Plant_Biotechnology

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					Breaking the Biological Barriers
to Plant Made Pharmaceuticals
            (PMPs)
                    (Nov. 02, 2007)

                    Kyun Oh Lee, Ph.D.
       Division of Applied Life Science & EB-NCRC
              Gyeongsang National University
Increasing demands for recombinant
proteins after human genome project




                         •   Therapeutic proteins
                         •   Diagnostic Proteins
                         •   Hormones
 Completed
 in 2003
                         •   Antibodies
                         •   Vaccines
                         •   Cosmetics Additives
                         •   Feed Additives
Protein-based therapeutics

    Biopharmaceutical                                           Year of approval   Condition or disease treated

    recombinant somatrem (human growth hormone) for injection   1985               human growth hormone (hGH) deficiency in children

                                                                1986               hairy cell leukemia

                                                                1988               genital warts

    recombinant interferon alfa-2b                              •1988              •Kaposi’s sarcoma

                                                                •1991              •hepatitis C

                                                                1992               hepatitis B

                                                                1986               hairy cell leukemia
    recombinant interferon alfa-2a
                                                                1988               Kaposi’s sarcoma

                                                                1986               reversal of kidney transplant rejection
    Muromonab-CD3
                                                                1993               reversal of heart and liver transplant rejection

    recombinant hepatitis B vaccine                             1986               hepatitis B prevention

    recombinant somatropin for injection                        1987               human growth hormone (hGH) deficiency in children

                                                                1987               acute myocardial infarction
    Alteplase
                                                                1990               acute massive pulmonary embolism

    Epoetin alfa (rEPO, Epogen)                                 1989               anemia of chronic renal failure

    recombinant hepatitis B vaccine                             1989               hepatitis B

    interferon alfa-n3                                          1989               genital warts

    adenosine deaminase                                         1990               severe immunodeficiency in infants

    interferon gamma-1b                                         1990               chronic granulomatous disease

                                                                1991               neutropenia caused by chemotherapy

    filgrastim (rG-CSF)                                         1994               bone marrow transplantation

                                                                1994               chronic, severe neutropenia

    sargramostim (yeast-derived GM-CSF)                         1991               bone marrow transplantation

    Aldesleukin (interleukin-2)                                 1992               renal cell carcinoma
Protein-based therapeutics

  Biopharmaceutical                                          Year of approval   Condition or disease treated

  Staumonab pendetide (OncoScint)                            1992               colorectal and ovarian cancers

  recombinant antihemophiliac factor (rAHF)                  1992               hemophilia A

  recombinant interferon beta-1b                             1993               relapsing, remitting multiple sclerosis

  dornase alpha (Pulmozyme)                                  1993               cystic fibrosis

  Pegaspargase                                               1994               lymphoblastic leukemia

  imiglucerase for injection (Cerezyme, recombinant
                                                             1994               Gaucher’s disease
  lucocerebrosidase)

  abciximab (ReoPro)                                         1994               prevention of blood clotting

  Humulin 70/30 (biosynthesized human insulin)               1996               diabetes mellitus

  Humatrope                                                  1996               adult- or childhood-onset growth hormone deficiency

  Serostim                                                   1996               AIDS wasting associated with catabolism, weight loss, or cachexia

  Saizen                                                     1996               human growth hormone deficiency in children

  Nutropin                                                   1996               Turner’s syndrome

  Infanrix (vaccine)                                         1997               diphtheria and tetanus toxoids absorbed

  coagulation factor IX (recombinant)                        1997               factor IX deficiencies (Christmas disease)

  Novolin 70/30 (biosynthesized human insulin)               1997               diabetes mellitus

  Velosulin human (semisynthesized purified human insulin)   1997               diabetes mellitus

  Genotropin                                                 1997               human growth hormone deficiency in adults

  Oprelvekin (Neumega)                                       1997               prevention of thrombocytopenia

  Rituximab (Rituxan)                                        1997               follicular B-cell non-Hodgkin’s lymphoma

  Becaplermin (Regranex Gel)                                 1997               diabetic foot ulcers

  daclizumab (Zenapax)                                       1997               acute renal allograft rejection

  Nutropin AQ                                                1997               human growth hormone deficiency in adults

  recombinant human insulin                                  1982               diabetes mellitus
2006 Top-selling Classes of Biological
Products

                                     2006 sales
 No.   Class of Products                              Selected Branded Products
                                     (US$ bln.)
 1     Erythropoietins               11.94            Aranesp, Procrit Eprex, Epogen, Neo-Recormon, ESPO
 2     Major cancer antibodies       10.62            Rituxan/MabThera, Herceptin, Avastin, Erbitux, Vectibix
 3     Anti-TNF antibodies           10.28            Enbrel, Remicade, Humira
                                                      Humalog, Humulin, Lantus, Levemir, Novorapid, Actrapid,
 4     Insulin and insulin analogs   8.97
                                                      Novolin
                                                      Novoseven, Kogenate, Helixate, Refacto, Advate, Recombinate,
 5     Rec. coagulation factors      4.71
                                                      Benefix
 6     Interferon beta               4.40             Avonex, Rebif, Betaferon / Betaseron
 7     G-CSF                         4.36             Neulasta, Neupogen, Neutrogin, GRAN
                                                      Genotropin, Norditropin, Humatrope, Nutropin, Saizen,
 8     Human growth hormone          2.47
                                                      Serostim
 9     Interferon alpha              2.26             Pegasys, Peg-Intron, Intron A
                                                      Cerezyme, Fabrazyme, Aldurazyme, Myozyme, Replagal,
 10    Enzyme Replacement            1.71
                                                      Naglazyme, Elaprase
 11    Antiviral antibody            1.1              Synagis
       Follicel stimulating
 12                                  1.04             Gonal-f, Puregon
       hormones

       Total:                        $63.83 (58조 원)

  Source: La Merie Business Intelligence, February 2007
Growth Rates (2006 vs 2005) of Top-
selling Classes of Biological Products


                                    56.8
                           60

                           50
      Growth Rate (%)




                           40

                                             27.5
                           30                        24.8          24.4

                                                                              17
                           20                                                          14.4
                                                                                                 11.4
                                                                                                             6.9      6.9   6.7
                           10

                               0




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Current production system for protein-
based therapeutics




                     Chinese Hamster Ovary (CHO) Cells
                     • Expensive
                     • Not economically viable for some
                        drugs
                     • Not safe from some pathogen
                        infection
                     • Need to develop new cost
                        efficient substituting systems
Plants: a solution to the shortfall in
manufacturing capacity for new drugs

      Molecule               Problematic                          Plant Solutions
                           Blood demand
                            increase; Donors
                            decrease                 Large scale production
     Hemoglobin            Serotypes                No concern about serotypes
                            incompatibility          No human pathogens
                           Safety concern
                            (HIV; HCV; HBV)
                           Expensive
                                                     Cost reduction
      Antibody             Production
                                                     Large scale production
                            capacity limited
      Enbrel
                           Capacity shortage        Large scale production
   (TNF blocker)
                           Worldwide
                            shortage (40% of
     Factor VIII                                     Large scale production: accessibility to all
                            hemophilia patients
 (Coagulation factor)                                 patients
                            have access to the
                            product)
                           Too expensive for
                                                     Cost reduction: accessibility for non
       Insulin              non industrialized
                                                      industrialized countries
                            countries
Plant molecular farming (Biopharming)?


  • The use of modified plants to produce therapeutically
    active proteins or industrial products
  • The end product of crop harvests is not food, feed or fi
    bers, but plant-made pharmaceuticals (PMPs)
  • Biopharming industry may be worth $100 billion by 2015

            ※벼 수매가격 ≤ 50,000원/40 kg
Plant Products


  I.  Plant-derived pharmaceuticals (non-GE)
      •   Over 120 pharmaceutical products currently in use are
          derived from plants, mainly from tropical forest species
  II. Plant-made pharmaceuticals and industrial products (GE)
      1. Pharmaceuticals
          •   Recombinant human proteins
          •   Therapeutic proteins
          •   Enzymes
          •   Antibodies (plantibodies)
          •   Vaccines
      2. Industrial products
          •   Proteins
          •   Enzymes
          •   Modified starches
          •   Oils
          •   Waxes
          •   Plastics
    Why use plants?


     1. Plants are the cheapest, most abundant source of protein on the
        planet: low cost
     2. Plant provide low upstream production costs : low cost
     3. Plants, as eukaryotes, can express and process most prokaryotic
        and eukaryotic proteins: stability
     4. Plant provide scalable production capacity and flexibility: easy
        scale up
     5. Plant seed permit stockpiling of inexpensive inventory: good
        storage
     6. Plants are free from animal and human pathogens: safety
                            Golgi complex                               Plant Golgi
                                           1,2-mannosidase I                     -1,2-Mannosidase I


                          UDP-GlcNAc                            UDP-GlcNAc        N-acetyl-glucosaminyl-
                                           GnT1                 Transporter       transferase I (GnT-I)
                          Transporter


                                           -mannosidase II                       -Mannosidase II
                                                                                       ⇒ Fuc-Transferase

                                                                              F
                                           GnT-II                                 GnT-II


                           UDP-Gal                                            F
                                           GalT                                   Xyl-Transferase
                           Transporter


                          CMP-NeuAc                                       F X
                                           SAT                                    N-acetyl-Glucosaminidase
                          synthetase

                                                                         F X
                              Complex Type glycan                      Complex Type
※벼 수매가격 ≤ 50,000원/40 kg
Comparison of pharmaceutical expression
systems

                                                                                                                                  Cost per
 Expression                 Advantages                             Disadvantages                         Applications
                                                                                                                                    gram
  system
              Established regulatory track; well-        Proteins not usually secreted;        Insulin (E. coli; Eli Lilly);
  Bacteria    understood genetics; cheap and easy to     contain endotoxins; no                growth hormone (Genentech);
              grow                                       posttranslational modifications       growth factor; interferon
                                                         Overglycosylation can ruin            Beer fermentation;
              Recognized as “safe;” long
                                                         bioactivity; safety; potency;         recombinant vaccines;
   Yeast      history of use; fast; inexpensive;
                                                         clearance; contains                   hepatitis B viral vaccine;
              posttranslational modifications                                                                                     $50–100
                                                         immunogens/antigens                   human insulin
                                                         Minimal regulatory track; slow
              Posttranslational modifications;           growth; expensive media;              Relatively new medium;
  Insect
              properly folded proteins; fairly high      baculovirus infection (extra          Novavax produces virus-like
   cells
              expression levels                          step); mammalian virus can infect     particles
                                                         cells
              Usually fold proteins properly; correct    Expensive media; slow growth;         Tissue plasminogen activator;
 Mammalian    posttranslation modifications; good        may contain allergens/                factor VIII (glycoprotein);        $500–
   cells      regulatory track record; only choice       contaminants; complicated             monoclonal antibodies              5,000
              for largest proteins                       purification                          (Hercepin)
                                                         Little regulatory experience;         Lipase (sheep, rabbits; PPL
              Complex protein processing; very high
 Transgenic                                              potential for viral contamination;    Therapeutics); growth
              expression levels; easy scale up; low-                                                                              $20–50
  animals                                                long time scales; isolation/GMPs      hormone (goats; Genzyme);
              cost production
                                                         on the farm                           factor VIII (cattle)
              Shorter development cycles; easy seed      Potential for new contaminants        Cholera vaccine (tobacco;
 Transgenic   storage/scaling; good expression levels;   (soil fungi, bacteria, pesticides);   Chlorogen, Inc.); gastric lipase
                                                                                                                                  $10–20
   plants     no plant viruses known to infect           posttranslational modifications;      (corn; Meristem); hepatitis B
              humans                                     contains possible allergens           (potatoes; Boyce Thompson)
 Note. Data from BioPharm International (2003).
Risks and concerns


  • Impact on the environment;
    - Food supply contamination; accidental or
    intentional gene flow, cross-pollination,
    - Outcrossing (non-target species near field
    sites e.g. butterflies, bees, etc)
  • Allergenicity, antigenicity, immunogenicity
  • Inappropriate use of plant by-products
  • Occupational exposure
  • Expression levels
  • Reproducibility
  • Differences in glycosylation patterns
  • Biological containment
  • Regulatory issues
State of the art


  • ~25 companies world wide are developing plants
    for molecular farming
  • 16 plant based products are under clinical trials
  • commercial products are launching
PMPs in or nearing clinical trials

 PMPs in clinical trial phase II

 Plant          Active substance            Use                                         Country, company

 Tobacco        Monoclonal antibody         Dental caries prophylaxis                   USA, Planet Biotechnology

 Maize          Enzyme, gastric lipase      Cystic fibrosis, pancreatitis               France, Meristem Therapeutics

 Tobacco        Antibody, cancer vaccine    Non-Hodgkin lymphoma                        USA, Large Scale Biology

 Duckweed       Alpha interferon            Hepatitis C                                 USA, Biolex

 Potato         Antigen                     Hepatitis B                                 USA, Arizona State University

 Arabidopsis    Protein, intrinsic factor   Diagnostic test, Absorption of vitamin 12   Denmark, Cobento Biotech

 Tobacco        Antibody                    Colds caused by rhinoviruses                USA, Planet Biotechnology
 other PMPs in clinical trial phase I

 Plant          Product/ active substance   Use                                         Country, company

 Lettuce        Vaccine                     Hepatitis B                                 Polish Science Academy

 Potato         Vaccine                     Norwalk virus                               USA, Arizona State University

 Spinach        Vaccine                     Rabies                                      USA, Thomas Jefferson University, Philadelphia

 Maize          Protein, lactoferrin        Dry eye syndrome                            France, Meristem Therapeutics

 Potato         Vaccine                     Travel diarrhoea                            USA, Arizona State University

 Mais           Vaccine                     Travel diarrhoea                            USA, Arizona State University

 Mais           Vaccine                     Travel diarrhoea                            USA, ProdiGene

 Duckweed       Alpha interferon            Hepatitis B and C                           USA, Biolex
 n.s.           Monoclonal antibody         Lessening the side-effects of               USA, Planet Biotechnology
                                            chemotherapy

 From: TAB report 104, 2005; Armin Spök, Molecular farming on the rise, 2006
Examples of current plant-made
pharmaceutical research


Alfalfa           Plasma proteins
                                                                                                                Eu, 24, 13%
Arabidopsis       Human intrinsic factor (vitamin B12 uptake)

                  Anti-HIV and anti-Herpes simplex antibodies, Microbicides for pulmonary
                  infection
                                                                                                       Canada, 51,
                  MABs for cancer, arthritis, and other autoimmune diseases like Crohn's disease                                                USA, 108,
Corn                                                                                                      28%
                  Vaccines for Hepatitis B transmission and Traveler's disease                                                                    59%
                  TGEV for animal health, Aprotinin for blood loss and heart surgery
                  Vaccines and antibodies for animal disease prevention, Antibodies
                                                                                                           Worldwide release application for PMP plants
Lemma             Human plasminogen for peripheral arterial occlusion, Alpha interferon

Moss              Factor IX for treatment of haemophilia B

                  Alternatives to antibiotics in poultry diets
Rice
                  Lysozyme for gastrointestinal health, topical infections and inflammations

Safflower         Pharmaceuticals and oil-body-based products for oral and dermal delivery                           Others
Spinach           Protective antigen for vaccine against Baccillus anthracis                                          17%
                                                                                                              Rice                              Maize
                  TGF-b glucocerebrosidase for Gaucher's disease                                              5%                                43%
                  Alpha galactosidase for enzyme replacement therapy
                  IgGs for the prevention of dental decay, prevention of common cold, and                    Soya
Tobacco                                                                                                       7%
                  neutralization of chemotherapeutic drug toxicity                                      Oilseed rape
                  GAD 7 cytokines for type 1 Diabetes, IL-10 for inflammatory bowel disease
                                                                                                             9%               Tobacco
                  Glycoprotein B of human cytomegalovirus (hCMV)
                                                                                                                               19%
                  Gastric lipase for cystic fibrosis
Tobacco, Corn
                  Lactoferrin for gastro-intestinal infection and dry eye syndrome                               The types of plant used for PMPs
Tomato, Potato,
                  Edible vaccines against E.coli, Norwalk virus, Hepatitis B, Antimicrobial peptides
Potato tuber

InfoSource is produced by Ag-West Biotech Inc 2003.
    Status of Biotech Pharmaceutical
    Production

•   Erythropoietein               •   Measles                      •   Zonna pellucida ZB3 Protein
•   Human Growth Factor           •   Aprotinin (Protease              (Contraceptive)
•   β-Interferon                      Inhibitor)                   •   Gastric Lipase (Cystic Fibrosis)
•   Malaria epitopes                                               •   Creatine Kinase
                                  •   α-Galactosidase (Fabry’s
•   Streptococcus Surface                                          •   Protein C (Anticoagulant)
                                      Disease)
    Antigen IgA (Dental Caries)                                    •   Neutropenia (Granulocyte
•   Carcinoembryonic Antigen      •   Interferon α 2a and 2b           Macrophage Colony Stimulating
                                  •   Personalized Non-Hodgkin’s       Factor)
•   Colon Cancer Antigens
                                      Lymphoma Vaccines            •   Epidermal Growth Factor
•   Interleukin 10 (Chrohn’s
    Disease; Inflammatory                                          •   α and β Hemoglobin
                                  •   Papilloma Virus Vaccines
    Bowel Syndrome)                                                •   Angiotensin Converting Enzyme
•   Glucocerebrosidase            •   Lysosomal Acid Lipase            (Hypertension)
    (Gaucher’s Disease)               (Woman’s Disease;            •   Insulin Like Growth Factor
•   Interleukin 4                     Atherosclerotic Plaques)         (Diabetes)
•   Urokinase (Breaks Blood       •   Personalized Vaccines for    •   Tissue Necrosis Factor (Rheumatoid
    Clots)                                                             Arthritis)
                                      Follicular Lymphoma
•   Human Serum Albumin                                            •   HIV-1 Peptide
                                  •   α Trichosanthin (HIV)        •   Lactoferrin
•   Rabies Antigens
•   Hepatitis B Surface           •   Diagnostics for Ovarian      •   Substance P (Neuropeptide)
    Antigen                           Cancer                       •   Animal Pharmaceuticals (Vaccines)
•   Rotavirus VP6                 •   Diagnostics for Ecclampsia   •   Feline Parvovirus (Panleukopenia)
•   Labile Eneterotoxin           •   Biomarkers for Alzheimers    •   Canine Parvovirus
    (Botulism)                        Disease                      •   Bovine Foot and Mouth
Commercial Pharma/Industrial Crop Products

                                                         Source of        Commercial            Company           Company Selling ²
     Product           Trade Name        Pharma Crop
                                                          Genes            Purpose ¹           Producing          (product number)
                                                                          Research and
     Aprotinin         AproliZean            Corn           Cow                               ProdiGene             Not available
                                                                         manufacturing
                                                                          Research and       Large Scale Bi   Sigma Chemical Company
     Aprotinin         Apronexin          Tobacco ³         Cow
                                                                         manufacturing           ology               (A6103)
                      Recombinant                                        Research and                         Sigma Chemical Company
      Avidin                                Corn          Chicken                             ProdiGene
                         avidin                                       diagnostic reagent                             (A8706)
  B-glucuronidase                                                        Research and
                      Not available         Corn          Bacteria                            ProdiGene             Not available
       (GUS)                                                          diagnostic reagent
                                                                           Research                           Sigma Chemical Company
     Trypsin            TrypZean            Corn            Cow                               ProdiGene
                                                                      and manufacturing                         (T3568 and T3449)
                                                                                                Ventria       Sigma Chemical Company
   Lactoferrin        Not available         Rice          Human            Research
                                                                                              Bioscience             (L4040)
                                                                                                Ventria       Sigma Chemical Company
    Lysozyme          Not available         Rice          Human            Research
                                                                                              Bioscience             (L1667)




 Notes
 (1)  Information on specific commercial uses of pharmaceutical and industrial crop products is not available to the public. Some have
      potential applications for human or veterinary medicine but none has been approved by the Food and Drug Administration, which
      regulates drugs.
 (2) Most of these products have been commercialized through Sigma Chemical Company, which specializes in products for research
      and diagnostic purposes. AproliZean and B-glucuronidase have been commercialized according to ProdiGene documents, but their
      current availability is unknown.
 (3) Unlike pharmaceutical corn, tobacco itself is not genetically engineered. Rather, a tobacco mosaic virus is engineered to contain
      a gene for aprotinin. The engineered virus then produces aprotinin in tobacco plants infected with the virus.
Representative diseases potentially
treatable with PMPs

          •   Alzheimer’s disease
          •   Arthritis
          •   Cancers
          •   Chronic obstructive pulmonary disease
          •   Crohn’s disease
          •   Cystic fibrosis
          •   Diabetes
          •   Heart disease
          •   Hepatitis C
          •   HIV
          •   Kidney disease
          •   Multiple sclerosis
          •   Obesity
          •   Spinal cord injuries    Source: Phillips 2003
The process of molecular farming


           Golgi complex
                          1,2-mannosidase I


         UDP-GlcNAc
                          GnT1
         Transporter


                          -mannosidase II



                          GnT-II


          UDP-Gal
                          GalT
          Transporter


         CMP-NeuAc
                          SAT
         synthetase


             Complex Type glycan




                                               Recombinant DNA   Transformation &
  Host development                                                                  Cultivation
                                               technology        regeneration




      Production of PMPs                                         Purification       Cell culture and harvest
Strategies for efficient Molecular
farming system: synthetic biology

     Synthetic Biology: the design and construction of new biological parts, devices,
      and systems, and the re-design of existing, natural biological systems for useful
      purposes
     For high yield protein production:
     Fast growing, high yield, stress and pathogen resistant host development
     Strong tissue specific promoter
     Gene silencing inhibition
     Codon usage optimization
     Using translation enhancer
     Efficient vector system development

     Efficient purification:
     Use of tag or fusion protein for efficient purification
     Secretion of protein using signal sequence
     Development of efficient protein purification technique

     Stable & safe protein:
     Glycosylation control and humanization
     ER stress (Unfolded Protein Response) control
     Use of chaperone and redox proteins
     Use of plant secretory and vacuolar systems
Plant expression systems for molecular
farming


  1.   Vegetative systems

  2.   Seed-based systems
      Seed have evolved to naturally store large amounts of
       lipid, protein and carbohydrate
      Seed desiccation allows intact storage for years
      Allow for an inventory of an intermediate between
       production and processing
      Uncouples harvesting from processing
Target product selection


  •   To use plants effectively we should choose:
     Products with very large volume applications (>100kg, eg.
      Lactoferrin, lysozyme)
     Products which are already well-known, even generics (eg. insulin,
      growth hormone – SemBioSys; α-interferon – BioLex)
     Products which have proven difficult to make in other systems (eg.
      sec lgAs – Planet Biotech )

  •   Products that do not make sense currently
     Products that are required in small volumes (eg. Erythropoietin-
      Amgen, approximately 8 ~ 10 kg per year = $3 billion)
     Products that require glycosylation for function (certain lgGs
      require sugars for effecter function)
     Products requiring unusual post-translational modifications (eg.
      amidation, sulfonation etc)
Suitable host system for protein production


5 weeks                    7 weeks




WT   Vector   8   10      WT   Vector   8   10
          Overexpressed              Overexpressed
Modifications of N-linked oligosaccharides
& protein N-glycosylation

Endoplasmic                              Dolichylphosphate
Reticulum

                                         N-Acetylglucosamine



                                         Mannose
Lumen


                                         Glucose

Cytoplasm

                                         Fucose



                                         Galactose

Golgi Complex
                                         Sialic acid



                                             Protein




                                              Polypeptide chain
N-glycosylation in ER; Glc3Man9GlcNAc2
glycans to Man8GlcNAc2 glycans

                                       Ribosome




                 Dolichol
                                                                    mRNA




                                   Oligosaccharyl                    -Glucosidase I
                                   Transferase          Calnexin
                                                        Folding      -Glucosidase II

                                   Endoplasmic
                                    Reticulum                         ER -Mannosidase



           Glc3Man9GlcNAc2
                                                                       Man8GlcNAc2


                                                       Golgi complex


                              : Folded peptide chain    : Glucose


                            : GlcNAc    : Mannose      : Phosphoric acid
Different N-glycosylation in Golgi-
complexes
                                                           Ribosome




                                        Dolichol
                                                                                      mRNA




                                                        Oligosaccharyl                   -Glucosidase I                      : Folded peptide chain
                                                        Transferase        Calnexin
                                                                                         -Glucosidase II
                                                                           Folding
                                                                                                                            : GlcNAc      : Mannose

                                                      Endoplasmic                                                           : Galactose    : Sialic acid
                                                                                         ER -Mannosidase
                                                      Reticulum
                                                                                                                            : Glucose     : Phosphoric acid
                              Glc3Man9GlcNAc2                                            Man8GlcNAc2




           Mammalian Golgi                                  Plant Golgi                                     Yeast Golgi
                     -1,2-Mannosidase I                               -1,2-Mannosidase I       -1,6-Mannosyl
                                                                                                 Transferase (OCH1)

                                                   UDP-GlcNAc          N-acetyl-glucosaminyl-
      UDP-GlcNAc
                     GnT-I                         Transporter         transferase I (GnT-I)
      Transporter
                                                                                                 -1,3-Mannosyl
                                                                                                 Transferase
                     -Mannosidase II                                  -Mannosidase II          (MNN1)
                                                                          ⇒ Fuc-Transferase                            -1,2-Mannosyl
                                                                                                                       Transferase
                                                                  F                              Mannose-6-            (MNN2,5?)
                     GnT-II                                            GnT-II
                                                                                                 Phosphate
                                                                                                 Synthesis
       UDP-Gal                                                    F                              (MNN4,6 and others)
                     GalT                                              Xyl-Transferase
       Transporter
                                                                                                                                  n

      CMP-NeuAc                                                  F X   N-acetyl-Glucosaminidase
                     SAT
      synthetase

                                                                 F X
            Complex Type                                 Complex Type                                       Mannan Type
Glucocerebrosidase production using
Arabidopsis and rice
Gaucher disease: most common lysosomal
storage disorder




                                    Found by Phillipe Gaucher in 1882




    Glucocerebrosidase deficiency
      (on chromosome 1, at 1q21)




                                            Clinical Features
Enzyme replacement therapy of Gaucher
disease

                                                 GC
                                                                                     (67,00 kDa)

                                                Neuraminidase


                                                 GC


                                               β-Galactosidase


                                                 GC


                                     β-N-acetylglucosaminidase


                                                 GC
                                                                           (60,43 kDa)

                                                GC
                                                      Glucocerebrosidase     Mannose
                                                       Fucose              Galactose
                                                      N-acetylglucosamine N-acetylneuraminic acid



                             1.   Ceredase®: a placentally-derived form, 1991
                             2.   Cerezyme®: a recombinant form, 1994
                             3.   Approved for type 1 Gaucher disease
                             4.   Targeting to the appropriate cells is difficult
                             5.   Does not provide a long life cure
                             6.   Too expensive.
        Developed by
   Roscoe O. Brady in 1973
Mannose-specific glycoprotein uptake of
macrophages via mannose-specific receptor

 CR             CR: Cysteine rich domain
                Mediates Ca2+ independent binding to sulphated N-linked
FNII            sugars

                FNII: Domain containing fibronectin type II repeats
            1
        2
            3   CRDs: Carbohydrate recognition domains
CRDs            Mediates Ca 2+ dependent binding to carbohydrate
        4
                Sugar specificity (monosacchride level):
            5
                L-Fuc=D-Man>D-GlcNac>>>D-Gal
        6
            7   CRD 4,5 and 7 are required for high affinity binding for
                complex sugars with terminal mannose residues
        8

 TM
  CT            TM and CT: Transmembrane domain and cytoplasmic tail
                Contain signals that mediate endocytosis and phagocytosis
Genetic  manipulation   of   plants  for
mannose-terminated glycan production
                                                   Ribosome




                                    Dolichol
                                                                            mRNA




                                                Oligosaccharyl               -Glucosidase I                                    : Folded peptide chain
                                                Transferase      Calnexin
                                                                             -Glucosidase II
                                                                 Folding
                                                                                                                             : GlcNAc      : Mannose

                                               Endoplasmic
                                                                             ER -Mannosidase                                : Galactose      : Sialic acid
                                               Reticulum
                                                                                                                             : Glucose     : Phosphoric acid
                          Glc3Man9GlcNAc2                                    Man8GlcNAc2



       Mammalian Golgi                         Genetically manipulated                          Plant Golgi
                 -1,2-Mannosidase I
                                                     plant Golgi                                             -1,2-Mannosidase I

                                                             -1,2-Mannosidase I                                      N-acetylglucosaminyl-
  UDP-GlcNAc                                                                           UDP-GlcNAc
                 GnTI                                                                                                 transferase I (GnTI)
  Transporter                                                                          Transporter


                 -Mannosidase II                                                                            -Mannosidase II
                                                 Mannose Type                                                   ⇒ Fuc-Transferase

                                                                                                         F
                 GnT-II                                                                                      GnT-II
                                                          X Δgnt1
   UDP-Gal                                                                                               F
   Transporter   GalT                                                                                        Xyl-Transferase


  CMP-NeuAc
                 SAT                                                                                 F X
  synthetase

                                                                                                     F
Different N-linked glycans structures


           α(2,3)
                     β(1,4)      β(1,2)
                                                                                 α(1,6)
                                          α(1,6)
                                                           β(1,4)     β(1,4)               Human
                                                                                          proteins
                                          α(1,3)

                     β(1,4)      β(1,2)
           α(2,6)




                     α(1,4)      β(1,2)

                                          α(1,6)
                        β(1,3)                             β(1,4)     β(1,4)               Plant
                                                                                          proteins
                                          α(1,3)
                                                         β(1,2)                  α(1,3)
                     α(1,4)      β(1,2)

                        β(1,3)



             ‘Lewis a’ epitope




                    GlcNAc        Man              Gal        NeuAC            Xyl        Fuc
Genetic manipulation of plants for
humanized glycan production

                                                                                                               α(1,6)

                                                                                                               α(1,3)
                                                                                                                           α(1,6)
 α(2,3)                                                                                                                               β(1,4)    β(1,4)           Plant
            β(1,4)      β(1,2)
                                                                                                                                                                proteins
                                                                  α(1,6)                                                   α(1,3)
                                 α(1,6)
                                             β(1,4)    β(1,4)               Human
                                                                           proteins
                                 α(1,3)
                                                                                                                                       Proteins from cgl (gnt1) plant
            β(1,4)      β(1,2)
 α(2,6)
                                                                                               α(1,4)             β(1,2)

                                                                                                       β(1,3)              α(1,6)
                                                                                                                                      β(1,4)    β(1,4)           Plant
                                                                                                                                                                proteins
          α(1,4)        β(1,2)                                                                                             α(1,3)

                                                                                                   α(1,4)         β(1,2)
                                 α(1,6)
               β(1,3)                       β(1,4)     β(1,4)               Plant                      β(1,3)
                                                                           proteins
                                 α(1,3)
                                           β(1,2)                 α(1,3)                           Proteins from β1,2-xylt, α1,3-fuct, (β1,3-galt, α1,4-fuct ) plant
           α(1,4)       β(1,2)

               β(1,3)
                                                                                          α(2,3)
                                                                                                      β(1,4)        β(1,2)
                                                                                                                                                           α(1,6)
                                                                                                                             α(1,6)
                                                                                                                                       β(1,4)     β(1,4)             Plant
                                                                                                                                                                    proteins
                                                                                                                             α(1,3)

                                                                                                      β(1,4)        β(1,2)
          GlcNAc         Man         Gal       NeuAC        Xyl            Fuc            α(2,6)


                                                                                      Proteins from β1,2-xylt, α1,3-fuct, (β1,3-galt, α1,4-fuct), β1,4-GalT, α1,6-FucT plant
Progress of genetic manipulation in
Arabidopsis



1. β1,2-xylt/α1,3-fuct-1: (completed)

2. β1,2-xylt/α1,3-fuct-2: (completed)

3. α1,3-fuct-1/α1,3-fuct-2: (completed)

4. β1,2-xylt/α1,3-fuct-1/α1,3-fuct-2: (completed)

5. β1,2-xylt/α1,3-fuct-1/α1,3-fuct-2/α1,4-fuct: (completed)

6. β1,2-xylt/α1,3-fuct-1/α1,3-fuct-2/β1,3-galt/α1,4-fuct:
                                                    (under construction)
Codon usage optimization for high
glucocerebrosidase expression in plants
    Strong seed specific promoter and signal
    sequence for efficient glycosylation


•    Pea vicilin promoter (7S storage                            Protein storage vacuole

     protein)
•    Rice 1Cys-Prx promoter (aleurone
     layer specific)
•    Rice amylase 3D (germination
                                                                                      Lytic vacuole
     specific)
•    Arabidopsis cruciferin3, 12S
     seed storage protein, contains an
     efficient vacuolar-targeting
     signal in the N-terminal region                                              Apoplasmic space

•    Rice a-globulin, 2S albumin-type
     seed storage protein, contains an
     efficient vacuolar-targeting
                                         The plant secretory and vacuolar systems. Indicated are
                                         endoplasmic reticulum (ER), Golgi, cell wall (CW), protein
     signal in the N-terminal region     storage vacuole (PSV) and lytic vacuole (LV).
                                         Plant Molecular Biology 38: 127–144, 1998.
•    Secretion signal sequences
Unfolded protein response (UPR) in yeast


                                                 Unfolded proteins,
                                                     ER stress


                           HAC1u


                   nucleus
                                             Ire1p

                                                       cytosol

                             HAC1i


                   UPR
                                                         Hac1p




        Induction of ER chaperones, glycosylation/modification,
        and secretion pathway genes
Transcriptional upregulation of ER
chaperones by Xbp1 (Hac1) and ATF6
                                                                                   •    An additional ER kinase
                     ER stress, Unfolded proteins                                       (PERK/PEK)
                                                                                   •    Alternative transcriptional
                                                                                        factors (ATF6, Xbp1)
                                                                                   •    ATF6 is a rER
                                                                                        transmembrane protein
                                                                                        with a luminal sensing
  S. cerevisiae                  Plants                                Mammals          domain, and a C-terminal
                                                                                        cytosolic transcription
                                                                                        transactivation domain
                                                                                        subject to release by
                                                                                        proteolysis by S1P/S2P
                             ATF6???                                       ATF6         proteases.

                   Ire1α Ire1β            Ire1α Ire1β
                                                           Other Ire1
      Ire1p                                              family members?
                                                                                       PEK-1
                                                                    Splicing of ATF6
                                                                    cytosolic domain
                  Xbp-1 mRNA???
                                                        Up-regulation of              elF-2α
                                                         Xbp-1 mRNA               phosphorylation
   HAC1 mRNA                              Xbp-1 mRNA
     splicing                                splicing


                                                                                   Attenuation of
                                                                                  protein synthesis

   Transcriptional upregulation of ER chaperones, glycosylation/modification,
                          and secretion pathway genes
Strategies for efficient Molecular
farming system: synthetic biology
     Synthetic Biology: the design and construction of new biological parts, devices,
      and systems, and the re-design of existing, natural biological systems for useful
      purposes
     For high yield protein production:
     Fast growing, high yield, stress and pathogen resistant host development
     Strong tissue specific promoter
     Gene silencing inhibition
     Codon usage optimization
     Using translation enhancer
     Efficient vector system development

     Efficient purification:
     Use of tag or fusion protein for efficient purification
     Secretion of protein using signal sequence
     Development of efficient protein purification technique

     Stable & safe protein:
     Glycosylation control and humanization
     ER stress (Unfolded Protein Response) control
     Use of chaperone and redox proteins
     Use of plant secretory and vacuolar systems

				
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