Molecular farming

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this presentation deals with Molecular Ph(f)arming, and bio-safety issues related to it. This was presented by me in credit seminar in the division of Agricultural physics, IARI, New Delhi.
the sources used are duly acknowledged in the figures and slides.

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Molecular farming

  1. 1. Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar
  2. 2. Definition: “ The use of whole organisms, organs, tissues or cells, or cell cultures, as bio-reactors for the production of commercially valuable products via recombinant DNA techniques.” Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar
  3. 3. A brief history of molecular farming 1986 First plant - derived recombinant therapeutic protein- human GH in tobacco & sunflower. (A. Barta, D. Thompson et al.) 1989 First plant - derived recombinant antibody – full-sized IgG in tobacco. (A. Hiatt, K. Bowdish) 1990 First native human protein produced in plants – human serum albumin in tobacco & potato. (P. C. Sijmons et al.) 1992 First plant derived vaccine candidate – hepatitis B virus surface antigen in tobacco. (H. S. Meson, D. M. Lam) 1992 First plant derived industrial enzyme – α-amylase in tobacco. (J.Pen, L. Molendijk et al.) 1995 Secretory IgA produced in tobacco. (J. K. Ma, A. Hiatt, M. Hein et al.) Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar
  4. 4. Continued…. 1996 First plant derived protein polymer – artificial elastin in tobacco. (X. Zhang, D. W. Urry, H. Daniel) 1997 First clinical trial using recombinant bacterial antigen delivered in a transgenic potato. (C. O. Tacket et al.) 1997 Commercial production of avidin in maize. (E. E. Hood et al.) 2000 Human GH produced in tobacco chloroplast. (J. M. Staub et al.) 2003 Expression and assembly of a functional antibody in algae. (S. P. Mayfield, S. E. Franklin et al.) 2003 Commercial production of bovine trypsin in maize. (S. L. Woodard et al.) Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar
  5. 5. Molecular Farming Strategy Clone a gene of interest Transform the host platform species Grow the host species, recover biomass Process biomass Purify product of interest Deliver product of interest Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar
  6. 6. Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar
  7. 7. Molecular Farming Hosts Bacteria Yeasts, (single celled fungi) Unicellular algae Mammalian, insect, plant, and filamentous fungal cell cultures Whole plants, ( corn, barley, rice, duckweed, moss protonema) Whole animals, (insects, birds, fish, mammals) Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar
  8. 8. Bacteria : Do not produce glycosylated full – sized antibodies. Contaminating endotoxin difficult to remove. Recombinant proteins often form inclusion bodies. Labour- and cost – intensive refolding in vitro necessary. Lower scalability Preferred for the production of small, aglycosylated proteins like Insulin, interferon-β. Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar
  9. 9. Animal Based Systems: Limited by legal and ethical restriction Require expensive equipment & media Delicate nature of mammalian cells Human pathogens and oncogenes Scaling up problems Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar
  10. 10. Plant Molecular Farming Significantly lower production cost than with transgenic animals, fermentation or bioreactors. Infrastructure & expertise already exists for the planting, harvesting & processing of plant material. Plants contain no known human pathogens (such as prions, virions, etc.) that could contaminate the final product. Higher plants generally synthesize proteins from eukaryotes with correct folding, glycosylation & activity. Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar
  11. 11. Continued…… Plant cells can direct proteins to environments that reduce degradation and therefore increase stability. Low ethical concerns. Easier purification (homologs don’t pose any purification challenge, e.g. serum proteins or antibodies). Versatile (production of a broad diversity of proteins).× Take more time to develop× Transgene & protein pollution Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar
  12. 12. Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar
  13. 13. Cost of Production: Antibodies Animal cell culture $ 333/g Transgenic milk $ 100/g Yeast cell culture $ 100/g Milled corn endosperm $ 0.2/g Enriched corn fraction $ 0.6/g Extracted corn fraction $ 2.1/g Moderate purity $ 3.3/g Rx purity $ 20 -200/g (Rainer Fischer; Stefan Schillberg) Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar
  14. 14. Expression systems for PMF Transgenic plants Plant - cell - suspension culture Transplastomic plants Transient expression system Hydroponic cultures Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar
  15. 15. Transgenic plants: Foreign DNA incorporated into the nuclear genome using  Agrobacterium tumefaciens  Particle bombardment Most common Long term non-refrigerated storage Scalability More ‘gene to protein’ time Biosafety concerns Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar
  16. 16. Plant cell suspension culture Culture derived from  transgenic explants  Transformation after desegregation Recombinant protein localization depends on  Presence of targeting / leader peptides in the recombinant protein  Permeability of plant cell wall for macromolecules Containment & production under GMP procedure Low scale up capacity Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar
  17. 17. Su-May Yu; Institute of Molecular Biology Academia SinicaPresentedNankang, Taipei in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar
  18. 18. Transplastomic plants: DNA introduced into chloroplast genome High transgene copy number No gene silencing Recombinant protein accumulate in chloroplast Natural transgene containment Long term storage not possible Long development time Limited use for production of therapeutic glycoproteins Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar
  19. 19. Transient expression systemApproaches: Biolistic delivery of ‘naked DNA’  Usually reaches only a few cells  Can be used for a rapid test for protein expression Agroinfiltration  Delivery of Agrobacterium in intact leaf tissue by vacuum infiltration  Targets many more cells in a leaf Infection with modified viral vectors Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar
  20. 20. Virus infected plants Gene of interest is cloned into the genome of a viral plant pathogen Infectious recombinant viral transcripts are used to infect plants Rapid & systemic infection High level production soon after inoculation Genetic modification of plant is entirely avoided Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar
  21. 21. Over view of transient-gene-expression approaches in plants R. Fischer and others(1999) Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar
  22. 22. Hydroponic culture A signal peptide is attached to the recombinant protein directing it to the secretory pathway Protein can be recovered from the root exudates (Rhizosecretion) or leaf guttation fluid (Phylosecretion) Technology being developed by the US biotechnology company Phytomedics Inc. Purification is easier Reduced fear of unintentional environmental release Expensive to operate hydroponic facilities Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar
  23. 23. Comparison of different production systems for expression of recombinant proteins S. Biemelt;U. Sonnewald (2004)Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar
  24. 24. Downstream processing & analysis of recombinant proteins from plants R. Fischer and others(1999) Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar
  25. 25. Choice of host speciesDepends on: Protein to be produced & its desired application Transformation efficiency Overall production cost Containment Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar
  26. 26. Comparison of various plant expression host speciesFeatures/ Organ Yield Storage/ Transform Production Specialtycrop protein -ation costs stabilityTobacco Leaf High Limited Well Good Nonfood/feed establishedAlfalfa Leaf High Limited Established Good Homogenous N glycosylation, use atmospheric N2Wheat Seed Good Optimal Inefficient OptimalMaize Seed High Optimal Established OptimalPea Seed Good Optimal Limited GoodRapeseed Seed Good Optimal Established Optimal Fusion with oleosin for easy purificationPotato Tuber Good Good Well Good establishedBanana Fruit Good Good Inefficient Good Can be eaten raw S. Biemelt;U. Sonnewald (2004) Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar
  27. 27. Classes of proteins within molecular farming Parental therapeutics and pharmaceutical intermediates Industrial proteins and enzymes Monoclonal antibodies Antigens for edible vaccines Biopolymers Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar
  28. 28. Therapeutic proteins produced in different plant hosts system Therapeutic protein Potential use hostα- and β- haemoglobin Blood substitute TobaccoHuman serum albumin(HAS) Blood substitute Potatoα-tricosanthin HIV Therapy tobaccoα- interferon Viral protection anticancer RiceEpidermal growth factor, Mitogen TobaccoErythropoietin, Tuber growthfactorHirudin Anticoagulant CanolaProtein C Anticoagulant TobaccoGlutamate decarboxylase Diabetes TobaccoHuman somatotropin Hypopituitary dwarfism TobaccoCalcitonin Paget disease, osteoporosis, potato parathyroid gland carcinoma A.S. Rishi; N.D.Nelson; A.Goyal (2001) Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar
  29. 29. Industrial enzymes & proteins produced in different plant host system Industrial enzymes Potential use Hostα- amylase Industry TobaccoPhytase Industry Alfalfa, TobaccoCellulase Industry Alfalfa, Tobacco, potatoManganese peroxidase Industry Alfalfa, Tobaccoβ- (1,4) xylanase Industry Tobacco, Canolaβ-(1,3-1,4)glucanase Industry Tobacco, BarleyAvidin Research reagent MaizeGlucuronidase Research reagent Maize A.S. Rishi; N.D.Nelson; A.Goyal (2001) Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar
  30. 30. Antibodies produced in different plant host system Potential use Antigen used HostHSV-2 Glycoprotein B of HSV SoybeanColon cancer Colon cancer antigen TobaccoDental care (tooth S. mutans antigen Tobaccodecay)Hodgkin’s lymphoma ScFv of IgG from mouse B-cell Tobacco lyphomaTumor associated ScFv84.64 against Cerealsmarker antigen carcinoembryogenic antigenResearch Human creatine kinase ArabidopsisPhytoremediation Atrazine TobaccoPlant protection Nematode antigen Tobacco A.S. Rishi; N.D.Nelson; A.Goyal (2001) Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar
  31. 31. Vaccines produced in different plant host systems Antigen Casual organism/Disease Host systemRabies virus glycoprotein Rabies Tomato, tobacco, spinachCapsid protein epitope Mink enteritis virus CowpeaSpike protein Piglet diarrhea TobaccoCT-B toxin Cholera PotatoLT-B toxin Travelers diarrhea PotatoHepatitis B surface antigen Hepatitis B Tobacco, PotatoHuman cytomegalovirus glycoprotein Human cytomegalovirus TobaccoBNorwalk virus antigen Gastrointestinal distress Tobacco, PotatoFoot & mouth disease antigen Foot and mouth disease CowpeaMalarial antigens Malaria TobaccoGp 41 peptide HIV-1 CowpeaHemagglutinin Influenza Tobaccoc-Myc cancer A.S. Rishi;Tobacco N.D.Nelson; A.Goyal (2001) Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar
  32. 32. Products on the market Avidin  Hood et al. (1997) reported the production of chicken egg white avidin in transgenic corn β-Glucuronidase  First reported to be produced commercially in transgenic corn (Kusnadi et al. 1998, Witcher et al. 1998) Trypsin (TrypZeanTM)  First large scale protein product from transgenic plant technology Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar
  33. 33. PMF products & producers M.E.Horn; S.L.Wooddard; J.A.Howard (2004) Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar
  34. 34. PMF LeadersCanada Medicago (Québec): alfalfa leaves for hemoglobin production. Sembiosys Genetics (Calgary): safflower for production of a fat- fighting peptide and somatotrophin. Plantigen (Ontario): trials of several plants for protein production.United States AtlaGen Bioscience (Morgan Hill, CA and Richland, WA): potato leaves. Ventria Bioscience: potato tubers. API: rice and other plants. CropTech: tobacco leaves for production of uronidase, irunosidase, glucocerebrosidase (for Gaucher’s disease) and vaccines. Dow AgroSciences: corn for production of vaccines and antibodies to prevent certain animal diseases. IPT (Monsanto): corn for antibody and somatotrophin production. Epicyte Pharmaceutical (San Diego): corn and rice seeds. Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar
  35. 35. Continued: Phytomedics: (Dayton, NJ): tobacco and tomatoes. Prodigene (College Station, TX): corn seeds for production of laccase, avidin, betaglucoronidase and aprotinin. Monsanto Protein Technologies: corn.Germany Planton: potato tubers. Greenovation: corn for production of factor IX for hemophilia B treatment. MPB Cologne: potato tubers, canola seedsFrance Meristem Therapeutics (Clermont-Ferrand): corn seeds, tobacco leaves for production of hemoglobin, gastric lipase, collagen, beta interferon, lactoferrin and albumin.Switzerland Syngenta: antibodies and others. Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar
  36. 36. Biosafety issues in molecular farmingPresented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar
  37. 37. Challenges Gene and protein pollution  Applied to all transgenic plants. Product safety  Applied to all pharmaceutical product. Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar
  38. 38. Transgene pollution – the problems Transgene pollution is the spread of transgenes beyond the intended genetically-modified species by natural gene flow mechanisms. Two classes of transgene pollution:  The possible spread of primary transgenes.  The possible spread of superfluous DNA sequences. Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar
  39. 39. Transgene pollution – the mechanismsVertical gene transfer Vertical gene transfer is the movement of DNA between plants that are at least partially sexually compatible. Most prevalent form of transgene pollution. Occurs predominantly via the dispersal of transgenic pollen. Also by seed dispersal. herbicide resistance genes have introgressed from transgenic oil seed rape to its weedy cousin Brassica campestris by hybridization (Mikkelsen et al.1996). Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar
  40. 40. Horizontal gene transfer Horizontal gene transfer is the movement of genes between species that are not sexually compatible and may belong to very different taxonomic groups. The process is common in bacteria, resulting in the transfer of plasmid-borne antibiotic resistance traits. Agrobacterium tumefaciens and related species represent a special case. Antibiotic resistance markers and transgenes encoding pharmaceutical proteins could be acquired by human pathogens. Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar
  41. 41. what not to look for in a platform crop for molecular farming Abundant pollen production Abundant seed production Small, easily dispersed seeds Important food/feed crop Widely planted throughout the world Often grown as open-pollinated varieties Spontaneously mates with wild relatives High frequency of gene flow by outcrossing Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar
  42. 42. Transgene pollution – possible solutions  Minimum required genetic modification.  Elimination of non-essential genetic information.  Containment of essential transgenes.  Alternative production systems  transient expression.  plant suspension cultures in sealed, sterile reactor vessels (Fischer et al., 1999a; Doran, 2000). Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar
  43. 43. Containment of essential transgenes  Physical or artificial  Maintained in green house  Concealing flowers/fruits in plastic bags in field  Isolation  Barrier crops  Biological containment Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar
  44. 44. Biological containment methods to prevent transgene pollution Use of self-pollinating crops, exploitation of cleistogamy Asynchronous flowering times, atypical growing seasons Use of crops lacking wild relatives that are compatible for hybridization Strengthening of hybridization barriers between compatible species Apomixis Interference with flower development Male sterility (interference with pollen development) Seed sterility (by ‘terminator technology’) Maternal inheritance (plastid transformation) Transgene integration on incompatible genomes Transgenic mitigation Conditional transgene excision Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar
  45. 45. Protein pollution – the problems Direct consumption (e.g. the ingestion of toxins by aphids, and knock- on effects to ladybirds and birds further up the food chain) By simple exposure to the plant (e.g. the effects of pollen on butterflies and moths) From the exudation of recombinant protein into the rhizosphere or leaf guttation fluid (most likely to affect microorganisms) By the consumption of dead and decaying plant material by saprophytes Waste plant material Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar
  46. 46. Protein pollution – possible solutions To a certain extent by physical containment Controlling gene expression  Restricting expression to particular tissue  To bring the transgene under inducible control as has been shown for recombinant ‘glucocerebrosidase’ (Cramer et al., 1999). Controlling protein accumulation and activity  protein can also be targeted to a specific intracellular compartment  Recombinant proteins can also be produced as inactive precursors that have to be processed by proteolytic cleavage before they attain full biological activity.  used for the expression of hirudin Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar
  47. 47. Product safety The purified protein may be contaminated with toxic substances from the plant or applied to the plant, e.g. plant derived metabolites, allergens, field chemicals (e.g. herbicides, pesticides, fungicides), fertilizers, dung and manure. The product itself, due to intrinsic properties, may be harmful. Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar
  48. 48. Conclusion: Use of virus infected plants is best approach for molecular farming Molecular farming provides an opportunity for the economical and large-scale production of pharmaceuticals, industrial enzymes and technical proteins that are currently produced at great expense and in small quantities. We must ensure that these benefits are not outweighed by risks to human health and the environment Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar
  49. 49. Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar

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