Martina Newell-McGloughlinDirector, UC Systemwide Biotech Research and Education Program

• Agronomic Traits– Biotic Stress

• Insect Resistance – Bt, cystatin • Disease Resistance

– Viral- coat protein protection (Papaya ringspot virus) – Bacterial, Fungal, Nematode (Rice blight, rice blast)

• Weed- herbicide tolerance (Striga, orobanchia) – ABiotic Stress

• Drought, Cold, Heat• Poor soil

– Salinity – tomatoes with transport protein– Aluminum -Citric acid

• Yield• Nitrogen Assimilation – nodulation by rhizobia, GDH metab eng• Starch Biosynthesis, O2 Assimilation, photosynthesis/Rubisco

• Quality Traits– Processing– Post harvest loss reduction– Reproduction: sex barriers, male sterility, seedless fruit– Nutrients (Nutraceuticals)

• Macro: Protein (Cassava-ASP), Carbohydrates, Fats, Fiber• Micro: vitamins, minerals, phytochemicals• Anti-nutrients: Phytase, Toxin removal

• Novel Crop Products– Proteins: nutraceuticals, therapeutics, vaccines

• Renewable resources: Biomass conversion, feedstocks, biofuels, phytoremediation

Crop Biotechnology

Source: ISAAA

•Biotech Crops 2008: 125 million hectares (310 million acres up 9.4%) X74 –

•25 countries (15 LDC) 12% increase over 2007, 13.3 M farmers (12 M 2007)

•90% resource-poor LDC farmers (12.3 M - 11.0 M 2007) most Bt cotton •New: Egypt, Burkina Faso, Paraguay , Uruguay. •India Bt cotton up to 7.4 M Ha.

– $44 billion 1996 to 2007, 44% yield gains, 56% reduction costs (including a 359,000 tonne a.i. in pesticides); gains of 141 million tons, would have required 43 million additional hectares

– Environmental pesticide footprint down by 15.4 %. GM reduction in 286 million kg of CO2 emissions equivalent to removing 6 M cars from the roads (Brookes 2008)

– HT- increase in no- till: reduction in erosion, soils much healthier, organic matter, less soil compaction, fuel use down by 20 gals/acre (Fawcett & Towery 2005 )

– CP papaya saved Hawaii papaya industry (and helped organic farmers!) may be the outcome for plum pox –C5 PTGS insurance against typhoid Mary in nurseries

Snap shot

• No natural resistance anywhere so could never breed resistance

• Removes viral reservoir thus protects all growers

Field trial of transgenic 'UH Rainbow' and 'UH SunUp' was established in Puna in 1995. Gonsalves, Mansardt, Ferreira

Plums highly resistant to PPV - System is totally resistant as virus is not harbored unknowingly – Tolerant non biotech trees can harbor virus

Farmers ChoiceFarmers ChoicePapaya ringspot virusPapaya ringspot virus

– China BT rice GM used pesticides fewer than 1/season; conventional rice used pesticides 3.7 times ( Rozell, 2005) Pesticides cost applied to the conventional rice was 8 to 10 times as high as GM. 80% reduction in pesticide use. Significant decrease in adverse health effects – Lives saved

– India Bt cotton decrease insecticide use 70% and increased productivity in 58% (737 kg/Ha)

– Organisms in “Bt crops” fields fared better in trials than those with insecticides Monarch butterflies increase

– Engineering Modified Bt Toxins to Counter Insect Resistance - pink bollworms cadherin mutation bypass with modifed Cry protein Science, Nov. 2007

– BT corn 90% reduction in mycotoxin fungal fumonisins - total US benefit estimated at $23m annually. (Wu, 2006)

– Blight-resistant potato (BASF -Rpi-blb1 and Rpi-blb2 NBS-LRR) -UI study concluded for the major potato-producing regions of the world would be $4.3 billion.

Snapshot

Number 2 in GM production.

17% of the global area of GM plants.

In 2007, 98% of soybean in Argentina was GM.

Yield have reached over 6 tonns per hectare

In 1994-95 production costs were 182 dolars/Ha; in 2007 are 117 dolars/Ha

In 1994-95 farmers spent 78 dolars/Ha in herbicides; today they spend 37 dolars/Ha and insecticide use has decrease 90%.

Economical benefits of GM soybean USD$ 20 billion + 1 million jobs

Problems, yes. Due to the economic success of GM soybean and maize, President Cristina Fernádez de Kirchne created a new tax on Gm soybean exports that producers oppose

The Argentinean Case: GM crops an engine of economical development

Between 2001-05 the chinese government spent 15 billion US dólars on AgBiotech projects; 2006-10 a 400% increase has been projected

National Biotechnology program work on the development of over 130 varieties of GM rice and 55 varieties of GM cotton

10 GM products have been aproved for human consumption (rice, maize, soybean and potato). Bt and disease resistent rice is commercially planted in China.

GM cotton was used by 7.1 million small farmers in 3.8 million hectares in 2007 with an economical benefit of USD$ 817 million in 2006.

China is awakening

Spain: farmer Jose Victor Nogues” After 5 years of GM crops in the area, most people can appreciate the huge benefits and lack of negative effects -Introducing GM maize was definitely the way forward

France: Thierry de l'Escaille, European Landowners' Organization, - “wide-scale adoption of these three biotech crops in Europe could significantly increase annual production, improve farmer income by more than 1 billion Euros (US$1.18 billion) and reduce spraying practices. With results like these, it's easy to understand why farmers want access to this new technology," said l'Escaille

Bt corn farmers earn about $85 more per acre, ISAAA (2006), while at the same time producing a healthier feed that is better for the environment.

Romania: Buzdugan farmers reported price premiums of up to 10 percent for biotech soybeans due to fewer weed impurities. Average price gain 2%. Production gain 29-33.5K M tonnes (16-19% ) Earnings increase 35 million and 62.4 million euros (2003).I can tell you that soybean farmers in Romania are very interested in biotech seeds," “Although the seeds are 10 to 15 percent more expensive, the income gains make the extra cost more than worthwhile”

– Transgenic breeding lines preferred because resistance can be introduced into commercial lines with greater speed. The BASF GM potatoes involves the use of two broad spectrum resistance genes, Rpi-blb1 and Rpi-blb2. These two genes have a structure associated with regulatory genes called nucleotide binding site-leucine rich repeat (NBS-LRR) class of regulatory proteins. Many disease resistance genes code for proteins of that class. Numerous plant NBS-LRR genes are present in the typical plant genome, each protein is specific for a particular pathogen signaling a defense response, frequently a localized plant cell death called hypersensitive response. The C terminus of the protein containing LRR recognizes a ligand feature of a pathogen activating the NBS signaling module to initiate the defense response. The blight fungus suppresses the potato defense genes in sensitive plants but is thwarted by successful defense genes.

– Blight-resistant potato (BASF -Rpi-blb1 and Rpi-blb2 NBS-LRR) -UI study concluded for the major potato-producing regions of the world would be $4.3 billion.

Blight-resistant potato

11

“Resistance” Genes natural and otherwise• Rootknot nematodes R in tomato (Mi) and (aphids). Alternate to

fumigation (Williamson UC Davis)

• Xylella fastidiosa Diffusible signal factor (Dsf) for disrupting Xf colonization, Inhibition of Xf polygalacturonase (PG). Targeting other Xf proteins required for virulence. protein/peptide-based inhibitors of pilins and adhesins – alternate to malathion vector control

• Xa21 rice R gene confers resistance to several Xoo. Defense response triggered by Xo molecule, AvrXa21. Transgenic more resistant due to copy number (Ronald)

• Apple Fireblight (E. amylovora) -antibiotic sprays, cecropin lytic peptide analog worked well in field trials (Norelli)

• Apple scab (V. inaequalis) Multiple applications of fungicides used exclusively to control this disease. 'McIntosh' trees expressing either the endo- or exochitinase gene or both increased resistance to apple scab

• Use of apoptosis inhibition to protect plants from mycotoxin damage (Gilchrist, UC Davis)

• Sclerotinia-resist sunflower oxalate oxidase Pioneer wheat

• Zinc Fingers Dow/Sangamo

Natural Disease Resistance Genes Have Been Cloned

Xa21 gene has been cloned from an African rice variety and introduced into modern cultivars to confer resistance to rice leaf blight. Pam Ronald, Plant Pathology, UC Davis

Use of apoptosis inhibition to protect plants from mycotoxin damage David Gilchrist, Plant pathology, UC Davis

David Gilchrist, Plant pathology, UC Davis

. Rice staple food for 2.4 billion people

• Fungal diseases destroy 50 million metric tons of rice per year; varieties being developed resistant to fungi - proteins with anti-fungal properties.

• Insects cause a 26 million tons loss of rice per year; insecticidal proteins environmentally friendly control.

• Viral diseases devastate 10 million tons of rice per year; Tungro virus genome transgenes defense systems. Cassava Mosaic Virus similar protection system as papaya working in Kenya

• Bacterial diseases cause comparable losses - transgenes such as cecropin lytic peptide basis for inbuilt resistance.

• Striga (Scrophulariaceae) is a• genus of obligate root-parasitic• flowering plants.

• All of the cultivated food-crop • cereals in Africa are parasitized • by one or more Striga spp.

• Striga spp. in the savanna • regions alone account for • $7 billion and are • detrimental to the lives of over

100 million African people.

STRIGA

Host plants release factors required by parasitic plants Striga causes massive losses to crops in Africa:control strategy to inactivate host recognition factors - John Yoder, UC Davis,

Host plants release factors required by parasitic plants: control strategy to inactivate host recognition factors

DMBQ

sorgoleone

Host plants Parasitic plants

maizecowpea

Arabidopsis

StrigaOrobancheTriphysaria

Seed germination

0 h 12 h 24 h

Haustorium development

+ Gene E+ Gene EControlControl+ Gene D+ Gene DControlControl

Abiotic Stress: Abiotic Stress: Drought, ColdHeat, SalinityDrought, ColdHeat, Salinity

Abiotic Stress: Abiotic Stress: Drought, ColdHeat, SalinityDrought, ColdHeat, Salinity

Drought Stressed RiceDrought Stressed Rice

Abiotic stress limiting factor for crops reaching genetic potential

Improved water conservation -Fewer crop losses -Higher yields on all acres through improved water utilization -Expand in drylands - Nuclear Factor Y B subunit

Crops limited by salinity on 40 % world's irrigated land (25 % US)

Cold: Engineering with COR15a Tf, role in freezing tolerance.

Plants engineered with Choline oxidase (codA) soil tolerated saline and cold

Homeodomain-leucine zipper (HD-Zip) transcription factors respond to H2O & osmotic stress, exongenous abscisic acid

Transport protein. Grow and fruit even in irrigation water that is > 50X saltier than normal. > 1/3 salty as seawater. Blumwald and Zhang)

Abiotic stress limiting factor for crops reaching genetic potential

Improved water conservation -Fewer crop losses -Higher yields on all acres through improved water utilization -Expand in drylands - Nuclear Factor Y B subunit

Crops limited by salinity on 40 % world's irrigated land (25 % US)

Cold: Engineering with COR15a Tf, role in freezing tolerance.

Plants engineered with Choline oxidase (codA) soil tolerated saline and cold

Homeodomain-leucine zipper (HD-Zip) transcription factors respond to H2O & osmotic stress, exongenous abscisic acid

Transport protein. Grow and fruit even in irrigation water that is > 50X saltier than normal. > 1/3 salty as seawater. Blumwald and Zhang)

- Improve Nitrogen Assimilation- Increase Sucrose hydrolysis,

Starch biosynthesis- Increase O2 availability - Modify photosynthesis

Yield Gene Control

Increased Yields

This short-day sorghum plant was used to map the Ma-1 gene (genes which modify photoperiodic behavior and thus maturity). This gene which works in other cereals would offer particular benefits to biomass and forage crops in which flowering is undesirable

BioFuelsBioFuels

• LDC 30 % global energy. Growth driven by population and economic . Of the world's 47 poorest countries, 38 are net oil importers, and 25 import all of their oil, consuming much of their national income to pay for it.

• The challenge: 5-10 times more efficient - 2001 $5/gal -2005 18c/gal• Biomass Conversion:Biomass Conversion: Organic polymeric material, lignin, starches

celluloses bioconverted ethanol; hemicellulose hydrolyzed to sugars, xylose , glucose .

• Modify Plants and algae to improve enzymatic conversion. • Modify enzymes to improve conversion and fermentation• Maize other cereals, Switch grass, elephant grass poplars• Biodiesel Biodiesel is biodegradable and non-toxic - alkyl esters made from the

transesterification of vegetable oils or animal fats., (60% less CO2)• Rapeseed, Botryococcus braunii (Bb) colloidal microalgae• Concerns: Food trade off – Efficiency of production – ecological impact

• Agronomic: HT, Disease resistance : increased productivity reduction of plantation establishment , reduced tree losses

• Poplar, Aspen, Pine, Walnut, Cottonwood trialed 57% timber• Cell Cycle: LFY gene, PTFL gene from Populus able to induce

early flowering in poplar.• Phytohormones increase size, biomass, wood quality. GA 20-

oxidase (AT) aspen fast growth (D/H) increas biomass (Eriksson)• Paper: low lignin, faster growing ( Sederoff/Chiang) • Biofuels: low lignin (ArborGen), faster growing • Concerns: robustness/health, gene flow, longevity, similar trees.

China lost track of Bt poplars.• Male Sterility/Flowering inhibition: restrict gene flow, grow

faster and produce more wood, since energy not wasted reproducing

• Tapetum barnase gene prevent pollen development promoters could lead to damage of non-reproductive tissue, (long generation times of conifers are considered).

• RNAi down regulation (full suppression often difficult)• Tissue specific cytotoxic stilbene synthase (STS) competes

chalcone synthase (CHS). Somatic cells STS no competition resveratrol anti-fungal (Höfig et al. 2006)

Seeing the wood for the Trees

• Simultaneously restores soil health and revegetates economical

• University of Georgia poplar trees with merA, from mercury-resistant bacteria. Soil-borne and selected for heavy metals. Absorb from soil, convert to a relatively inert form, and release as vapor. 10X Mercury removal

• Richard Meagher gene from the E. coli soybeans to Arabidopsis pumps arsenic from the soil and stores it in its leaves,

• Bioremediation: Wilfred Chen Riverside developing high-affinity microbial bioadsorbents for heavy metal removal using engineered E. coli with surface-expressed peptide analogues (ECs) of phytochelatin - microbial bioadsorbents - removal of heavy metals - cadmium, mercury, and lead.

Phytoremediation

Modify Fruit Ripening

Fruit ripening modified to Improve Quality and Reduce Postharvest Losses by altering the activity of cell wall enzymes, such as polygalacturonase, involved in softening and deterioration.

The biosynthesis of ethylene, the fruit-ripening hormone, has also been blocked in several ways to delay fruit ripening. Ethylene can then be applied to induce ripening when desired, as is currently done with tomatoes and bananas.

Caffeine-free coffee plants and controlled ripening coffee plants. Coffee and tea plants with improved disease resistance and tolerance of environmental stresses such as cold and drought. Controlling the ripening process will allow the farmer to both control and lengthen the harvest season.

25

Modern Biology / Genomics - A new Research-Paradigm in modern Biology

Genotype / Genes / DNA Inherited Information

Defining an Organism

Phenotype Appearance and Traits

of an Organism

Improved

Crop Plants Genomics

= the Totality of the Informationof all Genes and their Functions

From Genomics to Improved CropsFrom Genomics to Improved CropsThe 2 Phases of BiologyThe 2 Phases of Biology

B i o i n f o r m a t i c s

GeneRNAProteinsMetabolitesOrganism

DNA SequenceMap Transcriptome

Proteome

Metabolome

Profiling

PhenotypeGenotypeReverse Genetics

Forward Genetics

Phase 1

New Plant Traits

Improved Crops

Phase 2

Molecular Breeding

Transgenics Genomics Platform

Metabolic Pathways are the target for complex modifications

Genome

Transcriptome

Proteome

Metabolome

Unraveling protein collaborations could change the way pathways are manipulated for improvement of valuable traits in plants. Attempts to modify storage proteins or secondary metabolic pathways more successful than have alterations of primary and intermediary metabolism

Plant Metabolic Engineering Challenges

Transcription Factors! Maize C1 and R, reg flavonoids aleurone , accumul rate anthocyanins activating the entire path (Bruce al 2000)

Wheat Rescue ancient TFs NAC (Uauy 2006)

• ID rate-limiting steps in synthesis

• Target to channel metab flow into new paths,

• Gene-silencing reduce or eliminate undesirable comps, traits, or switch off genes to inc desirable

• Direct DNA cassette Paul Christou 2009

• Mod storage proteins or 2nd metabolic paths > success than alt 1ry and intermed metabolism (ILSI, 2004/ 2008).

Other Considerations•Site of synthesis•Site of activity•Charge and size•Redundancy

Enzymes and Intermediaries known- Little known on controls and integration

MetabolicPathway

Engineering

Beware the flux! High Lysine maize

• Photo: Fruc-1, 6-

Bisphosphatase Phosphoribulokinase ↓3X ↓10X, minor photosynth rate

• Plastid aldolase, catalyzes reverse reaction not subject to allosteric regulate, signif ↓↓ in photosynth rate & C partitioning (Haake et al., 1998)

• Best: Mod single genes or series ind enzy. steps.

• Conversion of exist comp to another rather than change flux through path

Many common food crops not perfect for nutritional requirements of humans or animals.

Proteins: Maize, wheat, Sweet potato and cassava

WHO: 800 million people suffer from malnutrition, Protein-energy malnutrition (PEM), the most lethal form, affects 1 in 4 children: 150 M (26.7%) underweight; 182 M stunted.

70% live in Asia, 26% Africa, 4% Latin America, Caribbean

• Grains low in Lysine – LDCs food - Feed Rations/pollution

• High Lysine maize: Use non feedback- enzyme (5X ppm)• N assimilation modified pathway GDH 12% increase protein

• SRP Nonallergenic Amaranthus Albumin for potato

• High Protein: Cytokinin rescue flower pair kernels fused single kernel two embryos - high protein/oil low CHO

• NAC Tfs (NAM) senescence and nutrient remobilization leaves to grains, RNAi delay senes 30% protein, Zn, Fe

• Artificial Proteins: • ASP-1-sweet potato 67% increase protein (EAA 80%)

Improved Nutritional Content

Improved Nutritional ContentCarbohydrates

• Starch High Amylose (resistant starch) inhibit 2 SBE• Sorbitol role in fruit carbon metabolism and affects quality

attributes sugar-acid balance and starch accum

• Wheat puroindoline genes in rice better starch/flourFibre – Humans increase

• Polymers, Inulins, Fructo-oligosaccharides (FOS)

• SC Fructans sucrose taste: GI Tract health- fermented colonic – bifidobacteria (compete pathogenic bacteria)SC Fatty acid – anticancer/ inhibit HMG-CoAR less LDL

• SC fructans 1-SST Jerusalem artichoke. 90% sucrose converted "fructan beets“ (Koops, 2000)

• Potato synthesize the full spectrum of inulins from globe artichoke roots

• Lignans: enterodiol/lactone estrogen-dependent cancer

Fibre – Animals Decrease

• Brown midrib (COMT)–Decreased lignin increase digestibility better feed conversion, livestock prefer (Sorghum)

Improved Nutritional Content

Oils and Fatty acids

• Altering chain length and saturation level•

• Novel genes to produce unusual fatty acids in oilseed

• MUFA: High Oleic Acid: more stable than PUFA heat/ oxidation resistant, little or no postrefining (hydrogenation): AS oleate desaturase soybean gave >80% oleic acid (23%), Less SF milk/meat of animals

• MCT: medical foods, ergogenic aids. Acyl-ACPT canola, increase in capric (C10) and caprylic (C8)

• High-CLA: Antiox- free radicals heart disease/cancer

• Omega -3 DHA-EPA “Fish Oil” CV/thrombosis/ Cancer/ Arthritis/ Cognitive/Mental/ premies - 6 Desaturase: Canola/soybean precursor SDA 3.6X ALA in generating EPA

• GLA safflower oil (C18:3n-6) anti-inflammatory effect, improved skin health and maintaining weight loss

• Sitostanol: phytosterol phospholipid Block cholesterol

Slide courtesy of Bruce Chassy

Improved Nutritional ContentMicro nutrients Vitamins/ minerals:

• Vit A Golden rice II -carotene-Rice 25X (CBP)• Biofortified cassva flour- Field trial Nigeria (Sayre)

• Vit B Folate increase in rice (pregnancy deficinicies)• Vit E tocopherol -TMT; Vit C increase corn DHARMinerals: Ferritin (bean S protein), Metallothionein (Rice,

wheat). Ca/proton antiporter (sCAX) Ca transport into vacuoles. Ca-fortified carrots enhanced absorption.

Multi vitamin Corn• Combinatorial direct DNA transformation rapid production

of multi-complex metabolic pathways • transferred 5 constructs controlled by different endosperm-

specific promoters into white maize. Different enzyme combinations show distinct metabolic phenotypes – resulting in

• 169X beta carotene (60 g/g v. 14 by breeding)• 6X vitamin C, and • 2X folate (Christou, 2009)

(bacteria)

(daffodil)

(daffodil)

Introduced enzyme

(maize)

Improved Nutritional Content

Functional components - greater than nutrient value alone

Phytochemicals: • Carotenoids: Golden Rice, Sweet Potato, Cassava - (sight, Carotenoids: Golden Rice, Sweet Potato, Cassava - (sight,

development)development)• Lycopene: polyamine Tomato – (reduce LDL, cancerLycopene: polyamine Tomato – (reduce LDL, cancer))• Isoflavones: genistein and daidzein; IsothiocyanatesIsoflavones: genistein and daidzein; Isothiocyanates• Phenolics: resveratrol antioxidant Sirtuins (anti-aging)Phenolics: resveratrol antioxidant Sirtuins (anti-aging)

polyphenol oxidase : help sequester protein during ensiling,polyphenol oxidase : help sequester protein during ensiling,• Gallic acid hydrolyzable tannins, sequester protein in the Gallic acid hydrolyzable tannins, sequester protein in the

rumen, more efficiently absorptionrumen, more efficiently absorption• Flavanols: Catechins, Flavones: quercetin (less adjuncts) Flavanols: Catechins, Flavones: quercetin (less adjuncts)

Anti-nutrients: Trypsin Inhibitors; oxalic acid; furans; Phytate, Bioavailability Phosphate, divalent ions: Phytase (Rice, alfalfa)

Allergens: soy P34 removal; peanut; gluten digestion

Toxins: Glycoalkaloid (potato) AS solanine• Cyanogenic glucoside (cassava) hydroxynitrile lyase

Ye et al. (2000) Science 287: 303-305.

“Golden” rice

• Over 120 million children worldwide are deficient in vitamin A. Rice engineered to for -carotene, converted to vit A in the body. This trait in rice cultivars distributed worldwide could prevent 1 to 2 M deaths each year.

• Golden Rice 23X rice 1.

Increased -Carotene in Rice Grains

(bacteria)

(daffodil)

Normal rice

(daffodil)

Introduced enzyme(source)

UCDAVIS

Ferritin, an iron-rich bean storage protein,

Phytase, an enzyme that breaks down phytate making Fe available, reabsorption of iron, a gene for a cystein-rich metallothionein-like protein has been engineered into rice

A lack of dietary iron, zinc and calcium results in unhealthy increases in cadmium uptake into the kidney and liver

Seeds Phytoene Synthase Canola

Carotenoids are Elevated 40 Times Oil

(maize)

“Golden” rice 2

38

Nutrigenomics Nutritional Genomics "Leave your drugs in the chemist's pot if you can heal the patient with food." (Hippocrates)

• Nutrigenomics refers to the prospective analysis of differences among nutrients in the regulation of gene expression

• Nutrigenomics/Nutritional Genomics/ Nutrigenetics?: Genetically based, nutrition intervention that maximizes the health and effectiveness of each individual.

• Monogenic Diseases: 97% of the “disease-associated” genes

• Phenylketonuria, phenylalanine hydroxylase PHE -> TYR. Leads to neurological damage and mental retardation. PHE restricted TYR-supplemented diets -no Aspartame!

• Lactose Intolerance – Juvenile enzyme active into adulthood

• Polygenic diseases obesity, cancer, diabetes, and cardiovascular diseases,

• SNP in haemochromatosis linked gene (HFE) risk,

• MTHFR PM higher intakes of folic acid serum homocysteine

• Most dietary effect specific interactions on molecular level, regulation of gene expression directly and indirectly activity of transcription factors

Nancy Fogg-Johnson: Nutrigenomics will revolutionize health and nutrition – It will inform how we prevent and treat disease and how food is grown, processed, and made. Eventually nutrigenomics will be able to discover diets that prevent or retard the onset of the most serious and widespread of today's killer diseases, like cancer, as well as degenerative diseases like Alzheimer's. When/if?

• Sitosterolemia (hyperabsorption of sterols hypercholesterolemia risk for atherosclerosis). Regulation of sterol uptake

• Mice was treated with a lipid metabolism-altering drug • DNA microarray used for expression profiling of various tissues. • Differential display with a control led to the discovery of an unknown gene. • Computer simulation found that two proteins gene regulated reverse transport of dietary sterols out of the apical surface of intestinal cells.

• Exploring human gene databases, found a human homologue • This explained why dietary sterols, which are structurally similar to cholesterol, are not absorbed in normal individuals.

• By scanning sitosterolemic individuals for this gene, it was found that all of them had a mutation in this gene responsible for their uncontrolled hyperabsorption of dietary sterols.

Nutrigenomics Nutritional Genomics •Number of genes regulate lipid metabolism/insulin sensitivity, affect susceptibility to T2 diabetes mellitus.

•SREBP-1c (sterol response element binding protein) mutations led to fatty livers, hypertriglyceridemia, severe insulin resistance, type 2 diabetes mellitus. One polymorph expression highly induced in mice on high fructose diets. Two missense mutations in exons domain of SREBP-1c were found in individuals displaying severe insulin resistance. Another association was found between an intronic single nucleotide polymorphism (C/T) between exons 18c and 19c and the onset of diabetes in men, but not in women.

•T2DM Asian/Hispanic populations insulin resistance rather than -cell dysfunction. In African-Americans the opposite.

•Hyperlipidemia: E4 allele in the apolipoprotein E higher LDL compared with the other (E1, E2, E3) for same fat intake levels

Nutrigenomics Nutritional Genomics •One single nucleotide polymorphism (-75 G/A) in the apolipoprotein A1 gene in women is associated with an increase in HDL . ApoA1 women showed increase in HDL with increase in PUFA compared to G variant taking similar amounts of PUFA.

•Haplotype (HapK) in leukotriene A4 hydrolase (LTA4H) risk of myocardial infarction (MI) European and African Americans. MI significantly greater in African-Americans HapK. (n6/n3)

•Barbecue Heterocyclic aromatic amines acetylated to reactive metabolites bind DNA - colon cancers. N-Acetyltransferase NAT is a phase II metabolism enzyme that exists in two forms: NAT1 and NAT2. HAA can be activated through acetylation to reactive metabolites which bind DNA and cause cancers. Only NAT2 fast acetylators can perform this acetylation. Studies have shown that the NAT2 fast acetylator genotype had a higher risk of developing colon cancer in people who consumed relatively large quantities of red meat.

• "Nutrigenetic Testing: Tests Purchased from Web Sites Mislead Consumers.“• Government Accountability Office (GAO) commercial "nutrigenetic" testing

dubious clinical validity of commercial genetic tests, and unethical practices • Investigators posed as 14 clients used the DNA from just 2 - man (48)-girl

(9mnth). Despite this, the test 'results' were contradictory and warned of risks for various conditions. osteoporosis, heart disease, diabetes and more. Affiliated companies then offer nutritional supplements to stave off these predicted sicknesses — but the pills turn out to be little more than multivitamins, offered with a hefty dose of misleading medical advice. Cost to you a mere $89 to $395! (Nature, 2006)

Caveat Emptor!

Will there be implications for your insurance if you have a susceptibility to heart disease? Will there be implications if you fail to follow a diet to retard the onset of symptoms?

Glycosylation Folding Volume System CommentsComplexity required

GlycosylationRequired

Protein Complexity

Protein Complexity

Volume< 100KGs

Volume<100KGs

Volume> 100KGs

Volume> 100KGs

Cell Culture

(mammalian, Insect, plant)

Microbes

Transgenic Animals

Transgenic Plants

YES

NO

HIGH

HIGH

LOW

LOW

• System well understood unproven

• Appropriate for Mabs, fusion proteins

• Well characterized System (Mammalian)

• Limited capacity Opt Manufacturing strategy

• System early unproven

• Appropriate for Mabs, new direction

• Well characterized System

• Excess capacity

How to Produce a Bio-therapeuticHow to Produce a Bio-therapeutic

The Cow Pock or the Wonderful Effects of the

New Inoculation! James Gillray (1757-1815) Vide--The Publications of ye Anti-Vaccine Society, June 12, 1802, Library of Medicine

Pasteurization Pasteurization

Vaccines create antibodies to neutralize the causative virus, bacteria or toxin.Antigens may be introduced by:Killed Vaccines

Killed vaccines designed to create antibodies without the negative effects of infection so are generally considered to be safe. However, during the inactivation process, some of the surface antigens needed to create the desired antibodies may be destroyed thus reducing their effectiveness.

Modified live vaccines contain an attenuated or weakened version of a disease agent. Modified live vaccines are effective but can negatively impact the health of the animal. Subunit vaccines use only the necessary parts of the virus to stimulate immunity. Unlike modified live vaccines, subunit vaccines stimulate the immune system to prevent disease without stressing the animal. And unlike killed vaccines, subunit vaccines do a better job of disease prevention as they only contain concentrated amounts of the target antigen. These qualities make subunit vaccines both safe and effective. Plant-made vaccines are a new type of subunit vaccines.

Rinder Pest

Devastating Disease 100% FatalExisting Vaccine: Problems transport, lack of refrigeration, and lack of a simple system for administration. Recombinant product, freeze-dried, abating problems with transportation and handling, administered to scarified skin to regenerate the serum.

The vaccinia virus is attenuated, The recombinant vaccine - two of the viral surface antigens H and F eliminates risk of contracting the disease it is easy to determine if the animal has been vaccinated and is not just a survivor.

Vaccination of cattle results in a high level of immunity, affording protection against test inoculations of 1000 times the lethal dose of rinderpest virus. Tilahun Yilma, UC Davis

• Supply the increasing demand for new biotech drugs (esp. antibodies)– 50 Mabs by 2010

• Significantly decrease unit costs

• Improve patients’ access to biotech medicines

Why Make Pharmaceuticals in

Plants?

• Plants are an efficient producer of proteins– Plants are scalable bioreactors– Plants provide cost advantages to mammalian cell culture systems– 3-5 times faster than mammalian systems

• Plant cells are similar to human cells– Similar protein synthesis machinery– Read the same genetic code– Assemble, fold and secrete complex proteins

Produce protein ( primarily Mabs)

• Correct post-translational modification mostly (unlike Microbes)

• No propagation of human pathogens or other mammalian contaminants; no other mammalian contaminants de novo;

• Asepsis can begin at purification, not inoculation (less go wrong)

• Scale-up utilizes the same technology used in agriculture to-day.

• Faster, cheaper, more convent, more efficient than CHO cells

Why Seeds?• Protein at the highest levels in the harvestable seed.

• Seeds are easier, more economical than whole plants to transport to a processing factory

• Proteins can be extracted and purified in prep. for packaging.

• Seeds can be stored for prolonged periods with protein intact.

• Hundreds of acres of protein-containing seeds could inexpensively double the production CHO bioreactor factory.

Why Plants?

Imbibe seeds to induce expression of -amylase promoter & production of desired protein.

Plants as Chemical Factories

Malting grain model (Ventria, Inc., CA and defunct attempt in MO, now Kansas)

Transform rice withdesired protein controlledby -amylase promoter in the aleurone layer. proteins lactoferrin and lysozyme

Extract and purify proteins from germinating seeds.

Grow rice crop in the field, harvest grain at maturity.

Ray Rodriguez, Molecular and Cellular Biology, UC Davis

Rice Lactoferin Lysozyme Peru 30% Less Diarrhea, Quicker recovery 3/6 days, 1/3 less recurrence

KAREN A. McDONALD/ BRYCE FALK Fellow: MICHAEL A. PLESHA

Plant Based Expression Systems for Efficient Production of Human

TherapeuticsHuman deficiency in alpha-1-antitrypsin (AAT) results in non-smoking related emphysema

The optimized human AAT gene has been cloned into Agrobacterium and is expressed using a chemically

inducible plant viral (CMV) replicon expression system

1a2a H

AAT

AAT

AAT

AAT

RNA 3a RNA AAT

CMV 2a CMV 2bPromoter

CMV 3a AATPromoter

Inducible Promoter CMV 1a

Replicase

Inducer

AAT expression kinetics via agroinfiltration of Nicotiana benthamiana plants will be optimized

Alternate scalable induction methods will be

evaluated

0

250

500

750

Negative Control(Healthy Plant)

pCMV-SPAAT +Inducer; Cotton

pCMV-SPAAT +Inducer; Syringe

Functional AAT (mg / kg FW tissue)

AAT is used as a model human protein; other proteins will

also be expressed and

optimized

Viruses PlantAb protected miceagainst genital herpes similar physical props to MCCremained stable in human exhibited no diff in affinity for binding, neutralizing HSV

Genetic Engineering Technology Allows Production of Novel Products

MetabolicPathways

Active VaccinesTransmissible gastroenteritis virusAntigenCholera/Hep B/banana

Acetylenic &Vernolic Acid Containing Chemical feedstocks

Passive Vaccines Ab enteric bacteria E.coli O157:H7 meat foodborne path

Polyhydroxybuterate biodegradable plastic

The Dow AgroSciences unique Concert™ Plant-Cell-Produced System uses plant cells, rather than whole plants, in a culture medium comprised primarily of water, sugar and salt. This eliminates the possibility of contamination and environment concerns. The Concert™ Plant-Cell-Produced System is totally bio-contained as all production is done in a sealed and sterile production process. This means only the required inputs get into the vaccine and only a safe, non-replicating vaccine comes out.

Alfalfa Plasma Proteins, Foot-and-mouth disease vaccine

Maize Anti- HIV and Anti herpes Simplex Antibodies Microbiocides for pulmonary infection Mabs for cancer, autoimmune disease RA, Vaccines hepatitis B, Norwalk virus (Travelers disease), Vaccines & Mabs for animal, Aprotinin for blood loss, Gastric lipase cystic fibrosis,

Lettuce Vaccines for Hepatitis B Moss Factor IX for hemophilia B Rice Lactoferrin Lysozyme for GI health, Alternatives to abs in

poultry diets, Topical infections, inflammations, B-cell lymphoma vaccine

Safflower Therapeutics and oil-based products for oral/dermal delivery Spinach Protective antigen for vaccine against Bacillus anthracis Soybean Tobacco extensin signal peptide - Anti-HSV-2 (IgG)

Tobacco Non-Hodgkins B-cell lymphoma, TGF-b glucocerebrosidase for Gauchers Syndrome , Alpha galactosidase for enzyme replacement therapy, IgGs for prevention of dental decay, common cold, GAD 7 cytokines for type 1 Diabetes, Colon cancer surface antigen – Fabrazyme fat-storage disorder

Tomato , Potato Banana (someday!)

Edible vaccines: Enterotoxigenic E. coli, Norwalk virus, Hepatitis B, Vibrio cholera, Rabies virus-intact Glycoprotein Antimicrobe peptides,

Wheat Carcinoembryonic antigen - Murine IgG signal peptidePotato Polyhydroxybuterate biodegradable plastic

• potential gene flow to food crops of the same species

• co-mingling of food and non-food crops

• worker exposure to plant material containing active pharmaceutical ingredients (APIs).

• ProdiGene paid $3 million bond for violations in Nebraska and Iowa reimburse the USDA for the costs, involved in disposing of the contaminated crops.

• Now making brazzein ( a sweet protein) in maize!

Concerns?

Physical and biologicalConfinement essentially means keeping the crop and its products on the

land where it was grown until removed for processing, with no inadvertent exposure to the public and minimal exposure of products to workers and the environment.

Identity Preservation: A Closed Loop SystemPreventing co-mingling or gene flow prime directive for industry as well as

regulatory agencies. (No StarLink®, repetition!).

Principles of Confinement

• Potential gene flow to food crops of the same species

• Vectors: Birds, insects, viruses

• Co-mingling of food and non-food crops

• Worker exposure active pharmaceutical ingredients (APIs).

Identity Preservation: A Closed Loop System

• Preventing co-mingling or gene flow prime directive for industry as well as regulatory agencies.

Physical and biological

• Confinement: physical, temporal and biological isolation, as well as appropriate spatial isolation, acreage limitations, dedicated equipment, facilities

• “Trap” Plant borders, Color coded kernels, Male sterile plants, work well - limited to a few species.

• Transformation plastid (not all pollen is plastid-free, illegit recomb none of the transgenes functional in nuclear genome)

• Genetic Use Restriction Technologies, or GURTs

Concerns/Confinement

Depending on the timing, expression of the lethal gene leads either to seeds that are incapable of germinating or to death of the seedling. During seed maturation stage lea promoters ( “late embryogenesis abundant”) activate the genes whose products control and accompany the drying process. Shortly after the seeds have swollen, germination-specific promoters activate the genes whose products break down substances from the storage tissue and transport them to the young seedling.T- GURT seed will germinate only when treated with this inductor chemical. V-GURT seed treated with an inductor that activates the lethal gene. Produce seeds that mature but that are no longer able to germinate and cannot therefore be used for re-sowing.

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