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1.
Plant Cell Rep ; 43(2): 43, 2024 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-38246952

RESUMO

KEY MESSAGE: Characterization of Physcomitrella 3'UTRs across different promoters yields endogenous single and double terminators for usage in molecular pharming. The production of recombinant proteins for health applications accounts for a large share of the biopharmaceutical market. While many drugs are produced in microbial and mammalian systems, plants gain more attention as expression hosts to produce eukaryotic proteins. In particular, the good manufacturing practice (GMP)-compliant moss Physcomitrella (Physcomitrium patens) has outstanding features, such as excellent genetic amenability, reproducible bioreactor cultivation, and humanized protein glycosylation patterns. In this study, we selected and characterized novel terminators for their effects on heterologous gene expression. The Physcomitrella genome contains 53,346 unique 3'UTRs (untranslated regions) of which 7964 transcripts contain at least one intron. Over 91% of 3'UTRs exhibit more than one polyadenylation site, indicating the prevalence of alternative polyadenylation in Physcomitrella. Out of all 3'UTRs, 14 terminator candidates were selected and characterized via transient Dual-Luciferase assays, yielding a collection of endogenous terminators performing equally high as established heterologous terminators CaMV35S, AtHSP90, and NOS. High performing candidates were selected for testing as double terminators which impact reporter levels, dependent on terminator identity and positioning. Testing of 3'UTRs among the different promoters NOS, CaMV35S, and PpActin5 showed an increase of more than 1000-fold between promoters PpActin5 and NOS, whereas terminators increased reporter levels by less than tenfold, demonstrating the stronger effect promoters play as compared to terminators. Among selected terminator attributes, the number of polyadenylation sites as well as polyadenylation signals were found to influence terminator performance the most. Our results improve the biotechnology platform Physcomitrella and further our understanding of how terminators influence gene expression in plants in general.


Assuntos
Briófitas , Bryopsida , Animais , Bryopsida/genética , Regiões 3' não Traduzidas , Agricultura Molecular , Expressão Gênica , Mamíferos
2.
Int J Mol Sci ; 24(12)2023 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-37373192

RESUMO

Immune checkpoint inhibitors (ICIs) are a class of immunotherapy agents capable of alleviating the immunosuppressive effects exerted by tumorigenic cells. The programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) immune checkpoint is one of the most ubiquitous checkpoints utilized by tumorigenic cells for immune evasion by inducing apoptosis and inhibiting the proliferation and cytokine production of T lymphocytes. Currently, the most frequently used ICIs targeting the PD-1/PD-L1 checkpoint include monoclonal antibodies (mAbs) pembrolizumab and nivolumab that bind to PD-1 on T lymphocytes and inhibit interaction with PD-L1 on tumorigenic cells. However, pembrolizumab and nivolumab are costly, and thus their accessibility is limited in low- and middle-income countries (LMICs). Therefore, it is essential to develop novel biomanufacturing platforms capable of reducing the cost of these two therapies. Molecular farming is one such platform utilizing plants for mAb production, and it has been demonstrated to be a rapid, low-cost, and scalable platform that can be potentially implemented in LMICs to diminish the exorbitant prices, ultimately leading to a significant reduction in cancer-related mortalities within these countries.


Assuntos
Antígeno B7-H1 , Nivolumabe , Nivolumabe/farmacologia , Receptor de Morte Celular Programada 1 , Agricultura Molecular , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Imunoterapia
4.
Trends Biotechnol ; 41(9): 1182-1198, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37012119

RESUMO

Many efforts have been put into engineering plants to improve crop yields and stress tolerance and boost the bioproduction of valuable molecules. Yet, our capabilities are still limited due to the lack of well-characterized genetic building blocks and resources for precise manipulation and given the inherently challenging properties of plant tissues. Advancements in plant synthetic biology can overcome these bottlenecks and release the full potential of engineered plants. In this review, we first discuss the recently developed plant synthetic elements from single parts to advanced circuits, software, and hardware tools expediting the engineering cycle. Next, we survey the advancements in plant biotechnology enabled by these recent resources. We conclude the review with outstanding challenges and future directions of plant synthetic biology.


Assuntos
Agricultura Molecular , Biologia Sintética , Engenharia Genética , Plantas/genética , Biotecnologia , Engenharia Metabólica
5.
Planta Med ; 89(10): 1010-1020, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-37072112

RESUMO

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


Assuntos
Doenças Transmissíveis , Agricultura Molecular , Animais , Plantas Geneticamente Modificadas/metabolismo , Biotecnologia/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Doenças Transmissíveis/diagnóstico , Mamíferos/metabolismo
6.
SLAS Technol ; 28(4): 278-291, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-36966988

RESUMO

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


Assuntos
Fator de Crescimento Epidérmico , Hidrogéis , Fator de Crescimento Epidérmico/farmacologia , Agricultura Molecular , Organoides , Ácido Hialurônico/farmacologia , Trifosfato de Adenosina
7.
Vaccine ; 41(13): 2261-2269, 2023 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-36868876

RESUMO

The outbreak of the SARS-CoV-2 global pandemic heightened the pace of vaccine development with various vaccines being approved for human use in a span of 24 months. The SARS-CoV-2 trimeric spike (S) surface glycoprotein, which mediates viral entry by binding to ACE2, is a key target for vaccines and therapeutic antibodies. Plant biopharming is recognized for its scalability, speed, versatility, and low production costs and is an increasingly promising molecular pharming vaccine platform for human health. We developed Nicotiana benthamiana-produced SARS-CoV-2 virus-like particle (VLP) vaccine candidates displaying the S-protein of the Beta (B.1.351) variant of concern (VOC), which triggered cross-reactive neutralising antibodies against Delta (B.1.617.2) and Omicron (B.1.1.529) VOCs. In this study, immunogenicity of the VLPs (5 µg per dose) adjuvanted with three independent adjuvants i.e. oil-in-water based adjuvants SEPIVAC SWETM (Seppic, France) and "AS IS" (Afrigen, South Africa) as well as a slow-release synthetic oligodeoxynucleotide (ODN) adjuvant designated NADA (Disease Control Africa, South Africa) were evaluated in New Zealand white rabbits and resulted in robust neutralising antibody responses after booster vaccination, ranging from 1:5341 to as high as 1:18204. Serum neutralising antibodies elicited by the Beta variant VLP vaccine also showed cross-neutralisation against the Delta and Omicron variants with neutralising titres ranging from 1:1702 and 1:971, respectively. Collectively, these data provide support for the development of a plant-produced VLP based candidate vaccine against SARS-CoV-2 based on circulating variants of concern.


Assuntos
Vacinas contra COVID-19 , COVID-19 , Coelhos , Animais , Humanos , SARS-CoV-2 , Agricultura Molecular , COVID-19/prevenção & controle , Adjuvantes Imunológicos , Anticorpos Neutralizantes , África do Sul , Anticorpos Antivirais , Glicoproteína da Espícula de Coronavírus/genética , Imunogenicidade da Vacina
8.
Int J Mol Sci ; 24(2)2023 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-36675043

RESUMO

Plant viruses have traditionally been studied as pathogens in the context of understanding the molecular and cellular mechanisms of a particular disease affecting crops. In recent years, viruses have emerged as a new alternative for producing biological nanomaterials and chimeric vaccines. Plant viruses were also used to generate highly efficient expression vectors, revolutionizing plant molecular farming (PMF). Several biological products, including recombinant vaccines, monoclonal antibodies, diagnostic reagents, and other pharmaceutical products produced in plants, have passed their clinical trials and are in their market implementation stage. PMF offers opportunities for fast, adaptive, and low-cost technology to meet ever-growing and critical global health needs. In this review, we summarized the advancements in the virus-like particles-based (VLPs-based) nanotechnologies and the role they played in the production of advanced vaccines, drugs, diagnostic bio-nanomaterials, and other bioactive cargos. We also highlighted various applications and advantages plant-produced vaccines have and their relevance for treating human and animal illnesses. Furthermore, we summarized the plant-based biologics that have passed through clinical trials, the unique challenges they faced, and the challenges they will face to qualify, become available, and succeed on the market.


Assuntos
Agricultura Molecular , Vírus de Plantas , Animais , Humanos , Plantas Geneticamente Modificadas/metabolismo , Vacinas Sintéticas , Vírus de Plantas/genética , Anticorpos Monoclonais/metabolismo
9.
Virology ; 578: 7-12, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36434906

RESUMO

Many plant virus-like particles (VLPs) utilized in nanotechnology are 30-nm icosahedrons. To expand the VLP platforms, we produced VLPs of Cytoplasmic type citrus leprosis virus (CiLV-C) in Nicotiana benthamiana. We were interested in CiLV-C because of its unique bacilliform shape (60-70 nm × 110-120 nm). The CiLV-C capsid protein (p29) gene was transferred to the pTRBO expression vector transiently expressed in leaves. Stable VLPs were formed, as confirmed by agarose gel electrophoresis, transmission electron microscopy and size exclusion chromatography. Interestingly, the morphology of the VLPs (15.8 ± 1.3 nm icosahedral particles) differed from that of the native bacilliform particles indicating that the assembly of native virions is influenced by other viral proteins and/or the packaged viral genome. The smaller CiLV-C VLPs will also be useful for structure-function studies to compare with the 30-nm icosahedrons of other VLPs.


Assuntos
Citrus , Vírus de RNA , Rhabdoviridae , Agricultura Molecular , Vírus de RNA/genética , Vírion/genética
11.
Molecules ; 27(24)2022 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-36558107

RESUMO

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


Assuntos
Crocus , Crocus/química , Fazendas , Hidroponia , Agricultura Molecular , Agricultura , Extratos Vegetais/química
12.
Biotechnol Bioeng ; 119(10): 2831-2841, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35822204

RESUMO

Hairy root systems have proven to be a viable alternative for recombinant protein production. For recalcitrant proteins, maximizing the productivity of hairy root cultures is essential. The aim of this study was to optimize a Brassica rapa rapa hairy root process for secretion of alpha- l-iduronidase (IDUA), a biologic of medical value. The process was first optimized with hairy roots expressing eGFP. For the biomass optimization, the highest biomass yields were achieved in modified Gamborg B5 culture medium. For the secretion induction, the optimized secretion media was obtained with additives (1.5 g/l PVP + 1 mg/l 2,4- d + 20.5 g/l KNO3 ) resulting in 3.4 fold eGFP secretion when compared to the non-induced control. These optimized conditions were applied to the IDUA-expressing hairy root clone, confirming that the highest yields of secreted IDUA occurred when using the defined additive combination. The functionality of the IDUA protein, secreted and intracellular, was confirmed with an enzymatic activity assay. A > 150-fold increase of the IDUA activity was observed using an optimized secretion medium, compared with a non-induced medium. We have proven that our B. rapa rapa hairy root system can be harnessed to secrete recalcitrant proteins, illustrating the high potential of hairy roots in plant molecular farming.


Assuntos
Produtos Biológicos , Brassica , Produtos Biológicos/metabolismo , Brassica/genética , Brassica/metabolismo , Agricultura Molecular , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
13.
Methods Mol Biol ; 2480: 49-60, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35616856

RESUMO

The production of recombinant proteins in seed crops has a long history and cereal grains are now one of the platforms in commercial use. Specific advantages include excellent storage properties, a well-developed endomembrane system with a high biosynthetic capacity and well-established cultivation procedures worldwide. However, the production of transgenic cereals is a time-consuming procedure and the lack of efficient transformation systems is still a significant bottleneck. Barley can be transformed at high efficiency but the protocols are genotype-dependent. Wheat is generally more challenging to transform, but considerable progress has been made in enhancing transformation efficiencies and in controlling transgene expression. In this chapter, we describe and discuss standard procedures for generating transgenic barley and wheat for the production of recombinant proteins.


Assuntos
Hordeum , Produtos Agrícolas/genética , Grão Comestível/genética , Hordeum/genética , Agricultura Molecular , Proteínas Recombinantes , Sementes/genética , Triticum/genética
14.
Methods Mol Biol ; 2480: 313-333, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35616870

RESUMO

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


Assuntos
Agricultura Molecular , Drogas Veterinárias , Canadá , Humanos , Plantas Geneticamente Modificadas/genética , Proteínas Recombinantes/genética
15.
Methods Mol Biol ; 2480: 335-342, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35616871

RESUMO

Academic scientists are increasingly engaged in translational research oriented toward bringing products and processes to commercial markets. They need to diligently analyze the intellectual property (IP) rights of others to avoid infringement, and use their own IP strategically. For this it is useful to perform a freedom-to-operate (FTO) analysis which includes searching the prior art and patent databases. This chapter outlines the principles of FTO analysis with a special focus on plant biotechnology.


Assuntos
Propriedade Intelectual , Agricultura Molecular , Biotecnologia , Liberdade , Plantas
16.
Methods Mol Biol ; 2456: 275-286, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35612749

RESUMO

Transient expression of recombinant proteins in plants is being used as a platform for production of therapeutic proteins. Benefits of this system include a reduced cost of drug development, rapid delivery of new products to the market, and an ability to provide safe and efficacious medicines for diseases. Although plant-based production systems offer excellent potential for therapeutic protein production, barriers, such as plant host defense response, exist which negatively impact the yield of product. Here we provide a protocol using tandem mass tags and mass spectrometry-based proteomics to quickly and robustly quantify the change in abundance of host defense proteins produced during the production process. These proteins can then become candidates for genetic manipulation to create host plants with reduced plant defenses capable of producing higher therapeutic protein yields.


Assuntos
Agrobacterium tumefaciens , Agricultura Molecular , Agrobacterium tumefaciens/metabolismo , Agricultura Molecular/métodos , Plantas/genética , Plantas Geneticamente Modificadas/genética , Proteômica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , /metabolismo
17.
Trends Biotechnol ; 40(10): 1248-1260, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35562237

RESUMO

Plant expression platforms are low-cost, scalable, safe, and environmentally friendly systems for the production of recombinant proteins and bioactive metabolites. Rice (Oryza sativa L.) endosperm is an ideal bioreactor for the production and storage of high-value active substances, including pharmaceutical proteins, oral vaccines, vitamins, and nutraceuticals such as flavonoids and carotenoids. Here, we explore the use of molecular farming from producing medicines to developing functional food crops (biofortification). We review recent progress in producing pharmaceutical proteins and bioactive substances in rice endosperm and compare this platform with other plant expression systems. We describe how rice endosperm could be modified to design metabolic pathways and express and store stable products and discuss the factors restricting the commercialization of transgenic rice products and future prospects.


Assuntos
Endosperma , Oryza , Carotenoides , Endosperma/genética , Endosperma/metabolismo , Flavonoides , Regulação da Expressão Gênica de Plantas , Agricultura Molecular , Oryza/genética , Oryza/metabolismo , Preparações Farmacêuticas/metabolismo , Proteínas de Plantas , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Proteínas Recombinantes/metabolismo , Vitaminas/metabolismo
18.
Curr Opin Chem Biol ; 68: 102133, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35378382

RESUMO

Molecular pharming relies on the integration of foreign genes into a plant system for production of the desired recombinant protein. The speed, scalability, and lack of contaminating human pathogens highlights plants as an enticing and feasible system to produce diverse protein-based products, including vaccines, antibodies, and enzymes. However, limitations of expression levels, host defense responses, and production irregularities underscore distinct areas for improvement within the molecular pharming pipeline. Within the past five years, mass spectrometry-based proteomics has begun to address these critical areas and show promise in advancing our understanding of the complex biological systems driving molecular pharming. Further, opportunities to leverage comprehensive proteome profiling have surfaced to meet good manufacturing practice regulations and move biopharmaceuticals derived from plants into mainstream production.


Assuntos
Agricultura Molecular , Proteômica , Humanos , Espectrometria de Massas , Plantas , Proteínas Recombinantes
19.
Nat Plants ; 8(3): 191, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35318446
20.
J Agric Food Chem ; 70(7): 2108-2116, 2022 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-35139640

RESUMO

The demand of functional food is increasing for improving human health. Plant molecular farming (PMF) employs plants as bioreactors for the production of pharmaceuticals. Now PMF has been used to produce antibodies, vaccines, and medicinal proteins, but it has not been well-studied for production of nutraceuticals and functional food. In this perspective, we extend the concept of PMF, present an updated overview of PMF for functional food development, including the progress, problem, and strategy, and then speculate how to use the PMF strategy to produce functional foods, especially with four major staple food crops (rice, wheat, maize, and soybean). Finally, we discuss the opportunities and challenges of PMF on functional food production in the future.


Assuntos
Alimento Funcional , Agricultura Molecular , Reatores Biológicos , Produtos Agrícolas , Humanos
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