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Recombinant Proteins in Plants :Problems and Prospects
 

Recombinant Proteins in Plants :Problems and Prospects

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The guest lecture delivered at Tamil Nadu Agricultural University on 6.2.2013 by Dr Gowda

The guest lecture delivered at Tamil Nadu Agricultural University on 6.2.2013 by Dr Gowda

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  • Here I would like to briefly explain how to make transgenic plant expressing vaccine antigens. First, a plant expression vector for antigen of choice is designed. Among various gene delivery methods, we mainly use agrobacterium-mediated transformation method, where the gene encoding the antigen will be inserted in a vectorderived from plant pathogen agrobacterium shown here, and then the vector is transferred to the bacteia. Now this bacteria can transfer the genes into chromosome of plant cells. Finally, transformed plant cells are selected and then regenerated to whole plants.
  • This technology is being used for several reasons. First, by introducing the transgene into a crop like corn, the farmer can use traditional production techniques to grow the crop. From the pharmaceutical side, the cost of producing the end product is greatly reduced compared to techniques currently in place.
  • Edible vaccines may be the most important and accepted biotech product.
  • The health and pharmaceutical industry uses biotech approaches for vaccine production. The normal bacteria in the mouth produces an acid that destroys enamel. The engineered version of the bacteria does not produce the acid. Children will be treated with the engineered version, which also has a gene that will destroy the other bacteria in the mouth, allowing it to become established.

Recombinant Proteins in Plants :Problems and Prospects Recombinant Proteins in Plants :Problems and Prospects Presentation Transcript

  • Recombinant Proteins in Plants Problems and Prospects Dr.P.H.Ramanjini Gowda Professor and Co ordinator Department of Biotechnology UAS.GKVK Bangalore 560065Guest lecture delivered at Tamil Nadu Agricultural University, Coimbatore on 6.2.2013
  • Why Do We Need Vaccines? Currently, we have vaccines for TWENTY-SIX different infectious diseases. The average American child receives about ten different vaccinations before the age of 2! Many different diseases are rarely seen anymore; in some cases the diseases have disappeared completely! Vaccinations save countless people all over the world from severe and even fatal diseases.
  • Beforethe 1700’s 1796 1970’s 1994 TodayThe Chinese Edward Smallpox Polio was Researchdeveloped Jenner was era- eradicated. is beingthe first developed dicated. done allform of the over thevaccines - smallpox world invariolation. vaccine order to from improve cowpox. vaccines.
  • Costs Vaccine production is not at all efficient for mass production.http://whyfiles.org/166plant_vaccines/2.html Their use in many parts of the world is limited because of the high costs.
  • “Combining a cost-effective productionsystem with a safe and efficaciousdelivery system, edible vaccinesprovide a compelling solution.”– Plants and Human Health: Delivery of Vaccines ViaTransgenic Plants (2003)
  • What exactly are“edible vaccines?” • Biopharmaceuticals • Plants or crops that produce human vaccines • The next generation of vaccines
  • Biopharming• Biopharming is the crop based production of industrial or therapeutic biomolecules.• Vaccines are the therapeutic biomolecules.• Plants are amenable for large scale biomass production.• Plants have good system of post translational modification of proteins.
  • Contd….• Complex multemeric proteins can be produced in plants.• The engineered edible vaccine can be consunmed orally without alteration.• The cost of vaccine is cheap since it needs no purification.• Needs no refrigeration.
  • Growing plants is much cheaperThe plants that produce the than producing vaccines.edible vaccines could begrown in third worldcountries. Targeted expression in plant storage tissues provides stability andAdvantages accumulation Plants are already regularly Agricultural used in pharmaceuticals, so products there are established can be purification protocols. transported around the Plants can’t host most world human pathogens, so the relatively vaccines won’t pose cheaply. dangers to humans.
  • Limitations• Low expression levels.• Glycosilation and post transcriptional modifications.• Animal and human studies are difficult.• Formulations of the vaccines.
  • For the last decade, scientists have known how to genetically engineer a plant to produce a desired protein. The two most common tools used to do this are:Cut out the selected Infect the plant withregion of the plasmid. the agrobacteria and DNA is coated on grow it in a medium. microscopically tiny gold Agrobacteria have a circular beads that are placed in a form of DNA called plasmids. vacuum chamber. The The plasmids are easily gene gun then allows manipulated because they compressed gas to expand, pushing the beads down naturally have two “cut” Grow the plant like Add the desired gene. until they hit a filter. The points where a gene can beregular crop. a DNA then flies off of the taken out and replaced with beads down into the tissue, one of the scientist’s choice. where some will enter a nucleus and become incorporated.
  • Plant-derived Vaccine StrategiesGene encoding an antigenic protein from a pathogen. Incorporate into a plant transformation vector for optimized expression in plant cells. Stable expression: Stable expression: Nuclear genome Chloroplast genome integration. integration. Integrate into a viral coding sequence for expression as a “by product” of viral replication. Transient expression: Modify viral genome to Infect plant to initiate adapt it into a plant viral replication. transformation vector for subsequent regulated release as a replicon in transgenic plants. Identify protective Create viral replicon coding epitope within sequence with epitope antigenic protein. fusions to coat protein.
  • Choice of the Plant System• Plant product should be eaten raw.• The plant is amenable for regeneration.• Should be rich source of protein.• Fast growing• Should grow under tough weather conditions.• Banana,cantaloupes,Peanut,Papaya.
  • Antigen Expression in PlantsThe cumulative number of antigens from pathogens of humans and/oranimals which have been expressed in plants, based upon published reports(original compilation of the reports was detailed in Khalsa G, Mason H, Arntzen C. Plant-derived vaccines: progress and constraints. In: R Fischer and S Schillberg (eds.) MolecularFarming: Plant-made Pharmaceuticals and Technical Proteins. John Wiley and Sons, Inpress in 2005).
  • Pharmaceutical Production in Plants Genetically modified plants have been used as “bioreactors” to produce therapeutic proteins for more than a decade. A recent contribution by transgenic plants is the generation of edible vaccines. Edible vaccines are vaccines produced in plants that can beadministered directly through the ingestion of plant materialscontaining the vaccine. Eating the plant would then confer immunityagainst diseases.Edible vaccines produced by transgenic plants areattractive for many reasons. The cost associatedwith the production of the vaccine is low,especially since the vaccine can be ingesteddirectly, and vaccine production can be rapidly upscaled should the need arises. Edible vaccine islikely to reach more individuals in developingcountries.The first human clinical trial took place in 1997.Vaccine against the toxin from the bacteria E.coliwas produced in potato. Ingestion of thistransgenic potato resulted in satisfactoryvaccinations and no adverse effects.
  • Edible VaccinesOne focus of current vaccine effort is on hepatitis B, a virus responsible forcausing chromic liver disease. Transgenic tobacco and potatoes were engineeredto express hepatitis B virus vaccine. During the past two years, vaccines againsta E.coli toxin, the respiratory syncytial virus, measles virus, and the Norwalkvirus have been successfully expressed in plants and delivered orally. Thesestudies have supported the potential of edible vaccines as preventive agents ofmany diseases.There is hope to produce edible vaccines in bananas, which are grown extensivelythroughout the developing world. Vol. 19, No. 3 Feb. 1, 1999
  • Transgenic Plants; Nuclear Transformation
  • Why use this technology?Familiar Production Systems • Genes introduced into field crops • New productions systems not needed • Producer can use traditional growing strategiesReduced End-Product Cost • Animal system: $1000 - $5000 per gram protein • Plant System: $1 - $10 per gram protein
  • Edible Vaccines – A Biopharming Dream Biotech Plants Serving Human Health Needs• A pathogen protein gene is cloned• Gene is inserted into the DNA of plant (potato, banana, tomato)• Humans eat the plant• The body produces antibodies against pathogen protein• Human are “immunized” against the pathogen• Examples: Diarrhea Hepatitis B Measles
  • Future Health-related Biotech Products Vaccines  Herpes  hepatitis C  AIDS  malaria Tooth decay  Streptococcus mutans, the mouth bacteria  releases lactic acid that destroys enamel  engineered Streptococcus mutans does not release lactic acid destroys the tooth decay strain
  • Rabies Neutralizing antibody titers(IU/ml)inmice immunized with plant extractsPlant Extract and route Neutralizing antibody titer on Key words Day 30 Day60Plant 1 IM 0.4 0.8 IM=Intramuscular IM+FA 0.7 1.2 IP=Intraperitoneal IP 0.6 1.0 FA=CompleatePlant 2 IM 0.5 1.0 Freaunds adjuvant IM+FA 1.0 1.5 ND=Not detected IP 0.8 1.2 IU=International unitsPlant 3 IM 0.7 1.2 IM+FA 1.2 1.6 IP 0.8 1.5Control Plant IM ND NDIM+FA ND NDIP ND ND
  • Protein expression in crop plants Earlier we have expressed ERA strain of rabies glycoprotein gene in Tobacco and Muskmelon and also obtained an Indian patent. The ERA strain obtained from Thomas Jefferson University has a patent on this gene. Hence, we have designed our own gene construct. The CVS glycoprotein gene will be subcloned to plant expression vector (pPS1) The pPS1 vector containing CaMV 35S promoter with the rabies glycoprotein gene will be transferred to Agrobacterium strain EHA105 and will be used for developing transgenic crop plants expressing Rabies Glycoprotein.
  • Alternative means of pilot productionProduction facility    bioreactor    refined product
  • Plants as bioreactors for pharmaceutical proteins PRESENT STATUS• Useful human proteins produced in plants – Human antibodies & other blood proteins – Protein and peptide hormones – Enzymes – Subunit vaccines• Proteins from plants are in the clinical pipeline – Human antibodies – Subunit vaccines – Enzymes• Regulatory Environment is evolving
  • Plants as bioreactors for pharmaceutical proteins FUTURE APPLICATIONS• Clinical unmet needs in cancer, infectious disease, cardiovascular disease, CNS disease, metabolic disorders, inflammatory disease, biowarfare agents• Options for injectable, oral and topical application• Treatment and prevention modalities
  • What is the challenge?• Developing drugs to treat human disease; protein based drugs are the fastest growing class of new drugs for treatment and prevention of human disease. But we face these barriers:• Capacity: – Insufficient capacity for drugs in the pipeline• Cost – Cost of goods – Capital for manufacturing facilities• Safety and Efficacy
  • Advantages of plants as bioreactors• Plants are the most efficient producers of proteins on earth – Plants are scalable bioreactors – Plants provide cost advantages• Plants cells are similar to human cells – Similar protein synthesis machinery – Read the same genetic code – Assemble, fold and secrete complex proteins
  • Antibodies:A Compelling Success Story• Inherently stable human proteins• High specificity; low toxicity• High drug approval rates• Injectable, topical and oral applications• Appropriate for chronic conditions• Potential long-lasting benefits
  • Antibodies: Natural Defense• Circulating antibodies protect us from invading viruses, bacteria and toxins• Secretory antibodies protect our vulnerable surfaces from pathogens and toxins, preventing entry and colonization• Passive antibodies in colostrum and milk provide passive immunity to neonates and infants• We make ~3g of antibodies a day• Like most animals, we surrender most of our antibodies to the environment
  • Antibodies: Natural DefenseORAL/GASTROINTESTINAL NASAL GENITO-URINARY TRACT PULMONARY TRACT TRACT
  • Emerging Antibody OpportunitiesTherapeutic areas requiring highquantities of antibodies, and low cost– Inflammatory diseases– CNS diseases– Cardiovascular diseases– Infectious diseases _ topical applications
  • Plant-produced Antibodies work• Anti-Streptococcus mutans (Guy’s 13) – Prevents dental caries in humans – Plant sIgA 10X more stable than IgG • Nature Medicine 1998• Anti-Herpes simplex virus (HSV8) – Prevents vaginal transmission of herpes – Proved in mice with rice and soybean PAb’s • Nature Biotechnology 1998
  • Plants Produce Assembled Antibodies Site of production: corn endosperm (starch and protein) Nature 342:76-78 (1989) Science 268: 716-9 (1995) Nature Biotechnology: 16:1361- 1364 (1998)
  • Comparison of Plant andMammalian Derived Antibodies• Peptide sequence: identical• Affinity: identical• Antibody types: Plant system more versatile – Can make any isotype including secretory IgA• Post-translational processing: different – core glycan identical, terminal sugar different – antigenicity & clearance: apparently identical
  • A Plant-produced antibody isscheduled for clinical development • Clinical trials planned to begin in 2003 • Clinical importance: herpes simplex virus – Over 50 million chronic sufferers – Over 1.5 million new cases/year in U.S. • Antibodies provide promising application in both prevention and treatment • Plant-produced antibodies are ideal – High quantities required – Scalable and have lower costs than traditional production
  • Capacity Shortage 60000 50000 40000Kg of MAb 30000 20000 10000 0 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 Mammalian Cell Culture Protein Capacity in Kg Optimistic MAb Demand (Dain Rauscher 00) Realistic MAb Demand (CSFB 01)
  • Processing Comparison*After harvest, the seeds can be stored indefinitely; therefore, when the protein isneeded, the purification process can begin immediately.Source: Cline, M.,”Plant-Made Pharmaceuticals: Overview of Technology andStewardship,” Fifth Biotechnology Roundtable, American Bar Association, St. Louis,May 2003.
  • Plant-derived pharmaceuticals have a full load of technology.Can the load be moved to benefit public health? • Finalize regulatory regime through commercialization • Currently pilot scale; commercial scale-up required • Secure public acceptance of technology
  • First Pant Made Drug on the Market• US FDA approved drug produced in carrots• The drug Taliglucerase alfa produced for the rare lysosomal storage disorder (Gaucher disease).• Israeli Biotech Protalix Biotherapeutics developed the method.• This drug can replace the avialble drug Cerezyme.
  • THANK YOU