Plant Molecular Farming
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US emergency labs ready to work on Ebola drugs

US emergency labs ready to work on Ebola drugs | Plant Molecular Farming | Scoop.it
All three US facilities established to quickly make vaccines and therapeutics in the event of a major public health threat say they are standing by to support any US government effort to scale up a treatment for Ebola.
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With plants as a distant afterthought.

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Ed Rybicki's curator insight, August 12, 2014 4:29 AM

"We are prepared to make any kind of vaccine," Giroir said, from the traditional kind grown in chicken eggs to newer varieties grown in mammalian or bacterial cells. "The whole idea is to take a process that may exist only on sticky notes at a small biotech company and scale it up as fast as possible"


Well, not ANY vaccine, obviously: why don't they just engage Medicago, or Kentucky BioProcessing or Fraunhofer USA for the plant-made stuff??

 
Plant Molecular Farming
A news site for the activities of members of the International Society for Plant Molecular Farming, as well as for papers of interest in the field
Curated by Ed Rybicki
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AzarGen’s case for a commercial plant-made pharmaceutical facility in South Africa

AzarGen’s case for a commercial plant-made pharmaceutical facility in South Africa | Plant Molecular Farming | Scoop.it
AzarGen, one of a few biopharming companies in the world focuses on developing human therapeutic proteins using advanced plant-based genetic engineering and synthetic biology techniques. Its leading product is a recombinant human surfactant protein, expected to increase the survivability of premature-born infants.
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With no local commercial biomanufacturing facility, in 2016 the company entered into a commercial agreement with American based bio-manufacturer iBio Inc. to develop and manufacture the surfactant protein for the treatment of neonatal respiratory distress syndrome (RDS).
“Synthetic biology and genetic engineering research, development and manufacturing initiatives and programmes that focuses on the biopharmaceutical needs and affordability for South Africans are vital,” says Dr. Mauritz Venter, Chief Executive Officer at AzarGen. Imported medicine, especially biologicals, are very expensive.
“If South Africa is to secure its future medicine supply, the country should invest in building its own commercial bio-manufacturing facility to produce biological drugs that are not just generic copies of the originator biopharmaceutical. We need a commercial, world-class biomanufacturing facility utilising an expression technology that will enable the local production of biobetters at the highest standards at significantly lower cost,” adds Dr. Venter.
Key factors to support local drug discovery and biomanufacturing.
A holistic approach to raise the awareness for affordable plant-based biological medicine.
A plant-made pharmaceutical platform is ideal for developing countries to establish a biopharmaceutical sector, not only for the production of biosimilars but for the production of novel biological drugs or modified derivatives of the originator product. The awareness of this ‘new industry’ potential can only be realised through collaborative efforts by government agencies, health systems, tertiary institutions, commercial networks and international partnerships. A biological drug with superior clinical efficacy at low cost can significantly improve the health status of citizens.
Increase investment and industry development support to capture commercial opportunities
An accredited large-scale commercial local biopharma operations where biobetters can be manufactured will enable (1) the production of high-quality-low-cost biological drugs that are clinically superior and safer than the originator drug, (2) rapid response production of vaccines and (3) boost local industrial development. Africa’s pharmaceutical market has high growth potential. Research released in 2015 by consultants McKinsey and Company predicts that the market will be worth $40 to $65-billion by 2020.
Unleash strong investment initiatives to support bioentrepreneurship and reduce unemployability of specialist science graduates.
The bio-economy strategy leans heavily towards boosting training and funding for students in life sciences but lacks a supporting framework that includes funding to stimulate entrepreneurial activities. This leads to high unemployability of life sciences graduates who face gloomy work prospects. Opportunities and spaces are needed where graduates can grow and hone their skills, and are supported to either pursue science entrepreneurship or find meaningful employment.
Advantages of a plant-based production platform versus mammalian and bacterial cells.
Plant-made manufactured biopharmaceuticals are not to be confused with medicinal plants or natural products.
“The plant applied is usually a tobacco variety, Nicotiana benthamiana, that is vacuum infiltrated with a modified Agrobacterium vector and the plant’s own protein-making machinery synthesizes the human therapeutic protein,” says Dr. Cobus Zwiegelaar, Chief Operating Officer at AzarGen. “The drug production process is safer, scalable and
potentially less costly - production, operating expenses and capital investments.”
The escalating cost of biological medicines not only poses growth risks for the national economy but puts South African healthcare at risk, especially biologicals to treat cancer and rare diseases.
“In plant manufactured pharmaceuticals safety is key as there is no animal or bacterial derived contamination. Plants enable the synthesis of complex biopharmaceutical proteins and production can easily scale to meet an increase in drug demand,” Dr. Zwiegelaar concludes.
Over the last six years, AzarGen has successfully secured multiple rounds of investment which has allowed the venture to achieve significant milestones:
Obtaining a Freedom-to-Operate analysis conducted by one of the world’s leading biotechnology legal advisory firms;
Producing novel synthetic promoters with enhanced protein expression capabilities;
Expressing and purifying human surfactant protein on a ‘proof-of-concept’ scale;
Successfully lodging patent applications in several international territories;
Securing funding from the Industrial Development Corporation (IDC) in 2011 and 2015 (current funding earmarked for scale-up production at iBio CMO) and
Successful application of iBio’s proprietary technology to achieve the first milestone in its commercial development agreement with iBio, Inc (USA).
Public and private sector representatives are invited to connect and engage with AzarGen to further explore the benefits that commercial plant-based biomanufacturing holds for South Africa. 
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This is a big, big deal - and I REALLY hope they pull it off; it'll make it that much easier for the rest of us!
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The sense behind retroviral anti-sense transcription

Retroviruses are known to rely extensively on the expression of viral proteins from the sense proviral genomic strand. Yet, the production of regulatory retroviral proteins from antisense-encoded viral genes is gaining research attention, due to their clinical significance. This report will discuss what is known about antisense transcription in Retroviridae, and provide new information about antisense transcriptional regulation through a comparison of Human Immunodeficiency Virus (HIV), Human T-cell Lymphotrophic Virus (HTLV-1) and endogenous retrovirus-K (ERVK) long terminal repeats (LTRs). We will attempt to demonstrate that the potential for antisense transcription is more widespread within retroviruses than has been previously appreciated, with this feature being the rule, rather than the exception.
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China scientist convicted in U.S. of theft of engineered rice

A federal jury on Thursday convicted a Chinese scientist in Kansas of conspiring to steal samples of a variety of genetically engineered rice seeds from a U.S. research facility, the U.S. Justice Department said, the latest attempt at agricultural theft linked to China.
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From Ventria Bioscience, no less - meaning molecular farming is suddenly worth industrial espionage?!  Watch this space...
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Rabies vaccine development by expression of recombinant viral glycoprotein

Rabies vaccine development by expression of recombinant viral glycoprotein | Plant Molecular Farming | Scoop.it
The rabies virus envelope glycoprotein (RVGP) is the main antigen of rabies virus and is the only viral component present in all new rabies vaccines being proposed. Many approaches have been taken since DNA recombinant technology became available to express an immunogenic recombinant rabies virus glycoprotein (rRVGP). These attempts are reviewed here, and the relevant results are discussed with respect to the general characteristics of the rRVGP, the expression system used, the expression levels achieved, the similarity of the rRVGP to the native glycoprotein, and the immunogenicity of the vaccine preparation. The most recent studies of rabies vaccine development have concentrated on in vivo expression of rRVGP by viral vector transduction, serving as the biotechnological basis for a new generation of rabies vaccines.

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Covers plant expression of rabies virus antigens, but then says this:
"cloned as a translational fusion product with the alpha mosaic virus (AlMV) coat protein (CP)” Alfalfa!! Idiot!
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Engineering Recombinant Virus-like Nanoparticles from Plants for Cellular Delivery

Engineering Recombinant Virus-like Nanoparticles from Plants for Cellular Delivery | Plant Molecular Farming | Scoop.it
Understanding capsid assembly following recombinant expression of viral structural proteins is critical to the design and modification of virus-like nanoparticles for biomedical and nanotechnology applications. Here, we use plant-based transient expression of the Bluetongue virus (BTV) structural proteins, VP3 and VP7, to obtain high yields of empty and green fluorescent protein (GFP)-encapsidating core-like particles (CLPs) from leaves. Single-particle cryo-electron microscopy of both types of particles revealed considerable differences in CLP structure compared to the crystal structure of infection-derived CLPs; in contrast, the two recombinant CLPs have an identical external structure. Using this insight, we exploited the unencumbered pore at the 5-fold axis of symmetry and the absence of encapsidated RNA to label the interior of empty CLPs with a fluorescent bioconjugate. CLPs containing 120 GFP molecules and those containing approximately 150 dye molecules were both shown to bind human integrin via a naturally occurring Arg-Gly-Asp motif found on an exposed loop of the VP7 trimeric spike. Furthermore, fluorescently labeled CLPs were shown to interact with a cell line overexpressing the surface receptor. Thus, BTV CLPs present themselves as a useful tool in targeted cargo delivery. These results highlight the importance of detailed structural analysis of VNPs in validating their molecular organization and the value of such analyses in aiding their design and further modification.

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Done by our friends from John Innes, et al.!
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The Cannabis Chronicles: To infinity (= legalisation) and beyond 

The Cannabis Chronicles: To infinity (= legalisation) and beyond  | Plant Molecular Farming | Scoop.it
With everything in place for the Dagga Couple to make history in the Pretoria High Court in July, the sucker’s bet for 2017 is that cannabis won’t be legalised this year. The million-dollar question for South Africa is therefore this: how do we legalise, reduce the size of the black market, and still retain the integrity of an informal economy that sustains vast swathes of our population? By KEVIN BLOOM.
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Two Novel DNAs That Enhance Symptoms and Overcome CMD2 Resistance to Cassava Mosaic Disease

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iBio Expands Product Development Capacity of cGMP Plant

iBio Expands Product Development Capacity of cGMP Plant | Plant Molecular Farming | Scoop.it
NEW YORK, NY -- (Marketwired) -- 11/10/16 -- iBio, Inc. (NYSE MKT: IBIO), a leading provider of plant-based biotechnology for developing and manufacturing biological products, announced completion of expanded product development capacity of its subsidiary, iBio CMO LLC, for the production of pharmaceutical proteins in hydroponically-grown green plants. This expansion enables iBio CMO's use of iBio proprietary technologies to conduct concurrent product development work for multiple clients in multiple product classes with time-saving transitions from laboratory-level feasibility and testing through cGMP production of active pharmaceutical ingredients for toxicology studies and phase 1 human clinical trials.

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Bean pod mottle virus: a new powerful tool for functional genomics studies in Pisum sativum

Bean pod mottle virus: a new powerful tool for functional genomics studies in Pisum sativum | Plant Molecular Farming | Scoop.it
Pea (Pisum sativum L.) is an important legume worldwide. The importance of pea in arable rotations and nutritional value for both human and animal consumption have fostered sustained production and different studies to improve agronomic traits of interest. Moreover, complete sequencing of the pea genome is currently underway and will lead to the identification of a large number of genes potentially associated with important agronomic traits. Because stable genetic transformation is laborious for pea, virus-induced gene silencing (VIGS) appears as a powerful alternative technology for determining the function of unknown genes. In this work, we present a rapid and efficient viral inoculation method using DNA infectious plasmids of Bean pod mottle virus (BPMV)-derived VIGS vector. Six pea genotypes with important genes controlling biotic and/or abiotic stresses were found susceptible to BPMV carrying a GFP reporter gene and showed fluorescence in both shoots and roots. In a second step, we investigated 37 additional pea genotypes and found that 30 were susceptible to BPMV and only 7 were resistant. The capacity of BPMV to induce silencing of endogenes was investigated in the most susceptible genotype using two visual reporter genes: PsPDS and PsKORRIGAN1 (PsKOR1) encoding PHYTOENE DESATURASE and a 1,4-β-D-glucanase, respectively. The features of the ‘one-step’ BPMV-derived VIGS vector include (i) the ease of rub-inoculation, without any need for biolistic or agro-inoculation procedures, (ii) simple cost-effective procedure and (iii) noninterference of viral symptoms with silencing. These features make BPMV the most adapted VIGS vector in pea to make low- to high-throughput VIGS studies.
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Now how about Bean yellow dwarf virus? Must try that....
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Making protective antibodies and other therapeutics in edible plant tissues for oral applications

Making protective antibodies and other therapeutics in edible plant tissues for oral applications | Plant Molecular Farming | Scoop.it
Although plant expression systems used for production of therapeutic proteins have the advantage of being scalable at a low price, the downstream processing necessary to obtain pure therapeutic molecules is as expensive as for the traditional Chinese hamster ovary (CHO) platforms. However, when edible plant tissues (EPTs) are used, there is no need for exhaustive purification, because they can be delivered orally as partially purified formulations that are safe for consumption. This economic benefit is especially interesting when high doses of recombinant proteins are required throughout the treatment/prophylaxis period, as is the case for antibodies used for oral passive immunization (OPI). The secretory IgA (SIgA) antibodies, which are highly abundant in the digestive tract and mucosal secretions, and thus the first choice for OPI, have only been successfully produced in plant expression systems. Here, we cover most of the up-to-date examples of EPT-produced pharmaceuticals, including two examples of SIgA aimed at oral delivery. We describe the benefits and drawbacks of delivering partially purified formulations and discuss a number of practical considerations and criteria to take into account when using plant expression systems, such as subcellular targeting, protein degradation, glycosylation patterns and downstream strategies, all crucial for improved yield, high quality and low cost of the final product.
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Chloroplast-made booster vaccine to confer immunity against different poliovirus serotypes

Chloroplast-made booster vaccine to confer immunity against different poliovirus serotypes | Plant Molecular Farming | Scoop.it
The WHO recommends complete withdrawal of oral polio vaccine (OPV) type 2 by April 2016 globally and replacing with at least one dose of inactivated poliovirus vaccine (IPV). However, high-cost, limited supply of IPV, persistent circulating vaccine-derived polioviruses transmission and need for subsequent boosters remain unresolved. To meet this critical need, a novel strategy of a low-cost cold chain-free plant-made viral protein 1 (VP1) subunit oral booster vaccine after single IPV dose is reported. Codon optimization of the VP1 gene enhanced expression by 50-fold in chloroplasts. Oral boosting of VP1 expressed in plant cells with plant-derived adjuvants after single priming with IPV significantly increased VP1-IgG1 and VP1-IgA titres when compared to lower IgG1 or negligible IgA titres with IPV injections. IgA plays a pivotal role in polio eradication because of its transmission through contaminated water or sewer systems. Neutralizing antibody titres (~3.17–10.17 log2 titre) and seropositivity (70–90%) against all three poliovirus Sabin serotypes were observed with two doses of IPV and plant-cell oral boosters but single dose of IPV resulted in poor neutralization. Lyophilized plant cells expressing VP1 stored at ambient temperature maintained efficacy and preserved antigen folding/assembly indefinitely, thereby eliminating cold chain currently required for all vaccines. Replacement of OPV with this booster vaccine and the next steps in clinical translation of FDA-approved antigens and adjuvants are discussed.
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Bidirectional cross-kingdom RNAi and fungal uptake of external RNAs confer plant protection

Bidirectional cross-kingdom RNAi and fungal uptake of external RNAs confer plant protection | Plant Molecular Farming | Scoop.it
Aggressive fungal pathogens such as Botrytis and Verticillium spp. cause severe crop losses worldwide. We recently discovered that Botrytis cinerea delivers small RNAs (Bc–sRNAs) into plant cells to silence host immunity genes. Such sRNA effectors are mostly produced by Botrytis cinerea Dicer-like protein 1 (Bc-DCL1) and Bc-DCL2. Here we show that expressing sRNAs that target Bc-DCL1 and Bc-DCL2 in Arabidopsis and tomato silences Bc-DCL genes and attenuates fungal pathogenicity and growth, exemplifying bidirectional cross-kingdom RNAi and sRNA trafficking between plants and fungi. This strategy can be adapted to simultaneously control multiple fungal diseases. We also show that Botrytis can take up external sRNAs and double-stranded RNAs (dsRNAs). Applying sRNAs or dsRNAs that target Botrytis DCL1 and DCL2 genes on the surface of fruits, vegetables and flowers significantly inhibits grey mould disease. Such pathogen gene-targeting RNAs represent a new generation of environmentally friendly fungicides.

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Antibodies from plants for bionanomaterials

Antibodies from plants for bionanomaterials | Plant Molecular Farming | Scoop.it
Antibodies are produced as part of the vertebrate adaptive immune response and are not naturally made by plants. However, antibody DNA sequences can be introduced into plants, and together with laboratory technologies that allow the design of antibodies recognizing any conceivable molecular structure, plants can be used as ‘green factories’ to produce any antibody at all. The advent of plant-based transient expression systems in particular allows the rapid, convenient, and safe production of antibodies, ranging from laboratory-scale expression to industrial-scale manufacturing. The key features of plant-based production include safety, speed, low cost, and convenience, allowing newcomers to rapidly master the technology and use it to its full advantage. Manufacturing in plants has recently achieved significant milestones and offers more than just an alternative to established microbial and mammalian cell platforms. The use of plants for product development in particular offers the power and flexibility to easily coexpress many different genes, allowing the plug-and-play construction of novel bionanomaterials, perfectly complementing existing approaches based on plant virus-like particles. As well as producing single antibodies for applications in medicine, agriculture, and industry, plants can be used to produce antibody-based supramolecular structures and scaffolds as a new generation of green bionanomaterials that promise a bright future based on clean and renewable nanotechnology applications.

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Virologica Sinica: special issue on haemorrhagic fever viruses

Virologica Sinica: special issue on haemorrhagic fever viruses | Plant Molecular Farming | Scoop.it

The first issue of 2017 is such a special issue with a focus on “Hemorrhagic fever viruses” (HFVs), organized by Prof. Zhihong HU and Prof. Jens KUHN. The reviews and research articles of the issue cover the recent progress on several important HFVs, such as Ebola virus, Dengue virus, Hantavirus, severe fever with thrombocytopenia syndrome virus and Crimean-Congo hemorrhagic fever virus, and we hope the readers would obtain the updated information through this issue. 


And we also have a call for papers on two special issues at this moment. The first one is on “Herpesviruses and antiviral strategies”, edited by Prof. Ke LAN and Prof. Min-Hua LUO. This special issue aims to summarize the recent findings in both laboratory and clinical studies, and can be utilized in the further antiviral therapeutics. The deadline for submission for this special issue is July 31, 2017. 


Our second special issue will be a focused issue to report the latest findings from a nation-wide surveillance project on “Viruses and their major natural hosts and vectors”. Professor Zhiming YUAN, and Ⅰ myself, will be the Co-Editors of the issue, and we hope to present the community of the findings of both field work and big data mining from this project. The deadline for submission is September 30, 2017. Besides, proposals for themed special issues are always welcome and appreciated.

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Greenovation Announces First Patient Dosed in Phase 1b Study with Moss-aGal

Press Release 
Greenovation Announces First Patient Dosed in Phase 1b Study with Moss-aGal 
 FREIBURG, Germany, March 15, 2017 
Greenovation Biotech GmbH announced today that it has dosed the first patient in a phase 1b study of its lead biopharmaceutical, moss-aGal, in adults with the inherited disorder, Fabry disease. Moss-aGal, a recombinant form of human alpha-galactosidase, has been developed by Greenovation as an enzyme replacement therapy (ERT) for patients with Fabry disease, a genetic lysosomal storage disorder. The phase 1b study, a single dose clinical trial, will assess the safety, pharmacokinetics and efficacy of moss-aGal on lyso-Gb3 levels in plasma and urine. The open-label, multicenter study is being conducted in Germany. "The first patient who received our drug moss-aGal tolerated the drug very well“ announces Thomas Frischmuth, PhD, CEO of Greenovation. “We are thrilled to see that our first moss-made biopharmaceutical is now in clinical testing and might potentially help improve patient’s quality of life in the future. This phase 1b study, which is the first clinical trial for Greenovation, represents a major milestone for the company and hopefully for Fabry patients.” 
 About Greenovation Biotech GmbH 
Greenovation develops next-generation therapeutics using its proprietary BryoTechnology platform. The company aims to optimize the production of highly-efficient glycoproteins for the treatment of rare diseases. Greenovation is a privately-owned biopharmaceutical company based in Heilbronn, Germany. It was founded in 1999 by Prof. Dr. Ralf Reski and Prof. Dr. Gunter Neuhaus. Today, Greenovation Biotech GmbH is majority-owned by Zukunftsfonds Heilbronn and L-EigenkapitalAgentur (Karlsruhe). About Fabry disease Fabry disease, a rare lysosomal storage disease, is caused by an inborn deficiency of the alpha-galactosidase (aGal) enzyme. A lysosomal enzyme, aGal breaks down a specific fatty acid, globotriaosylceramide (Gb3). In patients with Fabry disease, absence of aGal causes continuous accumulation of Gb3 in the cells. Symptoms include pain, and heart, skin and kidney complications, and can lead to organ failure. In enzyme replacement therapy, the missing enzyme is replaced with regular intravenous administrations of a biopharmaceutically-produced substitute. 
 Contact and further information: Greenovation Biotech GmbH Manon Kirstein, Tel. +49 761 470 99 0 
e-mail: mkirstein@greenovation.com 
 i.V. Manon Kirstein Head of Business Development Greenovation Biotech GmbH Branch Office: Hans-Bunte-Str. 19, 79108 Freiburg, Germany Main Office: Inselwiesenstr. 10, 74076 Heilbronn, Germany p + 49 761 470 99 111 f + 49 761 470 99 191 mkirstein@greenovation.com www.greenovation.com Managing Director: Thomas Frischmuth, PhD HRB 722568 AG Stuttgart, VAT-No. DE-812793603
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The possible function of endogenous Dioscorea bacilliform viruses

The complete genome sequence of Dioscorea bacilliform TR virus (DBTRV) was determined. The closest relatives of DBTRV are Dioscorea bacilliform AL virus (DBALV) and Dioscorea bacilliform RT virus 1 (D
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siRNA, is what they're saying?
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Plant-made virus shells could deliver drugs directly to cancer cells - American Chemical Society

Viruses are extremely efficient at targeting and delivering cargo to cells. In the journal ACS Nano, researchers report they have harnessed this well-honed ability — minus the part that makes us sick — to develop virus-like nanoparticles to deliver drugs straight to affected cells. In lab tests, they show that one such particle can be produced in plants and it ferries small molecules to cancer cells. For this work, Frank Sainsbury and colleagues copied the core protein shell of the Bluetongue virus, a pathogen that affects ruminant animals. Previous research has shown that the capsid is stable, has a large cavity for small molecules or proteins to pack into, and is easy to produce with high purity. The researchers wanted to try making the virus-shell nanoparticles using plants. This is an increasingly popular approach to producing pharmaceuticals as it minimizes possible contamination by human pathogens, which plants don’t carry. But first they needed to understand the structure of the shells. Using single particle cryo-electron microscopy, the team showed for the first time that the recombinant shell nanoparticles produced by plants were different from the natural virus capsid. With the nanoparticles’ detailed structure in hand, the researchers then genetically and chemically engineered them to their specifications, and loaded proteins and small molecules inside the shells. Lab testing showed that the plant-made virus particles, which naturally bind to receptors on cancer cells, were taken in by human breast cancer cells. The findings suggest the nanoparticles can potentially be used for the targeted delivery of drugs.

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Molecular farming is getting real, people...
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Plant-made Hemophilia Therapy Shows Promise

Plant-made Hemophilia Therapy Shows Promise | Plant Molecular Farming | Scoop.it
People with hemophilia require regular infusions of clotting factor to prevent them from experiencing uncontrolled bleeding.
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Great news! Mark up another success for molecular farming...B-)
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Lease out land, bring in GMOs, it’s the way out

Why need drastic action if East Africans are to eat
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Yup - one of the better ways to food security...
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Furin expression in N benthamiana leads to efficient processing of latent transforming growth factor-β1

Furin expression in N benthamiana leads to efficient processing of latent transforming growth factor-β1 | Plant Molecular Farming | Scoop.it
Transforming growth factor beta (TGF-β) is a signalling molecule that plays a key role in developmental and immunological processes in mammals. Three TGF-β isoforms exist in humans, and each isoform has unique therapeutic potential. Plants offer a platform for the production of recombinant proteins, which is cheap and easy to scale up and has a low risk of contamination with human pathogens. TGF-β3 has been produced in plants before using a chloroplast expression system. However, this strategy requires chemical refolding to obtain a biologically active protein. In this study, we investigated the possibility to transiently express active human TGF-β1 in Nicotiana benthamiana plants. We successfully expressed mature TGF-β1 in the absence of the latency-associated peptide (LAP) using different strategies, but the obtained proteins were inactive. Upon expression of LAP-TGF-β1, we were able to show that processing of the latent complex by a furin-like protease does not occur in planta. The use of a chitinase signal peptide enhanced the expression and secretion of LAP-TGF-β1, and co-expression of human furin enabled the proteolytic processing of latent TGF-β1. Engineering the plant post-translational machinery by co-expressing human furin also enhanced the accumulation of biologically active TGF-β1. This engineering step is quite remarkable, as furin requires multiple processing steps and correct localization within the secretory pathway to become active. Our data demonstrate that plants can be a suitable platform for the production of complex proteins that rely on specific proteolytic processing.
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Production of human glucocerebrosidase in glyco-engineered Nicotiana benthamiana plants

Production of human glucocerebrosidase in glyco-engineered Nicotiana benthamiana plants | Plant Molecular Farming | Scoop.it
For the production of therapeutic proteins in plants, the presence of β1,2-xylose and core α1,3-fucose on plants’ N-glycan structures has been debated for their antigenic activity. In this study, RNA interference (RNAi) technology was used to down-regulate the endogenous N-acetylglucosaminyltransferase I (GNTI) expression in Nicotiana benthamiana. One glyco-engineered line (NbGNTI-RNAi) showed a strong reduction of plant-specific N-glycans, with the result that as much as 90.9% of the total N-glycans were of high-mannose type. Therefore, this NbGNTI-RNAi would be a promising system for the production of therapeutic glycoproteins in plants. The NbGNTI-RNAi plant was cross-pollinated with transgenic N. benthamiana expressing human glucocerebrosidase (GC). The recombinant GC, which has been used for enzyme replacement therapy in patients with Gaucher's disease, requires terminal mannose for its therapeutic efficacy. The N-glycan structures that were presented on all of the four occupied N-glycosylation sites of recombinant GC in NbGNTI-RNAi plants (GCgnt1) showed that the majority (ranging from 73.3% up to 85.5%) of the N-glycans had mannose-type structures lacking potential immunogenic β1,2-xylose and α1,3-fucose epitopes. Moreover, GCgnt1 could be taken up into the macrophage cells via mannose receptors, and distributed and taken up into the liver and spleen, the target organs in the treatment of Gaucher's disease. Notably, the NbGNTI-RNAi line, producing GC, was stable and the NbGNTI-RNAi plants were viable and did not show any obvious phenotype. Therefore, it would provide a robust tool for the production of GC with customized N-glycan structures.
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A platform for the effective management of biological contaminants for the production of microalgae

A platform for the effective management of biological contaminants for the production of microalgae | Plant Molecular Farming | Scoop.it
Microalgal cultivation that takes advantage of solar energy is one of the most cost-effective systems for the biotechnological production of biofuels, and a range of high value products, including pharmaceuticals, fertilizers and feed. However, one of the main constraints for the cultivation of microalgae is the potential contamination with biological pollutants, such as bacteria, fungi, zooplankton or other undesirable microalgae. In closed bioreactors, the control of contamination requires the sterilization of the media, containers and all materials, which increases the cost of production, whereas open pond systems severely limits the number of species that can be cultivated under extreme environmental conditions to prevent contaminations. Here, we report the metabolic engineering of Chlamydomonas reinhardtii to use phosphite as its sole phosphorus source by expressing the ptxD gene from Pseudomonas stutzeri WM88, which encodes a phosphite oxidoreductase able to oxidize phosphite into phosphate using NAD as a cofactor. Engineered C. reinhardtii lines are capable of becoming the dominant species in a mixed culture when fertilized with phosphite as a sole phosphorus source. Our results represent a new platform for the production of microalgae, potentially useful for both closed photobioreactors and open pond systems without the need for using sterile conditions nor antibiotics or herbicides to prevent contamination with biological pollutants.
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