoxin in lettuce chloroplasts. Chloroplast genetic engineering offers several advantages, including high levels of transgene expression, multigene engineering in a single transformation event and maternal inheritance of the transgenes. Site specific integration of transgene into chloroplast genomes, and homoplasmy were confirmed. Immunotoxin levels reached up to 1.1% of total soluble protein or 33.7 µg per 100 mg of leaf tissue (fresh weight). We demonstrated that transgenic immunotoxin efficiently causes the inhibition of VEGFR2 overexpression, cell growth and proliferation. These results indicate that plant chloroplasts are a suitable compartment for synthesizing recombinant immunotoxins.
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Affiliation(s)
- Malihe Mirzaee
- Department of Plant Breeding & Biotechnology, Faculty of Agriculture, Tarbiat Modares University, P.O. Box 1497713111, Tehran, Iran
| | - Mokhtar Jalali-Javaran
- Department of Plant Breeding & Biotechnology, Faculty of Agriculture, Tarbiat Modares University, P.O. Box 1497713111, Tehran, Iran.
| | - Ahmad Moieni
- Department of Plant Breeding & Biotechnology, Faculty of Agriculture, Tarbiat Modares University, P.O. Box 1497713111, Tehran, Iran
| | - Sirous Zeinali
- Department of Molecular Medicine, Pasteur Institute of Iran, Tehran, Iran
| | - Mahdi Behdani
- Biotechnology Research Center, Biotechnology Department, Venom & Biotherapeutics Molecules Lab., Pasteur Institute of Iran, Tehran, Iran
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Tokuhara D. Challenges in developing mucosal vaccines and antibodies against infectious diarrhea in children. Pediatr Int 2018; 60:214-223. [PMID: 29290097 DOI: 10.1111/ped.13497] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/20/2017] [Revised: 12/14/2017] [Accepted: 12/26/2017] [Indexed: 12/24/2022]
Abstract
Infectious diarrhea in children can be life-threatening and imposes a large economic burden on healthcare systems, therefore more effective prophylactic and therapeutic drugs are needed urgently. Because most of the pathogens responsible for childhood diarrhea infect the gastrointestinal mucosa, providing protective immunity at the mucosal surface is an ideal way to control pathogen invasion and toxic activity. Mucosal (e.g. oral, nasal) vaccines are superior to systemic (subcutaneous or intramuscular) vaccination for conferring both mucosal and systemic pathogen-specific immune responses. Therefore, great efforts has been focused on the development of cost-effective mucosal vaccines for the past 50 years. Recent progress in plant genetic engineering has revolutionized the production of inexpensive and safe recombinant vaccine antigens. For example, rice plant biotechnology has facilitated the development of a cold-chain-free rice-based oral subunit vaccine against Vibrio cholerae. Furthermore, this technology has led to the creation of a rice-based oral antibody for prophylaxis and treatment of rotavirus gastroenteritis. This review summarizes current perspectives regarding the mucosal immune system and the development of mucosal vaccines and therapeutic antibodies, particularly rice-based products, and discusses future prospects regarding mucosal vaccines for children.
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Affiliation(s)
- Daisuke Tokuhara
- Department of Pediatrics, Osaka City University Graduate School of Medicine, Abenoku, Osaka, Japan
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32
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Bal J, Luong NN, Park J, Song KD, Jang YS, Kim DH. Comparative immunogenicity of preparations of yeast-derived dengue oral vaccine candidate. Microb Cell Fact 2018; 17:24. [PMID: 29452594 PMCID: PMC5815244 DOI: 10.1186/s12934-018-0876-0] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2017] [Accepted: 02/09/2018] [Indexed: 01/10/2023] Open
Abstract
BACKGROUND Dengue is listed as a neglected tropical disease by the Center for Disease Control and Preservation, as there are insufficient integrated surveillance strategies, no effective treatment, and limited licensed vaccines. Consisting of four genetically distinct serotypes, dengue virus (DENV) causes serious life-threatening infections due to its complexity. Antibody-dependent enhancement by pre-existing cross-reactive as well as homotypic antibodies further worsens the clinical symptoms of dengue. Thus, a vaccine conferring simultaneous and durable immunity to each of the four DENV serotypes is essential to restrict its escalation. In deeply affected resource-limited countries, oral vaccination using food-grade organisms is considered to be a beneficial approach in terms of costs, patient comfort, and simple logistics for mass immunization. The current study used a mouse model to explore the immunogenicity of an oral dengue vaccine candidate prepared using whole recombinant yeast cells (WC) and cell-free extracts (CFE) from cells expressing recombinant Escherichia coli heat-labile toxin protein B-subunit (LTB) fused to the consensus dengue envelope domain III (scEDIII). Mice were treated orally with recombinant WC and CFE vaccines in 2-week intervals for 4 weeks and changes in systemic and mucosal immune responses were monitored. RESULTS Both WC and CFE dosage applications of LTB-scEDIII stimulated a systemic humoral immune response in the form of dengue-specific serum IgG as well as mucosal immune response in the form of secretory sIgA. Antigen-specific B cell responses in isolated lymphoid cells from the spleen and Peyer's patches further indicated an elevated mucosal immune response. Cellular immune response estimated through lymphocyte proliferation assay indicated higher levels in CFE than WC dosage. Furthermore, sera obtained after both oral administrations successfully neutralized DENV-1, whereas CFE formulation only neutralized DENV-2 serotype, two representative serotypes which cause severe dengue infection. Sera from mice that were fed CFE preparations demonstrated markedly higher neutralizing titers compared to those from WC-fed mice. However, WC feeding elicited strong immune responses, which were similar to the levels induced by CFE feeding after intraperitoneal booster with purified scEDIII antigen. CONCLUSIONS CFE preparations of LTB-scEDIII produced strong immunogenicity with low processing requirements, signifying that this fusion protein shows promise as a potent oral vaccine candidate against dengue viral infection.
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Affiliation(s)
- Jyotiranjan Bal
- Department of Molecular Biology, Department of Bioactive Material Sciences, Institute for Molecular Biology and Genetics, Chonbuk National University, Jeonju, Jeollabuk-do, 54896, Republic of Korea
| | - Nguyen Ngoc Luong
- Department of Biology, College of Sciences, Hue University, Hue, Vietnam
| | - Jisang Park
- Department of Molecular Biology, Department of Bioactive Material Sciences, Institute for Molecular Biology and Genetics, Chonbuk National University, Jeonju, Jeollabuk-do, 54896, Republic of Korea
| | - Ki-Duk Song
- Department of Animal Biotechnology, The Animal Molecular Genetics and Breeding Center, Chonbuk National University, Jeonju, Jeollabuk-do, 54896, Republic of Korea
| | - Yong-Suk Jang
- Department of Molecular Biology, Department of Bioactive Material Sciences, Institute for Molecular Biology and Genetics, Chonbuk National University, Jeonju, Jeollabuk-do, 54896, Republic of Korea
| | - Dae-Hyuk Kim
- Department of Molecular Biology, Department of Bioactive Material Sciences, Institute for Molecular Biology and Genetics, Chonbuk National University, Jeonju, Jeollabuk-do, 54896, Republic of Korea.
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Sherman A, Biswas M, Herzog RW. Innovative Approaches for Immune Tolerance to Factor VIII in the Treatment of Hemophilia A. Front Immunol 2017; 8:1604. [PMID: 29225598 PMCID: PMC5705551 DOI: 10.3389/fimmu.2017.01604] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2017] [Accepted: 11/07/2017] [Indexed: 01/19/2023] Open
Abstract
Hemophilia A (coagulation factor VIII deficiency) is a debilitating genetic disorder that is primarily treated with intravenous replacement therapy. Despite a variety of factor VIII protein formulations available, the risk of developing anti-dug antibodies (“inhibitors”) remains. Overall, 20–30% of patients with severe disease develop inhibitors. Current clinical immune tolerance induction protocols to eliminate inhibitors are not effective in all patients, and there are no prophylactic protocols to prevent the immune response. New experimental therapies, such as gene and cell therapies, show promising results in pre-clinical studies in animal models of hemophilia. Examples include hepatic gene transfer with viral vectors, genetically engineered regulatory T cells (Treg), in vivo Treg induction using immune modulatory drugs, and maternal antigen transfer. Furthermore, an oral tolerance protocol is being developed based on transgenic lettuce plants, which suppressed inhibitor formation in hemophilic mice and dogs. Hopefully, some of these innovative approaches will reduce the risk of and/or more effectively eliminate inhibitor formation in future treatment of hemophilia A.
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Affiliation(s)
- Alexandra Sherman
- Department of Pediatrics, University of Florida, Gainesville, FL, United States
| | - Moanaro Biswas
- Department of Pediatrics, University of Florida, Gainesville, FL, United States
| | - Roland W Herzog
- Department of Pediatrics, University of Florida, Gainesville, FL, United States
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Genetic manipulations in crops: Challenges and opportunities. Genomics 2017; 109:494-505. [PMID: 28778540 DOI: 10.1016/j.ygeno.2017.07.007] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2017] [Revised: 07/21/2017] [Accepted: 07/25/2017] [Indexed: 01/01/2023]
Abstract
An alarming increase in the human population necessitates doubling the world food production in the next few decades. Although a number of possible biotechnological measures are under consideration, central to these efforts is the development of transgenic crops to produce more food, and the traits with which plants could better adapt to adverse environmental conditions in a changing climate. The emergence of new tools for the introduction of foreign genes into plants has increased both our knowledge and the capacity to develop transgenic plants. In addition, a better understanding of genetic modifications has allowed us to study the impact that genetically modified crop plants may have on the environment. This article discusses different techniques routinely used to carry out genetic modifications in plants while highlighting challenges with them, which future research must address to increase acceptance of GM crops for meeting food security challenges effectively.
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Dong Y, Li J, Yao N, Wang D, Liu X, Wang N, Li X, Wang F, Li H, Jiang C. Seed-specific expression and analysis of recombinant anti-HER2 single-chain variable fragment (scFv-Fc) in Arabidopsis thaliana. Protein Expr Purif 2017; 133:187-192. [PMID: 28286176 DOI: 10.1016/j.pep.2017.03.009] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/12/2016] [Revised: 11/20/2016] [Accepted: 03/08/2017] [Indexed: 12/11/2022]
Abstract
Antibodies to human epidermal growth factor receptor 2 (HER2) are a key element of breast cancer therapy; however, they are expensive to produce and their availability is limited. A seed-specific expression system can be used to produce recombinant proteins. We report a seed-specific expression system for the manufacture of anti-HER2 ScFv-Fc in Arabidopsis thaliana, driven by the Phaseolus vulgaris β-phaseolin promoter. Recombinant anti-HER2 ScFv-Fc was successfully and specifically expressed in seeds, and identified by protein analysis. The highest protein accumulation level, with a maximum of 1.1% of total soluble protein, was observed in mature seeds. We also demonstrated the anti-tumor potency of the plant-derived antibody against SK-BR-3 cells. These results suggest that seed-expression systems could contribute to the manufacture of commercial antibodies such as anti-HER2 ScFv-Fc.
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Affiliation(s)
- Yuanyuan Dong
- Ministry of Education Engineering Research Center of Bioreactor and Pharmaceutical Development, Jilin Agricultural University, Changchun 130118, China
| | - Jian Li
- Ministry of Education Engineering Research Center of Bioreactor and Pharmaceutical Development, Jilin Agricultural University, Changchun 130118, China
| | - Na Yao
- Ministry of Education Engineering Research Center of Bioreactor and Pharmaceutical Development, Jilin Agricultural University, Changchun 130118, China
| | - Dezhong Wang
- Ministry of Education Engineering Research Center of Bioreactor and Pharmaceutical Development, Jilin Agricultural University, Changchun 130118, China
| | - Xiuming Liu
- Ministry of Education Engineering Research Center of Bioreactor and Pharmaceutical Development, Jilin Agricultural University, Changchun 130118, China
| | - Nan Wang
- Ministry of Education Engineering Research Center of Bioreactor and Pharmaceutical Development, Jilin Agricultural University, Changchun 130118, China
| | - Xiaowei Li
- Ministry of Education Engineering Research Center of Bioreactor and Pharmaceutical Development, Jilin Agricultural University, Changchun 130118, China
| | - Fawei Wang
- Ministry of Education Engineering Research Center of Bioreactor and Pharmaceutical Development, Jilin Agricultural University, Changchun 130118, China
| | - Haiyan Li
- Ministry of Education Engineering Research Center of Bioreactor and Pharmaceutical Development, Jilin Agricultural University, Changchun 130118, China.
| | - Chao Jiang
- Ministry of Education Engineering Research Center of Bioreactor and Pharmaceutical Development, Jilin Agricultural University, Changchun 130118, China.
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Gerasymenko IM, Sheludko YV, Klebanovych AA, Rudas VA, Shakhovsky AM, Klein TM, Kuchuk NV. Comparison of effectiveness of 5'-regulatory sequences in transplastomic tobacco chloroplasts. Transgenic Res 2017; 26:65-75. [PMID: 27565642 DOI: 10.1007/s11248-016-9980-2] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2016] [Accepted: 08/18/2016] [Indexed: 11/30/2022]
Abstract
The development of tools which ensure the desired level of transgene expression in plastids is a prerequisite for the effective utilization of these plant organelles for the deployment of bioactive proteins. High-level accumulation of target proteins is considered as a positive feature of transplastomic plants, but excessive accumulation of foreign proteins may have deleterious effects on host plants. On the other hand, expression at low levels can result in ineffective phenotypes. We compared the effectiveness of different 5'-regulatory sequences in driving the expression of a reporter gene, β-glucuronidase (uidA), in tobacco chloroplasts. To achieve varying expression levels, we have chosen heterologous 5'-regulatory sequences which either differ significantly from their homologous counterparts or depend on specific nuclear encoded factors. The Medicago truncatula psbA promoter/5'-UTR supported the highest levels of protein accumulation, surpassing the other tested sequences by two to three orders of magnitude. The heterologous regulatory sequence of Phaseolus vulgaris rbcL gene was as efficient in tobacco chloroplasts as the corresponding homologous promoter/5'-UTR. The Arabidopsis thaliana ndhF promoter/5'-UTR supported as high reporter activity levels as the rbcL 5'-sequences, whereas the effectiveness of A. thaliana psbN promoter/5'-UTR was three fold lower. The characterized regulatory sequences can be utilized to establish transplastomic lines with desirable levels of target protein accumulation. The ability to control transgene expression should be useful for achieving appropriate levels of protein accumulation and thereby avoid their negative impacts on host plant physiology.
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Affiliation(s)
- I M Gerasymenko
- Institute of Cell Biology and Genetic Engineering, National Academy of Sciences of Ukraine, Zabolotnoho Str. 148, 03143, Kiev, Ukraine.
| | - Y V Sheludko
- Institute of Cell Biology and Genetic Engineering, National Academy of Sciences of Ukraine, Zabolotnoho Str. 148, 03143, Kiev, Ukraine
| | - A A Klebanovych
- Institute of Cell Biology and Genetic Engineering, National Academy of Sciences of Ukraine, Zabolotnoho Str. 148, 03143, Kiev, Ukraine
| | - V A Rudas
- Institute of Cell Biology and Genetic Engineering, National Academy of Sciences of Ukraine, Zabolotnoho Str. 148, 03143, Kiev, Ukraine
| | - A M Shakhovsky
- Institute of Cell Biology and Genetic Engineering, National Academy of Sciences of Ukraine, Zabolotnoho Str. 148, 03143, Kiev, Ukraine
| | - T M Klein
- DuPont Pioneer AgBiotech, DuPont Experimental Station, Wilmington, DE, USA
| | - N V Kuchuk
- Institute of Cell Biology and Genetic Engineering, National Academy of Sciences of Ukraine, Zabolotnoho Str. 148, 03143, Kiev, Ukraine
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Daniell H, Chan HT, Pasoreck EK. Vaccination via Chloroplast Genetics: Affordable Protein Drugs for the Prevention and Treatment of Inherited or Infectious Human Diseases. Annu Rev Genet 2016; 50:595-618. [PMID: 27893966 PMCID: PMC5496655 DOI: 10.1146/annurev-genet-120215-035349] [Citation(s) in RCA: 50] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Plastid-made biopharmaceuticals treat major metabolic or genetic disorders, including Alzheimer's, diabetes, hypertension, hemophilia, and retinopathy. Booster vaccines made in chloroplasts prevent global infectious diseases, such as tuberculosis, malaria, cholera, and polio, and biological threats, such as anthrax and plague. Recent advances in this field include commercial-scale production of human therapeutic proteins in FDA-approved cGMP facilities, development of tags to deliver protein drugs to targeted human cells or tissues, methods to deliver precise doses, and long-term stability of protein drugs at ambient temperature, maintaining their efficacy. Codon optimization utilizing valuable information from sequenced chloroplast genomes enhanced expression of eukaryotic human or viral genes in chloroplasts and offered unique insights into translation in chloroplasts. Support from major biopharmaceutical companies, development of hydroponic production systems, and evaluation by regulatory agencies, including the CDC, FDA, and USDA, augur well for advancing this novel concept to the clinic and revolutionizing affordable healthcare.
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Affiliation(s)
- Henry Daniell
- Department of Biochemistry, School of Dental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104;
| | - Hui-Ting Chan
- Department of Biochemistry, School of Dental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104;
| | - Elise K Pasoreck
- Department of Biochemistry, School of Dental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104;
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Ahmad N, Michoux F, Lössl AG, Nixon PJ. Challenges and perspectives in commercializing plastid transformation technology. JOURNAL OF EXPERIMENTAL BOTANY 2016; 67:5945-5960. [PMID: 27697788 DOI: 10.1093/jxb/erw360] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/29/2023]
Abstract
Plastid transformation has emerged as an alternative platform to generate transgenic plants. Attractive features of this technology include specific integration of transgenes-either individually or as operons-into the plastid genome through homologous recombination, the potential for high-level protein expression, and transgene containment because of the maternal inheritance of plastids. Several issues associated with nuclear transformation such as gene silencing, variable gene expression due to the Mendelian laws of inheritance, and epigenetic regulation have not been observed in the plastid genome. Plastid transformation has been successfully used for the production of therapeutics, vaccines, antigens, and commercial enzymes, and for engineering various agronomic traits including resistance to biotic and abiotic stresses. However, these demonstrations have usually focused on model systems such as tobacco, and the technology per se has not yet reached the market. Technical factors limiting this technology include the lack of efficient protocols for the transformation of cereals, poor transgene expression in non-green plastids, a limited number of selection markers, and the lengthy procedures required to recover fully segregated plants. This article discusses the technology of transforming the plastid genome, the positive and negative features compared with nuclear transformation, and the current challenges that need to be addressed for successful commercialization.
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Affiliation(s)
- Niaz Ahmad
- Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering, Jhang Road, Faisalabad, Pakistan
| | - Franck Michoux
- Alkion Biopharma SAS, 4 rue Pierre Fontaine, 91058 Evry, France
| | - Andreas G Lössl
- Department of Applied Plant Sciences and Plant Biotechnology, University of Natural Resources and Applied Life Sciences (BOKU), Vienna, Austria
| | - Peter J Nixon
- Department of Life Sciences, Sir Ernst Chain Building-Wolfson Laboratories, Imperial College, South Kensington Campus, London SW7 2AZ, UK
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Olejniczak SA, Łojewska E, Kowalczyk T, Sakowicz T. Chloroplasts: state of research and practical applications of plastome sequencing. PLANTA 2016; 244:517-27. [PMID: 27259501 PMCID: PMC4983300 DOI: 10.1007/s00425-016-2551-1] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/25/2016] [Accepted: 05/29/2016] [Indexed: 05/07/2023]
Abstract
This review presents origins, structure and expression of chloroplast genomes. It also describes their sequencing, analysis and modification, focusing on potential practical uses and biggest challenges of chloroplast genome modification. During the evolution of eukaryotes, cyanobacteria are believed to have merged with host heterotrophic cell. Afterward, most of cyanobacterial genes from cyanobacteria were transferred to cell nucleus or lost in the process of endosymbiosis. As a result of these changes, a primary plastid was established. Nowadays, plastid genome (plastome) is almost always circular, has a size of 100-200 kbp (120-160 in land plants), and harbors 100-120 highly conserved unique genes. Plastids have their own gene expression system, which is similar to one of their cyanobacterial ancestors. Two different polymerases, plastid-derived PEP and nucleus-derived NEP, participate in transcription. Translation is similar to the one observed in cyanobacteria, but it also utilizes protein translation factors and positive regulatory mRNA elements absent from bacteria. Plastoms play an important role in genetic transformation. Transgenes are introduced into them either via gene gun (in undamaged tissues) or polyethylene glycol treatment (when protoplasts are targeted). Antibiotic resistance markers are the most common tool used for selection of transformed plants. In recent years, plastome transformation emerged as a promising alternative to nuclear transformation because of (1) high yield of target protein, (2) removing the risk of outcrossing with weeds, (3) lack of silencing mechanisms, and (4) ability to engineer the entire metabolic pathways rather than single gene traits. Currently, the main directions of such research regard: developing efficient enzyme, vaccine antigen, and biopharmaceutical protein production methods in plant cells and improving crops by increasing their resistance to a wide array of biotic and abiotic stresses. Because of that, the detailed knowledge of plastome structure and mechanism of functioning started to play a major role.
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Affiliation(s)
- Szymon Adam Olejniczak
- Department of Genetics and Plant Molecular Biology and Biotechnology, The University of Lodz, Banacha Street 12/16, 90-237, Lodz, Poland.
| | - Ewelina Łojewska
- Department of Genetics and Plant Molecular Biology and Biotechnology, The University of Lodz, Banacha Street 12/16, 90-237, Lodz, Poland
| | - Tomasz Kowalczyk
- Department of Genetics and Plant Molecular Biology and Biotechnology, The University of Lodz, Banacha Street 12/16, 90-237, Lodz, Poland
| | - Tomasz Sakowicz
- Department of Genetics and Plant Molecular Biology and Biotechnology, The University of Lodz, Banacha Street 12/16, 90-237, Lodz, Poland
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Daniell H, Lin CS, Yu M, Chang WJ. Chloroplast genomes: diversity, evolution, and applications in genetic engineering. Genome Biol 2016; 17:134. [PMID: 27339192 PMCID: PMC4918201 DOI: 10.1186/s13059-016-1004-2] [Citation(s) in RCA: 840] [Impact Index Per Article: 93.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
Chloroplasts play a crucial role in sustaining life on earth. The availability of over 800 sequenced chloroplast genomes from a variety of land plants has enhanced our understanding of chloroplast biology, intracellular gene transfer, conservation, diversity, and the genetic basis by which chloroplast transgenes can be engineered to enhance plant agronomic traits or to produce high-value agricultural or biomedical products. In this review, we discuss the impact of chloroplast genome sequences on understanding the origins of economically important cultivated species and changes that have taken place during domestication. We also discuss the potential biotechnological applications of chloroplast genomes.
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Affiliation(s)
- Henry Daniell
- Department of Biochemistry, School of Dental Medicine, University of Pennsylvania, South 40th St, Philadelphia, PA, 19104-6030, USA.
| | - Choun-Sea Lin
- Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan
| | - Ming Yu
- Department of Biochemistry, School of Dental Medicine, University of Pennsylvania, South 40th St, Philadelphia, PA, 19104-6030, USA
| | - Wan-Jung Chang
- Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan
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Kwon KC, Daniell H. Oral Delivery of Protein Drugs Bioencapsulated in Plant Cells. Mol Ther 2016; 24:1342-50. [PMID: 27378236 PMCID: PMC5023392 DOI: 10.1038/mt.2016.115] [Citation(s) in RCA: 56] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2016] [Accepted: 05/29/2016] [Indexed: 12/11/2022] Open
Abstract
Plants cells are now approved by the FDA for cost-effective production of protein drugs (PDs) in large-scale current Good Manufacturing Practice (cGMP) hydroponic growth facilities. In lyophilized plant cells, PDs are stable at ambient temperature for several years, maintaining their folding and efficacy. Upon oral delivery, PDs bioencapsulated in plant cells are protected in the stomach from acids and enzymes but are subsequently released into the gut lumen by microbes that digest the plant cell wall. The large mucosal area of the human intestine offers an ideal system for oral drug delivery. When tags (receptor-binding proteins or cell-penetrating peptides) are fused to PDs, they efficiently cross the intestinal epithelium and are delivered to the circulatory or immune system. Unique tags to deliver PDs to human immune or nonimmune cells have been developed recently. After crossing the epithelium, ubiquitous proteases cleave off tags at engineered sites. PDs are also delivered to the brain or retina by crossing the blood–brain or retinal barriers. This review highlights recent advances in PD delivery to treat Alzheimer's disease, diabetes, hypertension, Gaucher's or ocular diseases, as well as the development of affordable drugs by eliminating prohibitively expensive purification, cold chain and sterile delivery.
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Affiliation(s)
- Kwang-Chul Kwon
- Department of Biochemistry, School of Dental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA
| | - Henry Daniell
- Department of Biochemistry, School of Dental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA
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Abstract
Plant-based vaccine technologies involve the integration of the desired genes encoding the antigen protein for specific disease into the genome of plant tissues by various methods. Agrobacterium-mediated gene transfer and transformation via genetically modified plant virus are the common methods that have been used to produce effective vaccines. Nevertheless, with the advancement of science and technology, new approaches have been developed to increase the efficiency of former methods such as biolistic, electroporation, agroinfiltration, sonication, and polyethylene glycol treatment. Even though plant-based vaccines provide many benefits to the vaccine industry, there are still challenges that limit the rate of successful production of these third-generation vaccines. Even with all the limitations, continuous efforts are still ongoing in order to produce efficient vaccine for many human and animals related diseases owing to its great potentials. This paper reviews the existing conventional methods as well as the development efforts by researchers in order to improve the production of plant-based vaccines. Several challenges encountered during and after the production process were also discussed.
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Xiao Y, Kwon KC, Hoffman BE, Kamesh A, Jones NT, Herzog RW, Daniell H. Low cost delivery of proteins bioencapsulated in plant cells to human non-immune or immune modulatory cells. Biomaterials 2016; 80:68-79. [PMID: 26706477 PMCID: PMC4706487 DOI: 10.1016/j.biomaterials.2015.11.051] [Citation(s) in RCA: 39] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2015] [Revised: 11/17/2015] [Accepted: 11/29/2015] [Indexed: 02/06/2023]
Abstract
Targeted oral delivery of GFP fused with a GM1 receptor binding protein (CTB) or human cell penetrating peptide (PTD) or dendritic cell peptide (DCpep) was investigated. Presence of GFP(+) intact plant cells between villi of ileum confirm their protection in the digestive system from acids/enzymes. Efficient delivery of GFP to gut-epithelial cells by PTD or CTB and to M cells by all these fusion tags confirm uptake of GFP in the small intestine. PTD fusion delivered GFP more efficiently to most tissues or organs than the other two tags. GFP was efficiently delivered to the liver by all fusion tags, likely through the gut-liver axis. In confocal imaging studies of human cell lines using purified GFP fused with different tags, GFP signal of DCpep-GFP was only detected within dendritic cells. PTD-GFP was only detected within kidney or pancreatic cells but not in immune modulatory cells (macrophages, dendritic, T, B, or mast cells). In contrast, CTB-GFP was detected in all tested cell types, confirming ubiquitous presence of GM1 receptors. Such low-cost oral delivery of protein drugs to sera, immune system or non-immune cells should dramatically lower their cost by elimination of prohibitively expensive fermentation, protein purification cold storage/transportation and increase patient compliance.
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Affiliation(s)
- Yuhong Xiao
- Department of Biochemistry, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA, USA
| | - Kwang-Chul Kwon
- Department of Biochemistry, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA, USA
| | - Brad E Hoffman
- Department of Pediatrics, College of Medicine, University of Florida, Gainesville, FL, USA
| | - Aditya Kamesh
- Department of Biochemistry, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA, USA
| | - Noah T Jones
- Department of Pediatrics, College of Medicine, University of Florida, Gainesville, FL, USA
| | - Roland W Herzog
- Department of Pediatrics, College of Medicine, University of Florida, Gainesville, FL, USA
| | - Henry Daniell
- Department of Biochemistry, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA, USA.
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Doron L, Segal N, Shapira M. Transgene Expression in Microalgae-From Tools to Applications. FRONTIERS IN PLANT SCIENCE 2016; 7:505. [PMID: 27148328 PMCID: PMC4840263 DOI: 10.3389/fpls.2016.00505] [Citation(s) in RCA: 92] [Impact Index Per Article: 10.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/12/2015] [Accepted: 03/29/2016] [Indexed: 05/17/2023]
Abstract
Microalgae comprise a biodiverse group of photosynthetic organisms that reside in water sources and sediments. The green microalgae Chlamydomonas reinhardtii was adopted as a useful model organism for studying various physiological systems. Its ability to grow under both photosynthetic and heterotrophic conditions allows efficient growth of non-photosynthetic mutants, making Chlamydomonas a useful genetic tool to study photosynthesis. In addition, this green alga can grow as haploid or diploid cells, similar to yeast, providing a powerful genetic system. As a result, easy and efficient transformation systems have been developed for Chlamydomonas, targeting both the chloroplast and nuclear genomes. Since microalgae comprise a rich repertoire of species that offer variable advantages for biotech and biomed industries, gene transfer technologies were further developed for many microalgae to allow for the expression of foreign proteins of interest. Expressing foreign genes in the chloroplast enables the targeting of foreign DNA to specific sites by homologous recombination. Chloroplast transformation also allows for the introduction of genes encoding several enzymes from a complex pathway, possibly as an operon. Expressing foreign proteins in the chloroplast can also be achieved by introducing the target gene into the nuclear genome, with the protein product bearing a targeting signal that directs import of the transgene-product into the chloroplast, like other endogenous chloroplast proteins. Integration of foreign genes into the nuclear genome is mostly random, resulting in large variability between different clones, such that extensive screening is required. The use of different selection modalities is also described, with special emphasis on the use of herbicides and metabolic markers which are considered to be friendly to the environment, as compared to drug-resistance genes that are commonly used. Finally, despite the development of a wide range of transformation tools and approaches, expression of foreign genes in microalgae suffers from low efficiency. Thus, novel tools have appeared in recent years to deal with this problem. Finally, while C. reinhardtii was traditionally used as a model organism for the development of transformation systems and their subsequent improvement, similar technologies can be adapted for other microalgae that may have higher biotechnological value.
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Matoba N. N-Glycosylation of Cholera Toxin B Subunit: Serendipity for Novel Plant-Made Vaccines? FRONTIERS IN PLANT SCIENCE 2015; 6:1132. [PMID: 26732492 PMCID: PMC4686596 DOI: 10.3389/fpls.2015.01132] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/16/2015] [Accepted: 11/29/2015] [Indexed: 05/28/2023]
Abstract
The non-toxic B subunit of cholera toxin (CTB) has attracted considerable interests from vaccinologists due to strong mucosal immunomodulatory effects and potential utility as a vaccine scaffold for heterologous antigens. Along with other conventional protein expression systems, various plant species have been used as production hosts for CTB and its fusion proteins. However, it has recently become clear that the protein is N-glycosylated within the endoplasmic reticulum of plant cells-a eukaryotic post-translational modification that is not present in native CTB. While functionally active aglycosylated variants have been successfully engineered to circumvent potential safety and regulatory issues related to glycosylation, this modification may actually provide advantageous characteristics to the protein as a vaccine platform. Based on data from our recent studies, I discuss the unique features of N-glycosylated CTB produced in plants for the development of novel vaccines.
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Waheed MT, Ismail H, Gottschamel J, Mirza B, Lössl AG. Plastids: The Green Frontiers for Vaccine Production. FRONTIERS IN PLANT SCIENCE 2015; 6:1005. [PMID: 26635832 PMCID: PMC4646963 DOI: 10.3389/fpls.2015.01005] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/17/2015] [Accepted: 10/30/2015] [Indexed: 05/10/2023]
Abstract
Infectious diseases pose an increasing risk to health, especially in developing countries. Vaccines are available to either cure or prevent many of these diseases. However, there are certain limitations related to these vaccines, mainly the costs, which make these vaccines mostly unaffordable for people in resource poor countries. These costs are mainly related to production and purification of the products manufactured from fermenter-based systems. Plastid biotechnology has become an attractive platform to produce biopharmaceuticals in large amounts and cost-effectively. This is mainly due to high copy number of plastids DNA in mature chloroplasts, a characteristic particularly important for vaccine production in large amounts. An additional advantage lies in the maternal inheritance of plastids in most plant species, which addresses the regulatory concerns related to transgenic plants. These and many other aspects of plastids will be discussed in the present review, especially those that particularly make these green biofactories an attractive platform for vaccine production. A summary of recent vaccine antigens against different human diseases expressed in plastids will also be presented.
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Affiliation(s)
- Mohammad T. Waheed
- Department of Biochemistry, Faculty of Biological Sciences, Quaid-i-Azam UniversityIslamabad, Pakistan
| | - Hammad Ismail
- Department of Biochemistry, Faculty of Biological Sciences, Quaid-i-Azam UniversityIslamabad, Pakistan
| | | | - Bushra Mirza
- Department of Biochemistry, Faculty of Biological Sciences, Quaid-i-Azam UniversityIslamabad, Pakistan
| | - Andreas G. Lössl
- Department of Applied Plant Sciences and Plant Biotechnology, University of Natural Resources and Applied Life SciencesTulln an der Donau, Austria
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Su J, Zhu L, Sherman A, Wang X, Lin S, Kamesh A, Norikane JH, Streatfield SJ, Herzog RW, Daniell H. Low cost industrial production of coagulation factor IX bioencapsulated in lettuce cells for oral tolerance induction in hemophilia B. Biomaterials 2015; 70:84-93. [PMID: 26302233 PMCID: PMC4562874 DOI: 10.1016/j.biomaterials.2015.08.004] [Citation(s) in RCA: 99] [Impact Index Per Article: 9.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/22/2015] [Revised: 08/01/2015] [Accepted: 08/04/2015] [Indexed: 01/13/2023]
Abstract
Antibodies (inhibitors) developed by hemophilia B patients against coagulation factor IX (FIX) are challenging to eliminate because of anaphylaxis or nephrotic syndrome after continued infusion. To address this urgent unmet medical need, FIX fused with a transmucosal carrier (CTB) was produced in a commercial lettuce (Simpson Elite) cultivar using species specific chloroplast vectors regulated by endogenous psbA sequences. CTB-FIX (∼1 mg/g) in lyophilized cells was stable with proper folding, disulfide bonds and pentamer assembly when stored ∼2 years at ambient temperature. Feeding lettuce cells to hemophilia B mice delivered CTB-FIX efficiently to the gut immune system, induced LAP(+) regulatory T cells and suppressed inhibitor/IgE formation and anaphylaxis against FIX. Lyophilized cells enabled 10-fold dose escalation studies and successful induction of oral tolerance was observed in all tested doses. Induction of tolerance in such a broad dose range should enable oral delivery to patients of different age groups and diverse genetic background. Using Fraunhofer cGMP hydroponic system, ∼870 kg fresh or 43.5 kg dry weight can be harvested per 1000 ft(2) per annum yielding 24,000-36,000 doses for 20-kg pediatric patients, enabling first commercial development of an oral drug, addressing prohibitively expensive purification, cold storage/transportation and short shelf life of current protein drugs.
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Affiliation(s)
- Jin Su
- Department of Biochemistry, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA, USA
| | - Liqing Zhu
- Department of Pediatrics, College of Medicine, University of Florida, Gainesville, FL, USA
| | - Alexandra Sherman
- Department of Pediatrics, College of Medicine, University of Florida, Gainesville, FL, USA
| | - Xiaomei Wang
- Department of Pediatrics, College of Medicine, University of Florida, Gainesville, FL, USA
| | - Shina Lin
- Department of Biochemistry, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA, USA
| | - Aditya Kamesh
- Department of Biochemistry, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA, USA
| | - Joey H Norikane
- Fraunhofer USA, Center for Molecular Biotechnology, Newark, DE, USA
| | | | - Roland W Herzog
- Department of Pediatrics, College of Medicine, University of Florida, Gainesville, FL, USA.
| | - Henry Daniell
- Department of Biochemistry, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA, USA.
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Daniell H, Streatfield SJ, Rybicki EP. Advances in molecular farming: key technologies, scaled up production and lead targets. PLANT BIOTECHNOLOGY JOURNAL 2015; 13:1011-2. [PMID: 26387508 PMCID: PMC4769792 DOI: 10.1111/pbi.12478] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/05/2023]
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Su J, Sherman A, Doerfler PA, Byrne BJ, Herzog RW, Daniell H. Oral delivery of Acid Alpha Glucosidase epitopes expressed in plant chloroplasts suppresses antibody formation in treatment of Pompe mice. PLANT BIOTECHNOLOGY JOURNAL 2015; 13:1023-32. [PMID: 26053072 PMCID: PMC4578979 DOI: 10.1111/pbi.12413] [Citation(s) in RCA: 45] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/23/2015] [Revised: 04/25/2015] [Accepted: 05/11/2015] [Indexed: 05/20/2023]
Abstract
Deficiency of acid alpha glucosidase (GAA) causes Pompe disease in which the patients systemically accumulate lysosomal glycogen in muscles and nervous systems, often resulting in infant mortality. Although enzyme replacement therapy (ERT) is effective in treating patients with Pompe disease, formation of antibodies against rhGAA complicates treatment. In this report, we investigated induction of tolerance by oral administration of GAA expressed in chloroplasts. Because full-length GAA could not be expressed, N-terminal 410-amino acids of GAA (as determined by T-cell epitope mapping) were fused with the transmucosal carrier CTB. Tobacco transplastomic lines expressing CTB-GAA were generated through site-specific integration of transgenes into the chloroplast genome. Homoplasmic lines were confirmed by Southern blot analysis. Despite low-level expression of CTB-GAA in chloroplasts, yellow or albino phenotype of transplastomic lines was observed due to binding of GAA to a chloroplast protein that has homology to mannose-6 phosphate receptor. Oral administration of the plant-made CTB-GAA fusion protein even at 330-fold lower dose (1.5 μg) significantly suppressed immunoglobulin formation against GAA in Pompe mice injected with 500 μg rhGAA per dose, with several-fold lower titre of GAA-specific IgG1 and IgG2a. Lyophilization increased CTB-GAA concentration by 30-fold (up to 190 μg per g of freeze-dried leaf material), facilitating long-term storage at room temperature and higher dosage in future investigations. This study provides the first evidence that oral delivery of plant cells is effective in reducing antibody responses in ERT for lysosomal storage disorders facilitating further advances in clinical investigations using plant cell culture system or in vitro propagation.
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Affiliation(s)
- Jin Su
- Department of Biochemistry, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA, USA
| | - Alexandra Sherman
- Department of Pediatrics, College of Medicine, University of Florida, Gainesville, FL, USA
| | - Phillip A. Doerfler
- Department of Pediatrics, College of Medicine, University of Florida, Gainesville, FL, USA
| | - Barry J. Byrne
- Department of Pediatrics, College of Medicine, University of Florida, Gainesville, FL, USA
| | - Roland W. Herzog
- Department of Pediatrics, College of Medicine, University of Florida, Gainesville, FL, USA
| | - Henry Daniell
- Department of Biochemistry, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA, USA
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Chan HT, Daniell H. Plant-made oral vaccines against human infectious diseases-Are we there yet? PLANT BIOTECHNOLOGY JOURNAL 2015; 13:1056-70. [PMID: 26387509 PMCID: PMC4769796 DOI: 10.1111/pbi.12471] [Citation(s) in RCA: 92] [Impact Index Per Article: 9.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/12/2015] [Revised: 08/12/2015] [Accepted: 08/14/2015] [Indexed: 05/13/2023]
Abstract
Although the plant-made vaccine field started three decades ago with the promise of developing low-cost vaccines to prevent infectious disease outbreaks and epidemics around the globe, this goal has not yet been achieved. Plants offer several major advantages in vaccine generation, including low-cost production by eliminating expensive fermentation and purification systems, sterile delivery and cold storage/transportation. Most importantly, oral vaccination using plant-made antigens confers both mucosal (IgA) and systemic (IgG) immunity. Studies in the past 5 years have made significant progress in expressing vaccine antigens in edible leaves (especially lettuce), processing leaves or seeds through lyophilization and achieving antigen stability and efficacy after prolonged storage at ambient temperatures. Bioencapsulation of antigens in plant cells protects them from the digestive system; the fusion of antigens to transmucosal carriers enhances efficiency of their delivery to the immune system and facilitates successful development of plant vaccines as oral boosters. However, the lack of oral priming approaches diminishes these advantages because purified antigens, cold storage/transportation and limited shelf life are still major challenges for priming with adjuvants and for antigen delivery by injection. Yet another challenge is the risk of inducing tolerance without priming the host immune system. Therefore, mechanistic aspects of these two opposing processes (antibody production or suppression) are discussed in this review. In addition, we summarize recent progress made in oral delivery of vaccine antigens expressed in plant cells via the chloroplast or nuclear genomes and potential challenges in achieving immunity against infectious diseases using cold-chain-free vaccine delivery approaches.
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Affiliation(s)
| | - Henry Daniell
- Correspondence (Tel 215 746 2563; fax 215 898 3695; )
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